Associations among pathogenic bacteria, parasites, and environmental and land use factors in multiple mixed-use watersheds

Over a five year period (2004-08), 1171 surface water samples were collected from up to 24 sampling locations representing a wide range of stream orders, in a river basin in eastern Ontario, Canada. Water was analyzed for Cryptosporidium oocysts and Giardia cyst densities, the presence of Salmonella enterica subspecies enterica, Campylobacter spp., Listeria monocytogenes, and Escherichia coli O157:H7. The study objective was to explore associations among pathogen densities/occurrence and objectively defined land use, weather, hydrologic, and water quality variables using CART (Classification and Regression Tree) and binary logistical regression techniques. E. coli O157:H7 detections were infrequent, but detections were related to upstream livestock pasture density; 20% of the detections were located where cattle have access to the watercourses. The ratio of detections:non-detections for Campylobacter spp. was relatively higher (>1) when mean air temperatures were 6% below mean study period temperature values (relatively cooler periods). Cooler water temperatures, which can promote bacteria survival and represent times when land applications of manure typically occur (spring and fall), may have promoted increased frequency of Campylobacter spp. Fifty-nine percent of all Salmonella spp. detections occurred when river discharge on a branch of the river system of Shreve stream order = 9550 was >83 percentile. Hydrological events that promote off farm/off field/in stream transport must manifest themselves in order for detection of Salmonella spp. to occur in surface water in this region. Fifty seven percent of L. monocytogenes detections occurred in spring, relative to other seasons. It was speculated that a combination of winter livestock housing, silage feeding during winter, and spring application of manure that accrued during winter, contributed to elevated occurrences of this pathogen in spring. Cryptosporidium and Giardia oocyst and cyst densities were, overall, positively associated with surface water discharge, and negatively associated with air/water temperature during spring-summer-fall. Yet, some of the highest Cryptosporidium oocyst densities were associated with low discharge conditions on smaller order streams, suggesting wildlife as a contributing fecal source. Fifty six percent of all detections of ≥2 bacteria pathogens (including Campylobacter spp., Salmonella spp., and E. coli O157:H7) in water was associated with lower water temperatures (<∼14 °C; primarily spring and fall) and when total rainfall the week prior to sampling was >∼27 mm (62 percentile). During higher water temperatures (>∼14 °C), a higher amount of weekly rainfall was necessary to promote detection of ≥2 pathogens (primarily summer; weekly rainfall ∼>42 mm (>77 percentile); 15% of all ≥2 detections). Less rainfall may have been necessary to mobilize pathogens from adjacent land, and/or in stream sediments, during cooler water conditions; as these are times when manures are applied to fields in the area, and soil water contents and water table depths are relatively higher. Season, stream order, turbidity, mean daily temperature, surface water discharge, cropland coverage, and nearest upstream distance to a barn and pasture were variables that were relatively strong and recurrent with regard to discriminating pathogen presence and absence, and parasite densities in surface water in the region.     

Landscape and seasonal factors influence Salmonella and Campylobacter prevalence in a rural mixed use watershed

Salmonella and Campylobacter prevalence in stream networks of the Satilla River Basin (SRB) were monitored monthly from August 2007 to August 2009 to study relationships between these pathogens and land use, presence of poultry houses and wastewater treatment plant (WWTP) discharge. Salmonella and Campylobacter were detected at all 10 stream sites and the three sites at the sole wastewater treatment plant (WWTP) in the study area. In all, 43% (129/299) and 62% (96/156) of samples were positive for Salmonella and Campylobacter, respectively, with detection frequency increasing in downstream sites with more poultry production and influence of WWTP discharge. Both Salmonella and Campylobacter detection frequencies were positively associated with the number of poultry houses in the subwatersheds, but agricultural land use as a proportion of the watershed was not a significant predictor of either pathogen. Fecal indicator bacterial levels were assessed and evaluated for their ability to predict the presence of pathogens. Of those examined, enterococci was most predictive; of the 129 samples positive for Salmonella, 88% (113/129) were detected when enterococci were above EPA single sample threshold (61 CFU 100 ml^-1); and of the 96 samples positive for Campylobacter, 90% (86/96) were detected when enterococci levels exceeded this level. Comparatively, Escherichia coli concentrations were above EPA single sample thresholds in 38% (49/129) of the positive Salmonella samples. Detection of the pathogens throughout the watershed indicated that there was potential for waterborne transmission especially in downstream areas that were more likely to have recreational users.     

Using SWAT, Bacteroidales microbial source tracking markers,and fecal indicator bacteria to predict waterborne pathogen occurrence in an agricultural watershed

Developing the capability to predict pathogens in surface water is important for reducing the risk that such organisms pose to human health. In this study, three primary data source scenarios (measured stream flow and water quality, modelled stream flow and water quality, and host-associated Bacteroidales) are investigated within a Classification and Regression Tree Analysis (CART) framework for classifying pathogen (Escherichia coli 0157:H7, Salmonella, Campylobacter, Cryptosporidium, and Giardia) presence and absence (P/A) for a 178 km² agricultural watershed. To provide modelled data, a Soil Water Assessment Tool (SWAT) model was developed to predict stream flow, total suspended solids (TSS), total N and total P, and fecal indicator bacteria loads; however, the model was only successful for flow and total N and total P simulations, and did not accurately simulate TSS and indicator bacteria transport. Also, the SWAT model was not sensitive to an observed reduction in the cattle population within the watershed that may have resulted in significant reduction in E. coli concentrations and Salmonella detections. Results show that when combined with air temperature and precipitation, SWAT modelled stream flow and total P concentrations were useful for classifying pathogen P/A using CART methodology. From a suite of host-associated Bacteroidales markers used as independent variables in CART analysis, the ruminant marker was found to be the best initial classifier of pathogen P/A. Of the measured sources of independent variables, air temperature, precipitation, stream flow, and total P were found to be the most important variables for classifying pathogen P/A. Results indicate a close relationship between cattle pollution and pathogen occurrence in this watershed, and an especially strong link between the cattle population and Salmonella detections.     

Bacteria,viruses, and parasites in an intermittent stream protected from and exposed to pasturing cattle: Prevalence,densities, and quantitative microbial risk assessment

Over 3500 individual water samples, for 131 sampling times, targeting waterborne pathogens/fecal indicator bacteria were collected during a 7-year period from 4 sites along an intermittent stream running through a small livestock pasture system with and without cattle access-to-stream restriction measures. The study assessed the impact of cattle pasturing/riparian zone protection on: pathogen (bacterial, viral, parasite) occurrence, concentrations of fecal indicators, and quantitative microbial risk assessments (QMRA) of the risk of Cryptosporidium, Giardia and Escherichia coli O157:H7 infection in humans. Methodologies were developed to compute QMRA mean risks on the basis of water samples exhibiting potentially human infectious Cryptosporidium and E. coli based on genotyping Crytosporidium, and E. coli O157:H7 presence/absence information paired with enumerated E. coli. All Giardia spp. were considered infectious. No significant pasturing treatment effects were observed among pathogens, with the exception of Campylobacter spp. and E. coli O157:H7. Campylobacter spp. prevalence significantly decreased downstream through pasture treatments and E. coli O157:H7 was observed in a few instances in the middle of the unrestricted pasture. Densities of total coliform, fecal coliform, and E. coli reduced significantly downstream in the restricted pasture system, but not in the unrestricted system. Seasonal and flow conditions were associated with greater indicator bacteria densities, especially in the summer. Norovirus GII was detected at rates of 7–22% of samples for all monitoring sites, and rotavirus in 0–7% of samples for all monitoring sites; pasture treatment trends were not evident, however. Seasonal and stream flow variables (and their interactions) were relatively more important than pasture treatments for initially stratifying pathogen occurrence and higher fecal indicator bacteria densities. Significant positive associations among fecal indicator bacteria and Campylobacter spp. detection were observed. For QMRA, adjusting for the proportion of Cryptosporidium spp. detected that are infectious for humans reduces downstream risk estimates by roughly one order of magnitude. Using QMRA in this manner provides a more refined estimate of beneficial management practice effects on pathogen exposure risks to humans.     

Impact of urbanization and agriculture on the occurrence of bacterial pathogens and stx genes in coastal waterbodies of central California

Fecal pollution enters coastal waters through multiple routes, many of which originate from land-based activities. Runoff from pervious and impervious land surfaces transports pollutants from land to sea and can cause impairment of coastal ocean waters. To understand how land use practices and water characteristics influence concentrations of fecal indicator bacteria (FIB) and pathogens in natural waters, fourteen coastal streams, rivers, and tidal lagoons, surrounded by variable land use and animal densities, were sampled every six weeks over two years (2008 & 2009). Fecal indicator bacteria (FIB; Escherichia coli and Enterococci) and Salmonella concentrations, the occurrence of Bacteroidales human, ruminant, and pig-specific fecal markers, E. coli O157:H7, and Shiga toxin (stx) genes present in E. coli, were measured. In addition, environmental and climatic variables (e.g., temperature, salinity, rainfall), as well as human and livestock population densities and land cover were quantified. Concentrations of FIB and Salmonella were correlated with each other, but the occurrence of host-specific Bacteroidales markers did not correlate with FIB or pathogens. FIB and Salmonella concentrations, as well as the occurrence of E. coli harboring stx genes, were positively associated with the fraction of the surrounding subwatershed that was urban, while the occurrence of E. coli O157:H7 was positively associated with the agricultural fraction. FIB and Salmonella concentrations were negatively correlated to salinity and temperature, and positively correlated to rainfall. Areal loading rates of FIB, Salmonella and E. coli O157:H7 to the coastal ocean were calculated for stream and river sites and varied with land cover, salinity, temperature, and rainfall. Results suggest that FIB and pathogen concentrations are influenced, in part, by their flux from the land, which is exacerbated during rainfall; once waterborne, bacterial persistence is affected by water temperature and salinity.     

Persistence of host-associated Bacteroidales gene markers and their quantitative detection in an urban and agricultural mixed prairie watershed

Microbial source tracking is an emerging tool developed to protect water sources from faecal pollution. In this study, we evaluated the suitability of real time-quantitative PCR (qPCR) Taqman assays developed for detection of host-associated Bacteroidales markers in a prairie watershed. The qPCR primers and probes used in this study exhibited high accuracy (88-96% sensitivity and ≥99% host specificity) in detecting Bacteroidales spp. that are associated with faeces from humans, ruminants, bovines, and horses. The ruminant- and human-associated markers were also found in high concentrations within individual faecal samples, ranging from 3.4 to 7.3 log₁₀ marker copy number g⁻¹ of individual host faeces. Following validation of host sensitivity and specificity, the host-associated Bacteroidales markers were detected in the Qu’Appelle Valley watershed of Saskatchewan, Canada which experiences a diversity of anthropogenic inputs. Concentrations of the ruminant marker were well-correlated with proximity to cattle operations and there was a correlation between the marker and Escherichia coli concentrations at these sites. Low concentrations of the human faecal marker were measured throughout the sampling sites, and may indicate a consistent influx of human faecal pollution into the watershed area. Persistence of each of the Bacteroidales host-associated marker was also studied in situ. The results indicated that the markers persist for shorter periods of time (99% decay in <8 days) compared with the conventional E. coli marker (99% decay in >15 days), suggesting they are effective at detecting recent faecal contamination events. The levels of Bacteroidales markers and E. coli counts did not correlate with the presence of the pathogenic bacteria, Salmonella spp. or Campylobacter spp. detected in the Qu’Appelle Valley. Collectively, the results obtained in this study demonstrated that the qPCR approach for detecting host-associated Bacteroidales spp. markers can be a useful tool in helping to determine host-specific impacts of faecal pollution into a prairie watershed.     

Seasonal relationships among indicator bacteria, pathogenic bacteria, Cryptosporidium oocysts, Giardia cysts, and hydrological indices for surface waters within an agricultural landscape

The South Nation River basin in eastern Ontario, Canada is characterized by mixed agriculture. Over 1600 water samples were collected on a bi-weekly basis from up to 24 discrete sampling sites on river tributaries of varying stream order within the river basin between 2004 and 2006. Water samples were analyzed for: densities of indicator bacteria (Escherichia coli, Clostridium perfringens, enterococci, total and fecal coliforms), the presence of pathogenic bacteria (Listeria monocytogenes, E. coli O157:H7, Salmonella spp., Campylobacter spp.), and densities of parasite Giardia cysts and Cryptosporidium oocysts. Relationships between indicator bacteria, pathogens, and parasite oocysts/cysts were overall weak, seasonally dependent, site specific, but primarily positive. However, L. monocytogenes was inversely related with indicator bacteria densities. Campylobacter, Salmonella, Giardia cysts and Cryptosporidium oocysts were most frequently detected in the fall. E. coli O157:H7 was detected at a very low frequency. Exploratory decision tree analyses found overall that E. coli densities were the most utilitarian classifiers of parasite/pathogen presence and absence, followed closely by fecal coliforms, and to a lesser extent enterococci and total coliforms. Indicator bacteria densities that classified pathogen presence and absence groupings, were all below 100 CFU per 100 mL⁻¹. Microorganism relationships with rainfall indices and tributary discharge variables were globally weak to modest, and generally inconsistent among season, site and microorganism. But, overall rainfall and discharge were primarily positively associated with indicator bacteria densities and pathogen detection. Instances where a pathogen was detected in the absence of a detectable bacterial indicator were extremely infrequent; thus, the fecal indicators were conservative surrogates for a variety of pathogenic microorganisms in this agricultural setting. The results from this study indicate that no one indicator or simple hydrological index is entirely suitable for all environmental systems and pathogens/parasites, even within a common geographic setting. These results place more firmly into context that robust prediction and/or indicator utility will require a more firm understanding of microorganism distribution in the landscape, the nature of host sources, and transport/environmental fate affinities among pathogens and indicators.     

Evaluation of host-specific Bacteroidales 16S rRNA gene markers as a complementary tool for detecting fecal pollution in a prairie watershed

Our ability to identify and eliminate fecal contamination of water, now and in the future, is essential to reduce incidences of waterborne disease. Bacterial source tracking is a recently developed approach for identifying sources of fecal pollution. PCR primers designed by Bernhard and Field [Bernhard, A.E., Field, K.G., 2000a. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66(10), 4571-4574] and Dick et al. [Dick, L.K., Bernhard, A.E., Brodeur, T.J., Santo Domingo, J.W., Simpson, J.M., Walters, S.P., Field, K.G., 2005. Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification. Appl. Environ. Microbiol. 71(6), 3184-3191] for the detection of human (HF183), pig (PF163) and ruminant (CF128) specific Bacteroidales 16s rRNA genetic markers were tested for their suitability in detecting fecal pollution in Saskatchewan, Canada. The sensitivity and specificity of these primers were assessed by testing eight raw human sewage samples and 265 feces from 12 different species in Saskatchewan. The specificity of each primer set was ≥94%. The accuracy of HF183 and PF163 to distinguish between the different species was 100%, whereas CF128 cross-reacted with 22% of the pig feces. Occurrence of the host-specific Bacteroidales markers and the conventional indicator Escherichia coli in relation to several enteropathogens was investigated in 70 water samples collected from different sites along the Qu'Appelle River (Saskatchewan, Canada). Human and ruminant fecal markers were identified in 41 and 14% of the water samples, respectively, whereas the pig marker was never detected in the river water. The largest concentrations in E. coli counts were concomitant to the simultaneous detection of HF183 and CF128. Thermotolerant Campylobacter spp., Salmonella spp. and Shiga toxin genes (stx1 and stx2)-positive E. coli (STEC) were detected in 6, 7 and 63% of the water samples, respectively. However, none of the stx positive water samples were positive for the E. coli O157:H7 gene marker (uidA). Odds ratios analysis suggests that CF128 may be predictive for the presence of Salmonella spp. in the river investigated. None of the fecal indicators were able to confidently predict the presence of thermotolerant Campylobacter spp. and STEC.     

Temporal flux and spatial dynamics of nutrients, fecal indicators, and zoonotic pathogens in anaerobic swine manure lagoon water

Confined animal feeding operations (CAFOs) often use anaerobic lagoons for manure treatment. In the USA, swine CAFO lagoon water is used for crop irrigation that is regulated by farm-specific nutrient management plans (NMPs). Implementation of stricter US environmental regulations in 2013 will set soil P limits; impacting land applications of manure and requiring revision of NMPs. Precise knowledge of lagoon water quality is needed for formulating NMPs, for understanding losses of N and C in ammonia and greenhouse gas emissions, and for understanding risks of environmental contamination by fecal bacteria, including zoonotic pathogens. In this study we determined year-round levels of nutrients and bacteria from swine CAFO lagoon water. Statistical analysis of data for pH, electrical conductivity (EC), inorganic and organic C, total N, water-soluble and total minerals (Ca, Cu, Fe, K, Mg, Mn, P, and Zn) and bacteria (Escherichia coli, enterococci, Clostridium perfringens, Campylobacter spp., Listeria spp., Salmonella spp., and staphylococci) showed that all differed significantly by dates of collection. During the irrigation season, levels of total N decreased by half and the N:P ratio changed from 9.7 to 2.8. Some seasonal differences were correlated with temperature. Total N and inorganic C increased below 19 °C, and decreased above 19 °C, consistent with summer increases in ammonia and greenhouse gas emissions. Water-soluble Cu, Fe, and Zn increased with higher summer temperatures while enterococci and zoonotic pathogens (Campylobacter, Listeria, and Salmonella) decreased. Although their populations changed seasonally, the zoonotic pathogens were present year-round. Increasing levels of E. coli were statistically correlated with increasing pH. Differences between depths were also found. Organic C, total nutrients (C, Ca, Cu, Fe, Mg, Mn, N, P, and Zn) and C. perfringens were higher in deeper samples, indicating stratification of these parameters. No statistical interactions were found between collection dates and depths.     

Evaluation of different analysis and identification methods for Salmonella detection in surface drinking water sources

The standard method for detecting Salmonella generally analyzes food or fecal samples. Salmonella often occur in relatively low concentrations in environmental waters. Therefore, some form of concentration and proliferation may be needed. This study compares three Salmonella analysis methods and develops a new Salmonella detection procedure for use in environmental water samples. The new procedure for Salmonella detection include water concentration, nutrient broth enrichment, selection of Salmonella containing broth by PCR, isolation of Salmonella strains by selective culture plates, detection of possible Salmonella isolate by PCR, and biochemical testing. Serological assay and pulsed-field gel electrophoresis (PFGE) can be used to identify Salmonella serotype and genotype, respectively. This study analyzed 116 raw water samples taken from 18 water plants and belonging to 5 watersheds. Of these 116, 10 water samples (8.6%) taken from 7 water plants and belonging to 4 watersheds were positive for a Salmonella-specific polymerase chain reaction targeting the invA gene. Guided by serological assay results, this study identified 7 cultured Salmonella isolates as Salmonella enterica serovar: Alnaby, Enteritidis, Houten, Montevideo, Newport, Paratyphi B var. Java, and Victoria. These seven Salmonella serovars were identified in clinical cases for the same geographical areas, but only one of them was 100% homologous with clinical cases in the PFGE pattern.     

Relationships between indicators,pathogens and water quality in an estuarine system

This study examined water and sediment samples for a range of indicator and pathogenic microorganisms from six sites in an urban estuary, Sydney, Australia. Water quality was affected by rainfall and sewage overflows which were associated with significant increases in the concentration of faecal coliforms, faecal streptococci, Clostridium perfringens spores, F-RNA bacteriophage, Aeromonas spp., Giardia and Cryptosporidium spp. However, in sediments, only faecal coliform concentrations were significantly increased by rainfall, although sewage overflow again resulted in increased concentrations of faecal coliforms, faecal streptococci, C. perfringens spores and Aeromonas. Isolation of Salmonella appeared to coincide with wet weather events and occasionally identical serotypes were detected in sediments at several locations within the estuary. However, isolations of enteric virus were sporadic and did not appear to be exclusively related to wet weather events. C. perfringens was identified as the most useful indicator of faecal pollution and was the only indicator significantly correlated to the presence of pathogenic Giardia (r = 0.41, p < 0.05) and the opportunistic bacterial genus Aeromonas (r = 0.39, p < 0.05). F-RNA bacteriophage was not significantly correlated with any of the pathogens examined.     

Seasonal stability of Cladophora-associated Salmonella in Lake Michigan watersheds

The bacterial pathogens Shigella, Salmonella, Campylobacter, and shiga toxin-producing E. coli (STEC) were recently found to be associated with Cladophora growing in southern Lake Michigan. Preliminary results indicated that the Salmonella strains associated with Cladophora were genetically identical to each other. However, because of the small sample size (n = 37 isolates) and a lack of information on spatial-temporal relationships, the nature of the association between Cladophora and Salmonella remained speculative. In this study, we investigated the population structure and genetic relatedness of a large number of Cladophora-borne Salmonella isolates from Lake Michigan (n = 133), as well as those isolated from stream and lake water (n = 31), aquatic plants (n = 8), and beach sands and sediments (n = 8) from adjacent watersheds. Salmonella isolates were collected during 2005-2007 between May and August from Lake Michigan beachsheds in Wisconsin, Illinois, and Indiana. The genetic relatedness of Salmonella isolates was examined by using the horizontal, fluorophore-enhanced rep-PCR (HFERP) DNA fingerprinting technique. While the Salmonella isolates associated with Cladophora exhibited a high degree of genetic relatedness (≥92% similarity), the isolates were not all genetically identical. Spatial and temporal relationships were evident in the populations examined, with tight clustering of the isolates both by year and location. These findings suggest that the relationship between Salmonella and Cladophora is likely casual and is related to input sources (e.g. wastewater, runoff, birds) and the predominant Salmonella genotype surviving in the environment during a given season. Our studies indicate that Cladophora is likely an important reservoir for Salmonella and other enteric bacterial pathogens in Lake Michigan beachsheds, which in turn may influence nearshore water quality.     

Bacterial pathogens in Hawaiian coastal streams--associations with fecal indicators, land cover, and water quality

This work aimed to understand the distribution of five bacterial pathogens in O’ahu coastal streams and relate their presence to microbial indicator concentrations, land cover of the surrounding watersheds, and physical-chemical measures of stream water quality. Twenty-two streams were sampled four times (in December and March, before sunrise and at high noon) to capture seasonal and time of day variation. Salmonella, Campylobacter, Staphylococcus aureus, Vibrio vulnificus, and V. parahaemolyticus were widespread —12 of 22 O’ahu streams had all five pathogens. All stream waters also had detectable concentrations of four fecal indicators and total vibrio with log mean ± standard deviation densities of 2.2 ± 0.8 enterococci, 2.7 ± 0.7 Escherichia coli, 1.1 ± 0.7 Clostridium perfringens, 1.2 ± 0.8 F⁺ coliphages, and 3.6 ± 0.7 total vibrio per 100 ml. Bivariate associations between pathogens and indicators showed enterococci positively associated with the greatest number of bacterial pathogens. Higher concentrations of enterococci and higher incidence of Campylobacter were found in stream waters collected before sunrise, suggesting these organisms are sensitive to sunlight. Multivariate regression models of microbes as a function of land cover and physical-chemical water quality showed positive associations between Salmonella and agricultural and forested land covers, and between S. aureus and urban and agricultural land covers; these results suggested that sources specific to those land covers may contribute these pathogens to streams. Further, significant associations between some microbial targets and physical-chemical stream water quality (i.e., temperature, nutrients, turbidity) suggested that organism persistence may be affected by stream characteristics. Results implicate streams as a source of pathogens to coastal waters. Future work is recommended to determine infectious risks of recreational waterborne illness related to O’ahu stream exposures and to mitigate these risks through control of land-based runoff sources.     

Quantification of pathogenic microorganisms and microbial indicators in three wastewater reclamation and managed aquifer recharge facilities in Europe

Managed Aquifer Recharge (MAR) is becoming an attractive option for water storage in water reuse processes as it provides an additional treatment barrier to improve recharged water quality and buffers seasonal variations of water supply and demand. To achieve a better understanding about the level of pathogenic microorganisms and their relation with microbial indicators in these systems, five waterborne pathogens and four microbial indicators were monitored over one year in three European MAR sites operated with reclaimed wastewater. Giardia and Cryptosporidium (oo)cysts were found in 63.2 and 36.7% of the samples respectively. Salmonella spp. and helminth eggs were more rarely detected (16.3% and 12.5% of the samples respectively) and Campylobacter cells were only found in 2% of samples. At the Belgian site advanced tertiary treatment technology prior to soil aquifer treatment (SAT) produced effluent of drinking water quality, with no presence of the analysed pathogens. At the Spanish and Italian sites amelioration of microbiological water quality was observed between the MAR injectant and the recovered water. In particular Giardia levels decreased from 0.24-6.14 cysts/L to 0-0.01 cysts/L and from 0.4-6.2 cysts/L to 0-0.07 cysts/L in the Spanish and Italian sites respectively. Salmonella gene copies and Giardia cysts were however found in the water for final use and/or the recovered groundwater water at the two sites. Significant positive Spearman correlations (p < 0.05, r_s range: 0.45-0.95) were obtained, in all the three sites, between Giardia cysts and the most resistant microbial markers, Clostridium spores and bacteriophages.     

Isolation and characterization of Bacteroides host strain HB-73 used to detect sewage specific phages in Hawaii

Previous studies have shown that Escherichia coli and enterococci are unreliable indicators of fecal contamination in Hawaii because of their ability to multiply in environmental soils. In this study, the method of detecting Bacteroides phages as specific markers of sewage contamination in Hawaii’s recreational waters was evaluated because these sewage specific phages cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17), were recovered from sewage samples in Europe and were reported to be effective in detecting phages from sewage samples obtained in certain geographical areas. However, GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple sources of sewage produced in Hawaii at high concentrations (5.2-7.3 × 10⁵ PFU/100 mL). These Bacteroides phages were considered as potential markers of sewage because they also survived for three days in fresh stream water and two days in marine water. Water samples from Hawaii’s coastal swimming beaches and harbors, which were known to be contaminated with discharges from streams, were shown to contain moderate (20-187 CFU/100 mL) to elevated (173-816 CFU/100 mL) concentrations of enterococci. These same samples contained undetectable levels (<10 PFU/100 mL) of F+ coliphage and Bacteroides phages and provided evidence to suggest that these enterococci may not necessarily be associated with the presence of raw sewage. These results support previous conclusions that discharges from streams are the major sources of enterococci in coastal waters of Hawaii and the most likely source of these enterococci is from environmental soil rather than from sewage.     

Escherichia coli, enterococci, and Bacteroides thetaiotaomicron qPCR signals through wastewater and septage treatment

Fecal indicators such as Escherichia coli and enterococci are used as regulatory tools to monitor water with 24 h cultivation techniques for possible input of sewage or feces and presence of potential enteric pathogens yet their source (human or animal) cannot be determined with routine methods. This critical uncertainty has furthered water pollution science toward new molecular approaches. Members of Bacteroides genus, such as Bacteroides thetaiotaomicron are found to have features that allow their use as alternative fecal indicators and for Microbial Source Tracking (MST). The overall aim of this study was to evaluate the concentration and fate of B. thetaiotaomicron, throughout a wastewater treatment facility and septage treatment facility. A large number of samples were collected and tested for E. coli and enterococci by both cultivation and qPCR assays. B. thetaiotaomicron qPCR equivalent cells (mean: 1.8 × 10⁷/100 mL) were present in significantly higher concentrations than E. coli or enterococci in raw sewage and at the same levels in raw septage. The removal of B. thetaiotaomicron target qPCR signals was similar to E. coli and enterococci DNA during the treatment of these wastes and ranged from 3 to 5 log₁₀ for wastewater and was 7 log₁₀ for the septage. A significant correlation was found between B. thetaiotaomicron marker and each of the conventional indicators throughout the waste treatment process for both raw sewage and septage. A greater variability was found with enterococci when compared to E. coli, and CFU and equivalent cells could be contrasted by various treatment processes to examine removal and inactivation via septage and wastewater treatment. These results are compared and contrasted with other qPCR studies and other targets in wastewater samples providing a view of DNA targets in such environments.     

Microbial risks associated with exposure to pathogens in contaminated urban flood water

Urban flood incidents induced by heavy rainfall in many cases entail flooding of combined sewer systems. These flood waters are likely to be contaminated and may pose potential health risks to citizens exposed to pathogens in these waters. The purpose of this study was to evaluate the microbial risk associated with sewer flooding incidents. Concentrations of Escherichia coli, intestinal enterococci and Campylobacter were measured in samples from 3 sewer flooding incidents. The results indicate faecal contamination: faecal indicator organism concentrations were similar to those found in crude sewage under high-flow conditions and Campylobacter was detected in all samples. Due to infrequent occurrence of such incidents only a small number of samples could be collected; additional data were collected from controlled flooding experiments and analyses of samples from combined sewers. The results were used for a screening-level quantitative microbial risk assessment (QMRA). Calculated annual risks values vary from 5 × 10⁻⁶ for Cryptosporidium assuming a low exposure scenario to 0.03 for Giardia assuming a high exposure scenario. The results of this screening-level risk assessment justify further research and data collection to allow more reliable quantitative assessment of health risks related to contaminated urban flood waters.     

Searching for Escherichia coli O157 in drinking and recreational waters in Northern Greece

Escherichia coli O157:H7 has been recognized in sporadic cases and outbreaks, as an important cause of bloody diarrhea and also a leading cause of acute renal failure in children. The purpose of this study was to investigate the presence of E. coli O157 in drinking and recreational water samples from Northern Greece. For this, a total of 1974 colonies of E. coli that were isolated from 1267 water samples were recultured on sorbitol-MacConkey agar. Out of them 121 were found non-sorbitol fermenting, but none had the somatic O157 antigen. This finding supports the hypothesis that drinking and recreational waters should not be considered possible routes of E. coli O157:H7 transmission in Northern Greece.     

Contamination of surface and potable water in South Asia by Salmonellae: Culture-independent quantification with molecular beacon real-time PCR

Low numbers (15-100 CFU) of Salmonella in food or water may pose a public health risk. The management of infections caused by Salmonella spp. during outbreaks or forecasting of contamination of aquatic resources largely depends on rapid, sensitive and accurate diagnostic in few hours. In this study, a real-time PCR assay in Molecular-Beacon format was developed and culture-independent quantitative enumeration of Salmonella spp. in surface and potable water is being reported for the first time from northern part of India. Molecular Beacon was designed in highly conserved region of invA gene (present in wide range of Salmonella serotypes including all subspecies) encoding an essential component of the invasion associated specialized type Ø protein secretion apparatus for detection of Salmonella spp. in water. The assay could detect directly 10 and 1 genomic equivalent of Salmonella typhimurium ATCC 14028 per PCR with detection probability of 100 and 20%, respectively. Further, the assay could detect 10 CFU/PCR or more of reference strain (S. typhimurium ATCC 14028) without any enrichment in the presence of 10⁸ CFU ml^− 1 of non-pathogenic E. coli (E. coli DH5α) with 100% detection probability. The assay could enumerate Salmonella spp. in surface (n = 40) and potable waters (n = 10) directly (without enrichment). Results indicate that northern India is at high risk of developing Salmonella borne infections. Further, real-time PCR assay in Molecular Beacon format can be used for identification of critical contamination points in natural water resources and potable water distribution systems, necessary to implement vaccination plan timely for prevention of waterborne outbreaks caused by Salmonella spp.     

Escherichia coli O157:H7 in drinking water from private water supplies in the Netherlands

The microbiological quality of drinking water from 144 private water supplies in the Netherlands was tested and additionally the occurrence of Escherichia coli O157 was examined. Faecal indicators were enumerated by using standard membrane filtration methods. The presence of E. coli O157 was determined using a specific enrichment method. Eleven percent of the samples contained faecal indicators whereas E. coli O157:H7 was isolated from 2.7% of the samples that otherwise met the drinking water standards. The E. coli O157 positive water supplies were located on camp-sites in agricultural areas with large grazer densities. Pulsed field gel electrophoresis (PFGE) analysis suggested that cattle might have been the cause of contamination. Our results indicate that compliance with microbiological quality standards obtained in routine monitoring does not always guarantee the absence of pathogens. The presence of pathogens such as E. coli O157 may suggest possible health consequences; however, a risk assessment process should be performed as the monitoring of both faecal indicator parameters and pathogens do not predict the effect of microbial contamination of drinking water on a population.