Legionella

Legionella is one of the emerging concerns of water quality in built water environments. Premise plumbing systems are among the recognised sources of infection. In the present study, colonisation of hot water networks in health care facilities, schools, hotels, private residences, office and industrial buildings was investigated. Data was analysed in connection with building and premise plumbing characteristics. Over 60% of all buildings were colonised by Legionella; counts were over 1000 CFU/L in 49%. The most prevalent type was Legionella pneumophila serogroup 2-14, isolated from 75% of the positive samples. Centrally produced hot water was found to be a key risk factor (46% of the samples were positive vs. 16% in individual systems); within this group the type of the building was less relevant. Colonisation levels in schools were similar to hotels or hospitals, representing a previously underestimated risk setting. Systems supplied by water from deep groundwater sources were significantly less likely to be colonised than more vulnerable sources (bank-wall filtration, surface water abstraction or karstic water; 28% vs. 51% positive), regardless of the type of treatment applied, including the presence of disinfection. The aggravating effect of larger, more complex and older buildings on colonisation was also confirmed. The present study represents the first baseline analysis, pre-empting regulation or monitoring requirements for Legionella. The prevalence of legionellae and the identified risk factors are indicative for other settings lacking targeted interventions. The statistically confirmed risk factors can serve as indicators for preliminary risk assessment and the prioritisation of interventions.

, we tested patients with severe respiratory illness for Legionella spp. infection and conducted a retrospective epidemiologic investigation. Of 1,805 patients tested, Legionella was detected in samples of 21 (1.2%); most were adults who had HIV or tuberculosis infections and were inappropriately treated for Legionella.

The electrochemical disinfection of germinated brown rice (GBR) circulating water and cooling tower water containing Legionella bacteria was investigated. Results showed the total aerobic plate counts (APC) in the treated GBR circulating water decreased significantly and the turbidity was largely improved at a pulse voltage of 1.0 kV; Legionella bacteria were also disinfected effectively at 1.0 kV. The disinfection was attributed to the synergistic effects of the oxide anode, the electric field, and the radicals formed during the electrochemical treatment. This suggests that electrochemical treatment could be applicable to the disinfection of water from other sources.

The study aim was to determine retrospectively whether the parallel use of 2 media [buffered charcoal yeast extract (BCYE) and medium of Wadowsky and Yee (MWY)] to isolate Legionella spp. from water samples taken from hospital water supply systems increased the sensitivity of the culture method as compared with methods/protocols in which only seeding on a selective medium is used. We analyzed the results obtained from 931 positive water samples. In 484 of the 931 positive water samples, Legionella spp. was isolated in the presence of other microorganisms; in 83% (400/484), we used MWY to count suspected colonies, which gave a lower number of unreadable plates. In the 447 samples containing only Legionella spp., the highest frequency of positive samples (93%, 418/447) was obtained with BCYE, whereas seeding on MWY yielded 78% (348/447) (P b 0.001). Evaluation of the influence of the media on the Legionella spp. counts obtained by the 2 media showed that BCYE agar produced significantly higher counts than MWY (P b 0.001). The major conclusions that may be drawn from our data are as follows: 1) BCYE gives a high recovery rate of positive samples (93%) and a much greater yield of Legionella spp. than MWY; 2) BCYE was necessary for the detection of non-L. pneumophila spp. which grew poorly on selective media; 3) selective media [MWY or GVPC (glycine, vancomycin, polymyxin B, and cycloheximide)] were necessary for the recovery of Legionella spp. when the non-selective medium (BCYE) was difficult to interpret because of contaminating background flora. The use of different media is recommended for routine water tests in hospitals.

A multiplex real-time PCR assay for detection of Legionella pneumophila and Legionella spp. and including an internal control was designed. Legionella species, L. pneumophila, and the internal control were detected simultaneously by probes labeled with 6-carboxy-fluorescein, hexachlorofluorescein, and indodicarbocyanine, respectively. Therefore, no postamplification analysis was required in order to distinguish the targets. The sensitivity of both assays was 2.5 CFU/ml,

The global outbreak of COVID-19 pandemic has forced the closure, or the reduction of the use of, many large facilities, as hospital wards at an unprecedented scale. Now, as many communities start planning for re-opening after the lockdown, previously abandoned buildings need to be prepared and cleared from becoming a breeding area for legionella or a source of infection for Legionnaires’ Disease (LD). Since LD attract up to 30% of sporadic cases of hospital-acquired pneumonia caused by legionellae and based on the literature reviewed, a multidisciplinary ‘communication model’ is proposed as an approach for enhancing the capacity of the hospitals, or any similar buildings, to mitigate any potential risk from legionella during the stages of re-opening. The approach takes into consideration all the challenges of LD, the legionella bacterium and the water stagnation. The main implication of this paper is that it brings different disciplines together to mitigate any possibilities of co-infections coming from the legionella, especially for the most vulnerable patients.

The aim of the present work was to validate the performances of a new molecular method comprehensive of water sample filtration, DNA extraction and Real-Time PCR for the quantification of Legionella spp. in clear water samples, in accordance with the recent ISO Technical Specification 12869:2012. All criteria and requirements were verified considering inclusivity and exclusivity, check of the calibration function, limit of detection and limit of quantification, recovery calculation, robustness and uncertainty of the entire method. The performances were validated as all parameters resulted to be in compliance with values detailed by the above mentioned standard. The described method proved to be specific, sensitive, accurate and it has been fully validated according to ISO/TS 12869:2012. The possibility of using a validated molecular method will improve the reliability of the results making it a promising tool that should be used in addition to cultural analysis. Moreover, these findings make it particularly suitable for a relatively inexpensive screening of water samples, reducing the turnaround time and the workload.

Legionella spp. are the causative agent of Legionnaire's disease and an opportunistic pathogen of significant public health concern. Identification and quantification from environmental sources is crucial for identifying outbreak origins and providing sufficient information for risk assessment and disease prevention. Currently there are a range of methods for Legionella spp. quantification from environmental sources, but the two most widely used and accepted are culture and real-time polymerase chain reaction (qPCR). This paper provides a review of these two methods and outlines their advantages and limitations. Studies from the last 10 years which have concurrently used culture and qPCR to quantify Legionella spp. from environmental sources have been compiled. 26/28 studies detected Legionella at a higher rate using qPCR compared to culture, whilst only one study detected equivalent levels of Legionella spp. using both qPCR and culture. Aggregating the environmental samples from all 28 studies, 2856/3967 (72%) tested positive for the presence of Legionella spp. using qPCR and 1331/3967 (34%) using culture. The lack of correlation between methods highlights the need to develop an acceptable standardized method for quantification that is sufficient for risk assessment and management of this human pathogen.

The Lascaux Cave contains a remarkable set of paintings from the Upper Palaeolithic. Shortly after discovery in 1940, the cave was modified for public viewing and, in 2001, was invaded by a Fusarium solani species complex. Benzalkonium chloride was used from 2001 to 2004 to eliminate the fungal outbreak. In this study, we carried out a sampling in most of the cave halls and galleries. Sequence analysis and isolation methods detected that the most abundant genera of bacteria were Ralstonia and Pseudomonas. We suggest that, as a result of years of benzalkonium chloride treatments, the indigenous microbial community has been replaced by microbial populations selected by biocide application.

Comparative sequence analysis of a 423-bp segment of the gyrA gene including a region homologous to the quinolone resistance-determining region (QRDR) of other species was evaluated as a novel typing method for Legionella strains. The study was performed with 29 reference strains representing 11 different Legionella species, with various serogroups, and with 13 clinical isolates of L. pneumophila. Pulsed-field gel electrophoresis and serotyping were employed for comparison of the clinical isolates. QRDR sequencing proved to be a highly discriminative tool for typing Legionellae, and permitted identification of species, serogroups and even different strains within serogroup 1. None of the isolates were resistant to quinolones in vitro and this correlated with dissence of mutations in the QRDR region. The data show that comparative sequence analysis of a short fragment of the gyrA gene is a potentially useful tool for typing of Legionella beyond the serogroup level. It is anticipated that mutations of the QRDR may arise in Legionella as a consequence of the introduction of quinolones as the agents of choice for the treatment of infections with this agent in immunocompromised patients. The employment of QRDR-typing maybe helpful in uncovering such mutations.

Availability of safe, pathogen-free drinking water is vital to public health; however, it is impossible to deliver sterile drinking water to consumers. Recent microbiome research is bringing new understanding to the true extent and diversity of microbes that inhabit water distribution systems. The purpose of this study was to determine how water chemistry in main distribution lines shape the microbiome in drinking water biofilms and to explore potential associations between opportunistic pathogens and indigenous drinking water microbes. Effects of disinfectant (chloramines, chlorine), water age (2.3 days, 5.7 days), and pipe material (cement, iron, PVC) were compared in parallel triplicate simulated water distribution systems. Pyrosequencing was employed to characterize bacteria and terminal restriction fragment polymorphism was used to profile both bacteria and eukaryotes inhabiting pipe biofilms. Disinfectant and water age were both observed to be strong factors in shaping bacterial and eukaryotic community structures. Pipe material only influenced the bacterial community structure (ANOSIM test, P < 0.05). Interactive effects of disinfectant, pipe material, and water age on both bacteria and eukaryotes were noted. Disinfectant concentration had the strongest effect on bacteria, while dissolved oxygen appeared to be a major driver for eukaryotes (BEST test). Several correlations of similarity metrics among populations of bacteria, eukaryotes, and opportunistic pathogens, as well as one significant association between mycobacterial and proteobacterial operational taxonomic units, provides insight into means by which manipulating the microbiome may lead to new avenues for limiting the growth of opportunistic pathogens (e.g., Legionella) or other nuisance organisms (e.g., nitrifiers).

The currently accepted culture techniques for the detection of Legionella spp. in water samples (AS/NZS 3896:1998 and ISO 11731 standard methods) are slow and laborious, requiring from 7 to 14 days for a result. We describe a fully validated rapid confirmation technique that uses real-time PCR incorporating the intercalating dye SYTO9 for the direct identification of primary cultures, significantly decreasing turnaround time and allowing faster remedial action to be taken by the industry.

Currently, the investigation of Legionella ecology falls into two distinct areas of research activity: (1) that Legionella multiply within water sources by parasitizing amoebic or ciliate hosts or (2) that Legionella grows extracellularly within biofilms. Less focus has been given to the overlaps that may occur between these two areas or the likelihood that Legionella employs multiple survival strategies to persist in water sources. It is likely that Legionella interacts with protozoa, bacteria, algae, fungi, etc., and biofilm components in a more complex fashion than multiplication or death due to the presence or absence of single components of these complex microbial systems. This paper addresses gaps that exist in the understanding of Legionella ecology and serves to pinpoint areas of future research. To assume that only one other class of organism is important to Legionella ecology may limit our understanding of how this bacterium proliferates in heated water sources and also limit our strategies for its control in the built environment.

Background: The prevention and control of legionellosis in hospital settings involves environmental sampling, among other measures. The data yielded by sampling constitute an important means of risk assessment and provide a valid basis on which to plan remedial (cleansing and disinfection) and preventive (maintenance) interventions. This retrospective study had 2 objectives: (1) to evaluate the utility of biofilm sampling at distal sites and (2) to identify an efficient environmental sampling strategy. Methods: Samples of hot water and biofilm were collected between June 1999 and March 2008 from 41 hospitals in Italy's Piemonte region. We analyzed results of the samples (water and biofilm) taken from the same site and results of the water samples taken from the recirculation loop and water samples taken from the distal sites during the same sampling run. Results: Microbiological analysis was performed on 3910 pairs of samples (water/biofilm). In 81% of the pairs, the results were concordant; in 17% of the pairs, Legionella was isolated only from the water samples, and in only 2% of the pairs was Legionella isolated from the biofilm sample alone. Data from 299 sampling runs show that 79% (236) of results from the water samples taken from the recirculation loop and water samples taken from the distal sites during the same sampling run were concordant, and 21% (63) were discordant. Conclusions: Our findings suggest that hospitals could safely adopt a simpler (water sampling only without biofilm sampling) and more efficient (monitoring of the entire system through sampling of recirculation loop water) environmental sampling policy.

Our laboratory has developed a novel real-time polymerase chain reaction (PCR) assay for the detection of Legionella pneumophila and differentiation from other Legionella spp. in clinical and environmental samples. The 23S rRNA gene was used as a target to detect all Legionella spp., and the mip gene was targeted for the specific detection of L. pneumophila in this multiplex Taqman® real-time PCR assay. The 23S rRNA gene is a novel target for Legionella testing; it detects all species and serogroups of Legionella without the contamination issues that accompany the use of the 16S rRNA gene as a target. This assay provides an analytical sensitivity of <1 colonyforming unit and a specificity of 100%. Because culture is important and provides a means for molecular typing via pulsed-field gel electrophoresis (PFGE), we developed a testing algorithm that includes both the new real-time PCR assay and culture for clinical and environmental samples and applied this algorithm during a period of 3 years. Of the 64 clinical samples received by our laboratory for Legionella testing during this period, PCR was found to be an essential diagnostic tool because only 13.3 % (2/15) clinical samples that were determined to be L. pneumophila were detected by culture during this period. Of the 276 environmental samples received for Legionella testing during this period, 140 were found to be positive for L. pneumophila. Of these 140 samples, 69.3% were detected by both PCR and culture methods, 29.3% were positive by PCR alone, and 1.4% were positive by culture methods alone. We feel these results indicate that our algorithm, including both PCR and culture, should be used for environmental samples. Among both the clinical and environmental Legionella samples identified by PCR, a subset was not suitable for culture because of issues of lengthy transport, antimicrobial treatment, or bacterial overgrowth. Samples like these are commonly submitted to our laboratory, so the use of our testing algorithm combining these methods is critical. We conclude that molecular and culture methods must be used in combination to provide the best and most comprehensive approach to laboratory detection and investigation of legionellosis.

A study of the quality of reclaimed water in treated effluent, after storage, and at three points in the distribution system of four plants in California, Florida, Massachusetts, and New York was conducted for 1 year. The plants had different treatment processes (conventional versus membrane bioreactor), production capacities, and methods for storage of the water, and the intended end uses of the water were different. The analysis focused on the occurrence of indicator bacteria (heterotrophic bacteria, coliforms, Escherichia coli , and enterococci) and opportunistic pathogens ( Aeromonas spp., enteropathogenic E. coli O157:H7, Legionella spp., Mycobacterium spp., and Pseudomonas spp.), as well as algae. Using immunological methods, E. coli O157:H7 was detected in the effluent of only one system, but it was not detected at the sampling points, suggesting that its survival in the system was poor. Although all of the treatment systems effectively reduced the levels of bacteria in the e...

Turkish bath or Moorish bath has a worldwide reputation and it is widely
frequented by Moroccans. This study was designed to determine, for the first time, the prevalence of colonization of Legionella species (L. spp.) in hot water collected from 51 Moorish baths “Hammams” and 11 showers of private apartments in Oujda city in the east of Morocco. Water sample analysis and Legionella identification was carried out according to the French Legionella spp. standard methods (AFNOR NF T90-431). Results showed that hot water from 37 Moorish baths (72.5%) contains L. spp. Twenty two baths (59.5%) of the positive baths were colonized by L. pneumpohila including the serogroup 1 (32.4%), and
sergoups 2-14 (56.7%). Fifteen positive baths (40.5%) were contaminated by Legionella species other than L. pneumophila. The colony count of L. pneumophila is less than 103 CFU/L, and remained below the tolerated threshold (a maximum of 990 CFU/L is reached in one bath). Seven domestic bathrooms (63.6%) were L. spp. positive. The most frequently isolated specie was L. pneumophila (71.4%), with most isolates belonging to serogroup 1 and 2-14 (4 baths /7 and 5 baths /7
respectively). A single water sample yielded a maximum colony count of 990 CFU/L of L. pneumophila. These findings show that hot water of Moorish baths and domestics showers are contaminated by Legionella. Although the degree of contamination did not reach the threshold level of infection, a constant monitoring of water should be put in place to control the risk of infection.

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)

The water used in dental unit waterlines (DUWLs) acts as a coolant for the high-speed equipments and as an irrigant during dental treatments. There are kind of water tanks. DUWLs provide a favorable environment for microbial biofilm and multiplation primarily due to the high surface in the tubing and the character of fluid dynamics in narrow, smooth-walled waterlines. Biofilms can harbour opportunist pathogens such as Legionella sp., Pseudomonas sp. Several studies have shown that DUWLs have high levels of microbial contamination. Presence of high level of microbial contamination is an important problem for dentists and dental patients who are immunocompromised. We collected water samples from DUWLs of 20 private dental offices. We have determined that only 2 (3.4%) out of 59 dental unit water samples were found to meet the standard (<200 CFU•ml −1) for DUWLs water quality by American Dental Association (ADA). Of the 59 water samples examined, 14 (24%) were positive for Pseudomonas sp. and 18 (30.5%) were positive for fungi. The most common 14 bacterial strains and seven fungi were isolated. Of bacterial strains, 57.1% were identified: Majority of the bacterial species isolated from our samples was identified as Pseudomonas fluorescens, Pasteurella haemolytica, Photobacterium damsela, Ochrobacter anthropi, Moraxella sp., Aspergillus flavus, Penicillium expansum. Legionella sp. were not detected in all water samples.

A microbiological environmental investigation was carried out in ten dental clinics in Italy. Microbial contamination of water, air and surfaces was assessed in each clinic during the five working days, for one week per month, for a three-month period. Water and surfaces were sampled before and after clinical activity; air was sampled before, after, and during clinical activity. A wide variation was found in microbial environmental contamination, both within the participating clinics and for the different sampling times. Before clinical activity, microbial water contamination in tap water reached 51,200 cfu/mL (colony forming units per milliliter), and that in Dental Unit Water Systems (DUWSs) reached 872,000 cfu/mL. After clinical activity, there was a significant decrease in the Total Viable Count (TVC) in tap water and in DUWSs. Pseudomonas aeruginosa was found in 2.38% (7/294) of tap water samples and in 20.06% (59/294) of DUWS samples; Legionella spp. was found in 29.96% (89/297) of tap water samples and 15.82% (47/297) of DUWS samples, with no significant difference between pre-and post-clinical activity. Microbial air contamination was highest during dental treatments, and decreased significantly at the end of the working activity (p b 0.05). The microbial buildup on surfaces increased significantly during the working hours. This study provides data for the establishment of standardized sampling methods, and threshold values for contamination monitoring in dentistry. Some very critical situations have been observed which require urgent intervention. Furthermore, the study emphasizes the need for research aimed at defining effective managing strategies for dental clinics.

We investigated Legionella and Pseudomonas contamination of hot water in a cross-sectional multicentric survey in Italy. Chemical parameters (hardness, free chlorine, and trace elements) were determined. Legionella spp. were detected in 33 (22.6%) and Pseudomonas spp. in 56 (38.4%) of 146 samples. Some factors associated with Legionella contamination were heater type, tank distance and capacity, water plant age, and mineral content. Pseudomonas presence was influenced by water source, hardness, free chlorine, and temperature. Legionella contamination was associated with a centralized heater, distance from the heater point >10 m, and a water plant >10 years old. Furthermore, zinc levels of <20 mg/L and copper levels of >50 mg/L appeared to be protective against Legionella colonization. Legionella species and serogroups were differently distributed according to heater type, water temperature, and free chlorine, suggesting that Legionella strains may have a different sensibility and resistance to environmental factors and different ecologic niches.

This study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae, and most Legionella species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 ؋ 10 ؊3 IFU, 5 ؋ 10 ؊2 color-changing units, and 1 CFU per reaction tube for C. pneumoniae, M. pneumoniae, and Legionella pneumophila, respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with C. pneumoniae infection, 9 samples from patients with M. pneumoniae infection, 19 samples from patients with Legionella species infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for C. pneumoniae and six were found to be positive for M. pneumoniae by using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniae and one additional sample positive for a Legionella species other than L. pneumophila; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.

Laboratories responsible for Legionella diagnostics around the world use a number of different culturing methods of non-equivalent sensitivities and specificities, to detect Legionella species in environmental samples. Specific countries usually standardize and use one approved method. For example, laboratories in Australia use the Australian Standard (AS) method and those in Europe, the International Standard method (ISO). However, no standard culturing methods have been established in South Africa to date. As a result, there is uncertainty about the true prevalence and most common species of Legionella present in the South African environment. In an attempt to provide guidelines for the development of a standard method specific for South Africa, the ISO, AS and a most probable number method were evaluated and compared. In addition, the effect of sample re-incubation with autochthonous amoebae on culture outcome was studied. Samples were collected from four environments, representing industrial water, mine water and biofilm. The samples were concentrated by membrane filtration and divided into three portions and cultured without pretreatment, after acid treatment and after heat treatment, on four culture media namely aBCYE, BMPA, MWY and GVPC agar. A selective approach, incorporating heat treatment, but not acid treatment, combined with culture on aBCYE and GVPC or MWY, was most appropriate for legionellae detection in the samples evaluated. Legionellae were cultured from 82% of the environmental samples we evaluated. In 54% of the samples tested, legionellae were present in numbers equal to or exceeding 10 2 colony-forming units per milliliter (cfu/ml). Legionella pneumophila serogroups (SGs) 1-14 were the most prevalent species and were present as single, or a combination of two or more SGs in a number of samples tested. Re-incubation of sample concentrates with autochthonous amoebae improved the culturability of legionellae in 50% of cultures on aBCYE and 25% on GVPC. r

Legionela spp. are ubiquitous in most environmental water sources; however, sewage treatment plants have not been examined as potential environmental reservoirs for these bacteria. This study used polymerase chain reaction, direct fluorescent-antibody staining, and culture methods to examine raw and treated sewage, ocean-receiving waters, and nearshore coastal environments for the presence of Legionella pneumophila and other Legionella spp. The study concluded that LegioneUla spp. are present in all phases of sewage treatment and that population numbers do not significantly decline through the treatment process. Ocean-receiving waters located 5 miles offshore, where the treated sewage is discharged, were found to contain LegioneUla spp., but ocean water between the discharge site and coastal bathing beaches was negative. This suggests that the Legionella spp. from the ocean discharge site were not reaching the nearshore beach waters. A flood control channel and river that entered the ocean were found to contain LegioneUa spp., and a nearby beach swimming area was also found to be positive, suggesting that land runoff from the flood control channel and river were the source of the Legionella spp. in the beach water samples that tested positive. 3618 on January 28, 2016 by guest http://aem.asm.org/ Downloaded from cleic Acids Res. 15:1335.

The existence and preliminary characterization of bacteriophages active against the Gram-negative human pathogen Legionella pneumophila, the causative agent of a very severe form of pneumonia, are reported. Four phages belonging to the family of the Myoviridae were isolated from various fresh water environments, and preliminary characterization showed that these crude preparations infect exclusively bacteria belonging to the genus Legionella. Standard phage amplification, purification, and characterization procedures were, however, not efficiently applicable making more research into these novel phages and their mechanism of infection necessary. The existence of Legionella bacteriophages is very promising for future applications such as the development of novel molecular tools, the design of new detection and typing methods, and the bioremediation of this environmental pathogen.

Cooling towers (CTs) are a leading source of outbreaks of Legionnaires&#39; disease (LD), a severe form of pneumonia caused by inhalation of aerosols containing Legionella bacteria. Accordingly, proper maintenance of CTs is vital for the prevention of LD. The aim of this study was to determine the distribution of Legionella in a subset of regionally diverse US CTs and characterize the associated microbial communities. Between July and September of 2016, we obtained aliquots from water samples collected for routine Legionella testing from 196 CTs located in eight of the nine continental US climate regions. After screening for Legionella by PCR, positive samples were cultured and the resulting Legionella isolates were further characterized. Overall, 84% (164) were PCR-positive, including samples from every region studied. Of the PCR-positive samples, Legionella spp were isolated from 47% (78), L. pneumophila was isolated from 32% (53), and L. pneumophila serogroup 1 (Lp1) was isolated...

Although the majority of cases of Legionnaires' disease (LD) are caused by Legionella pneumophila, an increasing number of other Legionella species have been reported to cause human disease. There are no clinical presentations unique to LD and hence accurate laboratory tests are required for early diagnosis. Therefore, we designed a real-time PCR assay that targets the 23S-5S rRNA intergenic spacer region (23S-5S PCR) and allows for detection of all Legionella species and discrimination of L. pneumophila from other Legionella species. In total, 271 isolates representing 50 Legionella species were tested and the assay was validated using 39 culture-positive and 110 culture-negative patient specimens collected between 1989 and 2006. PCR-positive results were obtained with all 39 culture-positive samples (100% sensitivity). Specimens that tested positive according to 23S-5S PCR, but were culture-negative, were further analysed by DNA sequencing of the amplicon or the macrophage infectivity potentiator (mip) gene. In addition to L. pneumophila, Legionella longbeachae, Legionella cincinnatiensis and Legionella micdadei were identified in the specimens. The assay showed a 7-log dynamic range displaying a sensitivity of 7.5 CFU/mL or three genome equivalents per reaction. Sixty-one specimens containing viruses or bacteria other than Legionellae were negative according to 23S-5S PCR, demonstrating its specificity. Use of this assay should contribute to the earlier detection of respiratory disease caused by Legionella species, as well as to increased rates of detection.

Although the majority of cases of Legionnaires' disease (LD) are caused by Legionella pneumophila, an increasing number of other Legionella species have been reported to cause human disease. There are no clinical presentations unique to LD and hence accurate laboratory tests are required for early diagnosis. Therefore, we designed a real-time PCR assay that targets the 23S-5S rRNA intergenic spacer region (23S-5S PCR) and allows for detection of all Legionella species and discrimination of L. pneumophila from other Legionella species. In total, 271 isolates representing 50 Legionella species were tested and the assay was validated using 39 culture-positive and 110 culture-negative patient specimens collected between 1989 and 2006. PCR-positive results were obtained with all 39 culture-positive samples (100% sensitivity). Specimens that tested positive according to 23S-5S PCR, but were culture-negative, were further analysed by DNA sequencing of the amplicon or the macrophage infectivity potentiator (mip) gene. In addition to L. pneumophila, Legionella longbeachae, Legionella cincinnatiensis and Legionella micdadei were identified in the specimens. The assay showed a 7-log dynamic range displaying a sensitivity of 7.5 CFU/mL or three genome equivalents per reaction. Sixty-one specimens containing viruses or bacteria other than Legionellae were negative according to 23S-5S PCR, demonstrating its specificity. Use of this assay should contribute to the earlier detection of respiratory disease caused by Legionella species, as well as to increased rates of detection.

The usefulness of two quantitative real-time PCR assays (qrt-PCRmip targeting Legionella pneumophila, and qrt-PCR16S targeting all Legionella species) performed on lower respiratory tract (LRT) samples for diagnostic and prognostic purposes in 311 patients hospitalized for community-acquired pneumonia (CAP) in Rhô ne-Alpes (France) was evaluated. The Now Legionella urinary antigen test (UAT) from Binax (Portland, ME, USA) was used as a reference test. Samples were divided into two groups. Group A included 255 CAP patients admitted to Chambery hospital in 2005 and 2006. The Now Legionella UAT was positive in 14 patients. Sensitivities, specificities, positive predictive and negative predictive values for both qrt-PCR tests were 63.6, 98.7, 77.7 and 97.4%, respectively. Group B included 56 consecutive legionellosis patients diagnosed during a 4-year period (2003-2006) at the Grenoble University Hospital. The qrt-PCR16S and qrt-PCRmip displayed a sensitivity of 82.14 and 80.4%, respectively. Among the 70 legionellosis cases, L. pneumophila serogroup 1 was isolated in 15;

Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.

Members of the Legionella genus find suitable conditions for their growth and survival in nuclear power plant cooling circuits. To limit the proliferation of Legionella pathogenic bacteria in nuclear power plant cooling circuits, and ensure that levels remain below regulatory thresholds, monochloramine treatment can be used. Although the treatment is highly effective, i.e. it reduces Legionella numbers by over 99%, Legionella bacteria can still be detected at low concentrations and rapid re-colonisation of circuits can occur after the treatment has ceased. The aim of this study was to develop an in vitro methodology for determining the intrinsic susceptibility of L. pneumophila strains, collected from various nuclear power plant cooling circuits subjected to different treatment conditions. The methodology was developed by using an original approach based on response surface methodology (RSM) combined with a multifactorial experimental design. The susceptibility was evaluated by the Ct factor. The susceptibility of environmental strains varies widely and is, for some strains, greater than that of known tolerant species; however, strain susceptibility was not related to treatment conditions. Selection pressure induced by monochloramine use did not result in the selection of more tolerant Legionella strains and did not explain the detection of Legionella during treatment or the rapid re-colonisation of cooling circuits after disinfection has ceased.

Nosocomial legionellosis is a growing concern worldwide. In Hungary, about 20% of the reported cases are health-care associated, but in the absence of legal regulation, environmental monitoring of Legionella is not routinely performed in hospitals. In the present study, 23 hospitals were investigated. The hot water distribution system was colonized by Legionella in over 90%; counts generally exceeded the public health limit value. Hot water temperature was critically low in all systems (&lt;45 °C), and large differences (3-38 °C temperature drop) were observed within buildings, indicating insufficient circulation. Most facilities were older than 30 years (77%); however, new systems (n = 3) were also shown to be rapidly colonized at low hot water temperature. Vulnerable source of drinking water, complex distribution system, and large volume hot water storage increased the risk of Legionella prevalence (OR = 28.0, 27.3, 27.7, respectively). Risk management interventions (including the...