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Using in situ subtropical aquatic mesocosms, fecal source (cattle manure versus sewage) was shown to be the most important contributor to differential loss in viability of fecal indicator bacteria (FIB), specifically enterococci in freshwater and Escherichia coli in marine habitats. In this study, sunlight exposure and indigenous aquatic microbiota were also important contributors, whose effects on FIB also differed between water types.

Human and ecosystem health can be damaged by fecal contamination of recreational waters. Microbial source tracking (MST) can be used to specifically detect domestic sewage containing human waste, thereby informing both risk assessment and remediation strategies. Previously, an inter-laboratory collaboration developed standardized PCR methods for a bacterial, an archaeal, and a viral indicator of human sewage. Here we present results for two subsequent years of field testing in fresh and salt water by five laboratories across the U.S. Gulf Coast (two in Florida and one each in Mississippi, Louisiana and Texas) using common standard operating procedures (SOPs) developed previously. Culturable enterococci were enumerated by membrane filtration, and PCR was used to detect three MST markers targeting domestic sewage: human-associated Bacteroides (HF183), Methanobrevibacter smithii and human polyomaviruses BK and JC (HPyVs). Detection of sewage markers in surface waters was significantly associated with higher enterococci levels and with exceedance of the recreational water quality standard in four or three regions, respectively. Sewage markers were frequently co-detected in single samples, e.g., M. smithii and HF183 were co-detected in 81% of Louisiana samples, and HPyVs and M. smithii were co-detected in over 40% of southwest Florida and Mississippi samples. This study demonstrates the robustness and inter-laboratory transferability of these three markers for the detection of pollution from domestic sewage in the waters impacting the Gulf of Mexico over a coastal range of over 1000 miles., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

Environmental Enterococcus spp. were compared by BOX-PCR genotyping and 16S rRNA gene sequencing to clarify the predictive relationship of BOX-PCR fingerprints to species designation. BOX-PCR and 16S rRNA gene relationships agreed for 77% of strains. BOX-PCR provided superior intraspecies discrimination but incorrectly identified some strains to the species level and divided some species into multiple groups.

Elevated concentrations of fecal indicator bacteria (FIB) in aquatic sediments and vegetation have prompted concern that environmental reservoirs of FIB disrupt the correlation between indicator organisms, pathogens and human health risks. FIB numbers, however, are typically normalized to volume of water or mass of substrate. Because these reservoirs tend to differ greatly in magnitude within and between water bodies, direct comparison between water column and benthic population sizes can be problematic. Normalization to a set volume of water or mass of substrate, e.g. cfu (100 ml)(-1) or cfu(100 g)(-1), can give a false picture of the relative contributions of various reservoirs to FIB numbers across the ecosystem, and of the potential for FIBs to trigger health advisories as they pass from one reservoir to another. Here, we normalized enterococci concentrations from water, sediment and submerged aquatic vegetation (SAV) to land surface area (m(2) ) to compare their relative importance in the entire system. SAV-associated enterococci comprised only 0-18% of the entire population, even though they displayed the highest concentrations of enterococci per unit mass. The largest proportion of the enterococci population was in the water column (4-77%) or sediments (20-95%), depending on the volume of each substrate available at a site and FIB concentrations within them. Models indicated that large shifts in the relative size of FIB populations in each substrate can result from changes in per cent SAV cover, water depth and depth of sediment colonization. It follows that high concentrations of FIB in sediments or SAV do not necessarily signify large environmental reservoirs of FIB that can affect the water column. Comprehensive analyses that include FIB measurements from water, SAV and sediment normalized to land surface area offer a more balanced perspective on total FIB numbers contained in various matrices of an aquatic system., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Water bodies, which are monitored for microbial water quality by quantification of faecal indicator organisms (IOs), can contain various zoonotic pathogens contributed by livestock waste and other sources. Sediments can serve as reservoirs of IOs and other enteric microorganisms, including pathogens. Agrochemicals may influence the survival of these microorganisms in water bodies impacted by livestock waste by enhancing or reducing their survival. Complex, 1100 l, freshwater mesocosms containing leaf litter, zooplankton, periphyton, phytoplankton, and invertebrate and vertebrate animals were used to investigate the response of Escherichia coli and enterococci to agrochemicals. Replicate tanks were treated with atrazine, malathion, chlorothalonil and inorganic fertilizer, either alone at 1× or 2× their expected environmental concentrations (EECs) or in pair-wise combinations at their EECs. IOs inoculated in sediment (∼10⁴ cfu per 100 ml) were enumerated over 28 days. IOs generally declined over time, but manova revealed that addition of fertilizer and atrazine resulted in significantly greater IO densities. Malathion, chlorothalonil and agrochemical concentration (1× vs 2×) did not significantly affect IO densities and no significant interactions between agrochemicals were noted. The augmentation of IO densities in sediments by fertilizer and atrazine may impact their reliability as accurate predictors of water quality and human health risk, and indicates the need for a better understanding of the fate of IOs and enteric pathogens in sediments exposed to agrochemicals., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Recent evidence of extended survival of fecal indicator bacteria in sediments and submerged aquatic vegetation (SAV) has raised concerns about using indicator bacteria to reliably detect fecal contamination. We monitored enterococci densities and population structure in water, sediment and SAV simultaneously at sites across a subtropical watershed (Tampa Bay, FL, USA) over one year to determine the extent to which each matrix serves as a potential reservoir of enterococci. SAV harbored significantly higher mean densities of enterococci than sediments, which harbored higher densities than water. Mean enterococci densities were also greater at sites located further upstream in the watershed. The population structure assessed by BOX-PCR genotyping was relatively dissimilar in each sample, although some similarities among samples suggested grouping by location. Strain diversity ranged from very high to negligible, with lowest overall diversity in lake samples taken during the summer. Several strains were highly abundant and cosmopolitan (found across sites, seasons, and matrices) and were identified by 16S rRNA gene sequencing as the Enterococcus species casseliflavus, faecalis, faecium, hirae, and mundtii. The proportional dominance of certain strains suggests the existence of persistent and possibly naturalized indicator bacteria populations that are not directly related to pollution events., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Enterococcus spp. are utilized worldwide as faecal indicator bacteria, but certain strains exhibit extended survival in environmental habitats and the factors influencing their persistence are poorly understood. We used flowing freshwater mesocosms to explore the effect of submerged aquatic vegetation (SAV) on the persistence of natural enterococci populations from a subtropical lake. The highest mean densities of culturable enterococci over 2 weeks occurred in SAV [8.6 x 10(2) colony-forming units (cfu) per 100 g wet weight], followed by sediments (1.3 x 10(2) cfu per 100 g) and water (18 cfu per 100 ml). However, due to relative differences in the total mass of each substrate in the entire system (water > sediments > SAV), SAV-associated enterococci represented only a minor proportion of the total population. Vegetated mesocosms harboured significantly higher mean cfu per mesocosm and cfu densities in sediments compared with their unvegetated counterparts, suggesting that SAV indirectly facilitates persistence in aquatic habitats. Populations were dominated (> 96%) by a single Enterococcus casseliflavus strain according to BOX-PCR genotyping, which did not change over the 10-month study and strongly suggests bacterial replication in the lake. The presence of such strains in the environment may represent highly competitive, naturalized and reproducing indicator bacteria populations that are not directly related to pollution events.

Fecal coliforms and enterococci are indicator organisms used worldwide to monitor water quality. These bacteria are used in microbial source tracking (MST) studies, which attempt to assess the contribution of various host species to fecal pollution in water. Ideally, all strains of a given indicator organism (IO) would experience equal persistence (maintenance of culturable populations) in water; however, some strains may have comparatively extended persistence outside the host, while others may persist very poorly in environmental waters. Assessment of the relative contribution of host species to fecal pollution would be confounded by differential persistence of strains. Here, freshwater and saltwater mesocosms, including sediments, were inoculated with dog feces, sewage, or contaminated soil and were incubated under conditions that included natural stressors such as microbial predators, radiation, and temperature fluctuations. Persistence of IOs was measured by decay rates (change in culturable counts over time). Decay rates were influenced by IO, inoculum, water type, sediment versus water column location, and Escherichia coli strain. Fecal coliform decay rates were significantly lower than those of enterococci in freshwater but were not significantly different in saltwater. IO persistence according to mesocosm treatment followed the trend: contaminated soil > wastewater > dog feces. E. coli ribotyping demonstrated that certain strains were more persistent than others in freshwater mesocosms, and the distribution of ribotypes sampled from mesocosm waters was dissimilar from the distribution in fecal material. These results have implications for the accuracy of MST methods, modeling of microbial populations in water, and efficacy of regulatory standards for protection of water quality.

Huntington Beach, California, one of the most popular surfing spots in the world, is plagued by sporadic, elevated levels of fecal bacteria. To assist with pollution source identification, we analyzed antibiotic resistance patterns (ARPs) of enterococci from four known sources (bird feces, urban runoff, coastal marsh sediment and sewage effluent from local sanitation district) and one unknown source (seawater) using seven antibiotics at four concentrations each. Of 2491 enterococci tested, all were resistant to at least one antibiotic at some level. Discriminant analysis indicated that the average correct classification rates for bird feces and urban runoff sources were above 80%. Sewage effluent contained mixed fecal sources. Sixty-four percent of the sewage isolates classified with the sewage category, while the other 35% of isolates were assigned evenly across the other three categories. When enterococci isolated from the seawater were classified using the known ARP database, it was evident that bird feces were the source of surf zone contamination on some days while the coastal salt marsh and sewage plume may have impacted the surf zone water quality to various degrees during other times.

Researchers isolated vancomycin-resistant strains of Enterococcus spp. from residential and hospital wastewater, sewage and chicken feces, but not from surface water or other animal feces. Vancomycin resistance was associated with the vanA and vanC genes.

Same-day microbial water quality assessments are not possible with standard methods, which increases the possibility of public exposure to fecal pathogens. This study examined the efficacy of high-volume hollow fibre ultrafiltration coupled to biosensor detection for enterococci in marine waters to allow same-day public notification of poor water quality. Fifty-six 100 l ultrafiltered samples and 100 ml grab samples were collected weekly from May to July 2007. Post-ultrafiltration processing included sonication and micron sieve passage to remove interfering particulates, followed by centrifugation for secondary concentration. Levels of enterococci in grab and ultrafiltration samples were determined by a standard method (EPA method 1600) for calculation of recovery efficiencies and concentration factors. Each final retentate was analysed with the RAPTOR evanescent wave biosensor. Enterococci levels increased over 26,000-fold in final retentates. Enterococci were detected when ambient concentrations exceeded the regulatory standard for a single sample (≥105 CFU/100 ml), and detection was highly correlated with breaches of the single-sample regulatory limit. The combined procedure required 2.5 h for detection compared with 24 h for EPA method 1600. This field study achieved rapid detection of enterococci by ultrafiltration, secondary concentration and biosensor analysis, and demonstrates its potential usefulness for water quality monitoring. [ABSTRACT FROM AUTHOR]

The validity of using indicator organisms (total and fecal coliforms, enterococci, Clostridium perfringens, and F-specific coliphages) to predict the presence or absence of pathogens (infectious enteric viruses, Cryptosporidium, and Giardia) was tested at six wastewater reclamation facilities. Multiple samplings conducted at each facility over a 1-year period. Larger sample volumes for indicators (0.2 to 0.4 liters) and pathogens (30 to 100 liters) resulted in more sensitive detection limits than are typical of routine monitoring. Microorganisms were detected in disinfected effluent samples at the following frequencies: total coliforms, 63%; fecal coliforms, 27%; enterococci, 27%; C. perfringens, 61%; F-specific coliphages, ∼40%; and enteric viruses, 31%. Cryptosporidium oocysts and Giardia cysts were detected in 70% and 80%, respectively, of reclaimed water samples. Viable Cryptosporidium, based on cell culture infectivity assays, was detected in 20% of the reclaimed water samples. No strong correlation was found for any indicator-pathogen combination. When data for all indicators were tested using discriminant analysis, the presence/absence patterns for Giardia cysts, Cryptosporidium oocysts, infectious Cryptosporidium, and infectious enteric viruses were predicted for over 71% of disinfected effluents. The failure of measurements of single indicator organism to correlate with pathogens suggests that public health is not adequately protected by simple monitoring schemes based on detection of a single indicator, particularly at the detection limits routinely employed. Monitoring a suite of indicator organisms in reclaimed effluent is more likely to be predictive of the presence of certain pathogens, and a need for additional pathogen monitoring in reclaimed water in order to protect public health is suggested by this study. [ABSTRACT FROM AUTHOR]

Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 μl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcusfaecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking ‘toolbox.’ [ABSTRACT FROM AUTHOR]

Sewage spills can release antibiotic-resistant bacteria into surface waters, contributing to environmental reservoirs and potentially impacting human health. Vancomycin-resistant enterococci (VRE) are nosocomial pathogens that have been detected in environmental habitats, including soil, water, and beach sands, as well as wildlife feces. However, VRE harboring vanA genes that confer high-level resistance have infrequently been found outside clinical settings in the United States. This study found culturable Enterococcus faecium harboring the vanA gene in water and sediment for up to 3 days after a sewage spill, and the quantitative PCR (qPCR) signal for vanA persisted for an additional week. Culturable levels of enterococci in water exceeded recreational water guidelines for 2 weeks following the spill, declining about five orders of magnitude in sediments and two orders of magnitude in the water column over 6 weeks. Analysis of bacterial taxa via 16S rRNA gene sequencing showed changes in community structure through time following the sewage spill in sediment and water. The spread of opportunistic pathogens harboring high-level vancomycin resistance genes beyond hospitals and into the broader community and associated habitats is a potential threat to public health, requiring further studies that examine the persistence, occurrence, and survival of VRE in different environmental matrices. [ABSTRACT FROM AUTHOR]

Enterococci are common, commensal members of gut communities in mammals and birds, yet they are also opportunistic pathogens that cause millions of human and animal infections annually. Because they are shed in human and animal feces, are readily culturable, and predict human health risks from exposure to polluted recreational waters, they are used as surrogates for waterborne pathogens and as fecal indicator bacteria (FIB) in research and in water quality testing throughout the world. Evidence from several decades of research demonstrates, however, that enterococci may be present in high densities in the absence of obvious fecal sources and that environmental reservoirs of these FIB are important sources and sinks, with the potential to impact water quality. This review focuses on the distribution and microbial ecology of enterococci in environmental (secondary) habitats, including the effect of environmental stressors; an outline of their known and apparent sources, sinks, and fluxes; and an overview of the use of enterococci as FIB. Finally, the significance of emerging methodologies, such as microbial source tracking (MST) and empirical predictive models, as tools in water quality monitoring is addressed. The mounting evidence for widespread extraenteric sources and reservoirs of enterococci demonstrates the versatility of the genus Enterococcus and argues for the necessity of a better understanding of their ecology in natural environments, as well as their roles as opportunistic pathogens and indicators of human pathogens. [ABSTRACT FROM AUTHOR]

Hollow-fiber ultrafiltration (HFUF) and PCR were combined to detect human-associated microbial source tracking marker genes in large volumes of fresh and estuarine Florida water. HFUF allowed marker detection when membrane filtration did not, demonstrating HFUF's ability to facilitate detection of diluted targets by PCR in a variety of water types. [ABSTRACT FROM AUTHOR]