Detection of Human-Derived Fecal Pollution in Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay

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 Enterococcus faecium 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.”     

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.     

Application of microbial source tracking methods in a Gulf of Mexico field setting

Aims:  Microbial water quality and possible human sources of faecal pollution were assessed in a Florida estuary that serves shellfishing and recreational activities.
Methods and Results:  Indicator organisms (IO), including faecal coliforms, Escherichia coli and enterococci, were quantified from marine and river waters, sediments and oysters. Florida recreational water standards were infrequently exceeded (6–10% of samples); however, shellfishing standards were more frequently exceeded (28%). IO concentrations in oysters and overlaying waters were significantly correlated, but oyster and sediment IO concentrations were uncorrelated. The human-associated esp gene of Enterococcus faecium was detected in marine and fresh waters at sites with suspected human sewage contamination. Lagrangian drifters, used to determine the pathways of bacterial transport and deposition, suggested that sediment deposition from the Ochlockonee River contributes to frequent detection of esp at a Gulf of Mexico beach.
Conclusions:  These data indicate that human faecal pollution affects water quality in Wakulla County and that local topography and hydrology play a role in bacterial transport and deposition.
Significance and Impact of the Study:  A combination of IO enumeration, microbial source tracking methods and regional hydrological study can reliably inform regulatory agencies of IO sources, improving risk assessment and pollution mitigation in impaired waters.     

Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters

Before new, rapid quantitative PCR (qPCR) methods for assessment of recreational water quality and microbial source tracking (MST) can be useful in a regulatory context, an understanding of the ability of the method to detect a DNA target (marker) when the contaminant source has been diluted in environmental waters is needed. This study determined the limits of detection and quantification of the human-associated Bacteroides sp. (HF183) and human polyomavirus (HPyV) qPCR methods for sewage diluted in buffer and in five ambient, Florida water types (estuarine, marine, tannic, lake, and river). HF183 was quantifiable in sewage diluted up to 10⁻⁶ in 500-ml ambient-water samples, but HPyVs were not quantifiable in dilutions of >10⁻⁴. Specificity, which was assessed using fecal composites from dogs, birds, and cattle, was 100% for HPyVs and 81% for HF183. Quantitative microbial risk assessment (QMRA) estimated the possible norovirus levels in sewage and the human health risk at various sewage dilutions. When juxtaposed with the MST marker detection limits, the QMRA analysis revealed that HF183 was detectable when the modeled risk of gastrointestinal (GI) illness was at or below the benchmark of 10 illnesses per 1,000 exposures, but the HPyV method was generally not sensitive enough to detect potential health risks at the 0.01 threshold for frequency of illness. The tradeoff between sensitivity and specificity in the MST methods indicates that HF183 data should be interpreted judiciously, preferably in conjunction with a more host-specific marker, and that better methods of concentrating HPyVs from environmental waters are needed if this method is to be useful in a watershed management or monitoring context.     

Distribution and diversity of the enterococcal surface protein (esp) gene in animal hosts and the Pacific coast environment

Aims:  This study sought to evaluate the distribution of the enterococcal surface protein ( esp ) gene in Enterococcus faecium in the Pacific coast environment as well as the distribution and diversity of the gene in Northern California animal hosts.
Methods and Results:  Over 150 environmental samples from the Pacific coast environment (sand, surf zone, fresh/estuarine, groundwater, and storm drain) were screened for the esp gene marker in E. faecium , and the marker was found in 37% of the environmental samples. We examined the host specificity of the gene by screening various avian and mammalian faecal samples, and found the esp gene to be widespread in nonhuman animal faeces. DNA sequence analysis performed on esp polymerase chain reaction amplicons revealed that esp gene sequences were not divergent between hosts.
Conclusions:  Our data confirm recent findings that the E. faecium variant of the esp gene is not human-specific.
Significance and Impact of the Study:  Our results suggest that the use of the esp gene for microbial source tracking applications may not be appropriate at all recreational beaches.     

Investigation of human sewage pollution and pathogen analysis at Florida Gulf coast beaches

Aims: Water quality at two Florida beaches was compared using faecal indicator bacteria measurements, microbial source tracking (MST) methods for detecting human source pollution and the assessment of pathogen presence. These values were also compared before and after remediation of wastewater infrastructure at one beach. Methods and Results: Faecal coliforms, Escherichia coli and enterococci were enumerated in estuarine water and sediment samples. PCR assays for the human‐associated esp gene of Enterococcus faecium and human polyomaviruses (HPyVs) were used to detect human sewage. Culturable Salmonella and enteric viruses were also analysed. MST identified human sewage contamination at one beach, leading to repair of a sewer main and relocation of portable restrooms. Exceedances of Florida recreational water regulatory standards were significantly reduced after remediation (by 52% for faecal coliforms and 39% for enterococci), and the frequency of detection of MST markers decreased. Coxsackie virus B4 and HPyVs were codetected following a major sewage spill, but Salmonella was not detected during the study. Conclusions: These data indicate that infrastructure remediation significantly reduced pollution from human sewage at the impacted beach. Significance and Impact of the Study: A comprehensive microbial water quality study that can identify contamination sources through the use of MST markers and close collaboration with local/and state agencies can result in tangible actions to improve recreational water quality and safety.     

A real-time polymerase chain reaction assay for quantitative detection of the human-specific enterococci surface protein marker in sewage and environmental waters

A real-time polymerase chain reaction (PCR) assay using SYBR Green I dye was developed to quantify the Enterococcus faecium enterococci surface protein (esp) marker in sewage (n = 16) and environmental waters (n = 16). The concentration of culturable enterococci in raw sewage samples ranged between 1.3 x 10(5) and 5.6 x 10(5) colony-forming units (cfu) per 100 ml. The real-time PCR detected 9.8 x 10(3)-3.8 x 10(4) gene copies of the esp marker per 100 ml of sewage. However, the concentration of culturable enterococci and the esp marker in secondary effluent was two orders of magnitude lower than raw sewage. Surface water samples were collected from a non-sewered catchment after storm events and the real-time PCR was applied to quantify the esp marker. Of the 16 samples tested, 6 (38%) were PCR-positive and the concentration of the esp marker ranged between 1.1 x 10(2) and 5.3 x 10(2) gene copies per 100 ml of water samples. The newly developed real-time PCR method was successfully used to quantify the esp marker in samples collected from sewage and environmental waters. The presence of the esp marker in water samples immediately after storm events not only indicated human faecal pollution but also provided evidence of the degree of human faecal pollution. To our knowledge, this is the first study that reports the use of a real-time PCR assay to quantify the esp marker in sewage and surface waters. Such study would provide valuable information for managers for the improved management of water quality.     

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Alternative Fecal Indicators and Their Empirical Relationships with Enteric Viruses,Salmonella enterica,and Pseudomonas aeruginosa in Surface Waters of a Tropical Urban Catchment

The suitability of traditional microbial indicators (i.e., Escherichia coli and enterococci) has been challenged due to the lack of correlation with pathogens and evidence of possible regrowth in the natural environment. In this study, the relationships between alternative microbial indicators of potential human fecal contamination (Bacteroides thetaiotaomicron, Methanobrevibacter smithii, human polyomaviruses [HPyVs], and F+ and somatic coliphages) and pathogens (Salmonella spp., Pseudomonas aeruginosa, rotavirus, astrovirus, norovirus GI, norovirus GII, and adenovirus) were compared with those of traditional microbial indicators, as well as environmental parameters (temperature, conductivity, salinity, pH, dissolved oxygen, total organic carbon, total suspended solids, turbidity, total nitrogen, and total phosphorus). Water samples were collected from surface waters of urban catchments in Singapore. Salmonella and P. aeruginosa had significant positive correlations with most of the microbial indicators, especially E. coli and enterococci. Norovirus GII showed moderately strong positive correlations with most of the microbial indicators, except for HPyVs and coliphages. In general, high geometric means and significant correlations between human-specific markers and pathogens suggest the possibility of sewage contamination in some areas. The simultaneous detection of human-specific markers (i.e., B. thetaiotaomicron, M. smithii, and HPyVs) with E. coli and enterococcus supports the likelihood of recent fecal contamination, since the human-specific markers are unable to regrow in natural surface waters. Multiple-linear-regression results further confirm that the inclusion of M. smithii and HPyVs, together with traditional indicators, would better predict the occurrence of pathogens. Further study is needed to determine the applicability of such models to different geographical locations and environmental conditions.     

Comparative survival of antibiotic-resistant and -sensitive fecal indicator bacteria in estuarine water.

The survival of antibiotic-resistant and -sensitive strains of Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Streptococcus equinus, and two environmental isolates, AP17 and AQ62, was examined in estuarine water. Each strain was rendered resistant to a combination of two antibiotics by serial passage in increasing concentrations of antibiotics. Cultures were incubated in filter-sterilized estuarine water for up to 7 days. Recovery was assessed by examining colony-forming ability on media with and without antibiotics. None of the antibiotic-resistant forms survived longer than its antibiotic-sensitive counterpart in estuarine water. Three of the resistant strains died off more rapidly than the antibiotic-sensitive wild type. Survival of the test bacteria in estuarine water was as follows: sensitive and resistant AQ62, resistant Escherichia coli less than sensitive Escherichia coli less than resistant AP17 less than resistant Enterococcus faecium less than sensitive AP17, sensitive and resistant S. equinus less than sensitive and resistant Enterococcus faecalis, sensitive Enterococcus faecium. The results supported the suggestion that fecal entercocci may serve as better indicators of fecal pollution than Escherichia coli in marine ecosystems. Moreover, the results indicated that the use of antibiotic-resistant mutants to follow the fate of bacteria in the environment is inappropriate without adequate studies to ensure that resistant and wild-type strains react similarly to environmental stressors.     

Arcobacter in Lake Erie beach waters: an emerging gastrointestinal pathogen linked with human-associated fecal contamination

The genus Arcobacter has been associated with human illness and fecal contamination by humans and animals. To better characterize the health risk posed by this emerging waterborne pathogen, we investigated the occurrence of Arcobacter spp. in Lake Erie beach waters. During the summer of 2010, water samples were collected 35 times from the Euclid, Villa Angela, and Headlands (East and West) beaches, located along Ohio's Lake Erie coast. After sample concentration, Arcobacter was quantified by real-time PCR targeting the Arcobacter 23S rRNA gene. Other fecal genetic markers (Bacteroides 16S rRNA gene [HuBac], Escherichia coli uidA gene, Enterococcus 23S rRNA gene, and tetracycline resistance genes) were also assessed. Arcobacter was detected frequently at all beaches, and both the occurrence and densities of Arcobacter spp. were higher at the Euclid and Villa Angela beaches (with higher levels of fecal contamination) than at the East and West Headlands beaches. The Arcobacter density in Lake Erie beach water was significantly correlated with the human-specific fecal marker HuBac according to Spearman's correlation analysis (r = 0.592; P < 0.001). Phylogenetic analysis demonstrated that most of the identified Arcobacter sequences were closely related to Arcobacter cryaerophilus, which is known to cause gastrointestinal diseases in humans. Since human-pathogenic Arcobacter spp. are linked to human-associated fecal sources, it is important to identify and manage the human-associated contamination sources for the prevention of Arcobacter-associated public health risks at Lake Erie beaches.     

Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards.     

Quantification of Human Polyomaviruses JC Virus and BK Virus by TaqMan Quantitative PCR and Comparison to Other Water Quality Indicators in Water and Fecal Samples

In the United States, total maximum daily load standards for bodies of water that do not meet bacterial water quality standards are set by each state. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have developed and optimized a TaqMan quantitative PCR (QPCR) assay based on the conserved T antigen to both quantify and simultaneously detect two HPyVs; JC virus and BK virus. The QPCR assay was able to consistently quantify ≥10 gene copies per reaction and is linear over 5 orders of magnitude. HPyVs were consistently detected in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5) and were not amplified in DNA extracted from 127 animal waste samples from 14 species. HPyV concentrations in sewage decreased 81.2 and 84.2% over 28 days incubation at 25 and 35°C, respectively. HPyVs results were compared to Escherichia coli, fecal coliform, and enterococci concentrations and the presence of three other human-associated microbes: Bacteroidetes, Methanobrevibacter smithii, and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples and were more human specific than the Bacteroidetes (HF183) or M. smithii. HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of contaminated waters and ultimately protect humans from exposure to pathogens.Maintaining healthy coastal water systems is essential, since poor water quality can have detrimental effects on mangroves, seagrass beds, coral reefs, the fishing and shellfish harvesting industries, and the health of recreational water users (1, 5, 15, 17, 20, 44). Since 1972 in the United States, each state has been required to set total maximum daily loads (TMDLs) for pollutants in water bodies according to section 303(d) of the Clean Water Act (50). The probability that microbial pathogens are present is estimated by enumerating indicator bacteria, which are shed in the feces of humans and most animals. The U.S. Environmental Protection Agency recommends using Escherichia coli and enterococci to assess the quality of freshwater and saline water, respectively (47); however, Florida currently uses fecal coliforms and enterococci as indicators of fecal pollution (42).When bacterial indicators exceed regulatory levels, a plan of action (TMDL implementation) must be developed to reduce pathogens. TMDL plans for “pathogen” reduction are particularly problematic because they rely upon surrogate indicator bacteria, which yield little or no insight as to the source of pollution. High indicator bacteria concentrations can be attributed to many sources, including agricultural runoff, storm water runoff, wildlife, pets, faulty septic systems (onsite wastewater treatment and disposal systems), and a failing central sewer infrastructure (5, 12, 28).To address the issue of source identification, methods have been developed in which the biochemistry or genetics of certain microorganisms are used to indirectly identify probable source(s) of fecal pollution, which is termed microbial source tracking (MST) (48). MST methods based on detection of a source-associated gene (marker) by PCR have proliferated over the past 10 years due to the additional information they can provide to watershed managers on fecal contamination sources (43). Although marker detection by endpoint (binary) PCR can give important insights on the source(s) of fecal contamination, quantitative measurements can provide information about the relative magnitude of contamination from various sources. Moreover, epidemiological studies on the correlation between recreational water use, microbial contamination, and the risk of illness will greatly benefit from the ability to quantify MST markers, rather than simply assessing binary (+/−) detection.Although many bacterial targets have been proposed for MST of human sewage (8, 39, 46a), fewer viral targets have been investigated (19, 24, 33). Polyomavirus is the sole genus in the family Polyomaviridae (22). These viruses have a 5-kbp double-stranded DNA genome surrounded by a 40- to 50-nm icosahedral capsid (38). The JCV and BKV human polyomaviruses (HPyVs) have similarly structured genomes that show ∼75% identity (21). BK virus (BKV) and JC virus (JCV) gained much attention in the late 1970s as the etiological agents of kidney nephritis (i.e., BKV reactivation in the kidneys) and progressive multifocal leukoencephalopathy (i.e., JCV reactivation in brain tissue) in the immunocompromised (16, 34). Serological studies have shown that >70% of adults harbor antibodies to BKV or JCV (27, 30, 44). These viruses are known for producing lifelong, asymptomatic viruria in immunocompetent individuals (37). In 2000 it was first suggested that JCV would be a useful indicator of human sewage in water (11). The obligate host specificity and abundance of BKV and JCV in municipal sewage has led to the successful use of these viruses to indicate human fecal pollution in environmental water samples (12, 29).Due to the health implications of BKV and JCV, several methods have been developed to rapidly detect either BKV or JCV in clinical samples (6, 31, 35, 56). However, from an MST standpoint, it is advantageous to target both BKV and JCV. BKV has been found in feces (54), and both viruses are excreted in the urine (6, 11, 37, 55, 60) either simultaneously or individually. The focus of this research was the modification of the previously developed nested PCR protocol for HPyVs detection (29) to a TaqMan quantitative PCR (QPCR) assay to simultaneously detect and quantify both BKV and JCV. Furthermore, we compared measurements obtained with the newly developed QPCR assay to those of other water quality indicators and MST markers. These indicators included bacterial indicator concentrations (49) and PCR detection of human-associated markers currently used for MST. These included human-associated Bacteroidetes (8), Methanobrevibacter smithii (46a), and adenovirus (36). To assess the potential of HPyVs to mimic the fate of pathogens in water, the persistence of all of the water quality indicators was assessed, and relationships between bacterial indicator organisms and MST markers in both human waste samples as well as contaminated environmental samples were examined.     

Lachnospiraceae and Bacteroidales alternative fecal indicators reveal chronic human sewage contamination in an urban harbor

The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the Lachnospiraceae family, which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci and the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.     

Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

Relationship between biofilm formation, the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium

One-hundred and twenty-eight enterococcal isolates were examined for their ability to form biofilm in relation to the presence of the gene encoding the enterococcal surface protein (esp), production of gelatinase and to the source of isolation. Neither esp nor gelatinase seemed to be required for biofilm formation: both Enterococcus faecalis and Enterococcus faecium did not show a correlation between the presence of either esp or the production of gelatinase and biofilm formation. However, in E. faecium while esp was found in isolates from either source, the presence of both esp and biofilm together was only found in strains from clinical settings, suggesting that there exists a synergy between these factors which serves as an advantage for the process of infection.