Pol. J. Environ. Stud. Vol. 26, No.

6 (2017), 2539-2543
DOI: 10.15244/pjoes/70000 ONLINE PUBLICATION DATE: 2017-10-13

Original Research
Seasonal Potential Transmission of Pathogens
Associated with Ground Drinking Water

Wiktor Halecki1,2*, Wiesław Barabasz3**

Department of Land Reclamation and Environmental Development, University of Agriculture,
1

Al. Mickiewicza 24/28, 30-059 Kraków, Poland

2
Department of Biometry and Forest Productivity, Faculty of Forestry, University of Agriculture in Krakow,
Al. 29 Listopada 46, 31-425 Kraków, Poland
3
Department of Microbiology, Faculty of Agriculture and Economics, University of Agriculture,
Mickiewicza 24/28, 30-059 Kraków, Poland

Received: 30 December 2016

Accepted: 29 March 2017

Abstract

The objective of this investigation was to assess the sanitary status of groundwater to ensure hygienic
requirement water standards. Pathogens in water may originate from animals and anthropogenic waste. In
a semi-rural area microorganisms are signiicant indicators connected with human activities in the under-
ground water. Notably, our results showed that a more eficient diagnostic group to determine the level of
contamination was proteolytic bacteria (p<0.01). Results showed that pathogenic bacteria such as Shigella
sp., Salmonella sp., Escherichia coli, and total coliforms were not detected. The highest number of Sta-
phylococcus sp. and fungal spores were found in the spring. Considerable additional research is needed to
appropriately measure microbial communities in underground water.

Keywords: diagnostic test, microbial indicators, seasonality, underground water, water pathogens

Introduction likewise the availability of oxygen as well as high content

of humus. Otherwise, underground water is sensitive to
Pathogenic microorganisms are suitable indicators contamination [7-8], especialy in households served with
of drinking water [1-4]. Determining underground either on-site wastewater systems or private wells in semi-
water is practical for a quality monitoring program [5]. rural areas [9]. Particularly in the spring, water circulation
Organic matter, ions, mineral form, nutrients, and toxic changes temperature and concentration of nutrients. For
substances inluence the growth of microorganisms that reason, the abundance of microorganisms luctuates
in the underground water [6]. It was shown that the in the water throughout the season. Overall, changes of
relationship between an individual feature of pathogenic temperature principally limit the growth of pathogenic
microorganisms and speciic hydrological parameters bacteria in the water [10]. In addition, pathogenic
requires appropriate micro-ecological conditions, microorganisms play a key role for the quality of water
and human consumption [11-12].
Local regulations restrict aquifer pollution. Our paper
focuses on reclaiming drinking water as established by the
*e-mail: [email protected] standard approach for common indicators of exposure to
**e-mail: [email protected]
2540 Halecki W., Barabasz W.

infection. Though disregarded in most empirical studies, Determining total viable count (TVC) of mesophilic
the major highlight of this research was to examine bacteria in 1 cm3 of water was applied onto nutrient agar,
potential health risks from the transmission of microbial MPA, after 48 hours of incubation at 37ºC. Gelatin was
pathogens. used to enumerate the total number of proteolytic bacteria
after 48 hours of incubation at 20ºC. Salmonella sp. and
Shigella sp. were incubated on SS medium at 37ºC after
Materials and Methods 48 hours. Chapman’s substratum was used to identify the
total number of Staphylococcus sp. after 48 hours of being
Study Area and Microbiological Analysis incubated at 37ºC. Microbiological examinations were
strictly performed in ive parallel repetitions from the
Water samples were collected between May 2011 same sample, and explicitly notiied as colony-forming
and January 2016 from suburban, semi-rural, and private units (CFU/cm3).
wells in: Rzezawa (10 m depth), Jodłówka (10 m), Krzec-
zów (6 m), Borek (6 m), and Łazy (8 m) located in Po- Statistical Analysis
land’s Małopolska Province. One plastic bottle (500 mL)
was chosen for each sample. Samples were tented to limit We carried out tests based on a diagnostic model that
the amount of intaken microorganisms throughout the was designed to classify the most accurate microbial
season. communities to detect contaminated water samples. We
Coliform bacteria were determined by presumptive procedured resampling of residuals data so that they
stage, conirmed stage, and completed test in the inal retained the structure of the interpolation of observations
reference method. A preliminary test involved inserting a that are less than or equal to particular values. Receiver
suitable volume of water sample to the substrate with LPB operating characteristic (ROC) was performed to reveal
Durham-tubes. Durham-tubes were also immersed in the signiicant differences between sensivity for mesophilic
medium to test for the production of the gas (hydrogen or and proteolytic microbial communities during the
carbondioxide). Durham tubes were inserted upside down season. Coeficient interval was measured by De Long
in the test tubes to detect gas production. Basal medium nonparametric method [15]. Comparison between the
containing a single disaccharide source – lactose – was numbers on each site was obtained using area under
used for this purpose. Agar medium was used to determine curve (AUC). Statistical calculations were surveyed using
the number of Escherichia coli and total coliforms after 24 PQ stat (version 5.1 Pl). Canoco for Windows (version
hours of incubation at 37ºC. To approve results for the 4.51) was applied to evaluate a potenial transmission
isolation of total coliforms, we additionaly incorporated of pathogens using the principal component analysis
samples of a previously prepared technique that exhibited a (PCA)  technique. All PCA loading is presented in
yellow-green metallic sheen on Endo’s medium subjected Table 1. Outlier data were rejected from auxiliary
to inal testing. Then we tried to detect colonies by color examination.
change of the pH indicator (colonies surrounded by dark
pink zone) when a suficient amount of common end-
products of bacterial fermentation acid was produced. The
total number of saprophytic fungal spores were incubated
at 28ºC after 72 hours of incubation.
Short-term experiments have indicated that warmer
temperatures can alter fungal biomass production. Spores
are associated with responses to warming by conducting
selection experiments. Spores are the most important
propagules for most fungi, and the impact these organisms
produce on their hosts will depend on the ability for fast
spore germination and colonization. Spore germination
may be affected by many factors – especially temperature
– which is required for growth. Fungi are prone to
temperature, and may adapt to warming from moderately
warm (16ºC) and warm (28ºC) in spore production
[13]. Speciically, we expected that 28ºC is considered
as representative value during all seasons in order to
formulate a predicting model under laboratory conditions.
Over 37ºC, fungi produce more spores per unit biomass
[14] and may not respond to our conclusions in this study.
Optimal mycelial growth of conidial germination is
initiated by germination of spores, and our standardization
can establish ield data for thousands of generations Fig.1. Diagnostic test between mesophilic and proteolytic
within a few months. bacteria.
Seasonal Potenial Transmission... 2541

Fig. 3. Mean number of Staphylococcus sp. in the sampling area.

Fig. 2. Box plot for the saprophytic fungal spores in 1 cm3 of

water from the sampling area.
level of contamination. We concluded that the amount of
Staphylococcus sp. was less abundant. Only in the spring
was the increase identiied (Fig. 3).
Results and Discussion PCA analysis revaled that Staphylococcus sp. made
no important contribution to the prediction of the density
Diagnostic Test of the faecal contamination indicators. 8 m depth has a
better value as a predictor of the presence of proteolythic
Only faecal and coagulant substances originating from bacteria and mesophylic bacteria (Fig. 4).
animal farms could initiate interaction with the microbial Two principal components were useful for presenting
community. Here, however, greater emphasis was placed the correlation between variables by the observation
on certain elements of the microbial community growing numbers. The irst principal component axis explained
at 20ºC, mostly Gram-negative bacteria. Results showed 58.15% of variance. The second component of axis
a response by pathogens to environmental factors. ROC explained 21.71% of variance (Fig. 4). Mesophilic bacteria
diagnosis (Fig. 1) was more appropriate to identify the and proteolytic bacteria represented the most important
sensitivity of proteolytic bacteria, and denoted signiicant factors (>0.6) quantiied by multivariate analysis
differences at p<0.01 (AUC = 0.54). (Table 1).
Identiication of Staphylococcus sp. is important for
Seasonal Variability diagnosing potentially pathogenic microorganisms in

Human households impact chemical luctuation,

especially for the development of pathogens during
anaerobic digestion treatment of cattle manure [16].
Efluents from biodigesters, which are widely spread into
farmland for agriculture use, is caused by local people
[15]. Microbial communities produce endotoxins such
as lipopolysaccharide leaching into underground water.
Moreover, the most responsive factor for microbial
communities is temperature. Therefore, diagnostic tests
require detection of pathogenic bacteria [17-18], and
focus on primarily fungal spores, which also should be
monitored. Numerical results released by the evaluation
of the total number of fungal spores varied during the
growing season. The major number of fungal spores
varied, particularly in the autumn (Fig. 2). A slightly
longer survival period of fungal spores was observed in
the underground water [19].
The increase of microorganisms growing at 20ºC
may transmit available organic matter in the water. Hence,
the signaling effects through the high contamination range Fig. 4. PCA for potenial transmission of pathogens in rural
of animal faecal matter was observed in the study area. wells. Open squares indicate autumn and closed squares indicate
The variability of Staphylococcus sp. also was examined spring. The summation includes the entire experimental data.
in our ield research and in effect was ascertained at The irst and the second principal components explain 80% of
each point in time, which was essential for assessing the the variance.
2542 Halecki W., Barabasz W.

Table 1. Rotated component loading matrix obtainted from pathogens associated with ground drinking water.
Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6
Staphylococcus sp. -0.01 -0.06 -0.04 -0.007 0.19 0.97
Mesophilic bacteria 0.76 0.63 -0.71 0.28 0.06 -0.003
Proteolytic bacteria -0.14 0.73 0.65 -0.07 0.03 0.07
6 m depth -0.01 0.07 0.08 0.18 0.95 -0.19
8 m depth 0.008 0.12 0.24 0.93 -0.20 0.05
10 m depth -0.98 -0.15 0.03 0.017 -0.009 -0.0006
Bold numbers indicate the most important factors of each principal component (> 0.6).

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