Levels Of Fecal Indicator Bacteria Research Articles

Spatio-Temporal Variation in the Exceedance of Enterococci in Lake Burley Griffin: An Analysis of 16 Years' Recreational Water Quality Monitoring Data.

Recreational waterbodies with high levels of faecal indicator bacteria (FIB) pose health risks and are an ongoing challenge for urban-lake managers. Lake Burley Griffin (LBG) in the Australian Capital city of Canberra is a popular site for water-based recreation, but analyses of seasonal and long-term patterns in enterococci that exceed alert levels (>200 CFU per 100 mL, leading to site closures) are lacking. This study analysed enterococci concentrations from seven recreational sites from 2001-2021 to examine spatial and temporal patterns in exceedances during the swimming season (October-April), when exposure is highest. The enterococci concentrations varied significantly across sites and in the summer months. The frequency of the exceedances was higher in the 2009-2015 period than in the 2001-2005 and 2015-2021 periods. The odds of alert-level concentrations were greater in November, December, and February compared to October. The odds of exceedance were higher at the Weston Park East site (swimming beach) and lower at the Ferry Terminal and Weston Park West site compared to the East Basin site. This preliminary examination highlights the need for site-specific assessments of environmental and management-related factors that may impact the public health risks of using the lake, such as inflows, turbidity, and climatic conditions. The insights from this study confirm the need for targeted monitoring efforts during high-risk months and at specific sites. The study also advocates for implementing measures to minimise faecal pollution at its sources.

Land use and rainfall influences on bacterial levels and sources in stormwater ponds.

Urban stormwater runoff is a known source of microbial contamination of stormwater ponds. However, less is known about the influences of land use and rainfall on microbial quality over time in these receiving waters. In this study, two fecal indicator bacteria (FIB), namely Escherichia coli and thermotolerant coliforms, were monitored in three stormwater ponds in Calgary, Alberta, Canada. The stormwater ponds were selected due to their potential as water sources for beneficial uses such as irrigation, which requires lower water quality than drinking water, thereby alleviating the pressure on the city's potable water demands. The selected stormwater ponds vary in size and shape, contribution catchment size, and percentages of several primary land use types. Microbial source tracking for human, dog, seagull, Canada goose, ruminant, and muskrat was also conducted to determine sources of bacterial contamination in the stormwater ponds. Sampling was conducted near the pond surface and adjacent to the shoreline, specifically near the outfalls that discharge stormwater runoff into the ponds and the inlets that convey water out of the ponds. Overall, the FIB concentrations in the vicinity of pond outfalls were significantly or relatively higher than those near pond inlets. The contamination in the McCall Lake and the Country Hills stormwater ponds showed higher amounts of human markers (40 to 60%) compared to the Inverness stormwater pond (< 20%), which coincided with their higher FIB concentration medians. The results revealed that stormwater drained from catchments with a higher percentage of commercial land use was more contaminated than those with primary residential land use, while the impacts of residential development on the FIB levels in the Inverness stormwater pond were not obvious. Furthermore, FIB concentrations in the ponds increased in response to both rain events and inter-event dry periods, with human-specific markers being predominant despite the high levels of animal markers during inter-event dry periods. Human-origin sources might be among the main microbial loading contributors in the pond catchments in general. All these findings can inform the development or improvement of measures for mitigating microbial pollution, strategies for reusing stormwater, and maintenance programs.

Scale of analysis drives the observed ratio of spatial to non-spatial variance in microbial water quality: insights from two decades of citizen science data.

While fecal indicator bacteria (FIB) testing is used to monitor surface water for potential health hazards, observed variation in FIB levels may depend on the scale of analysis (SOA). Two decades of citizen science data, coupled with random effects models, were used to quantify the variance in FIB levels attributable to spatial versus temporal factors. Separately, Bayesian models were used to quantify the ratio of spatial to non-spatial variance in FIB levels and identify associations between environmental factors and FIB levels. Separate analyses were performed for three SOA: waterway, watershed, and statewide. As SOA increased (from waterway to watershed to statewide models), variance attributable to spatial sources generally increased and variance attributable to temporal sources generally decreased. While relationships between FIB levels and environmental factors, such as flow conditions (base versus stormflow), were constant across SOA, the effect of land cover was highly dependent on SOA and consistently smaller than the effect of stormwater infrastructure (e.g. outfalls). This study demonstrates the importance of SOA when developing water quality monitoring programs or designing future studies to inform water management.

Bacteriological Assessment of Fecal Contamination in the Sediments of the Gulf of Annaba (Southern Mediterranean): A Preliminary Investigation

This study investigated the bacteriological and physicochemical quality of seawater and sediment samples collected from four sampling sites in the Gulf of Annaba (Northeastern Algeria) over a one-year period. Culture-based techniques were used to quantify and assess Fecal Indicator Bacteria (FIB) and potentially pathogenic bacteria. Additionally, various physicochemical parameters including temperature, pH, salinity, dissolved oxygen, and suspended solids were measured. The results revealed seasonal variations in the physicochemical variables, reflecting the influence of environmental conditions in the research area. The highest concentrations of FIB were observed in samples obtained from Sidi Salem and Rezgui Rachid, indicating a possible association with sewage contamination. Furthermore, the sediments collected from all sites exhibited higher levels of FIB and potentially pathogenic bacteria compared to the seawater samples, particularly during the summer and fall seasons.

Inclusion of hydrodynamic properties of bathing waters is critical in selecting faecal indicators to assess public health impacts of faecal contamination

The EU Bathing Water Directive (BWD) requires member states to assess bathing water quality according to the levels of faecal indicator bacteria (FIB) in designated bathing areas. However, this criterion has two significant limitations given that the BWD does not; (i) account for differences in hydrodynamic properties of bathing waters and, (ii) assumes that all faecal pathogens decay equally in aquatic environments. This study simulated sewage discharge events in three hypothetical aquatic environments characterised by different advection and dispersion parameters in the solute transport equation. Temporal changes in the downstream concentration of six faecal indicators were determined in simulations that utilised measured decay rates of each faecal indicator from a programme of controlled microcosm experiments in fresh and seawater environments. The results showed that the decay rates of faecal indicators are not a critical parameter in advection dominant water bodies, such as in fast-flowing rivers. Therefore, faecal indicator selection is less important in such systems and for these, FIB remains the most cost-effective faecal indicator to monitor the public health impacts of faecal contamination. In contrast, consideration of faecal indicator decay is important when assessing dispersion and advection/dispersion dominant systems, which would pertain to transitional (estuarine) and coastal waterbodies. Results suggest that the inclusion of viral indicators, such as crAssphage and PMMoV, could improve the reliability of water quality modelling and minimise the risk of waterborne illnesses from faecal contamination.

Land use as a critical determinant of faecal and antimicrobial resistance gene pollution in riverine systems

The WHO recognises antimicrobial resistance (AMR) as a global health threat. The environment can act as a reservoir, facilitating the exchange and the physical movement of resistance. Aquatic environments are at particular risk of pollution, with large rivers subject to pollution from nearby human, industrial or agricultural activities. The land uses associated with these activities can influence the type of pollution. One type of pollution and a likely contributor to AMR pollution that lowers water quality is faecal pollution. Both pose an acute health risk and could have implications for resistance circulating in communities. The effects of land use are typically studied using physiochemical parameters or in isolation of one another. However, this study aimed to investigate the impact of different land uses on riverine systems. We explored whether differences in sources of faecal contamination are reflected in AMR gene concentrations across agricultural and urban areas. Water quality from three rivers impacted by different land uses was assessed over one year by quantifying faecal indicator bacteria (FIB), microbial source tracking markers (MST) and AMR genes. In addition, a multiparametric analysis of AMR gene pollution was carried out to understand whether agricultural and urban areas are similarly impacted. Faecal indicators varied greatly, with the highest levels of FIB and the human MST marker observed in urban regions. In addition, these faecal markers correlated with AMR genes. Similarly, significant correlations between the ruminant MST marker and AMR gene levels in agriculture areas were observed. Overall, applying multiparametric analyses to include AMR gene levels, separation and clustering of sites were seen based on land use characterisation. This study suggests that differences in prescription of antimicrobials used in animal and human healthcare may influence environmental resistomes across agricultural and urban areas. In addition, public health risks due to exposure to faecal contamination and AMR genes are highlighted.

Fecal Enterococci Levels in Selected Tributaries of the Pampanga River Basin, Philippines, and Their Relation to Land Use

This study aims to generate data which can be used as a potential starting point for the updating of the Philippine Water Quality Criteria and the determination of the true impact of land use to the fecal contamination of the Pampanga River Basin (PRB), the largest subwatershed of Manila Bay. Levels of fecal indicator bacteria (FIB) were determined in the selected tributaries of the PRB, representing three land use categories, namely, the forest/woodland (control), agricultural and residential lands. FIB were quantified in order to investigate the potential contribution of the selected areas in the fecal contamination of the PRB. The study was conducted in 2021 covering March, May, June, July, and September to represent the dry (March and May) and wet (June, July, and September) seasons. Counts of FIB, namely thermotolerant coliform, E. coli, and enterococci were qualitatively correlated with the results of the ocular survey and key informant interview based on known fecal contributors and their relevant rainfall data. FIB counts of water bodies in the selected agricultural and residential land use categories had Geometric Mean (GM) counts that are statistically greater than those of bodies of water near the representative forest/woodland (control), and exceeded the acceptable GM limits for all FIB, regardless of the season. Notably, the GM values recorded for the waters near the selected forest/woodland (control) passed the water quality criteria for all measured FIB parameters for both seasons. Furthermore, enterococci levels in the control site were statistically lower during the wet season. These initial findings suggest that agricultural and residential land use categories could be major contributors to the unacceptable water quality of tributaries of the Pampanga River Basin. The prevalence of thermotolerant coliforms and E. coli was noted regardless of rainfall and land use, indicating these FIB may not be adequate as water quality indicators. With their ability to survive and persist in fecally contaminated sediments in water bodies and in nutrient-poor environments, enterococci could be more definitive indicators of fecal contamination and microbiological quality of environmental waters.

MICROBIOLOGICAL STUDY OF THE ANTHROPOGENIC PRESSURE ON THE YANTRA RIVER IN THE REGION OF VELIKO TARNOVO

The report discusses the microbiological status of the Yantra River in the region of the town of Veliko Tarnovo. Water samples collected at 11 points of the river stretch were analyzed for quantification of E. coli and coliforms, intestinal enterococci and heterotrophic bacteria. It was found increased levels of fecal indicator bacteria in the river waters, varying over time and along the riverside. The number of viable E. coli and enterococci increased under the influence of untreated wastewater influx and the Belitsa River.

Influence of temperature and water quality on the persistence of human mitochondrial DNA, human Hf183 Bacteroidales, fecal coliforms and enterococci in surface water in human fecal source tracking context.

Mitochondrial DNA (mtDNA) is used as a genetic marker to track fecal contamination in surface water. Its potential to effectively discriminate between the nonpoint sources of fecal pollution (e.g. human, livestock) in water environments is relevant for water quality management. However, there is a lack of knowledge about the environmental persistence of mtDNA in relation to those of other microbial parameters, such as fecal indicator bacteria (FIB). In this study, mesocosms composed of water collected from four rivers and tap water were spiked with raw wastewater to mimic human fecal contamination. Mesocosms composed of raw wastewater were also studied. The mesocosms were incubated at 4 °C or at 22 °C for 189 days, from which the levels of human mtDNA (HumtDNA) and human Bacteroidales (Hf183) were measured by qPCR. The levels of FIB (fecal coliforms and enterococci) and heterotrophs were determined by culture methods along with the determination of physicochemical attributes. The decay rates of the genetic markers and FIB were determined with first-order decay rate models. The decay rates of HumtDNA (0.004-0.059 d-1), Hf183 (0.007-0.082 d-1), and the two FIBs (0.005-0.066 d-1) were similar at 4 °C, while the genetic markers both had higher decay rates (0.013-0.919 d-1) at 22 °C. Different HumtDNA decay rates were observed between the river mesocosms (0.043-0.919 d-1) and the wastewater and tap water mesocosms (0.004-0.095 d-1). Covariations of pH and conductivity among the HumtDNA, Hf183 and FIB decay rates were observed. HumtDNA and Hf183 had similar environmental persistence, whereas fecal coliforms and enterococci persisted longer at 22 °C. Finally, HumtDNA had the same trends of persistence in the four river mesocosms, suggesting a relative stability of this marker in different rivers. Our results suggest that HumtDNA could be more suitable for tracking the source of a recent fecal contamination in complement to FIB.

Fecal indicator bacteria levels at a marine beach before, during, and after the COVID-19 shutdown period and associations with decomposing seaweed and human presence

Studies are limited that evaluate seaweed as a source of bacteria to beach waters. The objective of the current study was to evaluate whether seaweed, along with humans and other animals, could be the cause of beach advisories due to elevated levels of enterococci. The monitoring period occurred a year prior to and through the COVID-19 beach shutdown period, which provided a unique opportunity to evaluate bacteria levels during prolonged periods without recreational activity. Samples of water, sediment, and seaweed were measured for enterococci by culture and qPCR, in addition to microbial source tracking by qPCR of fecal bacteria markers from humans, dogs, and birds. During periods of elevated enterococci levels in water, these analyses were supplemented by chemical source tracking of human-associated excretion markers (caffeine, sucralose, acetaminophen, ibuprofen, and naproxen). Results show that enterococci with elevated levels of human fecal markers persist in the seaweed and sediment and are the likely contributor to elevated levels of bacteria to the nearshore waters. During the shutdown period the elevated levels of enterococci in the sediment were isolated to the seaweed stranding areas. During periods when the beaches were open, enterococci were distributed more uniformly in sediment across the supratidal and intertidal zones. It is hypothesized from this study that human foot traffic may be responsible for the spread of enterococci throughout these areas. Overall, this study found high levels of enterococci in decomposing seaweed supporting the hypothesis that decomposing seaweed provides an additional substrate for enterococci to grow.

Microbial Source Tracking as a Method of Determination of Beach Sand Contamination.

Beach sand may act as a reservoir for numerous microorganisms, including enteric pathogens. Several of these pathogens originate in human or animal feces, which may pose a public health risk. In August 2019, high levels of fecal indicator bacteria (FIB) were detected in the sand of the Azorean beach Prainha, Terceira Island, Portugal. Remediation measures were promptly implemented, including sand removal and the spraying of chlorine to restore the sand quality. To determine the source of the fecal contamination, during the first campaign, supratidal sand samples were collected from several sites along the beach, followed by microbial source tracking (MST) analyses of Bacteroides marker genes for five animal species, including humans. Some of the sampling sites revealed the presence of marker genes from dogs, seagulls, and ruminants. Making use of the information on biological sources originating partially from dogs, the municipality enforced restrictive measures for dog-walking at the beach. Subsequent sampling campaigns detected low FIB contamination due to the mitigation and remediation measures that were undertaken. This is the first case study where the MST approach was used to determine the contamination sources in the supratidal sand of a coastal beach. Our results show that MST can be an essential tool to determine sources of fecal contamination in the sand. This study shows the importance of holistic management of beaches that should go beyond water quality monitoring for FIB, putting forth evidence for beach sand monitoring.

Hurricane Harvey Impacts on Water Quality and Microbial Communities in Houston, TX Waterbodies.

Extreme weather events can temporarily alter the structure of coastal systems and generate floodwaters that are contaminated with fecal indicator bacteria (FIB); however, every coastal system is unique, so identification of trends and commonalities in these episodic events is challenging. To improve our understanding of the resilience of coastal systems to the disturbance of extreme weather events, we monitored water quality, FIB at three stations within Clear Lake, an estuary between Houston and Galveston, and three stations in bayous that feed into the estuary. Water samples were collected immediately before and after Hurricane Harvey (HH) and then throughout the fall of 2017. FIB levels were monitored by culturing E. coli and Enterococci. Microbial community structure was profiled by high throughput sequencing of PCR-amplified 16S rRNA gene fragments. Water quality and FIB data were also compared to historical data for these water body segments. Before HH, salinity within Clear Lake ranged from 9 to 11 practical salinity units (PSU). Immediately after the storm, salinity dropped to < 1 PSU and then gradually increased to historical levels over 2 months. Dissolved inorganic nutrient levels were also relatively low immediately after HH and returned, within a couple of months, to historical levels. FIB levels were elevated immediately after the storm; however, after 1 week, E. coli levels had decreased to what would be acceptable levels for freshwater. Enterococci levels collected several weeks after the storm were within the range of historical levels. Microbial community structure shifted from a system dominated by Cyanobacteria sp. before HH to a system dominated by Proteobacteria and Bacteroidetes immediately after. Several sequences observed only in floodwater showed similarity to sequences previously reported for samples collected following Hurricane Irene. These changes in beta diversity corresponded to salinity and nitrate/nitrite concentrations. Differential abundance analysis of metabolic pathways, predicted from 16S sequences, suggested that pathways associated with virulence and antibiotic resistance were elevated in floodwater. Overall, these results suggest that floodwater generated from these extreme events may have high levels of fecal contamination, antibiotic resistant bacteria and bacteria rarely observed in other systems.

Rainfall leads to elevated levels of antibiotic resistance genes within seawater at an Australian beach

Anthropogenic waste streams can be major sources of antibiotic resistant microbes within the environment, creating a potential risk to public health. We examined patterns in the occurrence of a suite of antibiotic resistance genes (ARGs) and their links to enteric bacteria at a popular swimming beach in Australia that experiences intermittent contamination by sewage, with potential points of input including stormwater drains and a coastal lagoon. Samples were collected throughout a significant rainfall event (40.8 mm over 3 days) and analysed using both qPCR and 16S rRNA amplicon sequencing. Before the rainfall event, low levels of faecal indicator bacteria and a microbial source tracking human faeces (sewage) marker (Lachno3) were observed. These levels increased over 10x following rainfall. Within lagoon, drain and seawater samples, levels of the ARGs sulI, dfrA1 and qnrS increased by between 1 and 2 orders of magnitude after 20.4 mm of rain, while levels of tetA increased by an order of magnitude after a total of 40.8 mm. After 40.8 mm of rain sulI, tetA and qnrS could be detected 300 m offshore with levels remaining high five days after the rain event. Highest levels of sewage markers and ARGs were observed adjacent to the lagoon (when opened) and in-front of the stormwater drains, pinpointing these as the points of ARG input. Significant positive correlations were observed between all ARGs, and a suite of Amplicon Sequence Variants that were identified as stormwater drain indicator taxa using 16S rRNA amplicon sequencing data. Of note, some stormwater drain indicator taxa, which exhibited correlations to ARG abundance, included the human pathogens Arcobacter butzleri and Bacteroides fragilis. Given that previous research has linked high levels of ARGs in recreationally used environments to antimicrobial resistant pathogen infections, the observed patterns indicate a potentially elevated human health risk at a popular swimming beach following significant rainfall events.

Molecular microbiological approaches reduce ambiguity about the sources of faecal pollution and identify microbial hazards within an urbanised coastal environment

Urbanised beaches are regularly impacted by faecal pollution, but management actions to resolve the causes of contamination are often obfuscated by the inability of standard Faecal Indicator Bacteria (FIB) analyses to discriminate sources of faecal material or detect other microbial hazards, including antibiotic resistance genes (ARGs). We aimed to determine the causes, spatial extent, and point sources of faecal contamination within Rose Bay, a highly urbanised beach within Sydney, Australia's largest city, using molecular microbiological approaches. Sampling was performed across a network of transects originating at 9 stormwater drains located on Rose Bay beach over the course of a significant (67.5 mm) rainfall event, whereby samples were taken 6 days prior to any rain, on the day of initial rainfall (3.8 mm), three days later after 43 mm of rain and then four days after any rain. Quantitative PCR (qPCR) was used to target marker genes from bacteria (i.e., Lachnospiraceae and Bacteroides) that have been demonstrated to be specific to human faeces (sewage), along with gene sequences from Heliobacter and Bacteriodes that are specific to bird and dog faeces respectively, and ARGs (sulI, tetA, qnrS, dfrA1 and vanB). 16S rRNA gene amplicon sequencing was also used to discriminate microbial signatures of faecal contamination. Prior to the rain event, low FIB levels (mean: 2.4 CFU/100 ml) were accompanied by generally low levels of the human and animal faecal markers, with the exception of one transect, potentially indicative of a dry weather sewage leak. Following 43 mm of rain, levels of both human faecal markers increased significantly in stormwater drain and seawater samples, with highest levels of these markers pinpointing several stormwater drains as sources of sewage contamination. During this time, sewage contamination was observed up to 1000 m from shore and was significantly and positively correlated with often highly elevated levels of the ARGs dfrA1, qnrS, sulI and vanB. Significantly elevated levels of the dog faecal marker in stormwater drains at this time also indicated that rainfall led to increased input of dog faecal material from the surrounding catchment. Using 16S rRNA gene amplicon sequencing, several indicator taxa for stormwater contamination such as Arcobacter spp. and Comamonadaceae spp. were identified and the Bayesian SourceTracker tool was used to model the relative impact of specific stormwater drains on the surrounding environment, revealing a heterogeneous contribution of discrete stormwater drains during different periods of the rainfall event, with the microbial signature of one particular drain contributing up to 50% of bacterial community in the seawater directly adjacent. By applying a suite of molecular microbiological approaches, we have precisely pinpointed the causes and point-sources of faecal contamination and other associated microbiological hazards (e.g., ARGs) at an urbanised beach, which has helped to identify the most suitable locations for targeted management of water quality at the beach.

An assessment of three methods for extracting bacterial DNA from beach sand

Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20-80gene copiesg-1 , recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches.

Bacterial Distribution and Community Structure in Beef Cattle Liver and Bile at Slaughter

In this study, the distribution of hygienic indicator bacteria in cattle livers and bile was examined at slaughterhouses. One hundred twenty-seven cattle livers with gallbladders were carefully eviscerated from carcasses at 10 slaughterhouses. Microbiological examination revealed that nine bile samples (7.1% prevalence) and 19 liver parenchyma samples (15.0% prevalence) were positive for Enterobacteriaceae (EB) with means ± standard deviations of 3.68 ± 4.63 log CFU/mL and 1.59 ± 2.47 log CFU/g, respectively; thus, bacterial contamination was apparent even at the postevisceration stage. Subsequently, 70 cattle livers were obtained at the postprocessing and storage stage from 7 of the 10 slaughterhouses. Microbiological analysis revealed significantly higher levels of EB in the liver parenchyma (3.00 ± 3.89 log CFU/g, P = 0.011) than those at the postevisceration stage, suggesting that bacterial dissemination and/or replication occurred in the liver parenchyma during processing and storage. According to 16S rRNA ion semiconductor sequencing analysis of representative samples from 12 cattle, Proteobacteria, Firmicutes, and Actinobacteria were dominant in both the parenchyma and bile in which EB and Escherichia coli were predominant among livers with higher EB levels. These results suggest that bile plays a role as a vehicle for bacterial transmission to the liver parenchyma. This study is the first to evaluate bacterial distribution and community structure in the liver and biliary microecosystem of cattle at slaughter. Our data support the use of EB testing of bile to screen cattle livers contaminated with high levels of fecal indicator bacteria.

Land Use, Weather, and Water Quality Factors Associated With Fecal Contamination of Northeastern Streams That Span an Urban-Rural Gradient

Fecal contamination of surface water has been associated with multiple enteric disease outbreaks and food recalls. Thus, it is important to understand factors associated with fecal contamination of agricultural water sources. Since fecal indicator bacteria (FIB) were used to monitor surface water for potential fecal contamination, the purpose of the present study was to characterize associations between environmental factors, and (i) FIB (E. coli, Enterococcus, and coliform) levels, and (ii) host-specific fecal marker detection. This study used data collected from 224 sites along 3 waterways, which spanned an urban-rural gradient around Syracuse, New York. Between 2008 and 2017, 2,816 water samples were collected, and E. coli, Enterococcus, and/or coliform concentrations were enumerated. Thirty-one samples were also tested for human and ruminant microbial source-tracking markers. Water quality (e.g., turbidity, nitrate) and weather data were also collected for each site. Univariable Bayesian regression was used to characterize the relationship between each microbial target and land use, water quality, and weather factor. For each model, probability of direction and region of practical equivalence overlap (ROPE) were calculated to characterize the association's direction and strength, respectively. While levels of different FIB were not correlated with each other, FIB levels were associated with environmental conditions. Specifically, FIB levels were also positively associated with temperature, nutrient and sediment levels. Log10 E. coli levels increased by 0.20 (CI = 0.11, 0.31) and log10 Enterococcus levels increased by 0.68 (CI = 0.08, 1.24) for each log10 increase in salinity and nitrate, respectively. These findings may indicate that similar processes drove microbial, sediment, and nutrient contamination of the sampled watersheds. While fecal contamination was strongly associated with land use, the direction of association varied between FIBs and the buffer distance used to calculate land use metrics. E. coli levels and human marker detection were positively associated with percent pasture cover within 122, 366, and 1,098 m of the sampling site, while Enterococcus and coliform levels were only associated with pasture cover within 1,098 m (not 122 or 366 m). Ruminant markers were positively associated with pasture cover within 122 m, but not 366 or 1,098 m. These findings highlight the importance of considering (i) adjacent land use (and associated non-point sources of contamination) when developing strategies for managing fecal hazards associated in agricultural and recreational water, and (ii) spatial scale (e.g., 122 vs. 1,098 m) when developing these strategies.

Rapid qPCR-Based Water Quality Monitoring in New York State Recreational Waters

Public swimming beaches often rely on culture-based methods to determine if fecal indicator bacteria (FIB) levels are greater than health risk-based beach action values (BAV). The slow turnaround time of culture-based assays can prevent effective beach closure and reopening decisions. Faster testing methods that can be completed on-site are needed. Additionally, beach closures are currently based on high FIB levels, but at-present there are no tools to examine the health risks to bathers from myriad pathogens (e.g., bacteria, viruses, protozoa) that may be present in recreational waters. Twelve New York State beaches (n = 9 freshwater and n = 3 marine) were monitored over the course of summer 2018, and two of the freshwater beaches were monitored in fall 2017 as part of a preliminary study. A rapid, in-field workflow for detecting fecal enterococci in water samples was tested using four assays on two Biomeme handheld devices. All Biomeme-based workflows involved in-field DNA extractions and qPCR using portable devices. Beach water samples were also analyzed using EPA-approved or EPA-based qPCR methods: two culture-based methods, Enterolert (targeting enterococci at freshwater and marine beaches) and Colilert (targeting E. coli at freshwater beaches); and one qPCR method based on EPA 1611.1. For low abundance pathogen quantification, nanoscale-qPCR was conducted in 2018 using the Pathogen Panel which targeted 12 viral, bacterial, and protozoal pathogens. In fall 2017, the qPCR-based methods performed similarly to Enterolert (r2 from 0.537 to 0.687) and correctly classified 62.5–75.0% of water samples for a BAV of 104 MPN per 100 ml. In summer 2018, the correlation between Enterococcus levels based on Biomeme qPCR and Enterolert varied substantially between the 12 beaches. Inclusion of diverse regions and beach types may have confounded the Biomeme qPCR results. The EPA 1611.1-based method showed a weak, significant correlation (r2 = 0.317, p = 0.00012) with Enterolert. Nanoscale-qPCR showed low-levels of pathogens present at all beach sites; but only three showed up with any substantial frequency, E. coli eae (25% of samples), norovirus (31.4%), and Giardia lamblia (11.4%). Preliminary studies to establish beach-specific correlation curves between rapid qPCR and Enterolert methods are needed before any qPCR assay is used to inform beach decisions.

Quantitative microbial human exposure model for faecal indicator bacteria and risk assessment of pathogenic Escherichia coli in surface runoff following application of dairy cattle slurry and co-digestate to grassland

Animal waste contains high numbers of microorganisms and therefore can present a potential biological threat to human health. During episodic rainfall events resulting in runoff, microorganisms in the waste and soil may migrate into surface runoff, contaminating surface water resources. A probabilistic human exposure (HE) model was created to determine exposure to faecal indicator bacteria (FIB): total coliforms (TC), E. coli and enterococci following application of bio-based fertiliser (dairy cattle slurry, digestate) to grassland; using a combination of experimental field results and literature-based data. This step was followed by a quantitative microbial risk assessment (QMRA) model for pathogenic E. coli based on a literature-based dose-response model. The results showed that the maximum daily HE (HEdaily) is associated with E. coli for unprocessed slurry (treatment T1) on day 1, the worst-case scenario where the simulated mean HEdaily was calculated as 2.84 CFU day −1. The results indicate that the overall annual probability of risk (Pannual) of illness from E. coli is very low or low based on the WHO safe-limit of Pannual as 10 −6. In the worst-case scenario, a moderate risk was estimated with simulated mean Pannual as 1.0 × 10 −5. Unpasteurised digestate application showed low risk on day 1 and 2 (1.651 × 10 −6, 1.167 × 10 −6, respectively). Pasteurised digestate showed very low risk in all scenarios. These results support the restriction imposed on applying bio-based fertiliser if there is any rain forecast within 48 h from the application time. This study proposes a future extension of the probabilistic model to include time, intensity, discharge, and distance-dependant dilution factor. The information generated from this model can help policymakers ensure the safety of surface water sources through the quality monitoring of FIB levels in bio-based fertiliser.

Indicator species characterization and removal in a detention pond in the Plaster Creek watershed

Microbial pathogen contamination is a leading cause of impairment for urban rivers and streams in Michigan. Reports on the ability of green infrastructure best management practices to remove microbial pathogens have been highly variable. This study evaluated the influence of a detention basin (Kreiser Pond) on microbial dynamics in the Plaster Creek watershed in West Michigan. High levels of fecal indicator bacteria and coliphage were documented in influent and effluent water, with significant increases in indicator microbe concentrations during storm events. In dry conditions, Kreiser Pond efficiently reduced the number of indicator microbes flowing through the basin. Rainfall volume had a greater influence on the diversity of bacteria than sampling location. Antibiotic resistance was prevalent in culturable E. coli from Kreiser Pond, demonstrating a potential public health risk and highlighting the need for identifying the ultimate sources of microbial pollution.