Culturable Fecal Indicator Bacteria Research Articles

Reduction of human fecal markers and enteric viruses in Sydney estuarine waters receiving wet weather overflows

The current microbial source tracking (MST) study tracked the reduction of the culturable fecal indicator bacteria enterococci, four human fecal markers (Bacteroides HF183, Lachnospiraceae Lachno3, cross-assembly phage (CrAssphage) and pepper mild mottle virus (PMMoV)) along with four enteric viruses - human adenovirus 40/41 (HAdV 40/41), enterovirus (EV), human norovirus GI (HNoV GI) and GII (HNoV GII) post wet weather overflows (WWOs) at two estuarine water sites from two depths under separate six-day sampling campaigns over seven and 12 days in Sydney, NSW, Australia. Neither HNoV GI nor GII was detected, while 13.9 % (10/72) of estuarine water samples had detections of EV. Quantifiable concentrations (0.64 to 2.00 log10 gene copies (GC)/100 mL) for HAdV 40/41 were returned from 65.2 % (47/72) of samples collected across the two sites and two depths with 30 quantifications recorded in the surface layer samples. In contrast the presence of HF183, Lachno3, CrAssphage, and PMMoV markers was observed in all 36 (100 %) estuarine water samples collected from the surface layer from both sites. Detection frequencies of these markers were slightly lower at 1 m above the bottom surface. The concentrations of the human fecal markers were compared to established gastrointestinal (GI) risk benchmarks. The concentrations of HF183, Lachno3 and CrAssphage marker only exceeded the GI risk benchmark until day 3, while concentrations of PMMoV marker were indicative of exceedance of the GI risk benchmark on day 7 post WWOs that was much longer than indicated by culturable enterococci concentrations that were within this GI risk benchmark by day 2 and day 4 for the two sites, respectively.

Human Fecal Contamination Corresponds to Changes in the Freshwater Bacterial Communities of a Large River Basin.

ABSTRACTMicrobial water quality is generally monitored by culturable fecal indicator bacteria (FIB), which are intended to signal human health risk due to fecal pollution. However, FIB have limited utility in most urbanized watersheds as they do not discriminate among fecal pollution sources, tend to make up a small fraction of the total microbial community, and do not inform on pollution impacts on the native ecosystem. To move beyond these limitations, we assessed entire bacterial communities and investigated how bacterial diversity relates to traditional ecological and human health-relevant water quality indicators throughout the Milwaukee River Basin. Samples were collected from 16 sites on 5 days during the summer, including both wet and dry weather events, and were processed by 16S rRNA gene amplicon sequencing. Historical water quality at each sampling location, as opposed to upstream land use, was associated significantly with bacterial community alpha diversity. Source partitioning the sequence data was important for determining water quality relationships. Sewage-associated bacterial sequences were detected in all samples, and the relative abundance of sewage sequences was strongly associated with the human Bacteroides fecal marker. From this relationship, we developed a preliminary threshold for human sewage pollution when using bacterial community sequence data. Certain abundant freshwater bacterial sequences were also associated with human fecal pollution, suggesting their possible utility in water quality monitoring. This study sheds light on how bacterial community analysis can be used to supplement current water quality monitoring techniques to better understand interactions between ecological water quality and human health indicators.IMPORTANCE Surface waters in highly developed mixed-use watersheds are frequently impacted by a wide variety of pollutants, leading to a range of impairments that must be monitored and remediated. With advancing technologies, microbial community sequencing may soon become a feasible method for routine evaluation of the ecological quality and human health risk of a water body. In this study, we partnered with a local citizen science organization to evaluate the utility of microbial community sequencing for identifying pollution sources and ecological impairments in a large mixed-use watershed. We show that changes in microbial community diversity and composition are indicative of both long-term ecological impairments and short-term fecal pollution impacts. By source partitioning the sequence data, we also estimate a threshold target for human sewage pollution, which may be useful as a starting point for future development of sequencing-based water quality monitoring techniques.

Comparative stability of assay results of enterococci measured by culture and qPCR over time in bathing beach waters

The diurnal presence of the culturable bacterial indicators of fecal contamination in the water environment has been shown to be highly variable over time due to natural die-off and injury from effects of sunlight and other environmental stressors. Molecular analytes of a quantitative polymerase chain reaction (qPCR) method for measuring fecal contamination degrade considerably slower than the alternative of culturable fecal indicator bacteria. The rapid qPCR method holds the promise of more timely notification decisions with respect to postings or closure being made on the basis of microbial water quality samples collected earlier on the same day. In the case of culture-based methods requiring a 24 h or longer incubation period, decisions must be based on samples collected no sooner than the previous day. To examine the effect of this lag in assay results, temporal stability of a molecular Enterococci target analyte with that of traditional culture-based cells is compared using data from USEPA studies conducted between 2003 and 2007 on seven freshwater and marine beaches that were impacted by publicly-owned treatment works. Generally, levels of the molecular indicator were more consistent throughout the day between 8:00 am and 3:00 pm. The difference in temporal consistency is even more pronounced when the 24-h lag in culture-based results is taken into account.

CrAssphage for fecal source tracking in Chile: Covariation with norovirus, HF183, and bacterial indicators

Anthropogenic fecal pollution in urban waterbodies can promote the spread of waterborne disease. The objective of this study was to test crAssphage, a novel viral human fecal marker not previously applied for fecal source tracking in Latin America, as a fecal pollution marker in an urban river in Chile. Human fecal markers crAssphage CPQ_064 and Bacteroides HF183, the human pathogen norovirus GII, and culturable fecal indicator bacteria (FIB) were quantified at six locations spanning reaches of the Mapocho River from upstream to downstream of Santiago, as well as in repeated sub-daily frequency samples at two urban locations. Norovirus showed positive correlation trends with crAssphage (τ = 0.57, p = 0.06) and HF183 (τ = 0.64, p = 0.03) in river water, but not with E. coli or enterococci. CrAssphage and HF183 concentrations were strongly linearly related (slope = 0.97, p < 0.001). Chlorinated wastewater effluent was an important source of norovirus GII genes to the Mapocho. Precipitation showed non-significant positive relationships with human and general fecal indicators. Concentrations of crAssphage and HF183 in untreated sewage were 8.35 and 8.07 log10 copy/100 ml, respectively. Preliminary specificity testing did not detect crAssphage or HF183 in bird or dog feces, which are predominant non-human fecal sources in the urban Mapocho watershed. This study is the first to test crAssphage for microbial source tracking in Latin America, provides insight into fecal pollution dynamics in a highly engineered natural system, and indicates river reaches where exposure to human fecal pollution may pose a public health risk.

Sewage-associated marker genes illustrate the impact of wet weather overflows and dry weather leakage in urban estuarine waters of Sydney, Australia

This study investigates the impact of wet weather overflows (WWOs) at three estuarine locations in Sydney, NSW, Australia. WWOs can occur when infiltration of stormwater leads to an excess volume of flow within the sewerage system, resulting in the release of diluted sewage into the environment. Sewage contamination poses a risk to human health due to the presence of pathogens. The magnitude of sewage contamination was monitored using established and novel sewage-associated marker genes, Bacteroides HF183, pepper mild mottle virus (PMMoV), crAssphage CPQ_056, Lachnospiraceae (Lachno3) marker genes along with culturable fecal indicator bacteria (FIB) Escherichia coli (E. coli) and enterococci. Water samples were collected at two water depths (0.5 m below the water surface and 1 m above the bottom surface) during one dry weather and two storm events. Analysis of sewage-associated marker genes showed greater (i.e., 3–5 orders of magnitude) concentrations in water samples collected during the storm events compared to dry weather event. Water samples were also analysed for four animal feces-associated marker genes targeting avian (GFD), dog (BacCan-UCD), cow (cowM2) and horse (HoF597) species to determine the extent of animal fecal contamination. Among the four marker genes, cowM2 and HoF597 could not be detected, while GFD marker gene was consistently present and BacCan-UCD was occasionally detected. Overall results suggested that after rainfall, untreated sewage from WWOs was present at sampling locations. In addition, microbial source tracking (MST) monitoring was able to distinguish the presence of a leaking sewer impacting on the recreational area during dry weather condition. This study demonstrated the capability of the MST monitoring approach to understand sources (sewage or animal) of fecal contamination. This capability will greatly enhance management decisions assisting in the prioritisation of remediation efforts of the sewerage system to improve estuarine bathing water quality and diminish human health risk.

Enhanced insights from human and animal host-associated molecular marker genes in a freshwater lake receiving wet weather overflows

This study investigated the magnitude of wet weather overflow (WWO)-driven sewage pollution in an urban lake (Lake Parramatta) located in Sydney, New South Wales, Australia. Water samples were collected during a dry weather period and after two storm events, and tested for a range of novel and established sewage- [Bacteroides HF183, crAssphage CPQ_056 and pepper mild mottle virus (PMMoV)] and animal feces-associated (Bacteroides BacCan-UCD, cowM2 and Helicobacter spp. associated GFD) microbial source tracking marker genes along with the enumeration of culturable fecal indicator bacteria (FIB), namely Escherichia coli (E. coli) and Enterococcus spp. The magnitude of general and source-specific fecal pollution was low in water samples collected during dry weather compared to storm events. The levels of HF183, crAssphage and PMMoV in water samples collected during storm events were as high as 6.39, 6.33 and 5.27 log10 GC/L of water, respectively. Moderate to strong positive correlations were observed among the quantitative occurrence of sewage-associated marker genes. The concentrations of HF183 and PMMoV in most storm water samples exceeded the risk benchmark threshold values established in the literature for primary contact recreators. None of the samples tested was positive for the cowM2 (cow) marker gene, while BacCan-UCD (dog) and GFD (avian) animal-associated markers were sporadically detected in water samples collected from both dry weather and storm events. Based on the results, the ongoing advice that swimming should be avoided for several days after storm events appears appropriate. Further research to determine the decay rates of sewage-associated marker genes in relation to each other and enteric viruses would help refine current advice. Microbial source tracking approaches employed in this study provided insights into sources of contamination over currently used FIB.

A culture-dependent and metagenomic approach of household drinking water from the source to point of use in a developing country

Rural households in developing countries rely on communal water supplies and household water frequently becomes contaminated following its collection, transportation and during its storage. Using culture-dependent and -independent techniques, we examined the changes in microbial water quality between communal tap water and household water storage in a rural area of Cameroon, Africa. The culturable fecal indicator bacteria (FIB) were used to assess the potential health risks associated with different household water storage conditions (e.g., type of container and open vs. closed container) and interventions (e.g., water storage days, cleaned on the last day of use, and hygiene practices). Only the amount of days the water was stored significantly differed (p-value < 0.05), which showed that potential health risks increased when water was stored for more than 3 days. The higher abundance of molecular FIB in biofilm than household water suggested that omnipresent biofilm in household water could potential health risk. The high-throughput sequencing revealed that the most abundant phylum was Proteobacteria, followed by Actinobacteria and Bacteroidetes in both the water and the biofilm samples. Bacterial genera seen in biofilm bacteria, such as Pseudomonas, Acinetobacter and Comamonas. Acinetobacter, Chryseobacterium, Stenotrophomonas and Corynebacterium, were relatively more abundant in the biofilm than in the water. Potential bacterial pathogens including Acinetobacter baumannii, Citrobacter freundii, Stenotrophomonas maltophilia and Haemophilus influenza, were detected in household water and biofilm. The microbial quality might be affected by water-storage time and households repeatedly using the same water storage containers without proper sanitization, triggering microbial regrowth and biofilm formation on water containers. Higher bacterial diversity and potentially pathogenic bacteria found in the biofilm samples of a household water supply are unhealthy for the house’s inhabitants. It is important to develop interventions aimed at preventing the formation of these dangerous biofilms in a communal water supply.

Extended persistence of general and cattle-associated fecal indicators in marine and freshwater environment

Fecal contamination of recreational waters with cattle manure can pose a risk to public health due to the potential presence of various zoonotic pathogens. Fecal indicator bacteria (FIB) have a long history of use in the assessment of recreational water quality, but FIB quantification provides no information about pollution sources. Microbial source tracking (MST) markers have been developed in response to a need to identify pollution sources, yet factors that influence their decay in ambient waters are often poorly understood. We investigated the influence of water type (freshwater versus marine) and select environmental parameters (indigenous microbiota, ambient sunlight) on the decay of FIB and MST markers originating from cattle manure. Experiments were conducted in situ using a submersible aquatic mesocosm containing dialysis bags filled with a mixture of cattle manure and ambient water. Culturable FIB (E. coli, enterococci) were enumerated by membrane filtration and general fecal indicator bacteria (GenBac3, Entero1a, EC23S857) and MST markers (Rum2Bac, CowM2, CowM3) were estimated by qPCR. Water type was the most significant factor influencing decay (three-way ANOVA, p: 0.006 to <0.001), although the magnitude of the effect differed among microbial targets and over time. The presence of indigenous microbiota and exposure to sunlight were significantly correlated (three-way ANOVA, p: 0.044 to <0.001) with decay of enterococci and CowM2, while E. coli, EC23S857, Rum2Bac, and CowM3 (three-way ANOVA, p: 0.044 < 0.001) were significantly impacted by sunlight or indigenous microbiota. Results indicate extended persistence of both cultivated FIB and genetic markers in marine and freshwater water types. Findings suggest that multiple environmental stressors are important determinants of FIB and MST marker persistence, but their magnitude can vary across indicators. Selective exclusion of natural aquatic microbiota and/or sunlight typically resulted in extended survival, but the effect was minor and limited to select microbial targets.

Microfluidic quantification of multiple enteric and opportunistic bacterial pathogens in roof-harvested rainwater tank samples.

Potable and non-potable uses of roof-harvested rainwater (RHRW) are increasing due to water shortages. To protect human health risks, it is important to identify and quantify disease-causing pathogens in RHRW so that appropriate treatment options can be implemented. We used a microfluidic quantitative PCR (MFQPCR) system for the quantitative detection of a wide array of fecal indicator bacteria (FIB) and pathogens in RHRW tank samples along with culturable FIB and conventional qPCR analysis of selected pathogens. Among the nine pathogenic bacteria and their associated genes tested with the MFQPCR, 4.86 and 2.77% samples were positive for Legionella pneumophila and Shigella spp., respectively. The remaining seven pathogens were absent. MFQPCR and conventional qPCR results showed good agreement. Therefore, direct pathogen quantification by MFQPCR systems may be advantageous for circumstances where a thorough microbial analysis is required to assess the public health risks from multiple pathogens that occur simultaneously in the target water source.

Influence of Bizerte city wastewater treatment plant (WWTP) on abundance and antibioresistance of culturable heterotrophic and fecal indicator bacteria of Bizerte Lagoon (Tunisia)

The waste water treatment plant (WWTP) of the city of Bizerte concentrates different types of chemical and biological pollutants in the Bizerte lagoon (Tunisia). Considering four upstream and downstream WWTP discharge stations, seventy nine, culturable bacterial strains were isolated and identified from water and sediment as fecal coliforms, fecal streptococci, pathogenic staphylococci and non-enterobacteriacea. Fecal coliforms were most abundant (2.5 105 bacteria/mg) in sediment of WWTP discharge. Leuconostoc spp (23.1%) and Chryseomonasluteola (23.1%) were the most prevalent culturable fecal indicator bacteria (FIB) isolated at the upstream discharge stations. However, Staphylococcus xylosus (13.9%) was the most prevalent culturable FIB isolated at the WWTP discharge stations. Moreover, high antibioticresistance phenotypes were present in all sampling stations, but especially in WWTP discharge station in both water and sediment. Resistance levels in water and sediment, respectively were amoxicillin (58.8%; 34.8%), penicillin (50%; 31.6%), oxacillin (60%; 33.3%), cefotaxim (55.2%; 39.1%), ceftazidim (66.7%; 50%), gentamycin (42.9%; 38.9%), tobramycin (50%; 25%), vancomycin (33.3; 71.4%), amikacin (66.7%; 0%) and ciprofloxacin (100%; 100%). Interestingly, ß-lactam antibiotic resistant FIB were mostly isolated from water as well as from sediments of upstream and WWTP discharge station. Canonical correspondence analysis CCA correlating antibiotic resistance profile with the abiotic data showed that, in water column, culturable bacterial strains isolated in upstream WWTP discharge stations were interestingly correlated with the resistance to amikacin, oxacillin, cefotaxim, ciprofloxacin and gentamycin, however, in sediment, they were correlated with the resistance to amoxicillin, oxacillin, céfotaxim and vancomycin. Serious ß-lactams and aminoglycosides acquired resistance appeared mainly in fecal streptococci and pathogen staphylococci groups.

On-farm treatment of wastewater used for vegetable irrigation: bacteria and virus removal in small ponds in Accra, Ghana

Many urban farmers in Accra collect irrigation water from streams and open drains, which they store in small, on-farm ponds before use. Given that this water can be highly contaminated with wastewater, another potential role of the ponds is to disinfect irrigation water prior to use. To better understand the factors influencing bacteria and virus removal in these small ponds, we investigated the removal of culturable fecal indicator bacteria (Escherichia coli and enterococci) and coliphage (F+ and somatic coliphage) in a single batch of water stored for 3 days. Sunlight exposure was found to be important for removal. Bacteria and coliphage removal rates were faster in shallow sun-exposed water than in deeper water, due to sunlight attenuation with depth. Bacteria removal rates varied depending on solar irradiation, and correlations between total daily UVB fluence and bacteria removal rates were observed. Coliphage removal was observed in sun-exposed water but not in dark controls that allowed for sedimentation, further highlighting the importance of sunlight-mediated processes. These small ponds appear to have similar disinfection processes to larger-scale waste stabilization ponds, but can have more efficient inactivation due to their shallow depth and operation as batch reactors. Design and management recommendations for on-farm ponds are discussed.

Biotic interactions and sunlight affect persistence of fecal indicator bacteria and microbial source tracking genetic markers in the upper Mississippi river.

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r(2), 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.

Decay of genetic markers for fecal bacterial indicators and pathogens in sand from Lake Superior

Beach sands impact water quality and pathogen loads, however, the comparative decay of the fecal indicator bacteria (FIB) Enterococcus spp. and Escherichia coli, and pathogens in freshwater sand have not been examined. In this study, freshwater sand microcosms were inoculated with sewage and pure cultures of bacterial pathogens to compare relative decay rates. The abundance of culturable Enterococcus spp. and E. coli, genetic markers for Enterococcus spp. (Entero1), total Bacteroides (AllBac), and human-specific Bacteroides (HF183), and genetic markers for the pathogens Campylobacter jejuni, methicillin-resistant Staphylococcus aureus (MRSA), Salmonella enterica subsp. enterica serovar Typhimurium, and Shigella flexneri were monitored over the course of two weeks using conventional culture methods and quantitative PCR (qPCR). The effect of moisture on the persistence of culturable FIB and all genetic markers was also determined. In addition, propidium monoazide (PMA) treatment was used to examine differences in the persistence of total genetic markers and those from live cells. Decay rates were statistically compared using Tukey's test. Moisture had a significant (p ≤ 0.05) effect on the decay rates of culturable indicator bacteria, total AllBac markers, and genetic markers for FIB, Salmonella, and MRSA from live cells. At 14% sand moisture, the decay rate of total markers was slower than that of live cells for all qPCR assays, but at 28% moisture, there was no difference in the decay rates of total and live markers for any assay. AllBac and MRSA markers increased in sand at 28% moisture, probably indicating cellular growth. Overall, culturable FIB and HF183 had decay rates that were most comparable to the bacterial pathogen markers examined in this study, whereas Entero1 and AllBac rarely exhibited decay rates similar to the bacterial pathogens in this study. The choice of FIB for assessment of fecal contamination in freshwater sand should take into account the pathogen of concern and sand moisture conditions.

Comparison of Bacterial Communities in Sands and Water at Beaches with Bacterial Water Quality Violations

Recreational water quality, as measured by culturable fecal indicator bacteria (FIB), may be influenced by persistent populations of these bacteria in local sands or wrack, in addition to varied fecal inputs from human and/or animal sources. In this study, pyrosequencing was used to generate short sequence tags of the 16S hypervariable region ribosomal DNA from shallow water samples and from sand samples collected at the high tide line and at the intertidal water line at sites with and without FIB exceedance events. These data were used to examine the sand and water bacterial communities to assess the similarity between samples, and to determine the impact of water quality exceedance events on the community composition. Sequences belonging to a group of bacteria previously identified as alternative fecal indicators were also analyzed in relationship to water quality violation events. We found that sand and water samples hosted distinctly different overall bacterial communities, and there was greater similarity in the community composition between coastal water samples from two distant sites. The dissimilarity between high tide and intertidal sand bacterial communities, although more similar to each other than to water, corresponded to greater tidal range between the samples. Within the group of alternative fecal indicators greater similarity was observed within sand and water from the same site, likely reflecting the anthropogenic contribution at each beach. This study supports the growing evidence that community-based molecular tools can be leveraged to identify the sources and potential impact of fecal pollution in the environment, and furthermore suggests that a more diverse bacterial community in beach sand and water may reflect a less contaminated site and better water quality.

Influence of resuspension on the fate of fecal indicator bacteria in large-scale flumes mimicking an oligotrophic river

In this study, large-scale flume systems simulating an oligotrophic river were used to explore the fate and transport of the fecal indicator bacteria (FIB) Escherichia coli and enterococci following a combined sewer overflow (CSO). Specifically, the removal pattern of FIB from the water column was examined as well as deposition onto the flume bed. Finally, the impact that a sudden increase in bed shear stress has on FIB in the water column was investigated. The large-scale flumes utilized in this study proved extremely useful for our investigations as they very closely approximated conditions within the Isar River (Munich, Germany). By using both natural substratum and fresh river water, as well as a flow velocity of nearly 1 m s−1 at a water depth of roughly 0.5 m, shear stresses typical of the Isar River (9 N m−2) were achieved. As a result, scaling effects were appreciably reduced. In our flume system, UV inactivation played only a minimal role in overall FIB removal. Therefore, we were able to more precisely investigate other mechanisms which result in FIB removal from the water column. From the two standard FIB removal experiments following a CSO, the removal rate coefficient (k) of 0.2 h−1 was identified for both E. coli and enterococci in the water column. An increase in the bed shear stress led to more than a 150% rise in total suspended solid (TSS) levels in the water column. These elevated TSS levels (≈50 mg l−1) increased the persistence of suspended FIB in the water column by 20 h (k = 0.05 h−1). This indicates that higher TSS loads resulting from resuspended bed sediments can significantly expand the area that is impacted by a CSO event. At lower TSS loads (<20 mg l−1) deposition onto the flume bed did not contribute significantly to FIB removal from the water column. Any deposition which did occur did not result in a net accumulation of culturable FIB in the benthic biofilm.

Association of Fecal Indicator Bacteria with Human Viruses and Microbial Source Tracking Markers at Coastal Beaches Impacted by Nonpoint Source Pollution

Water quality was assessed at two marine beaches in California by measuring the concentrations of culturable fecal indicator bacteria (FIB) and by library-independent microbial source tracking (MST) methods targeting markers of human-associated microbes (human polyomavirus [HPyV] PCR and quantitative PCR, Methanobrevibacter smithii PCR, and Bacteroides sp. strain HF183 PCR) and a human pathogen (adenovirus by nested PCR). FIB levels periodically exceeded regulatory thresholds at Doheny and Avalon Beaches for enterococci (28.5% and 31.7% of samples, respectively) and fecal coliforms (20% and 5.8%, respectively). Adenoviruses were detected at four of five sites at Doheny Beach and were correlated with detection of HPyVs and human Bacteroides HF183; however, adenoviruses were not detected at Avalon Beach. The most frequently detected human source marker at both beaches was Bacteroides HF183, which was detected in 27% of samples. Correlations between FIBs and human markers were much more frequent at Doheny Beach than at Avalon Beach; e.g., adenovirus was correlated with HPyVs and HF183. Human sewage markers and adenoviruses were routinely detected in samples meeting FIB regulatory standards. The toolbox approach of FIB measurement coupled with analysis of several MST markers targeting human pathogens used here demonstrated that human sewage is at least partly responsible for the degradation of water quality, particularly at Doheny Beach, and resulted in a more definitive assessment of recreational water quality and human health risk than reliance on FIB concentrations alone could have provided.

Test of Direct and Indirect Effects of Agrochemicals on the Survival of Fecal Indicator Bacteria

Water bodies often receive agrochemicals and animal waste carrying fecal indicator bacteria (FIB) and zoonotic pathogens, but we know little about the effects of agrochemicals on these microbes. We assessed the direct effects of the pesticides atrazine, malathion, and chlorothalonil and inorganic fertilizer on Escherichia coli and enterococcal survival in simplified microcosms held in the dark. E. coli strain composition in sediments and water column were positively correlated, but none of the agrochemicals had significant direct effects on E. coli strain composition or on densities of culturable FIBs. In a companion study, microcosms with nondisinfected pond water and sediments were exposed to or shielded from sunlight to examine the potential indirect effects of atrazine and inorganic fertilizer on E. coli. The herbicide atrazine had no effect on E. coli in dark-exposed microcosms containing natural microbial and algal communities. However, in light-exposed microcosms, atrazine significantly lowered E. coli densities in the water column and significantly increased densities in the sediment compared to controls. This effect appears to be mediated by the effects of atrazine on algae, given that atrazine significantly reduced phytoplankton, which was a positive and negative predictor of E. coli densities in the water column and sediment, respectively. These data suggest that atrazine does not directly affect the survival of FIB, rather that it indirectly alters the distribution and abundance of E. coli by altering phytoplankton and periphyton communities. These results improve our understanding of the influence of agricultural practices on FIB densities in water bodies impacted by agricultural runoff.

Comparison of Fecal Indicator Bacteria Densities in Marine Recreational Waters by QPCR

The US EPA is currently investigating the use of quantitative PCR (qPCR) analysis techniques to estimate densities of fecal indicator bacteria (FIB) in recreational waters. Present water quality guidelines, based on culturable FIB, prevent same day water quality determination, whereas results from qPCR-based approaches are available within several hours. Epidemiological studies at Publicly-Owned Treatment Works (POTW)-impacted freshwater beaches have also indicated correlations between qPCR determined Enterococcus densities and swimming-related illness rates. Similar qPCR assays are now available for several other accepted or emerging FIB groups. This study provides an initial assessment of qPCR estimated Enterococcus, Bacteroidales, E. coli and Clostridium spp. densities in marine water and sand samples collected over one summer from two POTW-impacted recreational beaches. Relative target sequence densities of these organisms in the samples did not correspond with their relative estimated cell densities. These observations were attributable to differences in target sequences recovered from the calibrator cells of the different types of organisms. Comparative cycle threshold (CT) qPCR analyses of whole cell calibrator samples provide a simple and standardizable approach for estimating both total cell and target sequence densities of different types of FIB in water. Cell density estimates obtained by this approach are subject to uncertainty due to potential variability in absolute numbers of target sequences in the target organisms under different physiological or environmental conditions, but still may allow for informative comparisons with the target sequence estimates.