Recreational Water Illnesses

Recreational
Water Illnesses
Edited by
Erica Leoni
Printed Edition of the Special Issue Published in
International Journal of Environmental Research and Public Health
www.mdpi.com/journal/ijerph
Recreational Water Illnesses
Recreational Water Illnesses
Special Issue Editor

Erica Leoni
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

Erica Leoni
University of Bologna
Italy
Editorial Office
MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland
This is a reprint of articles from the Special Issue published online in the open access journal
International Journal of Environmental Research and Public Health (ISSN 1660-4601) from 2018 to 2019
(available at: https://www.mdpi.com/journal/ijerph/special issues/Recreational Water Illnesses)
For citation purposes, cite each article independently as indicated on the article page online and as
indicated below:
LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Article Number,
Page Range.
ISBN 978-3-03897-578-6 (Pbk)

ISBN 978-3-03897-579-3 (PDF)

c 2019 by the authors. Articles in this book are Open Access and distributed under the Creative
Commons Attribution (CC BY) license, which allows users to download, copy and build upon
published articles, as long as the author and publisher are properly credited, which ensures maximum
dissemination and a wider impact of our publications.
The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons
license CC BY-NC-ND.
Contents
About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface to ”Recreational Water Illnesses” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Athena Mavridou, Olga Pappa, Olga Papatzitze, Chrysa Dioli, Anastasia Maria Kefala,
Panagiotis Drossos and Apostolos Beloukas
Exotic Tourist Destinations and Transmission of Infections by Swimming Pools and Hot
Springs—A Literature Review
Reprinted from: IJERPH 2018, 15, 2730, doi:10.3390/ijerph15122730 . . . . . . . . . . . . . . . . . 1
Lucia Bonadonna and Giuseppina La Rosa

A Review and Update on Waterborne Viral Diseases Associated with Swimming Pools
Reprinted from: IJERPH 2019, 16, 166, doi:10.3390/ijerph16020166 . . . . . . . . . . . . . . . . . . 21
Erica Leoni, Federica Catalani, Sofia Marini and Laura Dallolio

Legionellosis Associated with Recreational Waters: A Systematic Review of Cases and
Outbreaks in Swimming Pools, Spa Pools, and Similar Environments
Franciska M. Schets, Harold H. J. L. van den Berg, Harry Vennema, Manon T. M. Pelgrim,
Cees Collé, Saskia A. Rutjes and Willemijn J. Lodder
Norovirus Outbreak Associated with Swimming in a Recreational Lake Not Influenced by
External Human Fecal Sources in The Netherlands, August 2012
Xiaohong Wei, Juntao Li, Shuiping Hou, Conghui Xu, Hao Zhang, Edward Robert Atwill,
Xunde Li, Zhicong Yang and Shouyi Chen
Assessment of Microbiological Safety of Water in Public Swimming Pools in Guangzhou, China
Antonios Papadakis, Dimosthenis Chochlakis, Vassilios Sandalakis, Maria Keramarou,

Yannis Tselentis and Anna Psaroulaki
Legionella spp. Risk Assessment in Recreational and Garden Areas of Hotels
Anthony C. Otigbu, Anna M. Clarke, Justine Fri, Emmanuel O. Akanbi and Henry A. Njom
Antibiotic Sensitivity Profiling and Virulence Potential of Campylobacter jejuni Isolates from
Estuarine Water in the Eastern Cape Province, South Africa
Daniela E. Koeck, Stefanie Huber, Nadera Hanifi, Manfred Köster, Martina B. Schierling and
Christiane Höller
Occurrence of Antibiotic-Resistant Bacteria in Therapy Pools and Surrounding Surfaces
Mahbubul H. Siddiqee, Rebekah Henry, Rebecca Coulthard, Christelle Schang,

Richard Williamson, Rhys Coleman, Graham Rooney, Ana Deletic and David McCarthy
Salmonella enterica Serovar Typhimurium and Escherichia coli Survival in Estuarine Bank
Sediments
v
Asja Korajkic, Brian R. McMinn and Valerie J. Harwood
Relationships between Microbial Indicators and Pathogens in Recreational Water Settings
Laura M. Suppes, Kacey C. Ernst, Leif Abrell and Kelly A. Reynolds

Validation of Questionnaire Methods to Quantify Recreational Water Ingestion
Federica Valeriani, Lory Marika Margarucci and Vincenzo Romano Spica

Recreational Use of Spa Thermal Waters: Criticisms and Perspectives for Innovative Treatments
vi
About the Special Issue Editor
Erica Leoni is Full Professor of Hygiene and Public Health at the Alma Mater Studiorum, University
of Bologna (Italy). She is the academic referent of the Unit of Hygiene, Public Health, and
Medical Statistics of the Department of Biomedical and Neuromotor Sciences of Bologna University.
She obtained her degree in Biological Sciences and her degree in Medicine and Surgery from
the University of Bologna, and her PhD in Microbiology at the University of Parma (Italy).
She participated in continuative didactic activity in the fields of general and applied hygiene in
different Bachelor, Master, and PhD courses at the University of Bologna. She was President and
Coordinator of the Degree Course on Movement Sciences 2007–08 and 2012–13, and a member of the
College of Professors of the Doctorate in “Health, Safety, and Urban Greening”. She has supervised
numerous PhD and post-doctoral students.
Her research work focuses on public health, epidemiology, environmental health, and the
promotion of healthy lifestyles. She is a member of the scientific board of the work group in ”Motor
Sciences for Health” of the National Scientific Society of Hygiene and Preventive Medicine. As part
of this role, she is actively involved in the development of Italian multi-centre studies concerning the
safety of recreational environments and the promotion of healthy lifestyles, in particular, concerning
the promotion of physical activity for primary and tertiary prevention. With reference to the last,
she participates in a European multi-centre study funded by the European Community concerning
Adapted Physical Activity (APA) administered for the tertiary prevention of osteoporosis. In the
2016–2018 biennium, she has been part of the National Committee for the assignment of the National
Scientific Qualification of the Full/Associate University Professors for the subjects of Public Health,
Nursing, and Medical Statistics.
vii
Preface to ”Recreational Water Illnesses”
Swimming and other water-based exercises are excellent ways to practice physical activity and to
gain health and social benefits. However, recreational water use may expose people to different health
risks due to exposure to chemicals or pathogens. The safety of recreational aquatic environments
is affected by numerous variables such as water quality, the health conditions of users, and the
correct functioning of the technological systems used for water treatment. This Special Issue aims
to provide new knowledge on health risks related to recreational waters, together with the need
to update prevention strategies. Such an approach will be essential for addressing the challenges
posed by the increasing use of recreational waters by people with different age and health conditions.
The editor wishes to thank all the contributors and the support of the IJERPH editorial staff, whose
professionalism and dedication have made this Issue possible.
Erica Leoni
ix
International Journal of
Environmental Research
and Public Health
Review
Exotic Tourist Destinations and Transmission of
Infections by Swimming Pools and Hot Springs—A
Literature Review
Athena Mavridou 1, *, Olga Pappa 1,2 , Olga Papatzitze 1,3 , Chrysa Dioli 1 ,
Anastasia Maria Kefala 1 , Panagiotis Drossos 1 and Apostolos Beloukas 1,4
1 Department of Biomedical Sciences, University of West Attica, 12243 Egaleo, Greece;
[email protected] (O.P.); [email protected] (O.P.); [email protected] (C.D.);
[email protected] (A.M.K.); [email protected] (P.D.); [email protected] (A.B.)
2 Central Public Health Laboratory, Hellenic Centre of Disease Control and Prevention,
15123 Maroussi, Greece
3 West Attica General Hospital, “Santa Barbara”, 12351 Santa Barbara, Greece
4 Institute of Infection and Global Health, University of Liverpool, Liverpool L69 3BX, UK
* Correspondence: [email protected]
Received: 5 October 2018; Accepted: 29 November 2018; Published: 3 December 2018
Abstract: A growing number of people undertake international travel, and yet faster growth of
such travel is expected in the tropics. Information on the hazards presented by pool and hot spring
waters in tropical countries is very limited. This review aims to collate available information on pool
water quality, alongside data on cases and outbreaks associated with swimming in pools in tropical
regions affecting both local populations and travellers. Bacteria species commonly causing cases and
outbreaks in the tropics as well as elsewhere in the world were excluded, and the review focuses
on studies related to pathogens that, with the exception of Cryptosporidium, are unusual in more
temperate climates. Studies concerning subtropical countries were included in the light of climate
change. Diseases transmitted by vectors breeding in poorly maintained, neglected or abandoned
pools were also included. 83 studies dealing with Microsporidia, Leptospira spp., Schistosomas spp.,
Cryptosporidium spp., Acanthamoeba spp., Naegleria spp., Clostridium trachomatis, viruses, and vectors
breeding in swimming pool and hot tub waters, and fulfilling predefined criteria, have been included
in our survey of the literature. In conclusion, prevention strategies for pool safety in the tropics are
imperative. Public health authorities need to provide guidance to westerners travelling to exotic
destinations on how to protect their health in swimming pools.
Keywords: swimming pools; tropics; subtropics; pool assessment; infectious diseases
1. Introduction
An increasing number of people undertake international travel for professional, social, recreational
and humanitarian purposes. Nowadays, more people travel greater distances and at greater speed
than ever before, and this upward trend looks set to continue. Internationally, tourist arrivals have
increased from 25 million globally in 1950 to 1235 million in 2016 [1] with travel for leisure and pleasure
accounting for more than half of international tourism arrivals [2].
According to the research project “Tourism Towards 2030”, the number of international tourist
arrivals worldwide will increase by an average of 3.3% per year through to 2030. Greater growth is
expected to occur in the tropical Asian and the Pacific regions, where arrivals are forecast to reach 535
million in 2030 (+4.9% per year). Countries of the tropical zone, such as those located in the Middle
East and Africa regions are expected to double their arrival numbers during the same period, from 61
IJERPH 2018, 15, 2730; doi:10.3390/ijerph15122730 1 www.mdpi.com/journal/ijerph

IJERPH 2018, 15, 2730
and 50 million to 149 and 134 million, respectively [1]. According to World Tourism highlights, 2001
the fastest developing region continues to be East Asia and the Pacific [3].
From a public health perspective, travellers are exposed to a variety of health risks in unfamiliar
environments [2]. International travel can pose severe risks to health, depending both on travellers’
health needs and on the type of travel undertaken. Accidents continue to be the primary cause of
morbidity and mortality for international travellers, but infections also present an important health
risk. Moreover, travellers interact dynamically with microbes and places. Travellers can carry these
microbes and their genetic material, and, as Baker stated,” can play multiple roles with regard to
microbes, as victims, sentinels, couriers, processors, and transmitters of microbial pathogens” [4].
There is abundant information and guidance to travellers regarding precautions that need to
be taken in respect of food, drinking water and air quality in tropical destinations. Nevertheless,
information on the hazards presented by recreational and especially pool, spa and hot spring waters
as a mode of transmission of pathogens is very limited, even though numerous infectious agents may
threaten the health or comfort of pool and hot tub users [5]. As examples, the important World Health
Organization (WHO) document [2] attributes only half, out of 244, pages to precautions related to the
use of recreational waters [2]; the European Network on Imported Infectious Diseases Surveillance
(TravelHealthPro) does not mention recreational waters on their webpage, which provides guidance
to travellers on how to take care of their health [6]; the announced revision of the WHO Guidelines
for recreational waters [3] has been suspended [3]; Page et al. do not refer to pool water in their
outstanding review regarding attitudes of tourists towards water use in the developing world [7].
Climate changes are creating conditions in the subtropical zones similar to the tropics and
these geographical regions were included in the review. For the purposes of our review, we
considered both primary transmission from pool waters and secondary infections spread by the
pool users. Tropical diseases encompass all diseases that occur solely, or principally, in the tropics.
In practice, the term is often taken to refer to infectious diseases that thrive in hot, humid conditions,
such as malaria, leishmaniasis, schistosomiasis, onchocerciasis, lymphatic filariasis, Chagas disease,
African trypanosomiasis, and dengue [8]. Besides the “big three” diseases—malaria, tuberculosis,
HIV/AIDS—which are well known causes of major global mortality, morbidity and burden, the term
“neglected tropical diseases” has been introduced in the literature. They comprise a new field for
travellers’ health and the list includes 40 helminth, bacterial, protozoan, fungal, viral and ectoparasitic
infections affecting local populations in the tropics, which are strongly associated with poverty and
socio-ecological systems, but also presenting a serious health risk for travellers [9]. It is worth noting,
however, that many of the so-called “tropical” diseases are not transmitted through recreational waters.
Objective
This review aims at collating information on pool water quality, and cases and outbreaks related
to swimming in pools and hot springs in tropical and subtropical regions, and at carrying out a
search and review of papers dealing with hazards deriving from the use of pools in the tropical and
subtropical zones.
2. Materials and Methods
Search Strategy/Inclusion Criteria

Cochrane instructions for a systematic search that seeks to identify all studies dealing with
incidences originating in tropical and subtropical countries were followed (https://ph.cochrane.org/
sites/ph.cochrane.org/files/public/uploads/HPPH_systematic_review_handbook.pdf). The studies
were required to meet pre-defined eligibility criteria, a major one being transmission via pool, spa
or hot spring waters or the detection of the pathogens in such waters. Thus, studies reporting
pathogens without confirmed transmission through recreational waters were excluded. For instance,
the Chikungunya virus was the aetiological agent of an outbreak in Kenya in 2004, and major outbreaks
2
IJERPH 2018, 15, 2730
followed in Indian Ocean island countries such as Reunion, Mauritius, Comoros, Seychelles and
Madagascar in 2005 to 2006 [4,10]. Burkholderia pseudomallei has also caused melioidosis in the
tropics [11]. Nevertheless, so far neither of these two pathogens’ transmission has been confirmed to
involve pool waters.
Diseases transmitted by vectors breeding in poorly maintained, neglected or abandoned pools
were also included. As tourism presents seasonality a high number of pools stay inactive for several
months, often still containing the water of the past season. This situation encourages the proliferation
of pathogens and the extensive use of these waters by vectors in order to breed. Thousands of flooded
swimming pools were abandoned in New Orleans following Hurricane Katrina and provided a natural
experiment to examine colonization of a novel aquatic habitat by mosquito larvae and their aquatic
predators [12–15].
A large number of cases and outbreaks described in the literature surely derive from unidentified
sources, among which a number is likely to have been from swimming pools: reports of this kind or
with a preconceived bias regarding the mode of transmission were excluded from this review.
Bacteria genera such as Legionella, Salmonella and Pseudomonas, commonly causing cases and
outbreaks in the tropics, but also in the rest of the world [16,17] were excluded. The review endeavours
to focus on infections transmitted by pool waters and caused by pathogens that, with the exception
of Cryptosporidium spp. [18], are unusual in the moderate climates and common in the tropical and
subtropical countries, including southern regions of Japan and North Australia, as shown in Figure 1.
Figure 1. Map of the world indicating the tropical and subtropical zones.
Other eligibility criteria were: that the studies were published in English, though we did allow a
few notable exceptions to this rule; and that the publications in question were scientific papers and
reviews in scientific journals, national and international public health platforms, and journals and
platforms related to tourism (for example UNWTO). PubMed, Google Scholar, Science Direct, CDC,
ECDC and WHO platform and publications were systematically searched.
Further to the aforesaid eligibility criteria, we reviewed 83 studies on cases and outbreaks in
tropical countries, 45 studies on modes and trends of pathogen transmission and selected outbreaks
in western countries, and 3 studies on trends in the tourist industry. In addition, information was
harvested from official national and international websites. They are presented in groups according to
the pathogen involved. As mentioned above, viruses transmitted by vectors that breed in waters were
also considered as waterborne pathogens.
3
IJERPH 2018, 15, 2730
3. Results
3.1. Assessments of Swimming Pools in Developing Tropical Countries

The repeated reference to health problems deriving from the use of swimming pools could be
related to sub-optimal regulations, which do not address all factors contributing to the swimmers’
well-being in a particular geographical area, or are poorly applied. For instance, countries around the
Mediterranean basin are among the most popular tourist destinations. In a study, investigating pool
and spa regulations in these countries, the conclusions were that the Africa and Middle East countries
of this region possess satisfactory regulations comparable to the regulations of the European countries
in the same area [19]. Similar conclusions were drawn from a report commissioned by Clúster de la
Indústria Química de les Illes Balears in which a few countries worldwide were picked at random and
their regulations presented and compared. Some tropical countries seem to adapt their regulations
to specific issues. For instance, in the Mexican regulation, free living amoebae are included in the
standard as water quality indicators along with bacterial parameters [20]. According to the authors of
both reviews, the major question was whether regulations were applied, and if controls of the water
quality and hygiene in the facilities were carried out by authorities.
Table 1. Assessments of swimming pools (SPs) located in tropical and subtropical countries.
Location/Country Positive Results Ref.

The authors suggest artificial plastic SPs as a prophylactic measure against
North Africa, Egypt [21]
infection with schistosomiasis in developing countries.
In a survey of 2 SPs, which included 50 water samples Dermatophytes, Aspergyllus
North Africa, Assiut Town, Egypt [22]
sp., Penicillium, Altenaria, Syncephalastrium, Mucor were detected.
Assessment of the environmental and health aspects of some SPs. Presence of
North Africa, Alexandria, Egypt [23]
pathogens indicated.
Assessment of 5 SPs, 30 water samples. Compliance of pool water with
North Africa, Alexandria, Egypt regulations regarding bacterial indicators was 56.7%. In 10% of the samples [24]
Cryptosporidium oocysts and Giardia cysts were detected.
Middle East, Ein Feshka, Dead Sea, Medical report of 10 cases of Mycobacterium marinum mimicking leismaniasis.
[25]
Israel Most of the infections were contracted in natural bathing pools.
An assessment of 58 water samples, collected from 46 SPs. All unacceptable
Middle East, West Bank, Palestine [26]
according to regulations. 21/23 water samples were positive for Salmonella spp.
Assessment of 85 SPs in Amman. Compliance of the pools’ water with the
Middle East, Amman, Jordan [27]
microbial parameters was 56.5%.
In a survey of 3 SPs, 50 keratinophilic fungal species were recovered. The most
Middle East, Nablus district, frequently isolated species were Acremonium strictum & Cladosporium
[28]
Palestine cladiosporioides. The most abundant species were Acremonium strictum, and
Aspergillus flavus.
In a survey of 7 SPs, faecal coliforms, E. coli, total heterotrophic bacteria were
Sub Saharan Africa, Ghana recovered from all SPs; E. coli O157:H7 were recovered from 2 SPs. Antibiotic [29]
resistance tests revealed the highest resistance was in sulfamethoxazole (46%).
A survey of 39 municipal SPs revealed protozoa (12.8%), P. aeruginosa (69.2%),
Asia, Guangzhou, China total coliforms, E. coli (4%), Cryptosporidium & Giardia (12.8%), E. coli O157, [30]
Shigella, and Salmonella.
In a survey of 10 indoor SPs, 593 water and environmental samples (shower
Asia, Ahwaz Iran areas, dressing rooms, pool walls, slippers) revealed 372 saprophytic fungi [31]
species and 32 yeasts. The most common were Aspergillus & Penicillium.
In a seasonal assessment of 2 indoor SPs (459 pool water, shower & dressing
room samples) faecal coliform Pseudomonas aeruginosa, Legionella, Escherichia coli
Asia, Shahrekord City, Iran [32]
and Heterotrophic Plate Count values exceeded regulations. The most prevalent
fungi were in the showers, the most frequent being Aspergillus spp. (48.91%).
Information on the monitoring and the assessment of swimming pool waters in countries of the
tropical and subtropical zones is limited (Table 1). It is possible that monitoring is carried out in some
countries, but scarcely few data have been published. From the North Africa countries some studies
have been published from Egypt [21–24] starting in the 1960s. In the Middle East, certain subtropical
4
IJERPH 2018, 15, 2730
countries such as Israel, Palestine and Jordan provided some limited monitoring data [25–27] including
a study from Palestine on the presence of fungi in pool water and facilities [28]. Setsoafia Saba et
al. published a water quality assessment of swimming pools and the risk of spreading infections
in Ghana, which is one of the very rare publications in Sub-Saharan Africa [29]. From the Asian
countries, China [30], and most often Iran [31,32], have published assessments which include bacterial
indicators, some tropical parasites and fungi related to the sanitary quality in the facilities [31,32]. In
light of the above, the aim of the present review is to update our knowledge of waterborne outbreaks
in the tropical and subtropical zones of the main tropical pathogens transmitted through the use of
swimming pools, spas and hot tubs with a particular emphasis on tourist facilities.
3.2. Microsporidia
Microsporidia are newly emerging pathogens of humans and animals. They are tiny obligate
intracellular parasitic fungi and as such are often still managed by diagnostic parasitology laboratories.
Due to the small size of their spores and uncharacteristic staining properties they are difficult to detect.
Accordingly, epidemiological studies to elucidate the sources of human-pathogenic Microsporidia
and their routes of transmission are difficult to perform [33]. Faecal-oral transmission is the likely
route of infection in humans with intestinal microsporidiosis [34]. The last two decades have seen
several publications related to ocular microsporidiosis, in particular those forms affecting the cornea.
Both immunocompetent, immunocompromised and AIDS patients are vulnerable to the acquisition of
microsporidia and especially to keratoconjunctivitis, which is usually seen in immunocompromised
individuals or in contact lens wearers. The organism is widespread in the environment and is
considered a waterborne pathogen [34,35]. Exposure to soil, muddy water, and minor trauma are
possible risk factors.
An analysis of risk factors for microsporidiosis showed that swimming in pools comprises an
additional significant risk factor [36], even though conventional levels of chlorine (1–3 mg/L) used
in swimming pools where water temperatures normally reach or exceed 22 ◦ C should be adequate
to greatly reduce or eliminate the infectivity of microsporidial species E. intestinalis, E. hellem and
E. cuniculi spores after relatively short exposure times [37]. In Paris (France), in a survey of pools for
microsporidia, Cryptosporidium spp. and Giardia spp., microsporidia were detected in only one out of
48 water samples [38].
The tropics seem to host most of the cases of microsporidial keratitis. A high prevalence has
been documented in Singapore [39] and in India [40] and transmission through contact with water has
been suggested. The present review identified 2 published studies (Table 2) clearly relating infection
to the presence of microsporidia in pools in the tropics. In Paris, Curry et al. referred to a case of
an HIV-negative patient from Bangladesh with bilateral keratitis who was found to be infected with
a microsporidial parasite belonging to the genus Nosema. The patient had bathed in a rural pond 7
days prior to the development of ocular symptoms. Nosema parasites are common insect parasites
and the source of this microsporidial infection was possibly from mosquito larvae developing in the
pond in which the patient bathed [41]. In Taipei, Taiwan, a retrospective study included 10 eyes of
9 immunocompetent patients diagnosed with microsporidial keratitis. All of them were known to
contract this disease after bathing in hot springs. The nine patients travelled and bathed in at least four
different spa resorts located in two different areas [42].
3.3. Parasites
Waterborne parasitic protozoan diseases are distributed worldwide and comprise, in both
developed and developing countries, reasons for epidemic and endemic human suffering. Looking at
the trends of the prevalence of parasitic diseases in the developed world a significant decrease has
been observed, which may be attributed to the substantial improvements in data reporting and the
establishment of surveillance systems [43,44]. The highest prevalence of parasitic protozoan infections
is known to occur in developing countries due to lower hygiene standards. In addition, developing
5
IJERPH 2018, 15, 2730
countries that are more likely to be most affected by such waterborne disease outbreaks still lack reliable
surveillance systems, and an international standardization of surveillance and reporting systems has
yet to be established [45]. In 1999, the European Network on Imported Infectious Diseases Surveillance
(TropNetEurop) was set up in order to collate reliable data on imported infectious diseases to Europe
and assess trends over time [46].
A review by Lim et al. provided a comprehensive overview of the available data and studies
on waterborne parasite occurrences among the Association of Southeast Asian Nations (ASEAN),
which is comprised of ten member states (i.e., Brunei Darussalam, Cambodia, Indonesia, Lao People’s
Democratic Republic (PDR), Malaysia, Myanmar, the Philippines, Singapore, Thailand, and Vietnam)
with the aims of identifying ways in which to progress. Many of these countries are booming tourist
destinations. Swimming pools are included as a source of transmission. He points out the fact that
there are massive gaps of knowledge in the occurrence, morbidity and mortality associated with
parasitic diseases [47]. According to a review providing data related to neglected parasitic protozoa in
the tropics reporting that only an estimated 1% of global outbreaks of waterborne parasitic protozoa
outbreaks have occurred in Asia, it is evident that there is a paucity of information from this region
where organized mechanisms of documentation of parasitic infections or waterborne outbreaks are
lacking [48]. Cryptosporidium, Amoebae and Schistosoma spp. are the parasites with the highest public
health significance when swimming pools are the route of transmission.
3.3.1. Schistosoma spp.

Schistosomiasis is caused by diagenetic blood trematodes. The three main species infecting
humans are Schistosoma haematobium, S. japonicum, and S. mansoni. Two other species, more localized
geographically, are S. mekongi and S. intercalatum. Other species of schistosomes, which parasitize birds
and mammals, can cause cercarial dermatitis in humans [49]. Acute schistosomiasis was first described
in 1847 in the prefecture of Katayama, Hiroshima district, Japan. A woman brought to the region to be
married was found to become acutely unwell with a fever after she had been exposed to fresh water.
Snails in fresh waters contribute to the life cycle of Schistosoma as, under optimal conditions, the eggs
hatch and release miracidia, which swim and penetrate specific snail intermediate hosts [49].
Schistosomiasis has been rare in Europe and there is very limited published literature dealing
with relevant outbreaks. In Corsica one outbreak involving 120 people infected after swimming in a
fresh water swimming pool is one of the rare published cases [50]. Nevertheless, schistosomiasis is
increasingly imported into temperate climates by immigrants and travellers to endemic areas [51–54].
Schistosomiasis in returning travellers is one of the most common imported tropical infections with
potentially serious complications, which are preventable upon early diagnosis [55]. Human contact
with water is required for infection by schistosomes.
Grobusch et al. studied imported schistosomiasis in Europe by seeking data from TropNetEurop.
Three hundred and thirty-three reports of schistosomiasis have been analysed for their epidemiological
and clinical features. The majority of patients were of European origin (53%), who travelled
predominantly for tourism to endemic areas (52%). The majority of infections were acquired in
Africa; 92 (%) infections were attributed to Schistosoma haematobium [56]. However, in a 15-year
observational study at the Hospital for Tropical Diseases, London, the prevalence of schistosomiasis in
presenting travellers is decreasing with predominant species S. haematobium [55].
Schistosomiasis is one of the endemic diseases that take advantage of environmental modifications
due to water conveyance in the Saharan countries, for example Burgina Faso [57]. In Egypt, risk factors
for S. haematobium infection were male gender, an age <21 years old, living in small communities, and
exposure to canal water [58].
One of the first published reports on an epidemic of acute schistosomiasis concerned travellers
returning from Mali. Imported schistosomiasis acquired in the Dogon country in Mali, West Africa,
was first demonstrated in 1989 in three Spanish travellers [59]. More recently, 79 cases of acute
schistosomiasis were reported by the Hospital of Tropical Diseases, London, between 1998 and 2012.
6
IJERPH 2018, 15, 2730
Most of these cases were young, male travellers who acquired their infection in Lake Malawi (53%).
Most of the other cases were from West Africa, with only 13% acquiring their disease in East Africa,
one in North Africa (Libya), and two in the Middle East (Saudi Arabia, Yemen). Most were on holiday
(68%), while 16% had been working as volunteers. All of them reported contact fresh water in an area
where schistosomiasis is endemic [60].
The present review identified five published studies clearly regarding schistosomiasis transmitted
via swimming pools in the tropics, of which three were related to tourism (Table 2). In 1993, a
35-year-old Belgian woman was admitted to the University Hospital of Antwerp with schistosomiasis
symptoms. She had swum with a group of travellers in a water pool in the Dongon valley in Mali.
Sixty-two per cent (eight people) of the 13 travellers had acquired Schistosoma infection; seven of
them had developed Katayama syndrome. All travellers, except one, who acquired a Schistomoma
infection, had swum for at least 5 min in the pool [61]. In a study from the area of Belo Horizonte,
Brazil, a group of 18 individuals was included. They had the impression that the water was clean
and no snails were observed. S. mansoni was transmitted from non-symptomatic positive residents
through infected intermediate hosts to visitors. The visitors came from an urban area who had never
had contact with the disease before and who developed acute schistosomiasis [62]. Also in Brazil,
transmission occurred in a non-endemic area of Brazil, which became a new point of transmission
due to the immigration of infected workers [63]. In Upper Benue Valley in Cameroon, swimming
in a pool for the local population was significantly associated with schistosomiasis infection [64].
The Department of Infectious Diseases, University Hospital of Leiden, The Netherlands, reported an
outbreak of schistosomiasis among non-immunized travellers. Of 30 travellers in two consecutive
groups, 29 who had swum in freshwater pools in the Dogon area of Mali, West Africa, were monitored
for 12 months. Twenty-eight (97%) of those became infected; 10 (36%) of the 28 had cercarial dermatitis,
and in 15 (54%), Katayama fever developed [65].
3.3.2. Cryptosporidium spp.

Transmission of Cryptosporidium has been on the increase over the last two decades. Currently,
31 valid Cryptosporidium species have been recognized and of these more than 17 have been found
to infect humans. The most commonly reported species in humans worldwide are C. parvum and
C. hominis [66]. This parasite has a low infectious dose, a small size that enables it to bypass water
filtration systems, and resistance to chlorine disinfection at levels routinely used at swimming pools,
water parks, and interactive fountains. It is the leading cause of outbreaks associated with disinfected
recreational water and has also caused outbreaks in child care facilities. Cryptosporidium has the ability
to cause community-wide outbreaks when transmitted in these venues [67] Swimming pool associated
cases and outbreaks of cryptosporidiasis have been reported abundantly in the western world [68–72].
The burden of cryptosporidiosis is higher in tropical countries. In Australia, for instance,
cryptosporidiosis seems to be an endemic problem in warm, remote areas and in Aboriginal and
Torres Strait Islander population-dominated regions [73]. The most recent Global Burden of Disease
Study listed Cryptosporidium as an important cause of disease and death of children under 5 years
of age in Sub-Saharan Africa [74]. From 2004 to 2010, 199 outbreaks of human gastroenteritis due
to the waterborne transmission of 59 enteric parasitic protozoa were reported worldwide and of
these, Cryptosporidium spp. was the etiological agent in 60.3% of the outbreaks [60,61]. Bathing
in contaminated swimming and therapeutic pools is a major mode of waterborne transmission of
Cryptosporidium and other pathogens [75].
In a recently published review, Ryan et al. found that the necessary key barriers to limiting
swimming-pool associated outbreaks of cryptosporidiosis (lack of uniform national and international
standards, poor adherence and understanding of regulations governing staff and patron behaviour,
and low levels of public knowledge and awareness) are not widely applied [76]. The present review
identified three published studies clearly reporting cryptosporidiosis transmitted via swimming pools,
or reporting detection of Cryptosporidium in pool waters, in tropical countries (Table 2).
7
IJERPH 2018, 15, 2730
A study of 35 pools in Beijing, including some hotel pools, showed that 16.7% and 15% were
positive for Cryptosoridium oocysts and Giardia cysts, respectively [66]. Also, in the Philippines, in a
total of 33 water samples taken from various environmental sources, including swimming pools, 45.5%
were positive for Cryptosporidium. Two hundred seventy three children developed cryptosporidiosis
after using a pool. Later on the same children used 10 swimming pools in a different prefecture and
four of them were infected [77]. In Broom, Western Australia, another outbreak of cryptosporidiosis
involving children who swam at the public pool was described [78].
3.3.3. Acanthamoeba, Naegleria Species

Free-living amoebae belonging to the genera Acanthamoeba, Balamuthia, Naegleria and Sappinia
are important causes of disease in humans and animals. Naegleria fowleri produces an acute, and
usually lethal, central nervous system (CNS) disease called primary Amoebic meningoencephalitis.
Acanthamoeba spp. are opportunistic free-living amoebae capable of causing granulomatous amoebic
encephalitis (GAE) in individuals with compromised immune systems [79]. Acanthamoeba spp., the
Trojan horse of the microbial world, as it carries viruses, has two stages in its life cycle, an active
trophozoite stage that exhibits vegetative growth and a dormant cyst stage with minimal metabolic
activity. It is a causative agent of cutaneous lesions and sinus infections, vision-threatening keratitis
and a rare but fatal encephalitis, known as granulomatous amoebic encephalitis [80]. Acanthamoebae
and Naegleria fowleri are commonly found in warm freshwater environments such as hot springs, lakes,
natural mineral water, and resort spas frequented by tourists. In an early survey of 13 swimming pools
in Belgium, Acanthamoeba strains were detected in 43.6% of the samples [81]. Similarly, amoebae were
detected in 27/30 swimming pools in New York State [82]. Previously thought to be a rare condition,
the number of reported Primary Amoebic meningoencephalitis cases is increasing each year [83].
The present review identified 13 published studies reporting detection of free-living amoebae
in pool waters in tropical countries [77,84–95] (Table 2). The earlier survey appeared in 1983 [84] and
reported the presence of pathogenic and free-living amoebae in swimming pool waters of Mexico City.
Among the organisms isolated, in their cystic or in their trophic stage, were Naegleria fowleri Carter and
Acanthamoeba castellanii Douglas [84]. One study in Taiwan reported a fatality caused after swimming
in hot springs [85]. The most recent one was carried out in two swimming pools in Alexandria
(Egypt) [86].
3.4. Leptospira spp.

Leptospirosis also belongs to the spectrum of travel-related infections. Leptospirosis is a
bacterial zoonosis caused by host-dependent spirochetes of the genus Leptospira, which is widespread
throughout the world. Its main sources are rodents, particularly rats, which excrete the spirochete
Leptospira spp. in urine. Humans are infected by direct contact with urine of infected animals or by
contact with an infected environment such as surface water [96].
Leptospirosis is an important re-emerging tropical disease, especially in areas with a notable
military presence. Several epidemics of leptospirosis have been reported worldwide during the past
century, while leptospirosis is endemic in most of the urban areas in Southern and Western India,
where outbreaks usually occur after flooding caused by heavy seasonal rainfall [97]. Almost every
country in South and Southeast Asia, South and Central America and several island nations across
the world are endemic to leptospirosis [98]. It is endemic in Sub-Saharan Africa; however, for most
countries scarce epidemiological data, if any, exist.
The disease has been increasingly reported in travellers, particularly those travelling to tropical
areas, due to the development of fresh-water sports and leisure activities [99,100], Leptospirosis is
often reported in travellers to South Africa [96] and in travellers from Sub Saharan Africa. Returning
from a water sports holiday in South Africa, a 49-year old man presented with acute leptospirosis [101].
In a recently published review from Rajarata University, Sri Lanka, the authors pointed out that
a clear increase in the proportion of travel-associated leptospirosis over the time was observed.
8
IJERPH 2018, 15, 2730
According to their review, the countries with the highest number of cases detected in travellers
returning from endemic regions are the US, Netherlands, Japan, France, Germany and Australia;
among reports of systematically collected country level data, Israel reported the highest incidence
of travel associated leptospirosis (41.7%) [102]. In a hospital study in Paris, France, fifteen cases of
travel-related leptospirosis were reported. All travellers except one were returning from holidays
in the tropics (seven from SE Asia, three from Sub-Saharan Africa, two from Reunion Island). The
most frequent at-risk exposure was bathing in fresh water [103]. The clinical course of a leptospirosis
outbreak at the Hash House Harriers Club on Guam, Micronesia, in the western Pacific Ocean, has
been reported. Patients declared multiple exposures to wet river banks, mud, and swamps [104].
One case clearly connected a leptospirosis case to swimming in a pool (Table 2). A 25 year-old
German woman visiting the Dominican Republic and staying for 3 weeks in a village became infected
when swimming in the heavily chlorinated swimming pool during a trip to Samana [105].
3.5. Viruses
Viruses are considered a significant cause of recreationally associated waterborne diseases with a
number of relevant outbreaks in western countries [106–109]. However, they have been difficult to
document because of the wide variety of illnesses associated and limitations in detection methods.
Noroviruses are the largest cause of outbreaks with just under half of the outbreaks occurring in
swimming pools (49%) [110]. Some sporadic publications refer to transmission of Hepatitis A virus
(HAV) [111,112] and Echovirus 30 [113].
The present review identified six published studies reporting the viruses’ detection in swimming
pool waters in tropical countries [114–119] (Table 2). The earliest was a study of a primary school
outbreak of pharyngoconjunctival fever attributed to swimming in the swimming pool of a school
camp [114]. Swimming pool water contaminated with Human Adenovirus serotype 4 (HAdV-4) was the
most likely source of infection, although one instance of likely person-to-person transmission was
noted [119].
3.6. Indirect Role of Swimming Pools in Water Related Diseases

Vector-borne diseases are human illnesses caused by parasites, viruses and bacteria that are
transmitted by mosquitoes, sandflies, triatomine bugs, blackflies, ticks, tsetse flies, mites, snails and
lice, causing more than 700,000 deaths globally. All the major vector-borne diseases, together, account
for around 17% of all infectious diseases [8]. Swimming pools are considered major contributors to
the disease burden, as proliferation sites for mosquitoes like Anopheles, transferring Malaria; Aedes,
transferring Dengue, yellow fever and the Zika viruses; Culex transferring West Nile Fever. The latter
became a major problem after the first cases and epidemics in western countries, starting in New York
in 1999 [120]. Concerning the ecology of the Culex mosquitoes, Petersen et al., in their review of the
spread of West Nile Virus, included density of poorly maintained swimming pools in the critical factors
for Arvoviral proliferation [121]. Chen et al., in their review of the significance of the Zika virus as a
new public health concern, emphasize the need to eliminate standing waters outside homes, including
swimming pools [122].
Distribution of vector-borne diseases is determined by complex demographic, environmental
and social factors. Global travel and trade, unplanned urbanization and making the transmission
season longer or more intense or causing diseases to emerge in countries where they were previously
unknown [8]. The world risk maps, demonstrated that for dengue fever Sub-Saharan areas and the
central and northern countries of South America are the most dangerous zones [123]. Mackenzie et al.,
in his risk map, presents the spread and resurgence of Japanese encephalitis, West Nile and dengue
encompassing all continents except Antarctica [124]. An important outbreak of dengue fever occurred
recently in France [125], and of the West Nile virus in Greece [126].
Vectors are contributing to the dispersion of lethal diseases in the tropics, initially in agricultural
and rural areas [127,128]. There are reports of westerners traveling in Southeast Asian countries who
9
IJERPH 2018, 15, 2730
were infected by the Zika virus—an Australian traveller to Indonesia [129], a Canadian traveller to
Thailand [130]—and the trends of the disease alter as time goes by with the symptoms getting more
severe [131]. Nevertheless, in a review by De Sylva and Marshall dealing with the factors contributing
to urban malaria transmission in Sub-Saharan Africa, the authors concluded that “artificial rather than
natural vector breeding sites provide the most abundant sources of mosquito larvae in African urban
centres. Africa’s demography is rapidly changing with a fast increasing number of people moving
to urban areas”. According to the same review, urban malaria is considered an emerging problem in
Africa because the populations of most large African cities have grown exponentially over the last 30
years. Ninety-five artificial vector breeding sites are referred to in this review, including swimming
pools, in contrast with only 42 natural sites [132].
The present review identified five published studies reporting surveys of swimming pools in the
tropics as environments encouraging vector’s proliferation (Table 2). Impoinvil et al. conducted larval
surveys in habitats located in urban Malindi, Kenya, and, out of the 250 habitats sampled, 66 were
unused swimming pools. Of the 110 habitats found to be positive for mosquitoes, unused swimming
pools represented 42.7% and 148 anopheline pupae were found in eight of the 66 unused swimming
pools while none was found in the other habitats [133]. The same authors in an earlier study reported
that, from a total of 889 Anopheles and 7217 culicine immatures found in diverse water body types,
unused swimming pools comprised 61% of all water bodies found to serve as the main habitats for
Anopheles immatures [134]. Studies have been carried out in Dakar [135] and in Brazil [136].
3.7. Chlamydia Trachomatis

Trachoma, caused by Chlamydia trachomatis, has been noted throughout history as a significant
cause of blindness. Chlamydia trachomatis is one of four bacterial species in the genus and they
are obligatory intracellular parasites. Trachoma is considered a neglected tropical disease. The
causative organism is passed from person to person by flies, fomites and fingers, particularly among
preschool-aged children [137]. In 1998, the World Health Assembly adopted the goal of Global
Elimination of Trachoma as a cause of blindness; the year 2020 was set as the target date by a WHO
Alliance set up to support the elimination agenda [138].
Interestingly, Warren et al. notes the suggestion that the construction of well-maintained
and chlorinated public swimming pools could be a practical method to improve facial cleanliness
and disinfection and consequently reduce trachoma rates among children in remote Indigenous
communities in Australia [139]. Nevertheless, simultaneous use by travellers might enhance
transmission of Chlamydia trachomatis through the use of the pool, even though such a case has
not been reported to the present. As with all obligatory intracellular parasites, C. trachomatis is unlikely
to be affected by chlorination and thus survives well in pool water. Ozone only, in a concentration
of 4 ppm, was enough to inactivate C. trachomatis and C. pneumonia according to a study conducted
mostly for medical purposes [140].
Table 2. List of surveillance studies of swimming pools (SPs), and respective cases and outbreaks of
infections associated with swimming pools and hot springs in tropical and subtropical countries.
Microsporidia
Location/Country Type of Research Positive Results Reference
A man suffered from bilateral keratitis after
Report of an incident of a traveller from bathing in a rural pond. The patient was
Rural areas, Bangladesh [41]
Bangladesh returning to Paris, France found to be infected with a microsporidial
parasite belonging to the genus Nosema.
All patients were known to have contracted
Retrospective study of patients diagnosed
Taipei, Taiwan microsporidial keratitis after bathing in [42]
with microsporidial keratitis
hot springs.
10
IJERPH 2018, 15, 2730
Table 2. Cont.
Schistosoma spp.
8/13 travellers infected with Schistosoma. 5/8
Study of an acute schistosomiasis in Belgian
Dogon Valley, Mali travellers had experienced swimmer’s itch [55]
travellers returning from Dogon Valley, Mali
and developed Katayama syndrome.
Belo Horizonte, State of Study of an outbreak of acute schistosomiasis
17 cases infected with S. mansoni. [56]
Minas Gerais, Brazil in a holiday resort at an endemic area
Study of an outbreak where an area became
infected due to influx of infected workers 50 workers infected in the pool with
São João del Rei, Brazil [57]
from endemic areas, who infected water S. mansoni.
sources, including SPs
High prevalence of the disease depending on,
Upper Benue Valley, Study of the risk factors for human
among other factors, the intensity of contact [58]
North Cameroon schistosomiasis in the local population
with the water.
Study of an outbreak in two groups of 30
29 infected with S. intercalatum,
Dogon Valley, Mali Dutch travellers returning from Dogon area [59]
S. haematobium.
of Mali where they swam in fresh water pools
Cryptosporidium spp.
Survey of 35 randomly selected hotel SPs, 60 16.7% positive for Cryptosporidium, 15%
Beijing, China [66]
water samples positive for Giardia.
Various areas,
Survey of water sources including SPs 33% positive for Cryptosporidium. [77]
Philippines
Broome, Kimberley 11/18 cases swam in the public pool. In
region, Western Investigation of outbreak of cryptosporidiasis faecal and pool water samples [78]
Australia Cryptosporidium ominis was identified.
Acanthamoebae & Naegleria Species
All SPs were positive for Acanthamoebae. The
Mexico City, Mexico Survey of six swimming pools most commonly found were Amoebae of the [84]
species Naegleria gruberi Schardinger.
One fatal case of meningoencephalitis caused
Taichung, Taiwan Diagnosis of fatality by N. fowleri and transmitted in hot springs [85]
was reported.
Both SPs were positive for Acanthamoeba spp.
Alexandria, Egypt Survey of two SPs [86]
and Naegleria spp.
Survey of 14 pools. Four water samples and Acanthamoeba species were detected in all
Kuala Lumpur, Malaysia six samples using swabs were collected from sampling sites of all SPs, while Naegleria spp. [87]
each was detected in 3 sampling sites of 8 SPs.
All therapy tubs were positive for
Survey of three physiotherapy tubs and 11
Mexico City, Mexico Acanthamoeba spp., while 7/11 SPs were [88]
SPs
positive for Naegleria spp.
Amoebae were detected in 20% of the SPs.
4/65 water samples were positive for
Brazil, Porto Alegre Survey of 65 water samples from SPs [89]
Acanthamoeba spp. while 9/65 water samples
were positive for free-living amoebae.
49.2% of pool water samples were positive
Egypt, various locations Survey in various waters including two SPs [90]
for heat-tolerant Acanthamoeba spp.
8/72 water samples were positive for
Porto Alegre, Brazil Survey in pools and spas Acanthamoeba spp. distributed in group [91]
genotypes T3, T5, T4, T15.
Study of the pathogenicity of strains from 4/4 Acanthamoeba spp. isolates from pool
Brasilia District, Brazil [92]
environmental sources waters were pathogenic.
In 71.6% of water samples Acanthamoeba spp.
Survey of 110 water and soil samples
Ahwaz, Iran was detected SP isolates belong to [93]
including four SPs
T4 genotype.
33.3% of SP water samples were positive for
Various areas, Survey of rivers, ponds, dispensers, wells,
Acanthamoeba sp. While 9.1% of SP water [77]
Philippines taps, natural lakes and SPs
samples were positive for Naegleria spp.
Adana, Afyon, Kutahya, 42% of water samples were positive for
Mersin and Nigde Survey of hot springs and SPs Acanthamoeba sp. belonging to T3, T4, [94]
provinces, Turkey T5 genotypes.
Malaysia Peninsular A survey of recreational lakes, streams, SPs Naegleria sp was detected in all samples. [95]
11
IJERPH 2018, 15, 2730
Table 2. Cont.
Leptospira spp.
Various places, A German woman developed leptospirosis
Study of leptospirosis in travellers [105]
Dominican Republic after swimming in a chlorinated SP.
Viruses
Study of a primary school outbreak of
40% of the students infected by Adenovirus
Queensland, Australia pharyngo-conjunctival fever attributed to [114]
type 3.
swimming in the SP of a school camp
A study of the risk of infection of HAdVs
Pretoria, South Africa detected in a survey of 3 SPs, 92 water HAdVs were detected in 15 samples. [115]
samples
16 Acanthamoeba strains were detected,
Survey of SPs for the detection of
Porto Alegre, Brazil HAdVs were detected in 62.5% (10/16) of [116]
adenoviruses in Acanthamoeba strains
Acanthamoeba isolates.
Investigation of an outbreak related to 90 children & the SP water were positive for
South Africa [117]
swimming in the school camp pool Echovirus 3.
A study to determine the prevalence of
HAdVs were detected in 28.1% of the samples
HAdVs in hot springs. 57 hot springs and 14
Taiwan, various areas from hot springs and 21.4% of SP [118]
public SPs were investigated, 57 water
water samples.
samples
A study of an outbreak of 50 patients used the same SP. HAdV type 4
Beijing, China pharyngoconjunctival fever related to was identified from the patients and SP [119]
swimming in a University SP water samples.
Vectors
Anopheles gambiae proliferating in SPs.
A systematic review of the factors
Artificial rather than natural breeding sites
Malindi, Kenya contributing to urban transmission of malaria [132]
provide most abundant sources for
in Sub-Saharan Africa
mosquito larvae.
A study on larvae surveys in urban Unused SPs accounted for 42.7% of all 110
Malindi, Kenya environments and the productivity of unused positive habitats. Anopheles gambiae s.l. and [133]
SPs in relation to other habitats Culex quinquefasciatus were detected.
A study on the abundance of immature
Unused SPs comprised 21.7% of water bodies
Malindi, Kenya Anopheles and culicines in various water body [134]
serving as habitats for immature Anopheles.
types in the urban environment
355 private properties were visited, including
An entomological survey on the determinants
Dakar, Senegal SPs. Culicidae larvae were found in 80 (23%) [135]
of malaria transmission in the city of Dakar
and Anopheles larvae in 11 (3%).
A study on the evaluation of two sweeping
Sao Jose de Rio Preto, Aedes aegypti was harvested in various types
methods for estimating the number of [136]
Brazil of containers including SPs.
immature Aedes aegypti
4. Discussion
Global experience and research demonstrate that international travel can pose various risks to
health, depending both on the health needs of the traveller and on the type of travel undertaken.
Travellers may encounter sudden and significant changes in altitude, humidity, temperature and
exposure to a variety of infectious diseases, which can result in illness. In addition, serious health risks
may arise in areas where accommodation is of poor quality, hygiene and sanitation are inadequate,
medical services are not well developed and clean water is unavailable [2]. Trends in the West towards
novel, alternative “natural” ponds, deprived of disinfection with chemicals and using instead biological
processes for cleaning the organic compounds in the water are expanding rapidly. Many European
countries have adopted guidelines or regulations specific for natural ponds. These establishments
require careful, scientifically sound management [141]. Some or all of these factors apply in numerous
tropical and subtropical countries of the developing world. Also, the trend towards higher standard
accommodation in tourist establishments and more water-intense activities—including the use of
pools and hot springs—coincides with changes in the global climate system leading to declining water
12
IJERPH 2018, 15, 2730
resources and poorer water quality in many regions [142]. Tourists’ lack of awareness of their impact
on the environment becomes an added factor aggravating sustainability in certain destinations [7].
A host of viral, bacterial, fungal, helminth and protozoal diseases that occur mainly in the
tropics and subtropics remain neglected, and hence the phrase “neglected tropical diseases” is used
to characterize them [9]. The growing number of people travelling to the tropics means that it is
imperative that there be more effective management of various public health issues by local authorities
in the countries concerned, as well as by international bodies, tourist agencies and social groups, to
enhance prevention and protection. Travellers should be considered as an integral part of the global
surveillance network for emerging infections. Research and the knowledge gained can be used to
alert the global community to the presence or susceptibility patterns of pathogens in different regions;
to inform strategies that can be used to control infections in developing countries; and to prepare
travellers to those areas and guide the care for those returning [4].
Tropical diseases transmitted via swimming pools and hot spring waters are less well understood
than the hazards deriving from the use of unsafe food or drinking water. Also, secondary transmission
occurs when travellers return to their homeland. Today, transmission at home is frequently related to
climate change. Any climate change may alter the disease burden resulting from exposure to pathogens
transmitted through recreational waters. In a study examining the impact of temperature, humidity,
and precipitation on the incidence of reported West Nile virus infections in the US, increasing weekly
maximum temperature and weekly cumulative temperature were similarly and significantly associated
with a higher incidence of reported WNV infections [143].
Eighty three studies dealing with Microsporidia, Leptospira, Schistosoma, Cryptosporidium, Amoebae,
viruses, and vectors breeding in swimming pools and hot springs in the tropics, and fulfilling pre-set
criteria, have been included in this survey of the literature. The survey indicated that papers dealing
with the quality of pool waters in the tropics are scarce, and information about infected tourists using
pools is even less. The published literature, at least in international languages, presenting assessments
and surveys of swimming establishments in the developing world are also scarce. Reasons for this
neglect seem to be manifold including, as noted above, lesser awareness on this issue, not only in the
developing, but also in the developed world. On the other hand, it is true that cases and outbreaks have
been difficult to document because by its very nature travelling makes it difficult to follow up the cases
in question and to carry out an epidemiological investigation. An international collaboration on this
issue, along the lines of the European Legionnaires’ Disease Surveillance Network (ELDSNet), would
certainly be an important step forward. As emphasized in a review published by RIVM, it is becoming
imperative that recreational waterborne infectious diseases be prioritized and quantified [144].
Castor and Beach have made several recommendations for the prevention and control of disease
transmission in swimming venues. They recommend the redesign of aquatic facilities, increased
governmental oversight of swimming pool maintenance and training of staff, and education of the
public regarding healthy swimming habits. In addition, they recommend that high-risk groups, such
as the elderly and infirm and pregnant women, should be made aware of their increased risk of illness
as a result of swimming, even in apparently adequately disinfected swimming waters [145]. Tourists in
general, and in particular tourists travelling to the tropical and subtropical countries of the developing
world, should be included in the list of vulnerable citizens and provided with specific advice, services
and care.
5. Conclusions
There is abundant information and guidance to travellers regarding precautions that need to
be taken in respect of food, drinking water and air quality in tropical destinations. Nevertheless,
information on the hazards presented by recreational and especially pool, spa and hot spring waters
as a mode of transmission of pathogens is limited. The survey indicated that papers dealing with the
quality of pool waters in the tropics are scarce, and information about infected tourists using pools
is even less. In addition, the ongoing climate change may alter the disease burden resulting from
13
IJERPH 2018, 15, 2730
exposure to pathogens transmitted through recreational waters. Under the pressure of a growing
number of people undertaking international travel, and yet faster growth of such travel in the tropical
and subtropical zones, assessments of the swimming pool establishments, research and prevention
strategies for pool safety in the tropics are imperative. Public health authorities need to provide
guidance to westerners travelling to exotic destinations on how to protect their health in swimming
pools. An international collaboration on this issue would certainly be an important step forward.
Funding: This research received no external funding

Acknowledgments: We would like to thank John C. Davis, DipTransIoL for editing the English text.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. World Tourism Organization, Tourism Highlights. 2017. Available online: http://www2.unwto.org/
publication/unwto-tourism-highlights-2017 (accessed on 20 July 2018).
2. World Health Organization, International Travel and Health, 2012. Available online: http://www.who.int/
ith (accessed on 20 July 2018).
3. World Health Organization. Water, Sanitation and Health Team. Guidelines for Safe Recreational
Water Environments. Volume 2, Swimming Pools and Similar Environments; World Health Organization:
Geneva, Switzerland, 2006; Available online: http://www.who.int/iris/handle/10665/43336 (accessed on
20 July 2018).
4. Baker, D.M. Tourism and the health effects of infectious diseases: Are there potential risks for tourists? Int. J.
Saf. Secur. Tour./Hosp. 2015, 12, 1–17.
5. Barna, Z.; Kádár, M. The risk of contracting infectious diseases in public swimming pools. A review. Annali
dell Istituto Superiore di Sanità 2012, 48, 374–386. [CrossRef]
6. TravelHealthPro. Available online: https://travelhealthpro.org.uk/ (accessed on 24 July 2018).
7. Page, J.S.; Essex, S.; Causevic, S. Tourist Attitudes Towards Water Use in the Developing World: A
Comparative Analysis. Tour. Manag. Perspect. 2014, 10, 57–67. [CrossRef]
8. World Health Organization: WHO. Available online: www.who.int (accessed on 24 July 2018).
9. Utzinger, J.; Becker, S.L.; Knopp, S.; Blum, J.; Neumayr, A.L.; Keiser, J.; Hatz, C.F. Neglected tropical diseases:
Diagnosis, clinical management, treatment and control. Swiss Med. Wkly. 2012, 142, w13727. [CrossRef]
10. Charrel, R.N.; de Lamballerie, X.; Raoult, D. Chikungunya outbreaks—The globalization of vector borne
diseases. N. Engl. J. Med. 2007, 356, 769–771. [CrossRef]
11. Rammaert, B.; Beauté, J.; Borand, L.; Hem, S.; Buchy, P.; Goyet, S.; Guillard, B. Pulmonary melioidosis in
Cambodia: A prospective study. BMC Infect. Dis. 2011, 11, 126–174. [CrossRef]
12. Goodman, R.A.; Buehler, J.W. Delinquent Mortgages, Neglected Swimming Pools and West Nile Virus,
California. Emerg. Infect. Dis. 2009, 15, 508–509. [CrossRef]
13. Marten, G.; Harrison, C.; Nguyen, M.; Sacket, S.; Thompson, G.; Carroll, M.; Riegel, C. The Use of Gambusia
to Control Mosquito Larvae in Abandoned Swimming Pools: The New Orleans Experience. New Orleans
Mosquito, Termite & Rodent Control Board. 2013, pp. 1–70. Available online: http://www.gerrymarten.
com/publicatons/pdfs/GM_new-orleans-swimming-pools.pdf (accessed on 24 July 2018).
14. Horney, J.; Goldberg, D.; Hammond, T.; Stone, K.; Smitherman, S. Assessing the Prevalence of Risk Factors
for Neglected Tropical Diseases in Brazos County, Texas. PLoS Curr. 2017, 9. [CrossRef]
15. Caillouët, K.A.; Carlson, J.C.; Wesson, D.; Jordan, F. Colonization of abandoned swimming pools by larval
mosquitoes and their predators following Hurricane Katrina. J. Vector Ecol. 2008, 33, 166–172. [CrossRef]
16. Hlavsa, M.C.; Hill, R.V.; Beach, J.M. Immediate closures and violations identified during routine inspections
of Public Aquatic Facilities—Network for Aquatic Facility Inspection Surveillance, United States, 2013.
MMWR Surveill. Summ. 2016, 65, 1–26. [CrossRef]
17. Hlavsa, C.M.; Cikesh, L.B.; Roberts, A.V.; Kahler, M.A.; Marissa, M.; Hilborn, D.E.; Wade, J.T.; Roellig, M.D.;
Murphy, L.J.; Xiao, L.; et al. Outbreaks Associated with Treated Recreational Water—United States, 2000–2014.
MMWR Surveill. Summ. 2018, 67, 547–551.
18. Chalmers, R.M. Waterborne ourbreaks of cryptosporodiasis. Ann. Ist Super Sanita. 2012, 48, 429–446.
[CrossRef]
14
IJERPH 2018, 15, 2730
19. Mavridou, A.; Pappa, O.; Papatzitze, O.; Blougoura, A.; Drossos, P. An overview of pool and spa regulations
in Mediterranean countries with a focus on the tourist industry. J. Water Health 2014, 12, 359–371. [CrossRef]
20. CliQ: Towards a Clean Future. Swimming Pool Regulations and Pool Market Analysis in the Tourism Sector.
Available online: https://cliqib.org (accessed on 26 July 2018).
21. Abd-Rabbo, H. A new suggestion. Artificial plastic swimming pools as prophylactic measures against
infection with schistosomiasis in developing countries. J. Trop. Med. Hyg. 1968, 71, 18–19.
22. Maghazy, S.M.N.; Abdel-Mallek, A.Y.; Bagy, M.M.K. Fungi in Two Swimming Pools in Assiut Town, Egypt.
Zentralbl. Mikrobiol. 1989, 144, 213–216. [CrossRef]
23. Abdou, M.H.; Akel, M.M.; El-Shal, W.I.; El-Naggar, A.S. Study of the environmental health aspects of
swimming pools in Alexandria City. J. Egypt. Public Health Assoc. 2005, 80, 263–296.
24. Abd El-Salam, M.M. Assessment of water quality of some swimming pools: A case study in Alexandria,
Egypt. Environ. Monit. Assess. 2012, 12, 7395–7406. [CrossRef]
25. Even-Paz, Z.; Haas, H.; Sacks, T.; Rosenmann, E. Mycobacterium marinum skin infections mimicking
cutaneous leishmaniasis. Br. J. Dermatol. 1976, 94, 435–442. [CrossRef]
26. Al-Khatib, I.A.; Salah, S. Bacteriological and chemical quality of swimming pools water in developing
countries: A case study in the West Bank of Palestine. Int. J. Environ. Health Res. 2003, 13, 17–22. [CrossRef]
27. Rabi, A.; Khader, Y.; Alkafajei, A.; Aqoulah, A.A. Sanitary conditions of public swimming pools in Amman,
Jordan. Int. J. Environ. Res. Public Health 2008, 5, 152–157. [CrossRef]
28. Ali-Shtayeh, M.S.; Khaleel, T.K.; Jamous, R.M. Ecology of dermatophytes and other keratinophilic fungi in
swimming pools and polluted and unpolluted streams. Mycopathologia 2003, 156, 193–205. [CrossRef]
29. Courage Kosi Setsoafia Saba; Saviour Kojo Tekpor. Water Quality Assessment of Swimming Pools and Risk
of Spreading Infections in Ghana. Res. J. Microbiol. 2015, 10, 14–23. [CrossRef]
30. Wei, X.; Li, J.; Hou, S.; Xu, C.; Zhang, H.; Atwill, E.R.; Li, X.; Yang, Z.; Chen, S. Assessment of Microbiological
Safety of Water in Public Swimming Pools in Guangzhou, China. Int. J. Environ. Res. Public Health 2018, 15,
1416–1428. [CrossRef]
31. Rafiei, A.; Amirrajab, N. Fungal Contamination of Indoor Public Swimming Pools, Ahwaz, South-west of
Iran. Iran. J. Public Health 2010, 39, 124–128.
32. Fadaei, A.; Amiri, M. Comparison of Chemical, Biological and Physical Quality Assessment of Indoor
Swimming Pools in Shahrekord City, Iran in 2013. Glob. J. Health Sci. 2014, 7, 240–248. [CrossRef]
33. Rinder, H. Transmission of microsporidia to humans: Water-borne, food-borne, air-borne, zoonotic, or
anthroponotic? Southeast Asian J. Trop. Med. Public Health 2004, 35, 54–57.
34. Joseph, J.; Vemuganti, G.K.; Sharma, S. Microsporidia: Emerging ocular pathogens. Indian J. Med. Microbiol.
2005, 23, 80–91. [CrossRef]
35. Dowd, S.E.; Gerba, C.P.; Pepper, I.L. Confirmation of the human-pathogenic microsporidia Enterocytozoon
bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water. Appl. Environ. Microbiol. 1998, 64,
3332–3335.
36. Hutin, Y.J.; Sombardier, M.N.; Liguory, O.; Sarfati, C.; Derouin, F.; Modaï, J.; Molina, J.M. Risk factors for
intestinal microsporidiosis in patients with human immunodeficiency virus infection: A case-control study.
J. Infect. Dis. 1998, 178, 904–907. [CrossRef]
37. Li, X.; Fayer, R. Infectivity of microsporidian spores exposed to temperature extremes and chemical
disinfectants. J. Eukaryot. Microbiol. 2006, 53, S77–S79. [CrossRef]
38. Fournier, S.; Dubrou, S.; Liguory, O.; Gaussin, F.; Santillana-Hayat, M.; Sarfati, C.; Molina, J.M.; Derouin, F.
Detection of Microsporidia, cryptosporidia and Giardia in swimming pools: A one-year prospective study.
FEMS Immunol. Med. Microbiol. 2002, 33, 209–213. [CrossRef]
39. Sharma, S.; Das, S.; Joseph, J.; Vemuganti, G.K.; Murthy, S. Microsporidial keratitis: Need for increased
awareness. Surv. Ophthalmol. 2011, 56, 1–22. [CrossRef]
40. Vemuganti, G.K.; Garg, P.; Sharma, S.; Joseph, J.; Gopinathan, U.; Singh, S. Is microsporidial keratitis an
emerging cause of stromal keratitis? A case series study. BMC Ophthalmol. 2005, 17, 19–33. [CrossRef]
41. Curry, A.; Mudhar, H.S.; Dewan, S.; Canning, E.U.; Wagner, B.E. A case of bilateral microsporidial keratitis
from Bangladesh–infection by an insect parasite from the genus Nosema. J. Med. Microbiol. 2007, 56,
1250–1252. [CrossRef]
42. Fan, N.-W.; Wu, C.-C.; Chen, T.-L.; Yu, W.-K.; Chen, C.-P.; Lee, S.-M.; Lin, P.-Y. Microsporidial Keratitis in
Patients with Hot Springs Exposure. J. Clin. Microbiol. 2012, 50, 414–418. [CrossRef]
15
IJERPH 2018, 15, 2730
43. Karanis, P.; Kourenti, C.; Smith, H. Waterborne transmission of protozoan parasites: A worldwide review of
outbreaks and lessons learnt. J. Water Health 2007, 5, 1–38. [CrossRef]
44. Baldursson, S.; Karanis, P. Waterborne transmission of protozoan parasites: Review of worldwide
outbreaks—An update 2004–2010. Water Res. 2011, 45, 6603–6614. [CrossRef]
45. Efstratiou, A.; Ongerth, E.J.; Karanis, P. Waterborne transmission of protozoan parasites: Review of
worldwide outbreaks—An update 2011–2016. Water Res. 2017, 114, 14–22. [CrossRef]
46. TropNet: European Network for Tropical Medicine and Travel Health. Available online: http://www.
tropnet.net/ (accessed on 26 July 2018).
47. Lim, A.L.Y.; Nissapatorn, V. Transmission of waterborne parasites in the Association of Southeast Asian
Nations (ASEAN): Overview and direction forward. Food Waterborne Parasitol. 2017, 8–9, 75–83. [CrossRef]
48. Plutzer, J.; Karanis, P. Neglected waterborne parasitic protozoa and their detection in water. Water Res. 2016,
101, 318–332. [CrossRef]
49. Centers for Disease Control and Prevention (CDC). Available online: https://search.cdc.gov/search/?
√
query=schistosomes&sitelimit=&utf8= &affiliate=cdc-main (accessed on 26 July 2018).
50. Boissier, J.; Grech-Angelini, S.; Webster, B.L.; Allienne, J.F.; Huyse, T.; Mas-Coma, S.; Toulza, E.;
Barré-Cardi, H.; Rollinson, D.; Kincaid-Smith, J.; et al. Outbreak of urogenital schistosomiasis in Corsica
(France): An epidemiological case study. Lancet Infect. Dis. 2016, 16, 971–979. [CrossRef]
51. Meltzer, E.; Artom, G.; Marva, E.; Assous, M.V.; Rahav, G.; Schwartzt, E. Schistosomiasis among travelers:
New aspects of an old disease. Emerg. Infect. Dis. 2006, 12, 1696–1700. [CrossRef]
52. Clerinx, J.; Bottieau, E.; Wichmann, D.; Tannich, E.; Van Esbroeck, M. Acute schistosomiasis in a cluster
of travelers from Rwanda: Diagnostic contribution of schistosome DNA detection in serum compared to
parasitology and serology. J. Travel. Med. 2011, 18, 367–372. [CrossRef]
53. Marchese, V.; Beltrame, A.; Angheben, A.; Monteiro, G.B.; Giorli, G.; Perandin, F.; Buonfrate, D.; Bisoffi, Z.
Schistosomiasis in immigrants, refugees and travellers in an Italian referral centre for tropical diseases. Infect.
Dis. Poverty 2018, 7, 55–65. [CrossRef]
54. Röser, D.; Bjerrum, S.; Helleberg, M.; Nielsen, H.V.; David, K.P.; Thybo, S.; Stensvold, C.R. Adventure tourism
and schistosomiasis: Serology and clinical findings in a group of Danish students after white-water rafting
in Uganda. JMM Case Rep. 2018, 5, e005141. [CrossRef]
55. Coltart, C.E.; Chew, A.; Storrar, N.; Armstrong, M.; Suff, N.; Morris, L.; Chiodini, P.L.; Whitty, C.J.
Schistosomiasis presenting in travellers: A 15 year observational study at the Hospital for Tropical Diseases,
London. Trans. R. Soc. Trop. Med. Hyg. 2015, 109, 214–220. [CrossRef]
56. Grobusch, M.P.; Mühlberger, N.; Jelinek, T.; Bisoffi, Z.; Corachán, M.; Harms, G.; Matteelli, A.; Fry, G.;
Hatz, C.; Gjørup, I.; et al. Imported schistosomiasis in Europe: Sentinel surveillance data from TropNetEurop.
J. Travel Med. 2003, 10, 164–169. [CrossRef]
57. Kpoda, N.W.; Sorgho, H.; Poda, J.N.; Ouédraogo, J.B.; Kabré, G.B. Schistosomiasis caused by Schistosoma
mansoni in the Kou valley: Characterization of the transmission system and socioeconomic impact. C. R.
Biol. 2013, 336, 284–288. [CrossRef]
58. El-Khoby, T.; Galal, N.; Fenwick, A.; Barakat, R.; El-Hawey, A.; Nooman, Z.; Habib, M.; Abdel-Wahab, F.;
Gabr, N.S.; Hammam, H.M.; et al. The epidemiology of schistosomiasis in Egypt: Summary findings in nine
governorates. Am. J. Trop. Med. Hyg. 2000, 62, 88–99. [CrossRef]
59. Corachan, M.; Ruiz, L.; Valls, M.E.; Gascon, J. Schistosomiasis and the Dogon country (Mali). Am. J. Trop.
Med. Hyg. 1992, 47, 6–9. [CrossRef]
60. Logan, S.; Armstrong, M.; Moore, E.; Nebbia, G.; Jarvis, J.; Suvari, M.; Bligh, J.; Chiodini, P.L.; Brown, M.;
Doherty, T. Acute Schistosomiasis in Travelers:14 Years’ Experience at the Hospital for Tropical Diseases,
London. Am. J. Trop. Med. Hyg. 2013, 88, 1032–1034. [CrossRef]
61. Colebunders, R.; Verstraeten, T.; Van Gompel, A.; Van den Ende, J.; De Roo, A.; Polderman, A.; Visser, L.
Acute Schistosomiasis in Travelers Returning from Mali. J. Travel Med. 1995, 2, 235–238. [CrossRef]
62. Enk, J.M.; Amorim, A.; Schall, T.V. Acute schistosomiasis outbreak in the metropolitan area of Belo Horizonte,
Minas Gerais: Alert about the risk of unnoticed transmission increased by growing rural tourism. Mem. Inst.
Oswaldo Cruz. 2003, 98, 745–750. [CrossRef]
16
IJERPH 2018, 15, 2730
63. Lambertucci, J.R.; Drummond, S.C.; Voieta, I.; de Queiróz, L.C.; Pereira, P.P.; Chaves, B.A.; Botelho, P.P.;
Prata, P.H.; Otoni, A.; Vilela, J.F.; et al. An outbreak of acute Schistosoma mansoni Schistosomiasis in a
nonendemic area of Brazil: A report on 50 cases, including 5 with severe clinical manifestations. Clin. Infect.
Dis. 2013, 57, e1–e6. [CrossRef]
64. Ndassa, A.; Mimpfoundi, R.; Gake, B.; Paul Martin, M.V.; Poste, B. Risk factors for human schistosomiasis in
the Upper Benue valley, in northern Cameroon. Ann. Trop. Med. Parasitol. 2007, 101, 469–477. [CrossRef]
65. Visser, L.G.; Polderman, A.M.; Stuiver, P.C. Outbreak of schistosomiasis among travelers returning from
Mali, West Africa. Clin. Infect. Dis. 1995, 20, 280–285. [CrossRef]
66. Xiao, S.; Yin, P.; Zhang, Y.; Hu, S. Occurrence of Cryptosporidium and Giardia and the Relationship between
Protozoa and Water Quality Indicators in Swimming Pools. Korean J. Parasitol. 2017, 55, 129–135. [CrossRef]
67. Centers for Disease Control and Prevention: Parasites-Cryptosporodium (also known as ‘Crypto’). Available
online: https://www.cdc.gov/parasites/crypto/ (accessed on 26 July 2018).
68. Coetzee, N.; Edeghere, O.; Orendi, J.; Chalmers, R.; Morgan, L. A swimming pool-associated outbreak of
cryptosporidiosis in Staffordshire, England, October to December 2007. Euro Surveill. 2008, 13, 19028.
69. Insulander, M.; Lebbad, M.; Stenström, T.A.; Svenungsson, B. An outbreak of cryptosporidiosis associated
with exposure to swimming pool water. Scand. J. Infect. Dis. 2005, 37, 354–360. [CrossRef]
70. Shields, J.M.; Gleim, E.R.; Beach, M.J. Prevalence of Cryptosporidium spp. and Giardia intestinalis in
Swimming Pools, Atlanta, Georgia. Emerg. Infect. Dis. 2008, 14, 948–950. [CrossRef]
71. Wheeler, C.; Vugia, D.J.; Thomas, G.; Beach, M.J.; Carnes, S.; Maier, T.; Gorman, J.; Xiao, L.; Arrowood, M.J.;
Gilliss, D.; et al. Outbreak of cryptosporidiosis at a California waterpark: Employee and patron roles and the
long road towards prevention. Epidemiol. Infect. 2007, 135, 302–310. [CrossRef]
72. Ichinohe, S.; Fukushima, T.; Kishida, K.; Sanbe, K.; Saika, S.; Ogura, M. Secondary Transmission of
Cryptosporidiosis Associated with Swimming Pool Use. Jpn. J. Infect. Dis. 2005, 58, 400–401.
73. Lal, A.; Cornish, L.M.; Fearnley, E.; Glass, K.; Kirk, M. Cryptosporidiosis: A Disease of Tropical and Remote
Areas in Australia. PLoS Negl. Trop. Dis. 2015, 9, e0004078. [CrossRef]
74. GBD Mortality Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and
cause-specific mortality for 240 causes of death, 1990–2013: A systematic analysis for the Global Burden of
Disease Study 2013. Lancet 2015, 385, 117–171. [CrossRef]
75. Dale, K.; Kirk, M.; Sinclair, M.; Hall, R.; Leder, K. Reported waterborne outbreaks of gastrointestinal disease
in Australia are predominantly associated with recreational exposure. Aust. N. Z. J. Public Health 2010, 34,
527–530. [CrossRef]
76. Ryan, U.; Lawler, S.; Reid, S. Limiting swimming pool outbreaks of cryptosporidiosis—The roles of
regulations, staff, patrons and research. J. Water Health 2017, 15, 1–16. [CrossRef]
77. Onichandran, S.; Kumar, T.; Salibay, C.C.; Dungca, Z.J.; Tabo, A.H.; Tabo, N.; Tan, T.C.; Lim, A.Y.;
Sawangjaroen, N.; Phiriyasamith, S.; et al. Waterborne parasites: A current status from the Philippines.
Parasit. Vectors 2014, 7, 244–252. [CrossRef]
78. Ng-Hublin, J.S.; Hargrave, D.; Combs, B.; Ryan, U. Investigation of a swimming pool-associated
cryptosporidiosis outbreak in the Kimberley region of Western Australia. Epidemiol. Infect. 2015, 143,
1037–1041. [CrossRef]
79. Centers for Disease Control and Prevention: Parasites-Acantamobea. Available online: https://www.cdc.
gov/parasites/acanthamoeba/ (accessed on 26 July 2018).
80. Visvesvara, G.S.; Moura, H.; Schuster, F.L. Pathogenic and opportunistic free-living amoebae: Acanthamoeba
spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol. Med. Microbiol.
2007, 50, 1–26. [CrossRef]
81. De Jonckheere, J.F. Pathogenic free-living amoebae in swimming pools: Survey in Belgium. Ann. Microbiol.
1979, 130B, 205–212.
82. Lyons, T.B.; Kapur, R. Limax Amoebae in Public Swimming Pools of Albany, Schenectady, and Rensselaer
Counties, New York: Their Concentration, Correlations, and Significance. Appl. Environ. Microbiol. 1977, 33,
551–555.
83. Heggie, T.W. Swimming with death: Naegleria fowleri infections in recreational waters. Travel. Med. Infect.
Dis. 2010, 8, 201–206. [CrossRef]
84. Rivera, F.; Ramírez, P.; Vilaclara, G.; Robles, E.; Medina, F. A survey of pathogenic and free-living amoebae
inhabiting swimming pool water in Mexico City. Environ. Res. 1983, 32, 205–211. [CrossRef]
17
IJERPH 2018, 15, 2730
85. Su, M.Y.; Lee, M.S.; Shyu, L.Y.; Lin, W.C.; Hsiao, P.C.; Wang, C.P.; Lai, S.C. A Fatal Case of Naegleria fowleri
Meningoencephalitis in Taiwan. Korean J. Parasitol. 2013, 51, 203–206. [CrossRef]
86. Al-Herrawy, A.Z.; Khalil, M.I.; El-Sherif, S.S.; Omar, F.A.E.; Lotfy, W.M. Surveillance and Molecular
Identification of Acanthamoeba and Naegleria Species in Two Swimming Pools in Alexandria University.
Egypt. Iran. J. Parasitol. 2017, 12, 196–205.
87. Init, I.; Lau, Y.L.; Arin Fadzlun, A.; Foead, A.I.; Neilson, R.S.; Nissapatorn, V. Detection of free living amoebae,
Acanthamoeba and Naegleria, in swimming pools, Malaysia. Trop. Biomed. 2010, 27, 566–577.
88. Rivera, F.; Ramírez, E.; Bonilla, P.; Calderón, A.; Gallegos, E.; Rodríguez, S.; Ortiz, R.; Zaldívar, B.; Ramírez, P.;
Durán, A. Pathogenic and Free-living Amoebae Isolated from Swimming Pools and Physiotherapy Tubs in
Mexico. Environ. Res. 1993, 62, 43–52. [CrossRef]
89. Caumo, K.; Frasson, A.P.; Pens, C.J.; Panatieri, L.F.; Frazzon, A.P.; Rott, M.B. Potentially pathogenic
Acanthamoeba in swimming pools: A survey in the southern Brazilian city of Porto Alegre. Ann. Trop. Med.
Parasitol. 2009, 103, 477–485. [CrossRef]
90. Al-Herrawy, A.; Bahgat, M.; Mohammed, A.; Ashour, A.; Hikal, W. Morpho-Physiological and Biochemical
Criteria of Acanthamoeba spp. Isolated from the Egyptian Aquatic Environment. Iran. J. Parasitol. 2013, 8,
302–312.
91. Fabres, L.F.; Rosa Dos Santos, S.P.; Benitez, L.B.; Rott, M.B. Isolation and identification of Acanthamoeba spp.
from thermal swimming pools and spas in Southern Brazil. Acta Parasitol. 2016, 61, 221–227. [CrossRef]
92. Alves Dde, S.; Moraes, A.S.; Nitz, N.; de Oliveira, M.G.; Hecht, M.M.; Gurgel-Gonçalves, R.; Cuba, C.A.
Occurrence and characterization of Acanthamoeba similar to genotypes T4, T5, and T2/T6 isolated from
environmental sources in Brasília, Federal District, Brazil. Exp. Parasitol. 2012, 131, 239–244. [CrossRef]
93. Rahdar, M.; Niyyati, M.; Salehi, M.; Feghhi, M.; Makvandi, M.; Pourmehdi, M.; Farnia, S. Isolation and
genotyping of acanthamoeba strains from environmental sources in Ahvaz city, Khuzestan province,
southern Iran. Iran. J. Parasitol. 2012, 7, 22–26.
94. Evyapan, G.; Koltas, I.S.; Eroglu, F. Genotyping of Acanthamoeba T15: The environmental strain in Turkey.
Trans. R. Soc. Trop. Med. Hyg. 2015, 109, 221–224. [CrossRef]
95. Ithoi, I.; Ahmad, A.F.; Nissapatorn, V.; Lau, Y.L.; Mahmud, R.; Mak, J.W. Detection of Naegleria species in
environmental samples from peninsular Malaysia. PLoS ONE 2011, 6, e24327. [CrossRef]
96. de Vries, S.G.; Visser, B.J.; Nagel, I.M.; Goris, M.G.; Hartskeerl, R.A.; Grobusch, M.P. Leptospirosis in
Sub-Saharan Africa: A systematic review. Int. J. Infect. Dis. 2014, 28, 47–64. [CrossRef]
97. Patil, D.Y.; Dahake, R.V.; Chowdhary, A.S.; Deshmukh, R.A. Clinico-epidemiological observations of human
leptospirosis from Mumbai, India. J. Infect. Public Health 2017, 10, 247–248. [CrossRef]
98. Sehgal, S.C. Epidemiological patterns of leptospirosis. Indian J. Med. Microbiol. 2006, 24, 310–311. [CrossRef]
99. Gelman, S.S.; Gundlapalli, A.V.; Hale, D.; Croft, A.; Hindiyeh, M.; Carroll, K.C. Spotting the spirochete:
Rapid diagnosis of leptospirosis in two returned travelers. J. Travel Med. 2006, 9, 165–167. [CrossRef]
100. Leshem, E.; Segal, G.; Barnea, A.; Yitzhaki, S.; Ostfeld, I.; Pitlik, S.; Schwartz, E. Travel-Related Leptospirosis
in Israel: A Nationwide Study. Am. J. Trop. Med. Hyg. 2010, 82, 459–463. [CrossRef]
101. Pimenta, D.; Democratis, J. Risky behavior: A rare complication of an uncommon disease in a returning
traveler. BMJ Case Rep. 2013. [CrossRef]
102. Bandara, M.; Ananda, M.; Wickramage, K.; Berger, E.; Agampodi, S. Globalization of leptospirosis through
travel and migration. Glob. Health 2014, 10, 61–70. [CrossRef]
103. van de Werve, C.; Perignon, A.; Jauréguiberry, S.; Bricaire, F.; Bourhy, P.; Caumes, E. Travel-related
leptospirosis: A series of 15 imported cases. J. Travel Med. 2013, 20, 228–231. [CrossRef]
104. Brinker, A.J.; Blazes, D.L. An outbreak of Leptospirosis among United States military personnel in Guam.
Trop. Dis. Travel Med. Vaccines 2017, 3, 16. [CrossRef]
105. Grobusch, M.P.; Bollmann, R.; Schönberg, A.; Slevogt, H.; Garcia, V.; Teichmann, D.; Jelinek, T.; Flick, H.;
Bergmann, F.; Rosseau, S.; et al. Leptospirosis in Travelers Returning from the Dominican Republic. J. Travel
Med. 2003, 10, 55–58. [CrossRef]
106. Hauri, A.M.; Schimmelpfennig, M.; Walter-Domes, M.; Letz, A.; Diedrich, S.; Lopez-Pila, J.; Schreier, E. An
outbreak of viral meningitis associated with a public swimming pond. Epidemiol. Infect. 2005, 133, 291–298.
[CrossRef]
18
IJERPH 2018, 15, 2730
107. D’Angelo, L.J.; Hieholzer, J.C.; Keenlyside, R.A.; Anderson, L.J.; Martone, W.J. Pharyngoconjunctival fever
caused by adenovirus type 4: Report of a swimming pool-related outbreak with recovery of virus from pool
water. J. Infect. Dis. 1979, 140, 42–47. [CrossRef]
108. Maunula, L.; Kalso, S.; Von Bonsdorff, C.H.; Pönkä, A. Wading pool water contaminated with both
noroviruses and astroviruses as the source of a gastroenteritis outbreak. Epidemiol. Infect. 2004, 132,
109. Morbidity and Mortality Weekly Report (MMWR). An Outbreak of Norovirus Gastroenteritis at a Swimming
Club—Vermont. Wkly Rep. 2004, 53, 793–795. Available online: https://www.cdc.gov/mmwr/preview/
mmwrhtml/mm5334a5.htm (accessed on 30 July 2018).
110. Sinclair, R.G.; Jones, E.L.; Gerba, C.P. Viruses in recreational water-borne disease outbreaks: A review. J. Appl.
Microbiol. 2009, 107, 1769–1780. [CrossRef]
111. Mahoney, F.J.; Farley, T.A.; Kelso, K.Y.; Wilson, S.A.; Horan, J.M.; McFarland, L.M. An outbreak of hepatitis
A associated with swimming in a public pool. J. Infect. Dis. 1992, 165, 613–618. [CrossRef]
112. Tallis, G.; Gregory, J. An outbreak of hepatitis A associated with a spa pool. Commun. Dis. Intell. 1997, 21,
353–354.
113. Faustini, A.; Fano, V.; Muscillo, M.; Zaniratti, S.; La Rosa, G.; Tribuzi, L.; Perucci, C.A. An outbreak of aseptic
meningitis due to echovirus 30 associated with attending school and swimming in pools. Int. J. Infect. Dis.
2006, 10, 291–297. [CrossRef]
114. Harley, D.; Harrower, B.; Lyon, M.; Dick, A. A primary school outbreak of pharyngo-conjunctival fever
caused by adenovirus type 3. Commun. Dis. Intell. 2001, 25, 9–12.
115. Van Heerden, J.; Ehlers, M.M.; Grabow, W.O. Detection and risk assessment of adenoviruses in swimming
pool water. J. Appl. Microbiol. 2005, 99, 1256–1264. [CrossRef]
116. Staggemeier, R.; Arantes, T.; Caumo, K.S.; Rott, M.B.; Spilki, F.R. Detection and quantification of human
adenovirus genomes in Acanthamoeba isolated from swimming pools. An. Acad. Bras. Cienc. 2016, 88,
117. Yeats, J.; Smuts, H.; Serfontein, C.J.; Kannemeyer, J. Investigation into a school enterovirus outbreak using
PCR detection and serotype identification based on the 5’ non-coding region. Epidemiol. Infect. 2005, 133,
1123–1130. [CrossRef]
118. Shih, Y.J.; Tao, C.W.; Tsai, H.C.; Huang, W.C.; Huang, T.Y.; Chen, J.S.; Chiu, Y.C.; Hsu, T.K.; Hsu, B.M. First
detection of enteric adenoviruses genotype 41 in recreation spring areas of Taiwan. Environ. Sci. Pollut. Res.
Int. 2017, 24, 18392–18399. [CrossRef]
119. Li, J.; Lu, X.; Sun, Y.; Lin, C.; Li, F.; Yang, Y.; Liang, Z.; Jia, L.; Chen, L.; Jiang, B.; et al. A swimming
pool-associated outbreak of pharyngoconjunctival fever caused by human adenovirus type 4 in Beijing,
China. Int. J. Infect. Dis. 2018, 75, 89–91. [CrossRef]
120. Komar, N. West Nile virus: Epidemiology and ecology in North America. Adv. Virus Res. 2003, 61, 185–234.
121. Petersen, L.R.; Brault, A.C.; Nasci, R.S. West Nile Virus: Review of the Literature. JAMA 2013, 310, 308–315.
[CrossRef]
122. Chen, H.L.; Tang, R.B. Why Zika virus infection has become a public health concern? Chin. Med. Assoc. 2016,
79, 174–178. [CrossRef]
123. Rogers, D.J.; Wilson, A.J.; Hay, S.I.; Graham, A.J. The Global Distribution of Yellow Fever and Dengue. Adv.
Parasitol. 2006, 62, 181–220. [CrossRef]
124. Mackenzie, J.S.; Gubler, D.J.; Petersen, L.R. Emerging flaviviruses: The spread and resurgence of Japanese
encephalitis, West Nile and dengue viruses. Nat. Med. 2004, 10, 98–109. [CrossRef]
125. Succo, T.; Noel, H.; Nikolay, B.; Maquart, M.; Cochet, A.; Leparc-Goffart, I.; Catelinois, O.; Salje, H.; Pelat, C.;
de Crouy-Chanel, P.; et al. Dengue sero-survey after a 2-month long outbreak in Nîmes, France, 2015: Was
there more than met the eye? Euro Surveill. 2018, 23. [CrossRef]
126. Patsoula, E.; Vakali, A.; Balatsos, G.; Pervanidou, D.; Beleri, S.; Tegos, N.; Baka, A.; Spanakos, G.;
Georgakopoulou, T.; Tserkezou, P.; et al. West Nile Virus Circulation in Mosquitoes in Greece (2010–2013).
Biomed. Res. Int. 2016, 2016, 2450682. [CrossRef]
127. Coulibaly, B.; Kone, R.; Barry, M.B.; Emerson, B.; Coulibaly, M.B.; Niare, O.; Beavogui, A.H.; Traore, S.F.;
Vernick, K.D.; Riehle, M.M. Malaria vector populations across ecological zones in Guinea Conakry and Mali,
West Africa. Malar. J. 2016, 15, 191–201. [CrossRef]
19
IJERPH 2018, 15, 2730
128. Matthys, B.; Koudou, B.G.; N’Goran, E.K.; Vounatsou, P.; Gosoniu, L.; Koné, M.; Gissé, G.; Utzinger, J.
Spatial dispersion and characterization of mosquito breeding habitats in urban vegetable-production areas
of Abidjan, Côte d’Ivoire. Ann. Trop. Med. Parasitol. 2010, 104, 649–666. [CrossRef]
129. Kwong, J.C.; Druce, J.D.; Leder, K. Zika virus infection acquired during brief travel to Indonesia. Am. J. Trop.
Med. Hyg. 2013, 89, 516–517. [CrossRef]
130. Fonseca, K.; Meatherall, B.; Zarra, D.; Drebot, M.; MacDonald, J.; Pabbaraju, K.; Wong, S.; Webster, P.;
Lindsay, R.; Tellier, R. First case of Zika virus infection in a returning Canadian traveler. Am. J. Trop. Med.
Hyg. 2014, 91, 1035–1038. [CrossRef]
131. Salehuddin, A.R.; Haslan, H.; Mamikutty, N.; Zaidun, N.H.; Azmi, M.F.; Senin, M.M.; Syed Ahmad Fuad, S.B.;
Thent, Z.C. Zika virus infection and its emerging trends in Southeast Asia. Asian Pac. J. Trop. Med. 2017, 10,
132. De Silva, M.P.; Marshall, J.M. Factors Contributing to Urban Malaria Transmission in Sub-Saharan Africa:
A Systematic Review. J. Trop. Med. 2012, 2012, 819563. [CrossRef]
133. Impoinvil, D.E.; Mbogo, C.M.; Keating, J.; Beier, J.C. The role of unused swimming pools as a habitat
for Anopheles immature stages in urban Malindi, Kenya. J. Am. Mosq. Control Assoc. 2009, 24, 457–459.
[CrossRef]
134. Impoinvil, D.E.; Keating, J.; Mbogo, C.M.; Potts, M.D.; Chowdhury, R.R.; Beier, J.C. Abundance of immature
Anopheles and culicines (Diptera: Culicidae) in different water body types in the urban environment of
Malindi, Kenya. J. Vector Ecol. 2008, 33, 107–116. [CrossRef]
135. Gadiaga, L.; Machault, V.; Pagès, F.; Gaye, A.; Jarjaval, F.; Godefroy, L.; Cissé, B.; Lacaux, J.P.; Sokhna, C.;
Trape, J.F.; et al. Conditions of malaria transmission in Dakar from 2007 to 2010. Malar. J. 2011, 21, 312–328.
[CrossRef]
136. Dibo, M.R.; Fávaro, E.A.; Parra, M.C.; Santos, T.C.; Cassiano, J.H.; Deitz, K.V.; Pagliotto, A.M.; Zini, N.;
Benetti, D.R.; Chiaravalloti-Neto, F. Evaluation of two sweeping methods for estimating the number of
immature Aedes aegypti (Diptera: Culicidae) in large containers. Rev. Soc. Bras. Med. Trop. 2013, 46, 502–505.
[CrossRef]
137. Courtright, P.; Rotondo, L.; MacArthur, C.; Jones, I.; Weaver, A.; Negash, B.K.; Olobio, N.; Binnawi, K.;
Bush, S.; Abdala, M.; et al. Strengthening the links between mapping, planning and global engagement for
disease elimination: Lessons learnt from trachoma. Br. J. Ophthalmol. 2018, 102, 1324–1327. [CrossRef]
138. 51st World Health Assembly, Geneva, Resolution WHA51.11, Global Elimination of Blinding Trachoma,
1988. Available online: http://www.who.int/blindness/causes/WHA51.11/en/ (accessed on 30 July 2018).
139. Warren, J.M.; Birrell, A.L. Trachoma in remote Indigenous Australia: A review and public health perspective.
Aust. N. Z. J. Public Health 2016, 40, 48–52. [CrossRef]
140. Yamazaki, T.; Inoue, M.; Ogawa, M.; Shiga, S.; Kishimoto, T.; Hagiwara, T.; Matsumoto, T.; Hayashi, T.
Inactivation of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae by ozone. Lett. Appl.
141. Giampaoli, S.; Garrec, N.; Donzi, G.; Valeriani, F.; Erdinger, L.; Romano Spica, V. Regulations concerning
natural swimming ponds in Europe: Considerations on public health issues. J. Water Health 2004, 12, 564–572.
[CrossRef]
142. Gössling, S.; Peeters, P.; Hall, M.C.; Ceron, J.-P.; Dubois, G.; Lehmann, V.; Scot, D. Progress in Tourism
Management Tourism and water use: Supply, demand and security. An international review. Tour. Manag.
2012, 33, 1–15. [CrossRef]
143. Soverow, J.E.; Wellenius, G.A.; Fisman, D.N.; Mittleman, M.A. Infectious Disease in a Warming World: How
Weather Influenced West Nile Virus in the United States (2001–2005). Environ. Health Perspect. 2009, 117,
1049–1052. [CrossRef]
144. de Roda Husman, A.M.; Schets, F.M. Climate Change and Recreational Water-Related Infectious Diseases,
RIVM Publications, 2010. Available online: http://hdl.handle.net/10029/258107 (accessed on 30 July 2018).
145. Castor, M.L.; Beach, M.J. Reducing illness transmission from disinfected recreational water venues:
Swimming, diarrhea and the emergence of a new public health concern. Pediatr. Infect. Dis. J. 2004,
23, 866–870. [CrossRef]
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
20
and Public Health
Review
A Review and Update on Waterborne Viral Diseases
Associated with Swimming Pools
Lucia Bonadonna * and Giuseppina La Rosa *
Italian National Institute of Health, Viale Regina Elena, 299-00161 Rome, Italy
* Correspondence: [email protected] (L.B.); [email protected] (G.L.R.); Tel.: +39-06-49902317 (L.B.);
+39-06-49902718 (G.L.R.)
Received: 8 November 2018; Accepted: 5 January 2019; Published: 9 January 2019
Abstract: Infectious agents, including bacteria, viruses, protozoa, and molds, may threaten the health
of swimming pool bathers. Viruses are a major cause of recreationally-associated waterborne diseases
linked to pools, lakes, ponds, thermal pools/spas, rivers, and hot springs. They can make their way
into waters through the accidental release of fecal matter, body fluids (saliva, mucus), or skin flakes
by symptomatic or asymptomatic carriers. We present an updated overview of epidemiological data
on viral outbreaks, a project motivated, among other things, by the availability of improved viral
detection methodologies. Special attention is paid to outbreak investigations (source of the outbreak,
pathways of transmission, chlorination/disinfection). Epidemiological studies on incidents of viral
contamination of swimming pools under non-epidemic conditions are also reviewed.
Keywords: adenovirus; enterovirus; hepatitis A virus; norovirus; swimming pool; waterborne disease
1. Introduction
Swimming pools have been implicated in the transmission of infections. The risk of infection has
mainly been linked to fecal contamination of the water, generally due to feces released by bathers or to
contaminated source water. Failure in disinfection has been recorded as the main cause of many of the
outbreaks associated with swimming pools.
The majority of reported swimming pool-related outbreaks have been caused by enteric
viruses [1,2]. Sinclair and collaborators reported that 48% of viral outbreaks occur in swimming
pools, 40% in lakes or ponds, and the remaining 12% in fountains, hot springs, and rivers (4% each) [1].
Viruses cannot replicate outside their host’s tissues and cannot multiply in the environment.
Therefore, the presence of viruses in a swimming pool is the result of direct contamination by bathers,
who may shed viruses through unintentional fecal release, or through the release of body fluids
such as saliva, mucus, or vomitus [3]. Evidence suggests that skin may also be a potential source of
pathogenic viruses.

We carried out a comprehensive literature review aimed at investigating waterborne viral outbreaks
linked to swimming pools, to explore the etiological agents implicated, pathways of transmission,
associations between indicator organisms and disease, and key issues related to chlorination/disinfection
procedures. Viral outbreaks are summarized in Table 1. The presence of enteric viruses in swimming pools
under non-epidemic conditions was also reviewed. Different databases (Scopus, PubMed, and Google
Scholar) were accessed using the terms norovirus, Norwalk virus, adenovirus, enterovirus, echovirus,
coxsackievirus, and hepatitis A, in combination with terms recreation, swimming, pool, and water.

IJERPH 2019, 16, 166
3. Viral Outbreaks Related to Swimming Pools
3.1. Adenovirus Outbreaks (N◦ = 15)

Adenoviruses are the enteric viruses most commonly associated with swimming pool-related
outbreaks. Human adenoviruses (HAdVs) belong to the Adenoviridae family and are classified into
seven species (A to G) and more than 90 types [4]. HAdVs are of major public health importance
and can result in a variety of clinical manifestations, including gastroenteritis, respiratory, ocular
and urinary tract infections [5]. Illnesses are common and ubiquitous with a worldwide distribution.
HAdVs are highly stable in the environment and can survive for prolonged periods in water [6].
Transmission in swimming pools can occur by ingestion, direct contact with contaminated water,
or through the inhalation of aerosol [7].
A Brazilian study recently detected HAdVs in Acanthamoeba isolated from water samples collected
from swimming pools [8]. HAdVs were found in 62.5% (10/16) of amoebae with DNA copies up
to 5.1 × 105 per milliliter, suggesting that Acanthamoeba may act as a reservoir and promote HAdV
transmission through water.
The first swimming pool-related outbreak, published in 1953, described a 1951 outbreak in Greeley,
Colorado, which thus came to be known as the “Greeley epidemic”. The outbreak, involving 206
cases, caused a combination of symptoms, such as acute conjunctivitis, pharyngitis, muscle pain,
and fever [9]. Between 25% and 50% of children swimming in the pool were affected. The transmission
apparently occurred either by contact with contaminated objects, such as toys, or while swimming in a
pool. The water was heavily chlorinated, with residual chlorine being close to 0.4 parts per million.
No definite pathogen was identified at the time of publication. Serum samples from Greeley patients
were later tested and showed a specific neutralizing antibody response to HAdV type 3 [10].
In 1954, an epidemic of pharyngeal-conjunctival fever occurred in Washington, D.C.,
with symptoms similar to those of the Greeley epidemic [10]. Over 300 cases were documented,
with acute respiratory illness characterized by one or more of the following symptoms: fever,
pharyngitis, and conjunctivitis. Cases occurred in all age groups, but predominantly in children.
Adenovirus type 3 was isolated in 80 of 300 patients from eye, throat washings and stools. The disease
occurred at different sites: in a children’s summer day camp, in an orphanage, and in two residential
neighborhoods. The suspected, but never confirmed, source of the epidemic was a swimming pool,
even if cases due to direct contact were also recorded in houses and hospitals. The pool, chlorinated by
hand, showed a low level of residual chlorine. Bell and collaborators were the first to suggest the term
pharyngoconjunctival fever for this disease.
In August 1955, 112 cases of pharyngoconjunctival fever occurred in Toronto, linked to an indoor
swimming pool. Seventy-four of the cases were children who had swum in the pool, while the
others had swum in pools elsewhere, or had had direct contact with a case at home. Only one case
had no history of either swimming or direct contact. Most of the children had pharyngitis, fever,
malaise, and muscle pain. Conjunctivitis was absent or minimal in children, but was the main cause of
discomfort in adults [11].
Another outbreak of pharyngoconjunctival fever was documented in August and September 1959
in Saitama Prefecture, Japan, among students of a primary and a middle school [12]. Epidemiological
investigations suggested that the outbreak was mainly due to the contamination of a swimming pool
used by the students of both schools. A total of 358 students were affected: 248 primary school students
(attack rate, 20.6%) and 110 middle school students (attack rate, 19.2%). Laboratory findings suggested
that the epidemic was due to HAdVs 3 and 7.
Foy and coworkers described an outbreak of pharyngoconjunctival fever (45 cases) in two
swimming teams in Washington in 1966. Adenovirus type 3 was the etiological agent [13]. Most
of the infected children had fever, pharyngitis, conjunctivitis, and diarrhea. In adults, symptoms
were milder, with a high incidence of conjunctivitis. The attack rate was 65% and 67% for the two
teams, respectively. Children had swum in the early morning, when the chlorinator of the pool was
22
IJERPH 2019, 16, 166
switched off, to avoid eye irritation with chlorine. Within one week, 25 of the 36 exposed swimmers
became ill. Children swimming in the afternoon, when chlorination was still working, did not get sick.
The infection was shown to spread in families having index cases (20 infected contacts). Analyses of
water were done approximately 14 days after the presumed exposure. For this reason, attempts to
isolate the virus from the water failed.
An outbreak of acute conjunctivitis due to HAdV type 7 occurred in Kansas, USA in 1973: 44 cases
and one hospitalization were documented [14]. Eye symptoms predominated (red or pink eyes,
swollen eyes), but a variety of other signs were also noted (mainly fever, headache, and nausea).
A school swimming pool was identified as the source of infection. Chlorine concentration was low
due to an equipment failure. The epidemic was easily controlled by raising the pool’s chlorine level.
Unfortunately, tests for viruses and bacterial indicators were carried out after super-chlorination and
consequently results were negative.
In 1977, two outbreaks associated with swimming pools occurred in Georgia, USA. The first,
due to HAdV type 3, involved at least 105 cases [15], with patients showing different symptoms,
including sore throat, fever, headache, anorexia and conjunctivitis. In this case, a private swimming
pool was the source of infection. A temporary malfunction in the water filtration system of the pool
associated with inadequate chlorine levels was recorded. Both waterborne and person-to-person
transmission occurred. In the second outbreak, HAdV type 4 was recognized as the etiological agent
of pharyngoconjunctival fever in 72 persons [16]. An insufficient amount of chlorine was found in
the water of the pool. To stop the spread of infection, the pool was closed during the summer and
adequately chlorinated. Adenovirus was recovered from the water sampled from the pool.
In Oklahoma, USA, an outbreak of pharyngitis caused by HAdV type 7a was recorded in
1982 among 77 children attending a swimming pool [17]. Symptoms included conjunctivitis, fever,
sore throat, headache, and abdominal pain. Two cases were hospitalized with dehydration from
persistent vomiting. A malfunction of the automatic pool chlorinator was identified as the cause of the
outbreak. In fact, during the two weeks preceding the epidemic, its failure forced the pool operator to
manually add chlorine to the pool.
Another outbreak was recorded in 1995, in Greece, where 80 athletes under 18 years of age
presented with fever, conjunctivitis, sore throat, weakness, and abdominal pain, after swimming in a
pool [18]. Seven athletes were hospitalized. Virological analyses on clinical samples were not performed
and the illness was attributed to HAdV on the basis of clinical symptoms alone. Water samples from
both the pool and the distribution system were tested for HAdV, enterovirus, and hepatitis A virus by
molecular methods. The water of the pool tested positive for HAdV and negative for the other viruses,
demonstrating its role as the source of infection. Samples from the water system were negative for all
viruses tested. Chlorine levels were found to be low, probably due to a malfunctioning of the pool
chlorination system.
Five HAdV outbreaks associated with swimming were recorded in the 2000s.
In the year 2000, an outbreak of pharyngoconjunctival fever occurred in North Queensland,
Australia, where, after a school camp, 34 children aged 4–12, got sick [19]. In addition to primary cases
acquired at the camp (N◦ = 25), nine other cases were acquired within the households. The school camp
had a large saltwater swimming pool. Adenovirus 3 was isolated from eye and throat swabs. A PCR
analysis of water samples for HAdV did not yield positive results. It was, however, demonstrated that
the pool was not properly maintained, and that the level of residual chlorine was inadequate.
An outbreak of pharyngoconjunctival fever affecting 59 children under 15 was recorded in a
municipality of Northern Spain in July 2008 [20]. Forty-three cases were recognized as primary cases,
all of whom attended a municipal swimming pool. The remaining 15 children were secondary cases,
which had been in close contact with a primary case. Adenovirus type 4 was detected in pharyngeal
swabs. Electrical system failures causing the intermittent breakdown of the pool’s bromine dosing
pumps and the slowing down of water circulation were assumed to have been the cause of the outbreak.
Swimming was only allowed after the disinfection system was restored and appropriate concentrations
23
IJERPH 2019, 16, 166
of bromine were reached. Due to logistic problems, no water samples were taken from the swimming
pool for virological analysis.
In 2011, children (4–9 years old) who had attended a swimming training center in Eastern China
showed symptoms of pharyngoconjunctival fever. Adenovirus type 3 was recognized as the etiological
agent [21]. A total of 134 cases were confirmed from among 900 amateur swimmers, with an incidence
of 14.9%. Fourteen hospital admissions were documented. Fever, tonsillitis, sore throat, headache,
sneezing, cough, conjunctivitis, fatigue, and diarrhea occurred among the bathers. The low level of
residual chlorine in the water, along with excessive crowding in the pool were suggested as having
caused the epidemic.
In the same year, in a primary school in Taiwan, an outbreak of HAdV infection occurred among
373 students, with four hospitalizations [22]. Most of the students attended a swimming course in two
swimming facilities outside the school and presented with fever and symptoms of upper respiratory
tract infection. Other symptoms included diarrhea, vomiting, skin eruptions and conjunctivitis.
Throat swabs of affected students were tested for influenza virus, adenovirus, respiratory syncytial
virus, coronavirus, metapneumovirus, parainfluenza types 1–4, and herpes simplex virus. Samples
were found positive only for HAdV type 7. Water samples were not obtained from any of the facilities
for virological analysis.
In 2013, an outbreak of pharyngoconjunctival fever involved 55 people (49 students and six staff)
at a university in Beijing, China [23]. Fifty patients (91%) attending the same swimming pool two
weeks before the onset of symptoms were considered primary cases. The other five subjects (9%) who
had not swum in the pool were defined as secondary cases (person-to-person transmission). Human
AdV type 4 was identified from both eye and throat swabs of the patients and from concentrated
swimming pool water samples. Gene sequences obtained from the water samples exhibited a 100%
match with the sequences obtained from swab samples. Control measures included the emptying and
closing of the pool, and the disinfection with a high dose of sodium hypochlorite (500 mg/L).
3.2. Enterovirus Outbreaks (N◦ = 6)

Enterovirus is a genus in the family Picornaviridae, consisting of four human enterovirus species.
Enteroviruses can cause many illnesses, including paralysis, meningitis, and cardiomyopathy, although
most infections are asymptomatic or cause less severe conditions, such as colds and fever. A number
of reports have described enterovirus infections linked to swimming pools.
The first enterovirus swimming pool-related outbreak occurred in 1987, at a municipal pool
in Colorado, USA. Twenty-six children presented with fever along with at least one additional
symptom such as malaise, headache, stomachache, nausea, or diarrhea [24]. It was found that the
pool chlorination system was operating improperly, with chlorine levels close to zero. Stool specimens
collected from the children affected were tested for common enteric bacterial pathogens (Salmonella,
Shigella, Aeromonas, and Campylobacter), but not for viruses. Enterovirus was suggested as a likely
etiological agent based on clinical manifestations, course of disease, incubation time, and the exclusion
of likely bacterial pathogens.
An enterovirus outbreak occurred in Ireland in 1992, with 46 cases experiencing vomiting,
diarrhea, and headache after attending an outdoor swimming pool in a small seaside village.
One subject had vomited into the pool, and echovirus 30 was isolated from this case and from
six other cases. Chlorine levels were found to comply with health standards, but were inadequate to
contain the risk of infection from vomitus [25].
Another echovirus 30 outbreak occurred in Rome, Italy, in late 1997 [26]. Children from two schools
showed clinical manifestations after swimming in a pool. Twenty children had meningitis-like symptoms
(fever, headache, and vomiting), and six of them were hospitalized. Other 48 children had respiratory
symptoms consistent with enterovirus infection. Echovirus 30 was isolated from the cerebrospinal fluid
and stools of the hospitalized children. Based on the epidemiological characteristics, it was hypothesized
that person-to-person transmission occurred both at the swimming pool and in a number of classrooms.
24
IJERPH 2019, 16, 166
Data on chlorination at the time of the outbreak were not available. Virological analysis of pool water was
performed one month after the outbreak, but yielded no positive results.
In South Africa, an outbreak involving 90 children occurred following a summer camp in 2001 [27].
Camp activities included swimming and other aquatic sports. Symptoms included mainly headaches,
sore eyes, and/or abdominal discomfort, with one case of vomiting. Four children were hospitalized for
meningitis. Echovirus 3 was detected in cerebrospinal fluid and stool samples from symptomatic and
asymptomatic children. The presence of viruses in the pool was not investigated. Water contamination
was confirmed through a total coliform count.
In Germany, 215 cases of aseptic meningitis were recorded from July to October 2001 [28]. Swimming
in a public, nature-like pond was identified as a risk factor for disease. Up to 1500 people visited the pond
each day during the summer holidays. Echovirus 3 and 30 were detected in cerebrospinal fluid samples
taken from some of the patients. An echovirus 30 sequence obtained from one water sample collected
from the pond showed a high level of genetic similarity (99% nucleotide homology) with sequences
obtained from patient isolates.
In August 2003, an outbreak of meningitis occurred among campers staying at a campground in
Connecticut, USA [29]. A total of 12 cases of aseptic meningitis, four hospitalized patients and 24 cases
of enterovirus-like illness with symptoms such as headache, neck stiffness, photophobia, sore throat,
chills, or exanthema were identified. Echovirus serotype 9 was detected in cerebrospinal fluid samples
from three of the patients. The spread of the virus was associated with swimming in a crowded
pool, which had low chlorine levels. As a result, the pool water was intermittently contaminated
with enterovirus.
3.3. Hepatitis a Virus Outbreaks (N◦ = 3)

Hepatitis A is a virus causing mild to severe liver disease. Globally, there are an estimated 1.4 million
cases of hepatitis A every year. The virus is transmitted mainly via the fecal/oral route through the
ingestion of contaminated food and water, or through direct contact with an infected subject. There is
evidence to suggest that hepatitis A can be acquired by swimming in contaminated water.
In September 1979, an outbreak of hepatitis A affecting 56 children (5–17 years old) and causing
31 hospitalizations was recorded in Hungary [30]. All of the children swam in a pool at a summer
camp. The pool was a non-chlorinated thermal pool/spa, which was overcrowded during the month
of August. It was concluded that crowding and poor hygienic conditions, with a suspected accidental
fecal release, contributed to the outbreak.
Another outbreak of hepatitis A was described in the USA during 1989. It involved 20 cases,
probably associated with a public swimming pool [31]. It was hypothesized that a cross-connection
between a sewage line and the pool water intake line may have been the cause of the outbreak.
According to another hypothesis, it was a swimmer who contaminated the water in the pool. However,
disinfectant levels in the pools met local standards.
Seven hepatitis A cases among children from six families were documented in Australia in
1997 [32]. The children had attended an outdoor spa pool treated with hydrogen peroxide solution.
It was hypothesized that hepatitis A virus was shed by the index case in the spa pool, and subsequently
ingested by the others, who became secondary cases. Virological analyses of water samples were
not performed.
3.4. Norovirus Outbreaks (N◦ = 7)

Noroviruses (NoVs), formerly known as Norwalk-like viruses, are small viruses within the family
Caliciviridae, subdivided into at least seven genogroups (GI–VII), with GI, GII, and GIV infecting
humans. They are recognized as a major cause of sporadic and epidemic gastroenteritis in both
industrialized and non-industrialized countries.
Outbreaks have been associated with a variety of settings including childcare centers, hospitals,
nursing homes, cruise ships and restaurants. Noroviruses are mainly transmitted via the fecal-oral
25
IJERPH 2019, 16, 166
route through contaminated food or water. Norovirus-contaminated water—both recreational and

drinking water—can thus lead to waterborne infections. A number of NoV swimming-pool related
outbreaks have been described.
In 1977, an outbreak of acute gastroenteritis with the typical symptoms of vomiting, cramping,
nausea and diarrhea was documented among 103 students and teachers at a primary school in Ohio,
USA [33]. Serologic studies suggested infection by Norwalk virus to be the cause of the outbreak.
The first cases recorded were caused by swimming in a contaminated pool, and a person-to-person
transmission followed. The water of the pool tested negative for both bacterial and viral pathogens.
Water contamination was linked to both the pool chlorinator, which was accidentally turned off at the
time of the school visits, and a leak in the water supply pipes.
A large outbreak of gastroenteritis due to NoVs was recorded in July 2001 in Helsinki. It involved
242 people (children and adults) after bathing in an outdoor wading pool [34]. Norovirus and astrovirus
were detected in both patient stool samples and pool water, with identical nucleotide sequences.
The pool was found to be heavily contaminated with human fecal material carried from public toilets.
The pool water had been manually chlorinated three times per week, thus not continuously. To control
the outbreak, the pool was emptied, refilled, and the water was heavily chlorinated (up to 10 mg/L of
free chlorine). For the subsequent swimming season, the pool was equipped with both a continuous
chlorination system and a water filtration system.
In 2002, a NoV outbreak associated with a swimming pool was reported in Minnesota, USA.
Thirty-six persons of three different youth sports teams became ill after swimming in a hotel pool and
spa [29]. Unfortunately, there is no other information available on this epidemic.
In 2004, an acute gastroenteritis epidemic affected 53 people who had swum in a pool in Vermont,
USA. Vomiting and/or diarrhea occurred within 72 h of attending a private indoor pool. Specimens
tested positive for NoV. At the time of the inspection, no equipment failures or irregularities were
identified. Nevertheless, deficiencies in pool operation and maintenance, including poorly trained
operators, inadequate maintenance checks, failure to alert management, and insufficient record keeping
were reported [35].
Finally, Yoder and coworkers documented a NoV outbreak linked to a hotel pool in Wisconsin,
USA in 2006, with 18 persons exhibiting symptoms of gastroenteritis, related to inadequate disinfection
and continued use by ill swimmers [36].
Table 1. List of viral swimming pool-related outbreaks.

N◦People
Caption Etiological Agent Location Virus Identified in Pool Waters Year Reference
Affected
206 HAdV Colorado, USA Not tested 1951 [9]
>300 HAdV 3 Washington Not detected 1954 [10]
112 HAdV 3 Canada Not tested 1955 [11]
358 HadV 3 and 7 Japan Not tested 1959 [12]
No. Water analyses two weeks after the
45 HAdV 3 Washington 1966 [13]
exposure
No. Samples were taken after
44 HAdV 7 Kansas 1973 [14]
hyperchlorination of the pool
105 HAdV 3 Georgia Not tested 1977 [15]
Yes. First swimming-pool related outbreak in
Adenovirus 72 HAdV 4 Georgia 1977 [16]
which AdV was recovered from water samples
77 HAdV 7a Oklahoma Not tested 1982 [17]
Yes. Pool water samples tested found positive
80 Unknow Greece for AdVs, and negative for enteroviruses and 1995 [18]
hepatitis A virus
34 HAdV 3 Australia No 2000 [19]
59 HAdV 4 Spain Not tested 2008 [20]
134 HAdV 3 China Not tested 2011 [21]
373 HAdV 7 Taiwan Not tested 2011 [22]
Yes. Gene sequences obtained from the water
55 HAdV 4 China samples were 100% identical to the sequences 2013 [23]
obtained from the swab samples.
26
IJERPH 2019, 16, 166
Table 1. Cont.
N◦ People
Caption Etiological Agent Location Virus Identified in Pool Waters Year Reference
Affected
26 Enterovirus-like Colorado Not tested 1987 [24]
46 Echovirus 30 Ireland Not tested 1979 [25]
No. Virological analysis of pool waters was
68 Echovirus 30 Italy 1997 [26]
performed one month after the outbreak
Enterovirus Not tested. Unclean swimming-pool water
90 Echovirus 3 South Africa 2001 [27]
was confirmed by total coliform count
Yes. An echovirus 30 sequence obtained from
pond water showed 100% amino-acid
215 Echovirus 13 and 30 Germany 2001 [28]
homology with sequence obtained from
patient isolates
36 Echovirus 9 Connecticut Not tested 2003 [29]
56 - Hungary Not tested 1987 [30]
Hepatitis A virus 20 - Louisiana, USA Not tested 1989 [31]
7 - Australia Not tested 1997 [32]
No. Pool water were found negative for both
103 - Ohio 1977 [33]
bacterial and viral pathogens.
Yes. Identical sequence was detected in both
242 - Finland 2001 [34]
patient stool and pool water
Norovirus
36 - Minnesota Not known 2001–2002 [29]
53 - Vermont Not tested 2004 [35]
18 - Wisconsin Not known 2006 [36]
4. Occurrence of Enteric Viruses in Swimming Pools under Non-Epidemic Conditions

To date, a limited number of studies examined the extent of viral contamination in swimming
pools under non-epidemic conditions.
The first isolation of viruses from urban wading pools was documented in Albany, NY, USA in
1959 [37]. Two enteroviruses (echovirus 3 and echovirus 11) were identified from two chlorinated
pools, both filled with water from the municipal supply. The same echovirus strains were found to
be present in raw sewage sampled at the Albany treatment plant, reflecting widespread infection in
the community.
In Toronto, Canada, Coxsackievirus B1 was isolated from children with pleurodynia, myalgia,
and primary peritonitis during 1964. Examination for the virus content of a gauze swab, which was
placed daily in a wading pool with high bather load located in a congested city area, revealed the
presence of the same Coxsackievirus type [38].
In 1979 in Israel, swimming pool samples were found positive for enterovirus: three for Echovirus
7, two for Coxsackievirus B6, and one for Echovirus 6 [39]. Viruses were isolated from water samples
with no detectable fecal or total coliform bacteria.
Different enteroviruses were detected in swimming pools and wading pools equipped with gas
chlorine and sand/gravel filters in Texas, USA, in 1980 [40]. After virus concentration from water,
samples were assayed on cell culture and plaque assays. Enteroviruses were found in 10/14 (71%) of
the examined samples. Coxsackieviruses B3 and B4, poliovirus 1, and echovirus 7 were isolated in
pool waters. No correlation was found with total coliform bacteria, as six among the positive virus
samples were negative for coliforms. In three samples, viruses were detected in the presence of free
chlorine exceeding 0.4 ppm and in the absence of coliforms, indicating that viruses can survive low
levels of biocides in actively used pools. Cell cultures used in the study were suited for the isolation of
enteroviruses, but it is likely that other viruses, not capable of growing on those cell lines, could also
have been present in the water.
Three years later, in 1983, enteroviruses were detected in 28.4% of 116 water samples [41] collected
from three outdoor swimming pools. A direct correlation was established between viral and microbial
contamination, and the low exchange of water in the pools.
In 2004, van Heerden and coworkers detected HAdV in 12 of 64 samples (18.7%) from an
indoor swimming pool and in three of 28 samples (10.7%) from an outdoor swimming pool [7].
Quantitative data were also obtained by Real-time PCR. Application of these results in an exponential
27
IJERPH 2019, 16, 166
risk assessment model, assuming a daily ingestion of 30 mL of water during swimming, indicated a
daily risk of infection ranging from 1.92 × 10−3 to 3.69 × 10−3 . No acceptable microbial risk has thus
far been established for swimming pool water. However, pool water quality is generally considered
comparable to drinking water quality (absence of fecal indicators and pathogens). For this reason,
a maximum of one infection per 10,000 consumers per year has been recommended as an acceptable
level of microbial risk for swimming pools. The risk of HAdV infections calculated for the swimming
pool water in the study exceeded this acceptable risk.
More recently, in 2007 in Cyprus, HAdVs and enteroviruses were detected in public swimming
pools complying with bacteriological standards (such as fecal coliforms and enterococci) [42].
The investigation was performed over a period of 21 months, from April 2007 to December 2008.
A total of 126 samples were obtained from swimming pools located in five major cities. Bacteriological
marker analysis showed that 98% of pools complied with the national regulations. Enteroviruses were
identified in four swimming pools, one containing echovirus 18, two containing echovirus 30 and one
containing poliovirus Sabin 1. In four swimming pools, HAdVs were detected, all characterized as
type 41.
In 2013–2014, a study investigated the presence of human enteric viruses (adenovirus, norovirus,
and enterovirus) in indoor and outdoor swimming pool waters in Rome. Bacteriological parameters
(fecal indicator bacteria, heterotrophic plate count, Pseudomonas aeruginosa, and Staphylococcus aureus)
were also investigated [43]. Moreover, the study was the first to examine the occurrence of non-enteric
viruses in swimming pool waters: human papillomavirus (HPV) and human polyomavirus (HPyV).
Interestingly, enteric viruses were not detected, while both HPVs and HPyVs were identified in
9/14 swimming pool water samples, by means of molecular methods. Neither of these viruses
had previously been recognized as potential recreational waterborne pathogens, although the WHO
Guidelines for safe recreational water environments do include HPVs among non-fecally-derived
viruses as viruses associated with plantar warts [3]. A variety of HPVs and HPyVs were found in
another study investigating spa/pool waters in Rome [44].
Recently, disinfected water from sixteen pools and spa collected in Rome between 2015 and 2018
were examined for the presence of human enteric viruses (adenovirus, norovirus and enterovirus.
Viruses were detected in 25% of the analyzed samples by molecular methods: two samples were
positive for adenovirus (type 41) and three samples for norovirus GII (type GII.4) (Bonadonna et al.,
unpublished data).
5. Concluding Remarks
Starting with the first HAdV outbreak recorded in 1951, we reviewed all of the reports concerning
swimming-pool related viral illness. The data collected here confirm the involvement of viruses in
cases and outbreaks associated with swimming pool attendance.
A number of considerations emerge:
• The paper reviews 29 viral outbreaks due to adenovirus, enterovirus, hepatitis A virus,
and norovirus, accounting for more than 3000 cases. Nevertheless, there are likely to have
been many other undetected cases and outbreaks. In fact, waterborne diseases are difficult to
record because of their wide variety, the difficulty associating symptoms with water use/contact,
and the limitations of pathogen detection methods. In the studies described, viruses responsible
for reported cases were detected in pool waters only in 21% of the outbreaks, and were found to
match with viruses of clinical origin. Currently, better and more rapid methods for the detection
of viruses in water samples are available than in the past, resulting in better studies and improved
reporting of viral recreational outbreaks worldwide. This allows researchers to identify the causes
of outbreaks and possible contributing factors for them. An excellent model to follow is the
US-Waterborne Disease and Outbreak Surveillance System (WBDOSS) that has been collecting
and reporting data related to occurrences and causes of waterborne disease outbreaks associated
with drinking and recreational waters since 1971.
28
IJERPH 2019, 16, 166
• Some of the studies found that waters meeting state or local water quality requirements contained
enteric viruses and were the source of disease outbreaks, confirming that bacterial indicators are
unreliable indicators of the presence of viruses and that enteric viruses are important hazardous
waterborne pathogens. Indeed, despite the relatively low concentration of viruses in water,
they may nevertheless pose health risks due to their low infectious doses (10–100 virions).
• The human illnesses associated with enteric viruses in the reviewed studies were diverse: the most
commonly reported symptoms were gastroenteritis, respiratory symptoms, and conjunctivitis.
More severe symptoms were also documented, however, including hepatitis and central nervous
system infections (aseptic meningitis).
• The majority of the outbreaks described involved mainly children and young people less than
18 years of age. This may be attributable to differences in behaviors, susceptibility and/or immune
defenses between children and adults. Children are known to experience more severe symptoms
than adults.
• Low concentrations of disinfectant/disinfection malfunction in swimming pools were reported in
the vast majority of the outbreaks. Only in one case the concentration of biocide was considered high.
In light of the health hazards posed by swimming pools, it is essential to constantly monitor water
quality in swimming pools and to assess the effectiveness of treatment and disinfection processes
and compliance with standards. Specifically, appropriate chemical and microbial evaluation of water
quality should be carried out, especially when large numbers of bathers are expected to use the pools.
Overcrowding should in any case be prevented. Since the behavior of swimmers may affect water
quality, strict rules of behavior in the pool should be followed and enforced, including shower before
entering the water, wash hands after using the toilet, take children to bathroom before swimming, and,
importantly, avoid swimming while sick.
Author Contributions: L.B. and G.L.R. conceived and wrote the paper, and approved the submitted version.
Funding: This research received no external funding.
References
1. Sinclair, R.G.; Jones, E.L.; Gerba, C.P. Viruses in recreational water-borne disease outbreaks: A review.
J. Appl. Microbiol. 2009, 107, 1769–1780. [CrossRef] [PubMed]
2. Barna, Z.; Kadar, M. The risk of contracting infectious diseases in public swimming pools. A review. Ann. Ist.
Super. Sanità 2012, 48, 374–386. [CrossRef] [PubMed]
3. WHO: Guidelines for Safe Recreational Water Environments—Swimming Pools and Similar Environments.
Available online: http://wwwwho.int/iris/handle/10665/43336 (accessed on 8 November 2018).
4. Human Adenovirus Working Group. Available online: http://hadvwg.gmu.edu/ (accessed on 14 December
2018).
5. La Rosa, G.; Suffredini, E. Adenovirus. In Handbook of Foodborne Diseases, 1st ed.; Liu, D., Ed.; CRC Press:
Boca Raton, FL, USA, 2018.
6. Mena, K.D.; Gerba, C.P. Waterborne adenovirus. Rev. Environ. Contam Toxicol. 2009, 198, 133–167. [CrossRef]
[PubMed]
7. Van Heerden, J.; Ehlers, M.M.; Grabow, W.O.K. Detection and risk assessment of adenoviruses in swimming
pool water. J. Appl. Microbiol. 2005, 99, 1256–1264. [CrossRef] [PubMed]
8. Staggemeier, R.; Arantes, T.; Caumo, K.S.; Rott, M.B.; Spilki, F.R. Detection and quantification of human
adenovirus genomes in Acanthamoeba isolated from swimming pools. An. Acad. Bras. Cienc. 2016, 88
(Suppl. 1), 635–641. [CrossRef] [PubMed]
9. Cockburn, T.A. An epidemic of conjunctivitis in Colorado associated with pharyngitis, muscle pain,
and pyrexia. Am. J. Ophthalmol. 1953, 36, 1534–1539. [CrossRef]
10. Bell, J.A.; Rowe, W.P.; Engler, J.I.; Parrot, R.H.; Huebner, R.J. Pharyngoconjunctival fever; epidemiological
studies of a recently recognized disease entity. J. Am. Med. Assoc. 1955, 157, 1083–1092. [CrossRef]
29
IJERPH 2019, 16, 166
11. Ormsby, H.L.; Aitchison, W.S. The role of the swimming pool in the transmission of pharyngeal-conjunctival
fever. Can. Med. Assoc. J. 1955, 73, 864–866.
12. Fukumi, H.; Nishikawa, M.; Takemura, M.; Odaka, Y. isolation of adenovirus possessing both the antigens of
types 3 and 7. Jpn. J. Med. Sci. Biol. 1961, 14, 173–181. [CrossRef]
13. Foy, H.M.; Cooney, M.K.; Hatlen, J.B. Adenovirus type 3 epidemic associated with intermittent chlorination
of a swimming pool. Arch. Environ. Health 1968, 17, 795–802. [CrossRef]
14. Caldwell, G.G.; Lindsey, N.J.; Wulff, H.; Donnelly, D.D.; Bohl, F.N. Epidemic of adenovirus type 7 acute
conjunctivitis in swimmers. Am. J. Epidemiol. 1974, 99, 230–234. [CrossRef] [PubMed]
15. Martone, W.J.; Hierholzer, J.C.; Keenlyside, R.A.; Fraser, D.W.; D’Angelo, L.J.; Winkler, W.G. An outbreak of
adenovirus type 3 disease at a private recreation center swimming pool. Am. J. Epidemiol. 1980, 111, 229–237.
[CrossRef] [PubMed]
16. D’Angelo, L.J.; Hierholzer, J.C.; Keenlyside, R.A.; Anderson, L.J.; Martone, W.J. Pharyngoconjunctival fever
caused by adenovirus type 4: Report of a swimming pool-related outbreak with recovery of virus from pool
water. J. Infect. Dis. 1979, 140, 42–47. [CrossRef] [PubMed]
17. Turner, M.; Istre, G.R.; Beauchamp, H.; Baum, M.; Arnold, S. Community outbreak of adenovirus type 7a
infections associated with a swimming pool. South. Med. J. 1987, 80, 712–715. [CrossRef] [PubMed]
18. Papapetropoulou, M.; Vantarakis, A.C. Detection of adenovirus outbreak at a municipal swimming pool by
nested PCR amplification. J. Infect. 1998, 36, 101–103. [CrossRef]
19. Harley, D.; Harrower, B.; Lyon, M.; Dick, A. A primary school outbreak of pharyngoconjunctival fever caused
by adenovirus type 3. Commun. Dis. Intell. 2001, 25, 9–12. [PubMed]
20. Artieda, J.; Pineiro, L.; Gonzalez, M.; Munoz, M.; Basterrechea, M.; Iturzaeta, A.; Cilla, G. A swimming
pool-related outbreak of pharyngoconjunctival fever in children due to adenovirus type 4, Gipuzkoa, Spain,
2008. Eurosurveillance 2009, 14, 19125.
21. Xie, L.; Yu, X.F.; Sun, Z.; Yang, X.H.; Huang, R.J.; Wang, J.; Yu, A.; Zheng, L.; Yu, M.C.; Hu, X.W.; et al.
Two adenovirus serotype 3 outbreaks associated with febrile respiratory disease and pharyngoconjunctival
fever in children under 15 years of age in Hangzhou, China, during 2011. J. Clin. Microbiol. 2012, 50,
1879–1888. [CrossRef]
22. Wei, S.H. An Adenovirus Outbreak Associated with a Swimming Facility. SM Trop. Med. J. 2016, 1, 1007.
23. Li, J.; Lu, X.; Sun, Y.; Lin, C.; Li, F.; Yang, Y.; Liang, Z.; Jia, L.; Chen, L.; Jiang, B.; et al. A swimming
pool-associated outbreak of pharyngoconjunctival fever caused by human adenovirus type 4 in Beijing,
China. Int. J. Infect. Dis. 2018, 75, 89–91. [CrossRef]
24. Lenaway, D.D.; Brockmann, R.; Dola, G.J.; Cruz-Uribe, F. An outbreak of an enterovirus-like illness at a
community wading pool: Implications for public health inspection programs. Am. J. Public Health 1989, 79,
889–890. [CrossRef] [PubMed]
25. Kee, F.; McElroy, G.; Stewart, D.; Coyle, P.; Watson, J. A community outbreak of echovirus infection associated
with an outdoor swimming pool. J. Public Health Med. 1994, 16, 145–148. [CrossRef] [PubMed]
26. Faustini, A.; Fano, V.; Muscillo, M.; Zaniratti, S.; La Rosa, G.; Tribuzi, L.; Perucci, C.A. An outbreak of aseptic
meningitis due to Echovirus 30 associated with attending school and swimming in pools. Int. J. Infect. Dis.
2006, 10, 291–297. [CrossRef] [PubMed]
27. Yeats, J.; Smuts, H.; Serfontein, C.J.; Kannemeyer, J. Investigation into a school enterovirus outbreak using
PCR detection and serotype identification based on the 5’ non-coding region. Epidemiol. Infect. 2005, 133,
28. Hauri, A.M.; Schimmelpfennig, M.; Walter-Domes, M.; Letz, A.; Diedrich, S.; Lopez-Pila, J.; Schreier, E.
An outbreak of viral meningitis associated with a public swimming pond. Epidemiol. Infect. 2005, 133,
29. Yoder, J.S.; Blackburn, B.G.; Craun, G.F.; Hill, V.; Levy, D.A.; Chen, N.; Lee, S.H.; Calderon, R.L.; Beach, M.J.
Surveillance for waterborne-disease outbreaks associated with recreational water—United States, 2001–2002.
MMWR Surveill. Summ. 2004, 53, 23–45.
30. Solt, K.; Nagy, T.; Csohan, A.; Csanady, M.; Hollos, I. An outbreak of hepatitis A due to a thermal spa.
Bp. Kozeu. 1994, 26, 8–12.
31. Mahoney, F.J.; Farley, T.A.; Kelso, K.Y.; Wilson, S.A.; Horan, J.M.; McFarland, L.M. An outbreak of hepatitis
A associated with swimming in a public pool. J. Infect. Dis. 1992, 165, 613–618. [CrossRef]
30
IJERPH 2019, 16, 166
32. Tallis, G.; Gregory, J. An outbreak of hepatitis A associated with a spa pool. Commun. Dis. Intell. 1997, 21,
353–354.
33. Kappus, K.D.; Marks, J.S.; Holman, R.C.; Bryant, J.K.; Baker, C.; Gary, G.W.; Greenberg, H.B. An outbreak of
Norwalk gastroenteritis associated with swimming in a pool and secondary person-to-person transmission.
Am. J. Epidemiol. 1982, 116, 834–839. [CrossRef]
34. Maunula, L.; Kalso, S.; von Bonsdorff, C.H.; Ponka, A. Wading pool water contaminated with both
35. Podewils, L.J.; Zanardi, B.L.; Hagenbuch, M.; Itani, D.; Burns, A.; Otto, C.; Blanton, L.; Adams, S.; Monroe, S.S.;
Beach, M.J.; et al. Outbreak of norovirus illness associated with a swimming pool. Epidemiol. Infect. 2007,
135, 827–833. [CrossRef] [PubMed]
36. Yoder, J.S.; Hlavsa, M.C.; Craun, G.F.; Hill, V.; Roberts, V.; Yu, P.A.; Hicks, L.A.; Alexander, N.T.; Calderon, R.L.;
Roy, S.L.; et al. Surveillance for waterborne disease and outbreaks associated with recreational water use and
other aquatic facility-associated health events—United States, 2005–2006. MMWR Surveill. Summ. 2008, 57, 1–29.
[PubMed]
37. Kelly, S.; Sanderson, W.W. Enteric Viruses in Wading Pools. Public Health Rep. 1961, 76, 199–200. [CrossRef]
[PubMed]
38. McLean, D.M.; Larke, R.P.B.; McNaughton, G.A.; Best, J.M.; Smith, P. Enteroviral Syndromes in Toronto,
1964. Can. Med. Assoc. J. 1965, 92, 658–661. [PubMed]
39. Marzouk, Y.M.; Goyal, S.M.; Gerba, C. Relationship of viruses and indicator bacteria in water and wastewater
of Israel. Water Res. 1980, 14, 1585–1590. [CrossRef]
40. Keswick, B.H.; Gerba, C.P.; Goyal, S.M. Occurrence of enteroviruses in community swimming pools. Am. J.
Public Health 1981, 71, 1026–1030. [CrossRef]
41. Cotor, F.; Zavate, O.; Finichiu, M.; Avram, G.; Ivan, A. Enterovirus contamination of swimming pool water;
correlation with bacteriological indicators. Virologie 1983, 34, 251–256. [PubMed]
42. Bashiardes, S.; Koptides, D.; Pavlidou, S.; Richter, J.; Stavrou, N.; Kourtis, C.; Papageorgiou, G.T.;
Christodoulou, C.G. Analysis of enterovirus and adenovirus presence in swimming pools in Cyprus from
2007–2008. Water Sci. Technol. 2011, 63, 2674–2684. [CrossRef] [PubMed]
43. La Rosa, G.; Della Libera, S.; Petricca, S.; Iaconelli, M.; Briancesco, R.; Paradiso, R.; Semproni, M.; Di
Bonito, P.; Bonadonna, L. First detection of papillomaviruses and polyomaviruses in swimming pool waters:
Unrecognized recreational water-related pathogens? J. Appl. Microbiol. 2015, 119, 1683–1691. [CrossRef]
44. Di Bonito, P.; Iaconelli, M.; Gheit, T.; Tommasino, M.; Della Libera, S.; Bonadonna, L.; La Rosa, G. Detection
of oncogenic viruses in water environments by a Luminex-based multiplex platform for high throughput
screening of infectious agents. Water Res. 2017, 123, 549–555. [CrossRef] [PubMed]
31
and Public Health
Article
Legionellosis Associated with Recreational Waters:
A Systematic Review of Cases and Outbreaks in
Swimming Pools, Spa Pools,
and Similar Environments
Erica Leoni 1, * ID
, Federica Catalani 2 ID
, Sofia Marini 3 ID
and Laura Dallolio 1 ID
1 Unit of Hygiene, Public Health and Medical Statistics, Department of Biomedical and Neuromotor Sciences,
University of Bologna, via S. Giacomo 12, 40126 Bologna, Italy; [email protected]
2 School of Hygiene and Preventive Medicine, Department of Biomedical and Neuromotor Sciences,
University of Bologna, via S. Giacomo 12, 40126 Bologna, Italy; [email protected]
3 Department of Life Quality Studies, University of Bologna, Campus of Rimini; Corso d’Augusto 237,
47921 Rimini, Italy; sofi[email protected]
* Correspondence: [email protected]; Tel.: +39-051-20948-07
Received: 12 July 2018; Accepted: 25 July 2018; Published: 30 July 2018
Abstract: Legionella spp. is widespread in many natural and artificial water systems, such as hot
water distribution networks, cooling towers, and spas. A particular risk factor has been identified
in the use of whirlpools and hot tubs in spa facilities and public baths. However, there has been no
systematic synthesis of the published literature reporting legionellosis cases or outbreaks related
to swimming/spa pools or similar environments used for recreational purposes (hot springs, hot
tubs, whirlpools, natural spas). This study presents the results of a systematic review of the literature
on cases and outbreaks associated with these environments. Data were extracted from 47 articles,
including 42 events (17 sporadic cases and 25 outbreaks) and 1079 cases, 57.5% of which were
diagnosed as Pontiac fever, without any deaths, and 42.5% were of Legionnaires’ disease, with a
fatality rate of 6.3%. The results are presented in relation to the distribution of Legionella species
involved in the events, clinical manifestations and diagnosis, predisposing conditions in the patients,
favourable environmental factors, and quality of the epidemiological investigation, as well as in
relation to the different types of recreational water sources involved. Based on the epidemiological
and microbiological criteria, the strength of evidence linking a case/outbreak of legionellosis with a
recreational water system was classified as strong, probable, and possible; in more than half of the
events the resulting association was strong.
Keywords: Legionella spp.; Legionnaires’ disease; Pontiac fever; recreational water; hot tubs;
whirlpools; spa pools; swimming pools
1. Introduction
Legionellosis is a disease transmitted through the inhalation of particles of aerosolized water
contaminated by the opportunistic waterborne pathogen, Legionella spp. [1]. After the first recognition
of legionellosis in 1976, when 221 participants of the annual convention of the American Legion
contracted pneumonia and 34 of them died, surveillance systems were developed and implemented in
several countries [2]. Legionellosis surveillance is a current public objective: In 2015, according to the
European Centre for Disease Prevention and Control surveillance, 7034 cases were reported in Europe,
concerning 1.4 cases per 100,000 inhabitants [3].

IJERPH 2018, 15, 1612
The majority of outbreaks described in the literature are correlated to Legionella pneumophila,
in particular serogroup 1, but other serogroups and species were also associated to human disease,
such as L. micdadei (now classified as Tatlockia micdadei), L. dumoffii, and L. longbeachae [4]. The two
fundamental clinical pictures determined by these infective agents are Legionnaires’ disease (LD) and
Pontiac fever (PF): The former is generally characterized by an acute pneumonia and, rarely, by an
extrapulmonary disease; Pontiac fever is a mild, self-limiting, flu-like illness, which resolves in a
few days.
Legionella spp. are widely distributed in both natural (i.e., lakes, rivers, groundwater, thermal
water) and man-made aquatic environments, such as the water systems of hospitals, hotels, private
houses [5,6], cooling towers [7], dental units [8,9], and recreational [10,11] or therapeutic [12,13]
facilities. Any system or equipment which contains, stores, or re-circulates non-sterile water that can
be aerosolized is a source of legionellosis [14,15]. Considering these elements, the recreational use
of water is an important potential way of exposure to Legionella spp., especially in hot water pools
equipped with hydromassage systems. A recent review on outbreaks of LD and PF highlights that
14% of the reported outbreaks from 2006 to 2017 recognized pools or spas as an attributed or suspected
source [16]. The role of these recreational facilities appears even more significant if one considers the
growing popularity of private hot tubs and the increasing number of people frequenting public spa
pools and similar environments.
Generally, the outbreak analysis and control measures, specific for each exposure setting, are
essential tasks of Public Health Authorities, including outbreak surveillance and analysis specifically
dedicated to the recreational water context. Epidemiological knowledge about these themes must
be constantly updated. To our knowledge, no systematic synthesis or critical appraisal exists of the
published literature reporting sporadic cases or outbreaks of LD and/or PF associated with recreational
water. In the present study, we performed a systematic review and analysis of investigations
on legionellosis cases or outbreaks related to treated and untreated recreational water, including
natural waters, swimming pools, spa pools, and similar environments (hot tubs, whirlpools, hot
spring baths, etc.), in accordance with the definitions given for these environments by World Health
Organization (WHO) guidelines [17].

In line with the objective of the study, we set out to perform a systematic review of cases and
outbreaks of LD and PF associated with recreational aquatic environments, such as swimming and spa
pools or natural spas. The literature search was conducted in Medline, including publications from
1 January 1977 (since the disease was first described in 1976) to 31 May 2018, using the following search
terms: (Legionella OR legionellosis OR “Pontiac fever” OR “Legionnaires’ disease”) AND (case* OR
cluster* OR outbreak* OR infection* OR investigation OR surveillance) AND (“recreational water” OR
spa OR pool OR “swimming pool” OR “hot tub” OR whirlpool OR bath OR “swim spa” OR “turkish
bath” OR sauna OR Jacuzzi OR “natural spa” OR “hot spring” OR “thermal spring” OR “warm spring”
OR spring OR thermal). The literature search was conducted without language restrictions, on the
condition that the articles had an exhaustive abstract in English reporting the information of interest.
A further selection of relevant publications was performed using the inclusion and exclusion criteria
listed below.
Inclusion criteria:
• Primary studies describing cases/outbreaks of LD or PF originating from recreational water.
Exclusion criteria:
• Not recreational water (hot water system, cooling tower, fountain, network water, therapeutic
water, water births);
• environmental studies without cases;
• not primary studies;
33
IJERPH 2018, 15, 1612
• articles focused only on clinical and laboratory aspects;

• abstract not available/ not complete or not exhaustive;
• articles focused on pools used for display only (retail premises, fairs, exhibitions, shows);
• articles evaluating only microbiological risk assessment; and
• hot tubs or pools on cruise ships (due to a recently published systematic review) [18].
Two researchers independently screened titles and abstracts to identify potentially relevant articles
and to exclude articles incompatible with the first five exclusion criteria; any disagreements were
resolved by discussion with a third author. After the application of the first five exclusion criteria,
the full texts of the remaining articles were examined, and any publications exclusively focused on
display spas were then excluded, since this type of exposure in environments used for retail premises,
fairs, exhibitions, and shows is not directly linked to recreational use. The remaining articles were
assigned to three categories related to three different recreational facilities or sources of infection:
(a) Private hot tub and similar facilities;
(b) public pools and spas and similar facilities, generally supplied by municipal network water; and
(c) spa facilities supplied by natural water, or hot spring/thermal water. Subsequently, we applied
the last two exclusion criteria to each category.
Data extracted from these publications included: Year, country, case definition, clinical form,
type of event (sporadic case or outbreak), number of cases, attack rate, number of hospitalizations
and/or deaths, risk factors, laboratory diagnosis, Legionella spp. involved, environmental isolates and
concentrations (cfu/L), type of recreational water, water supply, and the type of epidemiological study
carried out (descriptive, analytical, presence/absence of environmental investigation). An event with
multiple cases (at least two) linked in space and time, with a suspected common source, was defined as
an outbreak. For each event (both sporadic cases and outbreaks), epidemiological and microbiological
criteria were adopted to characterize the strength of evidence linking the legionellosis event with the
suspected recreational water system. Table 1 summarizes these criteria.
Table 1. Strength of evidence linking a case/outbreak of legionellosis with a recreational water system.
Strength of Evidence Epidemiological and Microbiological Criteria
• An analytical epidemiological study demonstrates a significant association between

case/outbreak of legionellosis and exposure to the recreational water; and
• the same species and serogroups of Legionella spp. are isolated from the water system
at any concentration.
Strong Or
• Descriptive epidemiology suggests that the case/outbreak is related to the

recreational water and excludes obvious alternative explanations; and
• Legionella spp. are isolated from the water system at any concentration and
environmental isolates show identical genotype profiles of clinical isolates.
• An analytical epidemiological study demonstrates a significant association between

case/outbreak of legionellosis and exposure to the recreational water; and
• Legionella spp. are not isolated from the recreational water.
Or
Probable
• Descriptive epidemiology suggests that the case/outbreak is related to the
recreational water and excludes obvious alternative explanations; and
• the same species and serogroups of Legionella spp. are isolated from the water system
at any concentration.
• Descriptive epidemiology suggests that the case/outbreak is related to exposure to

Possible the recreational water and excludes obvious alternative explanations; and
• Legionella spp. are not isolated from the recreational water.
34
IJERPH 2018, 15, 1612
Data were analysed as the frequency distribution of the different variables included.
3. Results
Of the 326 articles retrieved from Medline, 259 were excluded for the following reasons:
99 investigations did not refer to recreational water, 82 were environmental studies without cases,
4 were not primary studies, 68 articles were focused only on clinical and laboratory aspects,
and 6 publications were in a language other than English and did not have an exhaustive English
abstract, as shown in Figure 1.
Figure 1. Flow chart of the selection process of articles.
At the end of the selection process, 47 articles were considered eligible for inclusion in the present
review, corresponding to 42 events. In four cases, different articles described varying aspects of the
same event, while two articles reported two and three different events, respectively. Among the
42 events of legionellosis, eight were linked to a hot tub/whirlpool/Japanese bath used in private
houses (Category A in Figure 1, in brief “private hot tub”), 22 were related to whirlpool spa/baths
in public centres and hotels (Category B in Figure 2, in brief “public spa pools”), and 12 to hot
spring/thermal spa pools (Category C in Figure 1, in brief “hot spring/thermal spa”).
The selected articles were published: Four in the 1980s, 16 in the 1990s, 19 in the 2000s, and three
from 2010 to 2018. In 11 articles, the authors did not report the date of onset. The events occurred in
different countries across the world, with the highest frequency of hot spring related events in Japan
(83.3%) and an overall highest frequency in Japan (18 events: 42.9%), followed by the USA (11 events:
26.2%), and the United Kingdom (4 events: 9.5%).
35
IJERPH 2018, 15, 1612
3.1. Legionellosis in Relation to Recreational Water Source

Table 2 shows all events and cases of legionellosis associated with recreational water systems,
distinguished per facility category. Of the 1079 total cases included in the 42 events, 57.5% were
diagnosed as PF, without any deaths, and 42.5% were of LD, with a fatality rate of 6.3%.
Table 2. Events of Pontiac fever (PF) and Legionnaires’ disease (LD) associated with recreational water.
Hot Spa Pools/Public Hot Spring Spa, Total

Tub/Whirlpool/Japanese Baths in Public Thermal Spa, Recreational
Characteristics of the Events
Bath in Private House Centres or Hotels Recreational Surface Waters
(8 Events) (22 Events) Water (12 Events) (42 Events)
Number of events with single cases 5 2 10 17
Number of outbreaks or events with
3 20 2 25
repeated cases a
Number of total cases 28 744 307 1079
Median number of cases per
6 (4–13) 23.5 (3–170) 148.5 (2–295) 23 (2–295)
outbreak (range)
Total number of PF cases (fatal cases) 22 (0) 598 (0) 0 620 (0)
Total number of LD cases (fatal cases) 6 (1) 146 (16) 307 (12) 459 (29)
Fatality rate on total cases (on LD cases) 3.6% (16.7%) 2.2% (11.0%) 3.9% (3.9%) 2.7% (6.3%)
Analytical epidemiology in outbreak
0 (0%) 8 (40.0%) 1 (50.0%) 9 (36.0%)
investigation (% of total outbreaks)
Events with environmental investigation
6 (75.0%) 20 (90.9%) 9 (75.0%) 35 (83.3%)
(% of total events)
Legionella spp. detected in environmental
4 (50.0%) 20 (90.9%) 8 (66.7%) 32 (76.2%)
water samples (% of total events)
Identical Legionella genotype in clinical
and environmental isolates (% of 1 (12.5%) 6 (27.3%) 7 (58.3%) 14 (33.3%)
total events)
Strength of evidence
Strong (%) 1 (12.5%) 15 (68.2%) 7 (58.3%) 23 (52.4%)
Probable (%) 3 (37.5%) 5 (22.7%) 1 (8.3%) 9 (21.4%)
Possible (%) 4 (50.0%) 2 (9.1%) 4 (33.3%) 10 (23.9%)
a 22 outbreaks and three events with repeated cases or cluster.
The private hot tubs were all supplied by municipal network water and were subjected to a
supplementary disinfection system only in two of the eight facilities involved in the legionellosis
events. Single cases occurred in five events (62.5%) corresponding to 17.9% of cases, while the
remaining three events were outbreaks with a low number of persons involved (from four to 13).
LD represented 21.4% of the cases, with a fatality rate of 16.7%.
Public spa pools were generally supplied by municipal network water and only three out of
22 facilities had their own supply system from groundwater (two spa pools) and mountain spring water
(one spa pool). In 54.5% of the facilities, water treatment included recycling, filtering, and chemical
disinfection with bromine (seven spa pools) or chlorine (five spa pools). In the remaining public spa
pools, water disinfection was not mentioned. Public spa pools were responsible for the highest number
of events (22), cases (744), and deaths (16). A sporadic case only occurred in 9.1% of the events, while
the remaining events were outbreaks often involving a high number of cases of up to 170 [19]. The LD
cases formed 19.6% of the total cases, with a fatality rate of 11.0%.
Hot spring/thermal spas were supplied by natural waters, i.e., hot springs and thermal waters.
This group also includes the only LD case associated with bathing in surface water. This was a fatal case
in a 27-year-old woman who had nearly drowned in estuarine water [20]. Water treatment and chlorine
disinfection were reported in only three out of the 11 hot spring/thermal water facilities (27.3%), while,
in one case, the authors specified that national regulations (France) precluded the addition of chemicals
to thermal spas to preserve the characteristics of the mineral water [21]. All cases linked to this
recreational water category were diagnosed as LD, with a fatality rate of 3.9%. Single cases occurred
in 83.3% of the events and only two outbreaks were reported. However, one of these was the largest
outbreak of LD associated with a hot spring bathhouse in Japan, with 295 cases, including confirmed
and probable cases [22].
36
IJERPH 2018, 15, 1612
3.2. Epidemiological Investigations

All the events with sporadic cases were studied by descriptive epidemiology. The epidemiological
investigations included an analytical study in 36.0% of outbreaks, with higher percentages in events
linked to public spa pools (40.0%) and hot spring/thermal water (50%), compared to private hot tubs
(no events with an analytical study). An environmental investigation was carried out in 83.3% of
events (private hot tubs and hot spring/thermal water: 75%; public spa pools: 90.9%) and allowed
the detection of Legionella spp. in 76.2% of the incriminated water sources and to evidence identical
molecular profiles of both clinical and environmental isolates in 33.3% of the events. Based on the
epidemiological and microbiological criteria specified in Table 1, the strength of evidence linking the
case/outbreak of legionellosis with the recreational water system was strong in 23 events (52.4%), with
percentages higher for public spa pools (68.2%) and hot spring/thermal water (58.3%) compared to
private hot tubs (12.5%). This was a consequence of the previously mentioned differences regarding
both the implementation of analytic epidemiology and the detection of environmental Legionella spp.,
which were carried out less frequently in private hot tub related events.
3.3. Events with Sporadic Cases of Legionellosis

Sporadic cases of legionellosis occurred in 17 distinct events, only one of PF [23] and 16 of LD
(Table 3), with a fatality rate of 29.4% (31.2% for LD cases). Most cases occurred in Japan (70.6%) [24–35],
and hot spring/thermal waters (56.2%) were the facilities most involved, followed by private hot tubs
(25%). Only two cases occurred in spa centres/public baths [35,36]. Four cases, three of which fatal,
were consequent to near drowning [20,32,35,37] and one case involved a 10-year-old girl, subjected to
immunosuppressive therapy for hemosiderosis after being exposed several times to the hot tub in her
maternal home [38].
Etiological diagnosis was confirmed by culture of clinical specimens in 75.0% of LD cases and
L. pneumophila was the species most frequently involved, in particular L. pneumophila SG 6 (31.2% of
LD cases). No differences were observed on the onset of cases in relation to the different concentrations
of legionellae detected from the suspected water sources. Genotyping of clinical and environmental
isolates was performed in seven out of 17 events. In accordance with the microbiological criteria
specified in Table 1, the strength of evidence linking the cases with the recreational water system was
strong in all the cases confirmed by molecular typing (43.7% of LD cases).
Table 3. Events with sporadic cases of Pontiac fever (PF) and Legionnaires’ disease (LD) associated
with recreational water.
Pontiac Fever Legionnaires’ Disease

(1 Event) a (16 Events) b
Number of cases (fatal cases) 1 (0) 16 (5)
Gender
Males 9
Females 6
Not reported 1 1
Median age (range) 37 56.5 (10–88)
Confirmation by culture in clinical specimen 0 12 (75.0%)
Legionella species and serogroup
L. pneumophila SG 1 0 3 (18.7%)
L. pneumophila (SG not reported) 1 (100%) 1 (6.2%)
L. rubrilucens 0 1 (6.2%)
37
IJERPH 2018, 15, 1612
Table 3. Cont.
Pontiac Fever Legionnaires’ Disease

(1 Event) a (16 Events) b
Environmental source
Private hot tub 1 4 (25.0%)
Public and hotel spa 0 2 (12.5%)
Hot spring/thermal spa 0 9 (56.2%)
Estuarine water 0 1 (6.2%)
Legionella colonization
<1000 cfu/L 0 2 (12.5%)
1000–10,000 cfu/L 0 2 (12.5%)
>10,000 cfu/L 0 2 (12.5%)
Not reported 1 (100%) 11 (68.7%)
Identical Legionella genotype in clinical and environmental isolates 0 7 (43.7%)
Strength of evidence
Strong (%) 0 7 (43.7%)
Probable (%) 1 (100%) 2 (12.5%)
Possible (%) 0 7 (43.7%)
a [23]; b [20,24–38].
3.4. Outbreaks of Legionellosis

A total of 25 outbreaks of legionellosis were found: 7 outbreaks of PF (Table 4), 11 outbreaks of
LD (Table 5), and 7 mixed events of PF and LD (Table 6). Among the LD events, two were repeated
cases on the same site, which occurred in different time periods (No. 2, 3 in Table 6), and one was a
long-lasting outbreak with three consecutive clusters (No. 10 in Table 6).
The total number of outbreak cases was 1062, of which 619 were PF cases (58.3%) and 443 were
LD cases (41.7%), with 24 deaths (total fatality rate: 2.3%, for LD: 5.4%). Most events occurred in public
spas (20/25 outbreaks, 80%), particularly in whirlpool spas of hotels or similar residential facilities,
such as inns and holiday resorts (11 of 25 outbreaks, 44%). The attack rate varied from 29.8% to 86.7%
for PF outbreaks and from 0.13% to 1.9% for LD outbreaks.
Etiological diagnosis was confirmed by culture of clinical specimens in 10 out of 11 outbreaks of
LD and in one out of seven mixed events of PF and LD (61.1% of total events with LD cases), while it
was never performed in PF outbreaks. L. pneumophila was the species most frequently involved, in
particular L. pneumophila SG 1 in 68% of total outbreaks (83.3% of outbreaks with LD cases) and SG
6 in 24% of total outbreaks (27.8% of outbreaks with LD cases). In three events, various species or
serogroups were identified as responsible for the disease by culture and/or serological assay.
Environmental isolates of Legionella spp. were obtained in 22 outbreaks (88%), in seven of which
various species or serogroups were detected (28%). Genotyping of clinical and environmental isolates
was performed in 10 events (40% of total outbreaks, 55.5% of outbreaks with LD cases). In accordance
with the epidemiological and microbiological criteria specified in Table 1, the strength of evidence
linking the outbreak with the recreational water system was strong in 16 events (64%).
38
Table 4. Outbreaks of Pontiac fever (PF) associated with recreational water.
Event No. Legionella spp.

No. of Cases Proportion Median Age Environmental Strength of
Country, Year Water System (Confirmed Diagnosis Based Attack Rate
(Fatal Cases) of Males (Range) Isolates (cfu/L) Evidence
(Reference) on)
1 L. pneumophila
L. pneumophila SG 6
Vermont, US, 1981 Inn whirlpool spa 34 (0) 45.9% 53.0% 27.9 SG 1,6 Strong
IJERPH 2018, 15, 1612
(antibody titre)
[39] L. dumoffii
2
Public whirlpool
Michigan, US, L. pneumophila SG 6 32 L. pneumophila
spa 14 (0) 29.8% 0 Strong
1982 (antibody titre) (25–39) SG 6
(women’s pool)
[40]
3
L. pneumophila
Colorado, US, Resort indoor L. pneumophila SG 6
13 (0) 38.0% na na SG 6 Strong
1992 whirlpool (antibody titre)
(>1,000,000)
[41]
L. pneumophila SG 1 negative
4 Private
(culture, antibody titre) na samples
Denmark, 1995 summerhouse 13 (0) 86.7% na Possible
L. micdadei (after whirlpool
[42] whirlpool
(antibody titre) cleaning)
5
39
whirlpool area: 66.0%
Wisconsin, US, Hotel whirlpool L. micdadei L. micdadei
45 (0) whirlpool users: na na Strong
1998 spa (antibody titre) (90,000/L)
71.0%
[43]
6 whirlpool area: 71.0%
Hotel whirlpool L. micdadei 41
Sweden, 1999 29 (0) whirlpool users: 37.9% negative samples Probable
spa (antibody titre) (21–57)
[44] 88.9%
7 Legionella non
Resort whirlpool L. pneumophila SG 1
England, 2008 6 (0) 86.0% 0 (24–37) pneumophila Probable
spa (antibody titre, urinary antigen)
[45] (100/L)
na: Not available; clinical and environmental isolates were never compared by molecular typing.
Table 5. Outbreaks of Legionnaires’ disease (LD) associated with recreational water.
Event No. Number of

Legionella spp. Proportion Median Age Environmental Strength of
Country, Year Water System Cases Attack Rate
(Diagnosis Based on) of Males (Range) Isolates (cfu/L) Evidence
(Reference) (Fatal Cases)
1
L. pneumophila SG 1 3
Vermont, US, 1987 Inn whirlpool spa na na na L. pneumophila SG 1,4 Strong
(culture, antibody titre) (0)
IJERPH 2018, 15, 1612
[46]
2
Netherlands Public spa sauna’s L. pneumophila SG 1 6 repeated cases males: 50
na 83.3% L. pneumophila SG 1 Strong
1992–96 footbath (culture) (2) females: 28
[47]
3 L. pneumophila SG
L. pneumophila SG 1 2 repeated cases 54.5
France 1994–97 Thermal spa na 50% 1,2,3,6,9,13 Strong
(culture) (1) (40–69)
[21] L. dumoffii
4
L. pneumophila SG 1 3 L. pneumophila
Japan, 1996 Public Japanese spa na na na Probable
(antibody titre) (0) SG 1
[48]
5 L. pneumophila SG 1,6
23 67 L. pneumophila SG 1
Japan, 2000 Public bath house (culture, antibody titre, urinary 0.13% 91.3% Strong
(2) (50–86) (880,000)
[27] antigen)
6 L. pneumophila SG 1 34 (20 65.0% L. pneumophila SG
62.2
40
Japan, 2000 Public bath house (culture, antibody titre, urinary confirmed) 0.20% (only 1,3,5,6 Strong
(27–85)
[49,50] antigen) (3) confirmed) (11400–84200)
L. pneumophila SG 1,8
295 (1,600,000)
7 L. pneumophila SG 1 64.5%
including 65 L. dumoffii
Japan, 2002 Hot spring bath (culture, antibody titre, urinary 1.5% (of 76 Strong
suspected cases (9–95) (5,200,000)
[22,51–55] antigen) examined)
(7) L. londiniensis
(15,000,000)
8
L. pneumophila SG 1 9 65 L. pneumophila SG 1
Japan, 2003 Public bath house 0.13% na Probable
(culture) (1) (52–82) (1,300,000)
[27]
9
L. pneumophila SG 1 3 50 L. pneumophila SG 1
France, 2010 Public whirlpool spa na 33.3% Strong
(culture, urinary antigen) (1) (30–70) (150,000)
[56]
Total: 44 (6)
tourists: 71.5
10 Cluster1: 21
L. pneumophila SG 1 hotel L. pneumophila SG 1
Spain, 2011–12 Hotel spa pool Cluster2: 2 na na Strong
(culture) workers: L. micdadei
[57] Cluster3: 3
49.5
Cluster4: 18
11
Spa house L. pneumophila SG 1,13 7 L. pneumophila
Japan, 2015 na 100% 66.3 Strong
(men’s pool) (culture) (0) SG 1,13
[58]
na: Not available; clinical and environmental isolates showed correlated molecular profiles in events No. 1, 2, 3, 5, 6, 7, 9, 10, and 11.
Table 6. Outbreaks of Pontiac fever (PF)/Legionnaires’ disease (LD) associated with recreational water.
Number of
Event No.
Legionella spp. Cases Proportion Median Age Environmental Strength of
Country, Year Water System Attack Rate
(Diagnosis Based on) PF + LD of Males (Range) Isolates (cfu/L) Evidence
(Reference)
(Fatal Cases)
1
IJERPH 2018, 15, 1612
L. micdadei 169 + 1 90.9% 32

Scotland, 1987–88 Hotel whirlpool spa 48.8% L. micdadei Probable
(antibody titre) (0) (LD: 0.5%) (2–72)
[19,59]
2
Private hot tub in L. pneumophila SG 1 5+1
Vermont US, 1991 na na na not investigated Possible
holiday home (antibody titre) (0)
[60]
PF: 12
3 L. pneumophila SG 6
22 + 2 22.0% (5–31)
Georgia US, 1999 Hotel whirlpool spa (culture, antibody titre, urinary na L. pneumophila SG 6 Strong
(0) (LD: 1.8%) LD: 66
[61] antigen)
(61–71)
4 L. micdadei L. micdadei
49 + 1 62.7% 20
Illinois US, 2002 Hotel spa area L. maceachernii 46% L. maceachernii Strong
(0) (LD: 1.2%) (2–58)
[62] (antibody titre) L. dumoffii
PF: 15
5
Hotel pool and hot L. pneumophila SG 1 101 + 6 33.7% PF: 43.6% (2–65)
41
Oklaoma US, 2004 L. pneumophila SG 1 Strong
tub area (antibody titre, urinary antigen) (0) (LD: 1.9%) LD: 100% LD: 6.5
[63]
(2–44)
6
L. pneumophila SG 1 116 + 2 PF: 41.4%
England, 2006 Leisure club spa pool na (18–85) L. pneumophila SG 1 Probable
(antibody titre, urinary antigen) (0) LD: 100%
[64]
7 PF: 54
Private outdoor L. pneumophila SG 1 3+1 PF: 66.7%
Netherlands, 2009 na (52–83) L. pneumophila SG 1 Probable
whirlpool spa (antibody titre, urinary antigen) (1 LD) LD: 0%)
[65] LD: 78
na: Not available; clinical and environmental isolates showed correlated molecular profiles in the event No. 3.
IJERPH 2018, 15, 1612
3.5. Patient Contributing Factors

PF cases showed no evidence of underlying risk factors. The median age of the PF patients,
when reported, varied from 12 to 54 years and, overall, males and females were affected with a
similar frequency.
LD patients were males in 60% of sporadic cases (Table 3) and in 71.9% of outbreaks, considering
only the events reporting gender distribution. The median age was 56.5 years (range: 10–88) in
sporadic cases and over 60 years in nine of the 13 LD outbreaks in which the age data was reported.
Patient risk factors and underlying medical conditions were specified in 24 of the 34 LD events (71.3%),
for a total of 155 cases. Figure 2 shows the occurrence of contributing factors and underlying medical
conditions in these patients. Heavy smoking was the most frequent risk factor (58.7% of patients)
and, among the underlying medical conditions, cardiovascular diseases (23.9%) and diabetes (11.0%)
had the highest prevalence. Four cases of Legionella pneumonia occurred after near drowning, one in
estuarine water and three in hot spring spas and public baths.
Drowning 2.6%
COPD 3.9%
Leukemia, malignancy 2.6%
Immunosuppressive therapy 3.9%
Diabetes 11.0%
Cardiovascular diseases 23.9%
Alc ohol abuse (also smokers) 1.9%
S mokers 58.7%
0 10 20 30 40 50 60 70
Figure 2. Distribution of underlying medical conditions and risk factors in 155 cases of
Legionnaires’ disease.
3.6. Environmental Contributing Factors

Excluding the only sporadic case related to estuarine water, environmental contributing factors
were investigated in 22 out of 41 events. In only one of these, no contributing environmental
conditions were found. In the other 21 events, inadequate water treatment and residual disinfectant
below the recommended levels were the most frequent factors that could have favoured the onset
of cases or outbreaks. The water temperature was reported in only four events and in three of
these the temperature was above 40 ◦ C (Figure 3). In PF events, the most frequent environmental
contributing factors were those related to plant maintenance and chemical treatment management (i.e.,
inappropriate residual disinfectant concentration), while the inadequacy or absence of the treatment
system was observed only for LD cases or outbreaks. This could be explained by the fact that many
LD events occurred in private hot tubs not subjected to a supplementary disinfection system.
Legionella spp. were isolated from the environmental samples of 32 facilities, at concentrations
higher than 103 cfu/L in water samples obtained from 11 of them (34.4%).
42
IJERPH 2018, 15, 1612
Temperature > 40°C 13.6%
Exc essive load 4.5%
No monitoring and rec ording of parameters 27.3%
Lac k of maintenanc e, inappropriate c leaning

18.2%
of filters
Tec hnic al failure or inedequac y of water
18.2%
c irc uit
Improper residual disinfec tant level 31.8%
Absent or inadequate treatment 31.8%
0 10 20 30 40 50
Figure 3. Distribution of environmental contributing factors in 22 recreational facilities associated with

legionellosis events.
4. Discussion
This review aimed to evaluate the cases and outbreaks of legionellosis associated with exposure
to recreational water since the disease was first described in 1976. Both sporadic cases and outbreaks
of LD and PF, described in the scientific literature, were included. Relevant findings from 47 articles
were synthesized, including 42 legionellosis events (17 sporadic cases and 25 outbreaks).
4.1. Temporal and Geographical Distribution

The events of legionellosis correlated with exposure to recreational water showed a
non-homogeneous distribution over time. In the 1980s, only four events were reported, probably
because, in these first years, there was a lower awareness of the problem and many cases were not
identified or associated with exposure to recreational water. In the 1990s and 2000s, the number
increased (16 and 19 events, respectively) and then declined in the years from 2010 until today
(only three reported in the literature). It could be hypothesized that the increase in knowledge and
awareness of risks associated with recreational water led to an improvement in the management and
maintenance and control measures, also after the issuing of international guidelines on the control
of legionellosis in recreational facilities. In 2006, the WHO Guidelines for safe recreational water
environments recommended the implementation of safety plans and adequate control measures
in pools and hot tubs [17]. Moreover, from 2005, the European Legionnaires’ Disease Surveillance
Network (ELDSNet, previously EWGLI), with respect to Legionella risk reduction in whirlpool spas,
recommended continuous treatment with 2–3 mg/L of chlorine or bromine, the checking of these levels
almost three times a day, the replacement of at least half of the water each day, sand filters backwashed
daily, and cleaning and disinfection of the whole system every day [66]. The implementation of these
measures could explain the reduction in the number of events in the most recent period.
The reported events of legionellosis involved 10 countries, with the highest number of events
(18) and cases (385) in Japan, where the habit of frequenting hot spring spas and public baths is very
widespread, following a long-established tradition in Japanese culture. Moreover, the average water
temperature in hot tubs in Japan usually ranges from 40 ◦ C to 43 ◦ C, which is higher than in Europe
(30–40 ◦ C) [27].
4.2. Clinical Features and Laboratory Evidence

This review includes both PF and LD events. PF cases totalled 620, only one of which was sporadic,
the others being included in 14 outbreaks. The number of PF cases related to recreational water is
probably underestimated: The benign nature of the disease, which often presents as an influenza-like
43
IJERPH 2018, 15, 1612
illness, means that the cases, especially when sporadic, are not identified as legionellosis and are,
therefore, not subjected to laboratory diagnosis. In the selected PF events, laboratory diagnosis was
performed only in outbreaks, and Legionella spp. were never culturally isolated. On the contrary,
in the events involving LD cases, cultural isolation from patients’ specimens allowed the species to be
identified in 75% of the sporadic cases and in 11 of the 18 outbreaks with LD cases (61.1%).
Among the different species and serogroups, L. pneumophila SG 1 (three sporadic cases and
15 outbreaks of LD) and SG 6 (five sporadic cases and two outbreaks of LD) were the agents most
frequently responsible, while, among the other species, L. micdadei was implicated in three outbreaks
of PF and two outbreaks of mixed PF and LD. In five events, various species or serogroups were
involved [27,30,42,58,62], one of which was the first case where the same genotype of L. rubrilucens
was isolated from the LD patient’s sputum and the hot spring water [30].
This review confirms certain known characteristics of the epidemiology of legionellosis. PF cases
showed no evidence of underlying risk factors and PF outbreaks had a high attack rate, with no
difference between males and females. On the contrary, LD cases prevalently involved males and
individuals presenting risk factors, such as smoking and all the underlying medical conditions that
reduce immune defenses. In LD outbreaks, the attack rate is low and the fatality rate is high (on
average, 6.3%, but up to 31.2% in events related to private hot tubs).
4.3. Recreational Water Facilities and Risk Assessment

Most events occurred in public spa pools (22 events, 744 cases). Of these, 10 were associated
with hotels or similar residential facilities and, therefore, fall within the surveillance system for
legionellosis linked to travel, which in Europe is carried out by the ELDSNet and coordinated by
ECDC. The recreational facilities supplied by natural water (hot spring, thermal water) were the setting
for 12 events, 10 of which with a single case. Most studies referring to hot spring/thermal spas (seven
out of 11) did not specify if the water was treated or untreated and how the facility was managed;
this is a limitation that makes it difficult to draw conclusions about the environmental conditions
contributing to these infections.
The recommended standards for Legionella spp. in hot tub water range from 0/100 mL to 1000/L
in different countries [67]. In the selected studies, the environmental isolates of Legionella spp. are
reported in 32 events, but only 13 specify the level of contamination, which ranges between 100 cfu/L
and >106 cfu/L. However, it should be noted that the isolation of Legionella spp. from environmental
samples was carried out after the legionellosis event had occurred and so the environmental conditions
may have changed. The lack of data on the Legionella concentrations in the water, and on the frequency
and duration of exposure, makes it difficult to perform a risk assessment. Various studies tried to
estimate the risk for Legionella infection due to spa pool use. Bouwknegt et al., (2013) estimated that the
infection risk for sitting in an active whirlpool for 15 min ranged from around 3% for a concentration
of 10 L. pneumophila cfu/L to up to 95% for >1000 cfu/L [68]. These findings suggest that a risk cannot
be excluded even in the presence of very low concentrations, and stricter requirements may be needed
to ensure adequate protection for users. Azima et al. (2013) suggested a reference value of <1 cfu/L,
which is less than the current detection limit [69].
4.4. Epidemiological Investigation and Strength of Evidence

The epidemiological investigation included an analytical study in nine outbreaks, four with a
case-control study and five with a retrospective cohort study. In all the events related to private hot
tubs, only descriptive epidemiology was carried out. This is justified by the difficulty in such events
to find a control group not exposed to the private hot tub. Also, sporadic cases were studied only
through descriptive epidemiology (case reports).
The environmental investigation was often delayed with respect to the event onset and, in some
cases, was made after control measures had already been adopted. These measures are specified
only in a limited number of articles and information is lacking on the follow-up procedures in almost
44
IJERPH 2018, 15, 1612
all the articles. Many studies do not report the environmental conditions that could have favoured
such infections. In 19 events, no information is available on the type of water treatment, the level
of residual disinfectant, or the state of maintenance of the facility. Only in three events is the water
temperature specified, a factor that, in these types of recreational facilities, plays a fundamental role
in the development of Legionella spp. and was probably co-responsible for three LD cases associated
with near drowning in hot spring spas and public baths [32,35,37]. Lying in or sitting up to the neck in
hot water (above 40 ◦ C), especially in combination with alcohol consumption, may cause drowsiness,
which may then lead to unconsciousness and, consequently, drowning [70].
Based on the selected criteria, the strength of evidence linking the cases/outbreaks to the
recreational water facilities was strong in 52.4% of events, probable in 21.4%, and possible in 23.9%.
Strong evidence was principally attributable to the results of analytical study in nine events, and to
the match of environmental and clinical isolates in 17 events. The comparison between strains of
environmental and clinical origin using molecular biology techniques was carried out at a very
high level of frequency, especially in cases concerning LD (43.7% of sporadic cases and 81.8% of LD
outbreaks).
4.5. Limitations
The present study was limited to articles published in English or with an exhaustive abstract in
English, and only peer-reviewed literature was considered. Furthermore, the legionellosis events that
are published represent only part of the overall number of cases: Larger LD outbreaks are more likely
to be published than sporadic cases and smaller events, especially of Pontiac fever. Also, the review
does not include cruise ship cases [18] and cases associated with display spa pools in retail premises,
fairs, exhibitions, and shows [71,72], which represent another important source of infection. Therefore,
the role of the recreational facilities as a source of infection is underestimated, also considering that in
many LD and PF cases the source of Legionella remains unknown [3,16].
The heterogeneity of epidemiological investigations, in terms of study design, sample size,
and information about the duration of exposure and environmental contributing factors, limited the
comparison of results. In particular, the lack of information about the treatment and management of
recreational facilities makes it difficult to exhaustively evaluate the role of environmental conditions.
5. Conclusions
Data extracted from the articles in this systematic review show that hot tubs, whirlpools, and spa
pools represent an important source of infection of Legionella spp., given the number of cases involved
(1079 from 1981 to 2015), the number of deaths (29), and the high percentage of events with strong
evidence of an association. On the contrary, the risk related to the natural recreational water of rivers
and lakes appears negligible: The only sporadic case reported is a case consequent to a near-drowning
in estuarine water [20].
Among the cases included in this review, PF cases were the most numerous and were caused
by a variety of species and serogroups: L. pneumopghila SG 6 and L. micdadei were the most often
responsible agents, while L. pneumophila SG 1 was responsible for most LD cases. Unlike PF cases,
LD cases prevalently involved individuals presenting risk factors, such as smoking, and underlying
medical conditions that reduce immune defenses.
Certain operating conditions that facilitate the formation of aerosol, such as the high temperature
of the water and the presence of hydromassage systems, are risk factors inherent to this kind
of recreational water. In hot tubs and similar facilities, it is impractical to maintain a water
temperature outside the range considered at risk. Therefore, other management strategies need
to be implemented, which may include appropriate design and adequate disinfection residual and
proper maintenance and cleaning of equipment as well as adequate ventilation. Features, such as
water sprays, should be periodically cleaned and flushed with a level of disinfectant adequate to
eliminate Legionella spp. [3,17,67]. In this review, the environmental conditions were described for
45
IJERPH 2018, 15, 1612
22 events, and in 21 of these (95.5%) at least one of the preventive measures recommended by the
various guidelines was not respected. Therefore, it seems important to increase collaboration between
the different professionals involved (public health experts, policy makers, facility managers, technical
staff, equipment manufacturers) to improve the knowledge of the operators and their awareness of the
risk and to favour compliance with control measures.
Author Contributions: E.L.: conception, design and supervision of the study; F.C., S.M., L.D.: selection and
analysis of the articles, collection of data of interest in a structured form. E.L. and L.D. drafted the manuscript.
All authors approved the final version of the manuscript.
Acknowledgments: The authors would like to thank Maria Cristina Labanti, Document Delivery Service,
Biblioteca Interdipartimentale di Medicina, Biblioteca Biomedica, Bologna University, who provided the full text
of some articles of interest.
Abbreviations
The following abbreviations are used in this manuscript:
LD Legionnaires’ Disease
PF Pontiac Fever
cfu colony forming unit
References
1. Muder, R.R.; You, V.L.; Woo, A.H. Mode of transmission of Legionella pneumophila: A critical review.
Arch. Int. Med. 1986, 146, 1607–1612. [CrossRef]
2. Springston, J.P.; Yocavitch, L. Existence and control of Legionella bacteria in building water systems: A review.
J. Occup. Environ. Hyg. 2017, 14, 124–134. [CrossRef] [PubMed]
3. ECDC. European Technical Guidelines for the Prevention, Control and Investigation of Infections Caused
by Legionella Species. June 2017. Available online: https://ecdc.europa.eu/sites/portal/files/documents/
Legionella%20GuidelinesFinal%20updated%20for%20ECDC%20corrections.pdf (accessed on 9 July 2018).
4. Cunha, B.A.; Cunha, C.B. Legionnaire’s Disease and its mimics: A clinical perspective. Infect. Dis. Clin.
N. Am. 2017, 31, 95–109. [CrossRef] [PubMed]
5. Borella, P.; Montagna, M.T.; Stampi, S.; Stancanelli, G.; Romano Spica, V.; Triassi, M.; Marchesi, I.;
Bargellini, A.; Tato, D.; Napoli, C.; et al. Legionella contamination in hot water of Italian hotels.
Appl. Environ. Microbiol. 2005, 71, 5805–5813. [CrossRef] [PubMed]
6. Leoni, E.; De Luca, G.; Legnani, P.; Sacchetti, R.; Stampi, S.; Zanetti, F. Legionella waterline colonization:
Detection of Legionella species in domestic, hotel and hospital hot water systems. J. Appl. Microbiol. 2005, 98,
373–379. [PubMed]
7. Walser, S.M.; Gerstner, D.G.; Brenner, B.; Höller, C.; Liebl, B.; Herr, C.E. Assessing the environmental health
relevance of cooling towers—A systematic review of legionellosis outbreaks. Int. J. Hyg. Environ. Health
2014, 217, 145–154. [CrossRef] [PubMed]
8. Dallolio, L.; Scuderi, A.; Rini, M.S.; Valente, S.; Farruggia, P.; Bucci Sabattini, M.A.; Pasquinelli, G.; Acacci, A.;
Roncarati, G.; Leoni, E. Effect of different disinfection protocols on microbial and biofilm contamination of
dental unit waterlines in community dental practices. Int. J. Environ. Res. Public Health 2014, 11, 2064–2076.
[CrossRef] [PubMed]
9. Ricci, M.L.; Fontana, S.; Pinci, F.; Fiumana, E.; Pedna, M.F.; Farolfi, P.; Sabattini, M.A.; Scaturro, M. Pneumonia
associated with a dental unit waterline. Lancet 2012, 379, 684. [CrossRef]
10. Donati, M.; Cremonini, E.; Di Francesco, A.; Dallolio, L.; Biondi, R.; Muthusamy, R.; Leoni, E. Prevalence of
Simkania negevensis in chlorinated water from spa swimming pools and domestic supplies. J. Appl. Microbiol.
2015, 118, 1076–1082. [PubMed]
11. Leoni, E.; Legnani, P.; Bucci Sabattini, M.A.; Righi, F. Prevalence of Legionella spp. in swimming pool
environment. Water Res. 2001, 35, 3749–3753. [CrossRef]
46
IJERPH 2018, 15, 1612
12. Leoni, E.; Sacchetti, R.; Zanetti, F.; Legnani, P.P. Control of Legionella pneumophila contamination in a system
for respiratory hydrotherapy with sulphurous spa water. Inf. Control Hosp. Epidemiol. 2006, 27, 716–721.
[CrossRef] [PubMed]
13. Leoni, E.; Sanna, T.; Zanetti, F.; Dallolio, L. Controlling Legionella and Pseudomonas aeruginosa re-growth in
therapeutic spas: Implementation of physical disinfection treatments, including UV/ultrafiltration, in a
respiratory hydrotherapy system. J. Water Health 2015, 13, 996–1005. [CrossRef] [PubMed]
14. Fields, B.S.; Benson, R.F.; Besser, R.E. Legionella and Legionnaire’s disease: 25 year of investigation.
Clin. Microbiol. Rev. 2002, 15, 506–526. [CrossRef] [PubMed]
15. Leoni, E.; Dallolio, L.; Sanna, T.; Stagni, F.; D’Alessandro, G.; Piana, G. Impact of a risk management plan
on Legionella contamination of dental unit water. Int. J. Environ. Res. Public Health 2015, 12, 2344–2358.
[CrossRef] [PubMed]
16. Hamilton, K.A.; Prussin, A.J.; Ahmed, W.; Haas, C.N. Outbreaks of Legionnaires’ Disease and Pontiac Fever
2006–2017. Curr. Environ. Health Rep. 2018, 5, 263–271.
17. WHO. Guidelines for Safe Recreational Water Environments. Volume 2, Swimming Pools and Similar Environments;
WHO: Geneva, Switzerland, 2006. Available online: http://www.who.int/water_sanitation_health/
bathing/srwe2full.pdf (accessed on 9 July 2018).
18. Mouchtouri, V.A.; Rudge, J.W. Legionnaires’ Disease in hotels and passenger ships: A systematic review of
evidence, sources, and contributing factors. J. Travel Med. 2015, 22, 325–337. [PubMed]
19. Goldberg, D.J.; Wrench, J.G.; Collier, P.W.; Emslie, J.A.; Fallon, R.J.; Forbes, G.I.; McKay, T.M.;
Macpherson, A.C.; Markwick, T.A.; Reid, D. Lochgoilhead fever: Outbreak of non-pneumonic legionellosis
due to Legionella micdadei. Lancet 1989, 1, 316–318. [CrossRef]
20. Faris, B.; Faris, C.; Schousboe, M.; Heath, C.H. Legionellosis from Legionella pneumophila serogroup 13.
Emerg. Infect. Dis. 2005, 11, 1405–1409. [CrossRef] [PubMed]
21. Molmeret, M.; Jarraud, S.; Mori, J.P.; Pernin, P.; Forey, F.; Reyrolle, M.; Vandenesch, F.; Etienne, J.; Farge, P.
Different growth rates in amoeba of genotypically related environmental and clinical Legionella pneumophila
strains isolated from a thermal spa. Epidemiol. Infect. 2001, 126, 231–239. [CrossRef] [PubMed]
22. Yabuuchi, E.; Agata, K. An outbreak of legionellosis in a new facility of hot spring bath in Hiuga City.
Kansenshogaku Zasshi 2004, 78, 90–98. [CrossRef] [PubMed]
23. Tolentino, A.; Ahkee, S.; Ramirez, J. Hot tub legionellosis. J. Ky. Med. Assoc. 1996, 94, 393–994. [PubMed]
24. Chiba, Y.; Okamoto, H.; Nagatomo, A.; Kunikane, H.; Watanabe, K. Legionnaires’ disease diagnosed by
bronchoalveolar lavage. Intern. Med. 1998, 37, 153–156. [CrossRef] [PubMed]
25. Ishikawa, A.; Okada, J.; Kondo, H.; Takayama, Y.; Sunagawa, K.; Enari, T.; Ishii, Y. Legionella pneumonia
which occurred in a private whirlpool bath user. Kansenshogaku Zasshi 2004, 78, 898–904. [CrossRef] [PubMed]
26. Ito, I.; Naito, J.; Kadowaki, S.; Mishima, M.; Ishida, T.; Hongo, T.; Ma, L.; Ishii, Y.; Matsumoto, T.;
Yamaguchi, K. Hot spring bath and Legionella pneumonia: An association confirmed by genomic
identification. Intern. Med. 2002, 41, 859–863. [CrossRef] [PubMed]
27. Kuroki, T.; Ishihara, T.; Ito, K.; Kura, F. Bathwater-associated cases of legionellosis in Japan, with a special
focus on Legionella concentrations in water. Jpn. J. Infect. Dis. 2009, 62, 201–205. [PubMed]
28. Kurosawa, H.; Fujita, M.; Kobatake, S.; Kimura, H.; Ohshima, M.; Nagai, A.; Kaneko, S.; Iwasaki, Y.;
Kozawa, K. A case of Legionella pneumonia linked to a hot spring facility in Gunma Prefecture, Japan. Jpn. J.
Infect. Dis. 2010, 63, 78–79. [PubMed]
29. Mashiba, K.; Hamamoto, T.; Torikai, K. A case of Legionnaires’ disease due to aspiration of hot spring
water and isolation of Legionella pneumophila from hot spring water. Kansenshogaku Zasshi 1993, 67, 163–166.
[CrossRef] [PubMed]
30. Matsui, M.; Fujii, S.; Shiroiwa, R.; Amemura-Maekawa, J.; Chang, B.; Kura, F.; Yamauchi, K. Isolation of
Legionella rubrilucens from a pneumonia patient co-infected with Legionella pneumophila. J. Med. Microbiol.
2010, 59, 1242–1246. [CrossRef] [PubMed]
31. Miyamoto, H.; Jitsurong, S.; Shiota, R.; Maruta, K.; Yoshida, S.; Yabuuchi, E. Molecular determination of
infection source of a sporadic Legionella pneumonia case associated with a hot spring bath. Microbiol. Immunol.
1997, 41, 197–202. [CrossRef] [PubMed]
32. Nozue, T.; Chikazawa, H.; Miyanishi, S.; Shimazaki, T.; Oka, R.; Shimazaki, S.; Miyamoto, S. Legionella
pneumonia associated with adult respiratory distress syndrome caused by Legionella pneumophila serogroup 3.
Intern. Med. 2005, 44, 73–78. [CrossRef] [PubMed]
47
IJERPH 2018, 15, 1612
33. Sasaki, E.; Kaida, H.; Izumikawa, K.; Izumikawa, K.; Hara, K.; Hirakata, Y.; Tomono, K.; Kohno, S. Two cases
of Legionella pneumophila pneumonia improved by parenteral ciprofloxacin administration. Nihon Kokyuki
Gakkai Zasshi 2003, 41, 211–218. [PubMed]
34. Shimizu, Y.; Nagase, K.; Kadono, K.N.; Funayama, Y.; Tsurushima, Y.; Hibino, T.; Kikuchi, H.; Nagase, S.;
Koyama, A. The haemodialysis patient who developed acute respiratory distress syndrome after a trip to a
hot spring spa. Nephrol. Dial. Transplant. 1999, 14, 455–457. [CrossRef] [PubMed]
35. Tokuda, H.; Yahagi, N.; Kasai, S.; Kitamura, S.; Otsuka, Y. A case of fatal pneumonia caused by
Legionella pneumophila serogroup 6 developed after drowning in a public bath. Kansenshogaku Zasshi 1997, 71,
36. Su, H.P.; Tseng, L.R.; Tzeng, S.C.; Chou, C.Y.; Chung, T.C. A legionellosis case due to contaminated spa
water and confirmed by genomic identification in Taiwan. Microbiol. Immunol. 2006, 50, 371–377. [CrossRef]
[PubMed]
37. Shiota, R.; Takeshita, K.; Yamamoto, K.; Imada, K.; Yabuuchi, E.; Wang, L. Legionella pneumophila
serogroup 3 isolated from a patient of pneumonia developed after drowning in bathtub of a hot spring spa.
Kansenshogaku Zasshi 1995, 69, 1356–1364. [PubMed]
38. Watson, A.M.; Boyce, T.G.; Wylam, M.E. Legionella pneumonia: Infection during immunosuppressive therapy
for idiopathic pulmonary hemosiderosis. Pediatr. Infect. Dis. J. 2004, 23, 82–84. [CrossRef] [PubMed]
39. Spitalny, K.C.; Vogt, R.L.; Orciari, L.A.; Witherell, L.E.; Etkind, P.; Novick, L.F. Pontiac fever associated with
a whirlpool spa. Am. J. Epidemiol. 1984, 120, 809–817. [CrossRef] [PubMed]
40. Mangione, E.J.; Remis, R.S.; Tait, K.A.; McGee, H.B.; Gorman, G.W.; Wentworth, B.B.; Baron, P.A.;
Hightower, A.W.; Barbaree, J.M.; Broome, C.V. An outbreak of Pontiac fever related to whirlpool use,
Michigan 1982. JAMA 1985, 253, 535–539. [CrossRef] [PubMed]
41. Miller, L.A.; Beebe, J.L.; Butler, J.C.; Martin, W.; Benson, R.; Hoffman, R.E.; Fields, B.S. Use of polymerase
chain reaction in an epidemiologic investigation of Pontiac fever. J. Infect. Dis. 1993, 168, 769–772. [CrossRef]
[PubMed]
42. Lüttichau, H.R.; Vinther, C.; Uldum, S.A.; Møller, J.; Faber, M.; Jensen, J.S. An outbreak of Pontiac fever
among children following use of a whirlpool. Clin. Infect. Dis. 1998, 26, 1374–1378. [PubMed]
43. Fields, B.S.; Haupt, T.; Davis, J.P.; Arduino, M.J.; Miller, P.H.; Butler, J.C. Pontiac fever due to
Legionella micdadei from a whirlpool spa: Possible role of bacterial endotoxin. J. Infect. Dis. 2001, 184,
44. Götz, H.M.; Tegnell, A.; De Jong, B.; Broholm, K.A.; Kuusi, M.; Kallings, I.; Ekdahl, K.A. Whirlpool associated
outbreak of Pontiac fever at a hotel in Northern Sweden. Epidemiol. Infect. 2001, 126, 241–247. [CrossRef]
[PubMed]
45. Modi, A.; Gardner, J.; Lighton, L.; Coetzee, N. Pontiac fever outbreak associated with a spa-pool, United
Kingdom, April 2008. Euro Surveill. 2008, 13, 18934. [PubMed]
46. Vogt, R.L.; Hudson, P.J.; Orciari, L.; Heun, E.M.; Woods, T.C. Legionnaires’ disease and whirlpool-spa.
Ann. Intern. Med. 1987, 107, 596. [CrossRef] [PubMed]
47. Den Boer, J.W.; Yzerman, E.; Van Belkum, A.; Vlaspolder, F.; Van Breukelen, F.J. Legionnaire’s disease and
saunas. Lancet 1998, 351, 114. [CrossRef]
48. Nakadate, T.; Yamauchi, K.; Inoue, H. An outbreak of Legionnaire’s disease associated with a Japanese spa.
Nihon Kokyuki Gakkai Zasshi 1999, 37, 601–607. [PubMed]
49. Nakamura, H.; Yagyu, H.; Kishi, K.; Tsuchida, F.; Oh-Ishi, S.; Yamaguchi, K.; Matsuoka, T. A large outbreak
of Legionnaires’ disease due to an inadequate circulating and filtration system for bath water, epidemiologic
manifestations. Intern. Med. 2003, 42, 806–811. [CrossRef] [PubMed]
50. Nakamura, H.; Yagyu, H.; Tsuchida, F.; Sudou, A.; Watanabe, O.; Kioi, K.; Kishi, K.; Oh-ishi, S.; Kiguchi, T.;
Yamaguchi, K.; et al. A major outbreak of Legionnaire’s disease due to a public bathhouse, clinical
examination. Nihon Kokyuki Gakkai Zasshi 2003, 41, 325–330. [PubMed]
51. Kawano, K.; Okada, M.; Kura, F.; Amemura-Maekawa, J.; Watanabe, H. Largest outbreak of legionellosis
associated with spa baths, comparison of diagnostic tests. Kansenshogaku Zasshi 2007, 81, 173–182. [CrossRef]
[PubMed]
52. Matsumoto, N.; Matsumoto, Y.; Ashitani, J.; Katoh, S.; Nakazato, M. An outbreak of Legionnaires’ disease
associated with a circulating bath water system at a public bathhouse. Nihon Kokyuki Gakkai Zasshi 2004, 42,
75–79. [PubMed]
48
IJERPH 2018, 15, 1612
53. Matsumoto, N.; Sasaki, T.; Nakao, H.; Katoh, T.; Fukuda, Y.; Nakazato, M.; Okayama, A. An outbreak of
Legionnaires’ disease associated with a circulating bathwater system at a public bathhouse. II: Radiological
findings of pneumonia. J. Infect. Chemother. 2008, 14, 123–129. [CrossRef] [PubMed]
54. Okada, M.; Kawano, K.; Kura, F.; Amemura-Maekawa, J.; Watanabe, H.; Yagita, K.; Endo, T.; Suzuki, S.
The largest outbreak of legionellosis in Japan associated with spa baths, epidemic curve and environmental
investigation. Kansenshogaku Zasshi 2005, 79, 365–374. [CrossRef] [PubMed]
55. Sasaki, T.; Matsumoto, N.; Nakao, H.; Katoh, T.; Fukuda, Y.; Nakazato, M.; Okayama, A. An outbreak of
Legionnaires’ disease associated with a circulating bathwater system at a public bathhouse. I: A clinical
analysis. J. Infect. Chemother. 2008, 14, 117–122. [PubMed]
56. Campese, C.; Roche, D.; Clément, C.; Fierobe, F.; Jarraud, S.; de Waelle, P.; Perrin, H.; Che, D. Cluster of
Legionnaires’ disease associated with a public whirlpool spa, France, April–May 2010. Euro Surveill. 2010,
15, 19602. [PubMed]
57. Sánchez-Busó, L.; Guiral, S.; Crespi, S.; Moya, V.; Camaró, M.L.; Olmos, M.P.; Adrián, F.; Morera, V.;
González-Morán, F.; Vanaclocha, H.; et al. Genomic investigation of a Legionellosis outbreak in a persistently
colonized hotel. Front. Microbiol. 2016, 6, 1556. [CrossRef] [PubMed]
58. Kuroki, T.; Amemura-Maekawa, J.; Ohya, H.; Furukawa, I.; Suzuki, M.; Masaoka, T.; Aikawa, K.; Hibi, K.;
Morita, M.; Lee, K.I.; et al. Outbreak of Legionnaire’s Disease caused by Legionella pneumophila serogroups 1
and 13. Emerg. Infect. Dis. 2017, 23, 349–351. [CrossRef] [PubMed]
59. Fallon, R.J.; Rowbotham, T.J. Microbiological investigations into an outbreak of Pontiac fever due to
Legionella micdadei associated with use of a whirlpool. J. Clin. Pathol. 1990, 43, 479–483. [CrossRef] [PubMed]
60. Thomas, D.L.; Mundy, L.M.; Tucer, P.C. Hot tub legionellosis. Legionnaires’ Disease and Pontiac Fever
after a point-source exposure to Legionella pneumophila. Arch. Intern. Med. 1993, 153, 2597–2599. [CrossRef]
[PubMed]
61. Benin, A.L.; Benson, R.F.; Arnold, K.E.; Fiore, A.E.; Cook, P.G.; Williams, L.K.; Fields, B.; Besser, R.E.
An outbreak of travel-associated Legionnaires disease and Pontiac fever, the need for enhanced surveillance
of travel-associated legionellosis in the United States. J. Infect. Dis. 2002, 185, 237–243. [CrossRef] [PubMed]
62. Huhn, G.D.; Adam, B.; Ruden, R.; Hilliard, L.; Kirkpatrick, P.; Todd, J.; Crafts, W.; Passaro, D.; Dworkin, M.S.
Outbreak of travel-related Pontiac fever among hotel guests illustrating the need for better diagnostic tests.
J. Travel Med. 2005, 12, 173–179. [PubMed]
63. Burnsed, L.J.; Hicks, L.A.; Smithee, L.M.; Fields, B.S.; Bradley, K.K.; Pascoe, N.; Richards, S.M.; Mallonee, S.;
Littrell, L.; Benson, R.F.; et al. Legionellosis outbreak investigation team. A large, travel-associated
outbreak of legionellosis among hotel guests, utility of the urine antigen assay in confirming Pontiac
fever. Clin. Infect. Dis. 2007, 44, 222–228. [CrossRef] [PubMed]
64. Foster, K.; Gorton, R.; Waller, J. Outbreak of legionellosis associated with a spa pool, United Kingdom.
Euro Surveill. 2006, 11, E060921.2. [CrossRef]
65. Euser, S.M.; Pelgrim, M.; den Boer, J.W. Legionnaires’ disease and Pontiac fever after using a private outdoor
whirlpool spa. Scand. J. Infect. Dis. 2010, 42, 910–916. [CrossRef] [PubMed]
66. Josef, C.A.; Lee, J.; van Wijngaarden, J.; Draser, V.; Castellanis Pastoris, M. European Guidelines for Control
and Prevention of Travel Associated Legionnaires’ Disease. The European Working Group for Legionella
Infections (EWGLI), 2005. Available online: http://www.legionellaonline.it/linee-guidaEWGLI_gen2005.
pdf (accessed on 9 July 2018).
67. WHO. Legionella and the Prevention of Legionellosis; WHO: Geneva, Switzerland, 2007; Available online:
http://www.who.int/water_sanitation_health/emerging/legionella.pdf (accessed on 9 July 2018).
68. Bouwknegt, M.; Schijven, J.F.; Schalk, J.A.; de Roda Husman, A.M. Quantitative risk estimation for a
Legionella pneumophila infection due to whirlpool use. Risk Anal. 2013, 33, 1228–1236. [PubMed]
69. Azuma, K.; Uchiyama, I.; Okumura, J. Assessing the risk of Legionnaires’ disease: The inhalation exposure
model and the estimated risk in residential bathrooms. Regul. Toxicol. Pharmacol. 2013, 65, 1–6. [CrossRef]
[PubMed]
70. Press, E. The health hazards of saunas and spas and how to minimize them. Am. J. Public Health 1991, 81,
49
IJERPH 2018, 15, 1612
71. Coetzee, N.; Duggal, H.; Hawker, J.; Ibbotson, S.; Harrison, T.G.; Phin, N.; Laza-Stanca, V.; Johnston, R.;
Iqbal, Z.; Rehman, Y.; et al. An outbreak of Legionnaires’ disease associated with a display spa pool in retail
premises, Stoke-on-Trent, United Kingdom, July 2012. Euro Surveill. 2012, 17, 20271. [PubMed]
72. Den Boer, J.W.; Yzerman, E.P.; Schellekens, J.; Lettinga, K.D.; Boshuizen, H.C.; Van Steenbergen, J.E.;
Bosman, A.; Van den Hof, S.; Van Vliet, H.A.; Peeters, M.F.; et al. A large outbreak of Legionnaires’ disease at
a flower show, the Netherland, 1999. Emerg. Infect. Dis. 2002, 8, 37–43. [CrossRef] [PubMed]
50
and Public Health
Article
Norovirus Outbreak Associated with Swimming in
a Recreational Lake Not Influenced by External
Human Fecal Sources in The Netherlands,
August 2012
Franciska M. Schets 1, *, Harold H. J. L. van den Berg 1 , Harry Vennema 1 , Manon T. M. Pelgrim 2 ,
Cees Collé 3 , Saskia A. Rutjes 1 and Willemijn J. Lodder 1
1 National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands;
[email protected] (H.H.J.L.v.d.B.); [email protected] (H.V.); [email protected] (S.A.R.);
[email protected] (W.J.L.)
2 Public Health Service Veiligheids-en Gezondheidsregio Gelderland-Midden,
Postbus 5364, 6802 EJ Arnhem, The Netherlands; [email protected]
3 Province of Gelderland, Postbus 9090, 6800 GX Arnhem, The Netherlands; [email protected]
* Correspondence: [email protected]; Tel.: +31-302743929
Received: 28 September 2018; Accepted: 9 November 2018; Published: 14 November 2018
Abstract: Swimming in fecally contaminated recreational water may lead to gastrointestinal illness.
A recreational water-associated outbreak of norovirus (NoV) infections affecting at least 100 people
in The Netherlands occurred in August 2012. Questionnaire responses from patients indicated
swimming in recreational lake Zeumeren as the most likely cause of illness. Most patients visited the
lake during the weekend of 18–19 August, during which the weather was exceptionally warm
(maximum temperatures 32–33 ◦ C), and visitor numbers elevated. Patients, mostly children,
became ill with gastroenteritis 1–6 days (median 2 days) after exposure. Four stool samples from
patients were NoV GI positive. Subsurface sandy soil from one of the beaches where most patients
swam was NoV GI positive; the water sample was negative. The epidemiological curve and the
timeline of investigation based on reported symptoms demonstrate the difficulty in discovering the
source in recreational water outbreaks. A NoV outbreak in a recreational lake that is not subjected
to external fecal contamination sources shows the need for active communication about human
shedding of viruses during and after diarrheal episodes and the advice to refrain from swimming,
even a few weeks after the symptoms have resolved.
Keywords: outbreak; recreational water; norovirus; swimming
1. Introduction
Noroviruses (NoV) are the leading cause of diarrhea in all age groups worldwide and they
are primarily transmitted through the fecal-oral route [1]. The viruses are extremely contagious,
with an estimated 50% infectious dose (ID50 ) as low as 2.6 (aggregated) viral particles [2]. Primary cases
often result from exposure to contaminated food or water, whereas person-to-person contact results in
further spread of the infection. Infected persons shed large numbers of viral particles into the environment,
and (low level) shedding may continue after symptoms have resolved [1,3]. Symptoms of NoV infections
are mainly projectile vomiting and watery non-bloody diarrhoea, but fever, abdominal cramping and
nausea do also occur. The incubation period is 10 to 51 h and illness is generally self-limiting, normally
lasting 2–3 days [1,4], but the duration of illness can be prolonged to 4–6 days in children younger than
11 years of age [5].

IJERPH 2018, 15, 2550
NoV have been genetically classified into five genogroups (G) [6]. GI and GII are primarily associated
with human disease [1]. The GII.4 cluster is responsible for most human norovirus outbreaks [7];
for example, over 80% of the outbreaks in the United States (1994–2006) were caused by strains
from this cluster [4]. GI strains are more often associated with waterborne outbreaks than other
genogroups [8]. NoV have been the cause of outbreaks related to contaminated municipal water supplies,
e.g., in Sweden [9], drinking water wells, e.g., in The Netherlands [10], ground water, e.g., in South
Korea [11], and various recreational water settings [12], including a fountain in Belgium, where Dutch
school children became ill during a school trip [13], a swimming pool [14] and a fresh water lake in the
United States [15], and a recreational lake in Finland [16]. A survey of recreational water associated viral
disease outbreaks, occurring between 1977 and 2006, identified noroviruses as the cause of the illness in
45% (n = 25) of the outbreaks [12]. The majority of these outbreaks resulted from exposure to lakes (56%,
n = 14). In a recently published study of bathing water related outbreaks in Finland that all occurred in
2014, epidemiological and microbiological data showed that NoV was the main causative agent [17].
This paper describes the occurrence and investigation of a recreational water associated outbreak
of NoV infections affecting at least 100 people in The Netherlands in August 2012.
2.1. Outbreak Description

On Monday, 20 August 2012, over 20 people reported gastro-intestinal complaints to the
province of Gelderland after swimming in recreational lake Zeumeren in Barneveld, The Netherlands.
During the week of 20–24 August, over 100 people reported the same with Public Health Service
Veiligheids-en Gezondheidsregio Gelderland-Midden (VGGM). All patients, who were mainly children,
had visited the lake during the previous weekend of 18 and 19 August. During that weekend,
the weather in The Netherlands was exceptionally warm with maximum temperatures of 32–33 ◦ C
(at weather station De Bilt, the station closest to Zeumeren) (www.knmi.nl). Because of the warm
weather, several thousands of people swam at Zeumeren during the weekend. Most patients became
ill one to one-and-a-half day after exposure to the lake water, and had swum at two of the ten possible
beaches surrounding the lake (Figure 1). The majority of the patients reported short-term symptoms
and recovered rapidly.
Figure 1. Recreational water lake Zeumeren, The Netherlands, with beaches 1—10 and sampling points.
A—C. Beaches 1—2 and 3 are the official beaches, which are sampled for monitoring according to the
EU BWD [18]. Sampling points for the outbreak investigation: A—water and subsurface sandy soil
sample taken, B and C—subsurface sandy soil samples taken.
52
IJERPH 2018, 15, 2550
Investigation of the outbreak started on Monday, 20 August 2012; as a precaution, an advice

against bathing in lake Zeumeren was set at the end of the afternoon on that same day. The advice
against bathing was removed on 14 September 2012 (Figure 2). Since occasionally swimmers were
observed in the lake after setting the advice against bathing, the case definition was defined as:
all people that visited lake Zeumeren between 15 August and 24 August 2012, and subsequently
developed gastro-intestinal symptoms.
Figure 2. Epidemiological curve, indicating the days on which the cases (ntotal = 45) were exposed and
their first day of illness, and the timeline of the outbreak investigation.
2.2. Site Description

Lake Zeumeren is located in a rural area (community Barneveld) in the center of The Netherlands.
It is a 32-hectare isolated fresh water lake with stagnant water, with groundwater and rainwater as
sources. The maximum water depth is 17 m and it has a sandy soil. Lake Zeumeren is an official bathing
site, where the water quality is monitored for fecal indicator parameters Escherichia coli and intestinal
enterococci, according to the European Bathing Water Directive (EU BWD) [18] with a fortnightly
frequency during the bathing season (1 May–1 October). The lake is surrounded by beaches, two of
which are identified as the official beaches, with demarcated bathing areas. At the two official beaches,
regular samples are taken for monitoring according to the EU BWD (Figure 1). The bathing zone
53
IJERPH 2018, 15, 2550
has a surface of 1.7 hectare, a maximum water depth of 1.5 m, and a total beach length of 530 m.
On-site facilities include toilets, showers, waste bins, and play equipment; the play equipment is
located on the dry land of beach 4 (Figure 1). Dogs are not allowed at the location during the bathing
season. The bathing water profile indicates that there is no connection to other water bodies, and that
the water quality is not influenced by wastewater treatment plants or run-off from agricultural land.
A substantial seagull population foraging at the lake may however occasionally influence the water
quality. According to the EU BWD, water quality at lake Zeumeren is classified as ‘excellent’.
2.3. Epidemiological Investigation

The province of Gelderland and the Public Health Service VGGM sent questionnaires by email
on 24 August 2012 to all patients with whom they had primary contact and for whom they had kept
records (n = 36). The patients were asked to answer the questions and to forward the questionnaire
to anyone they knew who had swum at the same site during the same period and had become ill
with the same symptoms. This approach was chosen in an attempt to rapidly identify the source of
the outbreak. Since there was no aim at performing a full case-control study, no matching controls
were sought. By allowing questionnaires to be forwarded, no records were (or could be) kept of the
number of questionnaires distributed. Apart from demographic questions about age and gender,
questionnaires included information requests about having visited lake Zeumeren (date between
15 August and 24 August 2012), having entered the water (including specification of beaches visited),
having swum (including duration of swimming), health conditions (choose from diarrhea, vomiting,
fever, fatigue, skin conditions, other), using the on-site toilets, and having bought and consumed food
and beverages on-site. Analysis of questionnaire responses was done by using SPSS (IBM, Armonk,
NY, USA, version 22).
2.4. Microbiological Investigation of Clinical Samples

Five patients were asked to send in stool samples; samples from four patients were received and
analyzed for the bacterial enteropathogens Salmonella, Shigella, Campylobacter, shiga toxin-producing
Escherichia coli (STEC) and Yersinia, and NoV at the medical microbiological laboratory of the Rijnstate
Hospital in Arnhem, The Netherlands, by using routine PCR. Later, the four samples were sent to
the National Institute for Public Health and the Environment (RIVM) for further testing. At RIVM,
the samples were analyzed for the presence of NoV by using a real-time RT-PCR assay, with primers,
probes and PCR conditions as previously described [19].
2.5. Environmental Investigation

In order to narrow down the outbreak and to identify the source, several leads were followed.
In response to the reported cases of gastroenteritis, water samples were taken from lake Zeumeren at
the two official beaches (Figure 1) on 20 August 2012 and were analyzed for fecal indicator parameters
E. coli and intestinal enterococci, by using the most probable number methods specified in the EU
BWD [18].
Although lake Zeumeren does not have a history of problems with cyanobacteria, the presence of
a faint greenish colouring of the water led to visual inspection for the presence of cyanobacteria and
sampling of the water at beach 4 (Figure 1) for determination of the dominant cyanobacterium genus
by microscopy and toxin levels by ELISA on Monday, 20 August 2012.
On Wednesday, 22 August, a woman reported to the province of Gelderland having fallen ill
after swimming in lake Zeumeren during the weekend prior to the outbreak weekend (11–12 August).
She attended a physician and had blood, urine and feces examined, which revealed a Campylobacter
jejuni infection that was treated with antibiotics. Although the woman did not meet the case definition,
it was decided to follow this lead because of the known nuisance caused by gulls at lake Zeumeren
that take over the beaches after the bathers have left. Water samples were taken at the two official
beaches on 23 August and analyzed for the presence of Campylobacter spp. by using a membrane
54
IJERPH 2018, 15, 2550
filtration method with incubation on Karmali Agar at 42 ± 0.5 ◦ C for 48 ± 2 h (in house method of Het
Waterlaboratorium, Haarlem, The Netherlands).
When the four analyzed stool samples appeared to be NoV positive, RIVM was contacted on
30 August and asked to take environmental samples for NoV analysis at lake Zeumeren. Sampling was
done on 3 September 2012. Water and subsurface sandy soil samples were only taken from the beaches
where most of the patients had stayed or swum during the weekend of 18 and 19 August as was
pointed out by the questionnaire results. One water sample was taken at sampling point A, and one
subsurface sandy soil sample was taken at each of the sampling points A–C (Figure 1).
The water sample (600 L) was taken by using an on-site filtration adsorption-elution procedure
for concentration of the sample [20,21]. The eluate was further concentrated by ultrafiltration under
high pressure (three bars) using a cellulose-acetate filter with a nominal molecular weight limit of
10,000 (Sartorius). The ultrafilter was rinsed with 3% beef extract (pH 9.0) resulting in the final
concentrate of approximately 40 mL which was stored at −70◦ C until further analysis. RNA extraction
was performed on 0.2, 1 and 5 mL of the concentrate as described by Rutjes et al. [21].
Subsurface sandy soil samples of approximately 50 g were taken by using a tubular soil sampler.
Subsequently, 10 mL 0.05 M glycine buffer pH 9.0 was added to 5.0 g of each sandy soil sample and
mixed at 500 rpm for 35 min at 4◦ C, after which it was allowed to settle for 10 min (in house method
RIVM). RNA extraction was done according to Rutjes et al. [21], by adding 20 and then 9 mL of
lysis buffer (Biomerieux, Boxtel, The Netherlands) to 5 mL and 1 mL volumes, respectively, of the
supernatant. Purification of the RNA extract was done by using a Qiagen RNeasy kit according to the
manufacturers’ instructions.
A real time reverse transcriptase PCR (RT-PCR) assay as described by Verhaelen et al. [19]
was performed to detect the presence of norovirus GI and GII RNA. The sequence of the detected
norovirus strain was determined by directly sequencing the purified RT-PCR product, which was
86 nucleotides long.
3. Results
3.1. Epidemiological Investigation

Public Health Service VGGM received 45 filled-in questionnaires that met the case definition.
Of the respondents, 56% (n = 25) were female. The age of the respondents ranged between 0 and
59 years of age, with more than half being children of 0–12 years of age (53%, n = 24), while the others
were adults of 18–59 years of age (47%, n = 21). All respondents visited lake Zeumeren between 15 and
19 August, but the majority were at the site on 18 August (89%, n = 40) and/or 19 August (49%, n = 22).
In total, 27 cases were exposed on more than one day during the specified period. All cases went into
the water for swimming and became ill afterwards, with health complaints starting 1–6 days (average
and median 2 days) after exposure to the bathing water and beaches at Lake Zeumeren and lasting
for 0.5–6 days (average and median 2 days). Reported health conditions were (multiple answers per
case allowed): diarrhea (84%, n = 38), vomiting (82%, n = 37), fatigue (49%, n = 22), and fever (16%,
n = 7). None of the cases reported skin conditions, and eight cases reported other complaints than
those indicated above, of which seven were related to gastro-intestinal discomfort. One case reported
a headache.
Forty-four of the cases indicated at which beach they had swum (multiple answers per case
were allowed), showing that most cases swam at beach 1 (70%, n = 31) and/or beach 2 (32%, n = 14).
Most cases (82%, n = 37) were in the water for 30 min to over an hour.
Fifty-eight percent (n = 26) of the cases used the permanent on-site toilets, the others did not.
Buying and consuming on-site sold food and beverages were not very common: 87% (n = 39) did not
buy or consume food and 98% (n = 44) did not buy or consume beverages.
55
IJERPH 2018, 15, 2550
3.2. Microbiological Investigation of Clinical Samples

The four investigated stool samples were positive with NoV GI, and not with any of the other
aetiologies. The fecal samples contained NoV genotype GI.Pd-GI.3. The partial genomic sequence
of the strain was submitted to Genbank under accession number MH828423. The size of the PCR
product was 1116 base pairs, including the primers. Sequence analysis yielded 1007 nucleotides,
733 overlapping with the typing region of open reading frame 1 (ORF1), and 275 overlapping with the
typing region of ORF2. Genotype assignment by phylogenetic analysis was supported by 100% and
99% bootstrap analysis, for ORF1 and ORF2, respectively. Typing from RT-PCR products is reliable if
the fragment has >100 nucleotides overlapping with both of the typing regions of ORF1 and ORF2.
3.3. Environmental Investigation

Water samples taken on 20 August 2012 showed levels of E. coli at the official beaches of 16 most
probable number (mpn)/100 mL at beach 1–2 and 6 mpn/100 mL at beach 3. Compared to previous
and later routine samplings during the same bathing season, slightly elevated levels of intestinal
enterococci at both beaches, 230 mpn/100 mL and 160 mpn/100 mL, respectively, were observed.
Examination of the samples taken for cyanobacteria analyses by microscopy demonstrated the
presence of Microcystis spp. at beach 4; intracellular microcystin levels were very low (<2 μg/L).
Campylobacter spp. were present in the water samples taken at the official beaches (one sample
per beach) on 23 August. Numbers appeared to be low, but could not be exactly determined due to
overgrowth of the culture medium. Further identification to the species level was not done.
NoV GI was detected in the subsurface sandy soil sample taken at sampling point A (beach 1).
The other subsurface sandy soil samples and the water sample were NoV negative. Sequencing of
the RT-PCR product from the subsurface sandy soil sample was impossible because the method for
typing of RT-PCR products is less sensitive than the detection method, and the amount of RNA in the
subsurface sandy soil sample appeared to be too low.
4. Discussion
Following the reporting of many cases of gastroenteritis that seemed related to exposure to
a recreational lake in The Netherlands during an exceptionally warm weekend in August 2012,
several leads were investigated in order to discover the etiological agent and the source of the outbreak.
Although the epidemiological investigation implicated the recreational lake as the most likely location
of contracting illness, the different directions in which the investigation went, based on reported
symptoms, demonstrate the difficulty in narrowing down the possible cause of recreational water
related outbreaks. The results of the examination of patient material can ease the search for the
etiological agent and the source; however, in this case, the reporting of a patient with a Campylobacter
infection in combination with the presence of a large seagull population resulted in choosing, what later
appeared to be, the wrong direction. Following several leads and having various patient and
environmental samples examined takes time, and in this case, led to the examination of water
and subsurface sandy soil samples only over two weeks after the outbreak presumably started,
thus diminishing the chances of detecting the etiological agent. Due to of the time gap between the
onset of the outbreak and the environmental sampling, subsurface sandy soil sampling was performed
in addition to the normal procedure of water sampling because sedimentation of virus particles during
the delay period could have resulted in the presence of virus particles in the subsurface sandy soil [22].
In this outbreak investigation, this appeared to be true and therefore subsurface sandy soil sampling
could be considered while environmental sampling in outbreak situations is delayed. However,
even if environmental samples are taken sooner, factors such as dispersion, dilution, sedimentation
and inactivation of the microorganisms in the water body may result in the inability to detect the
microorganisms that caused the outbreak [23]. The detection of NoV GI in the stool samples of outbreak
related patients and the detection of NoV GI in the subsurface sandy soil of the beach where most
56
IJERPH 2018, 15, 2550
patients swam support the probability that exposure to the lake resulted in the outbreak. NoV G
I strains have a stronger association with waterborne transmission than GII strains [8,24], possibly
because they are more stable in water than GII strains [8]. In a survey of recreational waters in Europe,
9.4% of 1440 samples were positive with NoV GI and GII [25], thus demonstrating the presence of NoV
in randomly selected recreational waters.
The epidemiological investigation showed that most of the patients were children. It has been
demonstrated that, while swimming, compared to adults, children spend more time in the water
and ingest a larger volume of water, thus increasing their exposure [26,27] and resulting in a higher
infection risk and attributable disease burden [28]. High exposure of children in combination with the
low infectious dose of NoV [2] and the incompletely developed immune system in young children
explain the high number of affected children. A possible bias is that parents are more likely to report
health complaints and sooner when their children are involved.
The detection of the same NoV genogroup in subsurface sandy soil and in patients two weeks
after the onset of the outbreak shows that NoV may be present in the environment for prolonged
time through accumulation in sediment [22], and may even be re-dispersed when bathers whirl the
soil. Although only RNA from virus particles was detected and no information about their infectivity
was available, these results indicate the value of a prolonged advice against bathing. Lake Zeumeren
is an isolated lake that is not subject to external sources of fecal contamination that may introduce
NoV, such as sewage water treatment plants, and the majority of the cases did not buy or consume
on-site sold food and beverages. Therefore, a food-related cause of the outbreak is unlikely and
the most plausible cause of contamination of the water is an infected human. Evidence of a single
person being responsible for contamination of the water was, however, not discovered, as is often the
case [8,24]. Prevention of outbreaks that have an infected human as the source can be done by active
communication of the long-standing advice of not to swim while having diarrhea, combined with the
information that viral shedding continues even after symptoms have resolved, although this does not
prevent occasional asymptomatic shedding [29].
5. Conclusions
An outbreak of gastrointestinal illness among over 100 visitors of a recreational lake in The
Netherlands during an exceptionally warm weekend was caused by NoV. The results of the
epidemiological investigation, combined with the detection of NoV GI in the stool samples of outbreak
related patients and in the subsurface sandy soil of the beach where most patients swam, suggest that
exposure to the recreational lake resulted in the outbreak. Although the primary contamination source
has not been discovered, the most likely cause of contamination of the water is an infected human,
because the lake is an isolated water body that is not subject to external sources of fecal contamination.
An outbreak like this warrants active communication about human shedding of viruses during and
after diarrheal episodes and the advice to refrain from swimming. The epidemiological curve and the
timeline of investigation based on reported symptoms demonstrate the difficulty in discovering the
source in recreational water outbreaks.
Author Contributions: Conceptualization, M.T.M.P., C.C. and F.M.S.; Methodology, H.H.J.L.v.d.B., W.J.L. and
H.V.; Investigation, F.M.S., H.H.J.L.v.d.B., M.T.M.P., W.J.L. and S.A.R.; Data Analysis, M.T.M.P., F.M.S., W.J.L.
and H.V.; Writing—Original Draft Preparation, F.M.S.; Writing—Review & Editing, F.M.S., H.H.J.L.v.d.B., H.V.,
M.T.M.P., C.C., S.A.R. and W.J.L.; Visualization, F.M.S. and M.T.M.P.; Supervision, F.M.S.
Acknowledgments: The authors thank the location manager of lake Zeumeren and the colleagues from the
province of Gelderland and Public Health Service VGGM involved in this outbreak investigation for their
contribution, Ana Maria de Roda Husman (RIVM) for critically reading of the manuscript and Leonard Bik for his
help with the figures.
57
IJERPH 2018, 15, 2550
References
1. Patel, M.M.; Hall, A.J.; Vinjé, J.; Parashar, U.D. Noroviruses: A comprehensive review. J. Clin. Virol. 2009, 44,
2. Teunis, P.F.M.; Moe, C.L.; Liu, P.; Miller, S.E.; Lindesmith, L.; Baric, R.S.; Le Pendu, J.; Calderon, R.L. Norwalk
virus: How infectious is it? J. Med. Virol. 2008, 80, 1468–1476. [CrossRef] [PubMed]
3. Atmar, R.L.; Opekum, A.R.; Gilger, M.A.; Estes, M.K.; Crawford, S.E.; Neill, F.H.; Graham, D.Y. Norwalk
virus shedding after experimental human infection. Emerg. Infect. Dis. 2008, 14, 1553–1557. [CrossRef]
[PubMed]
4. Glass, R.I.; Parashar, U.D.; Estes, M.K. Norovirus gastroenteritis. N. Engl. J. Med. 2009, 361, 1776–1785.
[CrossRef] [PubMed]
5. Rockx, B.; De Wit, M.; Vennema, H.; Vinjé, J.; De Bruin, E.; Van Duynhoven, Y.; Koopmans, M. Natural history
of human calicivirus infection: A prospective cohort study. Clin. Infect. Dis. 2002, 35, 246–253. [CrossRef]
[PubMed]
6. Zheng, D.P.; Ando, T.; Fankhauser, R.L.; Beard, R.S.; Glass, R.I.; Monroe, S.S. Norovirus classification and
proposed strain nomenclature. Virology 2006, 346, 312–323. [CrossRef] [PubMed]
7. Atmar, R.L.; Ramani, S.; Estes, M.K. Human noroviruses: recent advances in a 50-year history. Curr. Opin.
Infect. Dis. 2018, 31, 422–432. [CrossRef] [PubMed]
8. Matthews, J.E.; Dickey, B.W.; Miller, R.D.; Felzer, J.R.; Dawson, B.P.; Lee, A.S.; Rocks, J.J.; Kiel, J.; Montes, J.S.;
Moe, C.L.; et al. The epidemiology of published norovirus outbreaks: A review of risk factors associated
with attack rate and genogroup. Epidemiol. Infect. 2012, 140, 1161–1172. [CrossRef]
9. Riera-Montes, M.; Sjölander, K.B.; Allestam, G.; Hallin, E.; Hedlund, K.O.; Löfdahl, M. Waterborne norovirus
outbreak in a municipal drinking-water supply in Sweden. Epidemiol. Infect. 2011, 139, 1928–1935. [CrossRef]
[PubMed]
10. Ter Waarbeek, H.L.; Dukers-Muijrers, N.H.; Vennema, H.; Hoebe, C.J. Waterborne gastroenteritis outbreak at
a scouting camp caused by two norovirus genogroups: GI and GII. J. Clin. Virol. 2010, 47, 268–272. [CrossRef]
[PubMed]
11. Cho, H.G.; Lee, S.G.; Kim, W.H.; Lee, J.S.; Park, P.H.; Cheon, D.S.; Jheong, W.H.; Jho, E.H.; Lee, J.B.;
Paik, S.Y. Acute gastroenteritis outbreaks associated with ground-waterborne norovirus in South Korea
during 2008–2012. Epidemiol. Infect. 2014, 142, 2604–2609. [CrossRef] [PubMed]
12. Sinclair, R.G.; Jones, E.L.; Gerba, C.P. Viruses in recreational water-borne disease outbreaks: A review.
J. Appl. Microbiol. 2009, 107, 1769–1780. [CrossRef] [PubMed]
13. Hoebe, C.J.; Vennema, H.; De Roda Husman, A.M.; Van Duynhoven, Y.T. Norovirus outbreak among primary
schoolchildren who had played in a recreational water fountain. J. Infect. Dis. 2004, 189, 699–705. [CrossRef]
[PubMed]
14. Podewils, L.J.; Zanardi Blevins, L.; Hagenbuch, M.; Itani, D.; Burns, A.; Otto, C.; Blanton, L.; Adams, A.;
Monroe, S.S.; Beach, M.J.; et al. Outbreak of norovirus illness associated with a swimming pool.
Epidemiol. Infect. 2007, 135, 827–833. [CrossRef] [PubMed]
15. Zlot, A.; Simckes, M.; Vines, J.; Reynolds, L.; Sullivan, A.; Scott, M.K.; McLuckie, J.M.; Kromer, D.; Hill, V.R.;
Yoder, J.S.; et al. Norovirus outbreak associated with a natural lake used for recreation–Oregon, 2014.
Am. J. Transplant. 2015, 15, 2001–2005. [CrossRef] [PubMed]
16. Polkowska, A.; Räsänen, S.; Al-Hello, H.; Bojang, M.; Lyytikäinen, O.; Nuorti, J.P.; Jalava, K. An outbreak of
norovirus infections associated with recreational lake water in Western Finland, 2014. Epidemiol. Infect. 2018,
17. Kauppinen, A.; Al-Hello, H.; Zacheus, O.; Kilponen, J.; Maunula, L.; Huusko, S.; Lappalainen, M.;
Miettinen, I.; Blomqvist, S.; Rimhanen-Finne, R. Increase in outbreaks of gastroenteritis linked to bathing
water in Finland in summer 2014. Euro Surveill. 2017, 22, 30470. [CrossRef] [PubMed]
18. The European Parliament and the Council of the European Union. Directive 2006/7/EC of the European
Parliament and of the Council of 15 February 2006 concerning the management of bathing water quality and
repealing Directive 76/160/EEC. Off. J. Europ. Union 2006, L64, 37–51.
19. Verhaelen, K.; Bouwknegt, M.; Lodder-Verschoor, F.; Rutjes, S.A.; De Roda Husman, A.M. Persistence of
human norovirus GII.4 and GI.4, murine norovirus, and human adenovirus on soft berries as compared with
PBS at commonly applied storage conditions. Int. J. Food Microbiol. 2012, 160, 137–144. [CrossRef] [PubMed]
58
IJERPH 2018, 15, 2550
20. Van Olphen, M.; Kapsenberg, J.G.; Van de Baan, E.; Kroon, W.A. Removal of enteric viruses from surface
water at eight waterworks in The Netherlands. Appl. Environ. Microbiol. 1984, 47, 927–932. [PubMed]
21. Rutjes, S.A.; Italiaander, R.; Van den Berg, H.H.; Lodder, W.J.; De Roda Husman, A.M. Isolation and detection
of enterovirus RNA from large-volume water samples by using the NucliSens miniMAG system and
real-time nucleic acid sequence-based amplification. Appl. Environ. Microbiol. 2005, 71, 3734–3740. [CrossRef]
[PubMed]
22. Skraber, S.; Schijven, J.; Italiaander, R.; De Roda Husman, A.M. Accumulation of enteric bacteriophage in
fresh water sediments. J. Water Health 2009, 7, 372–379. [CrossRef] [PubMed]
23. Schets, F.M.; De Roda Husman, A.M.; Havelaar, A.H. Disease outbreaks associated with untreated
recreational water use. Epidemiol. Infect. 2011, 139, 1114–1125. [CrossRef] [PubMed]
24. Bitler, E.J.; Matthews, J.E.; Dickey, B.W.; Eisenberg, J.N.; Leon, J.S. Norovirus outbreaks: a systematic review
of commonly implicated transmission routes and vehicles. Epidemiol. Infect. 2013, 141, 1563–1571. [CrossRef]
[PubMed]
25. Wyn-Jones, A.P.; Carducci, A.; Cook, N.; D’Agostino, M.; Divizia, M.; Fleischer, J.; Gantzer, C.; Gawler, A.;
Girones, R.; Höller, C.; et al. Surveillance of adenoviruses and noroviruses in European recreational waters.
Water Res. 2011, 45, 1025–1038. [CrossRef] [PubMed]
26. Schets, F.M.; Schijven, J.F.; De Roda Husman, A.M. Exposure assessment for swimmers in bathing waters
and swimming pools. Water Res. 2011, 45, 2392–2400. [CrossRef] [PubMed]
27. DeFlorio-Barker, S.; Arnold, B.F.; Sams, E.A.; Dufour, A.P.; Colford, J.M., Jr.; Weisberg, S.B.; Schiff, K.C.;
Wade, T.J. 2017 Child environmental exposures to water and sand at the beach: Findings from studies of
over 68,000 subjects at 12 beaches. J. Expo. Sci. Environ. Epidemiol. 2017, 28, 93–100. [CrossRef] [PubMed]
28. Arnold, B.F.; Wade, T.J.; Benjamin-Chung, J.; Schiff, K.C.; Griffith, J.F.; Dufour, A.P.; Weisberg, S.B.;
Colford, J.M., Jr. Acute gastroenteritis and recreational water: highest burden among young US children.
Am. J. Public Health 2016, 106, 1690–1697. [CrossRef] [PubMed]
29. Teunis, P.F.; Sukhrie, F.H.; Vennema, H.; Bogerman, J.; Beersma, M.F.; Koopmans, M.P. Shedding of norovirus
in symptomatic and asymptomatic infections. Epidemiol. Infect. 2015, 143, 1710–1717. [CrossRef] [PubMed]
59
and Public Health
Article
Assessment of Microbiological Safety of Water in
Public Swimming Pools in Guangzhou, China
Xiaohong Wei 1,2,† , Juntao Li 1,† , Shuiping Hou 1 , Conghui Xu 1 , Hao Zhang 1 ,
Edward Robert Atwill 3,4 , Xunde Li 3,4 , Zhicong Yang 1, * and Shouyi Chen 1, * ID
1 Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China;
[email protected] (X.W.); [email protected] (J.L.); [email protected] (S.H.); [email protected] (C.X.);
[email protected] (H.Z.)
2 School of Public Health, Sun Yat-Sen University, Guangzhou 510030, China
3 Department of Population Health and Reproduction, University of California Davis, California,
CA 95616, USA; [email protected] (E.R.A.); [email protected] (X.L.)
4 Western Institute for Food Safety and Security, University of California Davis, California, CA 95616, USA
* Correspondence: [email protected] (Z.Y.); [email protected] (S.C.); Tel.: +86-138-2614-0717 (Z.Y.)
† These authors contributed equally to this article.
Received: 15 June 2018; Accepted: 3 July 2018; Published: 5 July 2018
Abstract: This study assessed microbiological safety of water from public swimming pools in
Guangzhou, China. Water samples from 39 outdoor municipal swimming pools were collected from
late June to early September, 2013 and subjected to detection of protozoa (Giardia and Cryptosporidium)
and bacteria (Pseudomonas aeruginos, total coliforms, E. coli, E. coli O157, Shigella, and Salmonella).
Cryptosporidium and Giardia were both detected in 5 (12.8%) swimming pools. Total coliforms were
detected in 4 (10.3%) samples with concentrations ranging from 1.3 to 154.0 MPN/100 mL while E. coli
was detected in 4 (10.3%) samples with concentrations ranging from 0.5 to 5.3 MPN/100 mL.
P. aeruginosa was detected in 27 (69.2%) samples but E. coli O157, Shigella and Salmonella were
not detected. Among these swimming pools, 9 (23%) met the Chinese National Standard of residual
chlorine levels and 24 (62%) were tested free of residual chlorine at least once. The multi-locus
sequence typing (MLST) analysis showed that all P. aeruginosa isolates belonged to new sequence
types (STs) with dominant ST-1764 and ST-D distributed in different locations within the area.
Some P. aeruginosa strains were resistant to medically important antibiotics. Results indicate potential
public health risks due to the presence of microbiological pathogens in public swimming pools in
this area.
Keywords: swimming pool; water; Giardia; Cryptosporidium; P. aeruginosa; antibiotic resistance;

multi-locus sequence typing
1. Introduction
Many microbiological pathogens, including bacteria, viruses, and parasites can cause waterborne
disease [1]. Waterborne disease is estimated to cause more than 2.2 million deaths per year and
many cases of illness every day, including diarrhea, gastrointestinal diseases and other systemic
illnesses [2,3]. Waterborne disease can have a significant impact locally and globally [4]. Waterborne
infections are transmitted in numerous ways, including but not limited to ingestion, airborne or contact
with contaminated water by a variety of infectious agents.
In addition to drinking water, which serves as a route for waterborne illness worldwide, especially
in developing countries [5], exposure to recreational water also frequently causes outbreaks of
waterborne diseases in both developed and developing countries [6–9]. For examples of recreational
water related outbreaks in developed countries, there have been reports of waterborne disease

IJERPH 2018, 15, 1416
associated with swimming pools in Australia [10], England and Wales [11] and other countries [12].
Most countries have programs for monitoring microbiological safety of swimming pool water.
However, diverse public health and economic conditions among countries result in different
microbiological and physical-chemical standards for swimming pool water even within countries of
the European Union [13]. In China, a National Standard for swimming pool water quality, entitled
“Hygienic Standard for Swimming Places” has been established (GB9667-1996). Major parameters
in the National Standard are water temperature (22–26 ◦ C), pH (6.5–8.5), turbidity (≤5 NTU), urea
(≤3.5 mg/L), free residual chlorine levels (0.3–0.5 mg/L), total bacteria count (≤1000 cfu/mL), total
coliforms (≤18 cfu/L). Although some recent works have studied water quality of swimming pools in
China, most of these studies have primarily focused on the basic parameters that are highlighted in the
National Standards, including pH, turbidity, urea content and total coliforms [14–16]. Literature on the
microbiological safety of swimming pool water in China has been notably sparse. Furthermore, from a
public health perspective it remains unknown if swimming pool water transmits antibiotic resistant
bacteria. By detection and analysis of key waterborne protozoa and bacteria, this study evaluated the
microbiological safety of water in outdoor public swimming pools in Guangzhou, which is a large
metropolitan area in southern China.
2.1. Swimming Pools Selection and Water Sample Collection

This survey was conducted between 20 June and 4 September 2013. Due to the potential differences
in public health conditions between suburban and urban districts within the Guangzhou area [17],
the suburban Baiyun district and the urban Haizhu district were chosen for sampling. In total 39 pools,
including 27 pools from the Baiyun district (21 residential pools, 6 pools from two water parks) and
12 pools from the Haizhu district (5 residential pools, 3 school pools, and 4 pools from one water park)
were enrolled in the study. All enrolled pools were outdoor public swimming pools. Three swimming
pools were exclusively for children while the remaining 36 pools were for both adults and children.
Water samples were collected between 10:00 a.m. and noon, a period prior to the pools being open
to swimmers. Using a submersible pump, 50 L water samples were pumped 10 cm from the bottom
of the pool into two sterile 25 L carboys. Four hundred milligrams of sodium thiosulfate (Na2 S2 O3 ,
XK13-001-00008, GB/T-637 standard, Guangzhou, China) was added to each carboy for dechlorination
of water. Carboys were placed in containers with ice and transported to the laboratory within two
hours and stored in a cold room (4 ◦ C) upon arrival at the laboratory and before processing.
A questionnaire was filled out for each swimming pool in order to support the microbiological
analysis. The questionnaires contained three sections that include general data on the swimming pools
(type, location, dimensions and depth), levels of residual chlorine, and management information (opening
time, hours of operation, availability of swimming-suit rentals, whether toddlers were required to wear
diapers during swimming, methods used to refresh water, frequency of testing residual chlorine levels).
2.2. Detection of Microbiological Pathogens from Water

With the aid of hollow fiber ultrafiltration as described by Rhodes et al. [18], the 50 L water sample
was filtered no later than 24 h after collection and concentrated to approximately 700 mL (retentate).
To test for Giardia and Cryptosporidium, 500 mL of the retentate was centrifuged at 4000 rcf at 25 ◦ C
for 15 min without breaking. Supernatant was discarded by aspiration and approximately 3 mL of
residual pellet was harvested. An immunomagnetic separation (IMS) procedure was carried out to
separate (oo) cysts from concentrated water samples using the Dynabeads GC-Combo kit (Invitrogen
Dynal AS, Oslo, Norway) according to the manufacturer’s instructions. The IMS was carried out
using a Bead Retriever with the procedure of ‘GC Combo’ (Invitrogen, Finland). Immunofluorescent
staining of final IMS products (approximately 50 μL) was performed using the Aqua-Glo G/C Direct
61
IJERPH 2018, 15, 1416
kit (Waterborne, New Orleans, LA, USA) according to the manufacturer’s instructions. Slides were
examined for (oo) cysts using an immunofluorescent microscope (Leica DM6000B).
All bacteria were detected according to China’s National Standard (GB) protocols. Specifically,
P. aeruginosa was detected using methods described in the Hygienic Standard for Cosmetics
(GB7918.4-2007, Ministry of Health, China). The methods used for detection of other bacteria were based
on the National Food Safety Standard (Ministry of Health, China), i.e., GB4789.3-2010 for total coliforms,
GB4789.38-2012 for E. coli, GB4789.36-2008 for E. coli O157, GB4789.5-2012 for Shigella and GB4789.4-2010
for Salmonella, respectively. The procedures for detection of these bacteria are described below.
To detect P. aeruginosa, 10 mL of retentate was resuspended in 90 mL of Soya Casein Digest
Lecithin Polysorbate (SCDLP) broth and incubated at 37 ◦ C for 24 h. Then, 100 μL of the culture was
streaked on cetrimide agar and incubated at 37 ◦ C for 24 h. For total coliforms, triplication of serially
diluted retentate (1, 0.1 and 0.01 mL) were resuspended in tubes of LST (Lauryl Sulfate Tryptose)
broth and incubated at 37 ◦ C for 48 h. The most probable number (MPN) was calculated according
to the number of tubes that generated positive reactions (generating bubbles). Similarly, triplication
of serially diluted culture (1, 0.1, and 0.01 mL) of LST broth generating bubbles were transferred into
E. coli broth and incubated at 44.5 ◦ C for 48 h for calculating MPN of E. coli. To detect E. coli O157,
25 mL of retentate was added to 225 mL of modified E. coli broth and incubated at 37 ◦ C for 24 h.
An aliquot of the culture was streaked onto E. coli O157 chromogenic medium and incubated at 37 ◦ C
for 24 h. Red or purple colonies were chosen to cultivate on TSI (Triple Sugar Iron) agar medium at
37 ◦ C for 24 h. For detection of Shigella, 25 mL of retentate was added to 225 mL of Shigella broth and
incubated at 42 ◦ C for 20 h. Then, an aliquot of the culture was streaked onto Shigella chromogenic
medium and incubated at 37 ◦ C for 48 h. Typical colonies were selected for cultivation on TSI (Triple
Sugar Iron) agar medium at 37 ◦ C for 24 h. For detection of Salmonella, 25 mL of retentate was added
to 225 mL of Buffered Peptone Water (BPW) and incubated at 37 ◦ C for 18 h. An aliquot of the BPW
culture was added to 10 mL of TTB (Tatrathionate Broth) and incubated at 42 ◦ C for 24 h. The culture
in TTB was then streaked onto Salmonella chromogenic medium and incubated at 37 ◦ C for 24 h. Purple
or red colonies were selected for incubation on TSI (Triple Sugar Iron) agar medium at 37 ◦ C for 24 h.
Isolates of P. aeruginosa, Shigella and Salmonella were confirmed biochemically and phenotypically
using the VITEK2 GN method (bioMériux, Marcy d’Etoile, France).
2.3. Testing P. aeruginosa Susceptibility to Antibiotics

P. aeruginosa susceptibility to antibiotics was tested using the VITEK2 AST-GN04 method
(bioMériux, Marcy d’Etoile, France). The selection of antimicrobial agents and interpretation of
minimum inhibitory concentration (MIC) (S = susceptible, IR = intermediate resistant, R = resistant)
were based on the Clinical and Laboratory Standards Institute [19]. The antimicrobial agents tested
are of medical importance, including piperacillin, piperacillin-tazobactam, ticarcillin-clavulanic acid,
ceftazidime, cefepime, imipenem, gentamicin, tobramycin, amikacin, levofloxacin and ciprofloxacin.
2.4. Multilocus Sequence Typing (MLST) of P. aeruginosa

Two or three typical P. aeruginosa colonies were picked and homogenized in 100 μL autoclaved
deionized water. The bacterial solutions were incubated at 100 ◦ C for 10 min followed by cooling
at 4 ◦ C for 3 min, then centrifuging at 12,000× g for 3 min. The primer pairs used for PCR and
sequencing are shown in Table 1. The amplification reactions and conditions for MLST of P. aeruginosa
were performed as described in the PubMLST (http://pubmlst.org/paeruginosa/info/primers.shtml).
Because the original temperature could not successfully amplify mutL, ppsA and trpE alleles, slightly
modified conditions (primer annealing temperature) were used to optimize amplification in each
reaction. The following adjusted PCR protocol was used: denaturing at 95 ◦ C for 1 min, 35 cycles of
1 min at 95 ◦ C (denaturing), 1 min at 55–57 ◦ C (primer annealing, 57 ◦ C for mutL and ppsA, 55 ◦ C
for trpE) and 1 min at 72 ◦ C (primer extension), and a final elongation step at 72 ◦ C for 10 min.
Sequencing of all the genes were performed by Life Biological Technology Company (Shanghai,
62
IJERPH 2018, 15, 1416
China). Sequences from the study and alleles and STs from the PubMLST (http://pubmlst.org/) were
used to construct a phylogenetic tree of P. aeruginosa isolates using MLST Data Analysis-Tree drawing
(https://pubmlst.org/paeruginosa/). A population snapshot was operated using the software eBRUST
V3 available at the website of http://eburst.mlst.net/v3/enter_data/comparative/.
Table 1. Primers used for multi-locus sequence typing (MLST) of P. aeruginosa isolates.
Primers Sequence (5 —3 )

Locus and Functions Size (bp)
Forward Reverse
Amplification ACCTGGTGTACGCCTCGCTGAC GACATAGATGCCCTGCCCCTTGAT 842
acsA
Sequencing GCCACACCTACATCGTCTAT GTGGACAACCTCGGCAACCT 390
Amplification TGGGGCTATGACTGGAAACC TAACCCGGTTTTGTGATTCCTACA 825
aroE
Sequencing ATGTCACCGTGCCGTTCAAG TGAAGGCAGTCGGTTCCTTG 495
Amplification CGGCCTCGACGTGTGGATGA GAACGCCTGGCTGGTCTTGTGGTA 940
guaA
Sequencing AGGTCGGTTCCTCCAAGGTC TCAAGTCGCACCACAACGTC 372
Amplification CCAGATCGCCGCCGGTGAGGTG CAGGGTGCCATAGAGGAAGTC 940
mutL
Sequencing AGAAGACCGAGTTCGACCAT ATGACTTCCTCTATGGCACC 441
Amplification ACCGCCACCCGTACTG TCTCGCCCATCTTGACCA 1042
nuoD
Sequencing ACGGCGAGAACGAGGACTAC TTCACCTTCACCGACCGCCA 366
Amplification GGTCGCTCGGTCAAGGTAGTGG GGGTTCTCTTCTTCCGGCTCGTAG 989
ppsA
Sequencing GGTGACGACGGCAAGCTGTA TCCTGTGCCGAAGGCGATAC 369
Amplification GCGGCCCAGGGTCGTGAG CCCGGCGCTTGTTGATGGTT 811
trpE
Sequencing TTCAACTTCGGCGACTTCCA GGTGTCCATGTTGCCGTTCC 441
2.5. Statistical Analysis

The Chi-square (χ2 ) test was applied to assess the potential differences in Giardia and
Cryptosporidium presence in water between suburban and urban swimming pools, because we found the
prevalence of Cryptosporidium was significantly higher in children hospitalized in suburban hospitals
than in urban hospitals in the same area in a previous study [17]. Logistic correlation was applied to
analyze whether the presence of P. aeruginosa was associated with the levels of free residual chlorine
in water.
3. Results
3.1. Characteristics of Swimming Pools

Outdoor swimming pools in the Guangzhou area mostly open between June and September.
The swimming water of all enrolled pools is refreshed by refilling the pools with tap water as
needed. No swimming suit rental was available in any of the surveyed swimming pools. Swimmers
brought their own swimming suits and children were not required to wear diapers when swimming.
The studied swimming pools had a diversity of shapes and surface areas that ranged from 100 to
1000 m2 . The depth of these pools ranged from 0.3 m to 2.0 m with 1.0 to 1.3 m for the majority of
the enrolled pools. All swimming pools used standard chlorination to disinfect water, and no further
treatment of the water had taken place. Among the pools, 24 (62%) checked free residual chlorine levels
once per day; 8 (20%) check twice per day; and 7 (18%) check 3–5 times per day. Free residual chlorine
levels were >0.5 mg/L in 18 (46%) swimming pools and <0.3 mg/L in 12 (31%) of the swimming pools.
The highest free chlorine level was 2.0 mg/L and the lowest free chlorine level was 0 mg/L. According
to the Chinese National Standard, ‘Hygienic Standard for Swimming Places’ (GB9667-1996, China) the
free residual chlorine level in swimming water should be between 0.3 mg/L and 0.5 mg/L. Our survey
results showed that only 9 (23%) of the studied swimming pools had free residual chlorine levels that
met this criteria.
3.2. Occurrence of Protozoa in Swimming Pool Water

Cryptosporidium and Giardia were both detected in 12.8% (5/39) of the enrolled swimming pools.
Among these positive pools, 2 pools were positive for Giardia only, another 2 were positive for
Cryptosporidium only, and 1 was positive for both Giardia and Cryptosporidium. Concentrations of (oo)
63
IJERPH 2018, 15, 1416
cysts in positive pool water were 0.03 cysts/L of Giardia and 0.03–0.14 oocysts/L of Cryptosporidium.
The proportional occurrences of both Giardia and Cryptosporidium were 25% (3/12) and 7.4% (2/27) in
pools in the Baiyun district (suburban) and the Haizhu district (urban), respectively. There was no
significant difference in the occurrence of Giardia and Cryptosporidium in swimming pools between
suburban and urban areas (p = 0.159).
3.3. Occurrence of Bacteria in Swimming Pool Water

Total coliforms were detected in 4 (10.3%) of 39 swimming pools, with concentrations of
1.3, 3.2, 154.0, and 154.0 MPN/100 mL, respectively. Concentrations of total coliform in 2 of the
positive pools were lower while that of the other 2 pools were higher than the 18 cfu/L specified
in the Chinese Standard. E. coli was also detected in the same 4 (10.3%) of 39 swimming pools,
with concentrations of 0.5, 1.3, 3.2, and 5.3 MPN/100 mL. Using the standard methods stated above,
pathogenic E. coli O157, Shigella and Salmonella were not detected in any of the enrolled swimming
pools. P. aeruginosa was detected in 27 (69.2%) of the swimming pools. Based on the Chinese National
Standard, the free residual chlorine levels in water of these swimming pools were divided into three
groups: <0.3 mg/L, 0.3–0.5 mg/L, and >0.5 mg/L. The proportional occurrence of P. aeruginosa in
each group is shown in Table 2. As shown in the table, the results of logistic correlation analysis using
<0.3 mg/L as the control group indicated that there was no significant relationship between the level
of free residual chlorine and the presence of P. aeruginosa. (p = 0.55 and 1.00, respectively).
Table 2. Occurrence of P. aeruginosa in swimming pool water with different residual levels of
free chlorine.
Residual Levels of Free Chlorine (mg/L) % (Positive/Total) Occurrence of P. aeruginosa p OR (95% CI)
<0.3 66.7 (8/12) - -
0.3 to 0.5 77.8 (7/9) 1.00 1.00 (0.21, 4.71)
>0.5 66.7 (12/18) 0.55 1.75 (0.28, 11.15)
Total 69.2 (27/39) - -
Note: OR = odds ratio, CI = confidence interval. <0.3 mg/L was used as control group.
3.4. MLST of P. aeruginosa

Among the 27 P. aeruginosa isolates, 17 isolates were positive for all the seven housekeeping genes,
therefore they were analyzed using MLST. One or more housekeeping genes in each isolate could not
be amplified or sequenced, as a result, 6 alleles of acsA, ppsA and trpE; 5 alleles of aroE, mutL and
nuoD; and 7 alleles of guaA were successfully sequenced (Table 3). The MLST analysis identified 11 STs
from the tested isolates, all were distinct from existing STs at (http://pubmlst.org/) and therefore
were novel STs. Using the curator Ellie Pinnock of PubMLST (http://pubmlst.org/), three ST types
have been named as ST-1764, ST-1765 and ST-1766 and the remaining types are designated as ST-A,
ST-B, ST-C, ST-D, ST-E, ST-F, ST-G and ST-H. According to the most stringent definition by the eBURST,
a ST group was defined as isolates sharing identical alleles at ≥6 of the 7 loci among isolates in a
group that belonged to a clonal complex. Otherwise it was singleton. Isolates stemming from a group
were considered as belonging to a single clonal complex. The STs with one and two distinct loci
compared to the founder strain were named single-locus variants (SLV) and double-locus variants
(DLV), respectively (Table 3). Phylogenetic analysis indicated that these STs from different pools and
different districts fell into the same branches with the exceptions of ST-D and ST-E in one branch,
ST-C and ST-1766 in one branch, and ST-A, ST-B, and ST-1764 in one branch (Figure 1). The type
and locations of these samples are listed in Table 3. To further explore the associations between the
P. aeruginosa isolates from current study and P. aeruginosa isolates from the database of PubMLST,
a population structure of P. aeruginosa isolates was created (Figure 2). As shown in the figure, STs
and isolates of P. aeruginosa from the current study were distinct from the 1833 STs at the PubMLST,
which supports the assertion of the existence of unique and novel STs of P. aeruginosa in swimming
pool water in the Guangzhou area.
64
Table 3. List of the alleles and bioinformatics analysis of STs of P. aeruginosa isolates from public swimming pools in Guangzhou, 2013.
acsA aroE guaA mutL nuoD ppsA trpE Group STs SLV DLV Freq Pool Type District
81 11 112 5 73 20 139 1 ST-A 1 1 1 Residential Haizhu
81 11 112 5 13 20 139 1 ST-B 2 0 2 Residential Baiyun
81 11 57 5 13 20 139 1 ST-1764 1 1 3 Residential Baiyun
IJERPH 2018, 15, 1416
5 3 95 5 93 6 47 2 ST-1766 1 0 2 Residential; Water park Baiyun; Haizhu

5 3 95 5 13 6 47 2 ST-C 1 0 3 Residential; Water park * Baiyun; Haizhu
70 5 72 2 3 20 26 3 ST-D 1 0 1 School Haizhu
70 5 72 2 3 4 26 3 ST-E 1 0 1 Water park Baiyun
5 5 57 13 13 74 3 Singleton ST-F - - 1 Water park Haizhu
83 5 9 3 13 10 3 Singleton ST-G - - 1 Residential Baiyun
32 13 24 13 13 6 25 Singleton ST-H - - 1 Water park Baiyun
39 6 4 14 61 15 2 Singleton ST-1765 - - 1 Residential Baiyun
* Represents two pools from the water park located in Haizhu district.
65
IJERPH 2018, 15, 1416
Figure 1. Phylogenetic tree of MLSTs of 17 P. aeruginosa isolates each from different swimming pools.
The red digits in the parentheses denote the numbers of the STs. The number of unmarked STs was 1.
Figure 2. Population structure of the 1833 STs listed in the P. aeruginosa PubMLST database and the
11 STs from this study. Note: The figure shows all BURST groups (connected STs), singleton STs,
ancestral founders (blue STs), and subgroup founders (yellow STs). Dots represent STs and lines
connect single-locus variants. Purple letters and digits represent STs of P. aeruginosa from the current
study and STs with green halos represent STs detected only in the current study. Line length and
singleton ST placement is arbitrary.
3.5. P. aeruginosa Susceptibility to Antibiotics

Susceptibility tests were performed on all the 27 P. aeruginosa isolates, each from different pools.
Six isolates (ID# 2, 16, 17, 32, 33 and 37) exhibited different levels of susceptibility (R, IR, or S) to tested
antibiotics while all the other isolates (IDs not provided) were susceptible to all tested drugs (Table 4).
Among the tested drugs, 2 isolates (#2 and #37) were resistant to piperacillin, 2 isolates (#17 and #33)
were resistant to imipenem, and 1 isolate (#17) was resistant to gentamicin (Table 4). The isolates #2,
#16, #17 and #37 corresponded to the MLST groups of ST-A, ST-1765, ST-1766, and ST-D, respectively.
All the rest of the isolates (not listed in Table 4) were susceptible to all tested drugs.
66
Table 4. Phenotypic antibiotic resistance traits of P. aeruginosa from public swimming pools in Guangzhou, 2013.
PENICILLINS B-LACTAM Cephems Carbapenems Aminoglycosides Fluoroquinolones

Isolates ID
Piperacillin Ticarcillin-Clavulanic Acid Tazobactam Ceftazidime Cefepime Imipenem Gentamicin Tobramycin Amikacin Ciprofloxacin Levofloxacin
2 R* S S S S S S S S S S
16 IR ** S S S S S IR S S S S
IJERPH 2018, 15, 1416
17 S *** S S S S R R S S S S
32 IR S S S S S IR S S S S
33 S S S S S R S S S S S
37 R S S S S S S S S S S
All other isolates S S S S S S S S S S S
* R = Resistant; ** IR = Intermediate Resistant; *** S = Susceptible.
67
IJERPH 2018, 15, 1416
4. Discussion
According to our observations during the survey and the survey results, overall there is a lack of
good practices in pool management, including water disinfection. The free residual chlorine levels of
18 (46%) swimming pools were greater than 0.5 mg/L, which is higher than that of China’s National
Standard for swimming pools. However, the standards for free residual chlorine for swimming pool
water in some European countries [13] are higher than the Chinese standard, which may increase the
efficiency of water disinfection. On the other hand, the pools open in June through September which is
the warmest season of the whole year in Guangzhou. UV radiation can lead to the formation of chlorate
in the water, which reduces the concentration of free chlorine available in water and subsequently
impacts the effects of chlorine disinfection. Also, because children were not required to wear diapers
when swimming, there was a potential of pool water contamination by children with diarrhea, which
is a reported route for swimming pool contamination with Cryptosporidium [20]. Finally, the manner
of refreshing pool water (adding water when necessary instead of replacing water completely) could
facilitate the accumulation of pathogens in water.
The overall prevalence of Giardia and Cryptosporidium were both 12.8% in swimming pools during
the study period. No significant differences in the occurrence of the two protozoal parasites were found
between pools in urban and suburban areas. This is converse to our previous findings that prevalence
of Cryptosporidium was significantly higher in children hospitalized in suburban hospitals than in
urban hospitals in the same area [17]. This is probably because of the different transmission routes of
Cryptosporidium in children patients living in urban and suburban areas. Due to limited numbers of
(oo) cysts detected from pool water we did not characterize the two parasites by genotyping, hence the
genotypes and zoonotic potential of the two parasites in pool water were unknown. In our previous
study we found that the dominant species of Cryptosporidium in children hospitalized for diarrhea in
the same area was C. parvum, a species that infects humans and a wide range of animals [17]. Also,
because of the limited numbers of oocysts detected in samples, we did not assess the viability of
oocysts, therefore, it was unknown if the oocysts detected in pool water were infective. However, it is
known that oocysts are resistant to chlorine and cause waterborne illness associated with swimming
pools [21,22]. Cryptosporidium and Giardia are infectious to people, especially children, the elderly,
others with weakened immunity (such as AIDS patients) and travelers, and may cause severe diarrhea
and even death [23,24]. The two parasites are major causes of waterborne parasitic infections all over
the world, including developed countries such as New Zealand, Australia and USA [25]. A challenge
to control these waterborne parasites is that chlorination is ineffective against (oo) cysts [25]. Although
UV irradiation [26] and ozone [27] show promising effects against (oo) cysts, these methods have not
been practical for disinfection of swimming pool water. As a result, waterborne Cryptosporidium and
Giardia associated with swimming occur frequently [28,29]. Therefore, it was not a surprise to detect
Cryptosporidium and Giardia in swimming pool water in the area, and the results demonstrated that
control of the two most common waterborne protozoa is a continuous task in Guangzhou area, as it
is elsewhere.
With respect to bacterial pathogens, both total coliform and E. coli were detected in approximately
10% of the enrolled swimming pools. These indicator organisms can potentially cause various diseases,
particularly in children under five years old and the elderly [6]. Presence of these organisms in water
often indicates the ineffectiveness of water disinfection [30], especially the pools with total coliform
concentrations higher than the National Standard in this study. The good news is that pathogenic
bacteria, E. coli O157, Shigella and Salmonella were not detected in all swimming pools, including those
10% of pools that were positive for total coliforms and E. coli. However, another pathogenic bacterium,
P. aeruginosa was found to be highly prevalent (69%) in studied swimming pools.
P. aeruginosa is ubiquitous in water and is resistant to chemical disinfectants such as chlorine [31].
P. aeruginosa cause a variety of disease including folliculitis, external otitis, keratitis, urinary infections
and gastrointestinal infections through exposure to skin, ears, eyes, urinary track, lungs and the
gut [12,30,32]. Seventy-nine percent of ear infections in swimmers were ascribed to P. aeruginosa with
68
IJERPH 2018, 15, 1416
symptoms ranging from earache to hearing loss [33]. According to a survey of waterborne diseases in
the USA between 1999 and 2008, P. aeruginosa was the second most common waterborne pathogenic
micro-organism after Cryptosporidium [6]. A study reported that P. aeruginosa was detected in 95 (59%)
of 161 samples of swimming pool filter backwash in Atlanta, Georgia, GA, USA [34]. In the present
study we detected similarly high occurrence (69%) of P. aeruginosa in public swimming pools in the
Guangzhou area. Also, the same STs or the same group of STs of P. aeruginosa were distributed among
different types of pools in different complexes, and in different locations (Table 3). Our MLST (Figure 1)
and population structure analysis (Figure 2) showed that STs of P. aeruginosa from swimming pools in
the studied area were distinct from strains from other areas and other types of samples, indicating that
unique P. aeruginosa strains were present in the aquatic environment in the area. Although our study
did not determine the routes of transmission of P. aeruginosa in the area, P. aeruginosa can be carried
on the equipment of swimmers [33]. Finally, several isolates of P. aeruginosa from swimming pools
were resistant to some medically important antibiotics such as piperacillin, imipenem, and gentamicin
(Table 4). This result is consistent with reports that P. aeruginosa isolates from water environments have
lower antibiotic resistance than those of clinical specimens [35]. However, the presence of antibiotic
resistant P. aeruginosa in swimming pool water indicates that swimming pool water can serve as a route
for transmitting antibiotic resistant bacteria and genes, hence presenting additional risks to swimmers
who use public swimming pools and workers who perform maintenance of the pools.
Waterborne illness associated with swimming pools is commonly attributed to fecal contamination
of water either by swimmers or animals that have access to outdoor pools [36,37]. Other non-fecal
substances, such as vomit, mucus, saliva and skin can also serve as sources of pathogenic
micro-organisms in swimming pools [38]. Although there are microbiological standards using total
bacteria count and total coliform for swimming water, research on microbiological safety associated
with pathogens in swimming pool water has been sparse in China. Our study showed the presence of
pathogens such as Cryptosporidium, Giardia and antibiotic resistant P. aeruginosa in public swimming
pools that potentially pose risks to public health. Data from our study and future similar studies will
provide a background for public health agencies and communities to improve the management of
public swimming pools in order to prevent waterborne illness associated with swimming pools.
5. Conclusions
This is the initial work of assessing microbiological safety of water in public swimming pools
in Guangzhou, China. The presence of Cryptosporidium, Giardia and antibiotic resistant P. aeruginosa
in swimming water indicate potential risks to public health in this area. Results suggest the need
of updating management of swimming pool facilities and treatment of swimming water in order to
prevent waterborne illness associated with swimming in the area.
Author Contributions: Conceptualization, E.R.A., X.L. and S.C.; Methodology, S.H. and C.X.; Data Curation,
X.W., J.L. and H.Z.; Data Analysis X.W. and X.L.; Draft Preparation, X.W.; Writing-Review & Editing, X.L. E.R.A.
and S.C.; Supervision, E.R.A., Z.Y., and S.C.
Funding: This work was supported by the University Outreach and International Programs (UOIP) Seed Grant
(2012) of University of California Davis and the Project for Key Medicine Discipline Construction of Guangzhou
Municipality (2013-2015-07).
Acknowledgments: The authors thank the Guangzhou Center for Disease Control and Prevention for its’ kind
support of this study.
References
1. Leclerc, H.; Schwartzbrod, L.; Dei-Cas, E. Microbial agents associated with waterborne diseases.
Crit. Rev. Microbiol. 2002, 28, 371–409. [CrossRef] [PubMed]
2. Bitton, G. Microbiology of Drinking Water Production and Distribution, 1st ed.; John Wiley & Sons, Inc.: Hoboken,
NJ, USA, 2014; p. 312.
69
IJERPH 2018, 15, 1416
3. WHO. Water Sanitation Hygiene. Available online: http://www.who.int/water_sanitation_health/en/

(accessed on 6 June 2018).
4. Alhamlan, F.S.; Al-Qahtani, A.A.; Al-Ahdal, M.N. Recommended advanced techniques for waterborne
pathogen detection in developing countries. J. Infect. Dev. Ctries. 2015, 9, 128–135. [CrossRef] [PubMed]
5. WHO. Global Water Supply and Sanitation Assessment 2000 Report. Available online: http://www.who.
int/water_sanitation_health/monitoring/jmp2000.pdf (accessed on 30 May 2018).
6. Barna, Z.; Kadar, M. The risk of contracting infectious diseases in public swimming pools. A review. Ann. Ist.
Super. Sanita 2012, 48, 374–386. [CrossRef] [PubMed]
7. WHO. Water Recreation and Disease. 2005. Available online: http://www.who.int/water_sanitation_
health/bathing/recreadis.pdf (accessed on 28 May 2018).
8. Giampaoli, S.; Romano Spica, V. Health and safety in recreational waters. Bull. World Health Organ.
2014, 92, 79. [CrossRef] [PubMed]
9. Mavridou, A.; Pappa, O.; Papatzitze, O.; Blougoura, A.; Drossos, P. An overview of pool and SPA regulations
[PubMed]
10. Waldron, L.S.; Ferrari, B.C.; Cheung-Kwok-Sang, C.; Beggs, P.J.; Stephens, N.; Power, M.L. Molecular
epidemiology and spatial distribution of a waterborne cryptosporidiosis outbreak in Australia.
11. Smith, A.; Reacher, M.; Smerdon, W.; Adak, G.K.; Nichols, G.; Chalmers, R.M. Outbreaks of waterborne
infectious intestinal disease in England and Wales, 1992–2003. Epidemiol. Infect. 2006, 134, 1141–1149.
[CrossRef] [PubMed]
12. Rice, S.A.; van den Akker, B.; Pomati, F.; Roser, D. A risk assessment of Pseudomonas aeruginosa in
swimming pools: A review. J. Water Health 2012, 10, 181–196. [CrossRef] [PubMed]
13. Dallolio, L.; Belletti, M.; Agostini, A.; Teggi, M.; Bertelli, M.; Bergamini, C.; Chetti, L.; Leoni, E. Hygienic
Surveillance in Swimming pools: Assessment of the Water Quality in Bologna Facilities in the Period
2010–2012. Microchem. J. 2013, 110, 624–628. [CrossRef]
14. Zhang, L.Y.; Chen, R.Y.; Li, Y. Investigation of various pathogenic bacteria in indoor swimming pool water.
Chin. J. Health Lab. Technol. 2008, 18, 335–337.
15. Zhang, H.X.; Li, D.; Guo, Q. Monitoring results of water quality of swimming pools in Dalian city during
2009–2010. Occup. Health 2011, 27, 2–3.
16. Zhou, W.M.; Han, R.P.; Qiu, Y.R. Detection result of water quality of swimming pools in Kunming City from
2009–2011. Occup. Health 2013, 29, 990–991.
17. Chen, X.; Atwill, E.R.; Zhong, F.; Wei, Y.; Hou, S.; Li, J.; Xu, C.; Xiao, C.; Yang, Z.; Li, X. Prevalence
and risk factors of Cryptosporidium infection in children with clinical diarrhea in Guangzhou, China.
J. Bacteriol. Parasitol. 2017, 8, 1–8. [CrossRef]
18. Rhodes, E.R.; Villegas, L.F.; Shaw, N.J.; Miller, C.; Villegas, E.N. A modified EPA Method 1623 that uses
tangential flow hollow-fiber ultrafiltration and heat dissociation steps to detect waterborne Cryptosporidium
and Giardia spp. J. Vis. Exp. 2012, 3791–4177. [CrossRef] [PubMed]
19. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; 23th Informational Supplement (Document
M100-S23); CLSI: Wayne, PA, USA, 2013.
20. Causer, L.M.; Handzel, T.; Welch, P.; Carr, M.; Culp, D.; Lucht, R.; Mudahar, K.; Robinson, D.; Neavear, E.;
Fenton, S.; et al. An outbreak of Cryptosporidium hominis infection at an Illinois recreational waterpark.
Epidemiol. Infect. 2006, 134, 147–156. [CrossRef] [PubMed]
21. MacKenzie, W.R.; Kazmierczak, J.J.; Davis, J.P. An outbreak of cryptosporidiosis associated with a resort
swimming pool. Epidemiol. Infect. 1995, 115, 545–553. [CrossRef] [PubMed]
22. Takagi, M.; Toriumi, H.; Endo, T.; Yamamoto, N.; Kuroki, T. An outbreak of cryptosporidiosis associated
with swimming pools. Kansenshogaku zasshi J. Jpn. Assoc. Infect. Dis. 2008, 82, 14–19. [CrossRef]
23. Gao, J.Z. Clinical Laboratory Parasitology; People’s Health Publishing House: Beijing, China, 2009.
24. White, G.C. Handbook of Chlorination and Alternative Disinfectants, 4th ed.; John Wiley & Sons Inc.: New York,
NY, USA, 1999.
25. Baldursson, S.; Karanis, P. Waterborne transmission of protozoan parasites: Review of worldwide
outbreaks—An update 2004–2010. Water Res. 2011, 45, 6603–6614. [CrossRef] [PubMed]
70
IJERPH 2018, 15, 1416
26. Hijnen, W.A.; Beerendonk, E.F.; Medema, G.J. Inactivation credit of UV radiation for viruses, bacteria and
protozoan (oo)cysts in water: A review. Water Res. 2006, 40, 3–22. [CrossRef] [PubMed]
27. Korich, D.G.; Mead, J.R.; Madore, M.S.; Sinclair, N.A.; Sterling, C.R. Effects of ozone, chlorine dioxide,
chlorine, and monochloramine on Cryptosporidium parvum oocyst viability. Appl. Environ. Microbiol.
1990, 56, 1423–1428. [PubMed]
28. Hall, V.; Taye, A.; Walsh, B.; Maguire, H.; Dave, J.; Wright, A.; Anderson, C.; Crook, P. A large outbreak of
gastrointestinal illness at an open-water swimming event in the River Thames, London. Epidemiol. Infect.
2017, 145, 1246–1255. [CrossRef] [PubMed]
29. Levy, D.A.; Bens, M.S.; Craun, G.F.; Calderon, R.L.; Herwaldt, B.L. Surveillance for waterborne-disease
outbreaks—United States, 1995–1996. MMWR CDC Surveill. Summ. Morb. Mortal. Wkly. Rep. CDC
Surveill. Summ. 1998, 47, 1–34.
30. McFeters, G.A.; Kippin, J.S.; LeChevallier, M.W. Injured coliforms in drinking water. Appl. Environ. Microbiol.
1986, 51, 1–5. [PubMed]
31. Craun, G.F.; Calderon, R.L.; Craun, M.F. Outbreaks associated with recreational water in the United States.
Int. J. Environ. Health Res. 2005, 15, 243–262. [CrossRef] [PubMed]
32. Mena, K.D.; Gerba, C.P. Risk assessment of Pseudomonas aeruginosa in water. Rev. Environ. Contam. Toxicol.
2009, 201, 71–115. [PubMed]
33. Hajjartabar, M. Poor-quality water in swimming pools associated with a substantial risk of otitis externa due
to Pseudomonas Aeruginosa. Water Sci. Technol. J. Int. Assoc. Water Pollut. Res. 2004, 50, 63–67. [CrossRef]
34. Hutcheson, C.; Cira, R.; Gaines, S.L.; Jones, K.R.; Howard, W.; Hornsby, D.; Redmond, M.; Rustin, C.;
Hlavsa, M.C.; Murphy, J.L.; et al. Microbes in Pool Filter Backwash as Evidence of the Need for Improved
Swimmer Hygiene—Metro-Atlanta, Georgia, 2012. MMWR-Morbid. Mortal. Wkly. Rep. 2013, 62, 385–388.
35. Reali, D.; Rosati, S. Antibiotic susceptibility and serotyping of Pseudomonas aeruginosa strains isolated
from surface waters, thermomineral waters and clinical specimens. Zent. Hyg. Umweltmed. 1994, 196, 75–80.
36. WHO. Microbial hazards. In Guidelines for Safe Recreational Water Environments. Swimming Pools and Similar
Environments; WHO Press: Geneva, Switzerland, 2006.
37. Nichols, G. Infection risks from water in natural and man-made environments. Euro Surveill. Bull. Eur. Mal.
Transm. Eur. Commun. Dis. Bull. 2006, 11, 76–78. [CrossRef]
38. Papadopoulou, C.; Economou, V.; Sakkas, H.; Gousia, P.; Giannakopoulos, X.; Dontorou, C.; Filioussis, G.;
Gessouli, H.; Karanis, P.; Leveidiotou, S. Microbiological quality of indoor and outdoor swimming pools in
Greece: Investigation of the antibiotic resistance of the bacterial isolates. Int. J. Hyg. Environ. Health 2008, 211,
71
and Public Health
Article
Legionella spp. Risk Assessment in Recreational and
Garden Areas of Hotels
Antonios Papadakis 1,2,† ID , Dimosthenis Chochlakis 1,3,† , Vassilios Sandalakis 1 ,
Maria Keramarou 1 , Yannis Tselentis 1 and Anna Psaroulaki 1,3, *
1 Department of Clinical Microbiology and Microbial Pathogenesis, School of Medicine, University of Crete,
Voutes—Staurakia, 71110 Heraklion, Crete, Greece; [email protected] (A.P.);
[email protected] (D.C.); [email protected] (V.S.); [email protected] (M.K.);
[email protected] (Y.T.)
2 Public Health Authority of Heraklion, 71201 Heraklion, Crete, Greece
3 Regional Laboratory of Public Health, School of Medicine, 71110 Heraklion, Crete, Greece
† These authors contributed equally to this work.
Received: 7 February 2018; Accepted: 22 March 2018; Published: 26 March 2018
Abstract: Several Travel-associated Legionnaires’ disease (TALD) cases occur annually in Europe.
Except from the most obvious sites (cooling towers and hot water systems), infections can also be
associated with recreational, water feature, and garden areas of hotels. This argument is of great
interest to better comprehend the colonization and to calculate the risk to human health of these
sites. From July 2000–November 2017, the public health authorities of the Island of Crete (Greece)
inspected 119 hotels associated with TALD, as reported through the European Legionnaires’ Disease
Surveillance Network. Five hundred and eighteen samples were collected from decorative fountain
ponds, showers near pools and spas, swimming pools, spa pools, garden sprinklers, drip irrigation
systems (reclaimed water) and soil. Of those, 67 (12.93%), originating from 43 (35.83%) hotels, tested
positive for Legionella (Legionella pneumophila serogroups 1, 2, 3, 6, 7, 8, 13, 14, 15 and non-pneumophila
species (L. anisa, L. erythra, L. taurinensis, L. birminghamensis, L. rubrilucens). A Relative Risk (R.R.)
> 1 (p < 0.0001) was calculated for chlorine concentrations of less than 0.2 mg/L (R.R.: 54.78),
star classification (<4) (R.R.: 4.75) and absence of Water Safety Plan implementation (R.R.: 3.96). High
risk (≥104 CFU/L) was estimated for pool showers (16.42%), garden sprinklers (7.46%) and pool
water (5.97%).
Keywords: Legionella; recreational water systems; risk; water safety plan; hotel
1. Introduction
Legionella bacteria live naturally in fresh water, as well as in artificial water systems such as hot
water tanks, hot tubs or spas, cooling towers, plumbing systems, and decorative pools or fountains [1,2].
Hotel gardens are, also, frequently irrigated with sprinklers and these may present an additional risk,
particularly if they utilize recycled grey-water or sewage-based water [3]. Legionella species are able to
reproduce at 25–43 ◦ C and able to survive at temperatures of up to 55–60 ◦ C, making it possible for
them to thrive even in hot water systems [4].
Two forms of legionellosis are caused by the Legionella pathogens: Legionnaires’ disease
(LD), presenting with pneumonia-like symptoms, and Pontiac fever, presenting with influenza-like
symptoms [5]. Legionnaires’ disease, a serious form of pneumonia, may be caused by any type of
Legionella bacteria, although L. pneumophila serogroup 1 is considered the most virulent of all species
and serogroups, causing approximately 75% of all Legionella infections [2,6,7]. Until now, more than
52 different species of Legionella with at least 73 different serogroups have been described, of which

IJERPH 2018, 15, 598
approximately 20 species have been associated with human disease [8–11]. The case-fatality ratio
of LD has been recorded in the order of 10–15%. Usually the incubation period ranges from two to
10 days, but in rare cases it may be longer, for up to 16–20 days after exposure [12,13]. Since LD is
normally acquired through the respiratory system by inhaling air that contains Legionella bacteria in an
aerosol, droplets with a diameter of less than 5 μm (~90% showers aerosols) may come into contact
with the lower human airways [8,14–16].
Travel-associated Legionnaires’ disease (TALD) corresponds to the cases of travelers who get
infected in the country they visit, but usually get diagnosed and/or report their infection back in the
country of their residence. These human cases do not include domestic ones, that is, cases in which
humans travel within their own country. Due to the long incubation period (2–10 days), travelers may
be exposed to Legionella bacteria in one country but develop symptoms and seek medical attention in
another (such as their home) country [17]. According to the 2015 report by the European Centre for
Disease Prevention and Control (ECDC), a total of 1141 human cases related to TALD were reported
during that year at the 28 EU Member States and Norway, with the number being 20% higher than that
in 2014; in 2014, a total of 953 human cases were reported (21% higher than the corresponding ones of
2013) [3]. The average risk to TALD ranged from 0.02 cases/million nights in the United Kingdom to
0.88 cases/million nights in Greece, according to a study carried out in 2009. In Greece, the pooled risk
of 1.68 cases/million nights when travelling to the country was the highest among the 10 European
countries [18,19].
Since 2004, the World Health Organization (WHO) has developed a Water Safety Plan (WSP)
approach according to its Guidelines for Drinking Water Quality, which is based on risk assessment
and risk management principles [20]. Together with the standard EN 15975-2 (concerning security
of drinking water supply), these guidelines are an internationally recognized principle on which the
production, distribution, monitoring and analysis of parameters in drinking water is based upon.
In Europe, the Commission Directive Council 98/83/EC (EU) and 2015/1787 align with the principles
and needs of the quality of water intended for human consumption. The WSP approach includes, also,
supporting programs such as verification monitoring, appropriate documentation and record-keeping,
training and communication [21,22].
The aims of the present study were: (1) culture and identify L. pneumophila and Legionella species
in environmental samples obtained from recreational areas and determine the frequency and
severity of colonization of Legionella in these sites; (2) estimate the risk factors associated with
Legionella colonization of recreational systems and (3) evaluate the implementation of WSPs to limit
Legionella colonization.
2.1. Inspections—Sample Collection

During the period of 2000–2017, the Local Public Health Authorities of the Island of Crete in
Greece inspected 119 hotels associated with TALD. Environmental samples from decorative fountain
ponds, showers near pools and spas, swimming pools, spa pools, garden sprinklers, drip irrigation
systems (reclaimed water) and soil were collected from each hotel, where applicable.
Specifically, as regards the reclaimed water from the drip irrigation systems, in all sample cases
it consisted of a product of secondary biological treatment followed by advanced treatment and
disinfection. According to the National legislation “Common Ministerial Decision (CMD) No 145116:
Measures, limits and procedures for reuse of treated wastewater (Issuing Institutions: Ministry of
Environment, Energy and Climate Change)”, this kind of water can be used unrestrictedly in irrigation
of gardens and in recreational areas of hotels provided that it fulfills the following criteria: (1) Total
coliforms (TC) (cfu/100 mL) ≤2 in 80% of samples and ≤20 in 95% of samples; (2) Biochemical oxygen
demand (BOD5 ) (mg/L) ≤10 in 80% of samples; (3) Total suspended solids (TSS) (mg/L) ≤2 in 80% of
73
IJERPH 2018, 15, 598
samples; (4) Turbidity (NTU) ≤2 median. The above criteria are met following secondary biological
treatment followed by advanced treatment and disinfection.
The sample sites were chosen based on the sites that could produce aerosols and on the sites
(rooms, areas and so on) potentially associated with a TALD case. The samples were collected according
to: (a) the guidelines for drinking-water quality (second edition) and (b) ISO 5667-2:1982—Part 2:
guidance on sampling techniques; since 2006, samples were collected following the ISO 19458:2006
Water quality—Sampling for microbiological analysis methodology. The samples were labeled and
temporarily stored in a cool box at a temperature of up to 5 (±3) ◦ C protected from direct light, before
being delivered to the laboratory immediately after the sampling (no more than 24 h).
2.2. Data Collection

Data on water temperature, pH, chlorine concentration, disinfection methodology, hotel star
rating, number of rooms/beds, implementation of a WSP, season and water supply were recorded.
Inspections were conducted following a checklist developed with some information such as name,
address of building, type of hot water production system, water disinfection system, periodicity and
type of water system maintenance and cleaning, water supplying and number of rooms and beds.
The temperatures were measured (two minutes after flushing) using a calibrated thermometer, placed
in the middle of the water stream. Free chlorine and pH were measured using a calibrated portable,
microprocessor-based meter. Samples were collected in 1 L sterile containers containing sufficient
sodium thiosulphate (20 mg) to neutralize any chlorine or other oxidizing biocides.
2.3. Plate Culture Method

The isolation of Legionella from water samples was performed by culture according to the
International Standard method ISO 11731 (1998) and ISO 11731-2 (2004). The latter ISO was implemented
from 2004 onwards. Briefly, water samples were concentrated by filtration and were re-suspended
in Distilled Deionized water. A volume of the suspension (200 μL) was spread on BCYE (Buffered
Charcoal Yeast Extract), BCY (Buffered Charcoal Yeast Extract without L-cysteine) and GVPC (Glycine
Vancomycin Polymyxin Cycloheximide) (Biomérieux, Craponne, France) Petri dishes: (a) directly after
filtration; (b) after incubation at 50 ◦ C for 30 min and (c) after the addition of an acid buffer (0.2 mol/L
solution of HCL, pH 2.2). The detection limit of the procedure was 50 CFU/L. The inoculated plates
were incubated for 10 days at 36 ± 1 ◦ C in 2.5% CO2 with increased humidity. Suspected colonies
were randomly chosen for subculture on BCY, BCYE and GVPC agar.
2.4. Typing of Legionella Isolates

Up until 2010, the isolated colonies were identified using an agglutination test (SLIDEX
Legionella-Kit, Biomérieux, Craponne, France), which allows for the discrimination of L. pneumophila
serogroup 1 from serogroups 2–14 and of L. anisa, while for the exact detection of each L. pneumophila
serogroup, individual latex polyclonal reagents were used (Pro-lab, Richmond Hill, ON, Canada).
2.5. Identification—MALDI-TOF Mass Spectrometry

From 2010 onwards, a MALDI Biotyper (Microflex LT MALDI-TOF mass spectrometer) (Bruker
Daltonics, Leipzig, Germany) equipped with a microSCOUT ion source was used for the identification
of individual Legionella colonies against its microbial database (v 3.1.2.0). Spectra were recorded
using the flexControl software with the default parameters for optimization set by the manufacturer
(Bruker Daltonics, Leipzig, Germany). For each spectrum, 240 laser shots were collected and analyzed
(6 × 40 laser shots from 120 different positions of the target spot). All identifications were evaluated
according to the manufacturer scoring scheme.
74
IJERPH 2018, 15, 598
2.6. Risk Assessment of Legionella Presence in Water Distribution Systems

To better calculate the risk, we used two different approaches. The first one was in accordance
with the recommendations of the European Legionnaires’ disease Surveillance Network (ELDSNet),
at which the microbiological results of the water samples were analytically and statistically analyzed
according to the number of Legionella bacteria in the water sample, which could represent a particular
risk to the human health. In particular, an insignificant risk was noted at ≤103 CFU/L, medium
risk at >103 CFU/L but <104 CFU/L, and high risk at ≥104 CFU/L. Especially for spa pools,
the recommendations point to the risk of legionellosis following Legionella sampling to low risk
(>102 but <103 ) and to high risk (≥103 CFU/L) [23]. A second inspection was performed at a hotel
when the Legionella count was ≥104 CFU/L in at least one sample or between >103 and ≤104 CFU/L
in more than two samples or between >103 and ≤104 CFU/L in at least one sample in concordance
with an aerobic count higher than 105 CFU/L.
The second approach for the assessment of the risk was based on a semi-quantitative risk matrix
approach, which was used to implement the water supply system risk assessment (Table 1). The risk
levels were divided into: high (≥20), medium (10–19), and low (<10). The risk (R) was evaluated
based on the following parameters: Likelihood (L) or the occurrence of accidents/damage, frequency
of the risk exposure, consequence or severity (S). The level of the risk was calculated as follows:
R = L × S [24,25].
Table 1. Presentation of the semi-quantitative risk matrix approach that was used to implement the
water supply system risk assessment. The risk levels were divided into: high (≥20), medium (10–19),
and low (<10). The risk (R) was evaluated based on the following parameters: Likelihood (L) or the
occurrence of accidents/damage, frequency of the risk exposure, consequence or severity (S). The level
of the risk was calculated as follows: R = L × S.
Severity or Consequence
Moderate
Minor (Short term
(Widespread
or localised, not Major (Potential Catastrophic
Insignificant aesthetic issues or
health related long-term health (Potential
(Wholesome water) long-term
non-compliance or effects) illness)
Rating: 1 non-compliance,
aesthetic) Rating: 8 Rating: 16
not heath related)
Rating: 2
Rating: 4
Most unlikely (Has not
taken place in the past and
it is highly improbable that 1 2 4 8 16
it will occur in the future)
Rating: 1
Unlikely (Is possible and
cannot be ruled out
2 4 8 16 32
completely)
Rating: 2
Likelihood or Foreseeable (Is possible and
Frequency under certain
3 6 12 24 48
circumstances could occur)
Rating: 3
Very likely (Has occurred in
the past and has the
4 8 16 32 64
potential to occur again)
Rating: 4
Almost certain (Has
occurred in the past and
5 10 20 40 80
could occur again)
Rating: 5
2.7. Instructions Given in Case of Positive Samples

According to the national guidelines, in samples which met the criteria of >103 to <104 , either:
(i) if a small proportion of samples (10–20%) were positive, the system was re-sampled. If a similar
count was recorded again, then a review of the control measures and risk assessment was carried out to
identify any remedial actions; (ii) if the majority of samples were positive, the system was considered
as colonized, even at a low level, with Legionella. Disinfection of the system was considered but an
75
IJERPH 2018, 15, 598
immediate review of control measures and a risk assessment was carried out to identify any other
remedial actions required. In samples which met the criteria of ≥104 : The system was re-sampled
and an immediate review of the control measures and risk assessment was carried out to identify any
remedial actions, including whether a disinfection of the whole system or affected area was necessary.
2.8. Implementation of a Water Safety Plan

As from 2005, a detailed standardized questionnaire (checklist, Appendix A) was used to evaluate
the risk associated with the non-implementation of a WSP. The checklist consisted of 42 scoring items
(11 of which were designated as “critical”), which were classified into seven categories: construction
and maintenance; cleaning and disinfection; cold-water distribution system; hot-water distribution
system; system protection cross-connections and backflow; record keeping; and on-site manually
conducted tests. The total negative score was calculated and classified qualitatively in the following
three categories: satisfactory result (0–7 points, <10% of the total negative score, no critical violation),
relatively satisfactory result (8–14 points, 11–20% of the total negative score, or a critical violation), and
unsatisfactory result (more than 14 points, >20% of the total negative score).

All statistical analyses were conducted using the IBM SPSS Statistics Version 24 statistical package,
the Epi-Info 2000 version 7.2.0.1 (Centers for Disease Control and Prevention, Atlanta, GA, USA) and
the MedCalc relative risk calculator statistical software free online version; Relative risk (R.R.) at a
95% confidence interval (CI). The analyses were calculated to assess categorical risk variables from
water distribution systems and hotel characteristics, associated with Legionellae-positive test results.
The results were considered statistically significant when the p value was <0.05 and highly significant
when the p value was <0.0001.
3. Results
3.1. Descriptive Data

Of the 518 samples collected, 67 (12.93%) originated from 43 (35.83%) hotels that tested positive
for Legionella species. The mean positivity was 9.41% (standard deviation of 11.91, max of 33.33%
min 0.00%) (Table 2). A second inspection was required for 49 hotels, a third for 16 hotels, a fourth
for five (5) hotels, while a fifth inspection was required for two (2) hotels. The repetitive inspections
were carried out in cases where the implementation of measures did not deliver the expected results
in terms of the presence of Legionella species at the sites tested and/or the risk factors (as these are
explained below) were repeatedly tested out of the limits.
In 14/119 (28.57%) of the hotels inspected, a cluster was noted (clusters were defined as the
presence of two or more cases having stayed overnight at the same accommodation site in the 14 days
before onset of illness and whose illness was within say the same two-year period) [23].
3.2. Isolation and Identification of L. pneumophila and Legionella Species in Environmental Samples Obtained
from Recreational Systems
Legionella was isolated from swimming pool showers, garden sprinklers, reclaimed water, swimming
pool water, decorative fountain, spa water and spa showers. On the contrary, no Legionella species were
isolated from shower heads, from Jacuzzis and from garden soil. All results are summarized in Table 2.
Legionella pneumophila serogroups 1, 2, 3, 6, 7, 8, 13, 14, 15 and 2–15 were detected. Of the
non-pneumophila species, L. anisa, L. erythra, L. taurinensis, L. birminghamensis, L. rubrilucens and
Legionella species were identified, the majority of which, belonged to the human pathogenic species
L. anisa. The CFU/L ranged from 50–350,000. The lowest CFU/L were detected in spa waters, while
the highest ones were identified in swimming pool showers and in garden sprinklers. All results are
summarized in Table 3.
76
Table 2. Collection sites and sites where Legionella species were detected. The percentages of the positive samples have been calculated based on the total number of
collected samples. The ranges have been assigned based on the European Centre for Disease Prevention and Control recommendations. The numbers at the left side
of the slash (/) correspond to the sample findings and the numbers at the right side of the slash (/) correspond to the hotel findings.
Samples/Hotels Range (CFU/L)

Original Sample Description
IJERPH 2018, 15, 598
Total No Positive ≤103 >103 and <104 ≥104

Reclaimed Water 13/9 3 (23.08%)/2 (2.22%) 2 (66.67%)/2 (22,22%) 1 (33.33%)/1 (11,11%) -
Decorative Fountains 45/24 3 (6.67%)/3 (12.50%) 1 (33.33%)/1 (4.17%) 2 (66.67%)/2 (8.33%) -
Shower Heads 2/1 0 - - -
Garden Sprinklers 37/21 15 (40.54%)/11 (52.38%) 6 (40%)/6 (28.57%) 4 (26.67%)/4 (19.05%) 5 (33.33%)/5 (23.81%)
Jacuzzi Water 15/10 0 - - -
Pool Water 107/61 5 (4.67%)/4 (6.56%) 1 (20%)/1 (1.64%) - 4 (80%)/3 (4.92%)
Spa Water 10/7 1 (10%)/1 (14.29%) 1/1 (100%) - -
Swimming pool Showers 271/104 38 (14.02%)/30 (28.85%) 17 (44.74%)/15 (50%) 10 (26.32%)/9 (30%) 11(28.95%)/10 (33.33%)
Spa Showers 16/7 2 (12.50%)/2 (28.57%) 2 (100%)/2 (28.57%) - -
Garden Soil 2/1 0 - - -
Total 518/119 67 (12.98%)/43 (36.13%) 30 (44.78%)/25 (21.01%) 17 (25.37%)/4 (11.76%) 20 (29.85%)/14 (11.76%)
77
Table 3. Legionella serogroups and species isolated and identified from recreational waters (Ranges as CFU/L).
Swimming Pool Water Spa Swimming Pool Shower Spa Shower Fresh Water from Garden Sprinklers Decoration Fountain Reclaimed Water
Legionella Serogroups/Species
Pos. Range Pos. Range Pos. Range Pos. Range Pos. Range Pos. Range Pos. Range
L.p. sg 1 1 700 5 350–1150 1 26,000
L.p. sg 2 4 100–2050
L.p. sg 3 3 50-650
L.p. sg 6 1 150 2 150–600
L.p. sg 7 5 200–3350
L.p. sg 8 2 50 1 300
L.p. sg 13 1 32,500
L.p. sg 14 1 50 3 150–100,000 1/1 150 3 250–13,000
L.p. sg 15
L.p. sg 2–15 8 50–100,000 4 13,500–200,000 1 1000
L. anisa 2 19,500–26,500 9 250–350,000 3 50–13,000 1 2500
L. erythra 1 650 3 400–13,000 1 200 1 1500
L. taurinensis 2 50 1 6500 1 2000
L. birminghamensis 1 650–8250 1 65,000
L. rubrilucens 1 26,000 3 50–6500
L. species 1 200,000 4 50–1000 1 1000 3 1000
Pos.: positive. L.p.: Legionella pneumophila. sg: serogroup.
IJERPH 2018, 15, 598
3.3. Univariate Examination of Risk Factors

The presence of Legionella was possibly associated (statistically significant correlation together
with a relative risk of >1) with: (a) the implementation of a WSP; (b) free chlorine <0.2 mg/L;
(c) hotel star classification of <4; (d) seasonal operation; (e) municipality hosting hotels population
of <104 people and (f) absence of an automated chlorination system together with free chlorine out
of range. The number of beds of >200, the number of rooms of >80 and the use of groundwater as a
source of water supply showed a statistically significant correlation in the absence of a relative risk >1.
On the other hand, no statistically significant correlation was calculated for cold water temperature of
>25 ◦ C, temperature of >20 ◦ C, the absence of an automated chlorination system, pH out of limits value
(pH within limits 7.0–7.8), the opening/closing period and the high season period (June–August).
All results are summarized in Table 4.
Table 4. Association of water distribution systems and hotel characteristics with Legionella colonization.
The terminology number needed to treat (NNT) has been used as proposed at the free online relative
risk calculator statistical software, MedCalc (Altman 1998). In our case, the term benefit is associated
with the number of additional inputs required for each parameter tested to get a positive association
with this parameter.
Risk Factors R.R. 95% CI z Statistic Statistic p Value NNT

Free chlorine <0.2 mg/L 54.78 20.47–148.04 7.94 <0.0001 (Harm) 1.85
No Water Safety Plan 3.96 2.32–6.75 5.04 <0.0001 (Harm) 6.06
Wrong implementation of WSP 3.78 1.42–10.08 2.66 0.0077 (Harm) 7.33
Number of rooms >80 0.29 0.17–0.47 4.83 <0.0001 (Benefit) 6.25
Number of beds >200 0.22 0.12–0.40 5.08 <0.0001 (Benefit) 5.89
Star classification <4 4.75 2.80–8.06 5.78 <0.0001 (Harm) 4.95
Groundwater as a source of water supply 0.27 0.13–0.58 3.34 0.0008 (Benefit) 5.70
No automated chlorination system and free chlorine <0.2 mg/L 5.16 2.58–10.31 4.65 <0.0001 (Harm) 3.60
pH out of limits and free chlorine <0.2 mg/L 4.52 1.71–11.96 3.04 0.0024 (Harm) 3.08
Population < 10,000 1.61 1.03–2.58 2.11 0.02 (Harm) 15.78
No automated chlorination system 1.93 0.71–5.25 1.29 0.04 (Harm) 12.57
Closing period 1.56 0.87–2.78 1.51 0.07 (Harm) 14.59
Seasonal operation 1.47 0.66–3.28 0.94 0.17 (Harm) 23.93
Cold water >25 ◦ C 1.38 0.82–2.64 1.29 0.14 (Harm) 18.17
Cold water >20 ◦ C 1.21 0.70–2.08 0.70 0.35 (Harm) 39.29
pH out of limits 0.68 0.2–1.98 0.69 0.25 (Benefit) 18.59
High season months 1.10 0.70–1.730 0.44 0.65 (Harm) 76.27
Opening period 1.03 0.35–3.05 0.06 0.45 (Harm) 214.76
Based on the risk assessment according to the guidelines of ECDC (low, medium and high for
CFU/L < 103 , 103 –103 and >104 respectively) a corresponding Figure 1 was built to demonstrate
the risk for each site tested. According to the semi-quantitative risk assessment a number of areas
were designated as low, medium and high according to the likelihood of any species been present,
the severity caused in the presence of any Legionella and the total risk (as a factor taking these
two parameters into consideration). Of the total sites tested, the garden sprinklers and the swimming
pool showers presented a higher risk when all three hazardous events were considered. As regards
the pool water, a high risk was calculated for the event of finding Legionella in the water system, only.
All results are summarized in Table 5.
Table 5. Risk assessment of the sites based on the likelihood for the presence of Legionella (*), the severity
raised if Legionella is present (ˆ) and the final risk score calculated (#). The final risk score was calculated
based on the findings of the previous two parameters.
Likelihood or Severity or
Area Hazard and Hazardous Event Risk Score # Risk Rating #
Frequency * Consequence ˆ
Event of finding Legionella in the water system 3 4 12 Medium
Reclaimed Water Inadequate disinfection method 3 4 12 Medium
Low chlorine residual in distribution systems 3 4 12 Medium
Event of finding Legionella in the water system 3 4 12 Medium
Decorative
Inadequate disinfection method 3 4 12 Medium
Fountains
Shower Heads Event of finding Legionella in the water system 2 4 8 Low
Event of finding Legionella in the water system 5 8 40 High
Garden
Inadequate disinfection method 5 8 40 High
Sprinklers
Low chlorine residual in distribution systems 5 8 40 High
78
IJERPH 2018, 15, 598
Table 5. Cont.
Likelihood or Severity or
Area Hazard and Hazardous Event Risk Score # Risk Rating #
Frequency * Consequence ˆ
Event of finding Legionella in the water system 2 4 8 Low
Jacuzzis Water Inadequate disinfection method 2 4 8 Low
Low chlorine residual in distribution systems 2 4 8 Low
Pool Water Inadequate disinfection method 2 8 16 Medium
Spa Water Inadequate disinfection method 2 4 8 Low
Swimming pool
Inadequate disinfection method 4 8 32 High
Showers
Low chlorine residual in distribution systems 4 8 32 High
Spa Showers Inadequate disinfection method 1 4 4 Low
Garden Soil Event of finding Legionella in the water system 1 4 4 Low
Figure 1. Risk assessment for Legionella contamination based on the CFU/L detected in each site tested.
3.4. Evaluation of the Implementation of WSPs

Data from the use of the checklist were collected from 51 hotels. The major recordings had to do
with water storage tank protection, clean showers, presence of the proper concentration of residual
chlorine, and water temperatures. All findings are summarized into Table 6.
Of the 51 hotels for which the questionnaire for the correct implementation of a WSP was filled in,
24 (36.4%) received a score B (relatively satisfactory) while 37 (63.7%) got a score C (unsatisfactory).
A higher colonization was recorded in the absence of a WSP (R.R. 3.04; p value <0.0001; CL 95%
1.73 to 5.34; z-statistic 3.87; NNT (Harm) 8.06) and in hotels where wrong implementation of WSP was
recorded (R.R. 3.78; p value 0.0077; CL 95% 1.42 to 10.08; z-statistic 2.66; NNT (Harm) 7.33).
79
IJERPH 2018, 15, 598
Table 6. Findings from the completion of the checklists at the 51 hotels. Only the items, for which a
deviation from the normal value was recorded and are presented herein.
Scoring Items %
No water storage tanks protection 100
* The showers are NOT clean and free of salts 81.2
* The residual chlorine <0.2 mg/L 72.8
The hot water temperature is <50 ◦ C. after 1 min of flowing 63.6
The cold-water temperature in taps is >25 ◦ C. after 2 min of flushing 54.5
Water store and circulation temp <60 ◦ C 54.5
The water exiting the heating unit <60 ◦ C and returning <50 ◦ C 54.5
The amount of stored water is >1 day 45.5
There is NO control book 45.5
* The outgoing temperature (cold) water from the tank is >25 ◦ C 36.3
There are leaks in the network 27.2
* NO random checks of water at least every 6 months 27.2
There is no thermal stratification of the water inside the heaters and storage water 27.2
The water distribution system cannot provide adequate water supply at peak times 18.2
There is a change (increase or decrease) in the consumption of the water 18.2
* Detected Legionella the last six months (at a concentration of more than 103 CFU/L) 18.2
The filters are NOT in good condition 9
* The network is NOT cleaned and disinfected when remained out of service >1 month 9
* The network and the tanks NOT cleaned with appropriate disinfectants at least annually 9
The water supply is interrupted for a long time 9
* The difference in temperature between 2 successive measurements of hot water is >10 ◦ C/1 min 9
There is a taste and odor problem 9
*: designates a critical item.
4. Discussion
Legionella is a naturally occurring microorganism found in freshwater environments and surface
waters where it exploits free-living amoeba cells for survival and multiplication, and, in that context, it
is not hard to be introduced in building water systems through the main community water supply
network. In fact, it has been stated that in the USA a percentage of 87.5% of Legionellosis outbreaks
has been related to community water systems [26]. Even though the colonization of a water system by
Legionella occurs frequently, this alone is not enough to pose a high risk to humans, unless the bacteria
population reaches high numbers and becomes dispersed through appropriate aerosolization. For such
conditions to take place, a series of variables must co-occur, such as the increased temperature of cold
water or decreased temperature of hot water (optimally 35–46 ◦ C), absence of water disinfectants
or under-treated water, out of range pH values (5.5–9.2), presence of substances like iron salts and
L -cysteine, as well as, co-existing and supporting microbial flora. Furthermore, other factors such
as the complexity of the architecture of the pipe system (high surface to volume ratio), presence of
blunt-end pipes, infrequently used pipe terminals, pipelines exposed to the sun heat, close proximity
of hot and cold-water pipelines, insufficient maintenance and end-points creating aerosols, have all
been evaluated to significantly affect the rate and extent of Legionella colonization in water systems [27].
All these factors may well be associated with the formation of biofilms, which can provide a means for
the survival and dissemination of the pathogen and undermine the efforts to eradicate bacteria from
water systems [28].
The presence of 1559 hotels and accommodation units in Crete with 85,407 rooms and 161,578 beds
(data drawn from the Hellenic National Service of Tourism for 2015), could not exclude the island from
the presence of TALDs [29]. The current research was focused mostly on the significance of sampling
from recreation areas and spas and in particular from pools, spas, jacuzzis, showers next to swimming
pools, shower heads and spa showers. The calculated relative risk of Legionella colonization was
less in swimming pools and in spas, due to the increased surveillance and implementation of proper
chlorination procedures, as opposed to the colonization in showers of swimming pools. The analyzed
data showed that the high chlorination, the mixed type of contamination and the lower temperatures in
80
IJERPH 2018, 15, 598
the pool are potential factors that may contribute to the prevention of any Legionella from reproducing
and reaching levels that would represent a health risk for bathers. The pool samples from which
Legionella were isolated, had significantly lower concentrations of residual free chlorine, in fact less
than the 0.4 mg/L required by the current regulations in Greece (0.4–0.7 mg/L). On the other hand,
no significant correlation between Legionella colonization and pH values was calculated in the current
study, agreeing with the study by Fragou et al., where the colonization of Legionella in 116 samples
collected from hotels and hospitals was evaluated against pH values [30]. In general, such conditions
support the growth of Legionella, while the frequent vigorous agitation that can occur in spas, pools and
whirlpool baths can form aerosols that could create airborne bacteria and transfer them to the lungs
of unsuspected individuals [31,32], even if the actual establishments are not actively used by these
humans [33]. This is, also, supported by the detection of Legionella even in more isolated water facilities
such as in spa and pools located on cruise ships that are actually one of the most common sources of
Legionella infection [32]. It seems that such aerosols-creating settings, along with the close proximity
of susceptible individuals, create a potential Legionella infectious environment. Such conditions are
recorded in showers, especially the ones that have their pipes or at least part of them exposed to
the sun heat. Similar results have been described from researches in Italy [4] a country that, like
Greece, is a major tourist destination and retains a Mediterranean climate. In fact it has been shown
that the risk for travelers is high in southeastern countries (including Greece) compared to countries
with moderate climates [19]. Hotel fountains creating aerosols certainly mimic the conditions met in
showers and in most cases they use recirculating water that is under a minimal degree of disinfection
supervision, if any at all. Therefore, they have already been linked to Legionellosis outbreaks since the
bacterium finds it easy to thrive in such conditions and disperse through the formed aerosols to the
surrounding environment, or at a much larger distance if favoring conditions exist (for example the
presence of wind) [34].
In our study, positive samples were recorded in 43/119 (36.13%) hotels, a result comparable to a
similar study in Turkey, where of the 52 hotels included in the study, negative samples for Legionella
were retrieved in 16 (30.8%) hotels [35]. According to the results of the current study, the seasonal
operation of hotels and their presence in municipalities with low population seems to play a role in the
increased risk of Legionella colonization; this finding has already been supported in a previous study
carried out in hotels in Greece [36].
Another point of interest was the high number of Legionella species isolated, indicating a
widespread dispersion of these microorganisms in the environment. In addition to the isolation
of L. pneumophila (serogroups 1, 2, 3, 6, 7, 8, 13, 14, 15, and 2–15), other potentially pathogenic
environmental species were also isolated, such as L. anisa, L. erythra, L. taurinensis, L. birminghamensis,
and L. rubrilucens, the agent responsible for Pittsburg pneumonia [37] which is similar to Pontiac
fever [38]. The presence of non-pneumophila species should not be underestimated since human cases
of infection by Legionella species other than L. pneumophila or even the second most common, L. anisa,
have been described worldwide, including the Island of Crete [39].
Legionella pneumophila serogroup 1 was detected in swimming pools and swimming pool showers
however the low number of samples collected did not allow us to make any correlation among the
positive samples, the detected serogroups and the sampling sites. The highest CFU/L was detected
for the L. pneumophila serogroup 14 (105 CFU/L) and for non-pneumophila species (200,000 CFU/L).
Nevertheless, a larger number of positive samples is required before coming to concrete conclusions on
a possible relationship between site of collection and number of bacteria. The prevalence of Legionella
species at certain environments has, also, been investigated by Litwin and colleagues [26] in which,
they isolated L. pneumophila serogroup 1 and 6, L. anisa, L. feeleii, and L. quateriensis in the water
reservoirs of recreational vehicles. As far as seasonality is concerned, in a similar research it was
claimed that the concentrations of Legionella recovered from swimming pools, like L. micdadei and
L. bozemanii, did not show any seasonal trend [4].
81
IJERPH 2018, 15, 598
As regards the use of reclaimed water, ingestion of enteric pathogens causing gastrointestinal
disease poses the greatest risk from exposure to wastewater however, inhalation of aerosols or dermal
contact can, also, lead to disease. Except from Vibrio cholerae, the rest of the enteric pathogens that
may cause gastrointestinal illnesses do not grow or survive for ever in water. On the other hand,
free-living pathogens, such as Legionella, can grow under favorable conditions in treated wastewater
and associated biofilms and, under certain circumstances, can survive within amoeba in water
distribution systems [40]. That is why, samples from garden drip irrigation systems should be collected
and tested in cases where reclaimed water is used and above all, it must be made clear that this type of
water should not be used for irrigation through spraying [41].
Concerning WSPs, they already exist in various contexts worldwide. For instance, WSP networks
are located in Africa, the Asia-Pacific region and in Latin America. WSPs have been designed in
order to monitor water utility and to ensure that its quality standard is consistently kept within
accepted limits [42]. In another study carried out in Germany, a decreased number of transgressions
was observed following implementation of WSP in all parts of a hospital, supporting its efficacy [43].
In a different study carried out in Iceland, the impacts of WSPs on drinking water quality and health
were evaluated and positive conclusions were drawn since implementation of WSPs resulted in
better compliance in drinking water, reduced Heterotrophic Plate Count (HPC) numbers in water and
decreased incidence of diarrhea in communities served by utilities implementing WSP [44]. It should
be noticed however, that it turns out that it not just the presence of a WSP that will ensure the
diminishing of the danger for a human infection by water-borne pathogens (including Legionella);
its proper implementation is sometimes a more crucial factor [45]. In fact, it is possible that the
improper implementation or the complete absence of a WSP (leading sometimes to temperature and
pH values out of range) may provide the ideal stress conditions for the Legionella bacteria to develop a
decreased sensitivity to antibiotics [46]. Of course, this is a huge aspect that needs further investigation.
Summing up, there is a need for awareness strategies in concordance with the operators of
accommodation sites for the prevention of human Legionella infections. Stronger evidence on the
source of infection can only be supported through enhanced standardization of Legionella investigation,
reporting and follow-up, together with the use of high discrimination laboratory techniques.
These factors could eventually lead to a more effective measure control [17]. Moreover, maintenance,
regular controls, interventions on the hydraulic system, and good hygiene practices together with the
assessment of risk analysis and establishment of a routine environmental microbiological surveillance
schemes should be implemented to minimize exposure to Legionella infection [47,48].
5. Conclusions
The presence of Legionella is one of the most serious microbiological risks according to the water
safety in distribution hotels. Knowing that Legionella bacteria can successfully thrive in natural
and artificial water environments with warm waters, certainly directs towards the investigation
of establishments with such conditions and with high human use or of those which present more
complicated constructions such as pools and spas.
Preventing recreational water LD is a multifaceted issue that requires both the development of
a WSP and, above all, its correct implementation, together with the participation of a large group of
people like hotel staff and national and local public health authorities.
Acknowledgments: We would like to thank all the environmental health inspectors of the Local Public Health
Authorities of the Crete Island who collected the environmental samples during all these years.
Author Contributions: Antonios Papadakis did the collection of the majority of the samples, the analysis of the
results and contributed on the writing of the article. Dimosthenis Chochlakis did the analysis of the samples and
contributed on the writing of the article. Vassilios Sandalakis did the analysis of the samples and contributed
on the writing of the article. Maria Keramarou did the analysis of the samples and the analysis of the results.
Yiannis Tselentis was in charge of the proper building and process of the study. Anna Psaroulaki was in charge of
the proper processing of the study and contributed on the writing of the article.
Conflicts of Interest: The authors have declared no conflicts of interest.
82
IJERPH 2018, 15, 598
Appendix A
Table A1. Detailed standardized questionnaire (checklist) used to evaluate risk associated with proper
or not implementation of a WSP. * Critical Control Point. The checklist has been adapted from the
European Guidelines for Prevention and Control of Travel Associated Legionnaires’ disease and from
the National School of Public Health 2004 Athens Olympic Games Checklist for building water systems.
Legionella Prevention and Management Checklist

Item to Check Yes No Remarks
A water safety plan is implemented according to the requirements of the European Legislation and
the proposals of the World Health Organization
Is there at least one named person responsible for Legionella control?
Responsible Person: (entity name)
Is this person properly trained in the control of Legionella and able to understand the system(s), risk
factors and control measures?
Is there a written Legionella control plan in place for each system which could pose a risk?
If the hotel is operating on a seasonal level, is the entire domestic water system disinfected with high
level (50 mg/L) chlorine for 2–4 h before the property is reopened?
Has there been any new pipe work or changes to existing pipe work in the last two years?
If yes, has it been checked to ensure that there are no pipes with intermittent or no water flow?
Was the system disinfected with high level chlorine or heat (60 ◦ C) following completion of the work?
General Testing Sites of the Water Network
1 The pressure on the meter is 1–12 atmospheres −1
2 The filters are in good condition −2
3 The sealing is in good condition −2
4 Absence of leaks on the network −2
The storage tank is maintained in good condition and no sediments are
5* −3
observed inside it
6 The water storage tanks have lids and wire mesh in each open air duct −1
7 The amount of water stored is no greater than one day’s use −1
The network is cleaned and disinfected when it is not under use for more than
8* −3
a month
The network and the tanks are cleaned with appropriate disinfectants at least
9* −3
once a year
10 The water supply is not interrupted for long periods of time −1
11 Unused taps are removed from the network −2
12 Checking water network diagrams
Cold Water Systems
13 Chillers are maintained in good condition −1
14 Chiller filters are maintained in good condition −1
Hot Water Systems
15 The system responds well at peak hours −1
16 There is no change (increase or decrease) in water consumption −1
17 * No standing water in piping for more than one week −3
18 * If No: Is a flushing procedure used? −3
19 * Shower heads and taps are clean and free from scale −3
Water Heating and Storage Appliances
20 The appliance is dried and checked −1
21 The appliance is cleaned if necessary −2
22 The hot water extraction duct is dried −1
23 The appliance is maintained in accepted sanitary condition −2
Faucets
24 Operated and maintained in accordance with the manufacturing instructions −2
Fire Protection Water Supply
25 No backflow of water fire extinguishing system in the water supply network −2
ANNEX I: Database Book
Are there regular records (logbook for example) of the critical monitoring
26 −2
activities kept on site (temperatures, chlorine levels, etc.)?
27 * Random checks of water at least every 6 months −3
28 In the control book (if any), there are no abnormal results −2
No Legionella detected the last six months (at a concentration of more than
29 * −3
1000 CFU/L)
ANNEX II: Recordings
30 * Cold water is maintained at temperatures below 25 ◦ C −3
31 The cold-water temperature in taps is <25 ◦ C after 2 min flushing −2
32 The hot water temperature is >50 ◦ C after 1 min flow −2
The difference in temperature between 2 successive measurements of hot
33 * −3
water is NOT > 10 ◦ C/1 min
34 Hot water store and circulation temp <60 ◦ C −2
83
IJERPH 2018, 15, 598
Table A1. Cont.
Legionella Prevention and Management Checklist

There is NO thermal stratification of the water inside the heaters and storage
35 −1
water
The water temperature is >60 ◦ C when exiting the heating unit and >50 ◦ C
36 −2
when returning to it
37 Is the pH maintained at 7.2–7.8? −2
38 * There is continuous treatment with chlorine at 0.2–0.5 mg/L? −3
39 There is no taste and odor problem −1
Inspection results: (A) satisfactory result (0 to 7 points, <10% of the total negative score, no critical violation);
(B) relatively satisfactory result (–8 to 14 points, 11–20% of the total negative score, or a critical violation);
(C) unsatisfactory result (more than 14 points, >20% of the total negative score).
References
1. Legionella. Developing a Water Management Program. Legionnaires. CDC. Available online: https://www.
cdc.gov/legionella/maintenance/wmp-toolkit.html (accessed on 6 December 2017).
2. Legionella and the Prevention of Legionellosis. Available online: http://apps.who.int/bookorders/anglais/
detart1.jsp?sesslan=1&codlan=1&codcol=15&codcch=633 (accessed on 7 December 2017).
3. Legionnaires’ Disease—Annual Epidemiological Report for 2015. Available online: http://ecdc.europa.eu/en/
publications-data/legionnaires-disease-annual-epidemiological-report-2015 (accessed on 6 December 2017).
4. Leoni, E.; Legnani, P.P.; Bucci Sabattini, M.A.; Righi, F. Prevalence of Legionella spp. in swimming pool
environment. Water Res. 2001, 35, 3749–3753. [CrossRef]
5. Prussin, A.J.; Schwake, D.O.; Marr, L.C. Ten questions concerning the aerosolization and transmission of
Legionella in the built environment. Build. Environ. 2017, 123, 684–695. [CrossRef] [PubMed]
6. Sikora, A.; Wójtowicz-Bobin, M.; Kozioł-Montewka, M.; Magryś, A.; Gładysz, I. Prevalence of
Legionella pneumophila in water distribution systems in hospitals and public buildings of the Lublin region of
eastern Poland. Ann. Agric. Environ. Med. AAEM 2015, 22, 195–201. [CrossRef] [PubMed]
7. Joseph, C. Surveillance of Legionnaires’ Disease in Europe. In Legionella; Marre, R., Abu Kwaik, Y., Bartlett, C.,
Cianciotto, N., Fields, B., Frosch, M., Hacker, J., Lück, P., Eds.; ASM Press: Washington, DC, USA, 2002;
pp. 311–317. [CrossRef]
8. Fields, B.S.; Benson, R.F.; Besser, R.E. Legionella and Legionnaires’ disease: 25 years of investigation.
Clin. Microbiol. Rev. 2002, 15, 506–526. [CrossRef] [PubMed]
9. McDade, J.E. Legionella and the Prevention of Legionellosis. Emerg. Infect. Dis. 2008, 14, 1006. [CrossRef]
10. Diederen, B.M.W. Legionella spp. and Legionnaires’ disease. J. Infect. 2008, 56, 1–12. [CrossRef] [PubMed]
11. Marrie, T. Legionella: Molecular microbiology. Emerg. Infect. Dis. 2009, 15, 139. [CrossRef]
12. European Legionnaires’ Disease Surveillance Network (ELDSNet). Available online: http://ecdc.europa.
eu/en/about-us/partnerships-and-networks/disease-and-laboratory-networks/eldsnet (accessed on
6 December 2017).
13. Seenivasan, M.H.; Yu, V.L.; Muder, R.R. Legionnaires’ disease in long-term care facilities: Overview and
proposed solutions. J. Am. Geriatr. Soc. 2005, 53, 875–880. [CrossRef] [PubMed]
14. Darquenne, C.; Prisk, G.K. Aerosol deposition in the human respiratory tract breathing air and 80:20 Heliox.
J. Aerosol Med. Off. J. Int. Soc. Aerosols Med. 2004, 17, 278–285. [CrossRef] [PubMed]
15. Respiratory Infection—Pennsylvania. Available online: https://www.cdc.gov/mmwr/preview/mmwrhtml/
lmrk053.htm (accessed on 6 December 2017).
16. Bollin, G.E.; Plouffe, J.F.; Para, M.F.; Hackman, B. Aerosols containing Legionella pneumophila generated by
shower heads and hot-water faucets. Appl. Environ. Microbiol. 1985, 50, 1128–1131. [PubMed]
17. Mouchtouri, V.A.; Rudge, J.W. Legionnaires’ disease in hotels and passenger ships: A systematic review of
evidence, sources, and contributing factors. J. Travel Med. 2015, 22, 325–337. [CrossRef] [PubMed]
18. Communicable Disease Threats Report, 12–18 November 2017, Week 46. Available online: http://ecdc.
europa.eu/en/publications-data/communicable-disease-threats-report-12-18-november-2017-week-46
(accessed on 6 December 2017).
19. Beauté, J.; Zucs, P.; de Jong, B. Risk for travel-associated Legionnaires’ disease, Europe, 2009. Emerg. Infect. Dis.
2012, 18, 1811–1816. [CrossRef] [PubMed]
84
IJERPH 2018, 15, 598
20. WHO. Guidelines for Drinking-Water Quality, Fourth Edition. Available online: http://www.who.int/
water_sanitation_health/publications/2011/dwq_guidelines/en/ (accessed on 7 December 2017).
21. EUR-Lex-31998L0083-EN-EUR-Lex. Available online: http://eur-lex.europa.eu/legal-content/EN/TXT/
?uri=CELEX%3A31998L0083 (accessed on 7 December 2017).
22. EUR-Lex-32015L1787-EN-EUR-Lex. Available online: http://eur-lex.europa.eu/legal-content/EN/TXT/
?uri=uriserv%3AOJ.L_.2015.260.01.0006.01.ENG (accessed on 7 December 2017).
23. European Technical Guidelines for the Prevention, Control and Investigation of Infections Caused by
Legionella Species. Available online: http://ecdc.europa.eu/en/publications-data/european-technical-
guidelines-prevention-control-and-investigation-infections (accessed on 7 December 2017).
24. EN 15975-2—European Standards. Available online: https://www.en-standard.eu/csn-en-15975-2-security-
of-drinking-water-supply-guidelines-for-risk-and-crisis-management-part-2-risk-management/?gclid=
EAIaIQobChMI4JCEqby72AIVWV8ZCh3-SgC7EAAYASAAEgLZofD_BwE (accessed on 3 January 2018).
25. WHO. Water Safety Plan Manual (WSP Manual). Available online: http://www.who.int/water_sanitation_
health/publications/publication_9789241562638/en/ (accessed on 3 January 2018).
26. Litwin, C.M.; Asebiomo, B.; Wilson, K.; Hafez, M.; Stevens, V.; Fliermans, C.B.; Fields, B.S.; Fisher, J.F.
Recreational vehicle water tanks as a possible source for legionella infections. Case Rep. Infect. Dis. 2013,
2013, 286347. [CrossRef] [PubMed]
27. Whiley, H.; Bentham, R.; Brown, M.H. Legionella persistence in manufactured water systems: Pasteurization
potentially selecting for thermal tolerance. Front. Microbiol. 2017, 8, 1330. [CrossRef] [PubMed]
28. Murga, R.; Forster, T.S.; Brown, E.; Pruckler, J.M.; Fields, B.S.; Donlan, R.M. Role of biofilms in the survival
of Legionella pneumophila in a model potable-water system. Microbiol. Read. Engl. 2001, 147, 3121–3126.
[CrossRef] [PubMed]
29. Chochlakis, D.; Sandalakis, V.; Panoulis, C.; Goniotakis, I.; Makridaki, E.; Tselentis, Y.; Psaroulaki, A. Typing
of Legionella strains isolated from environmental samples in Crete, Greece, during the period 2004–2011.
J. Water Health 2013, 11, 762–771. [CrossRef] [PubMed]
30. Fragou, K.; Kokkinos, P.; Gogos, C.; Alamanos, Y.; Vantarakis, A. Prevalence of Legionella spp. in water
systems of hospitals and hotels in south western Greece. Int. J. Environ. Health Res. 2012, 22, 340–354.
[CrossRef] [PubMed]
31. Campese, C.; Roche, D.; Clément, C.; Fierobe, F.; Jarraud, S.; de Waelle, P.; Perrin, H.; Che, D. Cluster of
Legionnaires’ disease associated with a public whirlpool spa, France, April–May 2010. Euro Surveill. 2010,
15, 19602. [PubMed]
32. Health Protection Surveillance Centre. National Guidelines for the Control of Legionellosis in Ireland, 2009; Health
Protection Surveillance Centre: Dublin, Ireland, 2009; ISBN 978-0-9551236-4-1.
33. Coetzee, N.; Duggal, H.; Hawker, J.; Ibbotson, S.; Harrison, T.G.; Phin, N.; Laza-Stanca, V.; Johnston, R.;
Iqbal, Z.; Rehman, Y.; et al. An outbreak of Legionnaires’ disease associated with a display spa pool in retail
premises, Stoke-on-Trent, United Kingdom, July 2012. Euro Surveill. 2012, 17, 20271. [PubMed]
34. McEvoy, M.; Batchelor, N.; Hamilton, G.; MacDonald, A.; Faiers, M.; Sills, A.; Lee, J.; Harrison, T. A cluster of
cases of legionnaires’ disease associated with exposure to a spa pool on display. Commun. Dis. Public Health
2000, 3, 43–45. [PubMed]
35. Erdogan, H.; Arslan, H. Colonization of Legionella species in hotel water systems in Turkey. J. Travel Med.
2007, 14, 369–373. [CrossRef] [PubMed]
36. Mouchtouri, V.; Velonakis, E.; Tsakalof, A.; Kapoula, C.; Goutziana, G.; Vatopoulos, A.; Kremastinou, J.;
Hadjichristodoulou, C. Risk factors for contamination of hotel water distribution systems by Legionella
species. Appl. Environ. Microbiol. 2007, 73, 1489–1492. [CrossRef] [PubMed]
37. Bäck, E.; Schvarcz, R.; Kallings, I. Community-acquired Legionella micdadei (Pittsburgh pneumonia agent)
infection in Sweden. Scand. J. Infect. Dis. 1983, 15, 313–315. [CrossRef] [PubMed]
38. Goldberg, D.J.; Wrench, J.G.; Collier, P.W.; Emslie, J.A.; Fallon, R.J.; Forbes, G.I.; McKay, T.M.; Macpherson, A.C.;
Markwick, T.A.; Reid, D. Lochgoilhead fever: Outbreak of non-pneumonic legionellosis due to Legionella micdadei.
Lancet Lond. Engl. 1989, 1, 316–318. [CrossRef]
39. Pasparaki, E.; Chochlakis, D.; Damianaki, A.; Psaroulaki, A. Severe community acquired pneumonia due to
Legionella maceachernii infection. Arch. Bronconeumol. 2015, 51, 97–98. [CrossRef] [PubMed]
85
IJERPH 2018, 15, 598
40. Marciano-Cabral, F.; Jamerson, M.; Kaneshiro, E.S. Free-living amoebae, Legionella and Mycobacterium in tap
water supplied by a municipal drinking water utility in the USA. J. Water Health 2010, 8, 71–82. [CrossRef]
[PubMed]
41. WHO. Potable Reuse. Available online: http://www.who.int/water_sanitation_health/publications/
potable-reuse-guidelines/en/ (accessed on 8 March 2018).
42. Lockhart, G.; Oswald, W.E.; Hubbard, B.; Medlin, E.; Gelting, R.J. Development of indicators for measuring
outcomes of water safety plans. J. Water Sanit. Hyg. Dev. 2014, 4, 171–181. [CrossRef] [PubMed]
43. Dyck, A.; Exner, M.; Kramer, A. Experimental based experiences with the introduction of a water safety plan
for a multi-located university clinic and its efficacy according to WHO recommendations. BMC Public Health
44. Gunnarsdottir, M.J.; Gardarsson, S.M.; Elliott, M.; Sigmundsdottir, G.; Bartram, J. Benefits of Water Safety
Plans: Microbiology, compliance, and public health. Environ. Sci. Technol. 2012, 46, 7782–7789. [CrossRef]
[PubMed]
45. Hadjichristodoulou, C.; Goutziana, G.; Mouchtouri, V.; Kapoula, C.; Konstantinidis, A.; Velonakis, E.;
Vatopoulos, A.; Kremastinou, J. Evaluation of standardized scored inspections for Legionnaires’ disease
prevention, during the Athens 2004 Olympics. Epidemiol. Infect. 2006, 134, 1074–1081. [CrossRef] [PubMed]
46. Sandalakis, V.; Chochlakis, D.; Goniotakis, I.; Tselentis, Y.; Psaroulaki, A. Minimum inhibitory concentration
distribution in environmental Legionella spp. isolates. J. Water Health 2014, 12, 678–685. [CrossRef] [PubMed]
47. De Filippis, P.; Mozzetti, C.; Amicosante, M.; D’Alò, G.L.; Messina, A.; Varrenti, D.; Giammattei, R.;
Di Giorgio, F.; Corradi, S.; D’Auria, A.; et al. Occurrence of Legionella in showers at recreational facilities.
48. Napoli, C.; Fasano, F.; Iatta, R.; Barbuti, G.; Cuna, T.; Montagna, M.T. Legionella spp. and legionellosis in
southeastern Italy: Disease epidemiology and environmental surveillance in community and health care
facilities. BMC Public Health 2010, 10, 660. [CrossRef] [PubMed]
86
and Public Health
Article
Antibiotic Sensitivity Profiling and Virulence
Potential of Campylobacter jejuni Isolates from
Estuarine Water in the Eastern Cape Province,
South Africa
ID
Anthony C. Otigbu * , Anna M. Clarke, Justine Fri, Emmanuel O. Akanbi and Henry A. Njom
Microbial Pathogenicity and Molecular Epidemiology Research Group (MPMERG), Department of Biochemistry
and Microbiology, Department of Biochemistry & microbiology, University of Fort Hare, Private Bag X1314,
Alice 5700, South Africa; [email protected] (A.M.C.); [email protected] (J.F.); [email protected] (O.E.A);
[email protected] (H.A.N.)
Received: 10 April 2018; Accepted: 3 May 2018; Published: 6 May 2018
Abstract: Campylobacter jejuni (CJ) is a zoonotic microbe and a major causative organism of diarrheal
infection in humans that often has its functional characteristics inactivated in stressed conditions.
The current study assessed the correlation between recovered CJ and water quality parameters
and the drug sensitivity patterns of the pathogen to frontline antibiotics in human and veterinary
medicine. Water samples (n = 244) from rivers/estuarines were collected from April–September
2016, and physicochemical conditions were recorded on-site. CJ was isolated from the samples using
standard microbiological methods and subjected to sensitivity testing to 10 antibiotics. Mean CJ
counts were between 1 and 5 logs (CFU/mL). Ninety-five isolates confirmed as CJ by PCR showed
varying rates of resistance. Sensitivity testing showed resistance to tetracycline (100%), azithromycin
(92%), clindamycin (84.2%), clarithromycin and doxycycline (80%), ciprofloxacin (77.8%), vancomycin
(70.5%), erythromycin (70%), metronidazole (36.8%) and nalidixic acid (30.5%). Virulence encoding
genes were detected in the majority 80/95, 84.2%) of the confirmed isolates from cdtB; 60/95 (63.2%)
from cstII; 49/95 (51.6%) from cadF; 45/95 (47.4%) from clpP; 30/95 (31.6%) from htrB, and 0/95
(0%) from csrA. A multiple resistance cmeABC active efflux pump system was present in 69/95 (72.6)
isolates. The presence of CJ was positively correlated with temperature (r = 0.17), pH (r = 0.02),
dissolved oxygen (r = 0.31), and turbidity (r = 0.23) but negatively correlated with salinity (r = −0.39)
and conductivity (r = −0.28). The detection of multidrug resistant CJ strains from estuarine water
and the differential gene expressions they possess indicates a potential hazard to humans. Moreover,
the negative correlation between the presence of the pathogen and physicochemical parameters such
as salinity indicates possible complementary expression of stress tolerance response mechanisms by
wild-type CJ strains.
Keywords: Campylobacter jejuni; physicochemical; virulence; drug resistance; estuary
1. Introduction
Campylobacter spp. are of the epsilonproteobacteria class of microorganism [1]. They are slow
growing, Gram-negative, spiral shaped, motile organisms, characterized by their microaerobic
nature [2]. They have been reported to be detected in greater quantities in diarrhea infections
in humans than any other enteric pathogen and they require less than 100 cells to infect a
host [3]. Campylobacteriosis is a chronic enteric infection primarily caused by cytotoxin-producing
Campylobacters that invade and colonize the gastrointestinal (GI) tract in humans [4]. It is a zoonotic
disease mainly transmitted via the consumption of poultry products [2], contact with pets and livestock,

IJERPH 2018, 15, 925
ingestion of water contaminated with human faeces originating from sewage, septic tanks, latrines
and even animal faeces or from raw milk [5]. In humans, the disease lasts between 4–7 days and is
characterized by acute enteritis, fever, vomiting and abdominal pain [4], with the danger of possibly
leading to some post-infectious neuropathic diseases, such as bacteraemia, Guillain–Barre syndrome
(GBS), reactive arthritis (ReA), and abortion [4,6]. Campylobacter jejuni and C. coli are the two widely
recognized pathogenic species of Campylobacter that cause diseases in humans [7]. A large number
of recorded campylobacteriosis outbreaks have also been traced to the ingestion of untreated surface
water contaminated through human activities or by avian wildlife faeces [8,9]. Recreational and
potable water are potential reservoirs of Campylobacter infection [5]. Therefore, the role of water in the
transmission of Campylobacter species is of significant importance.
1.1. Virulence Determinants

Campylobacter jejuni possesses some multiple cell surface expressive virulent factors responsible
for its high prevalence and pathogenicity compared to other enteric bacteria [3,10]. The secreted toxin
is either enterotoxic or cytotoxic in its mode of action [10]. The target sites of enterotoxins are the
cells lining the gastrointestinal (GI) tract of the host where its virulent effect is felt, resulting in either
cytotoxin-linked inflammatory diarrhea associated with fever or non-inflammatory diarrhea associated
with enterotoxins, characterized by non-leukocyte watery stools [4].
1.1.1. Cytolethal Distending Toxin (CDT)

The Cytolethal Distending Toxin (CDT) is an apoptosis triggering toxin produced by a group of
Gram negative bacteria including Campylobacter jejuni (CJ) [9]. This toxin plays an important role in the
host mucosal inflammatory response for interleukin-8 (IL-8) released by intestinal cells [10]. CDT is
suggested to have an AB2 tripartite structure with cdtB as the main effector, while cdtA and cdtC are
makeup units associated with cell membrane binding [11]. The cdtA protein has a molecular mass of
27 KDa, CdtB has a molecular mass of 29 KDa, and cdtC has a molecular mass of 20 KDa [11]. Subunit
A is the active unit directly responsible for DNA damage, while subunit B is a binding subunit that
helps to bind the toxin to the specific target cells which inhibit cdc2, causing cellular distention and
eventually death. The DNase activity of CDT is lethal—causing singular strand breakage with an
estimated lethal dose (LD) of 50 pg/mL [11]. The pivotal role played by in cell and DNA degradation
not only results in inflammatory diarrhea with faecal leukocytes but can potentially create lesions in
fragmented DNA strands that can promote cancer [4,9]. CDT has high cross species sequence similarity
and cdtB has the highest interspecies similarity [11]. It is, however, believed that some species lack
cdtB but still have the potential to cause symptoms in children less than 3 years old [11].
1.1.2. Campylobacter Invasion Antigens (ciaB)

This is a protein synthesized by CJ which facilitates invasion to epithelial cells [10] of
the gastrointestinal tract where it inflicts increased damage on the columnar epithelial cells,
leading to swelling and rounding of invaded cells as a result of the cytotoxin and enterotoxin
activities [8,10]. Campylobacter heat labile cytotonic (CTON) and cytotoxin (CTOX) are associated
with non-inflammatory and inflammatory diarrhea, respectively [11]. Cia proteins are suggested to
modify host cell regulatory pathways to promote CJ pathogenicity [12].
1.1.3. Fibronectin-Binding Protein (cadF)

This outer membrane conserved gene encodes a protein containing 326 amino acids of molar
mass 37 kDa and plays a vital role in adherence to intestinal epithelial cells [13]. Internalization of the
organism into its host is harnessed by the binding activity of cadF to the extracellular fibronectin [14].
However, studies have shown that there may be a reduction in cadF functionality when it assumes a
defensive viable but non-culturable (VBNC) state [14].
88
IJERPH 2018, 15, 925
1.1.4. Sialyltransferases (cstII)

This is an outer core structure carbohydrate called lipo-oligosaccharide (LOS) expressed by CJ
that evades detection by mimicry of the human gangliosides [15]. The mechanism of action of this
gene is providing the LOS with a protective barrier which facilitates its invasion of the epithelial cells
by portraying a resemblance to the human ganglioside in the vertebrate nerve cells, allowing the host’s
immune system to self-destruct its own ganglioside [15]. It is believed to trigger the development of
autoimmune diseases, such as Guillain–Barre Syndrome [16].
1.1.5. Post Transcriptional Regulator (csrA)

csrA gene is a carbon starvation regulator gene linked to the encoding of protein regulation which
plays a vital role in CJ’s ability to responsively regulate a stationary phase mechanism to withstand
hostile conditions [17]. Other associated virulence expressed by this gene is related to oxidative stress
survival, the adherence of intestinal epithelial cells and biofilm formation [18]. Biofilm formation
is an adaptive mechanism which complements the fragility of an organism when exposed to stress
conditions by triggering a switch into a VBNC state [19].
1.1.6. ATP-Dependent Endopeptidase Protease (clpP)

This protease subunit in the bacterial caseinolytic proteases (CLP) contributes to virulence via
energy formation through the degradation of virulence regulators [20], while indirectly playing
a triggering role in stress tolerance of the organism when subjected to stress conditions [21].
The association of clpP with clp ATPase subunits enhances the proteolytic activity of the enzyme
in the presence of ATP, producing a catalytic action [21]. In many pathogens, clpP enhances protein
induced growth, under conditions, such as high temperature and oxidative stress [22].
1.1.7. Periplasmic Chaperon (htrB)

This is a periplasmic chaperon gene that encodes an acyltransferase for lipid A synthesis [23,24].
Synthesis of this enzyme regulates the organism’s response to environmental changes [25]. It is,
however, interesting to note that the Campylobacter species shows varying diversities of infection
outcomes [26] attributable to differences in genetic composition [22].
1.2. Treatment and Drug Resistance

Campylobacter infection is, at times, regarded as self-limiting, but in the case of severe
complications, antibiotics are commonly recommended, especially for immunodeficient patients.
Antibiotic resistance among Campylobacter species has emerged as a global public health burden [4].
There are cases of growing resistance of Campylobacter spp. against the front line and alternative
treatment therapies, such as macrolides (erythromycin), tetracycline, fluoroquinolones and
aminoglycoside (gentamycin) [7,27]. The unregulated use of antimicrobial agents as food additives in
livestock in order to prevent and control infections and enhance growth rates [28,29] has contributed
to an increased resistance in microbes against multiple antibiotics [9]. The unregulated administration
of fluoroquinolones to poultry has contributed to increased resistance of CJ to fluoroquinolones in
industrialized regions [27].
A survey of the antimicrobial susceptibility of Campylobacter species isolated from poultry and pigs
was carried out in the Western Cape and Gauteng provinces of South Africa and the results displayed
clear traces of resistance to fluoroquinolones, macrolides and tetracycline antibiotics, while some of
the isolates displayed multidrug resistance [30]. These characteristic drug resistances were prominent
among two specific Campylobacter species, CJ and C. coli, which have very similar epidemiology, but
require biochemical tests to distinguish between them [30]. Previous studies have reported variation in
CJ sensitivity to erythromycin and ciprofloxacin; resistance rates of 79.2% were reported in Nigeria [31],
0% in Djibouti [14] and in Qatar, resistance rates of 63.2% to ciprofloxacin and 8.6% to erythromycin
89
IJERPH 2018, 15, 925
were shown [32]. Resistance to ciprofloxacin, another antimicrobial agent of consideration next to
erythromycin, has also been recorded in some other parts of the world [33]. Multidrug resistance
in Campylobacter is a widely studied area. Previous studies have suggested mutation as a factor
responsible for the acquisition of this characteristic [34–36]. Campylobacters have an innate resistance
trait in combination with externally acquired resistance traits to express virulence [35]. Mutation is
believed to play a role in the evolution of the cmeABC operon [15,36] in the multidrug efflux system.
Drug resistance has, however, been attributed to target modification-mediated enzymatic inactivation
and enhanced efflux [37].
2.1. Study Area

The Swartkops estuary (33◦ 52 S; 25◦ 38 E) was selected for this study, and it is one of the most
important estuaries in South Africa. It is also an important bird area (IBA) harbouring approximately
4000 migratory birds annually [38]. It is located close to the coastal city of Port Elizabeth in Nelson
Mandela Bay Municipality of the Eastern Cape Province. The river is approximately 134 km long,
while the estuary is approximately 16.4 km long with a permanent open connection into Algoa Bay
in the Indian Ocean [39]. The total catchment area of the Swartkops River (including the tributary)
is about 1360 km2 [39]. Surrounding areas in the catchment of the Swartkops River are used for
agriculture, while the lower reaches of the river and the estuary are surrounded by extensive human
development, including several industries [40].
2.2. Sampling and Isolation of CJ
Sampling and Isolation

The spot sampling method, as described by the JEEP92 project [41], was used. An Aestuaria
Bandi 410 vessel was used for sampling a total distance of 12.775 km of the Swartkops river estuary
between the six sample points (Figure 1). Triplicate water samples were collected against water flow
from surface level and at a depth below (3 m) using sterile bottles from each sampling point over a
6-month period (April–September 2016) covering three seasons (autumn, winter and spring) of the year
and transported at 4 ◦ C to the laboratory and analyzed within 5 h after collection. Physicochemical
parameters (temperature, pH, electrical conductivity, salinity and turbidity) of sample stations were
recorded in-situ [42] using the YSI 650 MDS multi-parameter reader at two levels (surface and bottom)
from each sampling point.
0DSRI6RXWK$IULFD
VKRZLQJ ORFDWLRQ RI
6ZDUWNRSV LQ WKH
( &
Figure 1. Location and GPS coordinates of the study sites in the Swartkops River estuary, Port Elizabeth.
Sampling locations within the estuary; Rowing Club (RC) (33◦ 50’14.60 S; 25◦ 34’14.37 E); Factory Dam
(FD) (33◦ 50’12.16 S; 25◦ 35’41.24 E); Redhouse Farm (RF) (33◦ 49’10.56 S; 25◦ 33’37.44 E); Bridge Canal
(BC) (33◦ 51’25.99 S; 25◦ 35’49.99 E); Despatch Mouth (DM) (33◦ 51’32.22 S; 25◦ 37’31.33 E); Tiger Bay
Canal (TB) (33◦ 52’0.26 S; 25◦ 36’22.32 E) (https://www.google.com.au/maps/).
90
IJERPH 2018, 15, 925
Bacteria cells were concentrated on a microfilter (0.65 μm pore size cellulose ester Millipore) from
raw water (100 mL; 10−1 ; 10−2 ) samples. The concentrated filter was aseptically folded and enriched in
20 mL nutrient broth supplemented with Preston Campylobacter selective supplement (SR0117-Oxoid)
with 5% lysed horse blood and incubated microaerobically at 37 ◦ C for 48 h. One hundred microlitres
of enrichment culture was sub-cultured to Campylobacter blood-free agar (CCDA; CM739; Oxoid)
containing CCDA selective supplement (SR155E; Oxoid) and incubated microaerobically using a
campy gas pack (5% O2, 10% CO2 , 85% N2 , CampyGen Oxoid) at 37 ◦ C for 72 h. All plating was carried
out in duplicate. Distinct presumptive colonies on each plate were counted by the surface count method
to determine the total viable Campylobacter counts (TVCC). Then, 8–10 different colonies per plate
were picked and subcultured on the selective medium for purity. A positive control (Campylobacter
jejuni ATCC 33560 strain) was included with each set of tests. Identification of positive isolates was
based on colony morphology, Gram-stain, no-growth in aerobic condition, hippurate hydrolysis and
oxidase tests.
2.3. PCR Confirmation of CJ and Detection of Virulence Genes

Genus confirmation was performed using the 23S rRNA gene [43], and species confirmation was
done using the hipO gene [44]. Screening for the pathogenic virulence genes, cdtB, cadF, cstII, csrA, htrB
and clpP, was performed on the CJ confirmed isolates. Genomic isolation from the confirmed isolates
was carried out using a commercial genomic DNA isolation kit (Qiagen Kit, Invitrogen, Thermo Fisher
Scientific, USA) according to the manufacturer’s instructions. Table 1 shows the primer sequences
(Inqaba biotech, South Africa), amplicon sizes and cycling conditions of the various genes used in this
study. The final concentration of the 25 μL PCR reaction consisted of 12.5 μL of the 2X master mix
(Sybrselect, USA) 0.5 μL forward and reverse primers, 6.5 μL molecular grade water and 5 μL template
DNA. The cycling conditions were as follows: initial denaturation was at 94 ◦ C for 5 min, and then
94 ◦ C for 30 s, with modifications in annealing temperatures specific to the primer pair (as given in
Table 1) for 5 min and extension at 72 ◦ C for 50 s. All PCR products were analyzed by electrophoresis
on 1.5% agarose gels (CSL-AG100, Cleaver Scientific Ltd. Warwickshire, UK) except for htrB and clpP
genes which were analyzed on 2% agarose gels. The gels were stained with ethidium bromide and
visualized with a UV transilluminator and photographed (Alliance 4.7).
Table 1. PCR primer sizes and annealing temperatures.
Annealing
Name of Gene Sequence (5 -3 ) Product (bp) References
Temperature (◦ C)
F-AATTGATGGGGTTAGCATTAGC
Campy 23S 316 55 [43]
R-CAACAATGGCTCATATACAACTGG
F-AGAGTTTGATCCTGGCTCAG
hipO 344 58 [44]
R-ACGGCTACCTTGTTACGACTT
F-CAC GGT TAA AAT CCC CTG CT
cdtB 495 52 [18]
R-GCA CTT GGA ATT TGC AAG GC
F-CGC ACC CAA TTT GAC ATA GAA
htrB 70 52 [45]
R-TTT TTA GAG CGC TTA GCA TTT GTC T
F-TCG GAG CAT TTT TGC TTA GTT G
clpP 90 52 [46]
R-CTC CAC CTA AAG GTT GAT GAA TCA T
F-CAC AGT CAG TGA AGG TGC TT
csrA 878 52 [47]
R-ACT CGC ACA ATC GCT ACT TC
F-CAG CTT TCT ATT GCC CTT GC
cstII 570 52 [18]
R-ACA CAT ATA GAC CCC TGA GG
F-TTGAAGGTAATTTAGATATG
cadF 400 42 [10]
R-CTAATACCTAAAGTTGAAAC
91
IJERPH 2018, 15, 925
2.4. Antimicrobial Sensitivity Testing

Confirmed CJ isolates were subjected to antimicrobial sensitivity testing with 10 antimicrobial
agents. The Kirby–Bauer disk diffusion method on Mueller–Hinton agar supplemented with 5%
horse blood, in accordance with Clinical and Laboratory Standards Institute guidelines [38] was
used to perform the antimicrobial profiling. An inoculum of each bacterial isolate was emulsified
in 3 mL of sterile normal saline (0.9%) in test tubes and the density was adjusted to 0.5 McFarland
standard (0.5 mL of 1% w/v BaCl2 and 99.5 mL of 1% v/v H2 SO4 ), equivalent to 1.0 × 108 cfu/mL.
The bacterial suspension was evenly spread on the Mueller–Hinton agar plates using sterile swab
sticks and allowed to dry. Antibiotic discs (Mast Diagnostics Ltd., UK) with the following drug
concentrations were selected for the assay: nalidixic acid (30 μg), ciprofloxacin (5 μg), azithromycin
(15 μg), doxycycline (30 μg), erythromycin (15 μg), clarithromycin (15 μg), vancomycin (15 μg),
tetracycline (30 μg), clindamycin (2 μg) and metronidazole (15 μg). Plates were incubated at 37 ◦ C
under microaerophilic conditions (5% O2 , 10% CO2 , 85% N2 ) with gas generator envelopes (CampyGen;
2.5 L Thermo Scientific, UK) for 48 h, and the diameter zones of inhibition were measured, and the
results were interpreted in accordance with the CLSI unit [48].
Detection of Multidrug Resistance Genes (cmeA, cmeB and cmeC)

Detection of the cmeA, cmeB and cmeC genes was determined by PCR, as described by [49],
with slight modifications. Table 2 shows the primer sequences (Inqaba biotech, South Africa). The final
concentration of the 25 μL PCR reaction consisted of 12.5 μL of the 2X master mix (Sybrselect, USA)
0.5 μL forward and reverse primers, 6.5 μL molecular grade water and 5 μL template DNA. The cycling
conditions were as follows: initial denaturation at 94 ◦ C for 7 min, followed by 94 ◦ C for 1 min,
annealing temperatures (see Table 2) for 1.5 min, extension at 72 ◦ C for 3 min and then, final extension
at 72 ◦ C for 5 min for 30 cycles. All PCR products were analyzed by electrophoresis on 1% agarose gel
(CSL-AG100, Cleaver Scientific Ltd. Warwickshire, UK). The gels were stained with ethidium bromide
and visualized with a UV transilluminator and photographed (Alliance 4.7). Campylobacter jejuni ATCC
33560 strains were used as the positive control.
Table 2. Primers used in the study.
Target Genes Primer Sequences 5’-3’ Annealing Temp (◦ C) Amplicon Size (bp)
F-TAGCGGCGTAATAGTAAATAAAC
CmeA 50 435
R-ATAAAGAAATCTGCGTAAATAGGA
F-AGGCGGTTTTGAAATGTATGTT
CmeB 50 444
R-TGTGCCGCTGGGAAAAG
F-CAAGTTGGCGCTGTAGGTGAA
CmeC 52 431
R-CCCCAATGAAAAATAGGCAGAGTA
3. Results
3.1. Physicochemical Analyses

The mean water temperature in the Swartkops river estuary for the sampled months was between
14.7 ◦ C and 15.6 ◦ C with the Despatch Mouth (DM) recording the highest mean temperature, while the
Rowing Club station (RC) had the lowest mean temperature. No clear-cut difference in pH values
was recorded for the sample stations, as the mean pH for all the stations ranged between 8.27 and
8.33. However, a high level of variation in the salinity level was recorded at all stations, with the mean
salinity ranging between 13.92 practical salinity units (psu) and 32.77 psu. Station E, which is the
dispatch point of Swartkops River to Algoa Bay recorded the highest salinity out of all the sampled
months, while the Rowing Club (RC) station recorded the lowest salinity. The average dissolved
oxygen (DO) concentrations were 53.83 mg/L and 62.9 mg/L, respectively. Station RC also recorded
the lowest DO reading, while the Tiger Bay (TB) station recorded the highest overall reading. In terms
92
IJERPH 2018, 15, 925
of turbidity, the Swartkops water was very turbid during the sampled seasons, ranging, on average,
between 4.2 Nephelometric Turbidity Units (NTU) and 66.9 NTU. The Factory Dam (FD) station
recorded the highest average turbidity (66.9 NTU), especially in July and August, while DM presented
more pristine water for all sample periods. The conductivity was between 20.9 ms/cm and 30 ms/cm
on average. A low coefficient of variability was observed for all sampled sites (Table 3).
Table 3. Physicochemical parameters of water at different sample stations.
Sample Salinity DO Turbidity Conductivity

Parameters Temp (◦ C) pH
Stations (psu) (mg/L) (NTU) (ms/cm)
M 14.7 8.32 13.9 53.8 37.2 20.9
RC
Cv 0.16 0.02 0.52 0.68 0.85 0.49
M 14.8 8.33 19.2 60.5 66.9 25.7
FD
Cv 0.16 0.01 0.4 0.67 0.74 0.43
M 14.9 8.27 14.9 55.3 48.4 22
RF
Cv 0.17 0.01 0.15 0.7 0.78 0.13
M 14.9 8.31 21.4 56.9 31.9 26.3
BC
Cv 0.15 0.02 0.5 0.69 0.4 0.55
M 15.6 8.29 32.8 58.9 4.2 26.3
DM
Cv 0.13 0.01 0.19 0.69 0.7 0.23
M 14.9 8.36 25.4 62.9 24.2 30
TB
Cv 0.15 0.01 0.38 0.68 1.5 0.44
M = mean; Cv = coefficient of variability; DO = dissolved oxygen.
3.2. PCR Confirmation of CJ and Detection of Virulence Genes

One hundred and twenty isolates were phenotypically confirmed as Campylobacteracea (Figure 2).
Further screening at the species level confirmed 95 isolates as C. jejuni (Figure 3) and the other
25 identified as C. coli (18) and C. upselensis (7). Determination of the occurrence of virulence genes in
the confirmed CJ isolates revealed the cdtB gene in 80/95 (84.2%) of the isolates, an indication that the
toxin production gene (cdtB) was the most prevalent virulence determinant. Forty-nine (52%) of the
isolates were identified as having adherence virulence genes (cadF), while 60/95 (63.2%) isolates tested
positive for the intestinal epithelial invasive virulence gene (cstII). This gene is also linked to the risk of
Gullian–Barre Syndrome (GBS) development. Thirty (31.6%) of the isolates were positive for the lipid
A synthesis gene (htrB) responsible for the adjustment of organisms to stressful external environmental
changes, while 45/95 (47.4%) of the isolates were identified as having the ATP dependent protease gene
(clpP), which is responsible for the degradation of damaged proteins due to unfavourable conditions.
The carbon starvation regulator gene (csrA), which is linked to cell division and the formation of
biofilm, was absent in all isolates. Isolates recovered from the Redhouse Farm (RF) and Bridge Canal
(BC) sampling sites were confirmed as housing all but the csrA gene which was absent in all isolates.
Table 4 shows the number of genes detected in confirmed CJ isolates.
Figure 2. PCR detection of 23S rRNA gene (316 bp). Lane 1 is themarker, Lanes 2–9 are test isolates,
Lane 10 is the negative control, and Lane 11 is the positive control.
93
IJERPH 2018, 15, 925

Figure 3. PCR detection of hipO gene (344 bp) for CJ confirmation. Lane 1 is the marker, Lane 2 is the
positive control, Lane 3 is the negative control, and Lanes 4–11 are the test isolates.
Table 4. Detection of pathogenic genes in confirmed C. jejuni (CJ) isolates.
No. of Isolates Confirmed No. of Isolates

Sample No. of
as Campylobacter Genus Confirmed as Genes Detected in CJ Isolates (% Positive)
Source Samples
for Samples (%) CJ (%)
Estuarine
244 23S rRNA HipO cdtB cadF cstII csrA htrB ClpP
water
120 (49.2) 95 (79.2) 80 (84.2) 49 (51.6) 60 (63.2) 0 (0) 30 (31.6) 45 (47.4)
3.3. Frequency of CJ Isolation

The frequency of bacterial isolation frequency at all sample sites for the sampling period was
recorded (Figure 4). The Despatch Mouth (DM) was the least Campylobacter-contaminated site, and the
most pristine with an isolation frequency of 33%. No Campylobacter count was recorded at DM for April,
May, June or July (autumn and winter), but Campylobacter were recorded in August and September
(spring). The Tiger Bay (TB) site appeared to be the most Campylobacter-contaminated site in the
Swartkops estuary with an isolation frequency of 100% during the sampled seasons. Campylobacter
counts were recorded for all sampling months, with higher readings in July, August and September.
However, the overall highest average Campylobacter counts were recorded at the Factory Dam (FD)
and Bridge Canal (BC) sites for the month of August. Consequently, very low counts were recorded in
April, May, and June (winter) for all sites.

Figure 4. Bacterial density at all sampling sites. * Mean values are expressed in log (cfu per 100 mL).
3.4. Physicochemical Parameters and Occurrence of C. jejuni

No significant positive correlation was observed between the population density of CJ and
temperature (r = 0.17), pH (r = 0.02), dissolved oxygen (r = 0.31), and turbidity (r = 0.23). A negative
correlation was observed with salinity (r = −0.39) and conductivity (r = −0.28). The correlation values
94
IJERPH 2018, 15, 925
were not statistically different for temperature, dissolved oxygen, salinity, turbidity and conductivity,
while they were statistically different for pH (Table 5).
Table 5. Correlation (Bravais Pearson) matrix between temperature, bacterial contamination, pH,
salinity, turbidity, dissolved oxygen and conductivity.
Temp PH Salinity DO Turbidity Conductivity CJ

Temp 1
pH −0.30 1
Salinity −0.60 0.46 1
DO 0.19 0.07 −0.31 1
Turbidity −0.08 −0.03 −0.12 −0.43 1
Conductivity −0.53 0.48 0.87 −0.32 0.08 1
CJ 0.17 0.02 −0.39 0.21 0.23 −0.28 1
3.5. Antimicrobial Sensitivity Testing and Prevalence of Multidrug Resistance (MDR) Efflux Pump Genes
The antibiotic sensitivity of 95 CJ isolates was profiled and revealed a higher degree of resistance
to tested antimicrobial agents. The highest resistance level, 95/95 (100%), was recorded for tetracycline,
followed by azithromycin (87/95, 92%), clindamycin (80/95, 84.2%), clarithromycin and doxycycline
(76/95, 80%), ciprofloxacin (78/95, 77.8%), vancomycin (67/95, 70.5%), and erythromycin (67/95, 70%),
with the lowest resistance levels recorded for metronidazole (35/95, 36.8%) and nalidixic acid (29/95,
30.5%). Nalidixic acid was the most effective antibiotic with a susceptibility of 57/95 (59%) (Figure 5).
The percentage incidences of multidrug resistance (MDR) efflux pump genes of C. jejuni are shown
in Table 6. A high (69/95, 72.6%) number of CJs expressed MDR efflux pump genes with only 6/95
(6.3%) not expressing them. Twenty isolates showed a disruption in the expression of all tripartite
efflux systems, which could trigger a malfunction of the cmeABC system.

Figure 5. Antimicrobial sensitivity Profile.
Table 6. Incidences of Campylobacter multidrug resistance (MDR) efflux pump genes.
No. of Isolates (n = 95)

Genes
a (%) b (%)
cmeA 11/20 (55) 9/20 (45)
cmeB 18/20 (90) 2/20 (20)
cmeC 14/20 (70) 7/20 (30)
cmeABC 69/95 (72.6) 6/95 (6.3)
a = positive isolates; b = negative isolates.
95
IJERPH 2018, 15, 925
4. Discussion
4.1. Survival of Organism

Estuaries are confluent ecosystems where a mixture of salty sea waters and rivers meet with
freshwater [42]. They are transition points from land to sea and freshwater to salt water and are rich in
organic contents [5]. The dynamic physicochemical nature of the ecosystem is peculiarly detrimental
to the survival of fastidious microbes such as Campylobacter [14]. However, Campylobacter spp. display
complex survival mechanisms by transiting to a stationary viable, but non-culturable, form (VBNC) for
survival [11]. The recovered strains in this study displayed differential gene expression, which could
be peculiarto wild-type CJ strains, and morphological evidence showed 66/95 (69%) without flagella.
This could be due to the expression of the flagella gene being switched off in a process known as
phase variation [49]. The carbon starvation regulator gene (csrA) which is linked to the encoding of
protein regulation was not detected in the study. The absence of csrA could have been complimented
by the presence of htrB and clpP genes. Other associated virulence expressed by this gene is related
to oxidative stress survival, adherence of intestinal epithelial cells and in biofilm formation [18,45].
Although the pathogenic potential of wild-type strains is debatable due to the adverse stress conditions
which can reduce their colonization and invasive abilities, their ability to live asymptomatically in their
hosts may not be fully ruled out, especially as this concerns externally acquired genes from surrounding
environs. This is indicated by the presence of the cstII gene in some of the strains (Figure 6). Some
of the test isolates lacked cdtB, and cadF genes and could, therefore, be considered non-pathogenic.
Most of the isolates lacked motility potential at the point of analysis, inferring the inactivation of the
racR gene [11]. The results as shown in Figure 7 strongly correlate with the inference drawn in a past
study suggesting that CJ could still retain its cadF adhesion functionality under stressed conditions [14].
Figure 6. PCR detection of cstII gene (570 bp) of CJ. Lane 1 is the marker, Lane 2 is the positive control,
Lane 3 is the negative control, and Lanes 4–13 are test isolates.
Figure 7. PCR detection of cadF gene (400 bp) of CJ: Lane 1 is the marker, Lane 2 is the positive control,
and Lanes 3–11 are test isolates.
The prevalence of the cytotoxin production gene (cdtB) in the confirmed isolates (Figure 8) shows
that the organism is capable of retaining its toxin production ability even in a starved state. The presence
of htrB and clpP genes (Figure 9) provides an extra boost in the organism’s aero-tolerance survival in
environmental waters. However, the survival of microorganisms in oxidative stress environments
has been attributed to their ability to develop specialized defensive mechanisms [11]. The existence
of CJ in the Swartkops could be as a result of oceanic effects rather than continental effects. The FD
sample site was the most polluted site in the Swartkops due to effluent discharges from the factory
directly to the river. Between FD and BC is the shallowest area; this may be due to high land surface
run-off into the river. This area has the highest population of migratory birds and fishing activities
96
IJERPH 2018, 15, 925
with the highest CJ density which strongly indicates that avian species are major reservoirs of the
organism. Seasonality, on the other hand, could also play a pivotal role in the survival and existence
of the organism, as larger population densities of the organism were recorded during spring and the
lowest population densities were recorded during winter [2,9]. The Swartkops river estuary has a
strong nutrient zonation, which is typified by the variation in salinity distribution in the estuary as
a result of upstream shift of salt and fresh waters, which may be responsible for the abundance and
distribution of species. Hence, this was the major reason for choosing to investigate the effect of the
physicochemical parameters on the survival of the organism. The salinity reading at DM was the
highest total average reading of all seasons, but barely affected the survival of the organism.

Figure 8. PCR detection of cdtB gene (495 bp) of CJ. Lane 1 is the marker, Lane 2 is the positive control,
Lane 3 is the negative control, and Lanes 4–12 are test isolates.
Figure 9. PCR detection of htrB (70bp) and clpP genes (90bp) of C. jejuni. Lane 1 is the marker, Lane 2
is the positive control, Lanes 3, 7, 9 and 10 are clpP positive isolates, and Lanes 4, 5, 6 and 8 are htrB
positive isolates.
4.2. Drug Resistance

Multiple resistances have been reported globally in both CJ and C. coli in human and
animal isolates. The resistance pattern was observed notably in tetracyclines, macrolides and
fluoroquinolones [1,7]. In South Africa, antimicrobials with broad-spectrum activity such as
tetracyclines are used in both the poultry and pig industries, as they are affordable and easy to
administer in food and water [30]. In a recent study, a high level of resistance to tetracyclines was also
revealed in Campylobacter spp. isolated from broilers and hens [1]. In this study, multidrug resistance
was observed in more than 70% of the isolates (Figure 10). The isolates harbored the Campylobacter
multidrug efflux pump (cmeABC) genes responsible for multidrug resistance [7]. Previous studies have
shown variation in the resistance pattern of CJ in South Africa [7,30]. Some studies revealed a high
resistance of the organism to fluoroquinolones, macrolides and tetracyclines [30], while some showed
high susceptibility to fluoroquinolones [1]. Resistance to fluoroquinolone is believed to develop more
rapidly in Campylobacter spp, than in other Gram-negative bacteria, mainly attributed to single-step
point mutations in gyrA [2]. However, these studies were carried out on CJ isolated from avians,
porcines and bovines. In this study, multidrug resistance was shown for fluoroquinolones, macrolides
and tetracyclines. A high resistance to tetracycline (100%) was observed and the highest susceptibility
(59%) was shown to nalidixic acid. Previous studies have suggested that aquatic environments (surface
and groundwater bodies) are perfect for horizontal gene exchange of mobile genetic elements (MGEs)
which results in antibiotic resistance [2].
97
IJERPH 2018, 15, 925
Figure 10. PCR detection of cmeABC in isolates. Lane 1 is the marker, Lane 2 is the positive control,
Lane 8 is the CmeA positive isolate (435 base pair, Lane 4 is the negative control, Lanes 5 and 6 are cmeB
positive isolates (444 bp), and Lane 3 is the cmeC positive isolate (431 bp).
5. Conclusions
This is the first study to report the occurrence of differential gene expressions in wild-type CJ
isolated from the Swartkops in the Eastern Cape Province. The results showed that the estuarine water
could potentially harbour multiple resistant CJ strains of public health concern among estuarine users.
Although the extent of their pathogenicity is not fully ascertained, it could be assumed that pathogens
with similar traits are likely to be found in other similar ecosystems.
6. Future Direction of Study

This study focused on the detection of active virulence-inducing genes and antimicrobial
sensitivity profiling of environmentally recovered CJ strains. More elaborate future studies should
include a comparative genomic analysis using whole genome sequencing (WGS) for other related
water sources of high importance around the Eastern Cape Province to fully understand the
pathophysiological mechanisms of recovered wild-type Campylobacter strains. Moreover, a comparative
study of the antimicrobial profile and analysis of expressed virulence of wild-type isolates and clinical
strains should be investigated. Other important virulence determinants which were not investigated
in this study such as racR and flaA genes associated with motility and cosR, rrpA or rrpB associated
with the oxidative stress response should be studied in wild-type isolates. It is also necessary to
conduct the same study on other closely-related organisms, such as Arcobacter species, to document
their pathogenicity.
Author Contributions: A.C.O. carried out the project experimental design, sample collection, performed the
experiments, analysed data, and drafted the manuscript, H.A.N. and J.F. contributed to data analysis, study design
and proof reading of manuscript, O.E.A. assisted in sample collection and proof reading of manuscript, A.M.C.
contributed to the study design and organisation of sampling.
Acknowledgments: This work was supported by the National Research Foundation (NRF) and the South African
Institute for Aquatic Biodiversity (SAIAB) under the NRF/SAIAB innovative scholarship.
References
1. Bester, L.; Essack, S. Prevalence of antibiotic resistance in Campylobacter isolates from commercial poultry
suppliers in KwaZulu-Natal, South Africa. J. Antimicrob. Chemother. 2008, 62, 1298–1300. [CrossRef]
[PubMed]
98
IJERPH 2018, 15, 925
2. Nichols, G.; Richardson, J.; Sheppard, S.; Lane, K.; Sarran, C. Campylobacter epidemiology: A descriptive
study reviewing 1 million cases in England and Wales between 1989 and 2011. BMJ 2012, 2, e001179.
[CrossRef] [PubMed]
3. Croinin, O.; Backert, S. Host epithelial cell invasion by Campylobacter jejuni: Trigger of zipper mechanism?
Research Advances in the study of Campylobacter, Helicobacter and related organisms. Front. Cell. Infect. Microbiol.
4. World Health Organization. The Increasing Incidence of Human Campylobacteriosis Report and Proceedings
of a WHO Consultation of Experts, Copenhagen, Denmark 21–25 November 2000; World Health Organization:
Copenhagen, Denmark; pp. 21–25.
5. EFSA (European Food Safety Authority); ECDC (European Centre for Disease Prevention and Control).
The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne
outbreaks in 2012. EFSA J. 2014, 12, 3547. [CrossRef]
6. Skirrow, M.; Blaser, M. Clinical aspects of Campylobacter infection. In Campylobacter, 2nd ed.; Nachamkin, I.,
Blaser, M.J., Eds.; ASM Press: Washington, DC, USA, 2000; pp. 69–88.
7. Engberg, J.; Aarestrup, F.; Taylor, D.; Gerner-Smidt, P.; Nachamkin, I. Quinolone and macrolide resistance
in Campylobacter jejuni and C. coli: Resistance mechanisms and trends in human isolates. Emerg. Infect. Dis.
2001, 7, 24–34. [CrossRef] [PubMed]
8. Sopwith, W.; Birtles, A.; Matthews, M.; Fox, A.; Gee, S.; Painter, M. Campylobacter jejuni multilocus sequence
types in humans, northwest England, 2003–2004. Emerg. Infect. Dis. 2006, 12, 1500–1507. [CrossRef]
[PubMed]
9. DefFraites, F.; Sanchez, L.; Brandt, A.; Kadlec, P.; Haberberger, L.; Lin, J.; Taylor, N. An outbreak of
Campylobacter enteritis associated with a community water supply on a US military installation. MSMR 2014,
21, 10–15.
10. Perera, V.; Nachamkin, I.; Ung, H.; Patterson, J.; McConville, M. Molecular mimicry in Campylobacter jejuni:
Role of the lipo-oligosaccharide core oligosaccharide in inducing anti-ganglioside antibodies. FEMS Immunol.
Med. Microbiol. 2007, 50, 27–36. [CrossRef] [PubMed]
11. Samosornsuk, W.; Asakura, M.; Yoshida, E.; Taguchi, T.; Eampokalap, B.; Chaicumpa, W.; Yamasaki, S.
Isolation and characterization of Campylobacter strains from diarrheal patients in Bangkok and its suburb in
Thailand. Jpn. J. Infect. Dis. 2015, 68, 209–215. [CrossRef] [PubMed]
12. Saouaf, J.; Li, B.; Zhang, G.; Shen, Y.; Furuuchi, N.; Hancock, W.; Greene, I. Deacetylase inhibition increases
regulatory T cell function and decreases incidence and severity of collagen-induced arthritis. Exp. Mol. Pathol.
2009, 87, 99–104. [CrossRef] [PubMed]
13. Sails, A.; Bolton, F.; Fox, A.; Wareing, D.; Greenway, D. Detection of Campylobacter jejuni and Campylobacter
coli in environmental waters by PCR enzyme- linked immunosorbent assay. Appl. Environ. Microbiol. 2002,
14. Patrone, V.; Campana, R.; Vallorani, L.; Dominici, S.; Federici, S.; Casadei, L.; Gioacchini, A.M.; Stocchi, V.;
Baffone, W. CadF expression in Campylobacter jejuni strains incubated under low-temperature water
microcosm conditions which induce the viable but non-culturable (VBNC) state. Antonie Van Leeuwenhoek
2013, 103, 979–988. [CrossRef] [PubMed]
15. Pérez-Boto, D.; López-Portolés, J.; Simón, C.; Valdezate, S.; Echeita, M. Study of the molecular mechanisms
involved in high-level macrolide resistance of Spanish Campylobacter jejuni and Campylobacter coli strains.
J. Antimicrob. Chemother. 2010, 65, 2083–2088.
16. Bach, J. Infections and autoimmune diseases. J. Autoimmun. 2005, 25, 74–80. [CrossRef] [PubMed]
17. Fields, J.; Thompson, S. Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation,
and host cell invasion. J. Bacteriol. 2008, 190, 3411–3416. [CrossRef] [PubMed]
18. González-Hein, G.; Huaracán, B.; García, P.; Figueroa, G. Prevalence of virulence genes in strains of
Campylobacter jejuni isolated from human, bovine and broiler. Braz. J. Microbiol. 2013, 44, 1223–1229.
19. Thakur, S.; Zhao, S.; McDermott, P.; Harbottle, H.; Abbott, J.; Gebreyes, W.; White, D. Antimicrobial resistance,
virulence, and genotypic profile comparison of Campylobacter jejuni and Campylobacter coli isolated from
humans and retail meats. Foodborne Pathog. Dis. 2010, 7, 835–844. [CrossRef] [PubMed]
20. Hlaváček, O.; Vachova, L. ATP- dependent proteinases in bacteria. Folia Microbiol. 2002, 47, 203–212.
[CrossRef]
99
IJERPH 2018, 15, 925
21. Frees, D.; Brøndsted, L.; Ingmer, H. Bacterial proteases and virulence. In Regulated Proteolysis in Microorg;
Springer Science: Dordrecht, The Netherlands, 2013; pp. 161–192.
22. Hughes, R.; Cornblath, D. Guillain-Barre´ syndrome. Lancet 2005, 366, 1653–1666. [CrossRef]
23. Parkhill, J.; Wren, B.W.; Mungall, K.; Ketley, J.M.; Churcher, C.; Basham, D.; Chillingworth, T.; Davies, R.M.;
Feltwell, T.; Holroyd, S.; et al. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals
hypervariable sequences. Nature 2000, 403, 665–668. [CrossRef] [PubMed]
24. Hausdorf, L.; Neumann, M.; Bergmann, I.; Sobiella, K.; Mundt, K.; Fröhling, A.; Schlüter, O.; Klocke, M.
Occurrence and genetic diversity of Arcobacter spp. in a spinach processing plant and evaluation of two
Arcobacter-specific quantitative PCR assays. Syst. Appl. Microbiol. 2013, 36, 235–243. [CrossRef] [PubMed]
25. Mihaljevic, R.; Sikic, M.; Klancnik, A.; Brumini, G.; Mozina, S.; Abram, M. Environmental stress factors
affecting survival and virulence of Campylobacter jejuni. Microb. Pathog. 2007, 43, 120–125. [CrossRef]
[PubMed]
26. Sheppard, S.; Dallas, J.; Strachan, N.; MacRae, M.; McCarthy, N.; Wilson, D.; Gormley, F.J.; Falush, D.;
Ogden, I.; Maiden, M.; et al. Campylobacter genotyping to determine the source of human infection.
Clin. Infect. Dis. 2009, 48, 1072–1078. [CrossRef] [PubMed]
27. Koolman, L.; Whyte, P.; Burgess, C.; Bolton, D. Distribution of virulence- associated genes in a selection of
Campylobacter isolates. Foodborne Pathol. Dis. 2016, 12, 424–432. [CrossRef] [PubMed]
28. Igimi, S.; Okada, Y.; Ishiwa, A.; Yamasaki, M.; Morisaki, N.; Kubo, Y.; Asakura, H.; Yamamoto, S.
Antimicrobial resistance of Campylobacter: Prevalence and trends in Japan. Food Addit. Contam. 2008,
25, 1080–1083. [CrossRef]
29. Rożynek, E.; Dzierżanowska-Fangrat, K.; Szczepańska, B.; Wardak, S.; Szych, J.; Konieczny, P.; Albrecht, P.;
Dzierżanowska, D. Trends in antimicrobial susceptibility of Campylobacter isolates in Poland (2000–2007).
Polskie Tow. Mikrobiol. Pol. Soc. Microbiol. 2009, 58, 111–115.
30. Jonker, A.; Picard, J. Antimicrobial susceptibility in thermophilic Campylobacter spp. isolated from pigs and
chickens in South Africa. J. S. Afr. Vet. Assoc. 2010, 81, 228–236. [CrossRef] [PubMed]
31. Coker, O.; Adefeso, O. The changing patterns of Campylobacter jejuni/coli in Lagos, Nigeria after ten years.
East African Med. J. 1994, 71, 437–440.
32. Ghunaim, H.; Behnke, J.; Aigha, I.; Sharma, A.; Doiphode, S.; Deshmukh, A.; Abu-Madi, M. Analysis of
Resistance to Antimicrobials and Presence of Virulence/Stress Response Genes in Campylobacter Isolates
from Patients with Severe Diarrhoea. PLoS ONE 2015, 10, e0119268. [CrossRef] [PubMed]
33. Nachamkin, I.; Mishu-Allos, B.; Ho, T. Campylobacter species and Guillain-Barre´ Syndrome. J. Clin. Microbiol.
1998, 11, 555–567.
34. Vacher, S.; Menard, A.; Bernard, E.; Santos, A.; Megraud, F. Detection of mutation associated with macrolide
resistance in thermophilic Campylobacter spp. By real-time PCR. Microb. Drug Resist. 2005, 11, 40–47.
[CrossRef] [PubMed]
35. Qin, S.; Wang, Y.; Zhang, Q.; Chen, X.; Shen, Z.; Deng, F. Identification of a novel genomic island conferring
resistance to multiple amino glycoside antibiotics in Campylobacter coli. Antimicrob. Agents Chemother. 2012,
36. Iovine, N. Resistance mechanisms in Campylobacter jejuni. Virulence 2013, 4, 230–240. [CrossRef] [PubMed]
37. Gibreel, A.; Taylor, D.E. Macrolide resistance in Campylobacter jejuni and Campylobacter coli.
J. Antimicrob. Chemother. 2006, 58, 243–255. [CrossRef] [PubMed]
38. Department of Water Affairs. Revision of general authorizations in terms of Section 39 of the National Water Act,
1998 (At No. 36 of 1998); Published under Government Notice 665 in Government Gazette 36820 No. 665;
Department of Water Affairs: Cape Town, South Africa, 2013.
39. Scharler, M.; Baird, D. A comparison of selected ecosystem attributes of three South African estuaries with
different freshwater inflow regimes using network analysis. J. Mar. Syst. 2005, 56, 283–308. [CrossRef]
40. Baird, D.; Marais, J.; Martin, P. The Swartkops Estuary: Proceedings of a Symposium at the University of Port
Elizabeth; Foundation for Research Development: Pretoria, South Africa, 2007; p. 8.
41. Heip, C.; Herman, P. Major biological processes in European tidal estuaries: A synthesis of the JEEP-92
Project. Hydrobiologia 1995, 311, 1–7. [CrossRef]
42. EPA. Guidelines for Water Reuse; EPA/625/R-04/108; Environmental Protection Agency, Municipal Support
Division Office of Wastewater Management Office of Water: Washington, DC, USA; Agency for International
Development: Washington, DC, USA, 2012.
100
IJERPH 2018, 15, 925
43. Vacher, S.; Menard, A.; Bernard, E.; Mégraud, F. PCR-Restriction Fragment Polymorphism
analysis for detection of point mutations associated with macrolide resistance in Campylobacter spp.
Antimicrob. Agents Chemother. 2003, 47, 1125–1128. [CrossRef] [PubMed]
44. Persson, S.; Olsen, K.E. Multiplex PCR for identification of Campylobacter coli and Campylobacter jejuni from
pure cultures and directly on stool samples. J. Med. Microbiol. 2005, 54, 1043–1047. [CrossRef] [PubMed]
45. Phongsisay, V. Campylobacter jejuni and the Guillain-Barré Syndrome. Ph.D. Thesis, RMIT University,
Melbourne, Australia, 2006.
46. Cohn, M.; Ingmer, H.; Mulholland, F.; Jørgensen, K.; Wells, J.; Brøndsted, L. Contribution of conserved
ATP-dependent proteases of Campylobacter jejuni to stress tolerance and virulence. Appl. Environ. Microbiol.
2007, 73, 7803–7813. [CrossRef] [PubMed]
47. Lin, J.; Overbye Michel, L.; Zhang, Q. CmeABC functions as a multidrug efflux system in Campylobacter
jejuni. Antimicrob. Agents Chemother. 2002, 46, 2124–2131. [CrossRef] [PubMed]
48. CLSI. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of infrequently Isolated or Fastidious
Bacteria, Approved Guidelines-2nd ed.; CLSI: Wayne, PA, USA, 2012.
49. De Vries, S.P.; Gupta, S.; Baig, A.; L’Heureux, J.; Pont, E.; Wolanska, D.P.; Maskell, D.J.; Grant, A.J. Motility
defects in Campylobacter jejuni defined gene deletion mutants caused by second-site mutations. Microbiology
2015, 161, 2316–2327. [CrossRef] [PubMed]
101
and Public Health
Article
Occurrence of Antibiotic-Resistant Bacteria in
Therapy Pools and Surrounding Surfaces
Daniela E. Koeck 1, *, Stefanie Huber 1 , Nadera Hanifi 1 , Manfred Köster 2 ,
Martina B. Schierling 1 and Christiane Höller 1
1 Bavarian Health and Food Safety Authority, Veterinärstraße 2, 85764 Oberschleißheim, Germany;
[email protected] (S.H.); Nadera.hanifi@lgl.bayern.de (N.H.);
[email protected] (M.B.S.); [email protected] (C.H.)
2 Bavarian Health and Food Safety Authority, 91058 Erlangen, Germany; [email protected]
* Correspondence: [email protected]; Tel.: +49-(0)9131-6808-5215
Received: 4 October 2018; Accepted: 21 November 2018; Published: 27 November 2018
Abstract: The number of patients colonized with antibiotic-resistant bacteria is increasing in health
care facilities. Because transmission of antibiotic-resistant bacteria is feared, there exist reports that the
affected patients are frequently excluded from hydrotherapy, which is a non-invasive and beneficial
treatment used for patients with different diseases. Data from the literature suggest that deficient
water disinfection measures exist, which are not always sufficient to kill all released bacteria. If
the pool water is not disinfected properly, it may also infect the bathers. Immunocompromised
patients are particularly susceptible to be infected with (antibiotic-resistant) bacteria. In order to
determine the distribution of antibiotic-resistant bacteria in the pool water treatment system and
the pool environment and to estimate the associated transmission risk we analyzed samples from
eleven health care facilities. Antibiotic-resistant bacteria were found in the water and surface samples
collected. One hundred and two antibiotic-resistant isolates from water samples and 307 isolates
from surrounding surfaces were obtained, respectively. The majority of the isolates belonged to
non-fermenting Gram-negative rods, like Pseudomonas spp. Some isolates were resistant to a wide
range of the tested antibiotics. The results indicate a relation between the number of isolates in
water samples and the number of patients using the pools in combination with deficiencies in water
treatment. In the pool environment the highest number of isolates was obtained from barefoot areas
and floor cleaning equipment.
Keywords: antibiotic resistance; hydrotherapy; pool; water; treatment; transfer
1. Introduction
Emerging and increasing antibiotic microbial resistance (AMR) represents one major threat to
human health in Europe and worldwide. Resistance to antibiotics is widely distributed among
Gram-positive and Gram-negative bacteria that may cause serious infections in humans, and AMR
is increasing in the EU, especially among Gram-negative bacteria. The major drivers behind
the occurrence and spread of AMR are the use of antimicrobial agents and the transmission of
antibiotic-resistant microorganisms between humans; between animals; and between humans, animals,
and the environment [1].
Bacteria that are resistant to three or more classes of antibiotics are called multidrug-resistant.
Infections with these bacteria are associated with increased morbidity, mortality, length of
hospitalization, and financial costs [2]. For a long time, methicillin-resistant Staphylococcus aureus
(MRSA) have been the main point of concern in public health, but during the last couple of years
extended-spectrum-ß-lactamase (ESBL)–producing bacteria have become a much more severe problem.
While MRSA are predominantly acquired in connection with medical treatments, the picture concerning

IJERPH 2018, 15, 2666
multidrug-resistant Gram-negative bacteria is much less clear and differs from species to species [3].
The production of ESBL is the most frequent resistance mechanism among Gram-negative bacteria.
The corresponding gene sequences of the ESBLs are mostly on mobile DNA elements and can be
transmitted horizontally, which contributes to the spread of these resistance genes [4]. Due to
the resistance of the microorganisms to β-lactam antibiotics and other classes of antibiotics such
as the fluoroquinolones (e.g., ciprofloxacin and levofloxacin), the therapeutic spectrum is strongly
restricted in the case of infection with ESBL-producing bacteria [5]. Options for treatment of patients
who are infected with multidrug-resistant bacteria are limited to only a few remaining last-line
antibiotics, such as carbapenems (e.g., imipenem, ertapenem, meropenem). However, the increasing
carbapenem-resistance limits options for the treatment of infected patients [6,7]. For the KRINKO
(German Commission on Hospital Hygiene and Infection Prevention) definition of multidrug-resistant
Gram-negative rods (MRGN), only resistance to antibiotics which are used as primary therapeutics for
severe infections (acylaminopenicillins, third and fourth generation cephalosporins, carbapenems, and
fluoroquinolones) has been included: 3MRGN (with resistance to three of the four antibiotic groups)
and 4MRGN (with resistance to four of the four antibiotic groups). Aminoglycosides, like amikacin,
gentamicin, and tobramycin, were not included in the KRINKO classification of multidrug-resistant
Gram-negative rods, as they are generally not used as monotherapeutics [8].
Hydrotherapy is a non-invasive and beneficial treatment for many patients, like patients with
chronic diseases, disabilities, or trauma. Very often those patients have a long history of medical
treatments, including antibiotic treatments. Thus, the probability that they have a disturbed microflora
with an increased rate of carriage of AMR and also a reduced colonization resistance against
AMR is relatively high [9]. Although pool water is usually disinfected, infections are known
to occur either due to deficiencies in water treatment or due to colonization of swimming pool
equipment [10–13]. Therapies performed in a swimming pool cause a large release of bacteria. Bathers
transfer approximately 105 –106 CFU per person in 15 min to the surrounding water body [14]. Bacteria
should be inactivated by disinfectants in the pool water; however, this is not always the case, because
they can be attached to particles or be protected by an EPS (extrapolymeric substance) and thus not
be exposed to the disinfectant. Bacteria can form biofilms on pool surfaces, especially in areas of
the pool where the concentration of disinfectant is low [15–17]. Additionally, they can be attached
to swimming aids, which are made from plastics, often foams, which provide a large surface. They
can also be found on tools for cleaning, where they are exposed occasionally but not permanently to
disinfectants. Pseudomonas aeruginosa, a species very often involved in biofilm formation, has been
commonly isolated from the pool environment [18,19].
Immunocompromised patients are particularly susceptible to infections with pathogens (including
antibiotic-resistant bacteria) via the mucous membranes and via penetration of bathing water into
auditory canals and the nasopharynx. Although some literature exists on antibiotic-resistant bacteria
in swimming pools [16,17,20], little is known about the prevalence of AMR. To this day, to our
knowledge, no information exists about the public health impact of therapy pools in the dissemination
of antibiotic-resistant bacteria. In order to assess the extent of contamination with antibiotic-resistant
bacteria, their distribution, and the associated transmission risk in clinical therapy pools, we performed
a study on this topic. The main objective of the project was to investigate the occurrence of
antibiotic-resistant bacteria in water of therapy pools, in filters, balance tanks, and on surrounding
surfaces. Factors contributing to their occurrence will be discussed with regard to details in pool
water treatment and disinfection, number of patients entering the pools, and usage of the pools for
other purposes. Finally, we derive recommendations for the management of patients colonized with
antibiotic-resistant bacteria in hydrotherapy pools.
103
IJERPH 2018, 15, 2666
2.1. Sampling
Eleven pre-selected therapy pools located in different hospitals in Bavaria were sampled in
accordance with the requirements of DIN EN ISO 19458 [21]. Bottles with capacities of 250 and
1000 mL, prepared with sufficient (100 mg/L) sodium thiosulfate (Na2 S2 O3 , sodium thiosulfate
pentahydrate, Merck, Darmstadt, Germany) for dechlorination, were used. Pool water and balance
tank water samples were collected from a depth of 30 cm, at a point about 40 cm away from the
basin edge, filtrate was taken from a sampling tap, and filter backwash water was taken directly
before the drain. The samples were transferred to the laboratory within 1–2 h from collection, using
appropriate insulated coolers, and they were processed immediately after arrival at the laboratory.
In addition to the water samples, samples from the surrounding surfaces (especially sanitary areas and
pool equipment) were taken with sterile, wet swabs, moistened with 0.9% NaCl (w/v). All sampling
sites are summarized in Table 1. The swabs were transported to the laboratory immediately after the
collection. In order to isolate microorganisms, the tips of the swabs were cut off and placed in tubes
containing 10 mL of sterile CASO-broth (BD BBL™ Trypticase™ Soy Broth, Becton Dickinson GmbH,
Heidelberg, Germany). The tubes were vortexed (for 2 min) to remove microbial cells from the swab
material and incubated for 24 h +/− 2 h at 37 ◦ C. Four samples of each sampling point (water and
surfaces) were collected over the course of one year (one sampling per quarter).
Table 1. Sampling sites of water samples (left column) and surrounding surfaces (right column).
Water Samples No. Volume Sampling Points in the Pool Surroundings No.
pool water 1 1000 mL swimming aid 1
filtrate 2 100 mL seats 2
balance tank water 3 100 mL spillway 3
filter backwash water 4 100 mL hand rail 4
toilet 5
shower rooms 6
barefoot areas 7
cleaning trolley 8
cleaning equipment 9
2.2. Determination of Water Quality Parameters According to DIN 19643-1

Typical water quality parameters (chemical and microbial) were determined from all water
samples according to DIN 19643-1 [21]. The parameter limits and all references for the methods used
are listed in Table 2.
For the measurement of chlorates and chlorites by LC-MS, a hypercarb column (100 × 2.1 mm,
5 μm, Thermo Scientific, Waltham, MA, USA) was used. The eluents used were deionate/methanol
95/5 with 1% (w/v) formic acid (A) and methanol with 1% (w/v) formic acid (B). The flow rate
was 0.3 mL/min with an injection rate of 10 μL. For subsequent mass spectrometry with the API
5500 device (SCIEX, Darmstadt, Germany), the software package Analyst 1.6.2 (SCIEX, Darmstadt,
Germany) was used. For the measurement, a three-point calibration with bracketing was performed.
The concentrations of the standards were 10, 50, and 100 μg/L. The samples were diluted according to
the calibration line. The internal standard used was 18 O3 -chlorate.
104
Table 2. Parameter limits are according to DIN 19643-1 [21]. Limits are only valid for pool water and filtrate (not for balance tank water and filter backwash water).
Microbial Parameter Method Parameter Limits Chemical Parameter Method Parameter Limits
Total heterotrophic counts at 36 ◦ C TrinkwV, appendix 5 <100 CFU/mL THM (mg/L) DIN EN ISO 10301:1997 <0.02
Escherichia coli DIN EN ISO 9308-2 <1 CFU/100 mL Bromate (mg/L) DIN EN ISO 15061 <2.0
Pseudomonas aeruginosa DIN EN ISO 16266 <1 CFU/100 mL Chlorate and chlorite (mg/L) LC-MS <30
Al (mg/L) DIN EN ISO 11885 <0.05
IJERPH 2018, 15, 2666
Fe (mg/L) DIN EN ISO 11885 <0.02

pH DIN EN ISO 10523 6.5–7.5
KS4.3 (mmol/L) DIN 38409-1 >0.7
l Nitrate (mg/L) EN ISO 10304:1995 <20
Ox. (mg/L) DIN EN ISO 8467 <0.75
Redox (mV) DIN 38404-6 >750
Free chlorine (mg/L) DIN EN ISO 7393-1 0.3–0.6
Bound chlorine (mg/L) DIN EN ISO 7393-1 <0.2
THM: Trihalogenmethane; LC-MS: Liquid chromatography–mass spectrometry.
105
IJERPH 2018, 15, 2666
To determine the acid capacity (KS4.3 ), the samples were titrated with 0.1 mol/L hydrochloric acid
until a pH of 4.3 was reached (DIN 38409-1). The necessary amount of acid was documented and the
acid capacity was calculated as follows:
a × 1000 × 0.1 × f
Ks = (1)
W
where Ks refers to acid capacity, a refers to the titrated volume in mL of 0.1 mol/L HCl, W refers to the
sample volume (100 mL), and f refers to the titer of 0.1 m HCl (=1.000).
The oxidation–reduction potential was recorded on site using the continuously operating
measuring systems the pools applied to control the necessary addition of chemicals. The measurement
was performed with platinum or gold electrodes against a silver/silver chloride reference electrode
and the measured voltage was converted to the standard hydrogen electrode.
2.3. Determination of the Total Number of CFU (Colony Forming Units) from the Pool Surroundings
Contact plates (ICRplus (Isolators and Clean Rooms) TSA (Tryptic Soy Agar) contact plates with
LTHThio neutralizers, Millipore, Darmstadt, Germany) were used to determine the total number of
CFU from the surrounding surfaces. All sampling sites are summarized in Table 1. The agar plates
were incubated for 24 h +/− 2 h at 37 ◦ C. It is important to ensure that sampling is carried out at
representative and similar sites to ensure comparability between the therapy pools. Based on the
DGfdB (German Society for Bathing) guideline 94.04 [22], it can be determined whether or not the
surface cleaning and disinfection was performed adequately.
2.4. Isolation of Antibiotic-Resistant Bacteria

The abovementioned water samples (1000 mL or 100 mL, respectively; Table 1) were filtrated
through sterile 0.45 μm membrane filters (Millipore, Darmstadt, Germany). Filters were subsequently
placed on MacConkey agar (Oxoid, Wesel, Germany) supplemented with cefotaxime (1 mg/L),
Brilliance carbapenem-resistant Enterobacteriaceae (CRE) Agar (Oxoid), ChromID™ VRE Agar
(bioMérieux, Nurtingen, Germany), and ChromIDTM MRSA SMART Agar (bioMérieux, Nurtingen,
Germany), and incubated for 24 h +/− 2 h at 37 ◦ C under aerobic conditions. Next, 200 μL from the
liquid enrichments of the culture swabs were plated on the same selective media and incubated for
24 h +/− 2 h at 37 ◦ C. One colony of each phenotype was picked from the selective plates, subcultured
on 10% defibrinated sheep blood agar (Oxoid), and incubated at 37 ◦ C for 24 h. Identification to
the species or genus level was performed using BD Phoenix™ 100 (Becton Dickinson Diagnostic
systems, Heidelberg, Germany) and MALDI-TOF-MS Microflex LT (Bruker, Bremen, Germany). The
MALDI Biotyper Real Time Classification System (Bruker) and the BD EpiCenterTM Software (Becton
Dickinson, Heidelberg, Germany) were used for the identification and taxonomical classification
of bacteria.
2.5. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing was performed by Phoenix™ Panels NMIC 448794 (Becton
Dickinson Diagnostic systems, Heidelberg, Germany) for Gram-negative bacteria, with 21 antimicrobial
substances (ampicillin, piperacillin, piperacillin-tazobactam, cefuroxime, cefotaxime, ceftazidime,
cefepime, imipenem, meropenem, aztreonam, gentamicin, tobramycin, amikacin, ciprofloxacin,
levofloxacin, trimethoprim-sulfamethoxazole, amoxicillin-clavulanic acid, ertapenem, fosfomycin+G6P,
tigecycline) and Panel NMIC 448796 for Gram-positive bacteria, with 23 antimicrobial substances
(penicillin G, ampicillin, oxacillin, cefoxitin, imipenem, clindamycin, erythromycin, vancomycin,
teicoplanin, linezolid, fusidic acid, rifampicin, nitrofurantoin, gentamycin, tobramycin, ciprofloxacin,
moxifloxacin, tetracycline, trimethoprim-sulfamethoxazole, daptomycin, fosfomycin, gentamicin-syn,
tigecycline) according to the manufacturer’s guidelines (Becton Dickinson Diagnostic Systems,
Heidelberg, Germany). Results of all antimicrobials tested were interpreted according to the European
106
IJERPH 2018, 15, 2666
Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (http://www.eucast.org/

clinical_breakpoints). The calculation of MIC50 (representing the minimal inhibitory concentration
(MIC) of 50% of the isolates) and MIC90 (representing the MIC of 90% of the isolates) were calculated
using the obtained values from Phoenix™.
2.6. Questionnaire
A standardized questionnaire was developed and given to the facilities operating the therapy
pools to document the technical details of the pool, their water treatment, cleaning procedures, and
frequency and duration of pool usage (Supplementary Materials S1).

Pearson’s χ2 test was used to test whether observed differences in contamination of water samples
and surface samples between the sampling locations were statistically significant. An interactive
calculation tool for chi-square tests (available from http://quantpsy.org) was employed for all statistical
analyses and the significance level was set at 95% (p ≤ 0.05) for all analyses.
3. Results
3.1. Water Quality Parameters According to DIN 19643-1

The number of bathers/patients using the pool clearly differed between the sampled facilities
(ranging from <50 patients per year to 35,000 patients per year). None of the pools exceeded the
maximal bathing load per hour (according to DIN 19643-1 [21]). The chemistry of the pool water
samples is shown in Table 3. Location six and eight were only sampled twice and location ten only
three times, because the bathing facilities were closed down during the study. The results of the filtrate,
balance tank water, and filter backwash water samples are not shown, since there are no standard
requirements specified in DIN 19643-1 [21]. The filtrate should only be investigated in case of problems
with the pool water. Most of the chemical parameters of the pool water met the requirements. Only the
acid capacity was often (59%) too low, which is an indicator for a low puffer capacity and may hinder
a proper flocculation. The aluminum concentration was above the limit value in 64% of the sampled
basins, which is an indication of a flocculation failure due to too much flocculant or an inadequate
flocculation (e.g., due to insufficient mixing). Furthermore, a relationship was observed between the
high number of visitors (indicated in grey) in pool number two and eight and a distinctly higher
concentration of bound and free chlorine.
The microbiological quality of the investigated pools was considered acceptable/unacceptable
according to the German standards DIN 19643 [21]. The limits are valid for pool water and filtrate
(not for balance tank water and filter backwash water). All pool water samples examined met
the microbiological standards specified in Table 2. We detected three exceedances in the filtrate:
>12,600/mL CFU total heterotrophic counts (36 ◦ C) and >200 CFU/100 mL P. aeruginosa in one sample
and again 108/mL CFU total heterotrophic counts (36 ◦ C) in another sample. Both samples came from
facility number ten. E. coli was not detected in any of the samples analyzed.
107
IJERPH 2018, 15, 2666
Table 3. Chemistry in pool water samples (according to DIN 19643-1) and number of visitors per year
(date from the questionnaire).
Hospital Number 1 2 3 4 5 6 7 8 9 10 11
Visitors/per year 2500 7200 4500 4500 10,800 1680 1450 35,000 3000 <50 300
THM (mg/L) 0.0 0.007 0.006 0.000 0.005 0.000 0.001 0.008 0.000 0.002 0.000
Bromate (mg/L) 0.055 <LOD <LOQ 0.009 0.011 0.054 0.003 0.005 0.005 0.004 0.005
Chlorate and chlorite
18.0 2.0 0.8 3.2 2.1 33.5 a 9.0 4.7 5.4 16.1 9.2
(mg/L)
Al (mg/L) 0.06 a 0.07 a <LOQ 0.07 a <LOQ 0.07 a <LOQ 0.08 a <LOQ 0.1 a 0.08 a
Fe (mg/L) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.0 <LOQ
pH 6.7 7.0 7.0 7.2 7.3 6.7 7.1 7.2 6.7 7.2 7.5
KS4.3 (mmol/L) 0.3 b 0.9 0.6 b 0.9 0.71 0.2 b 0.4 b 1.0 0.2 b 0.5 b 1.3
Nitrate (mg/L) 10.8 7.5 28.8 a 26.8 a 14.3 30.0 a <NG 3.5 13.3 30.7 a 14.3
Ox (mg/L) 0.7 0.5 0.7 0.5 1.4 <LOD 0.4 0.9 a 0.3 0.3 1.0 a
Redox (mV) 780.80 821.50 806.00 738.30 b 819.30 767.00 675.00 b 716.30 b 823.00 803.30 919.00
Free chlorine (mg/L) 0.51 0.72 a 0.40 0.27 b 0.50 0.33 0.54 0.77 a 0.46 0.49 0.59
Bound chlorine (mg/L) 0.11 0.15 0.06 0.06 0.06 0.01 0.03 0.25 a 0.04 0.02 0.02
<LOD = below limit of detection; <LOQ = below limit of quantification; a above requirements acc. DIN 19643-1;
b below requirements acc. DIN 19643-1 [21].
3.2. Determination of Total Heterotrophic Counts from the Pool Surroundings

An analysis of the samples revealed that 78% of the total plate count samples (370) showed
bacterial growth, i.e., were counted as positive (Table 4). One hundred and seventeen samples (32%)
exceeded the action value (800 CFU/100 cm2 ) from the DGfdB guideline 94.04 [22]. However, as
our samples were taken when bathers were present, these results should be interpreted with caution,
because the guideline values refer to areas after cleaning and/or disinfection in order to determine if
the surface cleaning and disinfection has been performed adequately.
Table 4. Number of positive samples from contact agar plates and percentage of positive samples
(n = 370).
Hospital Number 1 2 3 4 5 6 7 8 9 10 11 % Positive Samples

swimming aid 4 2 2 4 4 1 2 1 3 3 3 66
seats 4 4 0 4 4 2 4 2 4 3 3 77
spillway 1 3 4 1 4 1 1 1 1 2 1 45
hand rail 4 4 2 4 4 2 4 2 4 2 3 80
toilet 3 3 1 4 3 2 3 2 4 3 3 70
shower rooms 2 2 1 4 4 2 4 2 3 3 3 68
barefoot areas 4 3 3 4 3 2 4 1 4 3 3 77
cleaning trolley 3 3 4 3 2 1 3 1 3 2 ND 57
cleaning equipment 1 4 3 4 3 2 4 2 4 4 ND 70
% positive samples 72 78 56 89 86 83 81 78 83 69 70
ND = not determined.
3.3. Bacterial Isolates

A total of 307 isolates from 23% positive samples (growth on selective media) were obtained
from all surface samples (n = 371). A significant difference between the surface samples across the
sampled pools was observed (X2 = 200.138, p-value = 0). Most isolates were obtained from hospital
number eight, which was only sampled twice (Figure 1). Hospital number two and five, which
have the second and third highest number of pool visitors per year, also yielded a high number of
isolates. A relationship was observed between the high number of visitors (Table 3) and the number
of isolates. The highest number of isolates was obtained from barefoot areas (78) and floor cleaning
equipment (49).
108
IJERPH 2018, 15, 2666
Figure 1. Number of antibiotic-resistant isolates (growth on selective media) from the surrounding
surface samples (n = 371).
A total of 102 isolates from 32% positive samples (growth on selective media) were obtained from
all water samples (n = 155) (Figure 2). A significant difference between the water samples across the
sampled pools was observed (X2 = 52.778, p-value = 0.002). The two therapy pools with the highest
number of isolates (number five and number eight) also had the highest number of visitors, even if
pool number eight was only sampled twice. Most of the positive water samples were from the balance
water and filtrate; the pool water itself was contaminated less frequently. Isolates could be obtained
directly from the pool water in only pool number two, six, eight, nine, and ten.
Figure 2. Number of antibiotic-resistant isolates (growth on selective media) from the water samples
(n = 155).
109
IJERPH 2018, 15, 2666
Not all isolates could be classified up to species level (Figure 3). For genetically closely related
species the applied identification methods (BD Phoenix™ and MALDI-TOF-MS) only allowed
assignment to the genus level. The majority of the isolates belonged to the taxonomically heterogeneous
group of non-fermenting Gram-negative bacteria. Typical genera like Burkholderia spp., Moraxella
spp., Pseudomonas spp., Stenotrophomonas spp., and Sphingomonas spp. were found. Some of them
are typical water borne bacteria, like Pseudomonas spp. E. coli was not isolated and other coliform
bacteria were very rare. Acinetobacter spp. were also not isolated. The abundant Gram-positive
genera are mostly inhabitants of the natural skin flora (like Staphylococcus epidermidis) or environmental
bacteria (like Bacillus subtilis). There was only one S. aureus isolate from a handrail, which was oxacillin
sensitive. Some genera appeared in high abundances only in water samples (like Sphingomonas and
Sphingobacterium) and some were mainly found in environmental samples (like Stenotrophomonas,
Bacillus, Achromobacter, Ochrobactrum). Pseudomonas spp. is one of the most abundant genera in
both kinds of samples. Isolates from the pool water (n = 14) were mostly Pseudomonas spp. (n = 3),
Sphingobacterium spp. (n = 4), and Staphylococcus spp. (n = 4).
100%
18.6 14.2
90%
<2%
13.3
80% 2.9 Streptococcus
9.8 Stenotrophomonas
70% Staphylococcus
24.3
11.8 Sphingomonas
60% Sphingobacterium
Pseudomonas
5.9
50% Ochrobactrum
23.5 21.7 Microbacterium
40% Enterobacter
Cupriavidus
30% Chryseobacterium
2.6 Burkholderia
11.8 5.5
20% Brevundimonas
3.9
Bacillus
8.4
10% 4.9 Achromobacter
3.9
3.9 6.1
0%
Water samples % Surface samples %
Figure 3. Taxonomic profile up to genus level for all antibiotic-resistant isolates. Left column:
percentage of water isolates (n = 102); right column: percentage of isolates from surfaces (n = 307).
Other genera present in <2%. Identification to the species or genus level was performed using BD
Phoenix™ and MALDI-TOF-MS.
110
IJERPH 2018, 15, 2666
3.4. Antimicrobial Susceptibility

The isolated Gram-positive genera have no or very rare clinical relevance, therefore the antibiotic
resistance patterns (antibiograms) of Gram-positive isolates were not further analyzed. There were
abundant Gram-negative genera, like Pseudomonas, Stenotrophomonas, and Sphingomonas, which can
cause infections especially in immunocompromised patients; their antibiograms are shown in Table 5.
Sphingobacterium and Achromobacter are the two other abundant Gram-negative genera. These are
rarely involved in human infections; their resistance patterns are shown in Table 6. For these five
bacteria genera, the MIC50 (minimal inhibitory concentration (MIC) for 50% of the isolates) and the
MIC90 (MIC for 90% of the isolates) were calculated using the obtained values from the PhoenixTM
experiments (Tables 7 and 8).
The antibiograms of other Pseudomonas species (e.g., P. putida) isolates (n = 136), are not shown, as
they are seldom associated with infections in humans. The total abundance of all Pseudomonas aeruginosa
isolates was 5%. Among these isolates five were resistant to ciprofloxacin (MIC50: 0.25 μg/mL; MIC90:
2 μg/mL) and levofloxacin (MIC50: 1 μg/mL; MIC90: 4 μg/mL). Three P. aeruginosa isolates were
simultaneously resistant to imipenem (MIC50: 2 μg/mL; MIC90: 8 μg/mL), ertapenem (MIC50:
2 μg/mL; MIC90: 2 μg/mL) and all tested fluoroquinolones. But all P. aeruginosa isolates were
susceptible against piperacillin, ceftazidime and cefepime. The 39 Stenotrophomonas maltophilia
isolates were resistant to almost all tested antibiotic substances. All isolates were susceptible against
trimethoprim-sulfamethoxazole. The susceptibility against polymyxins was not investigated. Among
the 14 Sphingomonas paucimobilis isolates there existed a high diversity between the resistance patterns.
One isolate was susceptible to at least one antibiotic substance of all classes of antibiotics. The majority
of the isolates was resistant to all tested carbapenems and sensitive to other classes. But there was also
one isolate, which was resistant to 4 of 4 antibiotic groups from the KRINKO classification for MRGN.
The isolates from the genus Achromobacter could not be resolved up to species level, as the
databases lack appropriate reference spectra. All Sphingobacterium spp. isolates belong either to
the species S. spiritivorum or S. multivorum. Some isolates were resistant to a wide range of the
tested antibiotics.
111
Table 5. Antibiotic resistance patterns of all Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Sphingomonas paucimobilis isolates from water and
surface samples.
Class of Antibiotics Antibiotic

ampicillin r r r r r r r s r r r r r s s
Penicillins piperacillin s s s s r r r s s r s s r r s
IJERPH 2018, 15, 2666
piperacillin-tazobactam s s s s r r r s s s s s r r s
Penicillins/ Beta-lactamase inhibitor amoxicillin-clavulanic acid r r r r r r r r r r r r r r r
Cephalosporins (2nd) cefuroxime r r r r r r r r r r r r r r r
Cephalosporins (3rd) cefotaxime r r r r r r r r r r r r r r r
Cephalosporins (3rd) ceftazidime s s s s r r r s s s s s r s s
Cephalosporins (4th) cefepime s s s s r r r s s s s s r s s
imipenem s r s r r r r r r r r r r s s
Carbapenems
ertapenem r r r r r r r r r r r r r r r
gentamicin s s s r r r r r r r r r s s s
Aminoglycosides tobramycin s s s s r r r r r r r r s s r
amikacin s s s s r r r r r r r r s s s
ciprofloxacin s r r s s r r s s s s r r s s
Fluoroquinolones
112
levofloxacin s r r s s s r s s s s s r s s
Sulfonamide antibiotic trimethoprim-sulfam. r r r r s s s r r r s r s s s
Fosfomycin fosfomycin ND ND ND ND r r r s s r s r s r r
Glycylcycline tigecycline r r r r r r r ND ND ND ND ND ND ND ND
Number of Isolates 16 3 2 2 2 13 24 6 2 1 1 1 1 1 1
Species Pseudomonas aeruginosa (26) Stenotrophomonas maltophilia (39) Sphingomonas paucimobilis (14)
Susceptibility (s) and resistance (r) were automatically determined with the BD Phoenix™ System; ND = Not determined.
Table 6. Antibiotic resistance patterns of all Achromobacter spp. and Sphingobacterium spp. isolates from water and surface samples.

ampicillin r r ND r r r r r r r r r s
Penicillins piperacillin s s s s s s r s s r r r s
piperacillin-tazobactam s s s s s r s s s s r s s
IJERPH 2018, 15, 2666
Penicillins/Beta-lactamase inhibitor amoxicillin-clavulanic acid r r r r r r r r r r r r r

Cephalosporins (2nd) cefuroxime r r r r r r r r r r r r r
Cephalosporins (3rd) cefotaxime r r r r r r r r r r r r r
Cephalosporins (3rd) ceftazidime s s s s r r r s s r r r s
Cephalosporins (4th) cefepime s s s r r r r s s s r s s
imipenem ND ND ND ND ND ND ND r s s s r s
Carbapenems
ertapenem ND ND ND ND ND ND ND r r r r r r
gentamicin s s r r r r r s r r r r s
Aminoglycosides tobramycin s s r r r r r r r r r r r
amikacin s s s r r r r s r r r r s
113
ciprofloxacin s r r r r r r s s s s s s
Fluoroquinolones
levofloxacin s r s r r r s s s s s s s
Sulfonamide antibiotic trimethoprim-sulfam. s s s s s s s s s s s s s
Fosfomycin fosfomycin r r r r r r r r r r r r r
Glycylcycline tigecycline ND ND ND r r r ND ND ND r ND r ND
Number of Isolates 1 2 1 10 3 1 1 1 4 1 3 2 1
Species Achromobacter spp. (19) Sphingobacterium spp. (11)
Susceptibility (s) and resistance (r) was automatically determined with the BD Phoenix™ System; ND = Not determined.
Table 7. MIC50 and MIC90 values of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Sphingomonas paucimobilis isolates from water and surface areas.
Pseudomonas aeruginosa Stenotrophomonas maltophilia Sphingomonas paucimobilis

MIC50 MIC90 MIC50 MIC90 MIC50 MIC90
ampicillin 16 16 16 16 8 16
Penicillins piperacillin 4 8 32 32 4 32
IJERPH 2018, 15, 2666
piperacillin-tazobactam 4/4 8/4 32/4 32/4 4/4 29.2/4

Penicillins/beta-lactamase inhibitor amoxicillin-clavulanic acid 64/2 64/2 64/2 64/2 32/2 60.8/2
Cephalosporins (2nd) cefuroxime 16 16 16 16 16 16
Cephalosporins (3rd) cefotaxime 8 8 8 8 8 8
Cephalosporins (3rd) ceftazidime 2 4 8 16 4 8
Cephalosporins (4th) cefepime 4 4 16 16 1 2
imipenem 2 8 16 16 16 16
Carbapenems
ertapenem 2 2 2 2 2 2
gentamicin 1 2 8 8 8 8
Aminoglycosides tobramycin 1 1 8 8 8 8
amikacin 4 4 32 32 32 32
114
ciprofloxacin 0.25 2 2 2 0.5 0.9
Fluoroquinolones
levofloxacin 1 4 1 4 0.5 0.5
Sulfonamide antibiotic trimethroprim-sulfam. 4/76 8/76 1/19 1/19 4/76 7.6/76
Fosfomycin fosfomycin ND ND >64 >64 32 64
Glycylcycline tigecycline 4 4 1 2 ND ND
The MIC values used for calculation of the MIC50 and MIC90 were determined with the BD PhoenixTM System; ND = not determined.
Table 8. MIC50 and MIC90 values of Achromobacter spp. and Sphingobacterium spp. isolates from water and surface areas.
Achromobacter spp. Sphingobacterium spp.

MIC50 MIC90 MIC50 MIC90
ampicillin 16 16 16 16
Penicillins piperacillin 4 5.2 16 32
IJERPH 2018, 15, 2666
piperacillin-tazobactam 4/4 4/4 8/4 32/4

Penicillins/Beta-lactamase inhibitor amoxicillin-clavulanic acid 16/2 64/2 4/2 4.4/2
Cephalosporins (2nd) cefuroxime 16 16 16 16
Cephalosporins (3rd) cefotaxime 8 8 8 8
Cephalosporins (3rd) ceftazidime 8 16 4 16
Cephalosporins (4th) cefepime 16 16 1 16
imipenem ND ND 4 8
Carbapenems
ertapenem ND ND 2 2
gentamicin 8 8 8 8
Aminoglycosides tobramycin 8 8 8 8
115
amikacin 32 32 32 32
ciprofloxacin 2 2 0.25 0.5
Fluoroquinolones
levofloxacin 2 4 0.5 0.5
Sulfonamide antibiotic trimethoprim-sulfam. 1/19 1/19 1/19 1/19
Fosfomycin fosfomycin >64 >64 >64 >64
Glycylcycline tigecycline 2 2 1* 1*
The MIC values used for calculation of the MIC50 and MIC90 were determined with the BD PhoenixTM System; ND = not determined. * calculated from three values.
IJERPH 2018, 15, 2666
4. Discussion
The therapy pool with the highest number of isolates obtained directly from the pool water
and from the sampled surfaces had not only the highest number of visitors but also seemed to
have problems with the water treatment (high bound chlorine levels). The cleaning intervals of the
pool areas were the same between the different health care facilities (once per day, according to the
evaluation of the questionnaire); hence there is no detectable correlation between the number of isolates
and the cleaning interval. These results indicate a correlation of the incidence of antibiotic-resistant
bacterial isolates with the number of patients in combination with deficiencies in water treatment. The
isolation of potential human pathogens, particularly P. aeruginosa, S. maltophilia, and S. paucimobilis
strains, indicates that these inhabitants of the nosocomial environment may have been released by
bathers, with contact to surfaces in the surrounding of the pool and the hospital environment, after
entering the pool. P. aeruginosa can potentially cause disease in healthy humans, but more often it
colonizes immunocompromised patients, like those with cystic fibrosis or cancer [23]. P. aeruginosa is
intrinsically resistant to the majority of antimicrobial agents due to the low permeability of its outer
membrane and the constitutive expression of various efflux pumps. Any additional acquired resistance
severely limits the therapeutic options for treating serious infections. The antimicrobial groups that
remain active against the susceptible P. aeruginosa phenotype include some fluoroquinolones (e.g.,
ciprofloxacin and levofloxacin), aminoglycosides (e.g., gentamicin, tobramycin, and amikacin), some
beta-lactams (piperacillin- tazobactam, ceftazidime, cefepime, imipenem, doripenem, and meropenem),
and polymyxins (polymyxin B and colistin). Resistance of P. aeruginosa to these agents can be
acquired through one or more of several mechanisms, like the acquisition of plasmid-mediated
resistance genes coding for various β-lactamases and aminoglycoside-modifying enzymes [24,25].
Some strains that have been isolated exhibited resistance to essentially antipseudomonal antibiotics,
like fluoroquinolones and carbapenems.
Another problematic nosocomial pathogen is S. maltophilia, which is also naturally resistant
to many broad-spectrum antibiotics (including all carbapenems). S. maltophilia is the third most
common nosocomial pathogen with multi-drug-resistance [26]. S. maltophilia is often associated with
pulmonary infections, urinary tract infections, bloodstream infections, and colonization of individuals
with cystic fibrosis, especially in immunocompromised patients. The treatment of infected patients
is very difficult [27,28]. It can be considered positive that all S. maltophilia isolates were susceptible
against trimethoprim-sulfamethoxazole, as trimethoprim-sulfamethoxazole is still the treatment of
choice for suspected or culture-proven S. maltophilia infections [29]. If a patient is infected with one
of these strains, polymyxins may also be effective treatment options, though not without frequent
adverse effects.
Sphingomonas paucimobilis has been implicated in various types of clinical infection. Although
infections by S. paucimobilis are rarely serious and could be effectively treated with antibiotics,
S. paucimobilis is capable of causing active infections in humans [30,31]. However, 93% of the isolates
in this study were resistant to aminoglycosides, and one isolate was resistant to ceftazidime. Another
study describes carbapenems, against which all isolates were resistant, as the most effective therapy
for infections with S. paucimobilis [32]. Achromobacter spp. have been identified as opportunistic
human pathogens in people with certain immunosuppressive conditions, such as cystic fibrosis,
cancer, and kidney failure [33]. Notably, 80% of the isolates originated from barefoot areas or the
floor cleaning equipment, which indicates a transmission due to insufficient management of floor
cleaning equipment.
Like Achromobacter spp., Sphingobacterium spiritivorum/multivorum is rarely involved in human
infection. Sphingobacterium species are intrinsically resistant to many commonly used antibiotics
and can grow in antiseptics and disinfectants [34]. S. multivorum can produce an extended-spectrum
β-lactamase and a metallo-β-lactamase, conferring resistance to third-generation cephalosporins
and carbapenems, respectively [35]. Two isolates were both resistant to carbapenems and
third-generation cephalosporins.
116
IJERPH 2018, 15, 2666
In addition to these quite important human pathogens, some uncommon antibiotic-resistant

Gram-negative bacterial species, like Chryseobacterium indologenes and Ochrobactrum anthropi, were
isolated. The isolation of Chryseobacterium indologenes and Ochrobactrum anthropi from swimming pool
water was described previously by Papadopoulou et al. [17]. In this study, Ochrobactrum anthropi could
only be isolated from the surface samples in the pool surroundings and not from the water samples.
For both species, rare clinical significance and resistance to a wide variety of antimicrobial agents has
been reported [36,37].
However, whether such resistant strains can contaminate bathers and cause infection strongly
depends on the immune status of the patient. The study has revealed deficiencies in the operation of
the pools, although the extent to which immunocompetent patients may be at risk from multidrug
resistant bacteria in the pool water could not be determined. The question of whether patients who are
proven carriers of multidrug resistant bacteria may use therapy pools has to be clarified in individual
cases, taking the respective bacterial species into account. In the case of unsafe operation or outdated
technology and patients colonized with 4MRGN, a ban on use should be considered.
5. Conclusions
Despite the reduction of antibiotic-resistant bacteria due to water treatment and disinfection,
some antibiotic-resistant bacteria are still present in the water of therapy pools and on surrounding
surfaces. There they can potentially persist and infect other patients and staff alike. Adequate pool
water treatment and management of cleaning and cleaning equipment can prevent the transmission of
these bacteria. The capacity of the water treatment defines the maximum bathers load. This maximum
number of visitors should not be exceeded to ensure good water quality.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/15/12/2666/

s1, S1: questionnaire for the study “resistant pathogens in hospital swimming pools”, S2: raw data.
Author Contributions: All authors made substantial contributions to study conception and the interpretation of
data. The analyses were carried out by N.H., M.K., M.B.S., and D.E.K. and the drafting of the manuscript was
carried out by D.E.K. All authors read and approved the final version of the manuscript.
Funding: This study was not supported by any external funding.
Acknowledgments: The authors wish to thank Laura Perez Alleres, Marijana Duilo-Bening, Christiane Christian,
Sylvia Kühnel, Angelika Mahr, and Christiane Strauss for their excellent technical assistance.
References
1. European Centre for Disease Prevention and Control, European Food Safety Authority, European Medicines
Agency. ECDC/EFSA/EMA first joint report on the integrated analysis of the consumption of antimicrobial
agents and occurrence of antimicrobial resistance in bacteria from humans and foodproducing animals.
EFSA J. 2015, 13, 114. [CrossRef]
2. Cerceo, E.; Deitelzweig, S.B.; Sherman, B.M.; Amin, A.N. Multidrug-resistant gram-negative bacterial
infections in the hospital setting: Overview, implications for clinical practice, and emerging treatment
options. Microb. Drug Resist. 2016, 22, 412–431. [CrossRef] [PubMed]
3. Valenza, G.; Nickel, S.; Pfeifer, Y.; Eller, C.; Krupa, E.; Lehner-Reindl, V.; Holler, C. Extended-spectrum-
beta-lactamase-producing Escherichia coli as intestinal colonizers in the German community.
Antimicrob. Agents Chemother. 2014, 58, 1228–1230. [CrossRef] [PubMed]
4. Munita, J.M.; Arias, C.A. Mechanisms of antibiotic resistance. Microbiol. Spectr. 2016, 4. [CrossRef]
5. Ramphal, R.; Ambrose, P.G. Extended-spectrum beta-lactamases and clinical outcomes: Current data.
Clin. Infect. Dis. 2006, 42 (Suppl. 4), S164–S172. [CrossRef]
6. European Centre for Disease Prevention and Control. ECDC publishes a directory of online resources for
prevention and control of antimicrobial resistance and healthcare-associated infections. Eur. Surveill. 2014, 19.
[CrossRef]
117
IJERPH 2018, 15, 2666
7. World Health Organization. Critically Important Antimicrobials for Human Medicine, 5th Revision; World Health
Organization: Geneva, Switzerland, 2017.
8. Empfehlung der Kommission für Kranken- haushygiene und Infektionsprävention (KRINKO) beim
Robert Koch-Institut (RKI). Hygienemaßnahmen bei Infektionen oder Besiedlung mit multiresistenten
gramnegativen Stäbchen. Bundesgesundheitsbl 2012, 55, 1311–1354. [CrossRef] [PubMed]
9. Sassone-Corsi, M.; Raffatellu, M. No vacancy: How beneficial microbes cooperate with immunity to provide
colonization resistance to pathogens. J. Immunol. 2015, 194, 4081–4087. [CrossRef] [PubMed]
10. Hollyoak, V.; Allison, D.; Summers, J. Pseudomonas aeruginosa wound infection associated with a nursing
home’s whirlpool bath. Commun. Dis. Rep. CDR Rev. 1995, 5, R100–R102. [PubMed]
11. Rice, S.A.; van den Akker, B.; Pomati, F.; Roser, D. A risk assessment of Pseudomonas aeruginosa in swimming
pools: A review. J. Water Health 2012, 10, 181–196. [CrossRef] [PubMed]
12. Schlech, W.F., III; Simonsen, N.; Sumarah, R.; Martin, R.S. Nosocomial outbreak of Pseudomonas aeruginosa
folliculitis associated with a physiotherapy pool. CMAJ 1986, 134, 909–913. [PubMed]
13. Tate, D.; Mawer, S.; Newton, A. Outbreak of Pseudomonas aeruginosa folliculitis associated with a swimming
pool inflatable. Epidemiol. Infect. 2003, 130, 187–192. [CrossRef] [PubMed]
14. Elmir, S.M.; Wright, M.E.; Abdelzaher, A.; Solo-Gabriele, H.M.; Fleming, L.E.; Miller, G.; Rybolowik, M.;
Peter Shih, M.T.; Pillai, S.P.; Cooper, J.A.; et al. Quantitative evaluation of bacteria released by bathers in a
marine water. Water Res. 2007, 41, 3–10. [CrossRef] [PubMed]
15. Friedman, M.S.; Roels, T.; Koehler, J.E.; Feldman, L.; Bibb, W.F.; Blake, P. Escherichia coli O157:H7 outbreak
associated with an improperly chlorinated swimming pool. Clin. Infect. Dis. 1999, 29, 298–303. [CrossRef]
[PubMed]
16. Kahanov, L.; Kim, Y.K.; Eberman, L.; Dannelly, K.; Kaur, H.; Ramalinga, A. Staphylococcus aureus and
community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in and around therapeutic
whirlpools in college athletic training rooms. J. Athletic Train. 2015, 50, 432–437. [CrossRef] [PubMed]
17. Papadopoulou, C.; Economou, V.; Sakkas, H.; Gousia, P.; Giannakopoulos, X.; Dontorou, C.; Filioussis, G.;
Gessouli, H.; Karanis, P.; Leveidiotou, S. Microbiological quality of indoor and outdoor swimming pools in
Greece: Investigation of the antibiotic resistance of the bacterial isolates. Int. J. Hyg. Environ. Health 2008,
18. Pereira, S.G.; Paixao, J.; Leitao, R.; Cardoso, O. Pseudomonas aeruginosa in a hydropathic facility: Diversity,
susceptibility and imipenem resistance mutation. Lett. Appl. Microbiol. 2011, 53, 518–524. [CrossRef]
[PubMed]
19. Schets, F.M.; van den Berg, H.H.; Baan, R.; Lynch, G.; de Roda Husman, A.M. Pseudomonas aeruginosa
on vinyl-canvas inflatables and foam teaching aids in swimming pools. J. Water Health 2014, 12, 772–781.
[CrossRef] [PubMed]
20. Tolba, O.; Loughrey, A.; Goldsmith, C.E.; Millar, B.C.; Rooney, P.J.; Moore, J.E. Survival of epidemic strains of
healthcare (HA-MRSA) and community-associated (CA-MRSA) meticillin-resistant Staphylococcus aureus
(MRSA) in river-, sea- and swimming pool water. Int. J. Hyg. Environ. Health 2008, 211, 398–402. [CrossRef]
[PubMed]
21. DIN 19643:2012-11, Aufbereitung von Schwimm- und Badebeckenwasser—Teil 1: Allgemeine
Anforderungen. Available online: https://www.beuth.de/de/norm/din-19643-1/164174095 (accessed
on 27 November 2018).
22. DGfdB Guideline R 94.04:2013-12, Reinigung, Desinfektion und Hygiene in Bädern. Available
online: http://www.drnuesken.de/uploads/media/94.04_Reinigung__Desinfektion_und_Hygiene_in_
Baedern_Blaudruck_2013.pdf (accessed on 27 November 2018).
23. Botzenhardt, K.; Döring, G. Ecology and Epidemiology of Pseudomonas aeruginosa; Campa, M., Bendinelli, M.,
Friedman, H., Eds.; Springer: Boston, MA, USA, 1993.
24. Eurosurveillance editorial team. ECDC publishes 2014 surveillance data on antimicrobial resistance and
antimicrobial consumption in Europe. Eur. Surveill. 2015, 20. [CrossRef]
25. Strateva, T.; Yordanov, D. Pseudomonas aeruginosa—A phenomenon of bacterial resistance. J. Med. Microbiol.
2009, 58, 1133–1148. [CrossRef] [PubMed]
26. Denton, M.; Kerr, K.G. Microbiological and clinical aspects of infection associated with Stenotrophomonas
maltophilia. Clin. Microbiol. Rev. 1998, 11, 57–80. [CrossRef] [PubMed]
118
IJERPH 2018, 15, 2666
27. McGowan, J.E., Jr. Resistance in nonfermenting gram-negative bacteria: Multidrug resistance to the
maximum. Am. J. Infect. Control 2006, 34, S29–S37. [CrossRef] [PubMed]
28. Waters, V.G.M.; Soong, G.; Amin, S.; Ernst, R.; Prince, A. Immunostimulatory properties of the emerging
pathogen Stenotrophomonas maltophilia. Infect. Immun. 2007, 75, 1698–1703. [CrossRef] [PubMed]
29. Abbott, I.J.; Slavin, M.A.; Turnidge, J.D.; Thursky, K.A.; Worth, L.J. Stenotrophomonas maltophilia: Emerging
disease patterns and challenges for treatment. Expert Rev. Anti-Infect. Ther. 2011, 9, 471–488. [CrossRef]
[PubMed]
30. Lin, J.N.; Lai, C.H.; Chen, Y.H.; Lin, H.L.; Huang, C.K.; Chen, W.F.; Wang, J.L.; Chung, H.C.; Liang, S.H.;
Lin, H.H. Sphingomonas paucimobilis bacteremia in humans: 16 case reports and a literature review. J. Microbiol.
Immunol. Infect. 2010, 43, 35–42. [CrossRef]
31. Ryan, M.P.; Adley, C.C. Sphingomonas paucimobilis: A persistent Gram-negative nosocomial infectious
organism. J. Hosp. Infect. 2010, 75, 153–157. [CrossRef] [PubMed]
32. Bayram, N.; Devrim, I.; Apa, H.; Gulfidan, G.; Turkyilmaz, H.N.; Gunay, I. Sphingomonas paucimobilis
infections in children: 24 case reports. Mediterr. J. Hematol. Infect. Dis. 2013, 5, e2013040. [CrossRef]
[PubMed]
33. Liu, L.; Coenye, T.; Burns, J.L.; Whitby, P.W.; Stull, T.L.; LiPuma, J.J. Ribosomal DNA-directed PCR for
identification of Achromobacter (Alcaligenes) xylosoxidans recovered from sputum samples from cystic fibrosis
patients. J. Clin. Microbiol. 2002, 40, 1210–1213. [CrossRef] [PubMed]
34. Fass, R.J.; Barnishan, J. In vitro susceptibilities of nonfermentative Gram-negative bacilli other than
Pseudomonas aeruginosa to 32 antimicrobial agents. Rev. Infect. Dis. 1980, 2, 841–853. [CrossRef] [PubMed]
35. Blahova, J.; Kralikova, K.; Krcmery, V., Sr.; Kubonova, K. Hydrolysis of imipenem, meropenem, ceftazidime,
and cefepime by multiresistant nosocomial strains of Sphingobacterium multivorum. Eur. J. Clin. Microbiol.
Infect. Dis. 1997, 16, 178–180. [CrossRef] [PubMed]
36. Holmes, B.P.M.; Kiredjian, M.; Kersters, K. Ochrobactrum anthropi gen. nov., sp. nov. from Human Clinical
Specimens and Previously Known as Group Vd. Int. J. Syst. Bacteriol. 1998, 38, 406–416. [CrossRef]
37. Sudharani, A. Chryseobacterium indologenes bacteremia in a preterm baby. Indian J. Med. Microbiol. 2011, 29,
119
and Public Health
Article
Salmonella enterica Serovar Typhimurium and
Escherichia coli Survival in Estuarine Bank Sediments
Mahbubul H. Siddiqee 1,2 , Rebekah Henry 1 , Rebecca Coulthard 1 , Christelle Schang 1 ,
Richard Williamson 1 , Rhys Coleman 3 , Graham Rooney 3 , Ana Deletic 1 and
David McCarthy 1, *
1 Environmental and Public Health Microbiology Laboratory (EPHM LAB), Department of Civil Engineering,
Monash University, Melbourne, VIC-3168, Australia; [email protected] (M.H.S.);
[email protected] (R.H.); [email protected] (R.C.);
[email protected] (C.S.); [email protected] (R.W.);
[email protected] (A.D.)
2 Molecular and Environmental Microbiology Laboratory (MEM LAB), Department of Mathematics and
Natural Sciences, BRAC University, Dhaka 1212, Bangladesh
3 Melbourne Water Corporation, Docklands, VIC-3008, Australia;
[email protected] (R.C.); [email protected] (G.R.)
Abstract: Estuarine bank sediments have the potential to support the survival and growth of fecal
indicator organisms, including Escherichia coli. However, survival of fecal pathogens in estuarine
sediments is not well researched and therefore remains a significant knowledge gap regarding
public health risks in estuaries. In this study, simultaneous survival of Escherichia coli and a fecal
pathogen, Salmonella enterica serovar Typhimurium, was studied for 21 days in estuarine bank
sediment microcosms. Observed growth patterns for both organisms were comparable under four
simulated scenarios; for continuous-desiccation, extended-desiccation, periodic-inundation, and
continuous-inundation systems, logarithmic decay coefficients were 1.54/day, 1.51/day, 0.14/day,
and 0.20/day, respectively, for E. coli, and 1.72/day, 1.64/day, 0.21/day, and 0.24/day for
S. Typhimurium. Re-wetting of continuous-desiccated systems resulted in potential re-growth,
suggesting survival under moisture-limited conditions. Key findings from this study include:
(i) Bank sediments can potentially support human pathogens (S. Typhimurium), (ii) inundation
levels influence the survival of fecal bacteria in estuarine bank sediments, and (iii) comparable
survival rates of S. Typhimurium and E. coli implies the latter could be a reliable fecal indicator in
urban estuaries. The results from this study will help select suitable monitoring and management
strategies for safer recreational activities in urban estuaries.
Keywords: fecal indicator; fecal pathogen; waterborne pathogens; recreational risks; QMRA
1. Introduction
Urban estuaries deliver multiple benefits to communities, including access to aquatic recreation,
amenity, transport, and supporting cultural traditions. As a result, 22 of the largest cities in the world
are located adjacent to estuaries [1]. However, community benefits from estuaries are under increasing
threat from pollution due to progressive urbanization and population growth [2]. Fecal contamination
is one of the leading threats to public health in these systems, via exposure to harmful organisms
during aquatic recreation [3–5].
Monitoring of public health risks in estuaries has historically relied on fecal indicator organisms,
such as Escherichia coli and Enterococcus spp.—principally due to the practical limitations and costs

IJERPH 2018, 15, 2597
associated with measuring pathogens. In many cases, significant correlations have been observed
between health outcomes (i.e., illness rates) and indicator organism concentrations in marine waters
and some freshwater systems [6,7]. However, there is a paucity of epidemiological studies for estuarine
environments [8], meaning that the link between indicator organisms and human health outcomes in
these systems is more uncertain.
There are an increasing number of studies that show that fecal indicators can survive and
grow in estuarine systems, especially in their sediments where they are protected from competition
and predation and have a rich nutrient supply [9]. For example, Solo-Gabriele et al. (2000) and
Desmarais et al. (2002) found that coastal bank sediments are a suitable habitat for the survival and
growth of E. coli. Further, Solo-Gabriele et al. (2000) found that that growth of E. coli was a function
of soil moisture content and hence, different soil moisture scenarios (as a result of tidal fluctuation)
could potentially exert various degrees of stress, leading to differential survival of fecal organisms.
At the same time, the survival potential of pathogens in these sediments may indeed be different to
that of the indicator organism, E. coli. Moreover, the survival of fecal organisms in the bank sediments
of estuaries in temperate climates is not well understood.
There are only a handful of studies that have explored the survival of pathogens in estuarine
systems, and even fewer which have focused on estuarine sediments. Indeed, there are only two
reported studies which have explored pathogen survival within the sediments of estuaries [9,10].
This lack of pathogen-survival studies in these types of systems is especially important considering
the salient features of estuarine bank sediment as opposed to other soils in terms of its particle size
distribution, organic content, composition of minerals, and organic matters [11]. With others having
demonstrated extended survival and resuspension [12,13] of fecal indicator organisms from the bank
sediments, this paucity in pathogen-survival data leaves a critical knowledge gap regarding the
reliability of these indicators for predicting public health risk in these systems.
This study aims to determine: (a) Whether different sections of tidally-influenced estuarine bank
sediments (fully inundated, periodically inundated, occasionally inundated, and fully desiccated) can
support the extended survival of a human pathogen (Salmonella enterica serovar Typhimurium), and
(b) whether the survival of a commonly used fecal contamination indicator (E. coli) is comparable
to Salmonella Typhimurium. Salmonella Typhimurium is recommended by the US EPA as a suitable
pathogen for water-based monitoring [14] and it is also responsible for the majority of Salmonella-related
infections (nearly 80% in Australia) [15].
2.1. Bacterial Strains and Inoculum Development

In this study, Escherichia coli K1 was used as a model indicator organism and S. Typhimurium
NVSL 6993 as the model enteric pathogen. E. coli and S. Typhimurium were subcultured from
glycerol stock (maintained at −80 ◦ C) onto Eosine-Methylene-Blue (EMB) (Oxoid, UK) agar and
Xylose-Lysine-Deoxcolate (XLD) (Oxoid, UK) agar, respectively. Plate cultures were incubated
overnight at 37 ◦ C. Single colonies were isolated and transferred to 150 mL LB broth and incubated
for 12 h at 37 ◦ C with shaking (60 rpm). The cultures were pelleted at 4000 × g for 15 min (at 10 ◦ C).
Supernatants were discarded, and the pellets resuspended in 5 mL Phosphate Buffered Saline (PBS)
(1×; pH 7.4). Pelleting was repeated, the supernatant discarded, and the final pellet resuspended in
5 mL PBS (1×; pH 7.4).
2.2. Collection and Processing of Bank Sediment

The top 8 cm layer of bank sediments (20 kg) were collected at low tide from the Yarra River
estuary at South Yarra (Victoria, Australia). The sediment was mixed to ensure homogeneity followed
by sterilization by autoclaving before cooling it to room temperature. Since the main focus of this
study was to test the influence of inundation scenarios, autoclaving of the bank sediment before
121
IJERPH 2018, 15, 2597
inoculation with the two test microorganisms was done. This sterilization was essential to ensure
that the survival rates of E. coli and S. Typhimurium were not influenced by predation, competition,
or parasitism by native microorganisms present in natural sediments, which are known to have
differential effects on these two organisms in different scenarios [16–18]. The influence of autoclaving
on the nutritional properties of the sediment was tested prior to experimentation; no significant change
in concentrations of total phosphorus (TP) and total dissolved nitrogen (TN) was observed when
measured according to the Australian Laboratory Handbook of Soil and Water Chemical Methods, 1992
(see Figure A1 and Table A1). Changes in particle size distribution were also investigated using the
electro-resistance counting method [19] and no detectable change was observed between autoclaved
and non-autoclaved sediments.
The sterilized sediment was inoculated with both bacterial strains to reach a final concentration
of 107 g−1 (wet sediment) cells. After mixing, the inoculated sediment was distributed into sterilized
plastic containers by sub-sampling 10 × 100 g aliquots into 14 open plastic containers, using a
randomization approach [20], to achieve a 3 cm thick layer consisting of 1 kg of sediment.
2.3. Experimental Matrix

Four experimental configurations were investigated in triplicate to represent four typical exposed
tidally influenced bank sediment areas (Table 1): (i) continuous desiccation (CD), which received
no inundation and represented bank sediment above the high tide mark, which does not receive
any moisture from inundation; (ii) extended desiccation (ED), no inundation for the first seven days
followed by periodic inundation after this period and represented the section of bank sediment that
is only inundated during extreme conditions (including high flow periods and/environmental flow
releases); (iii) periodically inundated (PI), representing tidally influenced sediment between high and
low tide marks, which received the normal tidal of the condition of the Yarra estuary with a 12.4 h
inundation cycle; and (iv) continuously inundated (CI) systems, which were permanently inundated
with water and represent bank sediments just below the low tide mark. A non-inoculated control (NI)
was used to monitor for environmental contamination that may occur during the experiment as a
result of the open system design. Sediment containers were faced toward the sun with a slope of 7◦
(same as observed in the bank at the South Yarra site from where the sediment was collected).
Table 1. Experimental configurations tested for survival of E. coli and S. Typhimurium and
their properties.
Inoculation with E. coli

Configuration No. of Replicates Extent of Inundation
and S. Typhimurium
Continuous desiccation (CD) 3 Yes Never
Starts on Day 7; periodic
Extended desiccation (ED) 3 Yes
(cycles of 12.4 h)
Periodic inundation (PI) 3 Yes Periodic (cycles of 12.4 h)
Continuous inundation (CI) 3 Yes Continuous
Non-inoculated (NI) 2 No Periodic (cycles of 12.4 h)
2.4. Experimental Procedure

All configurations were placed outdoor (Figure 1) under a Perspex sheet to protect the
configurations from rain and direct fecal deposition by local wildlife. ED configurations were placed
on a high platform to ensure shorter exposure to inundations compared to the PI configurations.
The remaining configurations were placed on the low platforms in a randomized manner to account
for differences related to spatial distribution. ED, PI, and NI containers were connected with silicon
hoses to sterile plastic bottles (autoclaved) containing 1 L (each) of sterile semi-synthetic river water;
representing water quality parameters of the Yarra River during a similar dry weather period at the
same location. Data from a previous project (data not shown) was used to calculate target TP, TN, and
122
IJERPH 2018, 15, 2597
total suspended solid (TSS) concentrations (0.09 mgL−1 , 0.91 mgL−1 , and 23 mgL−1 , respectively).
The stock semi-synthetic water was made using autoclaved bed sediment (collected from the river
at South Yarra) to produce a final TSS concentration of 23 mgL−1 . As sediment alone was unable
to reproduce the target TN and TP concentrations, inorganic salts (KH2 PO4 for TP and a mixture of
KNO3 , NH4 Cl, and C6 H5 O2 N for TN) were added as outlined by Bratieres et al. (2008) to achieve the
target concentrations [21]. The stock water was stored in room temperature and periodically tested
to ensure sterility throughout experimentation. The water containers (all containing 1 L each) were
kept on a platform, which had a periodic vertical movement mimicking the tidal fluctuations of the
Yarra River (12.4 h cycle) covering the intended sediment configurations. The inundating water was
replaced every week.
Figure 1. Outdoor experimental set up for the bank sediment survival study, which is covered by a
transparent Perspex sheet; ED configurations placed on a higher platform and all the others on the
lower platforms. Bottles containing water for inundating sediments (see upper right corner) were
located on a vertically moving platform.
The survival experiment was conducted over 21 days. The first three days of the experiment
coincided with a heat wave experienced in Melbourne; on each day, the maximum atmospheric
temperature exceeded 40 ◦ C. The average daily maximum temperature during this experiment was
29.4 ◦ C and the average solar radiation was 25.6 MJ/m2 (BOM, Australia, 2014).
2.5. Collection and Processing of Samples

A total of 6 g of top layer sediment was sampled (in four sub-samples) from each configuration
on Days 0, 2, 7, 14, and 21. Further samples were collected from PI and ED on Day 1. The four
sub-samples were homogeneously mixed to make a uniform mixture. Moisture content measurements
were conducted on ~1.5 g of sediment following Standards Australia, (2005) [22]. For culture-based
assays, ~1.5 g of sediment was dissolved in 25 mL sterile de-ionized water and mixed for 1 min at
120 shakes min−1 to dislodge particle-bound bacteria. The homogenized mix was pelleted at 400× g
for 5 min. From the supernatant, a single 5 mL aliquot was diluted in 5 mL PBS (1×; pH 7.4) and
vortexed for 3 s. The diluted supernatants were then further diluted to a final concentration of 10−4 in
PBS (1×; pH 7.4).
2.6. Enumeration of E. coli and S. Typhimurium and Calculation of Die-Off Rates

A volume of 100 μL was plated, in triplicate, from the 10−3 and 10−4 dilutions on EMB agar for
E. coli, and on XLD agar for S. Typhimurium for culturable counts as two differential media are needed
to monitor two different organisms in the same system [23,24]. Plate counts were converted to cfu/g
(dry weight) based on the relevant dilution factor and corresponding moisture content. Colony counts
were then log-transformed and used to construct a line of best fit and the gradient of this line was used
to estimate the die-off rate [25]. In cases where no colony was observed in any of the triplicate plates,
data points were replaced with half of the detectable limit (i.e., 0.5 cfu/plate [26]). Data points that
123
IJERPH 2018, 15, 2597
fluctuated around the lowest detectable limit were not used to estimate die-off rates. Colony counts of
below 30 cfu/plate were considered for calculation of die-off rates, with possible uncertainties taken
into consideration during data analysis and interpretation. The overall die-off rates were calculated as
the logarithmic difference of initial and final numbers divided by the number of days elapsed.
3. Results and Discussion
3.1. The Impact of Experimental Design

Preliminary investigations demonstrated the insignificant impact that autoclaving had on
particle size distributions and nutrient contents. Furthermore, other analyses (Appendices A and B)
demonstrated that the process of autoclaving did not significantly impact microbial survival through
the re-release of nutrients from dead microorganisms. However, since the experiment was conducted
in an outdoor environment, it was influenced by the prevailing atmospheric conditions.
The initial phase of the experiment coincided with high atmospheric temperatures (>40 ◦ C) and
solar radiation, both known to reduce survival of E. coli and S. Typhimurium [11,26,27]. During this
initial period, a sharp decrease in moisture level was observed in configurations without inundation
(CD and ED). In fact, on Day 2, there was almost no moisture remaining in these systems (Figure 2).
Overall, the moisture content readings of different configurations were reduced significantly (from 41%
to as low as 10%) in all configurations, except for the CI system.
Another by-product of the heat wave was an increase in TP and TN concentration in the sediment
and inundating water (Tables A1 and A2). However, since the water was replaced weekly, exposure
to the increased nutrient levels was not persistent. Furthermore, the concentration of TN and TP
within the sediment (0.21 and 0.81 mg/g, respectively, at the beginning) showed very little change
(up to 0.4 mg/g for TP and up to 1.4 mg/g for TN) across the experimental period. Thus, it can
be assumed that the alteration in water column concentrations did not have a significant effect on
sediment survival rates.
50%
45%
Moisture content [% by weight]
40%
35%
30%
Extended Desiccation
25%
Continuous Inundation
20% Continuous Desiccation
15% Periodic Inundation
10%
5%
0%
0 5 10 15 20 25
Days since start of experiment
Figure 2. Levels of moisture content in four test sediment configurations during the experiment
(error bars represent standard deviations around the mean values for each of the configurations).
3.2. Survival of E. coli and S. Typhimurium

The results of the survival study indicate that both E. coli and S. Typhimurium are able to
survive for up to 21 days in all three configurations that received moisture (ED, PI, and CI).
Both microorganisms withstood the first 24 h of the experiment after sediment inoculation, which
was evident from the culture counts of the two tested configurations (PI and ED; Figure 3). Notably,
even though the atmospheric temperature reached 43 ◦ C within a few hours of inoculation, E. coli
124
IJERPH 2018, 15, 2597
counts increased marginally, and S. Typhimurium counts increased nearly 10 times of the initial
number. This initial growth could reflect the fact that fecal microorganisms are adapted to animal
gut environments (37 ◦ C for mammals and 42 ◦ C for birds). Sediment properties that are widely
known to influence bacterial survival include available nutrients [28], organic matter [29], particle
size [30], and clay content [31]. Among these, estuarine bank sediment is generally known to have
finer particles [32,33], as well as higher organic matter content [34], which can offer several advantages,
such as protection from UV light [35,36] and nutritional support. Therefore, it is perhaps not surprising
to see extended survival of fecal microorganisms, including the potential pathogen, in estuarine banks
even in unfavorable weather conditions. This, in turn, suggests that in the absence of other stressors,
S. Typhimurium can probably grow in estuarine bank sediments in high-temperature conditions
when moisture is available and potentially serve as a source of this pathogen to the water column
if re-suspended.
The initial phase of growth in the first 24 h was followed by a sharp die-off, consistent across
organisms and configurations (Figure 3). The CD system had the highest die-off rates for both types of
bacteria during the experiment; 1.54/day for E. coli and 1.72/day for S. Typhimurium. Since significant
losses in soil moisture took place during the early phase of the experiment (moisture content decreased
from 40% to <1%), these very fast die-off rates were consistent with the literature [11,37]. In addition,
high die-off rates might have been facilitated by very high air temperatures [38] coupled with high
radiation levels [39].
9 9
Extended Desiccation Extended Desiccation
8 Continuously Inundated 8 Continuous Inundation
Log S. Typhimurium (CFU/g)
7 Continuous Desiccation 7 Continuous Desiccation

Log E. coli (CFU/g)
Periodic Inundation Periodic Inundation

6 6
5 5
4 4
3 3
2 2
1 1
0 5 10 15 20 25 0 5 10 15 20 25
Days since start of the experiment Days since start of the experiment
(a) (b)
Figure 3. Survival of the test organisms; (a) E. coli K1 densities (cfu/g dw) and (b) S. Typhimurium
NVSL 6993 densities (cfu/g dw) in the four experimental configurations (error bars represent standard
deviations around the mean values of the replicates tested).
The two test organisms in the ED systems behaved similarly to the CD systems in the first
week of the experiment (Figure 3). The die-off rate for E. coli was calculated to be 1.51/day and for
S. Typhimurium it was 1.64/day. Interestingly, when inundation was restored to these systems after
Day 7 (although the ED system received a shorter duration of inundation compared to PI counterparts),
an increase in moisture content coincided with the detection of E. coli colonies on both Days 14 and
21 (in two of the three replicates on both days). S. Typhimurium was also detected on Day 21 in one
of the replicate boxes while the NI controls remained negative. An increase in moisture level due to
inundation restoration might have influenced this regrowth/recurrence of both test organisms.
The detection of culturable bacteria after a period of drying indicates that both bacteria may
survive either in dormant form or at least survive in very low densities. It is important to note that
although above the limit of detection, the colony counts were below 30 cfu/plate, and therefore may
have higher associated uncertainties. However, colonies were reproducibly isolated within the replicate,
suggesting a level of survival was possible. Dormancy and subsequent regrowth of S. Typhimurium
upon increasing moisture has previously been reported [40]. Also, desiccation has been shown for
both E. coli and S. Typhimurium to significantly enhance resistance towards environmental stressors,
125
IJERPH 2018, 15, 2597
including high temperature [41,42]. This could perhaps partly explain the recurrence of these two
organisms in the ED systems. Therefore, it is possible that bank sediments, which only receive
moisture very intermittently (i.e., bank sediments above the usual high-tide mark, which are inundated
only during king tides or high flows) could support the growth of viable fecal pathogens that, if
washed, could get resuspended back into the water column [43]. Resuspension could also occur due to
other natural and anthropogenic activities, like storms, floods, recreational activities, and commercial
dredging, which have all been known to cause resuspension of sediment borne bacteria and causing
elevated levels of fecal organisms [44,45]. Therefore, extended survival of potential fecal pathogens,
like S. Typhimurium, in estuarine bank sediment could comprise a potential human health risk.
PI systems had higher levels of survival for both E. coli and S. Typhimurium compared to the
desiccated CD and ED systems (Figure 3). For PI, the decay rate of E. coli was 0.14/day, while a slightly
higher decay rate was observed for S. Typhimurium (0.21/day). The decrease in cell counts after Day 1
may be associated with very high air temperatures and solar radiation, which also resulted in a very low
moisture content (close to 5%; Figure 2). It has previously been reported that Gram-negative bacteria
require a moisture content of 93% or more for optimal growth [46]. However, despite some variation
among the three biological replicates for the PI configurations, it was evident that the scenario still
supported the survival of both test organisms for up to three weeks. Therefore, under more favorable
conditions (lower atmospheric temperature, lower solar radiation, and higher moisture), it is likely
that survival of these fecal organisms (including potential pathogens) would be higher.
Survival rates of E. coli and S. Typhimurium in the PI configuration were found to be comparable.
This contradicts the notion that fecal pathogens cannot survive in estuarine sediments, and suggests
that E. coli could be a reliable indicator of this pathogen in this scenario. Furthermore, the extended
survival of a potential fecal pathogen in the tidally influenced zone of an estuarine bank could
have human health implications. This is especially the case for bank sediments, where natural and
anthropogenic activities (including tide, storm, flood, recreation, dredging, etc.) can readily cause
resuspension into the water column [10,45,47].
The CI systems showed slightly higher die-off rates when compared to PI (0.20/day for E. coli
and 0.24/day for S. Typhimurium). It was interesting to see higher die-off rates compared to those
of PI, especially since the organisms in the CI systems were exposed to higher moisture levels and
reduced solar irradiation. However, the water used for inundating the experimental containers was
stagnant in the CI configurations, which led to some interference due to the formation of algae after
the first week, and this may have resulted in a faster die-off. The algal growth resulted in lower TP
and TN concentrations in the inundating water in the CI systems compared to the other configurations
(Table A2).
In the estuarine context, better survival of E. coli in areas of coastal bank sediment with
intermittent drying effect compared to continuously moist sediment has previously been reported by
Solo-Gabriele et al. (2000). They argued that this better survival was due to having a better competitive
advantage over the predators existing in natural sediment. Although our study attempted to exclude
the biotic factors by sterilizing the soil beforehand, the CI systems with algal interference supports the
idea that algal bloom (which could occur in estuarine systems) could potentially reduce the survival
of fecal organisms in estuarine bank sediments. In this scenario, even under these algae-influenced
conditions, the survival rates of both organisms were again comparable, reinforcing the ability of
E. coli to represent S. Typhimurium in complex systems, including those with algae blooms. It is
also important to note that apart from the CI systems, no other configurations showed any sign of
algal interference.
This study was conducted under open atmospheric conditions, which led to some challenges,
including changing TP and TN concentrations. However, other studies have demonstrated that
increases in TP does not necessarily impact the survival of fecal organisms, like E. coli [48,49]. Likewise,
increased levels of organic or inorganic nitrogen do not necessarily impact the survival of fecal bacteria
in ambient waters [48]. Therefore, it can be assumed that the changes in TP and TN concentrations
126
IJERPH 2018, 15, 2597
did not significantly influence the die-off rates of the test organisms. In fact, conducting the study in
an ambient environment allowed a unique opportunity to explore the comparative survival of these
organisms under highly unfavorable conditions.
In combination, the survival data suggests, for the first time, that both E. coli and S. Typhimurium
were able to withstand severe environmental conditions in estuarine bank sediments. Overall, the
die-off rates observed in this study ranged from 0.14/day to 1.54/day for E. coli and 0.21/day to
1.72/day for S. Typhimurium. In three of the four configurations (i.e., CD, PI, CI), die-off rates for
the two organisms were similar. A wide range of die-off rates has been reported in soil sediment
systems for these organisms [27,40,50,51], and differences have been attributed to both biotic and
abiotic factors [11,38,49,52–54] that are also encountered in bank sediments.
This study investigated the influence of different bank sediment scenarios on the survival of
two fecal microorganisms in potentially extreme weather conditions with respect to temperature,
radiation, and desiccation. Our underlying hypothesis is that if the test organisms survive in this
study’s extreme climatic conditions, then they can also survive in the less extreme conditions that
exist in many systems year-round. With an absence of previous studies focusing on bank sediments
under similar experimental conditions, the results of this study significantly enhance our knowledge
of the survival of fecal organisms in estuarine bank sediments. Further, this study demonstrates that,
without studying fecal pathogen survival in all environments (estuarine bank sediment in this case),
perhaps it could be premature to conclude that E. coli is not a good indicator organism.
4. Conclusions
This study presented data on the survival of the faecal indicator organism, E. coli, and the human
fecal pathogen, S. Typhimurium, in estuarine bank sediments. Different degrees of tidal inundation
were observed to influence the survival for both test organisms, with periodic inundation, mimicking
natural tidal cycles, being the most conducive to persistence. While the simultaneous survival rates
for these two organisms were found to be comparable, for the first time, this experiment presents
evidence that S. Typhimurium may survive for over three weeks in estuarine bank sediments, which
could be of critical importance. This, in turn, highlights the potential of bank sediments as a source of
viable pathogens to the water column, especially when natural processes and anthropogenic activities
cause resuspension. Considering the comparable survival patterns of E. coli and S. Typhimurium in
the contrasting experimental conditions, E. coli cannot be excluded from being a reliable indicator of
public health risks associated with S. Typhimurium in estuarine bank sediments.
Author Contributions: Conceptualization: M.H.S., R.H., R.C. (Rebecca Coulthard), C.S. and D.M.; Methodology:
M.H.S., R.H., R.C. (Rebecca Coulthard), C.S. and D.M.; Validation: M.H.S., R.C. (Rebecca Coulthard), C.S., R.W.;
Formal analysis: M.H.S., R.C. (Rebecca Coulthard), C.S., D.M.; Writing—original draft preparation: M.H.S., D.M.;
Writing—review and editing: M.H.S., R.H., R.C. (Rhys Coleman), A.D. and D.M.; Supervision: R.H., A.D. and
D.M.; Funding acquisition: D.M., R.C. (Rhys Coleman) and G.R.
Funding: This research was funded by [Melbourne Water] and the [Australian Research Council], grant number
[LP120100718].
Acknowledgments: We gratefully acknowledge the timely help we received from Peter Kolotelo, Dusan Jovanovic,
Gayani Chandrasena, and Scott Coutts during various stages of the study.
Appendix A
Effect of Autoclaving
Autoclaving was done in this experiment to exclude the interference from predation and
competition by native organisms present in natural sediments. However, a combination of very high
temperature and pressure kills the naturally occurring microbes in the sediment and releases nutrients
into the sediment. Also, some other reports suggest that autoclaving can change sediment’s own
127
IJERPH 2018, 15, 2597
nutritional properties [42–44]. Therefore, it was essential to check if autoclaving was introducing any
bias towards higher or lower survival. Therefore, along with the experimental configurations (Table 1),
another set (triplicate) of sediments was augmented with a mixture of E. coli and S. Typhimurium
culture (each at a concentration of 109 g−1 wet sediment and hereby named as Nutritionally
Augmented) before autoclaving. These sediments were eventually inoculated with both test organisms
and were tested along the test configurations as mentioned above (Figure A1).
8 8
Periodic Inundation
Periodic Inundation
7 7 Nutritionally Augmented
Nutritionally Augmented
6 6
Log (CFU S. Typhimurium/g)

Log (CFU E.coli/g)
5 5
4 4
3 3
2 2
1 1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Days since start of the experiment Days since start of the experiment
(a) (b)
Figure A1. Influence of augmented nutrition on survival of (a) E. coli K1 and (b) S. Typhimurium NVSL
6993 (error bars represent standard deviations around the mean values for each of the configurations).
It was observed in this study that there was no obvious impact of nutritional augmentation on
E. coli or S. Typhimurium survival where additional microorganisms were added to the sediment before
autoclaving. This could suggest that the extra nutrition (if at all) available to E. coli and S. Typhimurium
through the autoclaving process did not cause significant bias in the observed die-off rates.
Appendix B
Changes in Sediments
Table A1. Mean (and relative standard deviations in %) of total phosphorus (TP) and total nitrogen
(TN) concentrations of sediment samples.
Time during Experiment E. coli (cfu/g dw) S. Typhimurium (cfu/g dw) TP (mg/g dw) TN (mg/g dw)
Original bank conditions—not inoculated, not autoclaved
Day = 0 540 Nil 0.21 * 0.77 *
Autoclaved, not inoculated
Day = 0 Nil Nil 0.18 * 0.78 *
Day = 21 (PI) Nil Nil 0.4 (12) 1.4 (5)
Autoclaved, spiked with E. coli, S. Typhimurim and mixed culture
Day = 0 ** ** 0.20 * 0.77 *
Day = 21 (PI) ** ** 0.4 (2) 1.3 (1)
Autoclaved, spiked with E. coli, S. Typhimurim
Day = 0 *** *** 0.21 * 0.81 *
Day = 21 (ED) *** *** 0.4 (7) 1.4 (3)
Day = 21 (CI) *** *** 0.4 (12) 1.3 (8)
Day = 21 (CD) *** *** 0.4 (4) 1.4 (0)
Day = 21 (PI) *** *** 0.4 (3) 1.4 (3)
* only one sample analysed, ** data presented in Appendix A, *** data presented in Section 3.2.
128
IJERPH 2018, 15, 2597
Table A2. Changes of nutrients in inundating water; means (and relative standard deviations in %) of
TP and TN concentration of weekly inflow samples.
Time during Experiment TP (mg/L) TN (mg/L) pH EC (mS/cm) Turbidity (NTU)

Fresh Inflow
0.13 (37) 0.9 (4) 6.6 (4) 0 (17) 2.14 *
Extended Desiccation
Day = 7 7.4 (2) 0.5 (8)
Day = 14 5.8 (6) 1.7 (6)
Day = 21 0.25 (48) 6.4 (48) 5.7 (7) 1 (38) 8.1 (62)
Non-Inoculated
Day = 7 0.27 (45) 13 (22) 7.2 (1) 2.5 (20)
Day = 14 0.17 (42) 4.2 (67) 6.1 (1) 0.3 (6)
Day = 21 5.8 (10) 2.7 (46) 4.4 (37)
Continuously Inundated
Day = 7 7.5 (0) 2.4 (10)
Day = 14 6.7 (2) 0.7 (1)
Day = 21 0.37 (25) 3.6 (19) 7.1 (0) 0.4 (4) 12.3 (44)
Periodically Inundated
Day = 7 0.27 (19) 10.83 (19) 6.7 (3) 1.9 (23)
Day = 14 0.2 (12) 5.8 (9) 6 (9) 0.7 (1)
Day = 21 0.25 (13) 12.2 (43) 5.8 (11) 1.7 (36) 4.4 (30)
Nutritionally Augmented
Day = 7 0.25 (43) 10.33 (49) 0.1 (1) 1.8 (46)
Day = 14 0.23 (11) 7.27 (12) 6.4 (9) 0.9 (23)
Day = 21 6.3 (14) 1.9 (7) 3.7 (27)
* Only one sample was tested against three for the others.
References
1. Ross, D.A. Introduction to Oceanography; Harper Collins College Publishers: New York, NY, USA, 1995;
ISBN 978-0-673-46938-0.
2. Mallin, M.A.; Williams, K.E.; Esham, E.C.; Lowe, R.P. Effect of human development on bacteriological water
quality in coastal watersheds. Ecol. Appl. 2000, 10, 1047–1056. [CrossRef]
3. Catalao-Dionisio, L.P.; Joao, M.; Ferreiro, V.S.; Fidalgo, M.L.; Garcia-Rosado, M.E.; Borrego, J.J. Occurrence
of Salmonella spp. in estuarine and coastal waters of Portugal. Antonie Van Leeuwenhoek 2000, 78, 99–106.
[CrossRef] [PubMed]
4. Samhan, F.A.; Kronlein, M.R.; Fakher, U.; Kronlein, C.; Stedtfeld, R.D.; Hashsham, S.A. Detection and
occurrence of indicator organisms and pathogens. Water Environ. Res. 2009, 81, 959–980. [CrossRef]
[PubMed]
5. Henry, R.; Schang, C.; Chandrasena, G.I.; Deletic, A.; Edmunds, M.; Jovanovic, D.; Kolotelo, P.; Schmidt, J.;
Williamson, R.; McCarthy, D. Environmental monitoring of waterborne Campylobacter: Evaluation of the
Australian standard and a hybrid extraction-free MPN-PCR method. Front. Microbiol. 2015, 6, 74. [CrossRef]
[PubMed]
6. Edberg, S.C.; Allen, M.J.; Smith, D.B.; Kriz, N.J. Enumeration of total coliforms and Escherichia coli from
source water by the defined substrate technology. Appl. Environ. Microbiol. 1990, 56, 366–369. [PubMed]
7. Odonkor, S.T.; Ampofo, J.K. Escherichia coli as an indicator of bacteriological quality of water: An overview.
Microbiol. Res. 2013, 4, e2. [CrossRef]
8. Australian Government. Guidelines for Managing Risks in Recreational Water; NHMRC: Canberra, Australia, 2008.
9. Schang, C.; Lintern, A.; Cook, P.L.M.; Osborne, C.A.; McKinley, A.; Schmidt, J.; Coleman, R.; Rooney, G.;
Henry, R.; Deletic, A.; et al. Presence and survival of culturable Campylobacter spp. and Escherichia coli in
temperate urban estuaries. Sci. Total Environ. 2016, 569–570, 1201–1211. [CrossRef] [PubMed]
129
IJERPH 2018, 15, 2597
10. Hood, M.A.; Ness, G.E. Survival of Vibrio cholerae and Escherichia coli in estuarine waters and sediments.
Appl. Environ. Microbiol. 1982, 43, 578–584. [PubMed]
11. Venkatramanan, S.; Ramkumar, T.; Anithamary, I. Distribution of grain size, clay mineralogy and organic
matter of surface sediments from Tirumalairajanar Estuary, Tamilnadu, east coast of India. Arab. J. Geosci.
2013, 6, 1371–1380. [CrossRef]
12. Solo-Gabriele, H.M.; Wolfert, M.A.; Desmarais, T.R.; Palmer, C.J. Sources of Escherichia coli in a Coastal
Subtropical Environment. Appl. Environ. Microbiol. 2000, 66, 230–237. [CrossRef] [PubMed]
13. Desmarais, T.R.; Solo-Gabriele, H.M.; Palmer, C.J. Influence of soil on fecal indicator organisms in a tidally
influenced subtropical environment. Appl. Environ. Microbiol. 2002, 68, 1165–1172. [CrossRef] [PubMed]
14. US-EPA. Fact Sheet: Final Third Drinking Water Contaminant Candidate List (CCL 3); Office of Water:
Washington, DC, USA, 2009.
15. OzFoodNet Working Group. Burden and Causes of Foodborne Disease in Australia: Annual Report of the
OzFoodNet Network, 2005; Office of Health Protection, Australian Government Department of Health and
Ageing: Canberra, Australia, 2006; Volume 30.
16. Chandran, A.; Mohamed Hatha, A.A. Relative survival of Escherichia coli and Salmonella typhimurium in a
tropical estuary. Water Res. 2005, 39, 1397–1403. [CrossRef] [PubMed]
17. Feng, F.; Goto, D.; Yan, T. Effects of autochthonous microbial community on the die-off of fecal indicators in
tropical beach sand. FEMS Microbiol. Ecol. 2010, 74, 214–225. [CrossRef] [PubMed]
18. Wanjugi, P.; Harwood, V.J. The influence of predation and competition on the survival of commensal and
pathogenic fecal bacteria in aquatic habitats. Environ. Microbiol. 2013, 15, 517–526. [CrossRef] [PubMed]
19. Vdović, N.; Obhođaš, J.; Pikelj, K. Revisiting the particle-size distribution of soils: Comparison of different
methods and sample pre-treatments. Eur. J. Soil Sci. 2010, 61, 854–864. [CrossRef]
20. McCarthy, D.T. Modelling microorganisms in urban stormwater. Ph.D. Thesis, Department of Civil
Engineering, Monash University, Melbourne, Australia, 2008.
21. Bratieres, K.; Fletcher, T.D.; Deletic, A.; Zinger, Y. Nutrient and sediment removal by stormwater biofilters:
A large-scale design optimisation study. Water Res. 2008, 42, 3930–3940. [CrossRef] [PubMed]
22. Australia Standard. AS 1289.2.1.1: Methods of Testing Soils for Engineering Purposes—Soil Moisture Content
Tests—Determination of the Moisture Content of a Soil-Oven Drying Method (Standard Method); SAI Global:
Sydney, Australia, 2005.
23. Roszak, D.B.; Grimes, D.J.; Colwell, R.R. Viable but nonrecoverable stage of Salmonella enteritidis in aquatic
systems. Can. J. Microbiol. 1984, 30, 334–338. [CrossRef] [PubMed]
24. Bogosian, G.; Sammons, L.E.; Morris, P.J.; O’Neil, J.P.; Heitkamp, M.A.; Weber, D.B. Death of the
Escherichia coli K-12 strain W3110 in soil and water. Appl. Environ. Microbiol. 1996, 62, 4114–4120. [PubMed]
25. Nguyen, H.T.M.; Le, Q.T.P.; Garnier, J.; Janeau, J.L.; Rochelle-Newall, E. Seasonal variability of faecal
indicator bacteria numbers and die-off rates in the Red River basin, North Viet Nam. Sci. Rep. 2016, 6, 21644.
[CrossRef] [PubMed]
26. Whitcomb, B.W.; Schisterman, E.F. Assays with lower detection limits: Implications for epidemiological
investigations. Paediatr. Perinat. Epidemiol. 2008, 22, 597–602. [CrossRef] [PubMed]
27. Mallmann, W.L.; Litsky, W. Survival of Selected Enteric Organisms in Various Types of Soil. Am. J. Public
Health Nations Health 1951, 41, 38–44. [CrossRef] [PubMed]
28. Pommepuy, M.; Guillaud, J.F.; Dupray, E.; Derrien, A.; Le Guyader, F.; Cormier, M. Enteric bacteria survival
factors. Water Sci. Technol. 1992, 25, 93–103. [CrossRef]
29. Pote, J.; Haller, L.; Kottelat, R.; Sastre, V.; Arpagaus, P.; Wildi, W. Persistence and growth of faecal culturable
bacterial indicators in water column and sediments of Vidy Bay, Lake Geneva, Switzerland. J. Environ. Sci.
2009, 21, 62–69. [CrossRef]
30. Garzio-Hadzick, A.; Shelton, D.R.; Hill, R.L.; Pachepsky, Y.A.; Guber, A.K.; Rowland, R. Survival of
manure-borne E. coli in streambed sediment: Effects of temperature and sediment properties. Water Res.
2010, 44, 2753–2762. [CrossRef] [PubMed]
31. Burton, G.A.; Gunnison, D.; Lanza, G.R. Survival of pathogenic bacteria in various freshwater sediments.
130
IJERPH 2018, 15, 2597
32. Malham, S.K.; Rajko-Nenow, P.; Howlett, E.; Tuson, K.E.; Perkins, T.L.; Pallett, D.W.; Wang, H.; Jago, C.F.;
Jones, D.L.; McDonald, J.E. The interaction of human microbial pathogens, particulate material and nutrients
in estuarine environments and their impacts on recreational and shellfish waters. Environ. Sci. Process. Impacts
2014, 16, 2145–2155. [CrossRef] [PubMed]
33. Perkins, T.L.; Clements, K.; Baas, J.H.; Jago, C.F.; Jones, D.L.; Malham, S.K.; McDonald, J.E. Sediment
composition influences spatial variation in the abundance of human pathogen indicator bacteria within an
estuarine environment. PLoS ONE 2014, 9. [CrossRef] [PubMed]
34. Gerba, C.P.; McLeod, J.S. Effects of sediments on the survival of Escherichia coli in marine waters.
35. Sinton, L.W.; Davies-Colley, R.J.; Bell, R.G. Inactivation of enterococci and fecal coliforms from sewage and
meatworks effluents in seawater chambers. Appl. Environ. Microbiol. 1994, 60, 2040–2048. [PubMed]
36. Davies-Colley, R.J.; Donnison, A.M.; Speed, D.J.; Ross, C.M.; Nagels, J.W. Inactivation of faecal indicator
micro-organisms in waste stabilisation ponds: Interactions of environmental factors with sunlight. Water Res.
1999, 33, 1220–1230. [CrossRef]
37. Byappanahalli, M.N.; Nevers, M.B.; Korajkic, A.; Staley, Z.R.; Harwood, V.J. Enterococci in the Environment.
Microbiol. Mol. Biol. Rev. 2012, 76, 685–706. [CrossRef] [PubMed]
38. Carlucci, A.F.; Pramer, D. An evaluation of factors affecting the survival of Escherichia coli in sea water. II.
Salinity, pH, and nutrients. Appl. Microbiol. 1960, 8, 247–250. [PubMed]
39. Korajkic, A.; McMinn, B.R.; Shanks, O.C.; Sivaganesan, M.; Fout, G.S.; Ashbolt, N.J. Biotic Interactions and
Sunlight Affect Persistence of Fecal Indicator Bacteria and Microbial Source Tracking Genetic Markers in the
Upper Mississippi River. Appl. Environ. Microbiol. 2014, 80, 3952–3961. [CrossRef] [PubMed]
40. Marsh, P.; Morris, N.Z.; Wellington, E.M.H. Quantitative molecular detection of Salmonella typhimurium in
soil and demonstration of persistence of an active but non-culturable population. FEMS Microbiol. Ecol. 1998,
27, 351–363. [CrossRef]
41. Chen, Z.; Diao, J.; Dharmasena, M.; Ionita, C.; Jiang, X.; Rieck, J. Thermal Inactivation of Desiccation-Adapted
Salmonella spp. in Aged Chicken Litter. Appl. Environ. Microbiol. 2013, 79, 7013–7020. [CrossRef] [PubMed]
42. Begley, M.; Hill, C. Stress adaptation in foodborne pathogens. Annu. Rev. Food Sci. Technol. 2015, 6, 191–210.
[CrossRef] [PubMed]
43. Frey, S.K.; Gottschall, N.; Wilkes, G.; Grégoire, D.S.; Topp, E.; Pintar, K.D.M.; Sunohara, M.; Marti, R.;
Lapen, D.R. Rainfall-induced runoff from exposed streambed sediments: An important source of water
pollution. J. Environ. Qual. 2015, 44, 236–247. [CrossRef] [PubMed]
44. An, Y.J.; Kampbell, D.H.; Breidenbach, G.P. Escherichia coli and total coliforms in water and sediments at lake
marinas. Environ. Pollut. 2002, 120, 771–778. [CrossRef]
45. Grimes, D.J. Release of Sediment-Bound Fecal Coliforms by Dredging. Appl. Microbiol. 1975, 29, 109–111.
[PubMed]
46. D’Aoust, J.Y.; Maurer, J. Salmonella species. In Food Microbiology: Fundamentals and Frontiers; Doyle, M.P.,
Beuchat, L.R., Eds.; ASM Press: Washington, DC, USA, 1997.
47. Stephenson, G.R.; Rychert, R.C. Bottom Sediment: A Reservoir of Escherichia coli in Rangeland Streams.
J. Range Manag. 1982, 35, 119–123. [CrossRef]
48. Chudoba, E.A.; Mallin, M.A.; Cahoon, L.B.; Skrabal, S.A. Stimulation of fecal bacteria in ambient waters
by experimental inputs of organic and inorganic phosphorus. Water Res. 2013, 47, 3455–3466. [CrossRef]
[PubMed]
49. Ferguson, C.M.; Coote, B.G.; Ashbolt, N.J.; Stevenson, I.M. Relationships between indicators, pathogens and
water quality in an estuarine system. Water Res. 1996, 30, 2045–2054. [CrossRef]
50. Tate, R.L. Cultural and Environmental Factors Affecting the Longevity of Escherichia coli in Histosols.
51. Zibilske, L.M.; Weaver, R.W. Effect of Environmental Factors on Survival of Salmonella typhimurium in Soil1.
J. Environ. Qual. 1978, 7, 593–597. [CrossRef]
52. Fujioka, R.S.; Hashimoto, H.H.; Siwak, E.B.; Young, R.H. Effect of sunlight on survival of indicator bacteria
in seawater. Appl. Environ. Microbiol. 1981, 41, 690–696. [PubMed]
131
IJERPH 2018, 15, 2597
53. Crane, S.R.; Moore, J.A. Modeling enteric bacterial die-off: A review. Water Air Soil Pollut. 1986, 27, 411–439.
[CrossRef]
54. Semenov, A.V.; van Overbeek, L.; Termorshuizen, A.J.; van Bruggen, A.H. Influence of aerobic and
anaerobic conditions on survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in
Luria-Bertani broth, farm-yard manure and slurry. J. Environ. Manag. 2011, 92, 780–787. [CrossRef] [PubMed]
132
and Public Health
Review
Relationships between Microbial Indicators and
Pathogens in Recreational Water Settings
Asja Korajkic 1, *, Brian R. McMinn 1 and Valerie J. Harwood 2
1 National Exposure Research Laboratory, Office of Research and Development, United States Environmental
Protection Agency, 26 West Martin Luther King Drive, Cincinnati, OH 45268, USA; [email protected]
2 Department of Integrative Biology, University of South Florida, 4202 East Fowler Ave, SCA 110, Tampa,
FL 33620, USA; [email protected]
Received: 16 November 2018; Accepted: 11 December 2018; Published: 13 December 2018
Abstract: Fecal pollution of recreational waters can cause scenic blight and pose a threat to public
health, resulting in beach advisories and closures. Fecal indicator bacteria (total and fecal coliforms,
Escherichia coli, and enterococci), and alternative indicators of fecal pollution (Clostridium perfringens
and bacteriophages) are routinely used in the assessment of sanitary quality of recreational waters.
However, fecal indicator bacteria (FIB), and alternative indicators are found in the gastrointestinal
tract of humans, and many other animals and therefore are considered general indicators of fecal
pollution. As such, there is room for improvement in terms of their use for informing risk assessment
and remediation strategies. Microbial source tracking (MST) genetic markers are closely associated
with animal hosts and are used to identify fecal pollution sources. In this review, we examine
73 papers generated over 40 years that reported the relationship between at least one indicator
and one pathogen group or species. Nearly half of the reports did not include statistical analysis,
while the remainder were almost equally split between those that observed statistically significant
relationships and those that did not. Statistical significance was reported less frequently in marine
and brackish waters compared to freshwater, and the number of statistically significant relationships
was considerably higher in freshwater (p < 0.0001). Overall, significant relationships were more
commonly reported between FIB and pathogenic bacteria or protozoa, compared to pathogenic
viruses (p: 0.0022–0.0005), and this was more pronounced in freshwater compared to marine.
Statistically significant relationships were typically noted following wet weather events and at
sites known to be impacted by recent fecal pollution. Among the studies that reported frequency
of detection, FIB were detected most consistently, followed by alternative indicators. MST markers
and the three pathogen groups were detected least frequently. This trend was mirrored by reported
concentrations for each group of organisms (FIB > alternative indicators > MST markers > pathogens).
Thus, while FIB, alternative indicators, and MST markers continue to be suitable indicators of fecal
pollution, their relationship with waterborne pathogens, particularly viruses, is tenuous at best
and influenced by many different factors such as frequency of detection, variable shedding rates,
differential fate and transport characteristics, as well as a broad range of site-specific factors such
as the potential for the presence of a complex mixture of multiple sources of fecal contamination
and pathogens.
Keywords: recreational water; fecal indicators; pathogens; relationships
1. Introduction
Approximately 39% of the United States (US) population and more than 50% of the global
population live near a coastal area [1,2]. Coastal tourism accounts for 85% of all tourism revenue
in the US [3], with the average beachgoer spending ~$35 per beach visit [4], resulting in a massive

IJERPH 2018, 15, 2842
contribution to local economy and national gross domestic product [5,6]. During 2013, approximately
10% of US beach samples (out of total 116,230 samples collected) at 3485 beaches exceeded the US
Environmental Protection Agency beach action value (BAV) for fecal indicator bacteria (FIB), indicating
unacceptable water quality [5]. Similarly, a more recent report for the European Union (EU) indicated
that ~15% of beach samples failed to meet the most stringent “excellent” quality standard at nearly
22,000 coastal beaches and inland sites across EU [7].
Because of a wide array of potential pathogens and typically low concentrations in environmental
waters, direct monitoring of waterborne pathogens can be costly, technically challenging, and in
some cases not feasible. Therefore, recreational waters are typically monitored for FIB levels instead.
Monitoring is intended to ensure that the water body is safe for human recreational contact, and the
resulting data are used to determine whether beach advisories or closures are needed. General FIB such
as total coliforms, fecal coliforms, Escherichia coli and enterococci have been used worldwide for over a
century in sanitary assessment of recreational waters [8–12]. The type of FIB measured and values used
in recreational water guidelines vary by country [13]. Other general fecal microorganisms, such as
Clostridium perfringens and various bacteriophages, are considered alternative indicator organisms,
and are also frequently measured in various water quality monitoring programs worldwide [13–19].
However, FIB and alternative indicator organisms are common inhabitants of gastrointestinal tracts
of mammals and birds [14,20], and their detection in environmental waters provides no information
about the source of pollution. Considering that ambient waters can be influenced by multiple point
and non-point pollution sources, identification of source is crucial for any remedial efforts and risk
assessment determinations since not all fecal sources pose the same risk to human health. For example,
human fecal pollution typically presents the greatest risk because of the possible presence of human
viral pathogens, while cattle manure may be a close second because of the possible presence of zoonotic
pathogens such as Cryptosporidium spp. and enteropathogenic E. coli [21]. Exposure to gull, chicken,
and pig feces carries a known risk, because of possible presence of zoonotic pathogens associated
with these animals including hepatitis E virus [22], Campylobacter spp., Brucella spp., pathogenic E. coli
and Salmonella spp. [23–25]. Microbial source tracking (MST) has emerged in response to a need to
identify the source(s) of fecal pollution to better safeguard human health and aid in remediation
efforts. The majority of MST genetic markers target the 16S rRNA gene of Bacteroides spp., although
some amplify other genes and, in some instances, viral targets [13,14]. Earlier technology centered on
end-point PCR, which provides a binary, presence/absence result, but more recent studies estimate
the concentration of a given MST genetic marker via real-time quantitative PCR (qPCR) [14]. Of note,
more rapid technology in lieu of molecular enrichment followed by qPCR is also being developed for
monitoring of Staphylococcus aureus and Pseudomonas aeruginosa, indicator species used to monitor the
water quality in swimming pools [26].
The majority of waterborne disease outbreaks associated with recreational use of untreated
waters (e.g., lakes and oceans) are caused by pathogenic microorganisms including bacteria, parasites,
and viruses, while chemicals (including toxins) accounted for approximately 6% of outbreaks with
confirmed etiology [27]. Among the pathogenic bacteria, virulent Escherichia coli serotypes (e.g.,
O157:H7), Campylobacter spp., Legionella spp., Shigella spp., Salmonella spp., and Pseudomonas spp. were
most commonly identified etiologic agents [28–32]. While other protozoan species are occasionally
identified as the cause (e.g., Naegleria spp.), [27,30], Cryptosporidium spp., followed by Giardia spp.
are etiological agents for the majority of recreational waterborne outbreaks [28,29,31,32]. Regarding
viral pathogens, noroviruses and adenoviruses were most frequently identified as causative agents
in outbreaks where etiology was confirmed [27,30,32,33]. In treated waters (e.g., swimming pools
and spas), Cryptosporidium spp. are most often identified as etiological agents [30–32], although
noroviruses and adenoviruses are becoming more frequently detected [33]. It is important to note
that etiological agents in nearly 30% of outbreaks in the US alone remain unidentified [27], and that
sporadic recreational waterborne illnesses not associated with outbreaks are excluded from this report.
134
IJERPH 2018, 15, 2842
Even though the concepts of FIB, alternative indicators, and MST markers were developed
to indicate fecal contamination and its sources, the same paradigm is often employed to indicate
pathogen presence under the assumption that indicators consistently covary with pathogen presence.
The goals of this review are: (1) to examine reported relationships between various indicators and
pathogen species to determine the feasibility of indicators as pathogen sentinels in recreational
waters; and (2) to identify factors that affect this relationship (or lack thereof). In addition, we
also queried epidemiological studies to determine which indicator(s) most commonly correlated
with illness in recreational waters. Our search criteria mandated that each study measured at least
one indicator (FIB or alternative) or MST marker along with at least one pathogen. We focused
on studies conducted in waters intended for primary human contact (e.g., swimming, wading,
diving, and surfing) such as beaches and swimming pools, but also included ambient waters
used for secondary or non-contact (e.g., boating, fishing) human activities. Our methodology for
collecting the manuscripts involved querying “PubMed” (www.ncbi.nlm.nih.gov/pubmed/) and
“Google Scholar” (https://scholar.google.com/) databases for following keywords: “recreational
water pathogens”, “recreational water viral pathogens”, “recreational water bacterial pathogens”,
“recreational water protozoan pathogens”, “recreational water fungal pathogens”, “swimming pools
pathogens”, “swimming pools viral pathogens”, “swimming pools bacterial pathogens, “swimming
pools protozoan pathogens” and “swimming pools fungal pathogens” regardless of the year published.
For the purposes of this review, a relationship is identified as a significant correlation (e.g., Pearson
Product Momentum Correlation, Wilcoxon signed-rank tests) and/or significant predictive relationship
(e.g., binary and other logistic regression modelling). Assumptions made in our analyses included
the following: (1) all measurement strategies yielded equivalent results (e.g., various culture-based,
molecular and microscopy were equally sensitive); and (2) data were not affected by characteristics
of the water samples (e.g., we assumed that the water chemistry did not influence performance
of the methods). In total, we collected 73 papers spanning over four decades of research from
25 countries: Argentina, Australia, Bolivia, Brazil, Canada, China, Cyprus, Democratic Republic of
Congo, France, Greece, Germany, Hungary, Iceland, Italy, Japan, Luxembourg, Netherlands, New
Zealand, Poland, Portugal, South Africa, Taiwan, United Kingdom, US, and Venezuela. The majority of
studies were conducted in freshwaters (lakes, rivers and streams), followed by marine/brackish
waters and swimming pools (Figure 1). Since some studies were conducted in both fresh and
marine/brackish waters, they were included in each water type in Figure 1. This resulted in a
total of 126 observations (i.e., report on a relationship between indicator and pathogen) since some
studies were conducted in both, marine and freshwater, and/or measured more than one type of
indicator or pathogen. The majority of observations (n = 52) did not report any relationship between
indicator(s) and pathogen(s), while those that did, were split into relationships that were statistically
significant (n = 30) and those that were not (n = 44). Statistically significant relationships (or the lack
thereof) and rationale for the observed trends are further examined in the following sections.
Figure 1. Documented relationships between various indicators and pathogens in freshwaters (n = 45),
marine/brackish waters (n = 29) and swimming pools (n = 3).
135
IJERPH 2018, 15, 2842
2. Relationships between Indicators and Pathogens in Recreational Waters

We queried studies conducted in marine, brackish, freshwater, and swimming pool waters
meeting our search criteria for FIB, alternative indicators, MST markers, and pathogen data. FIB levels
were typically reported as colony forming units (CFU), or most probable number (MPN), depending
whether studies measured concentrations using membrane filtration on selective-differential media or
defined substrate technology (e.g., Enterolert and Colilert), respectively (Tables 1 and 2). However,
a few studies quantified general FIB using qPCR and expressed concentrations usually as gene copies
per unit volume (Tables 1 and 2). Alternative indicators of fecal pollution such as C. perfringens
and different bacteriophages [34] were also of interest for inclusion to assess their reliability for
estimating pathogen presence compared to general FIB. C. perfringens was measured using membrane
filtration on selective-differential media with concentrations expressed as CFU per unit volume, while
bacteriophage concentrations were usually measured via single or double agar layer (SAL, DAL)
techniques, with data expressed as plaque forming units (PFU) per unit volume (Table 3).
Lastly, studies using molecular assays targeting MST markers were gathered to determine
any potential relationships with pathogenic organisms. Depending on the detection assay used,
MST data was reported as either presence/absence (end-point PCR) or as gene copies (qPCR)
per unit volume. Assays for general MST markers included those targeting 16S rRNA gene of
Bacteroidales spp. (i.e., Bac23F, GenBac3, AllBac), and pepper mild mottle viruses (PMMoV) (Table 4).
For human-associated MST markers, most assays targeted 16S rRNA or other functional genes
of Bacteroidales or Bacteroidales-like organisms (e.g., HF183, gyrB, Bfra, HF134, B. thetaiotamicron,
BacHum-UCD, B. dorei, B. uniformis, B. stericoris, HumM2, HumM19), as well as 16S rRNA of human
-associated C. coccoides, nifH gene of Methanobrevibacter smithii, and esp gene of Enterococcus faecium
(Table 4). Bovine/ruminant-associated MST markers typically target 16S rRNA genes of Bacteroidales
spp. (e.g., BacCow, CF128, and CF193) or toxin-genes of E. coli (e.g., LTIIa), while swine-associated
MST markers target Bacteroidales spp. (e.g., PF163) or E. coli (e.g., STII) (Table 4). Since pets and
waterfowl can influence water quality, dog-associated markers have been developed targeting 16S
rRNA of Bacteroidales spp. (BacCan and DogBac), as well as seagull associated markers targeting 16S
rRNA from Catelicoccus marimammalium or Bacteroides spp. (Gull) (Table 4).
We also gathered data for various bacterial, viral, and protozoan pathogens. For bacterial
pathogens, we collected data on 10 genera (Vibrio, Salmonella, Shigella, Mycobacteria, Pseudomonas,
Escherichia, Aeromonas, Campylobacter, Legionella, and Listeria). Measurement strategies ranged from
culture-based (data reported as CFU, MPN or presence/absence) to end-point PCR (presence/absence)
and qPCR (gene copies) (Tables 1–4). For viral pathogens, we collected data on eight different species
including enteroviruses, adenoviruses, noroviruses, hepatitis A and E, astroviruses, rotaviruses,
reoviruses, and sapoviruses. Viral data were expressed as MPN (infectious viruses, ICC-[RT]-PCR),
presence/absence (PCR) or gene copies (qPCR) (Tables 1–4). The most frequently measured protozoan
pathogens, Cryptosporidium spp. and Giardia spp. (oo)cysts, were usually enumerated using
immunomagnetic separation, followed by staining, although in some instances, qPCR was also
performed (Tables 1–4). Enterocytozoon bieneusi was measured using similar detection methods to that
of Cryptosporidium and Giardia (oo)cysts (Table 2), while two pathogenic amoeba species (Acanthamoeba
spp. and Naegleria fowleri) were reported as presence/absence (i.e., PCR) (Table 1). Lastly, Candida
spp. were enumerated using membrane filtration on selective-differential media and reported as
CFU (Table 2). Sections below summarize our results regarding relationships that FIB, alternative
indicators and MST markers have with pathogens and waterborne illness occurrence in freshwater,
marine/brackish waters and swimming pools.
3. FIB and Pathogens in Freshwater

The relationships between FIB and various pathogens in freshwater, and the individual studies
from which they were derived, are summarized in Table 1. Of the 41 studies, approximately one
third (n = 18) [35–52] did not report any relationship between indicators and pathogens measured.
136
IJERPH 2018, 15, 2842
Of the remaining 23 studies, thirteen reported positive relationship between at least one indicator
and one pathogen [53–65], while ten did not find significant relationships [66–75]. Please see Table 1
(“relationship” and “comments” columns) for summary of relationships and other comments regarding
studies that found no significant relationship or those that did not report it.
Studies that found significant, positive relationships most commonly reported it for
Cryptosporidium/Giardia (oo)cysts and pathogenic E. coli spp., followed by Salmonella spp. and
Campylobacter spp. (Table 1). Relationships were less frequently noted for Shigella spp. and
adenoviruses, as well as non-fecal pathogens such as Legionella spp. and Acanthamoeba spp. (Table 1).
E. coli was the FIB with the greatest number of significant relationships, followed by enterococci, fecal
and total coliforms (Table 1). Significant relationships were reported between E. coli and pathogenic
E. coli spp. (n = 7), Cryptosporidium/Giardia (oo)cysts (n = 6), Salmonella spp. (n = 6), Campylobacter
spp. (n = 4), and adenoviruses (n = 2) (Table 1). Enterococci also had the greatest number of
statistically significant relationships with pathogenic E. coli spp. (n = 5), Cryptosporidium/Giardia
(oo)cysts (n = 4), Salmonella spp. (n = 3), Campylobacter spp. (n = 2), and adenoviruses (n = 1) (Table 1).
Fecal coliforms correlated with pathogenic E. coli spp. (n = 5), followed by Salmonella spp. (n = 3),
Cryptosporidium/Giardia (oo)cysts (n = 2), and Campylobacter and Shigella spp. (n = 1 each) (Table 1).
Statistically significant relationships between total coliforms and different pathogens (pathogenic
E. coli spp., Salmonella spp., Campylobacter spp., Legionella, and Acanthamoeba spp.) were reported only
once for each pathogen (Table 1). The methodology employed did not appear to influence the outcome,
as significant relationships were not more likely when both indicator and pathogen were measured by
a similar technique (Table 1).
The frequency of significant relationships of FIB with bacterial or protozoan pathogens was
similar; however, significant relationships with viral pathogens were less frequent (Fisher’s exact test,
p: 0.0005–0.0022). While lack of relationship between FIB and pathogenic viruses is not surprising
given the enormous differences between the two groups in terms of persistence in the environment
and low levels of viral pathogens typically found in ambient waters, correlations between FIB and
protozoan pathogens are more difficult to understand. Interestingly, when Cryptosporidium and
Giardia spp. were detected, they were generally present in higher concentrations compared to viral
pathogens. Significant relationships were reported more commonly for rivers and streams compared
to lakes [58–60,62], but this trend was not statistically significant (Fisher’s exact test, two-tailed
p = 0.085). Correlations appeared to be influenced by weather conditions, as most occurred during wet
seasons and/or following rainfall events [53,61,63,67,75]. Not surprisingly, correlations were also more
likely following sewage spills and/or wastewater discharges [54,56,57,61] and in waters impacted
by agricultural operations [57,61], likely due to elevated FIB concentrations, greater likelihood of
pathogen presence and the potential for location to be dominated by single fecal source.
137
Table 1. Relationships between fecal indicator bacteria and various pathogens in freshwater.
Indicator(s) 1 Pathogen(s) 1 Location Relationship 2 Comments Reference

All three pathogens frequently co-detected.
a V. cholerae Salmonella spp.
b, b, Apies River and tributaries,
E. coli NR Concentrations of E. coli and pathogens higher during [35]
Shigella spp. b South Africa
wet season.
Cryptosporidium and Giardia oo(cysts)
Cryptosporidium and Sure River and tributaries, Cryptosporidium and Giardia oo(cysts) frequently co-detected.
IJERPH 2018, 15, 2842
E. coli a correlated with each other and with E. [53]

Giardia (oo)cysts c Luxembourg Concentrations highest during the wet season.
coli.
All three pathogens frequently co-detected.
M. avium d , P. aeruginosa d ,
E. coli d “Aohai See” Lake, China No significant correlation. Different seasonal patterns observed for E. coli and all [66]
Salmonella spp. d
three pathogens.
All three pathogens frequently detected in samples where
Campylobacter spp. a , Giardia and E. coli strongly correlated with the all E. coli > 550 CFU.
E. coli e Avon River, New Zealand [54]
Cryptosporidium (oo)cysts c three pathogens. Higher concentrations of all pathogens found in samples
during a large sewage discharge due to earthquake.
Giardia and Canals connecting Rapipat and Highest levels of FIB and pathogens found in the most
E. coli a NR [36]
Cryptosporidium (oo)cysts c Rangsit canals, Thailand populated area.
Correlations generally stronger in samples not impacted by
Chicago area waterways system,
E. coli e,d , Cryptosporidium and Stronger correlations between FIB and the wastewater effluent.
various rivers and lakes, USA [55]
enterococci e,d Giardia (oo)cysts c Giardia spp. than Cryptosporidium spp. Associations between pathogens and enterococci generally
states
stronger than with E. coli
Fecal coliforms e , St Joseph River and Galien River Two or more virulence genes frequently co-detected.
138
E. coli e and enterococci Pathogenic E. coli b watersheds in Michigan and No significant correlation. Samples with lower FIB levels, typically had a lower [67]
e Indiana, USA proportion of virulence genes.
Samples exceeding recreational water quality guidelines
Pathogenic E. coli b , more likely to contain pathogenic E. coli genes but not
Various streams in
E. coli e , enterococci e Cryptosporidium and Giardia FIB correlated with all pathogens. Cryptosporidium and Giardia (oo)cysts. [56]
Pennsylvania, USA
(oo)cysts c Affected by non-point source and run-off following snow
melt and rain events as well.
E. coli and Aeromonas spp. co-detected in all samples.
S. enterica d , Aeromonas spp. d , Ao, Hong and Tao lakes in
E. coli d No significant correlation. Higher concentrations of E. coli and pathogens in particle [68]
M. avium d , P. aeruginosa d Beijing, China
attached fraction of the sample.
Pseudomonas aeruginosa was co-detected with all FIB in
Total coliforms a ,
P. aeruginosa a Sauce Grande lagoon, Argentina NR all samples. [37]
E. colia , enterococci a
All FIB positively correlated with temperature.
Viruses co-detected in 26% of samples.
Total coliforms a , fecal Human enteroviruses b , Presence of viruses directly related to dissolved oxygen and
Altamaha River, USA No significant correlation. [69]
coliforms a adenoviruses b streamflow, but inversely related to temperature, rainfall in
the last 30 days.
Presence of Salmonella spp., Staphylococcus aureus and
enterococci frequently coincided with fecal coliform and E.
Total coliforms a , fecal
coli levels above 1000 MPN/100 mL.
coliforms a , Salmonella spp. b , S. aureus b Msunduzi River, South Africa NR [38]
Salmonella spp. not detected in drier, colder months when
E. coli a , enterococci e
fecal coliform and E. coli levels were below 1000 MPN/100
mL.
Table 1. Cont.

Multiple pathogenic genes co-detected in samples meeting
Various rivers in Georgia,
and exceeding FIB guidelines.
Kansas, Michigan, North
Fecal coliforms e , Only eaeA gene positively correlated Some pathogenic genes also detected in three samples that
Pathogenic E. coli b Carolina, New Jersey, Ohio, [57]
E. colie , enterococci e with FIB. met FIB guidelines.
South Dakota, Tennessee, Texas
All sites known to be impacted by human fecal pollution
IJERPH 2018, 15, 2842
and Virginia, USA

and agricultural operations (upstream).
Enterococci and Campylobacter spp. frequently co-detected,
but enterococci were also detected in samples negative
d Various ponds, rivers and creeks
Enterococci e Campylobacter spp. No significant correlation. for Campylobacter. [70]
in Florida, USA
Florida DOH guidelines would not indicate
Campylobacter spp. presence.
Infectious enteroviruses a , total
Infectious enteroviruses were not detected in samples with
Fecal coliforms e , enteroviruses d , hepatitis A d ,
Rivers in France NR elevated fecal coliforms concentrations. [39]
enterococci e Norwalk I and II d , astroviruses
d , rotaviruses d Fecal coliforms, but not enterococci fluctuated seasonally.
Most samples failed to meet Taiwan CDC guidelines of 0

d Hot spring recreational facilities, total coliforms per 100 mL.
Total coliforms e Legionella spp. NR [40]
Taiwan No Legionella spp. detected in two samples that met Taiwan
CDC coliform guidelines.
Pathogenic E. coli d ,
Shigella spp. d , Salmonella spp. d ,
Various pathogen genes frequently co-detected.
139
C. jejuni d ,
Various streams around Lake Temporal variation in pathogen concentration was observed
E. coli a L. pneumophila d , L. monocytogenes No significant correlation. [71]
d, Miyajimanuma, Japan with higher levels detected in colder months and when
geese were present (not significant).
V. cholerae d , and
V. parahaemolyticus d
Highest concentration of adenovirus found at two urban
Human adenovirus d , Giardia sites.
E. coli d Rivers in France NR [41]
and Cryptosporidium (oo)cystsc Generally higher concentration of Giardia compared to
Cryptosporidium.
Acanthamoeba spp. and Legionella spp.
Acanthamoeba spp. b,d , significantly associated with total Legionella detection was significantly correlated with water
e Puzih River and two hot springs
Total coliforms Naegleria spp. b,d , Legionella spp. coliforms in hot spring samples but not temperature and more likely in the presence of [58]
b,d recreational facilities, Taiwan
river. No significant correlation for Vermamoeba vermiformis.
Naegleria spp.
In ~1/3 of samples at least two viral targets were
Human adenoviruses b,d , Danube, Berettyo, Koros and
co-detected.
E. coli a , enterococci a noroviruses GII b , and Tisza Rivers and two rivulets NR [42]
~42%, 12% and 14% of designated recreational waters
enteroviruses b (Koloska and Keki), Hungary
contained adenoviruses, enteroviruses and noroviruses.
Higher geometric mean of FIB in Salmonella spp. positive
Significant correlation commonly
samples than in Salmonella spp. negative samples.
b Transitional and inland waters, observed in waters classified as “poor”
E. coli a , enterococci e Salmonella spp. Even though significant correlation was reported, Salmonella [59]
Portugal and “sufficient” but also seen in waters
was also detected in water samples with “good” and
classified as “good” or “excellent”
“excellent” water quality.
Table 1. Cont.

Shiga-toxin producing E. coli found in samples where E. coli
Sauce Chico River, El Belisario
Total coliforms e, E. coli counts generally exceeded or were close to the US EPA
e Pathogenic E. coli b Stream and San Bernardo Stream, NR [43]
recommended limits.
Argentina
High E. coli counts correlated with rainy season.
IJERPH 2018, 15, 2842
Rotaviruses detected most frequently, followed

by adenoviruses.
Rotavirus b , human Adenovirus
Total coliforms a, The majority of samples exceeded recommendations for
b , human Astrovirus b , Rivers and streams, Brazil No significant correlation. [73]
fecal coliforms e recreational waters from the standard methods used for the
Norovirus b
examination of water and wastewater of 5000 and 1000
MPN per 100 mL for total and fecal coliforms.
Samples met European Directive 2006/7/CE for E. coli
and enterococci.
e Lake Ma Vallee, Democratic
E. coli e , enterococci e Pseudomonas spp. NR Pseudomonas spp. detected at only one site which [44]
Republic of Congo
coincidentally had the highest E. coli and enterococci
concentrations.
Beach seasonal mean C, jejuni
abundance correlated with beach
Pathogenic E. coli d , C. jejuni d , Michigan, Superior, Huron and seasonal E. coli concentration and at one High degree of beach specific temporal variability in
E. coli a [60]
Shigella spp. d , Salmonella spp. d Erie lakes, USA beach pathogenic E. coli abundance was pathogenic gene concentrations.
positively correlated with daily E. coli
concentrations
140
Approximately 50% of pathogenic bacteria resistant to at
Pathogenic E. coli b , Shigella spp. The occurrence of pathogenic bacteria least two antibiotics.
Fecal coliforms a La Paz River basin, Bolivia [61]
b and Salmonella spp. b associated with fecal coliform densities. Pathogens were frequently detected during rainy season at
sites impacted by anthropogenic activities.
Only Salmonella spp., detected sporadically in waters with
E. coli a , enterococci a , Cryptosporidium spp. c , Salmonella
Lake Parramatta, Australia NR relatively low FIB concentrations. [45]
fecal coliforms a spp. b , Campylobacter spp. a
FIB concentration higher during wet weather.
Campylobacter spp. Legionella
d,
River, lake ponds and a wadi in Campylobacter spp. detected in all samples, Cryptosporidium
E. coli e spp. d , adenovirus d , NR [46]
Netherlands spp. never detected, other pathogens detected sporadically.
Cryptosporidium spp. d
Arboviruses, hepatitis A and E viruses and E. coli O157:H7
Campylobacter spp. b ,
were not detected in any of the samples. Rotavirus,
Salmonella spp. b , E. coli O157:H7
d , Cryptosporidium and Giardia norovirus, enterovirus Salmonella and Campylobacter spp.
Fecal coliforms e , detected sporadically.
(oo)cysts c , astroviruses b , Canals and lakes, Netherlands NR [47]
E. colie , enterococci e Infectious enteroviruses found in one sample.
hepatitis A b and E b viruses,
Low concentration of Cryptosporidium and Giardia (oo)cysts
rotavirus b , norovirus b ,
detected in samples that complied with the European
enterovirus b
bathing water legislation.
L. pneumophila detected in > 90% of samples.
Puzih River and hot spring Total coliforms and L. pneumophila
Total coliforms e L. pneumophila d L. pneumophila and total coliforms also correlated with [62]
recreational areas, Taiwan significantly correlated.
turbidity.
Table 1. Cont.

Abundance and distributions of shiga-toxin genes
highly variable.
E. coli e , enterococci e Shiga toxin genes d Lake Erie and tributaries, USA No significant correlation. The majority of samples positive for shiga toxin genes were [74]
below the advisory threshold levels for E. coli and
enterococci.
IJERPH 2018, 15, 2842
Cryptosporidium and
E. coli e , enterococci e , Higher concentrations of indicators and more frequent
Giardia (oo)cysts c , infectious Lake Carroll, Tampa, FL NR [48]
fecal coliforms e pathogen detection following rain events.
enteroviruses a , Salmonella spp a
Human adenovirus human d, Adenoviruses detected more frequently than enteroviruses,
Delaware Lake, Madison Lake
E. coli e enterovirus d , Norovirus GI and No significant correlation. followed by noroviruses. [72]
and East Fork Lake, USA
GII d Human adenovirus and enterovirus correlated.
All sampling sites achieved “sufficient” bathing water
quality for enterococci but not E. coli.
Campylobacter spp. a ,
E. coli total
a, With the exception of Aeromonas spp., detection of all other
Salmonella spp. a , P. aeruginosa a , River Ruhr and barrier lakes,
coliforms a , NR pathogens was sporadic. [49]
Cryptosporidium and Giardia Germany
enterococci e Precipitation preceding sampling event resulted in elevated
(oo)cysts c , Aeromonas spp. e
concentration of total coliforms, E. coli, enterococci,
Aeromonas spp. and Cryptosporidium and Giardia (oo)cysts.
High concentrations of Pseudomonas spp. detected in
E. coli e , enterococci e Pseudomonas spp. e , Norovirus d Geothermal pools, Iceland NR samples that also contained high FIB counts. [50]
Norovirus was not detected.
141
S. aureus Salmonella spp.
d, d,
E. coli d , enterococci d Prickett Creek, USA NR No correlation between E. coli and enterococci. [51]
noroviruses d
L. monocytogenes Salmonella spp.
b,
The fraction of samples that contained an indicator when
Total coliforms e , fecal b , E. coli O157:H7 b ,
South Nation River basin, Weak relationships, but mostly positive pathogen was detected was highest for the protozoan
coliforms e , Campylobacter spp. b , [63]
Canada (except L. monocytogenes). parasites.
E. coli e , enterococci e Cryptosporidium and
Relationships dependent on season and site.
Giardia (oo)cysts c
Various rivers and lakes in
Concentrations of all indicators
France, Germany, Italy, > 50% of samples positive for adenovirus.E. coli
E. coli a , enterococci a Human adenoviruses b correlated with frequency of [64]
Netherlands, Poland, United concentrations higher than enterococci.
adenovirus detection.
Kingdom
Various rivers and lakes in
Both viruses frequently detected in samples that met “good”
Adenoviruses d,f , norovirus GI b France, Germany, Italy,
E. coli a , enterococci a NR water quality guidelines for both E. coli and enterococci. [52]
and GII b Netherlands, Poland, United
Adenoviruses detected more frequently than noroviruses.
Kingdom
Total coliforms a , fecal Salmonella spp. f , pathogenic E. Significant but weak correlations Cryptosporidium and Giardia (oo)cysts detected in samples
coliforms a , coli f , Cryptosporidium and Giardia Wanzhou watershed, China between indicators and Salmonella spp. with low indicator concentrations. [65]
E. coli a , enterococci e (oo)cysts c and pathogenic E. coli. Concentrations of indicators influenced by rainfall.
Significant correlation between Cryptosporidium and
Cryptosporidium and Giardia
Total coliforms a Lake Tianjin, China No significant correlation Giardia (oocysts). [75]
(oo)cysts c
Giardia detected more frequently.
1 Data reporting: most probable number (MPN) a , Presence/absence b , total (oo)cysts c , gene copies d , colony forming units (CFU) e , Integrated cell culture (ICC)/MPN PCR f .
2 NR (not reported).
IJERPH 2018, 15, 2842
4. FIB and Pathogens in Marine and Brackish Water

Table 2 lists general FIB and pathogen relationships reported in the datasets analyzed for marine
and brackish waters. Of the 29 studies reviewed, almost half (n = 13) did not report statistical analysis of
the relationships [52,76–87]. Within the remaining studies, ten did not find a relationship [64,70,88–95]
while six reported a positive relationship between at least one indicator and one pathogen [59,96–100].
Statistical significance was reported less frequently in marine and brackish waters compared to
freshwater (17 vs. 44) and the proportion of statistically significant relationships (compared to
non-significant) was considerably higher in freshwater (Fisher’s exact test, p < 0.0001). Please see
Table 2 (“relationship” and “comments” columns) for a summary of relationships and other comments
regarding studies that found no significant relationship or those that did not report it.
Significant relationships with FIB were most commonly reported for Salmonella spp., followed
by adenoviruses, and Campylobacter spp., Vibrio spp., S. aureus, and protozoan pathogens (Table 2).
The most significant relationships with pathogens were reported for enterococci (n = 11), followed
by E. coli (n = 4), and fecal coliforms (n = 2). No statistically significant relationships were reported
for total coliforms. Significant relationships were reported between enterococci and adenoviruses
(n = 8), Salmonella spp. (n = 4), Cryptosporidium/Giardia (oo)cysts (n = 4), Campylobacter spp. (n = 3), and
Candida spp., Vibrio spp., S. aureus, noroviruses, and E. bieneusi (one observation each) (Table 2). E. coli
formed significant relationships with Salmonella spp. and Vibrio spp. (n = 1 each) and adenoviruses
(n = 2). Statistically significant relationships with fecal coliforms were reported only for adenoviruses
(28.6%, n = 7). The methodology employed did not appear to influence the outcome; significant
relationships were not more likely when both indicator and pathogen were measured by a similar
technique (Table 2).
As expected, FIB had more significant relationships with bacterial pathogens compared to viral
pathogens (Fisher’s exact, p = 0.0069), but there was no significant difference in other comparisons (i.e.,
FIB relationships with bacterial compared to protozoan pathogens, or FIB relationships with protozoan
compared to viral pathogens). Of note, FIB most likely to correlate with pathogens were enterococci,
which supports its recommended use to monitor marine recreational water quality. No clear trend for
different marine water types (e.g., brackish waters and coastal beaches) was observed with respect to
statistically significant indicator/pathogen relationships [59,96,98–101], suggesting that hydrological
factors play less of a role compared to freshwaters. Similar to freshwater, the common trend among
the studies reporting significant relationships was that they were conducted in waters impacted by
fecal contamination [96,98,99], and when bather numbers were high [97], conditions likely to result in
elevated FIB and pathogen levels.
142
Table 2. Relationships between fecal indicator bacteria and various pathogens in brackish and marine waters.

V. vulnificus b , S. aureus e ,
When HPyV, V. vulnificus, and Giardia spp. were detected so
Fecal coliforms e,
enterovirus d , norovirus d ,
were all indicators and alternative indicators.
E. coli e , enterococci hepatitis A virus d , Virginia Key Beach, Florida, USA NR [77]
a,d,e When FIB levels exceeded regulatory standards, HPyVs and
pathogens also detected.
(oo)cysts c
IJERPH 2018, 15, 2842
enterovirus d , norovirus d , When enterococci levels by qPCR and CS exceeded MDL,
Fecal coliforms e , E. Coastal Beaches, Miami Dade
hepatitis A virus d , NR Cryptospordium, Giardia, enteroviruses and V. vulnificus were [76]
coli e , enterococci d,e County, Florida, USA
Cryptosporidium and Giardia co-detected.
(oo)cysts c
Cryptosporidium and Giardia Presence of Cryptosporidium and Giardia were significantly
Enterococci e Coastal beaches, Venezuela No significant correlation. [88]
(oo)cysts c correlated.
e b Enterococci but not E. coli correlated Pathogens detected in waters of “good” and “excellent”
E. coli e, enterococci C. albicans b, Salmonella spp. Saronicos Gulf, Athens, Greece [96]
with Salmonella spp., but not C. albicans. quality.
Total coliforms a , fecal b Canals around Galveston Bay, Salmonella spp. detection occurred (nearly 100%) when FC
Salmonella spp. NR [78]
coliforms a TX, USA concentrations >2000/100 mL.
C. parvum c , G. lamblia c , G. Maryland, US Chesapeake Bay, C. parvum, G. duodenalis and E. bieneusi Correlations observed especially apparent with high bather
Enterococci a [97]
duodenalis c E. bieneusi c USA correlated with enterococci counts. numbers in water.
Enterococci a,d ,
E. coli L. pneumophila S. aureus
b, b,
a,d , MRSA b , adenovirus b , No indicator used had a significant correlation with GI
143
Malibu beach, California USA NR [79]
fecal coliforms a , total enterovirus d , Hepatitis A d , illness in swimmers or any reference pathogen.
coliforms a Norovirus d
Enterococci co-detected with pathogenic Campylobacter spp.,
Enterococci e Campylobacter spp. d , Florida, Quietwater Beach, USA No significant correlation. but levels of enterococci were not indicative of levels of [70]
Campylobacter present.
Salmonella spp. b , Campylobacter
spp. a , Cryptosporidium and Changes in FIB concentrations associated with changes in
E. coli a , enterococci a Estuaries, Melbourne Australia No significant correlation. [89]
Giardia (oo)cysts c , adenoviruses temperature, flow, humidity and rainfall.
a enteroviruses a
Total coliforms e , fecal

Southern California coastal 5 of 12 sites, FIB exceeded CA recreational water quality
coliforms e , E. coli e , Adenoviruses a No significant correlation. [90]
waters, USA limits.
enterococci e
FIB and viral pathogen detection associated with storm
Adenoviruses b , enteroviruses b , Rivers and creeks in California, events.
coliforms e , enterococci No significant correlation. [91]
e hepatitis A b USA Total and fecal coliforms correlated with each other but not
enterococci.
FIB concentrations showed strong seasonal pattern,
Total coliforms a , fecal
associated with winter storms
coliforms a , Enterovirus b , adenoviruses b Newport Bay, California, USA No significant correlation [92]
Total and fecal coliforms correlated with each other but not
enterococci a
enterococci.
Table 2. Cont.

Salmonella spp. not detected.
Fecal coliforms e,
Salmonella spp. b, enteric viruses Ben T. Davis and Bahia beaches,
b NR Coxsackie B4 detected following major sewage spill [80]
E. coli e , enterococci e Florida, USA
FIB correlated with rainfall.
Fecal coliforms e, Cryptosporidium and Giardia FIB co-detected with all samples positive for
Sarasota Bay, Florida, USA NR [81]
IJERPH 2018, 15, 2842
enterococci e (oo)cysts c enteroviruses a enteric pathogens.

Levels of FIB correlated with presence
e of Salmonella spp., especially in waters Salmonella spp. also detected in samples classified as “Good”
E. coli e , enterococci e Salmonella spp. Coastal Waters, Portugal [59]
deemed “poor” or “sufficient” or “Excellent”.
compared to “excellent”.
Presence of adenovirus strongly
Fecal coliforms e Hillsborough River and St. Johns Samples collected in waters with known human fecal
Adenovirus b correlated with concentrations of all [98]
E. coli e , enterococci e River, Florida, USA pollution, all FIB exceeded regulatory standards.
three FIB.
E. coli e ,
Hillsborough River, FL E. coli, enterococci and fecal coliforms All FIB concentrations exceeded regulatory standards in
fecal coliforms e , Adenovirus b [99]
Tampa Bay Beach, Florida, USA correlated with adenovirus. samples.
enterococci e
FIB concentrations frequently exceeded recreational water
Enterococci e ,
Avalon and Doheny Beaches, quality guidelines.
fecal coliforms e , Adenovirus b No significant correlation. [93]
California Adenoviruses frequently detected at Doheny beach, but not
total coliforms e , E. coli e
Avalon.
All samples with elevated FIB levels also had high levels of
144
Total coliforms e ,
somatic and F-specific phage present.
fecal coliforms e , Enteroviruses e Coastal beaches, Barcelona, Spain NR [82]
55% of samples having infectious virus beach quality was
enterococci e , E. coli e
within EU standards for levels of FIB.
Enterococci e ,
Enterovirus a , Cryptosporidium Viruses detected in samples where FIB levels were within
fecal coliforms a , St. Lucie Estuary, Florida, USA No significant correlation. [94]
and Giardia (oo)cysts c regulatory limits.
E. coli a , total coliforms a
Adenovirus b , Norovirus d ,
(oo)cysts c , FIB and pathogens co-detected
Enterococci a,d Coastal beaches, Florida, USA NR [83]
C. jejuni, Salmonella spp. b , S. Seawater samples taken near sewage discharges.
aureus b ,
E. coli 0157-H7 b
V. vulnificus e , Enterococci were co-detected with V. vulnificus and V.
Enterococci e Chesapeake, Bay, MD, USA NR [84]
V. parahaemolyticus e parahaemolyticus.
Table 2. Cont.

Enterococci were co-detected with V. vulnificus and V.
parahaemolyticus.
V. vulnificus e , All V. vulnificus isolates susceptible to 14 of 26 antibiotics
Enterococci e Chesapeake, Bay MD, USA NR [85]
V. parahaemolyticus e and V. parahaemolyticus to 11 of 26 antibiotics.
All samples positive for enterococci and Vibrio spp. and
IJERPH 2018, 15, 2842
within local recreational water quality guidelines.

All samples considered “good” quality based on local
coliforms e , E. coli e , Hepatitis A b , Norovirus GI b Coastal beaches, Lisbon, Portugal No significant correlation. [95]
recreational water quality guidelines.
enterococci e
S. aureus was found in 37% of total samples, 1.1% positive
Coastal beaches, Miami, Florida, for MRSA.
Enterococci a,e S. aureus e NR [86]
USA Enterococci had a positive correlation with reports of skin
illness.
Salmonella, Campylobacter and C. jejuni
positively associated with enterococci
Salmonella spp. Campylobacter
a, and marginally associated with S.
Detection of at least one pathogens occurred in 21 of 22
E.coli e , enterococci e spp. a , S. aureus e , V. vulnificus e , Hawaii streams, USA aureus. [100]
streams tested.
V. parahaemolyticus e V. vulnificus was positively associated
with all FIB, V. parahaemolyticus with E.
coli.
Fecal coliforms e , Enterovirus co-detected with fecal coliforms, enterococci.
145
Enteroviruses b Florida Keys, Florida, USA NR [87]
enterococci e No sites in violation of water quality standards.
Coastal beaches in Cyprus, Italy,
FIB levels significantly lower in seawater than in freshwater
E. coli a , enterococci a Human Adenovirus b Portugal, Spain and No significant correlation. [64]
samples.
United Kingdom
Beaches considered “clean” based on FIB levels were
Human adenovirus b norovirus b positive for both adenovirus and noroviruses.
E. coli e , enterococci e Portugal, Spain and United NR [52]
GI and GII Freshwater sites had higher frequency of virus detection
Kingdom
than marine sites.
1 Data reporting: most probable number (MPN) a , Presence/absence b , total (oo)cysts c , gene copies d , colony forming units or plaque forming units (CFU/PFU) e , Integrated cell culture
(ICC)/MPN PCR f . 2 NR (not reported).

IJERPH 2018, 15, 2842
5. Alternative Indicators and Pathogens in Marine, Brackish and Freshwater

Ten studies conducted in freshwater and fourteen studies conducted in brackish and marine
waters measured at least one alternative indicator and one pathogen. In freshwater, four studies
measured C. perfringens, five studies measured bacteriophage, and one study measured both (Table 3).
In brackish/marine waters, the majority (n = 12) of studies measured coliphage (somatic, F-specific),
followed by C. perfringens (n = 7), and phages infecting Bacteroides thetaiotaomicron (n = 1) (Table 3).
Similar to FIB, more statistically significant relationships were reported in freshwater compared
to brackish/marine waters (Fisher’s exact test, p = 0.0057). Please see Table 3 (“relationship” and
“comments” columns) for summary of relationships and other comments regarding studies that found
no significant relationship or those that did not report it.
In freshwater, half of studies (n = 5) did not report statistical analysis [41,45,47,49,102], four
reported at least one statistically significant relationship [54,63,64,103], while a single study reported a
non-significant relationship [39] (Table 3). Statistically significant relationships were reported more
frequently for C. perfringens, followed by F-specific and somatic coliphages (Table 3). C. perfringens
had positive relationships with Campylobacter spp. (n = 2), and Listeria spp., Salmonella spp. and
pathogenic E. coli spp. (one observation each). F-specific coliphage correlated with noroviruses,
Cryptosporidium/Giardia (oo)cysts, and Campylobacter spp. (one observation each) while somatic
coliphage correlated only with adenoviruses (n = 2). Similar to FIB, the observed correlations occurred
in waters affected by sewage discharge [54], following rainfall events [103], and were affected by
season and sampling site [63].
In marine and brackish waters, approximately, half of studies (n = 6) did not report any statistical
analysis [76,77,81,82,87,89], while three reported statistically significant relationships [79,90,100], and
five reported non-significant relationships [64,88,91,92,94]. Two studies found significant relationships
between F-specific coliphage and pathogens; one reported it with methicillin resistant S. aureus (MRSA),
and S. aureus at a marine beach affected by fecal-impacted freshwater intrusion [79], while a second
reported it for adenoviruses in water impacted by urban run-off [90] (Table 3). No studies noted
a significant relationship between somatic coliphage and pathogens (Table 3). Only a single study
conducted in Hawaii [100], a state that recommends using C. perfringens for monitoring ambient
waters [104], found a relationship between this indicator, and two pathogens (Campylobacter spp., and
V. parahaemolyticus) (Table 3). The methodology employed did not appear to influence the outcome in
marine or freshwaters; significant relationships were not more likely when both indicator and pathogen
were measured by a similar technique (Table 3). While there were insufficient data to perform statistical
analyses regarding relationship of alternative indicators and different pathogen groups, F-specific
coliphage tended to perform better compared to somatic coliphage and C. perfringens.
146
Table 3. Relationship of alternative indicators of fecal pollution and pathogens in freshwater and marine/brackish waters.

Freshwater
C. perfringens e , Campylobacter spp. a , Giardia and Avon River, Christchurch, New F-RNA more strongly correlated with F-RNA concentrations typically higher than C. perfringens.
[54]
F-RNA coliphage e Cryptosporidium (oo)cysts c Zealand all three pathogens than C. perfringens. Study conducted in river affected by sewage discharge.
IJERPH 2018, 15, 2842
Enterovirus genomes and somatic coliphage

Infectious enteroviruses total
a,
frequently co-detected.
enteroviruses d , hepatitis Ad ,
Somatic coliphage e Rivers in France No significant correlation. Infectious enteroviruses and hepatitis A, Norwalk I and II, [39]
Norwalk I and II d , astroviruses
d , rotaviruses d astroviruses, rotaviruses detected in only one or
two samples.
Highest concentration of protozoan parasites and
Human adenovirus d , Giardia C. perfringens found at the site with high proportion of
C. perfringens e Rivers in France NR [41]
and Cryptosporidium (oo)cysts c agricultural operations, forests and semi-natural
environments.
Noroviruses f , rotaviruses f , Both coliphages and all viruses co-detected in all samples.
Somatic and F+ Maas and Waal Rivers,
infectious reoviruses and NR Coliphage concentrations higher than pathogenic virus [102]
coliphage e Netherlands
enteroviruses a concentrations.
Higher statistical correlation observed between enteric
viruses than between enteric viruses and coliphages.
Somatic and F+ Noroviruses d , adenoviruses d , Marine Reservoir and tributaries, F+ coliphage positively correlated with Noroviruses most abundant, followed by rotaviruses.
[103]
coliphage e astroviruses d , rotaviruses d Singapore norovirus concentrations. Wet weather concentration of coliphage and viruses higher
147
than dry weather concentration, but difference is not
statistically significant.
No Cryptosporidium detected which coincided with the low
Cryptosporidium spp. c , Salmonella
C. perfringens e Lake Parramatta, Australia NR C. perfringens concentrations. [45]
spp. b , Campylobacter spp. a
C. perfringens concentration lower than FIB.
Campylobacter spp. a , Salmonella
spp. b , E. coli O157:H7 d ,
Somatic coliphage detected more frequently and at higher
Somatic and F+ concentrations compared to F+ coliphage.
(oo)cysts c , astroviruses b , Canals and lakes, Netherlands NR [47]
coliphages e Highest concentrations of bacteriophages occurred
hepatitis A and E viruses b ,
following a heavy rainfall.
rotavirus b , norovirus b ,
enterovirus a
Campylobacter spp. a , Salmonella
Concentrations typically lower and less variable compared
spp. a , P. aeruginosaa , River Ruhr and barrier lakes,
C. perfringens e NR to the FIB. [49]
Cryptosporidium and Giardia Germany
No association with precipitation.
(oo)cysts c , Aeromonas spp. e
Table 3. Cont.

L. monocytogenes b , Salmonella spp.
b , E. coli O157:H7 b ,
South Nation River basin, Positive, but weak relationships with
C. perfringens e Campylobacter spp. b , Correlations with FIB were also weak but positive. [63]
Canada pathogens.
IJERPH 2018, 15, 2842
(oo)cysts c
Various rivers and lakes in FIB showed better correlation with adenovirus than somatic
Concentrations of somatic coliphage
France, Germany, Italy, coliphage.
Somatic coliphage e Human adenoviruses b correlated with frequency of [64]
Netherlands, Poland, United Somatic coliphage concentrations comparable to E. coli
adenovirus detection.
Kingdom concentrations.
Marine, and brackish waters
enterovirus d , norovirus d , Higher concentrations in high tide samples as opposed to
C. perfringens e hepatitis A virus d , Virginia Key Beach, Florida, USA NR low tide. [77]
Cryptosporidium and Giardia Not correlated with FIB.
(oo)cysts c
enterovirus d , norovirus d ,
C. perfringens e , Coastal Beaches, Miami Dade High levels of C. perfringens also signaled high levels of all
hepatitis A virus d , NR [76]
F+ coliphage e County, Florida, USA FIB.
(oo)cysts c
148
Cryptosporidium and Giardia Detection of C. perfringens coincided with human-associated
C. perfringens a Coastal beaches, Venezuela No significant correlation. [88]
(oo)cysts c MST markers.
L. pneumophila b , S. aureus b ,
MRSA b , adenovirus b , F+ coliphage had strong association
F+ coliphage e Malibu beach, California USA F+ coliphage had strong association with GI illness. [79]
enterovirus d , Hepatitis A d , with MRSA and S. aureus presence.
Norovirus d
Salmonella spp. b , Campylobacter
Docklands, South Yarra and
C. perfringens e and spp. a , Cryptosporidium and Positive correlation between the presence of C. perfringens
Abbotsford estuaries, Melbourne NR [89]
F+ coliphage e Giardia (oo)cysts c , adenoviruses and F+ coliphage.
a , enteroviruses a Australia
Presence of adenovirus was

Somatic e and Southern California coastal
Adenoviruses a significantly correlated with F-specific No correlation between two coliphage types. [90]
F+ coliphage e waters, USA
coliphage.
Somatic coliphages detected more frequently than F+.
Somatic e and Adenoviruses b , enteroviruses b , Rivers and creeks in California, Somatic coliphage were not correlated with total coliforms,
No significant correlation. [91]
F+ coliphage e hepatitis A b USA but F+ coliphage were positively correlated with total/fecal
coliforms but not enterococci.
Peak concentrations of FIB and F+ coliphage associated with
F+ coliphage a Enterovirus b , adenoviruses b Newport Bay, California, USA No significant correlation. [92]
winter storms.
Alternative indicators for co-detected in samples positive
C. perfringens e and Cryptosporidium and Giardia for enteric pathogens.
Sarasota Bay, Florida, USA NR [81]
coliphage e (oo)cysts c , enteroviruses a Coliphage levels were significantly influenced by salinity
and turbidity.
Table 3. Cont.

Marine, and brackish waters
Enteroviruses were co-detected with FIB.
Somatic e and
Genogroup I and II F-specific RNA more common in
F+ coliphage e , phages
Enteroviruses e Coastal Waters, Portugal NR samples the others. [82]
IJERPH 2018, 15, 2842
infecting Bacteroides
Densities of somatic coliphage were higher than FIB
thetaiotaomicron GA17 e
densities and did not correlate with them.
Somatic coliphage concentrations higher than F+ coliphage.
Somatic e and Enterovirusa , Cryptosporidium
St. Lucie Estuary, Florida, USA No significant correlation. Somatic coliphage correlated with the total coliform [94]
F+ coliphage e and Giardia (oo)cysts c
concentrations.
Salmonella spp. a , Campylobacter
C. perfringens was marginally associated
C. perfringens e and spp. a , S. aureus e , Concentrations of C. perfringens and F+ coliphage
Hawaii streams, USA with Campylobacter spp. and [100]
F+ coliphage e V. vulnificus e , comparable.
V. parahaemolyticus.
V. parahaemolyticus e
C. perfringens e ,
Enteroviruses b Florida Keys, Florida, USA NR Enteroviruses co-detected with C. perfringens and coliphage. [87]
F+ coliphage e
Somatic coliphage e Human Adenovirus b Portugal, Spain and No significant correlation. Somatic coliphage concentrations lower than FIB. [64]
United Kingdom
1 Data reporting: most probable number (MPN) a , Presence/absence b , total (oo)cysts c , gene copies d , colony forming units or plaque forming units (CFU/PFU) e , Integrated cell culture
149
(ICC)/MPN (RT)PCR f . 2 NR (not reported).
IJERPH 2018, 15, 2842
6. MST Markers and Pathogens in Marine, Brackish and Freshwater

The number of studies that measured MST marker(s) along with at least one pathogen is
considerably smaller (n = 19; eight in freshwater, and 11 in brackish/marine/waters) compared
to studies measuring FIB or alternative indicators (Table 4). The majority of MST measurements
reported were for human-associated marker(s) (76.1%), followed by general MST markers (7.0%),
cattle and dog associated MST markers (5.6%), and seagull and swine-associated MST markers
(2.8%) (Table 4). Most frequently measured pathogens were viruses (adenovirus, enterovirus,
noroviruses, hepatitis, and infectious enteric viruses) and bacteria (E. coli, Campylobacter spp.,
Salmonella spp., V. vulnificus, S. aureus, and Legionella spp.) with 22 measurements each, while
Cryptosporidium and Giardia (oo)cysts were reported less frequently (n = 6) (Table 4). Irrespective
of the water type, nine of these studies did not report statistical analyses for relationships between MST
marker(s) and pathogens [51,76,77,79,80,83,86,88,105], and another seven reported non-significant
relationship [48,56,57,70,72,98,99]. The remaining two studies reported statistically significant
relationship [93,106] (Table 4). Please see Table 4 (“relationship” and “comments” columns) for
summary of relationships and other comments regarding studies that found no significant relationship
or those that did not report it.
Significant relationships between pathogens and human-associated MST markers were reported
for HF183 and adenoviruses, at a marine beach impacted by non-point source(s) [93], and between
HF183/HF134 and Campylobacter spp. in freshwater affected by livestock operations [106]. In the
same freshwater study, cattle-associated MST markers (CF128, CF193) correlated with E. coli O157:H7,
and Salmonella spp., while a general Bacteroidales MST marker (Bac32F) correlated with all three
pathogens [106]. The methodology employed did not appear to influence the outcome; in other words,
significant relationships were not more likely when both indicator and pathogen were measured by a
similar technique (Table 4). There were insufficient data regarding relationship of MST markers and
different pathogen groups (e.g., bacterial, viral and protozoan) to perform statistical analyses. While
it may seem counter-intuitive that MST markers (particularly human-associated subset), were not
generally correlated with pathogens, it is important to note that sensitivity and specificity of MST
markers varies greatly [14]. Furthermore, many pathogens reported in these studies are zoonotic,
making this relationship even more tenuous.
150
Table 4. Relationships between MST markers and various pathogens in in freshwater and marine/brackish waters.
MST marker 1 Pathogens 1 Location Relationship 2 Comments Reference

Freshwater
Pathogenic E. coli spp. a ,
Various streams in Pennsylvania, All MST markers detected more frequently in samples
esp a , LTIIa a , STII a Cryptosporidium and Giardia No significant correlation. [56]
USA exceeding recreational water quality guidelines.
(oo)cysts b
IJERPH 2018, 15, 2842
Various rivers in Georgia,

Kansas, Michigan, North
a esp was present in nine samples that met exceeding
esp a Pathogenic E. coli spp. Carolina, New Jersey, Ohio, No significant correlation. [57]
recreational water quality guidelines.
South Dakota, Tennessee, Texas
and Virginia, USA
c Various ponds, rivers and creeks Campylobacter and MST markers co-detected at only one site.
HPyV a , nifH a , HF183 a Campylobacter spp. No significant correlation. [70]
in Florida, USA HPyV and nif H were not detected during the study.
Human adenovirus c , human g-Bfra detected more frequently and at higher
Delaware Lake, Madison Lake
gyrB c , g-Bfra c enterovirus c , Norovirus GI and No significant correlation. concentrations than gyrB. [72]
and East Fork Lake, USA
GII c , porcine sapovirus c gyrB and g-Bfra frequently correlated.
Higher concentrations of indicators and more frequent
pathogen detection following rain events.
esp a , HPyV a , HF183 a (oo)cysts b , infectious Lake Carroll, Tampa, FL No significant correlation. [48]
d esp, but not HPyV or HF183 correlated with FIB (E. coli,
enteroviruses d , Salmonella spp.
enterococci, fecal coliforms).
Positive relationship between detection
of Bac32F and all pathogens. Bac32F detected most frequently, followed by CF128/193,
Positive relationship between PF163 and HF183/134
Bac32F CF128 CF193
a, a, a, E. coli O157:H7 Salmonella spp.
a, a, Little Bow and Oldman Rivers,
CF128/193 and E. coli O157:H7 and Pathogens were detected infrequently with Campylobacter [106]
151
HF134 a , HF183 a , PF163 a Campylobacter spp. a Canada
Salmonella spp. spp. most commonly detected. Water impacted by
Positive relationship between agricultural operations.
HF183/134 and Campylobacter spp..
S. aureus c , Salmonella spp. c ,
HPyV c , HF183 c , AllBac c Prickett Creek, USA NR Salmonella spp. were most frequently detected pathogen. [51]
noroviruses c
Precipitation and turbidity positively correlated with
esp a Infectious enteric viruses a Lake Michigan, USA NR [105]
viruses.
Marine and brackish waters
V. vulnificus c , S. aureus c ,
HPyV, V. vulnificus, Giardia spp. were co-detected with all
enterovirus c , norovirus c ,
a FIB and alternative indicators.
HPyV a, esp hepatitis A c virus, Virginia Key Beach, Florida, USA NR [77]
When FIB levels exceeded regulatory standards, HPyVs and
Cryptosporidium and
pathogens also detected.
Giardia (oo)cysts b
V. vulnificus c , S. aureus c ,
HPyV a , esp a , Bacteroides
enterovirus c , norovirus c , During rain event, DogBac was co-detected with
thetaiotaomicron a , Coastal Beaches, Miami Dade
hepatitis A c virus, NR Cryptospordium, Giardia, enteroviruses and V. vulnificus along [76]
BacHum-UCD a and County, Florida, USA
Cryptosporidium and with enterococci.
DogBac a
Giardia (oo)cysts b
Table 4. Cont.
MST marker 1 Pathogens 1 Location Relationship 2 Comments Reference

Marine and brackish waters
The levels of (oo) cysts varied with the extent of sewage
Cryptosporidium and
HF183 c , C. coccoides c Coastal beaches, Venezuela NR pollution and bather density. [88]
Giardia (oo)cysts b
HF183 and C. coccoides correlated with C. perfringens.
IJERPH 2018, 15, 2842
GenBac3 HF183
c, a,c ,
BacHum-UCD c , B. dorei a,c ,

HumM2 c , HF134 a ,
L. pneumophilaa , S. aureusa , MRSA a ,
HumM19 a , B. stericoris a , Human-associated MST markers were only predictive of
pathogenic E. coli a , adenovirus a ,
B. uniformis c , nifH a,c , esp c , Malibu beach, California USA NR illness at the site known to be impacted by human sewage [79]
enterovirus c , Hepatitis A c ,
HPyV a,c , Gull Bacteroides a , from faulty infrastructure.
Norovirus a
C. marimammalium c ,
BacCow-UCD c ,
BacCan-UCD c
c In some instances, MST markers were co-detected with

HPyV a , nifH a , HF183 a Campylobacter spp. Quietwater Beach, Florida, USA No significant correlation. [70]
Campylobacter especially following rain events.
Coxsackie virus B4 and HPyVs were co-detected following a
Ben T. Davis and Bahia beaches, major sewage spill.
esp a , HPyV a Salmonella spp. a , enteric viruses d NR [80]
Florida, USA Fecal coliform concentrations correlated with the esp marker
HPyV did not correlate with FIB.
Hillsborough River and St. Johns No significant correlation with HPyV,
HPyV c , HF183 c , nifH a Adenovirus a Adenovirus co-detected with HF183 and nifH. [98]
River, Florida, USA NR for HF183, nifH.
All FIB concentrations
152
Hillsborough River, St, Johns exceeded regulatory standards
GenBac3 a , HPyVc , HF183 a , No significant relationship with HPyV,
Adenovirus a River, Ben T. Davis beach, HF183 and nifH detected in 80% of [99]
nifH a other MST markers NR
Florida, USA samples, whereas adenoviruses were detected
in 60% of the samples.
Avalon and Doheny Beaches, At Doheny Beach HPyV and HF183 Adenovirus not detected at Avalon Beach, impacted by
HPyV c , HF183 a , nifH a Adenovirus a [93]
California, USA presence correlated with adenovirus. non-point source(s).
Adenovirus a , Norovirus,
PMMoV co-occurred with FIB, other MST markers and
PMMoV c , HF183 c , Cryptosporidium and Giardia
Coastal beaches, Florida, USA NR pathogens. [83]
BacHum-UCD c , esp a , nifH c (oo)cysts b , C. jejuni a , Salmonella
Seawater samples taken near sewage outfalls.
spp. a , S. aureus a , E. coli 0157-H7 a
BacHum-UCD c , HF183 c ,
Coastal beaches, Miami, Florida, Co-occurrence with S. aureus detection.
DogBac c , S. aureus e NR [86]
USA No correlation found with MST markers and skin illness.
C. marimammalium c
1 Data reporting: Presence/absence a, total (oo)cysts b, gene copies c, most probable number (MPN) d, colony forming units (CFU) e; 2 NR (not reported).
IJERPH 2018, 15, 2842
7. Various Indicators and Pathogens in Swimming Pools

Our search of literature for paired measurements of indicator(s) and pathogen(s) recorded for
swimming pools yielded considerably fewer studies (n = 3), compared to ambient waters. None of the
studies reported statistical analyses on relationships between indicators and pathogens. Two studies,
both conducted in Italy, were performed on pools that were in compliance with microbiological
requirements for E. coli, enterococci, P. aeruginosa, and S. aureus [107,108]. However, one study
detected infectious Simkania negevensis, a bacterium related to Chlamydia, in nearly 43% of samples,
while the second one measured Papillomaviruses in 64% of samples [108]. Interestingly, HPyVs
were co-detected with Papillomaviruses in all the samples [108]. L. pneumophila and enteric viruses
(adenovirus, norovirus, and enteroviruses) [108] were not detected. Examination of wading pools in
Finland during a gastroenteritis outbreak detected Norovirus GII and astrovirus in ~83% and ~33% of
samples, respectively [109]. E. coli was absent from samples collected ~2 weeks before the outbreak,
but high concentrations (370–24,000 CFU/100 mL) were measured in two samples taken during the
outbreak [109].
8. Relationship of Indicators with Illness

To identify associations between the presence of general FIB, alternative indicators or MST
markers with that of waterborne illness occurrence, various epidemiologic studies were collected
from existing literature dating back to the early 1990s. For inclusion, it was required that the study
measured at least one FIB, alternative indicator or MST marker (culture or molecular) in combination
with an epidemiological survey of resulting illness from the recreational water exposure. In total,
17 studies [76,79,86,110–124] met these criteria and were included in analyses. One study each was
conducted in Europe and Africa, and fifteen studies were conducted in the US. Since some of these
studies were conducted in more than one water type, this resulted in the inclusion of 20 freshwater
sites and 29 brackish/marine sites. Thirteen different microbiological assays were reported including
those targeting: enterococci, fecal and total coliforms, E. coli, somatic and F+ coliphage, as well as
various general and human-associated MST markers (Figure 2). In addition to gastrointestinal illnesses
characterized by symptoms of diarrhea, vomiting, and stomach cramps, other waterborne illnesses
included skin, ear and sinus infection [76,79,86,110–113,115–122,124]. For epidemiological studies,
assays targeting enterococci were the most commonly recorded, with 25 instances of measurements of
either culture based or molecular enterococci targets, followed by human-associated MST markers,
F+ coliphage, fecal coliforms, general MST markers, total coliforms, culturable E. coli, somatic coliphage
and finally E. coli qPCR signal (Figure 2).
Figure 2. Summary of epidemiological studies reporting on linkage between illness and various
indicator types.
153
IJERPH 2018, 15, 2842
Correlations between observed illness in these studies were most common with enterococci
(10 studies out of 17) [79,86,110,111,113,114,116,117,120,121], followed by F+ coliphage
(5 studies) [79,113,118,119,123] (Figure 2), suggesting that these two indicators may be better
predictors of waterborne illness occurrence. Fecal coliforms, human-associated MST markers (Bsteri,
BuniF2, and HF134), general MST marker (GenBac3), culturable E. coli, total coliforms, and somatic
coliphage were correlated with illness less frequently (Figure 2). Twenty-seven indicator measurements
across all studies were correlated with human illness, and 93% of these studies were conducted in
waters with known point or non-point source contamination, contaminated surface/ground water flow
or following wet weather events. Only six studies [79,86,117–119,124], all of which found relationship
between indicator and illness, measured pathogens, in addition to recording illness information, and
indicator organism concentrations. Only one of the six studies found a relationship between pathogens
and illness or indicator concentrations. This is not surprising since, in these studies, pathogens were
detected infrequently and at low concentrations. This illustrates the potential challenges of detecting
relationships between indicators and pathogens in the field even when health relationships were
observed with fecal indictors.
9. Factors that Influence Indicator and Pathogen Relationships

Most recreational waters at any given time are impacted by many different sources of fecal
contamination (e.g., treated and untreated wastewater, agricultural operations, stormwater, and
domestic and wildlife animals) and these influences can change depending on many different factors
including precipitation, tidal flow and wind direction. In addition, each fecal source has its own set
of indicators and the potential for different types of pathogens. Therefore, the more fecal sources
a recreational water is impacted by, the more challenging it will be to show correlations between
indicators and pathogens. Preceding sections described our findings regarding relationships between
indicators and pathogens in recreational waters, as well as relationships between indicators and illness.
Overall, FIB were better predictors of bacterial and protozoan pathogen presence (compared to viral),
relationships were more probable under scenarios where both indicator and pathogen were likely
to be present at higher concentrations, and enterococci and F-specific coliphage tended to be better
predictors of waterborne illness occurrence compared to other indicators. The following sections
examine various factors that are likely to influence the observed trends.
10. Detection Frequency and Concentrations of Indicators and Pathogens in Marine, Brackish
and Freshwaters
The observed relationships between indicators and pathogens can be influenced by logistical
factors that may confound determination of actual relationships, including study design and
methodological limitations. Study design determines the frequency at which a target (FIB, alternative
indicator, MST marker or pathogen) was measured (per study or cumulative multiple studies),
while methodology employed influences likelihood of detection. We compiled studies that reported
frequency of detection (or data that allowed calculation of frequency detection such as total number
of samples and samples positive) for at least one FIB/alternative indicator/MST marker and at least
one pathogen per sample(s), resulting in inclusion of 49 studies (Table 5). Microbial data collected
were first grouped according to indicator (FIB, alternative or MST) or pathogen type (bacterial, viral
or protozoan), and further organized according to the detection format employed (different types
of culture-based or molecular). Table 5 describes the detection frequency (per study and per total
cumulative samples) for microorganism targets (FIB, alternative indicators, MST, and pathogens) for
both freshwater and brackish/marine waters.
154
IJERPH 2018, 15, 2842
Table 5. Frequency (%) of detection of microorganisms over all eligible studies (those that included
data on individual observations). Detection frequency is expressed per study and for cumulative
samples across all studies. Studies with least one sample positive for the organism were scored positive
in the “per study” column.
Detection Frequency Detection Frequency Detection Frequency Detection Frequency

Organism
per Study (%) and n 2 per Sample 2 per Study (%) and n 2 per Sample 2
Freshwater Brackish/Marine
FIB
Total coliforms (MPN) 100% (4) 100% (275) N/A N/A
Total coliforms (CFU) 100% (3) 97.2% (1988) 100% (5) 99.7% (317)
Fecal coliforms (MPN) 100% (4) 100% (147) N/A N/A
Fecal coliforms (CFU) 100% (6) 96.7% (1726) 100% (11) 98.3% (524)
E. coli (MPN) 100% (8) 97.7% (1846) 100% (5) 100% (55)
E. coli (CFU) 100% (10) 90% (2530) 100% (8) 94.1% (406)
E. coli (Q) 100% (5) 89.6% (221) N/A N/A
Enterococci (MPN) 100% (2) 81.7% (301) 100% (5) 61.7% (162)
Enterococci (CFU) 100% (13) 96.6% (2584) 100% (13) 97.6% (705)
Enterococci (Q) 100% (2) 100% (302) 100% (3) 100% (34)
Alternative indicators
C. perfringens (CFU) 100% (4) 83.2% (1843) 100% (5) 61.6% (73)
C. perfringens (Q) 100% (2) 73.2% (56) N/A N/A
Somatic coliphage (PFU) 100% (4) 85.5% (394) 100% (1) 100% (20)
F+ coliphage (PFU) 100% (2) 93.2% (73) 100% (7) 34.4% (90)
F- coliphage (PFU) N/A N/A 100% (3) 28% (25)
B. fragilis phage (PFU) N/A N/A 100% (1) 16.7% (12)
B. thetaiotaomicron phage
N/A N/A 100% (1) 30% (20)
(PFU)
MST markers
GenBac3 (Q) 100% (1) 75% (8) N/A N/A
HF183 (E) N/A N/A 100% (3) 31.8% (255)
HF183 (Q) N/A N/A 100% (4) 25.5% (105)
BacHum-UCD (Q) N/A N/A 100% (1) 95.45% (22)
HPyV (E) 100% (1) 0% (18) 100% (4) 51.08% (204)
HPyV (Q) 100% (1) 100% 98) 100% (2) 12.2% (255)
C. coccoides Human (Q) N/A N/A 100% (1) 69.2% (13)
B. thetaiotamicron (E) N/A N/A 100% (1) 26.7% (15)
nifH (E) 100% (1) 0% (18) 100% (2) 2.8% (255)
nifH (Q) N/A N/A 100% (1) 100% (7)
gyrB (Q) 100% (1) 50.8% (65) N/A N/A
g-Bfra 100% (1) 92.3% (65) N/A N/A
esp (E) 100% (3) 6.2% (649) 80% (5) 19.12% (204)
LTII (E) 100% (1) 7.4% (217) N/A N/A
STII (E) 100% (1) 4.6% (217) N/A N/A
DogBac (Q) N/A N/A 100% (1) 86.7% (15)
Bacterial pathogens
E. coli O157:H7 (MPN) 100% (1) 0.6% (823) N/A N/A
E. coli O157:H7 (E) 100% (1) 13.4% (67) 0% (1) 0% (7)
Pathogenic E. coli (eae) (E) 100% (4) 53.7% (350) N/A N/A
Pathogenic E. coli (eae) (Q) 100% (1) 31.3% (32) N/A N/A
Pathogenic E. coli (stx1) (E) 100% (4) 7.9% (302) N/A N/A
Pathogenic E. coli (stx2) (E) 100% (3) 29.7% (350) N/A N/A
Salmonella spp. (MPN) 100% (6) 14% (1076) 33% (3) 8.7% (196)
Salmonella spp. (E) N/A N/A 100% (1) 28.6% (7)
Salmonella spp. (Q) 100% (4) 27.3% (1188) N/A N/A
S. aureus (MPN) 100% (1) 44.6% (112) 100% (2) 70.4% (27)
S. aureus (E) N/A N/A 100% (1) 57.1% (7)
S. aureus (Q) 100% (1) 37.5% (8) N/A N/A
Campylobacter spp. (MPN) 100% (3) 24.7% (1009) 100% (1) 30.9% (55)
Campylobacter spp. (E) N/A N/A 100% (1) 14.3% (7)
Campylobacter spp. (Q) 100% (3) 81.3% (80) 100% (1) 18.2% (11)
Pseudomonas spp. (MPN) 100% (2) 80.6% (191) N/A N/A
P. aeruginosa (Q) 100% (2) 39.6% (53) N/A N/A
Shigella spp. (E) 100% (1) 6.3% (48) N/A N/A
Shigella spp. (Q) 100% (2) 14.5% (1148) N/A N/A
Legionella spp. (E) 100% (2) 41.9% (217) N/A N/A
Legionella spp. (Q) 100% (1) 20% (30) N/A N/A
Listeria spp. (MPN) 100% (1) 18.7% (395) N/A N/A
V. cholerae (Q) 100% (2) 52.5% (1148) N/A N/A
V. vulnificus (Q) N/A N/A 100% (2) 44.4% (27)
Aeromonas spp. (Q) 100% (3) 100% (248) N/A N/A
M. avium (Q) 100% (2) 34.4% (64) N/A N/A
155
IJERPH 2018, 15, 2842
Table 5. Cont.
Detection Frequency Detection Frequency Detection Frequency Detection Frequency

Organism
per Study (%) and n 2 per Sample 2 per Study (%) and n 2 per Sample 2
Freshwater Brackish/Marine
Viral pathogens
Infectious enterovirus (MPN) 100% (3) 18.4% (158) 100% (3) 0% (27)
Enterovirus (E) 100% (3) 14.4% (222) 100% (3) 37.8% (45)
Enterovirus (Q) 100% (4) 29.1% (103) 33% (3) 14.8% (27)
Infectious reovirus (MPN) 100% (1) 21.9% (32) N/A N/A
Reovirus (Q) 100% (1) 100% (8) N/A N/A
Human adenovirus (MPN) N/A N/A 100% (1) 0% (27)
Human adenovirus (E) 100% (4) 42.2% (1118) 100% (6) 16.5% (309)
Human adenovirus (Q) 100% (5) 40.7% (214) 100% (1) 33.3% (12)
Astrovirus (E) 100% (1) 15.4% (52) N/A N/A
Astrovirus (Q) 100% (2) 17.3% (133) N/A N/A
Norovirus (E) 100% (3) 29.5% (112) 100% (1) 27.3% (22)
Norovirus (Q) 100% (1) 100% (8) 50% (2) 26.3% (19)
Norovirus GI (Q) 50% (4) 7.5% (213) N/A N/A
Norovirus GII (Q) 66.7% (3) 16.2% (198) N/A N/A
Rotavirus (E) 100% (3) 18.4% (141) N/A N/A
Rotavirus (Q) 66.7% (3) 33.8% (142) N/A N/A
Hepatitis A (E) N/A N/A 100% (1) 80% (10)
Hepatitis A (Q) 100% (1) 1.5% (68) 0% (1) 0% (12)
Bovine adenovirus (Q) 100% (1) 56.7% (30) N/A N/A
Protozoan pathogens
Acanthamoeba spp. (E) 100% (1) 24.6% (126) N/A N/A
Naegleria spp. (E) 100% (1) 13.5% (126) N/A N/A
Cryptosporidium spp. (M) 88.9% (9) 36.5% (1456) 60% (5) 23.2% (112)
Cryptosporidium spp. (E) N/A N/A 0% (1) 0% (12)
Cryptosporidium spp. (Q) N/A N/A 100% (1) 26.7% (15)
Giardia spp. (M) 100% (8) 63.7% (1426) 80% (5) 22.3% (112)
Giardia spp. (E) N/A N/A 100% (2) 16.7% (12)
Giardia spp. (Q) N/A N/A 100% (1) 33.3% (15)
Fungal pathogens
C. albicans (MPN) N/A N/A 100% (1) 45.4% (152)
1MPN, most probable number; CFU, colony forming units; PFU, plaque forming units; E, end-point PCR; Q, qPCR;
M, microscopy; 2 N/A, not available.
Each FIB was detected at least once in 100% of studies, which was true for most of the microbial
targets. General FIB were also the most frequently detected on a per sample basis, as they were found
in 94.2% of samples across 13,823 measurements in marine and freshwaters (Table 5). In freshwater,
detection frequency of FIB per sample was 95% across 11,920 measurements and it was somewhat
lower in brackish/marine waters (93% across 2203 measurements) (Table 5). Detection frequency
of alternative indicators per sample, irrespective of the water type, was considerably less than FIB,
averaging 60.6% (across 2606 measurements); the difference between water types was also more
pronounced than for FIB (freshwater detection frequency 83.7%/2366 measurements vs. marine
45.1%/240 samples) (Table 5). While the 2705 samples analyzed for MST markers in marine and
freshwaters were similar to alternative indicators, the overall detection frequency average (42.9%) was
considerably lower (Table 5). The frequency of detection and total number of samples collected in each
water type (37.4%/1355 in freshwater vs. 47.3%/1350 in marine water) was similar (Table 5).
Irrespective of the water type, bacterial pathogens were measured more often (9280 total samples),
compared to viral (3462) and protozoan (3400) pathogens, although the frequency of detection across
different pathogen groups was similar (33.8%, bacterial; 29.6%, viral; and 28.9%, protozoan (Table 5)).
There also appeared to be no appreciable difference in detection frequency between the water types for
any of the pathogen groups, although considerably more samples were collected in freshwater (Table 5).
For bacterial pathogens, frequency of detection across 8936 total samples collected in freshwater was
35.2%, compared to 30.3% in 344 marine/brackish water samples (Table 5). Similarly, viral pathogens
were detected in 33.1% freshwater samples (out of 2952) and 23.6% of 510 marine water samples
(Table 5). Lastly, protozoan pathogens were detected in 34.6% of 3,134 freshwater samples and 24.4%
of 266 marine water samples (Table 5).
156
IJERPH 2018, 15, 2842
A subset of studies examined (n = 33) reported concentration data in the body of the manuscript,
tables or supplemental materials, allowing graphs to be created displaying average densities per
organism and water type (Figures 3 and 4). Concentrations of indicators were on average 1–3 log10
higher than pathogen concentrations for both water types. Both indicators and pathogens in marine
waters were found at slightly lower levels (0.5–1 log10 ) than those observed in freshwater (Figures 3
and 4 and Table 6). Within an indicator group, concentrations of FIB ranged from not detected
(ND (observed only for enterococci)) to 5.39 log10 per 100 mL (Table 6), and total coliform levels
were the highest, followed by fecal coliforms, E. coli, and enterococci (Figures 3 and 4). Alternative
indicator concentrations were lower than FIB (ranging from ND–3.29 log10 per 100 mL (Table 6)),
and C. perfringens levels were higher than somatic and F-specific coliphage (Figures 3 and 4).
MST marker concentrations were reported less frequently and were more variable, ranging from
ND–2.50 log10 copies per 100 mL (Table 6 and Figures 3 and 4). Bacterial pathogen concentrations
(range: ND–5.09 log10 per 100 mL) were higher than viral (range: ND–1.58 log10 per 100 mL) and
protozoan pathogens (range: ND–1.93 log10 per 100 mL), in both marine and freshwater (Table 6,
Figures 3 and 4).
Figure 3. Mean concentration of FIB, alternative indicators, MST markers, bacterial, viral and
protozoan pathogens in freshwater. Error bars represent standard deviation (c, culture-based; Q,
qPCR; m, microscopy).
Figure 4. Mean concentration of FIB, alternative indicators, MST markers, bacterial, viral and protozoan
pathogens in marine and brackish waters. Error bars represent standard deviation (c, culture-based; Q,
qPCR; m, microscopy).
157
IJERPH 2018, 15, 2842
Table 6. Concentrations of various indicators and pathogens from select studies in marine/brackish
and freshwaters.
Range or Average per Study

Organism 1 Marine References 4 Freshwater References 4
(log10 per 100 mL) 2,3
FIB
Total coliforms (c) 2.67–3.89 N/A [37,75]
Fecal coliforms (c) 0.93–5.39 [76,77,80,87,94] [39,57,65,125]
E. coli (c) 0.59–3.51 [76,77,80,82,89,94,96,100] [36,37,44,46,50,51,54–57,65,75,125]
E. coli (q) 1.75–3.97 N/A [41,51,55,68]
Enterococci (c) ND–3.62 [70,76,77,80,82–85,87–89,94,96,100,101] [37,39,44,47,50,51,55–57,65,70]
Enterococci (q) 0.63–3.21 [76,77,83] [51,55]
Alternative indicators
C. perfringens (c) 0.20–2.33 [76,77,87–89,100] [54]
C. perfringens (q) 0.35 N/A [41]
Somatic coliphage (c) 0.61–3.29 [82,90,94] [39,102,103,126]
F-specific coliphage (c) ND–2.76 [76,82,89,90,94,100] [47,54,102,103]
MST markers
AllBac(q) 0.93 [83] [51]
PMMoV (q) ND [83] N/A
HF183 (q) ND–0.69 N/A [51]
HBBac (q) 0.72 [83] N/A
HPyV (q) 2.36 N/A [51]
esp (q) 0.06 [76] N/A
nifH (q) ND [83] N/A
DogBac (q) 2.50 [76] N/A
Bacterial pathogens
Campylobacter spp. (c) 1.05 N/A [54]
Campylobacter spp. (q) 0.74–2.29 N/A [46,70]
EHEC (q) 0.13 N/A [65]
Salmonella spp. (c) ND–2.36 [80,84,89,100] N/A
Salmonella spp. (q) 0.06–4.08 N/A [51,65,68]
S. aureus (c) 0.70–0.77 [76,77] N/A
S. aureus (q) 0.78 N/A [51]
Aeromonas spp. (q) 5.09 N/A [68]
Stx1 (q) 0.03 N/A [57]
Stx2 (q) 0.03 N/A [57]
Mycobacterium spp. (q) 3.41 N/A [68]
Pseudomonas spp. (c) 0.37–3.72 N/A N/A
Legionella spp (q) ND N/A [46]
V. vulnificus (c) 1.91–2.11 [84,100] N/A
V. parahaemolyticus (q) 1.01 [85] N/A
Viral pathogens
Enterovirus (c) ND–0.04 [80,82,87,89,94] [39,47,102]
Enterovirus (q) ND–0.56 [76,77] [39]
Adenovirus (q) 0.08–1.02 N/A [41,46,103]
Astrovirus (q) 1.48 N/A [103]
Norovirus (q) ND–1.40 [77,94] [51,102]
Norovirus GI (q) ND–1.02 N/A [50,103]
Norovirus GII (q) 1.58 N/A [103]
Rotavirus (q) 1.41–1.53 N/A [102,103]
Reovirus (c) 0.18–0.31 N/A [47,102]
Hepatitis A (q) ND [77] N/A
Protozoan pathogens
E. bieneusi (m) 0.12 [101] N/A
Giardia spp.(m) ND–1.93 [76,83,88,89,94,96,127] [36,41,47,54–56,65,75]
Cryptosporidium spp. (m) ND–0.73 [76,77,83,88,89,94,96,127] [36,41,47,54–56,65,75]
1 c, culture; q, qPCR; m, microscopy. 2 Range is provided when more than one study measured a given parameter,
while average per study is provided when a single study measured a given parameter. Units include: CFU, MPN,
PFU, gene copies or total (oo)cysts. 3 ND, not detected; 4 N/A, not available.
As evidenced by the examples above, readily detected microorganisms are more likely to be
measured and frequency of detection of a given microorganism is influenced by the concentration and
distribution of the target in the sample types tested, as well as the limit of detection of the method used.
Culture methods such as membrane filtration can have a low limit of detection, e.g., 1 CFU/100 mL,
and can reliably detect FIB in water samples with minimal contamination. Conversely, pathogens are
generally present sporadically and in lower levels than fecal indicators. These types of targets require
high-throughput filtration methods that can achieve large concentration factors, with the tradeoff that
limits of detection are generally quite high. In addition, the volumes sampled, and the concentration
158
IJERPH 2018, 15, 2842
strategy used can vary between studies and can affect the sensitivity of a given method. These logistical
factors frequently result in unbalanced comparisons in which the indicator organism is frequently
detected, but the pathogen is not. Therefore, the disconnect between indicators and pathogens may
not be due to a true lack of relationship in many cases, but to methodology that is much more suited to
detecting indicators than pathogens.
11. Microbial Levels in Fecal Material

The observed relationships between indicators and pathogens can also be affected by factors
intrinsic to the organisms themselves, including levels in various hosts, as well as shedding frequencies
and duration. FIB are commensal inhabitants of the GI tract of humans and other animals and as such
are shed continually in feces. Levels of fecal coliforms, E. coli, and enterococci typically found in human
feces range 105 –109 CFU per gram [13], while levels detected in untreated wastewater are somewhat
lower (105 –108 CFU per 100 mL) [13,128]. The concentration of FIB in animal excreta is lower still,
ranging from 104 to 107 CFU per gram, depending on the animal host [13]. Alternative indicators are
also commensal organisms of the GI tract but are typically found in lower concentrations and are more
influenced by diets and physiologies of the host [13,14,128]. For example, C. perfringens levels in animal
and human feces range from undetectable to 108 CFU per gram [13], while coliphages were absent
from some animal feces and primary wastewater effluents [128,129] and typically did not exceed
~103 PFU/mL of untreated wastewater [128]. MST markers target different fecal microorganisms
that are strongly associated with particular hosts [14] and the human-associated subset is reported
to range from 103 to 1010 gene copies per gram of feces or 100 mL of untreated wastewater, while
animal-associated MST markers range from 104 –109 gene copies per gram of feces depending on the
sensitivity of individual markers and geographic region [13].
Pathogens may cause symptomatic or asymptomatic infection of their human and animal hosts.
Shedding rates can vary widely, although levels found in the wastewater are typically several orders
of magnitude lower than any indicator species [128,130–133] likely due to the sporadic nature of
pathogen occurrence and detection compared to indicators. Additionally, only a small part of the
population is infected with pathogens at any given time, resulting in considerable variation in the
levels of pathogens, particularly when originating from relatively small populations. Differential
shedding of pathogens from infected hosts is also contributing to the occurrence of pathogens in
recreational waters. For example, shedding rates for human viral pathogens can be as high as 1011
viral particles per gram of feces in the case of adenoviruses [134], while shedding rates of bacterial
pathogens are typically lower [133], as cattle excreting >104 CFU per gram of feces E. coli O157:H7
are considered to be “super-shedders” [135]. Cryptosporidium and Giardia (oo)cyst shedding rates by
the infected individuals can range from 106 to 1011 per gram of feces [132] and are typically higher
in animal hosts compared to human [131,136], although not all (oo)cysts excreted by animals are
zoonotic [131,137].
Shedding duration of viruses can vary from weeks to months [133], and some viruses display
distinct seasonal trends (e.g., infectious enteroviruses are more prevalent in wastewater in summer
and early fall) [134]. Similar to pathogenic viruses, excretion of (oo)cysts is typically long term [132].
Shedding duration of bacterial pathogens is shorter with median values typically reported to be
~2 weeks, although in some instances it can last considerably longer [138,139]. Similar to viral
and protozoan pathogens, shedding is affected by many different factors including diet and age of
the host [140,141], temperature [140], as well as composition of gut microbiome [142]. Infectious
dose of different pathogens is also variable and typically the lowest for viruses [134,143], medium
range for protozoan pathogens and generally highest for bacterial pathogens [143], although E. coli
O157:H7, with a low infectious dose, is an exception [144]. The infectious dose of viral, bacterial and
protozoan pathogens is dependent on many factors, including individual strains and health status of
the host [134,143].
159
IJERPH 2018, 15, 2842
12. Susceptibility to Environmental Stressors

While wastewater treatment processes generally result in some removal of indicators and
pathogens [128,145], sanitary sewer and combined sewer overflows, along with other infrastructure
failures can result in release of indicators and pathogens into ambient waters. In addition, different
indicator and pathogen groups exhibit variable susceptibilities to disinfection strategies. Bacteria
are generally susceptible to chlorination and UV treatment [146]. Protozoa and viruses are typically
most susceptible to UV treatment [146,147], with the notable exception of adenoviruses [148]. Once
indicators and pathogens are released into ambient waters, a new panoply of biotic and abiotic
environmental factors affects fate and transport characteristics, including ambient sunlight, indigenous
microbiota (i.e., predation and competition interactions), temperature, salinity, nutrient levels, location
(water column vs. sediment), source of fecal pollution and resilience of individual organisms.
Ambient sunlight and associated UV radiation typically act to increase the decay rates, although
the magnitude of this effect is influenced by the environmental conditions [149] and measurement
strategies [150,151]. For example, viable cells and culturable/infectious organisms typically display
the effects of UV damage more readily than their corresponding nucleic acids. Interactions with
indigenous microbiota also increase decay rates, although this was predominantly shown for FIB, MST
markers and some bacterial pathogens, (e.g., [152–155]) with inconclusive data for other organisms
(e.g., various bacteriophages and C. parvum [156,157]). Influx of nutrients (in the form of organic carbon,
nitrogen and phosphorus) can result in extended persistence [158–160], and potentially mitigate the
effects of biotic interactions [161] but this assertion was not tested in detail for organisms other
than culturable FIB. Temperature and location affect decay rates of most organisms tested (e.g., FIB,
bacteriophage, viral pathogens, MST markers) almost unilaterally with greater persistence at lower
temperatures [150,162–166] and in the sediments and sands compared to the water column (recently
reviewed in [167]).
Similar to the effect of ambient sunlight, salinity (and the associated ionic content of brackish
and marine waters) affected the decay rates of culturable/infectious FIB, alternative indicators, MST
markers and pathogens more so than their corresponding nucleic acids [168–174]. The effect of
source of fecal pollution has been studied on FIB and MST markers, and indicators originating from
ruminants are more persistent compared to those from other fecal sources (e.g., dog, seagull, and
human) [169,175–179], although different human sources (e.g., feces, septage, and sewage) elicit
different decay rates [152]; analogous information for alternative indicators and pathogens is still
missing. Finally, studies that compared decay of various indicators to pathogens directly under the
same experimental conditions are rare and report conflicting results. For instance, in one study, E. coli
O157:H7 persisted longer than FIB (e.g., E. coli and enterococci) in freshwater [180], but another group
reported similar trend in the freshwater sediments but not the water column [181]. Another group
reported no difference in decay between FIB, various MST markers and C. jejuni, S. enterica and
adenovirus in freshwater [151]. Others reported considerably faster decay of C. jejuni (but not C. coli or
Salmonella spp.) than FIB and MST markers, irrespective of the water type [171]. As exemplified above,
variable responses of different indicator and pathogen groups to these stressors and the resulting
differential decay rates further confound the indicator paradigm.
13. Conclusions
FIB and alternative indicator organisms (C. perfringens and coliphages) have been used for over a
century, and continue to be used today as indicators of general fecal pollution in many applications,
including the assessment of sanitary quality of recreational waters [8]. MST markers are used to
identify source(s) of fecal pollution and are a more recent addition to the monitoring toolbox available
to water quality managers and other practitioners in the field [14]. The goal of this review is to
two-fold. Our primary objective was to examine reported relationships between various indicators
and pathogens in recreational waters to determine the value of different indicators as surrogates for
160
IJERPH 2018, 15, 2842
pathogen presence. Secondly, we aimed to more closely inspect different factors that may have an
impact on this relationship.
The majority of the studies either did not report a relationship, or they reported a statistically
non-significant relationship. Among the studies that observed statistically significant relationships,
it was considerably more common in freshwater compared to marine waters. General FIB tended to
form statistically significant relationships more commonly with bacterial and protozoan pathogens,
(compared to viral pathogens) and this difference was statistically significant. Alternative indicators
and MST markers correlated with pathogens less frequently, although it occurred more in freshwater
than marine/brackish waters. Overall, statistically significant relationships were detected more
frequently in waters known to be impacted by fecal pollution and following wet weather events, both
scenarios under which indicators and pathogens are more likely to be co-detected.
Among factors influencing these relationships frequency of detection and variable concentrations
of indicators and pathogens were identified as major contributing factor. Not surprisingly, general FIB
were measured and detected more frequently than any other indicator or pathogen (generally in >90%
of samples) and were also reported at higher concentrations, irrespective of the water type. Alternative
indicators were also frequently detected in samples (>70%), while MST markers were measured and
detected less frequently, and in lower concentrations than FIB or alternative markers (frequently in
<10% of samples). Pathogen detection frequencies were similarly low. Low frequency of detection
affects the ability to establish relationships between the frequently-detected and infrequently-detected
analytes, as the dataset becomes left-censored (biased toward non-detects values). What looks like
“absence” is frequently an artifact of comparing an analyte with high density (e.g., FIB) with one of
low density (e.g., pathogen). Better concentration and recovery methods for the infrequently-detected
analytes may provide a more realistic picture of the relationships among these various microorganisms
in environmental waters. Finally, concentrations in feces and wastewater, shedding rates and patterns
of various indicator and pathogen groups differ, as do their fate and transport characteristics in
secondary habitats. Indicators are typically present in higher concentrations than any of the pathogen
groups, and are also shed constantly, or more frequently, compared to pathogens. Upon entry into the
secondary habitats, a host of biotic and abiotic factors differentially affects persistence of indicators and
pathogens, further confounding the indicator–pathogen paradigm. Lastly, another important factor
impacting the ability to establish relationships between indicators and pathogens is the realization
that most locations are impacted by multiple sources of fecal contamination. Although it is difficult to
measure the impact of multiple fecal inputs, tools such as sanitary surveys and GIS mapping have
the ability to indicate potential point and non-point sources of fecal pollution and future MST studies
should improve our understanding of the impacts of multiple fecal sources.
To further our understanding of indicator and pathogen relationships, future studies measuring
these microorganisms in recreational waters should evaluate and report the existence (or lack thereof)
of such relationships. Other considerations include careful selection of targeted pathogens and
methodology used to quantify them. Furthermore, providing the data on a per sample basis (rather
than descriptive statistics of a dataset) in at least supplementary materials, will enable metanalyses,
which may yield a more robust estimate of a true state of indicator/pathogen paradigm. Lastly,
while standardized and sensitive methods exist for FIB detection and enumeration in recreational
waters, analogous procedures for alternative indicators, MST markers and pathogens are still missing.
Standardization of detection and quantification methods suitable for each indicator/pathogen group
can enable more accurate evaluation of any statistically significant relationships between these
two groups.
Author Contributions: Conceptualization, A.K., B.R.M. and V.J.H.; Investigation, A.K. and B.R.M.; Data Curation,
A.K. and B.R.M.; Writing-Original Draft Preparation, A.K. and B.R.M.; Writing-Reviewing & Editing, A.K. and
V.J.H.; Visualization, A.K.
161
IJERPH 2018, 15, 2842
Disclaimer: The United States Environmental Protection Agency through its Office of Research and Development
funded and managed the research described here. It has been subjected to Agency’s administrative review and
approved for publication. The views expressed in this article are those of the author(s) and do not necessarily
represent the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
References
1. Kummu, M.; de Moel, H.; Ward, P.J.; Varis, O. How close do we live to water? A global analysis of population
distance to freshwater bodies. PLoS ONE 2011, 6, e20578. [CrossRef] [PubMed]
2. National Oceanic and Atmospheric Administration. State of the Coast: National Coastal Population Report.
Population Trends from 1970 to 2020; National Oceanic and Atmospheric Administration: Silver Springs, MD,
USA, 2013.
3. Dorfman, M.H.; Stoner, N.; Rosselot, K.S. Testing the Waters: A Guide to Water Quality at Vacation Beaches;
Natural Resources Defense Council: New York, NY, USA, 2014.
4. Rabinovici, S.J.; Bernknopf, R.L.; Wein, A.M.; Coursey, D.L.; Whitman, R.L. Economic and health risk
trade-offs of swim closures at a lake Michigan beach. Environ. Sci. Technol. 2004, 38, 2737–2745. [CrossRef]
[PubMed]
5. Devine, J. The Impacts of Beach Pollution; Natural Resources Defense Council: New York, NY, USA, 2014.
6. Penn, J.; Hu, W.; Cox, L.; Kozloff, L. Values for recreational beach quality in Oahu, Hawaii. Mar. Resour. Econ.
2016, 31, 47–62. [CrossRef]
7. European Environment Agency. European Bathing Water Quality in 2017; European Environment Agency:
Luxembourg, Luxembourg, 2018.
8. American Water Works Association. Water Quality and Treatment: A Handbook of Community Water Supplies,
4th ed.; McGraw-Hill: New York, NY, USA, 1990.
9. World Health Organization. Guidelines for Safe Recreational Water Environments—Volume 2 Swimming Pools
and Similar Environments; World Health Organization: Geneva, Switzerland, 2006.
10. World Health Organization. Gudelines for Safe Recreational Water Environments, Volume 1: Coastal and Fresh
Waters; World Health Organization: Geneva, Switzerland, 2003.
11. Environmental Protection Department. Water Quality Criteria/Standards Adopted in the Asia Pacific Region;
Asia Pacific Economic Cooperation Secretariat: Singapore, 2003.
12. European Environment Agency. Directive 2006/7/EC of the European Parliament and of the Council of February
2006 Concerning the Management of Bathing Water Quality and Repealing Directive 76/160/EEC; European
Environment Agency: Geneva, Switzerland, 2006.
13. Harwood, V.; Shanks, O.; Korajkic, A.; Verbyla, M.; Ahmed, W.; Iriate, M. General and Host-Associated Bacterial
Indicators of Faecal Pollution; Global Water Pathogen Project; Michigan State University: E. Lansing, MI,
USA, 2017.
14. Harwood, V.J.; Staley, C.; Badgley, B.D.; Borges, K.; Korajkic, A. Microbial source tracking markers for
detection of fecal contamination in environmental waters: Relationships between pathogens and human
health outcomes. FEMS Microbiol. Rev. 2014, 38, 1–40. [CrossRef] [PubMed]
15. Department of Environment and Conservation. Western Australian Guidelines for Biosolids Management;
European Environment Agency: Geneva, Switzerland; Perth, Australia, 2012.
16. North Carolina Environmental Quality. North Carolina Adm. Code 15A NCAC 2U Reclaimed Water;
North Carolina Department of Environment and Natural Resources: Raleigh, NC, USA, 2011.
17. Queensland Government Environmental Protection Agency. Queensland Water Recycling Guidelines;
Queensland Government Environmental Protection Agency: Brisbane, Australia, 2005.
18. United States Environmental Protection Agency. Review of Coliphages as Possible Indicators of Fecal
Contamination for Ambient Water Quality; United States Environmental Protection Agency: Washington,
DC, USA, 2015.
19. United States Environmental Protection Agency. 2016 Coliphage Experts Workshop: Discussion Topics and
Findings; United States Environmental Protection Agency: Washington, DC, USA, 2016.
20. Nguyen, K.H.; Senay, C.; Young, S.; Nayak, B.; Lobos, A.; Conrad, J.; Harwood, V.J. Determination of wild
animal sources of fecal indicator bacteria by microbial source tracking (MST) influences regulatory decisions.
162
IJERPH 2018, 15, 2842
21. Soller, J.A.; Schoen, M.E.; Varghese, A.; Ichida, A.M.; Boehm, A.B.; Eftim, S.; Ashbolt, N.J.; Ravenscroft, J.E.
Human health risk implications of multiple sources of faecal indicator bacteria in a recreational waterbody.
22. Forni, D.; Cagliani, R.; Clerici, M.; Sironi, M. Origin and dispersal of hepatitis E virus. Emerg. Microbes Infect.
23. Salines, M.; Andraud, M.; Rose, N. Combining network analysis with epidemiological data to inform
risk-based surveillance: Application to hepatitis E virus (HEV) in pigs. Prev. Vet. Med. 2018, 149, 125–131.
[CrossRef] [PubMed]
24. Lama, J.K.; Bachoon, D.S. Detection of Brucella suis, Campylobacter jejuni, and Escherichia coli strains in feral
pig (Sus scrofa) communities of Georgia. Vector-Borne Zoonot. Dis. 2018, 18, 350–355. [CrossRef]
25. Migura-Garcia, L.; Ramos, R.; Cerda-Cuellar, M. Antimicrobial resistance of salmonella serovars and
campylobacter spp. Isolated from an opportunistic gull species, yellow-legged gull (Larus michahellis).
J. Wildl. Dis. 2017, 53, 148–152. [CrossRef] [PubMed]
26. Valeriani, F.; Giampaoli, S.; Romano Spica, V. The molecular enrichment approach for the identification of
microbiological indicators in recreational waters. Microchem. J. 2014, 112, 70–74. [CrossRef]
27. Graciaa, D.S.; Cope, J.R.; Roberts, V.A.; Cikesh, B.L.; Kahler, A.M.; Vigar, M.; Hilborn, E.D.; Wade, T.J.;
Backer, L.C.; Montgomery, S.P.; et al. Outbreaks associated with untreated recreational water—United States,
2000–2014. Am. J. Transplant. 2018, 18, 2083–2087. [CrossRef] [PubMed]
28. Hlavsa, M.C.; Roberts, V.A.; Anderson, A.R.; Hill, V.R.; Kahler, A.M.; Orr, M.; Garrison, L.E.; Hicks, L.A.;
Newton, A.; Hilborn, E.D.; et al. Surveillance for waterborne disease outbreaks and other health events
associated with recreational water—United States, 2007–2008. MMWR Surveill. Summ. 2011, 60, 1–32.
[PubMed]
29. Hlavsa, M.C.; Roberts, V.A.; Kahler, A.M.; Hilborn, E.D.; Wade, T.J.; Backer, L.C.; Yoder, J.S. Recreational
water-associated disease outbreaks—United States, 2009–2010. MMWR Morb. Mortal. Wkly. Rep. 2014, 63,
6–10. [PubMed]
30. Hlavsa, M.C.; Roberts, V.A.; Kahler, A.M.; Hilborn, E.D.; Mecher, T.R.; Beach, M.J.; Wade, T.J.; Yoder, J.S.
Outbreaks of illness associated with recreational water—United States, 2011–2012. MMWR Morb. Mortal.
Wkly. Rep. 2015, 64, 668–672. [PubMed]
31. Dale, K.; Kirk, M.; Sinclair, M.; Hall, R.; Leder, K. Reported waterborne outbreaks of gastrointestinal disease
in Australia are predominantly associated with recreational exposure. Aust. N. Z. J. Public Health 2010, 34,
32. Guzman-Herrador, B.; Carlander, A.; Ethelberg, S.; Freiesleben de Blasio, B.; Kuusi, M.; Lund, V.; Lofdahl, M.;
MacDonald, E.; Nichols, G.; Schonning, C.; et al. Waterborne outbreaks in the Nordic countries, 1998 to 2012.
Eurosurveillance 2015, 20, 21160. [CrossRef]
33. Sinclair, R.G.; Jones, E.L.; Gerba, C.P. Viruses in recreational water-borne disease outbreaks: A review. J. Appl.
34. Ahmed, W.; Goonetilleke, A.; Gardner, T. Alternative indicators for detection and quantification of faecal
pollution. Water 2008, 39, 46–49.
35. Abia, A.L.K.; Ubomba-Jaswa, E.; Genthe, B.; Momba, M.N.B. Quantitative microbial risk assessment (QMRA)
shows increased public health risk associated with exposure to river water under conditions of riverbed
sediment resuspension. Sci. Total Environ. 2016, 566–567, 1143–1151. [CrossRef]
36. Diallo, M.B.; Anceno, A.J.; Tawatsupa, B.; Houpt, E.R.; Wangsuphachart, V.; Shipin, O.V. Infection risk
assessment of diarrhea-related pathogens in a tropical canal network. Sci. Total Environ. 2008, 407, 223–232.
[CrossRef] [PubMed]
37. Fernandez, C.; Salerno, C.M.; Paoloni, J.D.; Laurent, G.C. Water quality in a lagoon in the southeast pampa
region of Argentina. Rev. Argent. Microbiol. 2007, 39, 51–56. [PubMed]
38. Gemmell, M.E.; Schmidt, S. Is the microbiological quality of the Msunduzi River (KwaZulu-Natal, South
Africa) suitable for domestic, recreational, and agricultural purposes? Environ. Sci. Pollut. Res. Int. 2013, 20,
39. Hot, D.; Legeay, O.; Jacques, J.; Gantzer, C.; Caudrelier, Y.; Guyard, K.; Lange, M.; Andreoletti, L. Detection
of somatic phages, infectious enteroviruses and enterovirus genomes as indicators of human enteric viral
pollution in surface water. Water Res. 2003, 37, 4703–4710. [CrossRef]
163
IJERPH 2018, 15, 2842
40. Hsu, B.M.; Chen, C.H.; Wan, M.T.; Cheng, H.W. Legionella prevalence in hot spring recreation areas of Taiwan.
41. Jacob, P.; Henry, A.; Meheut, G.; Charni-Ben-Tabassi, N.; Ingrand, V.; Helmi, K. Health risk assessment related
to waterborne pathogens from the river to the tap. Int. J. Environ. Res. Public Health 2015, 12, 2967–2983.
[CrossRef] [PubMed]
42. Kern, A.; Kadar, M.; Szomor, K.; Berencsi, G.; Kapusinszky, B.; Vargha, M. Detection of enteric viruses in
hungarian surface waters: First steps towards environmental surveillance. J. Water Health 2013, 11, 772–782.
[CrossRef]
43. Marucci, P.L.; Olivera, N.L.; Brugnoni, L.I.; Sica, M.G.; Cubitto, M.A. The occurrence of Shiga toxin-producing
Escherichia coli in bathing water of the Sierra de la Ventana region, Buenos Aires Province, Argentina.
Environ. Monit. Assess. 2011, 175, 1–8. [CrossRef]
44. Mwanamoki, P.M.; Devarajan, N.; Thevenon, F.; Atibu, E.K.; Tshibanda, J.B.; Ngelinkoto, P.; Mpiana, P.T.;
Prabakar, K.; Mubedi, J.I.; Kabele, C.G.; et al. Assessment of pathogenic bacteria in water and sediment
from a water reservoir under tropical conditions (Lake Ma Vallee), Kinshasa Democratic Republic of Congo.
Environ. Monit. Assess. 2014, 186, 6821–6830. [CrossRef]
45. Roser, D.J.; Davies, C.M.; Ashbolt, N.J.; Morison, P. Microbial exposure assessment of an urban recreational
lake: A case study of the application of new risk-based guidelines. Water Sci. Technol. 2006, 54, 245–252.
[CrossRef]
46. Sales-Ortells, H.; Agostini, G.; Medema, G. Quantification of waterborne pathogens and associated health
risks in urban water. Environ. Sci. Technol. 2015, 49, 6943–6952. [CrossRef] [PubMed]
47. Schets, F.M.; van Wijnen, J.H.; Schijven, J.F.; Schoon, H.; de Roda Husman, A.M. Monitoring of waterborne
pathogens in surface waters in Amsterdam, the Netherlands, and the potential health risk associated with
exposure to Cryptosporidium and Giardia in these waters. Appl. Environ. Microbiol. 2008, 74, 2069–2078.
[CrossRef] [PubMed]
48. Staley, C.; Reckhow, K.H.; Lukasik, J.; Harwood, V.J. Assessment of sources of human pathogens and fecal
contamination in a florida freshwater lake. Water Res. 2012, 46, 5799–5812. [CrossRef] [PubMed]
49. Strathmann, M.; Horstkott, M.; Koch, C.; Gayer, U.; Wingender, J. The River Ruhr—An urban river under
particular interest for recreational use and as a raw water source for drinking water: The collaborative
research project “Safe Ruhr”—Microbiological aspects. Int. J. Hyg. Environ. Health 2016, 219, 643–661.
[CrossRef] [PubMed]
50. Thorolfsdottir, B.O.; Marteinsson, V.T. Microbiological analysis in three diverse natural geothermal bathing
pools in iceland. Int. J. Environ. Res. Public Health 2013, 10, 1085–1099. [CrossRef]
51. Weidhaas, J.; Anderson, A.; Jamal, R. Elucidating waterborne pathogen presence and aiding source
apportionment in an impaired stream. Appl. Environ. Microbiol. 2018, 84, AEM-02510. [CrossRef]
52. Wyn-Jones, A.P.; Carducci, A.; Cook, N.; D’Agostino, M.; Divizia, M.; Fleischer, J.; Gantzer, C.; Gawler, A.;
Girones, R.; Holler, C.; et al. Surveillance of adenoviruses and noroviruses in European recreational waters.
Water Res. 2011, 45, 1025–1038. [CrossRef]
53. Burnet, J.B.; Penny, C.; Ogorzaly, L.; Cauchie, H.M. Spatial and temporal distribution of Cryptosporidium and
Giardia in a drinking water resource: Implications for monitoring and risk assessment. Sci. Total Environ.
2014, 472, 1023–1035. [CrossRef]
54. Devane, M.L.; Moriarty, E.M.; Wood, D.; Webster-Brown, J.; Gilpin, B.J. The impact of major earthquakes and
subsequent sewage discharges on the microbial quality of water and sediments in an urban river. Sci. Total
Environ. 2014, 485–486, 666–680. [CrossRef]
55. Dorevitch, S.; Doi, M.; Hsu, F.C.; Lin, K.T.; Roberts, J.D.; Liu, L.C.; Gladding, R.; Vannoy, E.; Li, H.;
Javor, M.; et al. A comparison of rapid and conventional measures of indicator bacteria as predictors
of waterborne protozoan pathogen presence and density. J. Environ. Monit. 2011, 13, 2427–2435. [CrossRef]
56. Duris, J.W.; Reif, A.G.; Krouse, D.A.; Isaacs, N.M. Factors related to occurrence and distribution of selected
bacterial and protozoan pathogens in pennsylvania streams. Water Res. 2013, 47, 300–314. [CrossRef]
[PubMed]
57. Haack, S.K.; Duris, J.W.; Fogarty, L.R.; Kolpin, D.W.; Focazio, M.J.; Furlong, E.T.; Meyer, M.T. Comparing
wastewater chemicals, indicator bacteria concentrations, and bacterial pathogen genes as fecal pollution
indicators. J. Environ. Qual. 2009, 38, 248–258. [CrossRef]
164
IJERPH 2018, 15, 2842
58. Ji, W.T.; Hsu, B.M.; Chang, T.Y.; Hsu, T.K.; Kao, P.M.; Huang, K.H.; Tsai, S.F.; Huang, Y.L.; Fan, C.W.
Surveillance and evaluation of the infection risk of free-living amoebae and Legionella in different aquatic
environments. Sci. Total Environ. 2014, 499, 212–219. [CrossRef] [PubMed]
59. Mansilha, C.R.; Coelho, C.A.; Reinas, A.; Moutinho, A.; Ferreira, S.; Pizarro, C.; Tavares, A. Salmonella:
The forgotten pathogen: Health hazards of compliance with European bathing water legislation. Mar. Pollut.
Bull. 2010, 60, 819–826. [CrossRef] [PubMed]
60. Oster, R.J.; Wijesinghe, R.U.; Haack, S.K.; Fogarty, L.R.; Tucker, T.R.; Riley, S.C. Bacterial pathogen gene
abundance and relation to recreational water quality at seven Great Lakes beaches. Environ. Sci. Technol.
2014, 48, 14148–14157. [CrossRef] [PubMed]
61. Poma, V.; Mamani, N.; Iniguez, V. Impact of urban contamination of the La Paz River basin on thermotolerant
coliform density and occurrence of multiple antibiotic resistant enteric pathogens in river water, irrigated
soil and fresh vegetables. Springerplus 2016, 5, 499. [CrossRef] [PubMed]
62. Shen, S.M.; Chou, M.Y.; Hsu, B.M.; Ji, W.T.; Hsu, T.K.; Tsai, H.F.; Huang, Y.L.; Chiu, Y.C.; Kao, E.S.;
Kao, P.M.; et al. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR
(Taqman) assay. Pathog. Glob. Health 2015, 109, 236–241. [CrossRef] [PubMed]
63. Wilkes, G.; Edge, T.; Gannon, V.; Jokinen, C.; Lyautey, E.; Medeiros, D.; Neumann, N.; Ruecker, N.; Topp, E.;
Lapen, D.R. Seasonal relationships among indicator bacteria, pathogenic bacteria, Cryptosporidium oocysts,
Giardia cysts, and hydrological indices for surface waters within an agricultural landscape. Water Res. 2009,
64. Wyer, M.D.; Wyn-Jones, A.P.; Kay, D.; Au-Yeung, H.K.; Girones, R.; Lopez-Pila, J.; de Roda Husman, A.M.;
Rutjes, S.; Schneider, O. Relationships between human adenoviruses and faecal indicator organisms in
European recreational waters. Water Res. 2012, 46, 4130–4141. [CrossRef] [PubMed]
65. Xiao, G.; Wang, Z.; Chen, J.; Qiu, Z.; Li, Y.; Qi, J.; Liu, W.; Shu, W. Occurrence and infection risk of waterborne
pathogens in Wanzhou watershed of the Three Gorges Reservoir, China. J. Environ. Sci. 2013, 25, 1913–1924.
[CrossRef]
66. Cui, Q.; Fang, T.; Huang, Y.; Dong, P.; Wang, H. Evaluation of bacterial pathogen diversity, abundance and
health risks in urban recreational water by amplicon next-generation sequencing and quantitative PCR.
J. Environ. Sci. 2017, 57, 137–149. [CrossRef] [PubMed]
67. Duris, J.W.; Haack, S.K.; Fogarty, L.R. Gene and antigen markers of Shiga-toxin producing E. coli from
Michigan and Indiana river water: Occurrence and relation to recreational water quality criteria. J. Environ.
Qual. 2009, 38, 1878–1886. [CrossRef] [PubMed]
68. Fang, T.; Cui, Q.; Huang, Y.; Dong, P.; Wang, H.; Liu, W.T.; Ye, Q. Distribution comparison and risk assessment
of free-floating and particle-attached bacterial pathogens in urban recreational water: Implications for water
quality management. Sci. Total Environ. 2018, 613–614, 428–438. [CrossRef] [PubMed]
69. Fong, T.T.; Griffin, D.W.; Lipp, E.K. Molecular assays for targeting human and bovine enteric viruses in
coastal waters and their application for library-independent source tracking. Appl. Environ. Microbiol. 2005,
70. Hellein, K.N.; Battie, C.; Tauchman, E.; Lund, D.; Oyarzabal, O.A.; Lepo, J.E. Culture-based indicators of fecal
contamination and molecular microbial indicators rarely correlate with Campylobacter spp. in recreational
waters. J. Water Health 2011, 9, 695–707. [CrossRef] [PubMed]
71. Ishii, S.; Nakamura, T.; Ozawa, S.; Kobayashi, A.; Sano, D.; Okabe, S. Water quality monitoring and risk
assessment by simultaneous multipathogen quantification. Environ. Sci. Technol. 2014, 48, 4744–4749.
[CrossRef] [PubMed]
72. Lee, C.S.; Lee, C.; Marion, J.; Wang, Q.H.; Saif, L.; Lee, J. Occurrence of human enteric viruses at freshwater
beaches during swimming season and its link to water inflow. Sci. Total Environ. 2014, 472, 757–766.
[CrossRef]
73. Miagostovich, M.P.; Ferreira, F.F.; Guimaraes, F.R.; Fumian, T.M.; Diniz-Mendes, L.; Luz, S.L.; Silva, L.A.;
Leite, J.P. Molecular detection and characterization of gastroenteritis viruses occurring naturally in the
stream waters of manaus, central Amazonia, Brazil. Appl. Environ. Microbiol. 2008, 74, 375–382. [CrossRef]
[PubMed]
74. Smith, C.J.; Olszewski, A.M.; Mauro, S.A. Correlation of shiga toxin gene frequency with commonly used
microbial indicators of recreational water quality. Appl. Environ. Microbiol. 2009, 75, 316–321. [CrossRef]
165
IJERPH 2018, 15, 2842
75. Xiao, S.; Zhang, Y.; Zhao, X.; Sun, L.; Hu, S. Presence and molecular characterization of Cryptosporidium and
Giardia in recreational lake water in Tianjin, China: A preliminary study. Sci. Rep. 2018, 8, 2353. [CrossRef]
76. Abdelzaher, A.M.; Wright, M.E.; Ortega, C.; Hasan, A.R.; Shibata, T.; Solo-Gabriele, H.M.; Kish, J.; Withum, K.;
He, G.; Elmir, S.M.; et al. Daily measures of microbes and human health at a non-point source marine beach.
77. Abdelzaher, A.M.; Wright, M.E.; Ortega, C.; Solo-Gabriele, H.M.; Miller, G.; Elmir, S.; Newman, X.; Shih, P.;
Bonilla, J.A.; Bonilla, T.D.; et al. Presence of pathogens and indicator microbes at a non-point source
subtropical recreational marine beach. Appl. Environ. Microbiol. 2010, 76, 724–732. [CrossRef] [PubMed]
78. Goyal, S.M.; Gerba, C.P.; Melnick, J.L. Occurrence and distribution of bacterial indicators and pathogens in
canal communities along the Texas coast. Appl. Environ. Microbiol. 1977, 34, 139–149. [PubMed]
79. Griffith, J.F.; Weisberg, S.B.; Arnold, B.F.; Cao, Y.; Schiff, K.C.; Colford, J.M., Jr. Epidemiologic evaluation of
multiple alternate microbial water quality monitoring indicators at three California beaches. Water Res. 2016,
80. Korajkic, A.; Brownell, M.J.; Harwood, V.J. Investigation of human sewage pollution and pathogen analysis
at Florida Gulf coast beaches. J. Appl. Microbiol. 2011, 110, 174–183. [CrossRef] [PubMed]
81. Lipp, E.K.; Farrah, S.A.; Rose, J.B. Assessment and impact of microbial fecal pollution and human enteric
pathogens in a coastal community. Mar. Pollut. Bull. 2001, 42, 286–293. [CrossRef]
82. Moce-Llivina, L.; Lucena, F.; Jofre, J. Enteroviruses and bacteriophages in bathing waters. Appl. Environ.
Microbiol. 2005, 71, 6838–6844. [CrossRef] [PubMed]
83. Rosario, K.; Symonds, E.M.; Sinigalliano, C.; Stewart, J.; Breitbart, M. Pepper mild mottle virus as an indicator
of fecal pollution. Appl. Environ. Microbiol. 2009, 75, 7261–7267. [CrossRef] [PubMed]
84. Shaw, K.S.; Rosenberg Goldstein, R.E.; He, X.; Jacobs, J.M.; Crump, B.C.; Sapkota, A.R. Antimicrobial
susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus recovered from recreational and commercial
areas of Chesapeake Bay and Maryland Coastal Bays. PLoS ONE 2014, 9, e89616. [CrossRef]
85. Shaw, K.S.; Sapkota, A.R.; Jacobs, J.M.; He, X.; Crump, B.C. Recreational swimmers’ exposure to
Vibrio vulnificus and Vibrio parahaemolyticus in the Chesapeake Bay, Maryland, USA. Environ. Int. 2015,
74, 99–105. [CrossRef]
86. Sinigalliano, C.D.; Fleisher, J.M.; Gidley, M.L.; Solo-Gabriele, H.M.; Shibata, T.; Plano, L.R.; Elmir, S.M.;
Wanless, D.; Bartkowiak, J.; Boiteau, R.; et al. Traditional and molecular analyses for fecal indicator bacteria
in non-point source subtropical recreational marine waters. Water Res. 2010, 44, 3763–3772. [CrossRef]
[PubMed]
87. Wetz, J.J.; Lipp, E.K.; Griffin, D.W.; Lukasik, J.; Wait, D.; Sobsey, M.D.; Scott, T.M.; Rose, J.B. Presence,
infectivity, and stability of enteric viruses in seawater: Relationship to marine water quality in the Florida
keys. Mar. Pollut. Bull. 2004, 48, 698–704. [CrossRef] [PubMed]
88. Betancourt, W.Q.; Duarte, D.C.; Vasquez, R.C.; Gurian, P.L. Cryptosporidium and Giardia in tropical
recreational marine waters contaminated with domestic sewage: Estimation of bathing-associated disease
risks. Mar. Pollut. Bull. 2014, 85, 268–273. [CrossRef] [PubMed]
89. Henry, R.; Schang, C.; Kolotelo, P.; Coleman, R.; Rooney, G.; Schmidt, J.; Deletic, A.; McCarthy, D.T. Effect
of environmental parameters on pathogen and faecal indicator organism concentrations within an urban
estuary. Estuar. Coast. Shelf Sci. 2016, 174, 18–26. [CrossRef]
90. Jiang, S.; Noble, R.; Chu, W. Human adenoviruses and coliphages in urban runoff-impacted coastal waters
of southern California. Appl. Environ. Microbiol. 2001, 67, 179–184. [CrossRef] [PubMed]
91. Jiang, S.C.; Chu, W. Pcr detection of pathogenic viruses in southern California urban rivers. J. Appl. Microbiol.
2004, 97, 17–28. [CrossRef]
92. Jiang, S.C.; Chu, W.; He, J.W. Seasonal detection of human viruses and coliphage in Newport Bay, California.
Appl. Environ. Microbiol. 2007, 73, 6468–6474. [CrossRef]
93. McQuaig, S.; Griffith, J.; Harwood, V.J. Association of fecal indicator bacteria with human viruses and
microbial source tracking markers at coastal beaches impacted by nonpoint source pollution. Appl. Environ.
94. Ortega, C.; Solo-Gabriele, H.M.; Abdelzaher, A.; Wright, M.; Deng, Y.; Stark, L.M. Correlations between
microbial indicators, pathogens, and environmental factors in a subtropical estuary. Mar. Pollut. Bull. 2009,
58, 1374–1381. [CrossRef]
166
IJERPH 2018, 15, 2842
95. Silva, A.M.; Vieira, H.; Martins, N.; Granja, A.T.; Vale, M.J.; Vale, F.F. Viral and bacterial contamination in
recreational waters: A case study in the Lisbon bay area. J. Appl. Microbiol. 2010, 108, 1023–1031. [CrossRef]
96. Efstratiou, M.A.; Tsirtsis, G. Do 2006/7/EC European Union Bathing Water Standards exclude the risk of
contact with Salmonella or Candida albicans? Mar. Pollut. Bull. 2009, 58, 1039–1044. [CrossRef] [PubMed]
97. Graczyk, T.K.; Sunderland, D.; Awantang, G.N.; Mashinski, Y.; Lucy, F.E.; Graczyk, Z.; Chomicz, L.;
Breysse, P.N. Relationships among bather density, levels of human waterborne pathogens, and fecal coliform
counts in marine recreational beach water. Parasitol. Res. 2010, 106, 1103–1108. [CrossRef] [PubMed]
98. McQuaig, S.M.; Scott, T.M.; Harwood, V.J.; Farrah, S.R.; Lukasik, J.O. Detection of human-derived fecal
pollution in environmental waters by use of a PCR-based human polyomavirus assay. Appl. Environ.
99. McQuaig, S.M.; Scott, T.M.; Lukasik, J.O.; Paul, J.H.; Harwood, V.J. Quantification of human polyomaviruses
JC virus and BK virus by taqman quantitative pcr and comparison to other water quality indicators in water
and fecal samples. Appl. Environ. Microbiol. 2009, 75, 3379–3388. [CrossRef] [PubMed]
100. Viau, E.J.; Goodwin, K.D.; Yamahara, K.M.; Layton, B.A.; Sassoubre, L.M.; Burns, S.L.; Tong, H.I.; Wong, S.H.;
Lu, Y.; Boehm, A.B. Bacterial pathogens in Hawaiian coastal streams—Associations with fecal indicators,
land cover, and water quality. Water Res. 2011, 45, 3279–3290. [CrossRef] [PubMed]
101. Graczyk, T.K.; Sunderland, D.; Tamang, L.; Shields, T.M.; Lucy, F.E.; Breysse, P.N. Quantitative evaluation
of the impact of bather density on levels of human-virulent microsporidian spores in recreational water.
102. Lodder, W.J.; de Roda Husman, A.M. Presence of noroviruses and other enteric viruses in sewage and
surface waters in the Netherlands. Appl. Environ. Microbiol. 2005, 71, 1453–1461. [CrossRef] [PubMed]
103. Rezaeinejad, S.; Vergara, G.G.; Woo, C.H.; Lim, T.T.; Sobsey, M.D.; Gin, K.Y. Surveillance of enteric viruses
and coliphages in a tropical urban catchment. Water Res. 2014, 58, 122–131. [CrossRef]
104. Fung, D.Y.C.; Fujioka, R.; Vijayavel, K.; Sato, D.; Bishop, D. Evaluation of fung double tube test for
Clostridium perfringens and Easyphage test for F-specific RNA coliphages as rapid screening tests for fecal
contamination in recreational waters of Hawaii (vol 15, pg 217, 2007). J. Rapid Methods Autom. Microbiol.
2007, 15, 411. [CrossRef]
105. Wong, M.; Kumar, L.; Jenkins, T.M.; Xagoraraki, I.; Phanikumar, M.S.; Rose, J.B. Evaluation of public
health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human-specific
bacteriological marker. Water Res. 2009, 43, 1137–1149. [CrossRef]
106. Walters, S.P.; Gannon, V.P.; Field, K.G. Detection of Bacteroidales fecal indicators and the zoonotic pathogens
E. coli 0157:H7, Salmonella, and Campylobacter in river water. Environ. Sci. Technol. 2007, 41, 1856–1862.
[CrossRef] [PubMed]
107. Donati, M.; Cremonini, E.; Di Francesco, A.; Dallolio, L.; Biondi, R.; Muthusamy, R.; Leoni, E. Prevalence of
Simkania negevensis in chlorinated water from spa swimming pools and domestic supplies. J. Appl. Microbiol.
2015, 118, 1076–1082. [CrossRef] [PubMed]
108. La Rosa, G.; Della Libera, S.; Petricca, S.; Iaconelli, M.; Briancesco, R.; Paradiso, R.; Semproni, M.;
Di Bonito, P.; Bonadonna, L. First detection of papillomaviruses and polyomaviruses in swimming pool
waters: Unrecognized recreational water-related pathogens? J. Appl. Microbiol. 2015, 119, 1683–1691.
[CrossRef] [PubMed]
109. Maunula, L.; Kalso, S.; Von Bonsdorff, C.H.; Ponka, A. Wading pool water contaminated with both
110. Wade, T.J.; Calderon, R.L.; Brenner, K.P.; Sams, E.; Beach, M.; Haugland, R.; Wymer, L.; Dufour, A.P. High
sensitivity of children to swimming-associated gastrointestinal illness: Results using a rapid assay of
recreational water quality. Epidemiology 2008, 19, 375–383. [CrossRef]
111. Yau, V.M.; Schiff, K.C.; Arnold, B.F.; Griffith, J.F.; Gruber, J.S.; Wright, C.C.; Wade, T.J.; Burns, S.; Hayes, J.M.;
McGee, C.; et al. Effect of submarine groundwater discharge on bacterial indicators and swimmer health at
Avalon Beach, CA, USA. Water Res. 2014, 59, 23–36. [CrossRef]
112. Napier, M.D.; Haugland, R.; Poole, C.; Dufour, A.P.; Stewart, J.R.; Weber, D.J.; Varma, M.; Lavender, J.S.;
Wade, T.J. Exposure to human-associated fecal indicators and self-reported illness among swimmers at
recreational beaches: A cohort study. Environ. Health 2017, 16, 103. [CrossRef]
167
IJERPH 2018, 15, 2842
113. Benjamin-Chung, J.; Arnold, B.F.; Wade, T.J.; Schiff, K.; Griffith, J.F.; Dufour, A.P.; Weisberg, S.B.;
Colford, J.M., Jr. Coliphages and gastrointestinal illness in recreational waters: Pooled analysis of six
coastal beach cohorts. Epidemiology 2017, 28, 644–652. [CrossRef]
114. Arnold, B.F.; Wade, T.J.; Benjamin-Chung, J.; Schiff, K.C.; Griffith, J.F.; Dufour, A.P.; Weisberg, S.B.;
Colford, J.M., Jr. Acute gastroenteritis and recreational water: Highest burden among young US children.
Am. J. Public Health 2016, 106, 1690–1697. [CrossRef]
115. Wade, T.J.; Sams, E.A.; Beach, M.J.; Collier, S.A.; Dufour, A.P. The incidence and health burden of earaches
attributable to recreational swimming in natural waters: A prospective cohort study. Environ. Health 2013,
12, 67. [CrossRef]
116. Colford, J.M., Jr.; Schiff, K.C.; Griffith, J.F.; Yau, V.; Arnold, B.F.; Wright, C.C.; Gruber, J.S.; Wade, T.J.; Burns, S.;
Hayes, J.; et al. Using rapid indicators for enterococcus to assess the risk of illness after exposure to urban
runoff contaminated marine water. Water Res. 2012, 46, 2176–2186. [CrossRef] [PubMed]
117. Wade, T.J.; Sams, E.; Brenner, K.P.; Haugland, R.; Chern, E.; Beach, M.; Wymer, L.; Rankin, C.C.; Love, D.;
Li, Q.; et al. Rapidly measured indicators of recreational water quality and swimming-associated illness at
marine beaches: A prospective cohort study. Environ. Health 2010, 9, 66. [CrossRef] [PubMed]
118. Colford, J.M., Jr.; Wade, T.J.; Schift, K.C.; Wright, C.; Griffith, J.F.; Sandhu, S.K.; Weisberg, S.B. Recreational
Water Contact and Illness in Mission Bay, California; Southern California Coastal Water Research Project: Costa
Mesa, CA, USA, 2005.
119. Colford, J.M., Jr.; Wade, T.J.; Schiff, K.C.; Wright, C.C.; Griffith, J.F.; Sandhu, S.K.; Burns, S.; Sobsey, M.;
Lovelace, G.; Weisberg, S.B. Water quality indicators and the risk of illness at beaches with nonpoint sources
of fecal contamination. Epidemiology 2007, 18, 27–35. [CrossRef] [PubMed]
120. Wade, T.J.; Calderon, R.L.; Sams, E.; Beach, M.; Brenner, K.P.; Williams, A.H.; Dufour, A.P. Rapidly measured
indicators of recreational water quality are predictive of swimming-associated gastrointestinal illness.
Environ. Health Perspect. 2006, 114, 24–28. [CrossRef]
121. Arnold, B.F.; Schiff, K.C.; Ercumen, A.; Benjamin-Chung, J.; Steele, J.A.; Griffith, J.F.; Steinberg, S.J.; Smith, P.;
McGee, C.D.; Wilson, R.; et al. Acute illness among surfers after exposure to seawater in dry- and wet-weather
conditions. Am. J. Epidemiol. 2017, 186, 866–875. [CrossRef] [PubMed]
122. Vonschirnding, Y.E.R.; Kfir, R.; Cabelli, V.; Franklin, L.; Joubert, G. Morbidity among bathers exposed to
polluted seawater—A prospective epidemiologic-study. S. Afr. Med. J. 1992, 81, 543–546.
123. Lee, J.V.; Dawson, S.R.; Ward, S.; Surman, S.B.; Neal, K.R. Bacteriophages are a better indicator of illness
rates than bacteria amongst users of a white water course fed by a lowland river. Water Sci. Technol. 1997, 35,
124. Van Asperen, I.A.; Medema, G.; Borgdorff, M.W.; Sprenger, M.J.W.; Havelaar, A.H. Risk of gastroenteritis
among triathletes in relation to faecal pollution of fresh waters. Int. J. Epidemiol. 1998, 27, 309–315. [CrossRef]
125. Schets, F.M.; Schijven, J.F.; de Roda Husman, A.M. Exposure assessment for swimmers in bathing waters
and swimming pools. Water Res. 2011, 45, 2392–2400. [CrossRef]
126. Schets, F.M.; De Roda Husman, A.M.; Havelaar, A.H. Disease outbreaks associated with untreated
recreational water use. Epidemiol. Infect. 2011, 139, 1114–1125. [CrossRef]
127. Grenier, J.L.; Davis, J.A. Water quality in south San Francisco Bay, California: Current condition and potential
issues for the South Bay Salt Pond Restoration Project. Rev. Environ. Contam. Toxicol. 2010, 206, 115–147.
[PubMed]
128. McMinn, B.R.; Ashbolt, N.J.; Korajkic, A. Bacteriophages as indicators of faecal pollution and enteric virus
removal. Lett. Appl. Microbiol. 2017, 65, 11–26. [CrossRef] [PubMed]
129. McMinn, B.R.; Korajkic, A.; Ashbolt, N.J. Evaluation of Bacteroides fragilis GB-124 bacteriophages as novel
human-associated faecal indicators in the United States. Lett. Appl. Microbiol. 2014, 59, 115–121. [CrossRef]
[PubMed]
130. Eftim, S.E.; Hong, T.; Soller, J.; Boehm, A.; Warren, I.; Ichida, A.; Nappier, S.P. Occurrence of norovirus in raw
sewage—A systematic literature review and meta-analysis. Water Res. 2017, 111, 366–374. [CrossRef]
131. Li, J.; Wang, H.; Wang, R.; Zhang, L. Giardia duodenalis infections in humans and other animals in China.
Front. Microbiol. 2017, 8, 2004. [CrossRef]
132. Rose, J.B.; Huffman, D.E.; Gennaccaro, A. Risk and control of waterborne cryptosporidiosis. FEMS Microbiol.
Rev. 2002, 26, 113–123. [CrossRef]
168
IJERPH 2018, 15, 2842
133. Sinclair, R.G.; Choi, C.Y.; Riley, M.R.; Gerba, C.P. Pathogen surveillance through monitoring of sewer systems.
Adv. Appl. Microbiol. 2008, 65, 249–269.
134. Ganesh, A.; Lin, J. Waterborne human pathogenic viruses of public health concern. Int. J. Environ. Health Res.
2013, 23, 544–564. [CrossRef]
135. Matthews, L.; Low, J.C.; Gally, D.L.; Pearce, M.C.; Mellor, D.J.; Heesterbeek, J.A.; Chase-Topping, M.;
Naylor, S.W.; Shaw, D.J.; Reid, S.W.; et al. Heterogeneous shedding of Escherichia coli O157 in cattle and its
implications for control. Proc. Natl. Acad. Sci. USA 2006, 103, 547–552. [CrossRef]
136. Daniels, M.E.; Shrivastava, A.; Smith, W.A.; Sahu, P.; Odagiri, M.; Misra, P.R.; Panigrahi, P.; Suar, M.;
Clasen, T.; Jenkins, M.W. Cryptosporidium and Giardia in humans, domestic animals, and village water
sources in rural india. Am. J. Trop. Med. Hyg. 2015, 93, 596–600. [CrossRef]
137. Oates, S.C.; Miller, M.A.; Hardin, D.; Conrad, P.A.; Melli, A.; Jessup, D.A.; Dominik, C.; Roug, A.; Tinker, M.T.;
Miller, W.A. Prevalence, environmental loading, and molecular characterization of Cryptosporidium and
Giardia isolates from domestic and wild animals along the central California Coast. Appl. Environ. Microbiol.
2012, 78, 8762–8772. [CrossRef] [PubMed]
138. Vonberg, R.P.; Hohle, M.; Aepfelbacher, M.; Bange, F.C.; Campos, C.B.; Claussen, K.; Christner, M.;
Cramer, J.P.; Haller, H.; Hornef, M.; et al. Duration of fecal shedding of shiga toxin-producing Escherichia coli
O104:H4 in patients infected during the 2011 outbreak in Germany: A multicenter study. Clin. Infect. Dis.
2013, 56, 1132–1140. [CrossRef] [PubMed]
139. Lanzas, C.; Warnick, L.D.; James, K.L.; Wright, E.M.; Wiedmann, M.; Grohn, Y.T. Transmission dynamics of a
multidrug-resistant Salmonella typhimurium outbreak in a dairy farm. Foodborne Pathog. Dis. 2010, 7, 467–474.
[CrossRef]
140. Venegas-Vargas, C.; Henderson, S.; Khare, A.; Mosci, R.E.; Lehnert, J.D.; Singh, P.; Ouellette, L.M.; Norby, B.;
Funk, J.A.; Rust, S.; et al. Factors associated with Shiga toxin-producing Escherichia coli shedding by dairy
and beef cattle. Appl. Environ. Microb. 2016, 82, 5049–5056. [CrossRef]
141. Procter, T.D.; Pearl, D.L.; Finley, R.L.; Leonard, E.K.; Janecko, N.; Reid-Smith, R.J.; Weese, J.S.; Peregrine, A.S.;
Sargeant, J.M. A cross-sectional study examining Campylobacter and other zoonotic enteric pathogens in dogs
that frequent dog parks in three cities in south-western ontario and risk factors for shedding of Campylobacter
spp. Zoonoses Public Health 2014, 61, 208–218. [CrossRef] [PubMed]
142. Xu, Y.; Dugat-Bony, E.; Zaheer, R.; Selinger, L.; Barbieri, R.; Munns, K.; McAllister, T.A.; Selinger, L.B.
Escherichia coli O157:H7 super-shedder and non-shedder feedlot steers harbour distinct fecal bacterial
communities. PLoS ONE 2014, 9, e98115. [CrossRef]
143. Leclerc, H.; Schwartzbrod, L.; Dei-Cas, E. Microbial agents associated with waterborne diseases. Crit. Rev.
144. Hara-Kudo, Y.; Takatori, K. Contamination level and ingestion dose of foodborne pathogens associated with
infections. Epidemiol. Infect. 2011, 139, 1505–1510. [CrossRef] [PubMed]
145. Harwood, V.J.; Levine, A.D.; Scott, T.M.; Chivukula, V.; Lukasik, J.; Farrah, S.R.; Rose, J.B. Validity of the
indicator organism paradigm for pathogen reduction in reclaimed water and public health protection.
Appl. Environ. Microbiol. 2005, 71, 3163–3170. [CrossRef]
146. Hijnen, W.A.; Beerendonk, E.F.; Medema, G.J. Inactivation credit of uv radiation for viruses, bacteria and
protozoan (oo)cysts in water: A review. Water Res. 2006, 40, 3–22. [CrossRef]
147. Nasser, A.M. Removal of Cryptosporidium by wastewater treatment processes: A review. J. Water Health 2016,
148. Eischeid, A.C.; Meyer, J.N.; Linden, K.G. Uv disinfection of adenoviruses: Molecular indications of DNA
damage efficiency. Appl. Environ. Microbiol. 2009, 75, 23–28. [CrossRef] [PubMed]
149. Nelson, K.L.; Boehm, A.B.; Davies-Colley, R.J.; Dodd, M.C.; Kohn, T.; Linden, K.G.; Liu, Y.; Maraccini, P.A.;
McNeill, K.; Mitch, W.A.; et al. Sunlight-mediated inactivation of health-relevant microorganisms in water:
A review of mechanisms and modeling approaches. Environ. Sci. Process. Impacts 2018, 20, 1089–1122.
[CrossRef] [PubMed]
150. Bae, S.; Wuertz, S. Decay of host-associated bacteroidales cells and DNA in continuous-flow freshwater and
seawater microcosms of identical experimental design and temperature as measured by PMA-qPCR and
qPCR. Water Res. 2015, 70, 205–213. [CrossRef] [PubMed]
169
IJERPH 2018, 15, 2842
151. Bae, S.; Wuertz, S. Survival of host-associated bacteroidales cells and their relationship with Enterococcus spp.,
Campylobacter jejuni, Salmonella enterica serovar typhimurium, and adenovirus in freshwater microcosms as
measured by propidium monoazide-quantitative pcr. Appl. Environ. Microbiol. 2012, 78, 922–932. [CrossRef]
[PubMed]
152. Wanjugi, P.; Sivaganesan, M.; Korajkic, A.; Kelty, C.A.; McMinn, B.; Ulrich, R.; Harwood, V.J.; Shanks, O.C.
Differential decomposition of bacterial and viral fecal indicators in common human pollution types.
Water Res. 2016, 105, 591–601. [CrossRef]
153. Korajkic, A.; McMinn, B.R.; Shanks, O.C.; Sivaganesan, M.; Fout, G.S.; Ashbolt, N.J. Biotic interactions and
sunlight affect persistence of fecal indicator bacteria and microbial source tracking genetic markers in the
upper Mississippi River. Appl. Environ. Microbiol. 2014, 80, 3952–3961. [CrossRef]
154. Kreader, C.A. Persistence of pcr-detectable bacteroides distasonis from human feces in river water.
Appl. Environ. Microbiol. 1998, 64, 4103–4105.
155. Dick, L.K.; Stelzer, E.A.; Bertke, E.E.; Fong, D.L.; Stoeckel, D.M. Relative decay of bacteroidales microbial
source tracking markers and cultivated Escherichia coli in freshwater microcosms. Appl. Environ. Microbiol.
2010, 76, 3255–3262. [CrossRef]
156. Booncharoen, N.; Mongkolsuk, S.; Sirikanchana, K. Comparative persistence of human sewage-specific
enterococcal bacteriophages in freshwater and seawater. Appl. Microbiol. Biotechnol. 2018, 102, 6235–6246.
[CrossRef]
157. Chauret, C.; Nolan, K.; Chen, P.; Springthorpe, S.; Sattar, S. Aging of Cryptosporidium parvum oocysts in river
water and their susceptibility to disinfection by chlorine and monochloramine. Can. J. Microbiol. 1998, 44,
158. McCrary, K.J.; Case, C.L.H.; Gentry, T.J.; Aitkenhead-Peterson, J.A. Escherichia coli regrowth in disinfected
sewage effluent: Effect of doc and nutrients on regrowth in laboratory incubations and urban streams.
Water Air Soil Pollut. 2013, 224, 1412. [CrossRef]
159. Shelton, D.R.; Pachepsky, Y.A.; Kiefer, L.A.; Blaustein, R.A.; McCarty, G.W.; Dao, T.H. Response of coliform
populations in streambed sediment and water column to changes in nutrient concentrations in water.
160. Gregory, L.F.; Karthikeyan, R.; Aitkenhead-Peterson, J.A.; Gentry, T.J.; Wagner, K.L.; Harmel, R.D. Nutrient
loading impacts on culturable E. coli and other heterotrophic bacteria fate in simulated stream mesocosms.
161. Wanjugi, P.; Fox, G.A.; Harwood, V.J. The interplay between predation, competition, and nutrient levels
influences the survival of Escherichia coli in aquatic environments. Microb. Ecol. 2016, 72, 526–537. [CrossRef]
[PubMed]
162. Sinton, L.W.; Davies-Colley, R.J.; Bell, R.G. Inactivation of enterococci and fecal coliforms from sewage and
meatworks effluents in seawater chambers. Appl. Environ. Microbiol. 1994, 60, 2040–2048. [PubMed]
163. Sinton, L.W.; Finlay, R.K.; Lynch, P.A. Sunlight inactivation of fecal bacteriophages and bacteria in
sewage-polluted seawater. Appl. Environ. Microbiol. 1999, 65, 3605–3613.
164. Sokolova, E.; Astrom, J.; Pettersson, T.J.; Bergstedt, O.; Hermansson, M. Decay of bacteroidales genetic
markers in relation to traditional fecal indicators for water quality modeling of drinking water sources.
Environ. Sci. Technol. 2012, 46, 892–900. [CrossRef]
165. Ngazoa, E.S.; Fliss, I.; Jean, J. Quantitative study of persistence of human norovirus genome in water using
TaqMan real-time RT-PCR. J. Appl. Microbiol. 2008, 104, 707–715. [CrossRef]
166. Wait, D.A.; Sobsey, M.D. Comparative survival of enteric viruses and bacteria in Atlantic ocean seawater.
Water Sci. Technol. 2001, 43, 139–142. [CrossRef]
167. Whitman, R.; Harwood, V.J.; Edge, T.A.; Nevers, M.; Byappanahalli, M.; Vijayavel, K.; Brandao, J.;
Sadowsky, M.J.; Alm, E.W.; Crowe, A.; et al. Microbes in beach sands: Integrating environment, ecology and
public health. Rev. Environ. Sci. Biotechnol. 2014, 13, 329–368. [CrossRef] [PubMed]
168. Green, H.C.; Shanks, O.C.; Sivaganesan, M.; Haugland, R.A.; Field, K.G. Differential decay of human faecal
bacteroides in marine and freshwater. Environ. Microbiol. 2011, 13, 3235–3249. [CrossRef] [PubMed]
169. Korajkic, A.; McMinn, B.R.; Harwood, V.J.; Shanks, O.C.; Fout, G.S.; Ashbolt, N.J. Differential decay of
enterococci and Escherichia coli originating from two fecal pollution sources. Appl. Environ. Microbiol. 2013,
170
IJERPH 2018, 15, 2842
170. Jeanneau, L.; Solecki, O.; Wery, N.; Jarde, E.; Gourmelon, M.; Communal, P.Y.; Jadas-Hecart, A.; Caprais, M.P.;
Gruau, G.; Pourcher, A.M. Relative decay of fecal indicator bacteria and human-associated markers:
A microcosm study simulating wastewater input into seawater and freshwater. Environ. Sci. Technol.
2012, 46, 2375–2382. [CrossRef] [PubMed]
171. Nayak, B.; Weidhaas, J.; Harwood, V.J. LA35 poultry fecal marker persistence is correlated with that of
indicators and pathogens in environmental waters. Appl. Environ. Microbiol. 2015, 81, 4616–4625. [CrossRef]
172. Hurst, C.J.; Gerba, C.P. Stability of simian rotavirus in fresh and estuarine water. Appl. Environ. Microbiol.
1980, 39, 1–5.
173. Lo, S.; Gilbert, J.; Hetrick, F. Stability of human enteroviruses in estuarine and marine waters. Appl. Environ.
Microbiol. 1976, 32, 245–249.
174. Fayer, R.; Graczyk, T.K.; Lewis, E.J.; Trout, J.M.; Farley, C.A. Survival of infectious cryptosporidium parvum
oocysts in seawater and eastern oysters (Crassostrea virginica) in the chesapeake bay. Appl. Environ. Microbiol.
1998, 64, 1070–1074.
175. Korajkic, A.; McMinn, B.R.; Ashbolt, N.J.; Sivaganesan, M.; Harwood, V.J.; Shanks, O.C. Extended persistence
of general and cattle-associated fecal indicators in marine and freshwater environment. Sci. Total Environ.
2019, 65, 1292–1302. [CrossRef]
176. Perkins, T.L.; Perrow, K.; Rajko-Nenow, P.; Jago, C.F.; Jones, D.L.; Malham, S.K.; McDonald, J.E. Decay
rates of faecal indicator bacteria from sewage and ovine faeces in brackish and freshwater microcosms with
contrasting suspended particulate matter concentrations. Sci. Total Environ. 2016, 572, 1645–1652. [CrossRef]
177. Curtis, K.; Michael Trapp, J. Examining the colonization and survival of E. coli from varying host sources
in drainage basin sediments and stormwater. Arch. Environ. Contam. Toxicol. 2016, 71, 183–197. [CrossRef]
[PubMed]
178. Cloutier, D.D.; McLellan, S.L. Distribution and differential survival of traditional and alternative indicators
of fecal pollution at freshwater beaches. Appl. Environ. Microbiol. 2017, 83, e02881-16. [CrossRef] [PubMed]
179. Anderson, K.L.; Whitlock, J.E.; Harwood, V.J. Persistence and differential survival of fecal indicator bacteria
in subtropical waters and sediments. Appl. Environ. Microbiol. 2005, 71, 3041–3048. [CrossRef] [PubMed]
180. Jenkins, M.B.; Fisher, D.S.; Endale, D.M.; Adams, P. Comparative die-off of Escherichia coli O157:H7 and fecal
indicator bacteria in pond water. Environ. Sci. Technol. 2011, 45, 1853–1858. [CrossRef] [PubMed]
181. Wanjugi, P.; Harwood, V.J. The influence of predation and competition on the survival of commensal and
pathogenic fecal bacteria in aquatic habitats. Environ. Microbiol. 2013, 15, 517–526. [CrossRef] [PubMed]
171
and Public Health
Article
Validation of Questionnaire Methods to Quantify
Recreational Water Ingestion
Laura M. Suppes 1, * , Kacey C. Ernst 2 , Leif Abrell 3 and Kelly A. Reynolds 2
1 Environmental Public Health Program, The University of Wisconsin-Eau Claire, 105 Garfield Avenue,
Eau Claire, WI 54702, USA
2 Mel and Enid Zuckerman College of Public Health, The University of Arizona, P.O. Box 245163,
Tucson, AZ 85724, USA; [email protected] (K.C.E.); [email protected] (K.A.R.)
3 Department of Soil, Water & Environmental Science, The University of Arizona, Gould-Simpson Building
Room 611, 1040 East 4th Street, Tucson, AZ 85721, USA; [email protected]
Received: 8 September 2018; Accepted: 19 October 2018; Published: 1 November 2018
Abstract: Swimming pool water ingestion volumes are necessary for assessing infection risk from
swimming. Pool water ingestion volumes can be estimated by questionnaire or measuring a chemical
tracer in swimmer urine. Questionnaires are often preferred to the chemical tracer method because
surveys are less time consuming, but no research exists validating questionnaires accurately quantify
pool water ingestion volumes. The objective of this study was to explore if questionnaires are
a reliable tool for collecting pool water ingestion volumes. A questionnaire was issued at four
pool sites in Tucson, Arizona to 46 swimmers who also submitted a urine sample for analyzing
cyanuric acid, a chemical tracer. Perceived ingestion volumes reported on the questionnaire were
compared with pool water ingestion volumes, quantified by analyzing cyanuric acid in swimmer
urine. Swimmers were asked if they swallowed (1) no water or only a few drops, (2) one to two
mouthfuls, (3) three to five mouthfuls, or (4) six to eight mouthfuls. One mouthful is the equivalent of
27 mL of water. The majority (81%) of swimmers ingested <27 mL of pool water but reported ingesting
>27 mL (“one mouthful”) on the questionnaire. More than half (52%) of swimmers overestimated
their ingestion volume. These findings suggest swimmers are over-estimating pool water ingestion
because they perceive one mouthful is <27 mL. The questionnaire did not reliably collect pool water
ingestion volumes and should be improved for future exposure assessment studies. Images of the
ingestion volume categories should be included on the questionnaire to help swimmers visualize the
response options.
Keywords: pool water ingestion; recreational water; swimming pool; risk assessment
1. Introduction
The annual number of Recreational Water Illness (RWI) outbreaks associated with treated
recreational water venues (“pools”) in the U.S. has increased since 1978 when reporting was initiated
(pools are defined as swimming pools, spas, interactive fountains, wading pools and dive pools) [1–3].
RWIs range from acute gastrointestinal illness (AGI), skin infection or rash to acute respiratory illness
(ARI). The majority of outbreaks are associated with AGI, which accounted for 81% of outbreaks during
summer months in 2011–2012 [4]. Most AGI outbreaks in treated recreational water are associated
with ingesting Cryptosporidium. Cryptosporidium has been detected in treated recreational water and
associated with outbreaks internationally [5–8]. From 2000–2014, Cryptosporidium caused 58% of
treated recreational water outbreaks in the U.S. [9]. The volume of pool water ingested by swimmers is
necessary to quantify infection risk from enteric pathogens like Cryptosporidium [10]. Risk assessment
can help identify unsafe swimming behaviors, at-risk populations, and priority hazards to direct the

IJERPH 2018, 15, 2435
development of pool safety guidelines. Recognizing the need for accurate data collection tools for
swimming pool risk assessment, this study compared perceived ingestion volumes reported on a
questionnaire to pool water ingestion volumes quantified by analyzing cyanuric acid in swimmer
urine. The questionnaire merged information and survey questions collected and developed by the
Centers for Disease Control and Prevention (CDC), the U.S. Environmental Protection Agency (USEPA),
and academic researchers to assess a variety of swimmer exposures. The objective was to determine if
questionnaires are a reliable tool for collecting pool water ingestion volumes.
One primary exposure related to risk of RWI is ingestion of water. Previously, the World Health
Organization (WHO) used questionnaires to estimate swimming ingestion rates and found swimmers
reported swallowing 20–50 mL/h [11]. These self-reported values, however, are underestimated
when compared to ingestion ranges found in other studies applying quantitative measurement
techniques. Thus, the WHO questionnaire may not accurately capture pool water ingestion magnitudes
among swimmers.
Ingestion can be quantified using methods that compare cyanuric acid in urine and pool
water. Cyanuric acid is added as a chlorine stabilizer to outdoor pool water, and when ingested,
passes through the human body unmetabolized [12]. Controlled studies show 98% of cyanuric acid
ingested is excreted in a 24 h period [12]. Using this technique, researchers Dufour et al. and Suppes
et al. showed swimmers ingested between 0–154 mL/h and 0–105.5 mL/h, respectively [13,14].
Information on perceived ingestion by study participants was not collected in the Dufour study,
but was collected by Suppes et al. using the questionnaire discussed in this article (see Supplementary
Materials). The questionnaire asked swimmers how much pool water was ingested during a timed
swim. The current article is one part of the Suppes et al. study and describes how accurately swimmers
perceive pool water ingestion by comparing reported to measured volumes. Our findings demonstrate
swimmers perceive higher ingestion exposures than in reality, which explains why self-reported
ingestion estimates are different than measured estimates.
2.1. Questionnaire Development

The CDC and USEPA websites and peer-reviewed literature were searched for pool outbreak
survey tools, tools developed in response to outbreaks, and tools designed to capture swimmer
exposures [15,16]. The CDC National Outbreak Reporting System (NORS) is available for reporting
nationwide waterborne disease outbreaks and includes exposure questions related to recreational
water. In-depth survey tools are also available through the CDC that collect data on swimmer
activity, gastrointestinal symptoms, confounding exposures, pool operations and maintenance, and are
designed to be administered by outbreak investigators [15]. Surveys intended to collect additional
exposure information, such as potential disinfection by-product exposures, were reviewed from the
USEPA assessment tool SWIMODEL among others [15].
Exposure risk factors relative to swimmer behavior and pool maintenance from the CDC surveys,
SWIMODEL, and peer-reviewed literature were compiled and organized into a draft questionnaire.
Three panels were assembled to review the draft for comprehensiveness and to recommend formatting
and included (1) six experts from the swimming pool industry; (2) an international group of
nine microbiologists, exposure scientists, and epidemiologists; and (3) an internal University of
Arizona panel of six respiratory health, epidemiology, exposure science, and public health specialists.
Meetings with each panel were held once and lasted 1–2 h following advance reviews of the
questionnaire. Individual communication with panel members by email or phone occurred throughout
the questionnaire development process. Questions from the draft were entered into DatStat Illume
Survey Developer Gateway Version 5.1.1.17347 (Seattle, WA, USA). The questionnaire was further
evaluated by the external review panel for errors and comprehensiveness prior to use. A modified
version of the questionnaire can be viewed in Table S1 of the Supplementary Materials.
173
IJERPH 2018, 15, 2435
The question used in this study to estimate pool water ingestion by “mouthfuls/swim” was
developed by Schets et al. and was selected over other surveys based on recommendations from
the expert questionnaire review committees [17]. Other surveys used specific volume classifications,
like “teaspoon”, that may have been difficult for younger participants in this study to interpret.
The Schets study quantified the average volume in one mouthful (27 mL), which allowed measured
volumes in the present study to be categorized into “mouthfuls/swim”. Swimmers were asked on our
questionnaire if they swallowed (1) no water or only a few drops, (2) one to two mouthfuls, (3) three to
five mouthfuls, or (4) six to eight mouthfuls. Using data from the Schets study indicating an average
mouthful is 27 mL, qualitative variables from our questionnaire were converted to quantitative volumes.
Despite the Schets study defining “no water to a few drops” as 0–5 mL, swimmers with measured
ingestion between 0–26 mL were categorized as: “1: no water or only a few drops”. There was
no qualitative ingestion category in the Schets study representing 6–26 mL. The other categories
were: 27–54 mL (one to two mouthfuls), 55–135 mL (three to five mouthfuls), and 136–216 mL (six to
eight mouthfuls).
2.2. Data Collection

This research was approved by the University of Arizona Human Subjects Research and
Institutional Review Board (project number: 12-0272-12). The questionnaire was issued to 46 swimmers
June–September 2013 in Tucson, Arizona, recruited at two outdoor public pools and two outdoor
private pools. Swimmers arriving at the pools on data collection days were approached by a member
of the research team, given details of the study’s objectives, and asked if they would participate by
completing a questionnaire after swimming and submitting a 24 h urine sample to quantify pool
water ingestion. Urine samples were preserved then cleaned by solid phase extraction and analyzed
using ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for
cyanuric acid. Pool water samples were collected at each pool site on the day swimmers were recruited,
transferred on ice, and preserved along with urine samples. Cyanuric acid was quantified in pool
water using UHPLC-MS/MS. Pool water ingestion volumes were calculated using cyanuric acid
concentrations in urine and pool water [13] (Equation (1)). Detailed results from the 24 h urine sample
portion of this study are published elsewhere [14].
μg μg
water ingestion (L) = ([cyanuric acid]urine ( L ) ÷ [cyanuric acid]pool water ( L )) × urine volume (L) (1)
All swimmers, regardless of age, gender, or other factors, were approached and asked to
participate. Swim duration for all participants was recorded on the questionnaire. Participants accessed
the questionnaire either on-site using tablets, electronic or smart phones, or on a personal computer
through email. Questionnaires were completed within six hours of swimming.
3. Results
Thirty-eight of 46 participants had usable water ingestion values for analysis. Four did not submit
a questionnaire, one submitted a urine sample less than the accepted volume threshold, and three urine
samples had signal-to-noise ratios <3, which indicates a measurement below the analytical equipment
limit of detection (UHPLC-MS/MS). The percent recoveries of cyanuric acid from urine and pool water
were 6% and 112%, respectively. Table 1 summarizes the study population.
Table 2 illustrates the number of swimmers who correctly and incorrectly reported the volume
range of pool water ingested during swimming. Sixteen of 38 swimmers (42%) correctly reported
their ingestion volume, 20/38 (52%) overestimated the amount of pool water ingested and 2/38
(5%) underestimated their ingestion volume. Thirty-one of 38 swimmers (81%) actually ingested
0–26 mL of water, but only 11/38 swimmers (29%) correctly reported ingesting 0–26 mL. All swimmers
(11/11) who reported ingesting “no water to a few drops” did ingest water within the volume range
categorized as “no water to a few drops” (0–26 mL). Four of 20 swimmers who reported ingesting
174
IJERPH 2018, 15, 2435
“one to two mouthfuls” actually ingested pool water within the volume range “one to two mouthfuls”
(27–54 mL). Only one swimmer reported ingesting “three to five mouthfuls”, but six actually did
ingest pool water within this volume range (55–135 mL). No swimmers ingested or reported ingesting
136–216 mL.
Table 1. Age and gender distributions of study participants.
Participant Demographics n = 38 (%)

Age
≤18 years 17 (44.7)
>18 years 21 (55.2)
Gender
Male 25 (65.7)
Female 13 (34.2)
Table 2. Number of swimmers reporting and actually ingesting pool water amounts within each
volume range listed on the questionnaire (n = 38).
No Water–Few One to Two Three to Five Six to Eight

Drops Mouthfuls Mouthfuls Mouthfuls
(0–26 mL) (27–54 mL) (55–135 mL) (136–216 mL)
No water–few drops 11 † 14 6 0
Measured One to two mouthfuls 0 4† 0 0

Ingestion * Three to five mouthfuls 0 2 1† 0
Six to eight mouthfuls 0 0 0 0
* Measured ingestion values have been categorized using mouthful volumes characterized by Schets et al. [17].
† Study participants correctly reporting ingestion volume.
4. Discussion
Developers of the question used on our survey found the average volume of one mouthful to
be 27 mL, which was used in this study to categorize measured ingestion volumes to mouthfuls.
The majority (81%) of swimmers actually ingested <27 mL of pool water but reported ingesting >27 mL
(one mouthful) on the questionnaire. More than half (52%) of swimmers overestimated their ingestion
volume across all volume categories. These findings suggest swimmers are overestimating pool water
ingestion because they perceive one mouthful to be <27 mL. The lack of accurate reporting of ingestion
volumes using a question recommended by experts suggests a need for improving questionnaire
techniques to assess recreational water ingestion. Since there is uncertainty about the volume of
water in one mouthful, the questionnaire can be improved by including images of a one-cup/250 mL
measuring glass with one to eight mouthfuls of liquid (Figure 1). Eight was the maximum number of
mouthfuls on the questionnaire. The questionnaire can also be improved by changing the “no water
to a few drops” category to “less than one mouthful” for consistency in questionnaire response
options. Including Figure 1 would help swimmers visualize the ingestion volume categories to reduce
inaccurate reporting.
Inconsistencies in method performance between this study and similar studies [13,18] and low
recoveries of cyanuric acid in urine indicate a need for improving techniques to quantify pool water
ingestion. Using comparable methods, Dorevitch et al. recovered 32.7% of cyanuric acid from swimmer
urine and 96.5–99% of cyanuric acid from pool water [18]. Dufour et al. did not report recovery
efficiencies for cyanuric acid in urine or pool water using a similar method [13]. Recovery of cyanuric
acid in urine and pool water was 6% and 112%, respectively, in the current study. Like this study,
Dorevitch et al. calculated pool water ingestion using Equation (1) and did not adjust cyanuric acid in
pool water to account for the lower recovery in urine. Self-reported pool water ingestion quantities
from a questionnaire by Dorevitch et al. were also compared to measured pool water ingestion
175
IJERPH 2018, 15, 2435
quantities. To be consistent with Dorevitch and Dufour, no percent recovery adjustments were made to
cyanuric acid in urine or pool water before analyzing measured and self-reported pool water ingestion
in this study. Measured ingestion estimates could be higher than reported in all three studies, but exact
pool water ingestion quantities cannot be estimated without a method that consistently recovers 100%
of cyanuric acid in urine. Cyanuric acid extraction efficiencies are dependent on the solid phase
extraction technique and analytical instrument. A more detailed comparison and discussion of method
performance and limitations between this study and others is published elsewhere [14].

Figure 1. The figure illustrates one to eight mouthfuls of liquid in a one-cup/250 mL measuring glass,
assuming one mouthful is equal to 27 mL of liquid [17].
5. Conclusions
This study highlights the need for improved questionnaire techniques to assess recreational water
ingestion. Our findings demonstrate swimmers perceive higher ingestion exposures than in reality,
which explains why self-reported ingestion estimates are different than measured estimates from
previous studies. Since there is uncertainty about the volume of water in one mouthful, researchers
who use this question technique in the future should include images of a one-cup/250 mL measuring
glass with one to eight mouthfuls of liquid to help swimmers visualize the ingestion volume categories.
The questionnaire category “no water to a few drops” should be changed to “less than one mouthful”
to be consistent with other response options on the questionnaire. The altered questionnaire should be
validated to ensure ingestion volumes are accurately reported.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/15/11/2435/

s1.
Author Contributions: Conceptualization, K.A.R.; methodology, K.A.R.; software, K.C.E. and L.A.; validation,
L.M.S., K.C.E., L.A., and K.A.R.; formal analysis, L.M.S.; investigation, K.A.R. and L.M.S.; resources, L.M.S., K.C.E.,
L.A., and K.A.R.; data curation, L.M.S.; writing—original draft preparation, L.M.S.; writing—review and editing,
L.M.S. and K.A.R.; visualization, K.A.R.; supervision, K.A.R.; project administration, K.A.R.; funding acquisition,
K.A.R.
Funding: Funding for this research was provided by the National Swimming Pool Foundation and Research
Foundation for Health and Environmental Effects.
Acknowledgments: Questionnaire development was made possible with assistance from Kristen Pogreba-Brown
from the University of Arizona’s Foodborne Illness Outbreak Investigation Team. Training in video surveillance
methods was provided by Paloma Beamer at the University of Arizona’s College of Public Health. Meredith Lisse
and Leena Patel in the Mel and Enid Zuckerman College of Public Health assisted with water sample collection
and analysis, site and participant recruitment, and urine processing. Analyses in the Arizona Laboratory for
Emerging Contaminants were supported by NSF CBET 0722579. The researchers would also like to thank all those
who assisted in the questionnaire review and all volunteers who donated their time to participate in this study.
Conflicts of Interest: The authors declare no conflicts of interest.
176
IJERPH 2018, 15, 2435
References
1. Craun, G.F.; Calderon, R.L.; Craun, M.F. Outbreaks associated with recreational water in the United States.
Int. J. Environ. Health Res. 2005, 15, 243–262. [CrossRef] [PubMed]
2. Hlavsa, M.C.; Roberts, V.A.; Hill, V.R.; Kahler, A.M.; Hilborn, E.D.; Wade, T.J.; Backer, L.C.; Yoder, J.S.
Recreational water-associated disease outbreaks—United States, 2009–2010. Morb. Mortal. Wkly. Rep. 2014,
63, 6–10.
3. Yoder, J.S.; Hlavsa, M.C.; Craun, G.F.; Hill, V.; Roberts, V.; Yu, P.A.; Hicks, L.A.; Alexander, N.T.; Calderon, P.L.;
Roy, S.L.; et al. Surveillance for waterborne disease and outbreaks associated with recreational water use
and other aquatic facility-associated health events—United States, 2005–2006. Morb. Mortal. Wkly. Rep. 2008,
57, 1–29.
4. Hlavsa, M.C.; Roberts, V.A.; Kahler, A.M.; Hilborn, E.D.; Mecher, T.R.; Beach, M.J.; Wade, T.J.; Yoder, J.S.
Outbreaks of illness associated with recreational water—United States, 2011–2012. Morb. Mortal. Wkly. Rep.
2015, 64, 668–672.
5. Abd El-Salam, M.M. Assessment of water quality of some swimming pools: A case study in Alexandria,
Egypt. Environ. Monit. Assess. 2012, 12, 7395–7406. [CrossRef] [PubMed]
6. Ehsan, M.A.; Casaert, S.; Levecke, B.; Van Rooy, L.; Pelicaen, J.; Smis, A.; Claerebout, E. Cryptosporidium and
Giardia in recreational water in Belgium. J. Water Health 2015, 13, 870–878. [CrossRef] [PubMed]
7. Lemmon, J.M.; McAnulty, J.M.; Bawden-Smith, J. Outbreak of cryptosporidiosis linked to an indoor
swimming pool. Med. J. Aust. 1996, 165, 613–616. [PubMed]
8. Polus, M. The occurrence of parasitic intestinal protozoa in swimming pools and other water recreation
facilities in Cracow. Przem. Chem. 2006, 95, 107–109.
9. Hlavsa, M.C.; Cikesh, B.L.; Roberts, V.A.; Kahler, A.M.; Vigar, M.; Hilborn, E.D.; Wade, T.J.; Roellig, D.M.;
Murphy, J.L.; Xiao, L.; et al. Outbreaks of illness associated with treated recreational water—United States,
2000–2014. Morb. Mortal. Wkly. Rep. 2018, 67, 547–551. [CrossRef] [PubMed]
10. Suppes, L.M.; Canales, R.A.; Gerba, C.P.; Reynolds, K.A. Cryptosporidium risk from swimming pool
exposures. Int. J. Hyg. Environ. Health 2016, 219, 915–919. [CrossRef] [PubMed]
11. World Health Organization Guidelines for Safe Recreational Water Environments, Volume 2: Swimming Pool
and Similar Environments. Available online: www.who.int/water_sanitation_health/bathing/srwe2full.pdf
(accessed on 29 November 2016).
12. Allen, L.M.; Briggle, T.V.; Pfaffenberger, C.D. Absorption and excretion of cyanuric acid in long distance
swimmers. Drug Metab. Rev. 1982, 13, 499–516. [CrossRef] [PubMed]
13. Dufour, A.P.; Evans, O.; Behymer, T.D.; Cantu, R. Water ingestion during swimming activities in a pool:
A pilot study. J. Water Health 2006, 4, 425–430. [CrossRef] [PubMed]
14. Suppes, L.M.; Abrell, L.; Dufour, A.P.; Reynolds, K.A. Assessment of swimmer behaviors on pool water
ingestion. J. Water Health 2014, 112, 150–152. [CrossRef] [PubMed]
15. Centers for Disease Control and Prevention Recreational Water Illness Response Toolkit. Available online: http:
//www.cdc.gov/healthywater/emergency/toolkit/rwi-outbreak-toolkit.html (accessed on 29 November 2016).
16. United States Environmental Protection Agency Swimmer Exposure Assessment Model. Available online:
https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/swimmer-exposure-assessment-
model-swimodel (accessed on 29 November 2016).
17. Schets, F.M.; Schijven, J.F.; Husman, A.M.D. Exposure assessment for swimmers in bathing waters and
swimming pools. Water Res. 2011, 45, 2392–2400. [CrossRef] [PubMed]
18. Dorevitch, S.; Panthi, S.; Huang, Y.; Li, H.; Michalek, A.M.; Pratap, P.; Wroblewski, M.; Liu, L.; Scheff, P.A.;
Li, A. Water ingestion during water recreation. Water Res. 2011, 45, 2020–2028. [CrossRef] [PubMed]
177
and Public Health
Review
Recreational Use of Spa Thermal Waters: Criticisms
and Perspectives for Innovative Treatments
Federica Valeriani, Lory Marika Margarucci and Vincenzo Romano Spica *
Public Health Unit, University of Rome “Foro Italico”, Rome 00135, Italy; [email protected] (F.V.);
[email protected] (L.M.M.)
Abstract: Natural spa springs are diffused all over the world and their use in pools is known since
ancient times. This review underlines the cultural and social spa context focusing on hygiene
issues, public health guidelines and emerging concerns regarding water management in wellness or
recreational settings. The question of the "untouchability" of therapeutic natural waters and their
incompatibility with traditional disinfection processes is addressed considering the demand for
effective treatments that would respect the natural properties. Available strategies and innovative
treatments are reviewed, highlighting potentials and limits for a sustainable management. Alternative
approaches comprise nanotechnologies, photocatalysis systems, advanced filtration. State of the
art and promising perspectives are reported considering the chemical-physical component and the
biological natural complexity of the spa water microbiota.
Keywords: recreational water; spa; thermal water; innovative treatment
1. Introduction
Natural spa springs are used for recreational purposes or wellness applications and are available
globally [1–3]. Especially in the Mediterranean basin, these waters have been exploited and valorized
for health and recreational purposes since ancient times [4–7]. After inheriting the approach to health
and well-being from the Greek culture, the Romans magnified this opportunity of personal and
social care through the realization of the monumental thermae publicae, with major spa buildings that
included areas for baths, gardens, stadiums, gyms, restrooms and spaces for massages or health-related
activities [7]. Over the centuries and different cultures, spas have maintained a significant role for
promoting health in the community. Nowadays, the increase in wellness awareness and fitness
expectations has led to the exploitation of thermal waters and extended spa businesses, based on the
notion of a joint interaction between natural resources and manmade enterprises [8]. Collectively, the
spa economy is estimated at $94 billion, with a consistent growth perspective in the coming decades [9].
Indeed, the global wellness economy had amplified the demand and the offer of products or services
based on mineral waters, sea and hot spring resources [8,9]. Specifically, the spa and recreational
thermal water tourism mainly flows towards Europe, mostly in German-speaking and Mediterranean
countries, but also in North America and Southeast Asia [8–11]. The application of thermal waters in
swimming pools, spa and wellness centers represents a renewed and promising tool for prevention,
rehabilitation, and health promotion, providing possible physical, mental or social benefits to patients
and several groups of people [1,12]. The general context of spa environments can support a holistic
approach to health promotion, also through the exposure to natural open-spaces, the presence of water
itself, the access to physical activities, physiotherapies, and health education opportunities. Even if
additional evidence-based data are needed, several studies have shown the therapeutic role of mineral
elements and other chemical compounds present in thermal waters [13]. The treatments with mineral

IJERPH 2018, 15, 2675
thermal water or mud proved effective in pain relief and function restoration, impacting also on quality
of life: several parameters of clinical interest, and other key issues were reported to play a role in several
rheumatologic diseases e.g., knee and hand osteoarthritis, chronic low back pain, rheumatoid arthritis,
and osteoporosis) compared to baseline and non-mineral similar treatments [14–16]. The thermal
waters and spas have ancient roots in history and still today represent a promising opportunity for
physical and social well-being but require surveillance to assure appropriate hygiene standards to the
final aim of reducing hazards and maximizing benefits.
The water of natural spas should be of satisfactory microbiological quality and must be adequately
managed to control the exposure of bathers and personnel to infectious agents. Indeed, the literature
describes individual cases or outbreaks associated with the use of swimming/spa pools or similar
environments, such as hot springs, hot tubs, whirlpools, natural spas, for recreational, wellness or
therapeutic purposes [17–21]. Knowledge on pool uses and on composition of the water that supplies
the spa is needed for an effective and appropriate management. Indeed, the peculiar and typical
composition of each thermal water represents an interesting richness and a potentially beneficial
property for health, but it also implies additional difficulties in defying the correct management,
treatment and monitoring of that specific water in a defined application, such as aerosol, beverages
lavender or in pool [22,23]. Based on their geological composition, natural waters may be enriched
with several salts and ions, such as sulfur, halogens from group 17 of the periodic table, e.g., chlorine
(Cl), bromine (Br), iodine (I), or alkaline earth metals comprising group 2 of the periodic table,
e.g., magnesium (Mg) or calcium (Ca) [23,24]. Therefore, in natural spa pools, the water should be left
untreated for assuring the specific composition, maintaining the original properties and the potential
health benefits [10,11,24–27]. However, it is well known that pools and spas can present a considerable
source of infection and other threats to human health [22,28,29]. In particular, several bacteria
such as Legionella, Pseudomonas, Mycobacteria, as well as protozoa such as amoebae, algae and other
microorganisms can naturally proliferate in the conditions characteristic of thermal waters and, if not
managed properly, can become a hazard for users [17–21]. This problem represents a dilemma between
treating or not treating natural spa waters and induces several pool managers to add disinfectants
into the natural solutions highly rich in salts, resigning the original water properties in favor of safety,
even if there is a lack of knowledge on the chemical risks related to the use of disinfectants in these
waters. Several alternative strategies have been proposed and the recent progress in nanotechnologies
is contributing to the field, leading to the introduction of innovative water treatment strategies for
thermal waters and spa contexts [30]. The objective of this review is to consider issues related to thermal
spa waters within the field of the recreational uses in pools, showing homologies and differences from
a public health point of view and perspectives for innovative treatments.
2. Spa Trends in the World

The last century saw massive changes and new trends in international health-tourism, where,
alongside the traditional health services, thermal-tourism and wellness-fitness became increasingly
popular [31,32]. The spa industry has grown by 7.7% annually, from $60 billion in 2007 to $94 billion in
2013, representing the fastest growing subsector in health tourism and leisure sector [33,34]. Several
countries reported an increase in spa economy; the spa services in Europe are mostly related to
health and healing while spa tourism in the US is more oriented towards the affirmation of a healthy
lifestyle [8,31,34]. In this area, Europe maintains a clear leadership; however, the Asia-Pacific region,
particularly Thailand, China and Australia, have great potentials and resources for the growth of the
wellness spa tourism market, especially due to the price reasonableness of the exclusive services [34–36].
Exposure to spa waters and related environments is involving a growing number of people all over
the world, posing new question related to safety and public health issues.
The increasing interest in thermal waters and spa resources is also reflected in the scientific
literature. Indeed, research related to “thermal waters” or “medicinal waters” or “spa salus per
aquam” has increased over the last 50 years (Figure 1). The first publication dated 1853 and it already
179
IJERPH 2018, 15, 2675
underlined how thermal water properties cannot be not altered in any way by treatments [37]. Later,
several authors have investigated the application of thermal water medicine and the nature of spa
waters [38,39]. The use of thermal waters for therapeutic purposes has always aroused a continuous
interest and debate all over the world, being dependent on the detailed physicochemical pattern of the
water joined with the specific indication for a treatment in a defined pathological condition [40,41].
Following the number of publications from different regions, it can be noticed how the interest in this
topic is mainly concentrated in Europe (42.3%), Asia (26.3) and Africa (21.7%). Regardless the complex
and heterogeneous debate on evidence-based therapeutic applications, spa waters represent a current
major approach to wellness worldwide. Their frequent use in pools often is a challenge for public
health authorities, both at cultural and technological level.
Figure 1. Number of publication entries in Medline (PubMed trend from 1853 to 2018, last access
10/2018). Publication entries were searched with the query: “thermal waters” OR “medicinal waters”
OR “spa salus per aquam”.
3. The Question about “Identity” and “Untouchability” of Spa and Medicinal Natural Waters
The growing popularity of swimming and other water activities for sports, fitness, therapy,
wellness or relaxation and amusement has increased the diffusion of swimming pools as well as
specific-use pools, such as spa pools, hot tubs, whirlpool bath, and natural spa pools [22,25,42].
The term “spa” is an acronym for salus per aquam, meaning health through water [5]. The common
terms associated with spa pools include hot tubs, whirlpool bath, and natural spa pools; all these types
of pools imply different water management and are designed for different wellness, therapeutic or
recreational purposes. Most spa pools (>98%) resigned their natural properties, being disinfected by
the addition of traditional chemicals with high oxidation potential, such as chlorine [25]. The treatment
choice as well as the risk assessment process must consider the nature of the water that supplies
the pool plant [25]. In natural spa pools, indeed, the water should remain untreated because the
claimed beneficial effects are supposed to derive from their unique chemical-physical properties
(Table 1) [22,25,43].
Moreover, in order to characterize these spring waters, also the biological component plays
a role. Spa waters, indeed, contain a metabolically versatile microflora that is characterized by
specialized bacteria belonging to that ecological niche, within a defined range of chemical and physical
parameters [43,44]. The biotic and abiotic components of these ecological niches have been deeply
studied, representing a mine for the identification of unknown and/or extremophile species within the
complex microbial community [44–47]. Today, it is possible to characterize this microbial community
by a massive-sequencing approach, describing a spa water microbiota and defining a “microbial
signature” that can be sampled and typed from the original spring source up to the pool facility
180
IJERPH 2018, 15, 2675
and final user’s applications [48–50]. This approach was already applied in different spa pools and
can provide further perspectives for the characterization of spa waters in recreational or wellness
uses, adding a candidate new biological parameter to the traditional chemical-physical ones: the
microbiota as a novel marker for public health [49–52]. Interestingly, it is now possible to associate
water properties to its microflora component, e.g., presence of H2 S and communities of sulphate
reducing bacteria, temperature and thermophiles, iron (Fe) and iron (III)-reducing bacteria, unravelling
biochemical pathways and considering water as an active biological fluid [24,53,54]. The microbiota
itself and the analysis of microflora biodiversity by Next Generation Sequencing (NGS), can support
the proposal of a modern classification of waters and their properties, opening up new perspectives
for the development of appropriate strategies for managing hygiene by respecting chemical, physical
and also microbiological natural components [10,50,53–56]. This approach is very promising but still
limited not just by the need of wet-laboratory equipment, protocols or qualified personnel because
several external services are available and more and more affordable. Most of all the bottleneck is
determined by the requirement of dedicated bioinformatic tools, such as database driven software
to analyze the data obtained from massive sequencing and transfer information in an appropriate
form to address public health questions. In order to collect and map information from different
springs, a dedicated database for spa microbiota was developed and made accessible online to the
collaborative research network at www.mfATLAS.it [50]. This tool was designed within a project
focused on studying the biological component in spa water springs and pools (Figure 2). It is open
to collaborations to analyze and host data from additional sampling points all over the world and
the relative metadata, further expanding the atlas-map and database. The availability of this massive
sequencing approach and bioinformatic support can improve knowledge on the natural microflora
inhabiting thermal spring waters, their geographical distribution, providing also information for the
identification of new species and their potential role in the field of wellness, therapeutics in that
spa facility, or for other biotechnology applications [11–15,55]. Based on the available version of the
mfAtlas database and in according with the observations from other studies and the Earth Microbiome
Project, the percentage of unknown species in is still high, covering about 10–70% of the spa water
microbiota component [50,56]. Previously, the access to unravelling these complex environmental
communities was strongly restricted by the available culture-based methods or classical sequencing of
libraries after cloning steps, rather than the massive sequencing approach that today is rapidly and
successfully diffusing in different fields [56–60]. NGS revealed as a promising strategy not only in
characterizing the natural microflora of spa waters, but also the presence of microbiological markers,
pathogens or the effectiveness of disinfection and other water treatments [56–59]. This novel approach
to solve hygiene questions is based on the genetic analysis of water biodiversity, starting from the DNA
of its microflora (mfDNA) [52]. It is opening promising perspectives for understanding the beneficial
potentials of spa waters, their fingerprint and their “untouchability” based on the respect of chemical,
physical, and also biological components.
Table 1. Classification of natural mineral waters based on fixed residue at 180 ◦ C and chemical
composition, according the 2009/54/EC Directive [43].
Classification of Mineral Waters

Classification according to fixed residue at 180◦ Classification according to chemical composition
Very low mineral content waters (Fixed residue <50 mg/L) Bicarbonate waters (>600 mg/L)
Low mineral content waters (Fixed residue 50–500 mg/L) Calcic waters (>150 mg/L)
Medium mineral content waters (Fixed residue 500–1500 mg/L) Chloride waters (>200 mg/L)
Rich mineral content water (Fixed residue <1500 mg/L) Ferrous waters (>1 mg/L)
Fluorurate waters (>1 mg/L)
Magnesiac waters (>50 mg/L)
Sulphated waters (>200 mg/L)
Sodium-Rich waters (>200 mg/L)
181
IJERPH 2018, 15, 2675
Figure 2. The mfAtlas database: presently, the database is accessible to the research network at
www.mfATLAS.it. The database is designed to be further extended to harbour information such as
water management, environmental and epidemiological data, international legislations.
Treatments for Spa and Medicinal Natural Waters: Limits and Perspectives
Several alternative water treatments were considered to assure the original properties and
composition of the water, reducing the adverse effects on bathers and environments, to the final
aim of offering sustainable solutions for spa waters in pools [61–63]. This challenging objective shares
principles and problems also with the management of other kinds of waters, so that a success in this
field can impact in other areas of water hygiene and conversely.
Otherwise, traditional disinfection approaches, e.g., chlorine, not only affect the harmful
microorganisms present in the pool but also destroy the beneficial chemical-physical composition
and the natural microflora, adulterating the therapeutic proprieties of these waters. The organic
and inorganic compounds present in these waters can react with chemical substances used for
disinfection, such as chloride/bromide-based chemicals, generating potentially toxic secondary
products [24,63–66]. Nonetheless, the equilibrium between maintaining the natural proprieties of the
thermal waters in pools and minimizing the microbial risk for people is not easy and can be achieved
by considering several candidate approaches [22,54,66]. Firstly, a possible solution can be based on
a personalization of the hygiene approach. The waters for natural spas, indeed, should be carefully
tested for satisfactory microbiological quality before designing and constructing the water-plant,
adopting a kind of individualized strategy based on the specific composition and destination use of
that water in that plant, following a dedicated water safety plan [10,22,25,66–69]. In vitro models and
protocols can prove useful in comparing materials or treatment methods [70]. The main task of water
management is to achieve a satisfactory control on exposure of the bathers to infectious agents or
other health risks, realizing an effective prevention of disease and accidents [22]. The high salinity
or high temperature of spa waters represents an additional challenge for the pipeline plant and its
maintenance, e.g., due to corrosion and concretion. Managing natural spa pools should follow the
guidelines for traditional pools, but additional concerns have to be highlighted, so that alternative
strategies may also be considered if proven to be effective and acceptable [25,47,68–70].
Even if spa waters should not be treated in order to try to maintain their original properties,
however, sometimes they are used as a common source of water for just filling a pool for recreational
purposes. Regarding the use of traditional pool disinfection of spa waters e.g., by chlorination, several
inconveniences can occur in addition to the generation of known but also of unexpected Disinfection
by-products (DBP) [65,66,69]. DBP are derived after the interaction with organic materials following
an already well-known process, but the scenario is complicated by the presence of other chemicals
naturally presents in the spa water or introduced by the bathers [22,65,71]. Oxidation in presence
of an already high salinity can favor precipitates and concretions or induce unexpected toxic DBP
with undesired -and often unpredictable-effects on bathers [71]. Moreover, the high temperature
and the intense aeration due to frequent bubbling in this kind of pools, considerably can increase
the evaporation of the active chemicals. Therefore, the disinfectant active doses are often poorly
182
IJERPH 2018, 15, 2675
quantified, and “classic” chemicals generally may show a very irregular efficiency in spa pools.
The type, form and use of each disinfectant need to be chosen with respect to the specific requirements
of the pool [68,71–74]. Pool size itself may represent a critical parameter [22,25]. The choice of the
disinfection strategy must be made after consideration of the efficacy of the specific product under
the specific circumstances of use and the feasibility of a monitoring of the disinfectant levels in that
pool [22,25,75–77]. Table 2 lists the several types of disinfecting agents and their advantages and
limits of use in swimming pools as well as their applications in spa pools. Chlorine is inexpensive
and relatively convenient to produce, store, transport, and apply. It provides rapid and long-lasting
bactericidal effects but is limited mainly because of the formation of potentially toxic DBPs, such
as trihalomethanes, halomethanes (THMs), haloacetic acids, halonitromethanes, haloacetonitriles,
chloramines, and chlorophenols [22,25,65,72–80]. For example, the levels of potentially toxic DBPs
tend to be higher in hot tubs, due to recirculation and smaller volumes but also because acceptable
thresholds tend to be more elevated than in swimming pools [73–75]. However, when treating natural
spring waters with chemical disinfection, whatever the final use, their natural characteristics are
modified [22,25]. In order to avoid adulteration of the natural properties of spa pools, a commonly
adopted alternative is based on dilution of pollutants by the frequent replacement of pool water. This
may be feasible for small pools and when a large reservoir is available, but it can become unsustainable
on the long term, due to the risk of depleting the aquifer.
Ozonation or ultraviolet (UV) irradiation represent additional solutions that are known and
already well engineered and tested. Even if effective they can be demanding to maintain during time
and both methods have no residual disinfection activity in the pool water [80–89]. Recently, advanced
oxidation processes (AOPs) have shown a demonstrated efficacy in the treatment of organic pollutants
in aquatic environments, but AOP technologies involve the generation of nonspecific hydroxyl radicals
and the production of activated compounds involved in THM formation in the post-chlorination
step, thus increasing the potential for DBPs formation [61,76,77,81]. UV irradiation is effective for
controlling resistant microorganisms, such as Cryptosporidium parvum and Giardia lamblia [83–85].
This physical treatment seems cost-competitive in terms of improving the quality of swimming pool
water; however, it has several limitations, including the life spam of the lamp and the potential
formation of nitrogenous-based DBPs [61,82–91]. Bromine-based disinfectants may provide rapid
and long-lasting disinfection effects, but they are more difficult to manage [61,92]. Several studies
raised the problem of DBPs and reported eye or skin irritation due to bromine-based disinfectants [92].
The use of bromine-based disinfectants is generally not very feasible for outdoor pools and spas also
because the bromine residue can get rapidly depleted in sunlight [25,92]. Copper/silver ionization was
also proposed based on experimental observations on the effectiveness of silver nanoparticles (NPs)
on harmful microorganisms, but several limits were reported including toxicity [61,92]. Hydrogen
peroxide is a broad-spectrum disinfectant usually supplied as a solution to be dosed or added to spa
pools; it is generally prepared by stabilizing ion-based chemistry [93]. The limitation in using hydrogen
peroxide is the requirement of high concentrations depending on the condition of the facility; therefore,
hydrogen peroxide disinfection was suggested only for small pools [25].
The World Water Development Report 2018 has outlined innovative natural treatments [94].
In this document nature-based solutions (NBS) are defined as a potential contribution to solving or
overcoming the major water management problems or technical challenges [22,25,95–98]. A revolution
in water treatment technologies is occurring and novel treatments based on physical methods are now
considered and studied [96,97]. Membrane filtration has largely replaced granular filtration, and UV
irradiation is enabling reduction in the use of or even elimination of classic disinfection chemicals,
such as chlorine and its derivatives [97,99,100]. Ultrafiltration membranes are widely used in water
treatment because of their favourable characteristics, such as easy modularization and improvement
in water quality. A main limitation is membrane fouling that induces a reduction of membrane flux,
an increase in energy consumption and in the consequent costs for water treatment [98].
183
Table 2. Several types of antimicrobial agents and their candidate applications in SPA pools: main advantages and limits for swimming pool uses.
Disinfection Solution Advantages Limits SPA Pool Applications References

In hot tubs, acceptable free chlorine levels tend to
Inexpensive and relatively convenient
The formation of potentially toxic DBPs, such as be higher than in swimming pools.
to produce, store, transport and use.
Chlorine-based THMs, HAAs, HANs, THAs and CAMs. Moreover, due the chemical characteristics of
Provides rapid and long-lasting [22,25,65,72–80]
disinfectant Presence of chlorine-resistant microorganisms such thermal water, the reaction between chemical
disinfection effects.
IJERPH 2018, 15, 2675
as Cryptosporidium parvum and Giardia lamblia. compound and disinfection agents can lead the
Residual disinfectant activity in pool.
increase the potentially toxic DBPs.
Toxic and explosive; heavier than air.
AOPs have recently shown successes in the
Highly effective, no smell. Risks and adverse health effects for the operator.
treatment of organic pollutants in aquatic
Can reduced the formation of Lack of residual disinfection proprieties; (usually
Ozone environments, involving the generation of [61,81]
potentially toxic disinfection joined with chlorine).
non-specific OH radicals.
by-products (DBPs). Production of activated compounds suitable for
A de-ozonization step is needed.
THMs formation in the post-chlorination step.
Physical treatment without adding
UV radiation can be proposed to reduce the risk
chemicals to the water.
of infection by dermatophytes eventually present
Ultraviolet (UV) Effective for the control of resistant The formation of nitrogenous-based DBPs (HANs)
in swimming pools that use thermal water. [61,81–90]
irradiation microorganisms including protozoa Lack of residual disinfection proprieties.
Cost-competitive with chlorine to improve the
such as Cryptosporidium parvum and
quality of swimming pool water.
Giardia lamblia.
It is difficult to dissolve and must be inserted into
Inexpensive and relatively convenient The use of bromine-based disinfectants is
the pool through an automatic feeder.
Bromine-based to apply. generally not practical for outdoor pools and
184
DBP [25,61,92]
disinfectant Provides rapid and long-lasting spas because the bromine residual is depleted
There are reports that is associated with eye and skin
disinfection effects. rapidly in sunlight.
irritation.
Copper/silver ionization was Silver is a broad-spectrum disinfectant usually
proposed for treatment of swimming Low effectiveness supplied as a solution to be dosed or added to
Stabilised silver/copper pool water: protocols and devices are Limited information on toxicity of ion forms and the spa-pool system. [25,61,92]
available. interaction with other chemicals. Higher concentrations may be required
No pH adjustment is required. depending on the condition of the facility.
Hydrogen peroxide can be used with silver and
With hydrogen peroxide the by-products are not
Effective copper ions (low levels of the silver and copper):
Hydrogen peroxide problematic but it can generate toxic radical [25,93]
Low pollution on water. proper consideration to replacement of water for
compounds.
preventing excessive build-up of the ions.
Note: Disinfection byproducts (DBPs); Hypochlorous acid (HOCl); Trihalomethanes (THMs); Haloacetonitriles (HANs); Hydroxyl radical (OH); Advanced oxidation processes (AOPs).
IJERPH 2018, 15, 2675
Rapid advances in nanotechnologies have encouraged the development of industrial applications

of manufactured nanoparticles (NPs) in a wide range of commercial products, such as drugs, paints,
electronics, foods, or cosmetics [101]. However, laws or guidelines regarding the applications of
NPs still are lacking and additional research on safety performance standards is required [101,102].
Table 3 summarizes the main innovative strategies and the possible applications of NPs in spa pools.
For example, nanoscale chitosan and its derivatives were proposed also in water treatment, because
of their antimicrobial effects on bacteria, viruses, fungi, and bacteriophages, through damaging cell
membrane or chelating trace metals [101–106]. Chitosan is currently used in personal care products and
biomedical products, as microbicide in agriculture and food wraps, and as a flocculant in water and
wastewater treatments [102–106]. It is a promising compound for low-cost and low-tech disinfection
applications and it was suggested for applications in developing countries, but it has several limitations
including its deterioration under different conditions [107]. A promising scenario is coming from
the use of light of different wavelength through photocatalysis processes [108]. In principle, this
approach can allow water treatment just by light and air, through the production of free radicals.
Metal oxide semiconductor NPs with a wide band gap are the basic materials used in heterogeneous
photocatalysis method. They can accelerate the degradation of pollutants under solar illumination [108].
Titanium dioxide (TiO2 ) and zinc oxide (ZnO) NPs are among the most extensively used metal
NPs [107,108]. TiO2 is already present in different products including foods additives and cosmetics,
paints and also coating for pools [70,109]. Silver and copper metals have been also considered for their
antibacterial properties within nanotechnological applications. Other kind of nanoparticles, Silver
NPs (AgNPs), have been developed since late 1800s and have been registered with the Environmental
Protection Agency for use as swimming pool algaecides since 1954 and as drinking water filters since
1970s [108–111].
AgNPs exhibit a strong and broad-spectrum antimicrobial activity and showed no harmful effects
on humans [112]. They are currently being used in the development of point-of-use water disinfection
systems and antibiofouling surfaces [113]. An innovative scenario is offered by the availability of
Carbon nanotubes (CNTs), that have been reported to induce DNA damages and cytotoxic effects
in prokaryotic cells, consequently disrupting the microbial diversity and community structure [114].
Several possible toxicological mechanisms of CNTs on microorganisms have been proposed, among
which the disruption of the cell membrane integrity, that is considered a key mechanism in this
antimicrobial process [114–116]. In the 21st century, advances made in the synthesis of carbon-based
nanomaterials have resulted in the development of graphene–carbon nanotubes [117]. Notably, the
three-dimensional graphene and graphene oxide-based nanostructures exhibit a large surface area and
sorption sites that provide a high adsorption capacity to efficiently extract pollutants and inactivate
viruses or bacteria in water [118,119].
In conclusion, advancements in the field are in continuous progress and several alternatives
have been proposed for water treatments, opening promising perspectives also for thermal spa
pools. Due to the heterogeneity of thermal spa waters and their peculiar requirements for the
therapeutic use in spa facilities, presently it seems still difficult to find a single strategy for all different
situations. Therefore, more than a unique ideal solution, the optimal strategy should be searched in the
combination of different methods, following an individualized approach based on water properties,
plant characteristics, destination use of the spa pool.
185
Table 3. Current and potential applications of antimicrobial nanomaterials.
Nature of Disinfection Type

Nanomaterials CAS NUMBER Antimicrobial Mechanism Current Applications Potential Future Applications in SPA Pools References
Physical Chemical
Portable water filters, clothing,
An alternative to traditional chemical
Silver nanoparticles AgNPs can disrupt the outer medical devices, coatings for
(AgNPs)
7440-22-4 5 membrane of target cells. washing machines, refrigerators,
disinfectants that are prone to generate harmful [101,108–111]
disinfection by-products
IJERPH 2018, 15, 2675
and food containers

Membrane damage, chelation of Personal care products,
They are promising for low-cost and low-tech
trace metals. Nano-scale chitosan microbicide in agriculture and
disinfection applications.
Chitosan 9012-76-4 5 and derivatives exhibit biomedical products, food wraps,
In water filtration, chitosan combined with sand
[101–106]
antimicrobial effects towards biomedical, flocculants in water
filtration removes up to 99% of turbidity.
bacteria, viruses, fungi. and wastewater treatment
Graphene oxide (GO) and
silver-graphene oxide (Ag-GO)
are used in various fields, such
DNA damages and cytotoxic In aquatic ecosystems, the stability of
as biotechnology and
effects towards prokaryotic cells nanomaterials is affected by the water
environmental engineering, due
Graphene oxide 1034343-98-0 5 5 and detrimentally change the
to their unique material
chemistry parameters of the receiving aquatic [114–119]
microbial diversity and environments such as ionic strength, natural
properties, including
community structures organic matters and pH
hydrophilicity, high surface area,
mechanical strength, and
antibacterial activity
186
H2 S killed microorganisms
through inducing oxidative Restore the normal bacteriostatic nature of the
H2 S 7783-06-4 5 stress by inhibiting antioxidant
None
thermal water
[24]
enzymes
The applicability is in evaluation. The presence
Production of Reactive Oxygen Air purifiers, water treatment
of some inorganic ions can be problem, because
Nano TiO2 13463-67-7 5 5 Species (ROS), cell membrane systems for organic contaminant
reduce the performance of TiO2 in water
[108,109]
and cell wall damage degradation.
treatment.
Ultrafiltration allowed the
Ultrafiltration can be selected as an alternative
removal of suspended matter, as
Ultrafiltration - 5 well as a part of the organic
Water treatment, swimming pool treatment process because of its ability to [97–100]
remove bacteria and viruses.
matter
Intracellular accumulation of
Antibacterial creams, lotions and
nanoparticles, cell membrane
ZnO 1314-13-2 5 5 damage, H2 O2 production,
ointment, deodorant, Surface coating [108,109]
self-cleaning glass and ceramics
release of Zn2+ ions
IJERPH 2018, 15, 2675
4. Guidelines and Regulations on Thermal Spa Water Pools

The global scenario of the international regulations in the spa field is very heterogeneous and
reflects the socio-economics and culture from the different countries (Table 4). In the USA, local
and state regulations consider the routine inspection of aquatic environments for preventing risks
and accidents [120]. Deaths due to pool entrapment have led to the enactment of the Virginia
Graeme Baker Pool and Spa Safety Act [121,122]. The act outlines provisions to minimize the risk
of entrapment, including mandatory requirements with respect to vacuum covers, pool barriers,
and main drain requirements. For hygienic aspects, U.S. states have separate law or guidelines
(e.g., Alabama and Kansas), although the point of reference remains the guidelines issued by the
World Health Organization (WHO) [22,122]. In fact, since 1994, WHO had been promoting the
development of guidelines for the use of recreational waters; these guidelines have now evolved
to safety guidelines for recreational aquatic environments [22]. In Canada, British Columbia health
authorities approve and inspect pools, hot tubs and other facilities to ensure safety in construction and
operation, according to the Pool Regulation under the Public Health Act [123]. Hygienic-sanitary safety
of thermal plants is addressed in the 2007 legislation, which has established the quality criteria for
waters, including spas, and obligations for pool managers, following the Health Canada’s Guidelines
for Canadian Recreational Water Quality. These guidelines address potential health hazards, such
as infections transmitted by disease-causing microorganisms, as well as aesthetics and nuisance
conditions [124]. The Pool Standards have set specific technical thresholds pertaining to water quality
and facility operations requirements under the Public Swimming Pools Regulation as well as other
requirements, including: operator and user education, recirculation systems, water chemistry and
microbiology indicators, water quality monitoring, anti-entrapment policies and other plans related
to pool safety [125]. The standards also include a protocol for the management of contaminated
public swimming pool water and the calculations for maximum bather load and flow rates through
anti-entrapment suction outlets. These standards were developed in consultation with the pool
industry, pool operators, and public health officials and recently revised in 2018 [125]. Australian
swimming pool regulations was established in 1990 and revised in 1992, focusing also on spa pools,
swimming pools and similar environments but does not include baths [126,127]. In the recent years,
some regulations and technical standards have been introduced with regard to spas [128,129]. Europe
is a rich continent in terms of natural hot springs, harbouring over 5000 springs and facilities, very
popular since ancient times [8,56]. However, the European legislative situation in this matter seems very
fragmented and inconsistent. A comprehensive community directive is still missing. The European
reference 76/160/EEC concerning bathing water, as amended by 2006/7/EC, is not applicable to the
spa and swimming pool waters or to the confined waters subjected to treatment or used for therapeutic
purposes [130,131]. Austria had already issued regulations concerning swimming pools with the goal
of preventing the spread of waterborne diseases [132]. Subsequent changes extended the scope of
the legislation, but only in 2012 a new law has been passed regarding the technical and operational
requirements for the water quality of traditional pools and bathing water, whirlpools, small natural
pools, and ancillary facilities used for recreational or therapeutic activities [133–137]. The government
of the Brussels-Capital region and the Walloon region in Belgium have issued specific ordinances
on sectoral conditions related to swimming pools, saunas, and general artificial reservoirs designed
for therapeutic, recreational, or sporting activities, but not domestic facilities [138–140]. In France,
the current legislative framework related to swimming pools is based on the Public Health Code
about the care and rehabilitation in thermal pools with an independent section concerning traditional
pools [141,142]. Furthermore, in 2010, the Agency for Environmental Health and Safety (ANSES)
published a document on health risks in swimming pools, indicating the thermal pools as “atypical”
pools [143]. In 2013, ANSES published the part II of this document, focusing on hot tubs (“bains à
remous”) [144]. In England, Health and Safety Executive is the authority overlooking the pools of
local authorities and schools. This institute together with the Health Protection Agency published
the guidelines concerning the control of the risk of infections in thermal pools [145]. These protocols
187
IJERPH 2018, 15, 2675
have been designed to improve the understanding about microbiological risks associated with the use
of thermal baths and to provide advice on risk management [25,145]. Similarly, in Ireland, a country
with a large number of geothermal pools, every swimming pool is a public spa that is managed in
compliance with the Safety, Health and Welfare at Work Act of 2005 as well as a specific set of guidelines
published to provide administrators with criteria and detailed information for management [146,147].
In Germany, the technical provisions for the management of swimming pools are summarized
in the DIN 19643, which guarantees hygiene safety in swimming pools, saunas, whirlpools, and
spas [148,149]. Periodically, the Federal Environment Agency publishes recommendations for health
managers and authorities [150]. Recently, new developments in swimming pool hygiene required a
revision of the standard series DIN 19643, with the introduction of new treatment processes based on
ultrafiltration [151]. Czech Republic has a legislation concerning the spa sector, but it is not extended
to the recreational applications of these waters or to the sanitary-hygiene related issues [152]. More
specific standards concerning the management of water for therapeutic uses and thermal spas, are also
available [153,154]. Iindependent standards are available in Portugal for specific regulations in spa
facilities, focusing on licenses, organization, management, and control and attention is dedicated to the
quality of public pools. However, Portugal uses a directive issued by the Council National Quality that
does not apply to thermal pools for therapeutic use, indicating to other specific regulations [155,156].
In Slovakia, a country with a great spa tradition, water quality used for swimming is managed by the
Public Health Authority and 36 regional health authorities, which overlook the pools supplied with
thermal water [157]. In Spain, pool regulations are available since 1987 and the health authorities refer
to the WHO guidelines [158–160]. The Ministry of Health, Social Services and Equality of Spain has
developed the Real Decreto project to establish the water quality criteria for public pools, spas, private
pools, and water parks but excludes natural pools and thermal waters used for medical therapeutic
purposes [161]. An innovative appearance of this law is the consideration of water safety plans.
Finland has included recommendations for public baths, spas, and swimming pools in the application
law for the European directive n.2006/7/CE [162,163]. Other countries, namely Cyprus, Bulgaria, and
Norway, have considered a different approach, not including this 2006 Directive along the specific
rules for pools [164–166]. In Italy, the recreational use of spas has extensively increased in the last few
years, but no specific guidelines have been established, yet. In fact, more recently, the Italian legislation
started referring to the law of “the reorganization of the thermal system” established in 2000 for
thermal waters [167]. This law reports the definition of thermal waters and the provisions concerning
bottling and permitted uses but does not deal with the hygiene aspects related to the recreational use.
The current legislation concerning swimming pools is the January 16th Agreement 2003 between the
Minister of Health, the Regions, and the Autonomous Provinces of Trento and Bolzano [168]. This
document is under revision and a public consultation was performed in 2016 [169]. The Agreement
specifies that the swimming facilities can be supplied with different types of water, including thermal
waters, but postponing the discipline of the latter to specific regional measures; moreover, additional
guidelines for the spa hygiene are available for preventing Legionnaire’s. This document has a chapter
fully dedicated to swimming pool measures that underlines the need for adequate design of pool spa
facilities because no specific treatment of these waters is allowed [170]. In summary, the complexity of
the argument in the different countries does not seem to have fostered the development of unequivocal
rules and shared strategies. Future joined projects and consensus documents would be welcome and
useful on a local and international scale.
188
Table 4. International guidelines, regulation and recommendation regarding recreational water environments.
Country Law References

New South Wales Consolidated Acts. Swimming Pools Act 1990 n. 31.
Australia New South Wales Consolidated Acts. Swimming Pools Act 1992 n. 49. [126–129]
Standard. Spa Pools Part 1: Public spas. 2007
IJERPH 2018, 15, 2675
Pool Water Quality and Operational Guidelines.

Bundesgesetzblatt für die Republik Österreich 1978; 167:3053–63.
Mitteilungen der Österreichischen Sanitätsverwaltung, 1992;93(11):358.
Austria [132–136]
Bundesgesetzblatt für die Republik Österreich. 1996; 212:4617-24.
Mitteilungen der Österreichischen Sanitätsverwaltung. 1997;98(5):228–32.
Gesamte Rechtsvorschrift für Bäderhygienegesetz, Fassung vom 28.10.2012.
Belgio. Arrêté du Gouvernement wallon portant conditions sectorielles relatives aux bassins de natation.
Belgium [138–140]
Belgio. Arrêté du Gouvernement de la Région de Bruxelles-Capitale fixant des conditions d'exploitation pour les bassins de natation.
Belgio. Arrete´ du Gouvernement de la Region de Bruxelles-Capitale fixant la liste des installations de classe IB, II et III en execution de l’article 4 de
l’ordonnance du 5 juin 1997 relative aux permis d’environnement.
Bulgary D’rzaven vestnik 1994; 65:1–14. [165]
Règlement de sécurité, Fédération de natation du Québec (natation en bassin)
Canada [123–125]
189
Guidelines for Canadian Recreational Water Quality
Alberta Health Pool Standards
Ciprium Ciprium Government Law N. 55(I)/92 [164]
Decree of Ministry of Health No.423/2001—On Spas and Sources
Czech Republic [152–154]
Decree of Ministry of Health No.252/2004—Requirements on Cold and Hot Water in Health Care and Accommodation Facilities
Decree of Ministry of Health No.135/2004—Requirements on Swimming Pools, Saunas and Outdoor Playgrouds.
Finlands Författningssamling 2008/70.
Finland [162,163]
Finlands Författningssamling 2014/47
Code de la santé publique, 2010. Section V: Surveillance des établissements thermaux.
France Code de la santé publique, 2010. Section I: Normes d'hygiène et de sécurité applicables aux piscines et baignades aménagées [141–144]
Afsset Evaluation des risques sanitaires liés aux piscines Partie I: piscines réglementées. Saisine Afsset «2006/11». Rapport final. 2010
Anses. Évaluation des risques sanitaires liés aux piscines Partie II: bains à remous. 10.13140/RG.2.1.2182.7043.
Management of Spa Pools: Controlling the Risk of Infection. Health Protection Agency. March 2006.
England [25,145]
Health and Safety Executive (HSE). The control of Legionella and other infectious agents in spa-pool systems.
Table 4. Cont.
Country Law References

DIN 19643. Aufbereitung von Schwimm- und Badebeckenwasser–Teil 1: Allgemeine Anforderungen.Beuth,Berlin
Germany Hygienische Anforderungen an Kleinbadeteiche. Empfehlung des Umweltbundesamtes. Bundesgesundhbl [148–151]
Bundesgesundheitsbl-Gesundheitsforsch-Gesundheitsschutz
IJERPH 2018, 15, 2675
DIN 19643. Aufbereitung von Schwimm- und Badebeckenwasser—Teil 4: Verfahrenskombinationen mit Ultrafiltration
Law of 24 October 2000, n. 323. Reorganization of the thermal sector. Official Gazette November 8, 2000, n. 261.
Italy [167–170]
Agreement between the Minister of Health, the Regions and the Autonomous Provinces of Trento and Bolzano G.U. March 3, 2003: 45, n. 51.
Guidelines with indications on legionellosis for managers of tourist accommodation and thermal facilities G.U. n 28 Febrary 5, 2005
Safety, Health and Welfare at Work Act”, 2005. Health and Safety Authority
Ireland [146,147]
Swimming Pool Safety Guidelines. Irish Water Safety, ILAM and Swim Ireland. 2010.
Norway Norsk Lovtidend, 1 sezione. 1996;11:767–73. [166]
Ministério da saúde Decreto-lei n. 142. 11 giugno 2004
Portugal [155,156]
Directiva Conselho Nacional da Qualidade "A qualidade nas piscinas de uso público". n.º 23, 1993.
Slovakia Zbierka zàkonov Slovenskej Republiky 1994;77:1350-1370. [157]
Boletìn Oficial del Ministerio de Sanidad y Consumo 1987;19:1147-52.
Spain Boletìn Oficial del Ministerio de Sanidad y Consumo 1998, 80. por el que se regulan las condiciones higiénico–sanitarias de piscinas de uso colectivo. [158–161]
190
Boletin Oficial orden 1319/2006
Real Decreto 742/2013
CDC's Model Aquatic Health Code
USA [120,122]
Virginia Graeme baker Pool and Spa Safety Act
Dedicated law and guidelines for U.S. STATES
IJERPH 2018, 15, 2675
5. Conclusions
Hot spring waters represent a unique natural fluid that humans have used since ancient times
for health and recreational purposes. Spa facilities are present all over the world denoting a relevant
resource for business that involves a large and growing number of users. The safeguard of the natural
composition of spa waters clashes with the need of appropriate treatments in pools. Innovative
strategies have been proposed, but further studies and validations are required. In addition to
traditional chemical-physical parameters, the possibility to characterize the biological component is
opening new perspectives for the classification and fingerprinting of spa waters through mfDNA
(microflora DNA) analysis and the definition of spa microbiota patterns. Recent advancements in
massive sequencing and bioinformatics are supporting this process, providing new tools for hygiene
and knowledge on properties of spa water. The heterogeneity of spa waters and their uses may suggest
an individualized approach to design and carry on a sustainable management through dedicated
technical solutions and water safety plans. Public health regulations for the use of spa waters in pools
are mainly lacking and a consensus at international level would be needed and welcome for providing
agreements and shared guidelines.
Author Contributions: V.R.S.: conception, design and supervision of the study; F.V., L.M.M.: selection and
analysis of the articles, collection of data of interest in a structured form; F.V., V.R.S.: Writing-Original Draft
Preparation. All authors approved the final version of the manuscript.
Funding: This research was supported by Fondazione per la Ricerca Scientifica Termale (FORST) grants, grant
number 1121.
References
1. Giampaoli, S.; Romano Spica, V. Health and safety in recreational waters. Bull World Health Organ. 2014, 92,
79. [CrossRef] [PubMed]
2. Van Tubergen, A.; van der Linden, S. A Brief History of Spa Therapy. Ann. Rheum. Dis. 2002, 61, 273–275.
[CrossRef] [PubMed]
3. Routh, H.B.; Bhowmik, K.R.; Parish, L.C.; Witkowski, J.A. Balneology, mineral water, and spas in historical
perspective. Clin. Dermatol. 1996, 14, 551–554. [CrossRef]
4. Frosh, W.A. “Taking the waters”—Springs, wells and spas. FASEB J. 2007, 21, 1948–1950. [CrossRef] [PubMed]
5. Frost, G.J. The spa as a model of an optimal healing environment. J. Altern. Complement. Med. 2004, 10, 85–92.
[CrossRef]
6. Croutier, A.L. Taking the Waters: Spirit, Art, Sensuality, 1st ed.; Abbeville Press: NewYork, NY, USA, 1992.
7. Jackson, R. Waters and spas in the classical world. Med. History 1990, 34, 1–13. [CrossRef]
8. Global Wellness Institute. Global Spa & Wellness Economy Monitor Global Wellness Institute. 2014. Available
online: www.gsws.org (accessed on 29 May 2018).
9. Mavridou, A.; Pappa, O.; Papatzitze, O.; Blougoura, A.; Drossos, P. An overview of pool and spa regulations
[PubMed]
10. Giampaoli, S.; Valeriani, F.; Romano Spica, V. Thermal water for recreational use: Overview of international
standards. Igiene e Sanita Pubblica 2012, 68, 863–873. [PubMed]
11. Özkuk, K.; Uysal, B.; Ateş, Z.; Ökmen, B.M.; Sezer, R.; Dilek, G. The effects of inpatient versus outpatient spa
therapy on pain, anxiety, and quality of life in elderly patients with generalized osteoarthritis: A pilot study.
Int. J. Biometeorol. 2018, 62, 1823–1832. [CrossRef] [PubMed]
12. Morer, C.; Roques, C.F.; Françon, A.; Forestier, R.; Maraver, F. The role of mineral elements and other chemical
compounds used in balneology: Data from double-blind randomized clinical trials. Int. J. Biometeorol. 2017,
13. Harzy, C.; Ghani, N.; Akasbi, N.; Bono, W.; Nejjari, C. Short- and long-term therapeutic effects of thermal
mineral waters in knee osteoarthritis: A systematic review of randomized controlled trials. Clin. Rheumatol.
2008, 28, 501–507. [CrossRef] [PubMed]
191
IJERPH 2018, 15, 2675
14. Güleç, A.T. Natural Thermal Spa Water Versus Hyperthermic Tap Water for Treatment of Recalcitrant Hand
Warts in Organ Transplant Recipients: A Patient-Blinded, Comparative Preliminary Study. Exp. Clin. Transp.
2018, 16, 189–193.
15. Matsumoto, S. Evaluation of the Role of Balneotherapy in Rehabilitation Medicine. J. Nippon Med. Sch. 2018,
16. Baron, P.A.; Willeke, K. Respirable droplets from whirlpools: Measurements of size distribution and
estimation of disease potential. Environ. Res. 1986, 39, 8–18. [CrossRef]
17. Insler, M.S.; Gore, H. Pseudomonas Keratitis and Folliculitis from Whirlpool Exposure. Am. J. Ophthalmol.
1986, 101, 41–43. [CrossRef]
18. Briancesco, R.; Meloni, P.; Semproni, M.; Bonadonna, L. Non-tuberculous mycobacteria, amoebae and
bacterial indicators in swimming pool and spa. Microchem. J. 2014, 113, 48–52. [CrossRef]
19. Jernigan, D.B.; Hofmann, J.; Cetron, M.S.; Nuorti, J.P.; Fields, B.S.; Benson, R.F.; Breiman, R.F.; Lipman, H.B.;
Carter, R.J.; Genese, C.A.; et al. Outbreak of Legionnaires’ disease among cruise ship passengers exposed to
a contaminated whirlpool spa. Lancet 1996, 347, 494–499. [CrossRef]
20. Silverman, A.R.; Nieland, M.L. Hot tub dermatitis: A familial outbreak of Pseudomonas folliculitis. J. Am.
Acad. Dermatol. 1983, 8, 153–156. [CrossRef]
21. Leoni, E.; Catalani, F.; Marini, S.; Dallolio, L. Legionellosis Associated with Recreational Waters: A Systematic
Review of Cases and Outbreaks in Swimming Pools, Spa Pools, and Similar Environments. Int. J. Environ.
Res. Public Health 2018, 15, 1612. [CrossRef] [PubMed]
22. World Health Organization (WHO). Guidelines for Safe Recreational Water Environments; WHO Press: Geneva,
Switzerland, 2006; Volume 2, Available online: http://apps.who.int/iris/bitstream/10665/43336/1/
9241546808_eng.pdf (accessed on 29 May 2018).
23. Andreassi, L.; Flori, L. Mineral water and spas in Italy. Clin. Dermatol. 1996, 14, 627–632. [CrossRef]
24. Giampaoli, S.; Valeriani, F.; Gianfranceschi, G.; Vitali, M.; Delfini, M.; Festa, M.R.; Bottari, E.; Romano Spica, V.
Hydrogen sulfide in thermal spring waters and its action on bacteria of human origin. Microchem. J. 2013,
108, 210–214. [CrossRef]
25. Health and Safety Executive (HSE). The Control of Legionella and Other Infectious Agents in Spa-Pool
Systems. 2014. Available online: www.hse.gov.uk/pubns/books/hsg282.htm (accessed on 29 May 2018).
26. Signorelli, C.; Pasquarella, C.; Saccani, E.; Sansebastiano, G. Treatment of thermal pool waters. Ig Sanita
Pubbl. 2006, 62, 539–552. [PubMed]
27. Fazlzadeh, M.; Sadeghi, H.; Bagheri, P.; Poureshg, Y.; Rostami, R. Microbial quality and physical-chemical
characteristics of thermal Springs. Environ. Geochem. Health 2016, 38, 413–422. [CrossRef] [PubMed]
28. Barna, Z.; Kádár, M. The risk of contracting infectious diseases in public swimming pools. A review.
Annali Dell’istituto Superiore di Sanita 2012, 48, 374–386. [CrossRef] [PubMed]
29. Ferretti, E.; Fantuzzi, G.; Romano Spica, V.; Caroli, S.; Bonadonna, L. Fifth International
Conference Swimming Pool & Spa. ISTISAN Congressi 13/C1 ISSN 0393-5620. Rome. 2013.
Available online: https://www.researchgate.net/publication/236157805_Free-living_amoebae_and_enteric_
protozoa_isolated_in_swimming_pool (accessed on 19 November 2018).
30. Chong, M.N.; Jin, B.; Chow, C.W.; Saint, C. Recent developments in photocatalytic water treatment
technology: A review. Water Res. 2010, 44, 2997–3027. [CrossRef] [PubMed]
31. Csirmaz, É.; Pető, K. International Trends in Recreational and Wellness Tourism. Procedia Econ. Finance 2015,
32, 755–762. [CrossRef]
32. Atanga Adongo, C.; Amuquandoh, F.E.; Amenumey, E.K. Modelling spa-goers’ choices of therapeutic
activities. J. Hosp. Tour. Manag. 2017, 31, 105–113. [CrossRef]
33. Loureiro, S.M.C.; Almeida, M.; Rita, P. The effect of atmospheric cues and involvement on pleasure and
relaxation: The spa hotel context. Int. J. Hosp. Manag. 2013, 35, 35–43. [CrossRef]
34. McCarthy, J. Global spa & wellness trends Psychology of Spas and Wellbeing. 2017. Available online:
http://psychologyofwellbeing.com/201701/2017-global-spa-wellness-trends.html (accessed on 29 May 2018).
35. Dryglas, D.; Salamaga, M. Segmentation by push motives in health tourism destinations: A case study of
Polish spa resorts. J. Destin. Market. Manag. 2018, 9, 234–246. [CrossRef]
36. Han, H.; Kiatkawsin, K.; Kim, W.; Lee, S. Investigating customer loyalty formation for wellness spa:
Individualism vs. Collectivism. Int. J. Hosp. Manag. 2017, 67, 11–23. [CrossRef]
192
IJERPH 2018, 15, 2675
37. Tunstall, J. Clinical remarks upon the effects of the bath thermal waters in the treatment of chronic
rheumatism. Assoc. Med. J. 1853, 1, 8–10. [CrossRef] [PubMed]
38. Brues, C.T. Observations on the Fauna of Thermal Waters. Proc. Natl. Acad. Sci. USA 1924, 10, 484–486.
[CrossRef] [PubMed]
39. Oliveira, D. Indications & contraindications of thermal waters. Rev. Bras. Med. 1957, 14, 635–638. [PubMed]
40. Alberti, S.; Tonolo, A.; De Felip, G. Preliminary observations on the microbial flora of thermal waters of
Viterbo in the nature of sulfobacteria. Rendiconti-Istituto Superiore di Sanita 1959, 22, 1018–1024. [PubMed]
41. Araujo, A.R.T.S.; Sarraguça, M.C.; Ribeiro, M.P.; Coutinho, P. Physicochemical fingerprinting of thermal
waters of Beira Interior region of Portugal. Environ. Geochem. Health. 2017, 39, 483–496. [CrossRef] [PubMed]
42. Directive 2009/54/EC of the European Parlament and the Concil of 18 June 2009 on the Exploitation and
Marketing of Natural Mineral Water. Available online: https://eur-lex.europa.eu/legal-content/en/ALL/
?uri=CELEX%3A32009L0054 (accessed on 19 November 2018).
43. Karakaya, M.Ç.; Doğru, M.; Karakaya, N.; Kuluöztürk, F.; Nalbantçılar, M.T. Radioactivity and hydrochemical
properties of certain thermal Turkish spa waters. J. Water Health 2017, 15, 591–601. [CrossRef] [PubMed]
44. Gomes, C.; Carretero, M.I.; Pozo, M.; Maraver, F.; Cantista, P.; Armijo, F.; Legido, J.L.; Teixeira, F.;
Rautureau, M.; Delgado, F. Peloids and pelotherapy: Historical evolution, classification and glossary.
Appl. Clay Sci. 2013, 75, 28–38. [CrossRef]
45. Valeriani, F.; Biagini, T.; Giampaoli, S.; Crognale, S.; Santoni, D.; Romano Spica, V. Draft Genome Sequence
of Tepidimonas taiwanensis Strain VT154-175. Genome Announc. 2016, 4, e00942-16. [CrossRef] [PubMed]
46. Yang, L.; Muhadesi, J.B.; Wang, M.M.; Wang, B.J.; Liu, S.J.; Jiang, C.Y. Thauera hydrothermalis sp. nov.; a
thermophilic bacterium isolated from hot spring. Int. J. Syst. Evol. Microbiol. 2018, 68, 3163–3168. [CrossRef]
[PubMed]
47. Jiang, X.; Takacs-Vesbach, C.D. Microbial community analysis of pH 4 thermal springs in Yellowstone
National Park. Extremophiles 2017, 21, 135–152. [CrossRef] [PubMed]
48. Borella, P.; Montagna, M.T.; Romano-Spica, V.; Stampi, S.; Stancanelli, G.; Triassi, M.; Marchesi, I.; Bargellini, A.;
Neglia, R.; Paglionico, N.; et al. Relationship between mineral content of domestic hot water and microbial
contamination. J. Trace Elem. Med. Biol. 2003, 17, 37–43. [PubMed]
49. Romano Spica, V. Advances in Microbiota Knowledge and NGS Technologies: Perspectives for Surveillance
in Recreational Waters. ICSPS 2019. Available online: https://8thswimpoolspa.sciencesconf.org/resource/
page/id/20 (accessed on 19 November 2018).
50. Valeriani, F.; Protano, C.; Gianfranceschi, G.; Leoni, E.; Galasso, V.; Mucci, N.; Vitali, M.; Romano Spica, V.
Microflora Thermarum Atlas project: Biodiversity in thermal spring waters and natural SPA pools. Water Sci.
Technol. Water Supply 2018, 18, 1472–1483. [CrossRef]
51. Valeriani, F.; Agodi, A.; Casini, B.; Cristina, M.L.; D’Errico, M.M.; Gianfranceschi, G.; Liguori, G.; Liguori, R.;
Mucci, N.; Mura, I.; et al. Potential testing of reprocessing procedures by real-time polymerase chain reaction:
A multicenter study of colonoscopy devices. Am. J. Infect. Control 2018, 46, 159–164. [CrossRef] [PubMed]
52. Giampaoli, S.; Berti, A.; Valeriani, F.; Gianfranceschi, G.; Piccolella, A.; Buggiotti, L.; Rapone, C.; Valentini, A.;
Ripani, L.; Romano Spica, V. Molecular identification of vaginal fluid by microbial signature. Forensic Sci.
Int. Genet. 2012, 6, 559–564. [CrossRef] [PubMed]
53. Fortney, N.W.; He, S.; Converse, B.J.; Boyd, E.S.; Roden, E.E. Investigating the Composition and Metabolic
Potential of Microbial Communities in Chocolate Pots Hot Springs. Front. Microbiol. 2018, 9, 2075. [CrossRef]
[PubMed]
54. Amin, A.; Ahmed, I.; Salam, N.; Kim, B.Y.; Singh, D.; Zhi, X.Y.; Xiao, M.; Li, W.J. Diversity and Distribution
of Thermophilic Bacteria in Hot Springs of Pakistan. Microb Ecol. 2017, 74, 116–127. [CrossRef] [PubMed]
55. The Earth Microbiome Project. Available online: www.earthmicrobiome.org (accessed on 19 November 2018).
56. Valeriani, F.; Crognale, S.; Protano, C.; Gianfranceschi, G.; Orsini, M.; Vitali, M.; Romano Spica, V.
Metagenomic analysis of bacterial community in a travertine depositing hot spring. New Microbiol. 2018, 41,
126–135. [PubMed]
57. Paduano, S.; Valeriani, F.; Romano Spica, V.; Bargellini, A.; Borella, P.; Marchesi, I. Microbial biodiversity of
thermal water and mud in an Italian spa by metagenomics: A pilot study. Water Sci. Technol. Water Supply
2018, 18, 1456–1465. [CrossRef]
193
IJERPH 2018, 15, 2675
58. Baron, J.L.; Vikram, A.; Duda, S.; Stout, J.E.; Bibby, K. Shift in the microbial ecology of a hospital hot water
system following the introduction of an on-site monochloramine disinfection system. PLoS ONE 2014, 9,
102679. [CrossRef] [PubMed]
59. Ma, X.; Baron, J.L.; Vikram, A.; Stout, J.E.; Bibby, K. Fungal diversity and presence of potentially pathogenic
fungi in a hospital hot water system treated with on-site monochloramine. Water Res. 2015, 71, 197–206.
[CrossRef] [PubMed]
60. Valeriani, F.; Giampaoli, S.; Buggiotti, L.; Gianfranceschi, G.; Romano Spica, V. Molecular enrichment for
detection of S. aureus in recreational waters. Water Sci. Technol. 2012, 66, 2305–2310. [CrossRef] [PubMed]
61. Tartanson, M.A.; Soussan, L.; Rivallin, M.; Chis, C.; Penaranda, D.; Lapergue, R.; Calmels, P.; Faur, C. A new
silver based composite material for SPA water disinfection. Water Res. 2014, 63, 135–146. [CrossRef] [PubMed]
62. Guida, M.; Di Onofrio, V.; Gallè, F.; Gesuele, R.; Valeriani, F.; Liguori, R.; Romano Spica, V.; Liguori, G.
Pseudomonas aeruginosa in Swimming Pool Water: Evidences and Perspectives for a New Control Strategy.
Int. J. Environ. Res. Public Health 2016, 13, 919. [CrossRef] [PubMed]
63. Varga, C.; László, M.; Gerencsér, G.; Gyöngyi, Z.; Szendi, K. Natural UV-protective organic matter in thermal
water. J Photochem Photobiol B. 2015, 144, 8–10. [CrossRef] [PubMed]
64. Valeriani, F.; Protano, C.; Vitali, M.; Romano Spica, V. Swimming attendance during childhood and
development of asthma: Meta-analysis. Pediatr. Int. 2017, 59, 614–621. [CrossRef] [PubMed]
65. Carter, R.A.A.; Joll, C.A. Occurrence and formation of disinfection by-products in the swimming pool
environment: A critical review. J. Environ. Sci. 2017, 58, 19–50. [CrossRef] [PubMed]
66. Cortés, C.; Marcos, R. Genotoxicity of disinfection byproducts and disinfected waters: A review of recent
literature. Mutat. Res. 2018, 831, 1–12. [CrossRef] [PubMed]
67. WHO. Water Safety in Buildings. Available online: http://www.who.int/water_sanitation_health/
publications/2011/9789241548106/en/ (accessed on 29 May 2018).
68. Napoli, C.; Giampaoli, S.; Gallè, F.; Frangella, C.; Di Onofrio, V.; Bonadonna, L.; Romano Spica, V.; Liguori, G.
World Health Organization document “water safety in buildings”: Italian translation. Igiene e Sanita Pubblica
2012, 68, 613–624. [PubMed]
69. Lee, J.; Jun, M.-J.; Lee, M.-H.; Lee, M.-H.; Eom, S.-W.; Zoh, K.-D. Production of various disinfection
byproducts in indoor swimming pool waters treated with different disinfection methods. Int. J. Hyg.
Environ. Health 2010, 213, 465–474. [CrossRef] [PubMed]
70. Valeriani, F.; Gianfranceschi, G.; Vitali, M.; Protano, C.; Romano Spica, V. Development of the laboratory
prototype “CavyPool” for assessing treatments and materials for swimming pools. Annali di Igiene Medicina
Preventiva e di Comunita 2017, 29, 548–560. [PubMed]
71. Fantuzzi, G.; Aggazzotti, G.; Righi, E.; Predieri, G.; Castiglioni, S.; Riva, F.; Zuccato, E. Illicit drugs and
pharmaceuticals in swimming pool waters. Sci. Total Environ. 2018, 635, 956–963. [CrossRef] [PubMed]
72. Tang, H.L.; Xie, Y.F. Biologically active carbon filtration for haloacetic acid removal from swimming pool
water. Sci. Total Environ. 2016, 541, 58–64. [CrossRef] [PubMed]
73. Zwiener, C.; Richardson, S.D.; De Marini, D.M.; Grummt, T.; Glauner, T.; Frimmel, F.H. Drowning in
disinfection byproducts? Assessing swimming pool water. Environ. Sci. Technol. 2007, 41, 363–372. [CrossRef]
[PubMed]
74. Manasfi, T.; Méo, M.D.; Coulomb, B.; Giorgio, C.D.; Boudenne, J.-L. Identification of disinfection by-products
in freshwater and seawater swimming pools and evaluation of genotoxicity. Environ. Int. 2016, 88, 94–102.
[CrossRef] [PubMed]
75. Kim, H.; Shim, J.; Lee, S. Formation of disinfection by-products in chlorinated swimming pool water.
Chemosphere 2002, 46, 123–130. [CrossRef]
76. Liu, R.; Tian, C.; Hu, C.; Qi, Z.; Liu, H.; Qu, J. Effects of bromide on the formation and transformation of
disinfection by-products during chlorination and chloramination. Sci. Total Environ. 2018, 625, 252–261.
[CrossRef] [PubMed]
77. Glauner, T.; Kunz, F.; Zwiener, C.; Frimmel, F.H. Elimination of swimming pool water disinfection byproducts
with advanced oxidation processes (AOPs). Acta Hydrochim. Hydrobiol. 2005, 33, 585–594. [CrossRef]
78. Lee, J.; Ha, K.-T.; Zoh, K.-D. Characteristics of trihalomethane (THM) production and associated health risk
assessment in swimming pool waters treated with different disinfection methods. Sci. Total Environ. 2009,
194
IJERPH 2018, 15, 2675
79. Hang, C.; Zhang, B.; Gong, T.; Xian, Q. Occurrence and health risk assessment of halogenated disinfection
byproducts in indoor swimming pool water. Sci. Total Environ. 2016, 543, 425–431. [CrossRef] [PubMed]
80. Daiber, E.J.; DeMarini, D.M.; Ravuri, S.A.; Liberatore, H.K.; Cuthbertson, A.A.; Thompson-Klemish, A.;
Byer, J.D.; Schmid, J.E.; Afifi, M.Z.; et al. Progressive increase in disinfection byproducts and mutagenicity
from source to tap to swimming pool and spa water: Impact of human inputs. Environ. Sci. Technol. 2016, 50,
81. Hansen, K.M.S.; Spiliotopoulou, A.; Cheema, W.A.; Andersen, H.R. Effect of ozonation of swimming pool
water on formation of volatile disinfection by-products—A laboratory study. Chem. Eng. J. 2016, 289, 277–285.
[CrossRef]
82. Spiliotopoulou, A.; Hansen, K.M.S.; Andersen, H.R. Secondary formation of disinfection by-products by UV
treatment of swimming pool water. Sci. Total Environ. 2015, 520, 96–105. [CrossRef] [PubMed]
83. Hijnen, W.A.M.; Beerendonk, E.F.; Medema, G.J. Inactivation credit of UV radiation for viruses, bacteria and
protozoan (oo)cysts in water: A review. Water Res. 2006, 40, 3–22. [CrossRef] [PubMed]
84. Craik, S.A.; Weldon, D.; Finch, G.R.; Bolton, J.R.; Belosevic, M. Inactivation of Cryptosporidium parvum
oocysts using medium- and low-pressure ultraviolet radiation. Water Res. 2001, 35, 1387–1398. [CrossRef]
85. Chang, J.C.H.; Ossoff, S.F.; Lobe, D.C.; Dorfman, M.H.; Dumais, C.M.; Qualls, R.G.; Johnson, J.D. UV inactivation
of pathogenic and indicator microorganisms. Appl. Environ. Microbiol. 1985, 49, 1361–1365. [PubMed]
86. Cheema, W.A.; Manasfi, T.; Kaarsholm, K.M.S.; Andersen, H.R.; Boudenne, J.-L. Effect of medium-pressure
UV-lamp treatment on disinfection by-products in chlorinated seawater swimming pool. Sci. Total Environ.
2017, 599–600, 910–917. [CrossRef] [PubMed]
87. Li, J.; Blatchley, E.R., III. UV photodegradation of inorganic chloramines. Environ. Sci. Technol. 2009, 43,
88. Cimetiere, N.; De Laat, J. Effects of UV-dechloramination of swimming pool water on the formation of
disinfection by-products: A lab-scale study. Microchem. J. 2014, 112, 34–41. [CrossRef]
89. Afifi, M.Z.; Blatchley, E.R., III. Effects of UV-based treatment on volatile disinfection byproducts in a
chlorinated, indoor swimming pool. Water Res. 2016, 105, 167–177. [CrossRef] [PubMed]
90. Sisti, M.; Pieretti, B.; De Santi, M.; Brandi, G. Inactivation of pathogenic dermatophytes by ultraviolet irradiation
in swimming pool thermal water. Int. J. Environ. Health Res. 2014, 24, 412–417. [CrossRef] [PubMed]
91. Florentin, A.; Hautemanière, A.; Hartemann, P. Health effects of disinfection by-products in chlorinated
swimming pools. Int. J. Hyg. Environ. Health 2011, 214, 461–469. [CrossRef] [PubMed]
92. World Health Organitation. Alternative Drinking-Water Disinfectants: Bromine, Iodine and Silver.
Available online: http://www.who.int/water_sanitation_health/publications/alternative-disinfectants/en/
93. Schwake, A.; Ross, B.; Cammann, K. Chrono amperometric determination of hydrogen peroxide in swimming
pool water using an ultramicroelectrode array. Sens. Actuators B Chem. 1998, 46, 242–248. [CrossRef]
94. World Water Development Report 19 March, 2018. Available online: http://www.unwater.org/publications/
world-water-development-report-2018/ (accessed on 19 November 2018).
95. Inoue, T.; Inoue, S.; Kubota, K. Bactericidal activity of manganese and iodide ions against Staphylococcus
aureus: A possible treatment for acute atopic dermatitis. Acta Derm. Venereol. 1999, 79, 360–362. [PubMed]
96. Akiyama, H.; Yamasaki, O.; Tada, J.; Kubota, K.; Arata, J. Antimicrobial effects of acidic hot-spring water on
Staphylococcus aureus strains isolated from atopic dermatitis patients. J. Dermatol. Sci. 2000, 24, 112–118.
[CrossRef]
97. Barbot, E.; Moulin, P. Swimming pool water treatment by ultrafiltration–adsorption process. J. Membr. Sci.
2008, 314, 50–57. [CrossRef]
98. Gitis, V.; Hankins, N. Water treatment chemicals: Trends and challenges. J. Water Process Eng. 2018, 25, 34–38.
[CrossRef]
99. Yao, K.M.; Habibian, M.T.; O’Melia, C.R. Water and waste water filtration. Concepts and applications.
Environ. Sci. Technol. 1971, 5, 1105–1112. [CrossRef]
100. Wang, X.; Ma, B.; Bai, Y.; Lan, H.; Liu, H.; Qu, J. The effects of hydrogen peroxide pre-oxidation on
ultrafiltration membrane biofouling alleviation in drinking water treatment. J. Environ. Sci. 2018, 73, 117–126.
[CrossRef] [PubMed]
195
IJERPH 2018, 15, 2675
101. Li, Q.; Mahendra, S.; Lyon, D.Y.; Brunet, L.; Liga, M.V.; Li, D.; Alvarez, P.J. Antimicrobial nanomaterials
for water disinfection and microbial control: Potential applications and implications. Water Res. 2008, 42,
102. Gazit, E. Self-assembled peptide nanostructures: The design of molecular building blocks and their
technological utilization. Chem. Soc. Rev. 2007, 36, 1263–1269. [CrossRef] [PubMed]
103. Badawy, M.E.I.; Rabea, E.I.; Rogge, T.M.; Stevens, C.V.; Steurbaut, W.; Hofte, M.; Smagghe, G. Fungicidal
and insecticidal activity of O-acyl chitosan derivatives. Polym. Bull. 2005, 54, 279–289. [CrossRef]
104. Rabea, E.I.; Badawy, M.E.; Stevens, C.V.; Smagghe, G.; Steurbaut, W. Chitosan as antimicrobial agent:
Applications and mode of action. Biomacromolecules 2003, 4, 1457–1465. [CrossRef] [PubMed]
105. Chirkov, S.N. The antiviral activity of chitosan (review). Appl. Biochem. Microbiol. 2002, 38, 1–8. [CrossRef]
106. Don, T.M.; Chen, C.C.; Lee, C.K.; Cheng, W.Y.; Cheng, L.P. Preparation and antibacterial test of chitosan/
PAA/PEGDA bilayer composite membranes. J. Biomater. Sci. Polym. Ed. 2005, 16, 1503–1519. [CrossRef]
[PubMed]
107. Rani, D.; Singla, P.; Agarwal, J. ‘Chitosan in water’ as an eco-friendly and efficient catalytic system for
Knoevenagel condensation reaction. Carbohydr. Polym. 2018, 202, 355–364. [CrossRef] [PubMed]
108. Ibrahim, M.M.; Asal, S. Physicochemical and photocatalytic studies of Ln3+ -ZnO for water disinfection and
wastewater treatment applications. J. Mol. Struct. 2017, 1149, 404–413. [CrossRef]
109. Jeon, S.K.; Kim, E.J.; Lee, J.; Lee, S. Potential risks of TiO2 and ZnO nanoparticles released from sunscreens
into outdoor swimming pools. J. Hazard. Mater. 2016, 317, 312–318. [CrossRef] [PubMed]
110. World Health Organization (WHO) and International agency for Research on Cancer (IARC). Monographs
on the Evaluation of Carcinogenic Risks to Humans. Carbon Black, Titanium Dioxide and Talc. Vol, 93, Lyon
France. Available online: https://monographs.iarc.fr/wp-content/uploads/2018/06/mono93.pdf (accessed
on 29 May 2018).
111. Seltenrich, N. Nanosilver: Weighing the risks and benefits. Environ. Health Perspect. 2013, 121, 220–225.
[CrossRef] [PubMed]
112. Nowack, B.; Krug, H.F.; Height, M. 120 years of nanosilver history: Implications for policy makers.
Environ. Sci. Technol. 2011, 45, 1177–1183. [CrossRef] [PubMed]
113. Gehrke, I.; Geiser, A.; Somborn-Schulz, A. Innovations in nanotechnology for water treatment.
Nanotechnol. Sci. Appl. 2015, 8, 1–17. [CrossRef] [PubMed]
114. Shrestha, B.; Acosta-Martinez, V.; Cox, S.B.; Green, M.J.; Li, S.; Canas-Carrell, J.E. An evaluation of the impact
of multiwalled carbon nanotubes on soil microbial community structure and functioning. J. Hazard. Mater.
2013, 261, 188–197. [CrossRef] [PubMed]
115. Shvedova, A.A.; Pietroiusti, A.; Fadeel, B.; Kagan, V.E. Mechanisms of carbon nanotube-induced toxicity:
Focus on oxidative stress. Toxicol. Appl. Pharmacol. 2012, 261, 121–133. [CrossRef] [PubMed]
116. Van Aken, B. Gene expression changes in plants and microorganisms exposed to nanomaterials. Curr. Opin.
Biotechnol. 2015, 33, 206–219. [CrossRef] [PubMed]
117. Zhu, B.; Xia, X.; Zhang, S.; Tang, Y. Attenuation of bacterial cytotoxicity of carbon nanotubes by riverine
suspended solids in water. Environ. Pollut. 2018, 234, 581–589. [CrossRef] [PubMed]
118. Ko, K.; Kim, M.J.; Lee, J.Y.; Kim, W.; Chung, H. Effects of graphene oxides and silver-graphene oxides on
aquatic microbial activity. Sci. Total Environ. 2019, 651, 1087–1095. [CrossRef] [PubMed]
119. Shen, L.; Jin, Z.; Wang, D.; Wang, Y.; Lu, Y. Enhance wastewater biological treatment through the bacteria
induced graphene oxide hydrogel. Chemosphere 2018, 190, 201–210. [CrossRef] [PubMed]
120. Centers for Disease Control and Prevention (CDC). The Model Aquatic Health Code (MAHC): An
All-inclusive Model Public Swimming Pool and Spa Code. Available online: https://www.cdc.gov/mahc/
index.html (accessed on 29 May 2018).
121. Virginia Graeme baker Pool and Spa Safety Act Guidelines for Entrapment Hazards: Making Pools and Spas
Safer; Virginia Graeme Baker Pool and Spa Safety Act, Title 14 of the U.S. Energy Independence and Security
Act; U.S. Consumer Product Safety Commission: Washington, DC, USA, 2007. Available online: https:
//www.govtrack.us/congress/bills/110/hr6/text (accessed on 29 May 2018).
122. Centers for Disease Control and Prevention (CDC). Available online: http://www.cdc.gov/healthywater/
swimming/pools/regulation/index.html (accessed on 29 May 2018).
123. Règlement de Sécurité, Fédération de Natation du Québec (Natation En Bassin) Janvier 2009. Available
online: http://www.fnq.qc.ca/doc/doc/00000519_safety.pdf (accessed on 29 May 2018).
196
IJERPH 2018, 15, 2675
124. Guidelines for Canadian Recreational Water Quality—Third Edition, Published by Authority of the Minister
of Health. Available online: http://www.healthcanada.gc.ca/ (accessed on 29 May 2018).
125. Alberta. Health Pool Standards July 2014 (Amended January 2018). Available online: https://open.alberta.
ca/publications/9781460137215 (accessed on 29 May 2018).
126. Australia. New South Wales Consolidated Acts. Swimming Pools Act 1990 n. 31. Available online:
http://www.austlii.edu.au/au/legis/nsw/num_act/spa1990n31219.pdf (accessed on 29 May 2018).
127. Australia. New South Wales Consolidated Acts. Swimming Pools Act 1992 n. 49. Available online:
http://classic.austlii.edu.au/au/legis/nsw/consol_act/spa1992192/ (accessed on 29 May 2018).
128. Standards Australia. Swimming and Spa Pools Part 1: Public spas2007 (Reconfirmed 2016), Standards
Australia. Available online: https://www.standards.org.au/standards-catalogue/sa-snz/building/cs-034/
as--2610-dot-1-2007 (accessed on 29 May 2018).
129. Queensland Department of Health. Swimming and Spa Pool Water Quality and Operational Guidelines;
Queensland Department of Health, Australian Capital Territory, 2004. Available online: https://www.
health.qld.gov.au/__data/assets/pdf_file/0021/444612/guidelines-pool-spa.pdf (accessed on 29 May 2018).
130. Council Directive 76/160/EEC of 8 December 1975 Concerning the Quality of Bathing Water. GUCE 8
December 1975 n. L 76. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:
31976L0160 (accessed on 29 May 2018).
131. Directive 2006/7/EC of the European Parliament and of the Council of 15 February 2006 Concerning
the Management of Bathing Water Quality and Repealing Directive 76/160/EEC. Available online: https:
//eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32006L0007 (accessed on 29 May 2018).
132. Verordnung des Bundesministers für Gesundheit und Umweltschutz vom 26 Juli 1978 über Hygiene in
Bädern. Bundesgesetzblatt für die Republik Österreich 1978, 167, 3053–3063.
133. Volksgesundheitsamt, Oberster Sanitätsrat. Mitteilungen der Österreichischen Sanitätsverwaltung 1992, 93, 358.
134. Austria. Bundesgesetzblatt für die Republik Österreich. 1996, 212, 4617–4624. Available online: https:
//ris.bka.gv.at/Dokumente/BgblPdf/1996_658_0/1996_658_0.pdf (accessed on 29 May 2018).
135. Volksgesundheitsamt, Oberster Sanitätsrat. Mitteilungen der Österreichischen Sanitätsverwaltung 1997, 98,
228–232.
136. Austria. Gesamte Rechtsvorschrift für Bäderhygienegesetz, Fassung vom 28.10.2012. Available
online: https://hygiene.medunigraz.at/fileadmin/institute-oes/hygiene/pdf/wasseruntersuchungen/
downloads/Bäderhygienegesetz.pdf (accessed on 29 May 2018).
137. Giampaoli, S.; Garrec, N.; Donzé, G.; Valeriani, F.; Erdinger, L.; Romano Spica, V. Regulations concerning
natural swimming ponds in Europe: Considerations on public health issues. J. Water Health. 2014, 12,
138. Belgium. Arrêté du Gouvernement Wallon Portant Conditions Sectorielles Relatives Aux Bassins De Natation.
March 13, 2003 (Repealing 26.05.2004 e M.B. 30.01.2007). Available online: http://formpe.environnement.
wallonie.be/html/CS%20Piscines%20(cl_2).pdf (accessed on 29 May 2018).
139. Belgium. Arrêté du Gouvernement de la Région de Bruxelles-Capitale Fixant des Conditions d’exploitation
Pour les Bassins de Natation. October 10 Ottobre 2002. Available online: https://vlex.be/vid/arr-conditions-
exploitation-bassins-natation-29702709 (accessed on 29 May 2018).
140. Belgium. Arrete´ du Gouvernement de la Region de Bruxelles-Capitale Fixant la Liste des Installations
de Classe IB, II et III en Execution de l’article 4 de L’ordonnance du 5 juin 1997 Relative aux Permis
D’environnement. 4 March 1999. Available online: http://www.etaamb.be/fr/arrete-du-gouvernement-de-
la-region-de-bruxellescapit_n1999031224.html (accessed on 29 May 2018).
141. Francia. Code de la Santé Publique. 2010. Section 5: Surveillance des Etablissements Thermaux. Article
R1322-45 a R1322-51 (Dernière Modification: 6 octobre 2018). Available online: http://www.codes-et-lois.fr/
code-de-la-sante-publique/article-r1322-47 (accessed on 29 May 2018).
142. Francia. Code de la Santé Publique, 2010. Section I: Normes d’hygiène et de Sécurité Applicables aux
Piscines et Baignades Aménagées—Articles D1332-1 a D1332-13 (Dernière Modification: 6 octobre 2018).
Available online: https://www.legifrance.gouv.fr/affichCode.do?idSectionTA=LEGISCTA000006190970&
cidTexte=LEGITEXT000006072665&dateTexte=20080921 (accessed on 29 May 2018).
143. Francia. Afsset Evaluation des Risques Sanitaires liés Aux Piscines Partie I: Piscines Réglementées.
Saisine Afsset «2006/11». Rapport Final. 2010. Available online: https://www.anses.fr/fr/system/files/
EAUX2007sa0409Ra.pdf (accessed on 29 May 2018).
197
IJERPH 2018, 15, 2675
144. Boudenne, J.-L. Évaluation des Risques Sanitaires liés aux Piscines Partie II: Bains à Remous.
10.13140/RG.2.1.2182.7043. 2015. Available online: https://www.researchgate.net/publication/282245336_
Evaluation_des_risques_sanitaires_lies_aux_piscines_Partie_II_bains_a_remous (accessed on 29 May 2018).
145. Health Protection Agency. Management of Spa Pools: Controlling the Risk of Infection; Health Protection Agency:
London, UK, March 2006.
146. Ireland. Safety, Health and Welfare at Work Act. 2005. Health and Safety Authority. Available online:
http://www.irishstatutebook.ie/eli/2005/act/10/enacted/en/print (accessed on 29 May 2018).
147. Ireland. Swimming Pool Safety Guidelines. Irish Water Safety, ILAM and Swim Ireland. 2010. Available
online: http://www.irelandactive.ie/contentfiles/Swimming-Pool-Safety-Guidelines1.pdf (accessed on 29
May 2018).
148. Germany. DIN 19643. Aufbereitung von Schwimm-und Badebeckenwasser–Teil 1: Allgemeine
Anforderungen.Beuth ; Englischer Titel: Treatment of the water of swimming-pools and baths—Part 1:
General requirements Ausgabedatum 1997-04 1997, Ausgabe:1997-04. Available online: https://www.beuth.
de/de/norm/din-19643-1/2936483 (accessed on 29 May 2018).
149. Germany. Hygienische Anforderungen an Kleinbadeteiche. Empfehlung des Umweltbundesamtes.
Bundesgesundhbl Gesundheitsschutz 2003, 46, 527–529. Available online: https://link.springer.com/article/10.
1007/s00103-003-0627-0 (accessed on 29 May 2018). [CrossRef]
150. Germany. Empfehlung des Umweltbundesamtes. Bundesgesundheitsbl-Gesundheitsforsch-Gesundheitsschutz
2006, 49, 926–937. Available online: https://doi.org/10.1007/s00103-006-0030-8 (accessed on 29 May 2018).
151. Germany. DIN 19643. Aufbereitung von Schwimm-und Badebeckenwasser—Teil 4:
Verfahrenskombinationen Mit Ultrafiltration. Englischer Titel: Treatment of water of swimming
pools and baths—Part 4: Combinations of process with ultrafiltration. 2018. Available online:
https://www.beuth.de/de/norm/din-19643-4/164174207 (accessed on 29 May 2018).
152. Czech Republic. Decree of the Ministry of Health 423/2001—On Spas and Sources. Available online:
http://www.mzcr.cz/obsah/souvisejici-legislativa_1757_3.html (accessed on 29 May 2018).
153. Czech Republic. Decree of Ministry of Health 252/2004 (update 70/2018)—Requirements on Cold and
Hot Water in Health Care and Accommodation Facilities. Available online: http://www.mzcr.cz/obsah/
souvisejici-legislativa_1757_3.html (accessed on 29 May 2018).
154. Czech Republic. Decree of Ministry of Health 135/2004—Requirements on Swimming Pools, Saunas
and Outdoor Playgrouds. Available online: https://publications.europa.eu/en/publication-detail/-/
publication/b52fa07c-8be9-454f-a2f0-417ae2d9f798 (accessed on 19 November 2018).
155. Portugal. Ministério da saúde Decreto-lei n. 142. 11 giugno 2004. Diário da República n. 136/2004, Série I-A
de 2004-06-11. Available online: https://dre.pt/web/guest/pesquisa/-/search/286109/details/normal?q=
142%2F2004 (accessed on 29 May 2018).
156. Conselho Nacional da Qualidade. Directiva CNQ n.º 23/93 de 24 de Maio—A qualidade das piscinas de uso
público. Lisboa, 1993.
157. Matisova, E. Report on Monitoring of Hygienic Situation on Natural and Artificial Pools in Year 2004; Authority of
Public Health of the Slovak Republic: Batislava, 2004; Available online: http://www.dinax.hu/dok/spa_
conference_2005_HUN/02/03.pdf (accessed on 29 May 2018).
158. Spain. Boletìn Oficial del Ministerio de Sanidad y Consumo. 1987, 19, pp. 1147–1152. Available online:
https://www.boe.es (accessed on 19 November 2018).
159. Spain. Boletìn Oficial del Ministerio de Sanidad y Consumo 1998, 80, por el que se regulan las condiciones
higiénico–sanitarias de piscinas de uso colectivo. Available online: http://www.bocm.es/boletin/CM_
Boletin_BOCM/19980527_B/12400.pdf (accessed on 19 November 2018).
160. Spain. La Orden 1319/2006 de 27 de junio de la Consejería de Sanidad y Consumo de la Comunidad de
Madrid, por la que se establecen los criterios que permitan establecer los niveles de formación del personal
que preste sus servicios como socorrista en piscinas, instalaciones acuáticas y medio natura. Available online:
http://www.bocm.es/boletin/CM_Boletin_BOCM/20060714_B/16600.pdf (accessed on 19 November 2018).
161. Spain. Real Decreto 742/13 de 27 de Septiembre, Por el que se Establecen los Criterios Técnico-Srios de
las Piscinas. Available online: https://www.boe.es/boe/dias/2013/10/11/pdfs/BOE-A-2013-10580.pdf
198
IJERPH 2018, 15, 2675
162. Finlands Författningssamling: Ålands landskapsregerings beslut om kvalitetskrav på och kontroll av vattnet vid
allmänna ytvattenbadplatser; Official publication: Ålands Författningssamling (ÅFS), Number: 2008/70;
Available online: https://eur-lex.europa.eu/legal-content/EN/NIM/?uri=CELEX:32006L0007 (accessed on
19 November 2018).
163. Finlands Författningssamling: Ålands landskapsregerings beslut om kvalitetskrav på och kontroll av vattnet vid
allmänna ytvattenbadplatser (2014/47) 16/10/2014; Official publication: Ålands Författningssamling (ÅFS),
Number: 2014/47; Available online: https://eur-lex.europa.eu/legal-content/EN/NIM/?uri=CELEX:
32006L0007 (accessed on 19 November 2018).
164. Ciprium Government. Law N. 55(I)/92 (2). 1992, Ciprium. Available online: www.cyprus.gov.cy (accessed
on 19 November 2018).
165. Bulgar. D’rzaven Vestnik. 1994, 6, pp. 1–14. Available online: http://lexbg/laws (accessed on 29 May 2018).
166. Norway. Norsk Lovtidend, section 1. 1996, 11, pp. 767–773. Available online: https://lovdata.no/register/
lovtidend (accessed on 19 November 2018).
167. Italy. Law of 24 October 2000, n. 323. Reorganization of the Thermal Sector. Official Gazette November 8,
2000, n. 261. (GU Serie Generale n.261 del 08-11-2000). Available online: http://www.gazzettaufficiale.it/
eli/id/2000/11/08/000G0377/sg (accessed on 19 November 2018).
168. Italy. Agreement between the Minister of Health, the Regions and the Autonomous Provinces of Trento
and Bolzano Concerning the Sanitary-Hygiene Aspects for the Construction, Maintenance and Supervision
of Swimming Pools for Swimming (16.01.2003). G.U. March 3, 2003: 45, n. 51. Available online: http:
//www.salute.gov.it/imgs/C_17_normativa_1911_allegato.pdf (accessed on 19 November 2018).
169. Valeriani, F.; Briancesco, R.; Sanzari, S.; Gianfranceschi, G.; Ferretti, E.; Bonadonna, L.; Romano Spica, V.
Some considerations on revision of legislation Hygiene-sanitary for the management of swimming pools for
swimming-pool use and the National Consultation of the Ministry of Health. Igiene e Sanita Pubblica 2017, 73,
247–266. [PubMed]
170. Italy. Guidelines with Indications on Legionellosis for Managers of Tourist Accommodation and Thermal
Facilities (13/01/2005). Official Gazette February 4, 2005, n. 28. Available online: http://www.salute.gov.it/
portale/documentazione/p6_2_2_1.jsp?id=2362 (accessed on 19 November 2018).
199
MDPI
St. Alban-Anlage 66
4052 Basel
Switzerland
Tel. +41 61 683 77 34
Fax +41 61 302 89 18
www.mdpi.com
International Journal of Environmental Research and Public Health Editorial Office

E-mail: [email protected]
www.mdpi.com/journal/ijerph
MDPI
St. Alban-Anlage 66
4052 Basel
Switzerland
Tel: +41 61 683 77 34
Fax: +41 61 302 89 18
www.mdpi.com ISBN 978-3-03897-579-3

Recreational Water Illnesses

Uploaded by

Copyright:

Available Formats

Recreational Water Illnesses

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Recreational Water Illnesses

Uploaded by

Copyright:

Available Formats

What is the document about?

What is the document about?

What environments are discussed as locations for recreational water illnesses?

What environments are discussed as locations for recreational water illnesses?

Recreational

Special Issue Editor

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

ISBN 978-3-03897-578-6 (Pbk)

About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface to ”Recreational Water Illnesses” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Lucia Bonadonna and Giuseppina La Rosa

Erica Leoni, Federica Catalani, Soﬁa Marini and Laura Dallolio

Antonios Papadakis, Dimosthenis Chochlakis, Vassilios Sandalakis, Maria Keramarou,

Mahbubul H. Siddiqee, Rebekah Henry, Rebecca Coulthard, Christelle Schang,

Laura M. Suppes, Kacey C. Ernst, Leif Abrell and Kelly A. Reynolds

Federica Valeriani, Lory Marika Margarucci and Vincenzo Romano Spica

Received: 5 October 2018; Accepted: 29 November 2018; Published: 3 December 2018

Keywords: swimming pools; tropics; subtropics; pool assessment; infectious diseases

IJERPH 2018, 15, 2730; doi:10.3390/ijerph15122730 1 www.mdpi.com/journal/ijerph

2. Materials and Methods

Search Strategy/Inclusion Criteria

3.1. Assessments of Swimming Pools in Developing Tropical Countries

Location/Country Positive Results Ref.

3.3.1. Schistosoma spp.

3.3.2. Cryptosporidium spp.

3.3.3. Acanthamoeba, Naegleria Species

3.4. Leptospira spp.

3.6. Indirect Role of Swimming Pools in Water Related Diseases

3.7. Chlamydia Trachomatis

Funding: This research received no external funding

Received: 8 November 2018; Accepted: 5 January 2019; Published: 9 January 2019

2. Materials and Methods

IJERPH 2019, 16, 166; doi:10.3390/ijerph16020166 21 www.mdpi.com/journal/ijerph

3. Viral Outbreaks Related to Swimming Pools

3.1. Adenovirus Outbreaks (N◦ = 15)

3.2. Enterovirus Outbreaks (N◦ = 6)

3.3. Hepatitis a Virus Outbreaks (N◦ = 3)

3.4. Norovirus Outbreaks (N◦ = 7)

route through contaminated food or water. Norovirus-contaminated water—both recreational and

Table 1. List of viral swimming pool-related outbreaks.

4. Occurrence of Enteric Viruses in Swimming Pools under Non-Epidemic Conditions

Received: 12 July 2018; Accepted: 25 July 2018; Published: 30 July 2018

IJERPH 2018, 15, 1612; doi:10.3390/ijerph15081612 32 www.mdpi.com/journal/ijerph

2. Materials and Methods

• articles focused only on clinical and laboratory aspects;

Strength of Evidence Epidemiological and Microbiological Criteria

• An analytical epidemiological study demonstrates a signiﬁcant association between

• Descriptive epidemiology suggests that the case/outbreak is related to the

• An analytical epidemiological study demonstrates a signiﬁcant association between

• Descriptive epidemiology suggests that the case/outbreak is related to exposure to

Figure 1. Flow chart of the selection process of articles.

3.1. Legionellosis in Relation to Recreational Water Source

Hot Spa Pools/Public Hot Spring Spa, Total

3.2. Epidemiological Investigations

3.3. Events with Sporadic Cases of Legionellosis

Pontiac Fever Legionnaires’ Disease

Pontiac Fever Legionnaires’ Disease

3.4. Outbreaks of Legionellosis

Event No. Legionella spp.