Geospatial Patterns of Antimicrobial Resistance Genes in the US EPA National Rivers and Streams Assessment Survey.

Antimicrobial resistance (AR) is a serious global problem due to the overuse of antimicrobials in human, animal, and agriculture sectors. There is intense research to control the dissemination of AR, but little is known regarding the environmental drivers influencing its spread. Although AR genes (ARGs) are detected in many different environments, the risk associated with the spread of these genes to microbial pathogens is unknown. Recreational microbial exposure risks are likely to be greater in water bodies receiving discharge from human and animal waste in comparison to less disturbed aquatic environments. Given this scenario, research practitioners are encouraged to consider an ecological context to assess the effect of environmental ARGs on public health. Here, we use a stratified, probabilistic survey of nearly 2000 sites to determine national patterns of the anthropogenic indicator class I integron Integrase gene (intI1) and several ARGs in 1.2 million kilometers of United States (US) rivers and streams. Gene concentrations were greater in eastern than in western regions and in rivers and streams in poor condition. These first of their kind findings on the national distribution of intI1 and ARGs provide new information to aid risk assessment and implement mitigation strategies to protect public health.

Persistence of fecal indicator bacteria and associated genetic markers from wastewater treatment plant effluents in freshwater microcosms.

Limited information exists on the environmental persistence of genetic markers for fecal indicator bacteria (FIB) in treated wastewaters. Here, the decay rate constants of culturable cells and genetic markers for four diverse groups of FIBs, such as enterococci, Clostridium, Escherichia coli, and Bacteroides, were investigated in freshwater microcosms seeded with disinfected and non-disinfected secondary-treated wastewaters. Decay rate constants of genetic markers and culturable cells varied significantly among the different FIB groups. Water temperatures (winter vs. fall/spring/summer) significantly affected the decay of all genetic marker and cell types; however, genetic marker decay were not found to be significantly different in disinfected (chlorination/ultraviolet) and non-disinfected wastewater-seeded microcosms or, for example, lake- and river-receiving waters. No evidence was seen that decay rate constants of FIB genetic markers from treated wastewater were substantially different from those observed in similar, previously reported microcosm studies using raw sewage. Unexpected relationships between decay rate constants of genetic markers and culturable cells of Bacteroides were observed. Results suggest that decay rate constants of FIB genetic markers determined from other studies may be applicable to treated wastewaters. Results of this study should be informative for ongoing efforts to determine the persistence of FIB genetic markers relative to surviving pathogens after wastewater treatment.

A Constructed Wetland for Treatment of an Impacted Waterway and the Influence of Native Waterfowl on its Perceived Effectiveness.

A constructed, variable-flow treatment wetland was evaluated for its ability to reduce microbial loads from the Banklick Creek, an impacted recreational waterway in Northern Kentucky. For this study, levels of traditional (Escherichia coli and enterococci measured by culture and molecular techniques) and alternative fecal indicators (infectious somatic and F+ coliphage, Clostridium spp. and Clostridium perfringens by culture), potential pathogens (molecular signal of Campylobacter spp.) as well as various microbial source tracking (MST) markers (human fecal marker HF183 and avian fecal marker GFD) were monitored during the summer and early fall through five treatment stages within the Banklick Creek Wetland. No difference in concentrations of traditional or alternative fecal indicators were observed in any of the sites monitored. Microbial source tracking markers were employed to identify sources of fecal contamination within the wetland. Human marker HF183 concentrations at beginning stages of treatment were found to be significantly higher (P value range: 0.0016-0.0003) than levels at later stages. Conversely, at later stages of treatment where frequent bird activity was observed, Campylobacter and avian marker (GFD) signals were detected at significantly higher frequencies (P value range: 0.024 to <0.0001), and both signals were strongly correlated (P = 0.0001). Our study suggests constructed wetlands are an effective means for removal of microbial contamination in ambient waters, but reliance on general fecal indicators is not ideal for determining system efficacy or assessing appropriate remediation efforts.

Exposure to human-associated fecal indicators and self-reported illness among swimmers at recreational beaches: a cohort study.

BACKGROUND: Fecal indicator bacteria used to assess illness risks in recreational waters (e.g., Escherichia coli, Enterococci) cannot discriminate among pollution sources. To address this limitation, human-associated Bacteroides markers have been proposed, but the risk of illness associated with the presence of these markers in recreational waters is unclear. Our objective was to estimate associations between human-associated Bacteroides markers in water and self-reported illness among swimmers at 6 U.S. beaches spanning 2003-2007. METHODS: We used data from a prospectively-enrolled cohort of 12,060 swimmers surveyed about beach activities and water exposure on the day of their beach visit. Ten to twelve days later, participants reported gastroinestinal, diarrheal, and respiratory illnesses experienced since the visit. Daily water samples were analyzed for the presence of human-associated Bacteroides genetic markers: HF183, BsteriF1, BuniF2, HumM2. We used model-based standardization to estimate risk differences (RD) and 95% confidence intervals (CI). We assessed whether the presence of Bacteroides markers were modifiers of the association between general Enterococcus and illness among swimmers using interaction contrast. RESULTS: Overall we observed inconsistent associations between the presence of Bacteroides markers and illness. There was a pattern of increased risks of gastrointestinal (RD = 1.9%; 95% CI: 0.1%, 3.7%), diarrheal (RD = 1.3%; 95% CI: -0.2%, 2.7%), and respiratory illnesses (RD = 1.1%; 95% CI: -0.2%, 2.5%) associated with BsteriF1. There was no evidence that Bacteroides markers acted as modifiers of Enterococcus and illness. Patterns were similar when stratified by water matrix. CONCLUSIONS: Quantitative measures of fecal pollution using Bacteroides, rather than presence-absence indicators, may be necessary to accurately assess human risk specific to the presence of human fecal pollution.

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods.

There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria proposed in this study should help scientists, managers, reviewers, and the public evaluate the technical quality of future findings against an established benchmark.

Evaluation of quantitative polymerase chain reaction assays targeting Mycobacterium avium, M. intracellulare, and M. avium subspecies paratuberculosis in drinking water biofilms.

Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples.

Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples.

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

Influence of wastewater disinfection on densities of culturable fecal indicator bacteria and genetic markers.

The US Environmental Protection Agency has proposed the use of quantitative polymerase chain reaction (qPCR) as a rapid alternative analytical method for monitoring recreational water quality at beaches. For qPCR to be considered for other Clean Water Act purposes, such as inclusion in discharge permits and use in Total Maximum Daily Load calculations, it is necessary to understand how qPCR detectable genetic markers are influenced by wastewater disinfection. This study investigated genetic markers for Escherichia coli, Enterococcus, Clostridium spp., Bacteroides, total Bacteroidales, as well as the human-associated Bacteroides markers, HF183 and HumM2, to determine which, if any, were influenced by disinfection (chlorination or ultraviolet light) of effluents from secondary wastewater treatment in different seasons. The effects of disinfection on culturable enterococci, E. coli, Bacteroides, and C. perfringens were also compared to their associated genetic markers. Disinfection of secondary treatment effluents significantly reduced culturable fecal indicator bacteria (FIB) but not genetic marker densities. No significant differences were observed in the responses of FIB culture and genetic marker densities to type of disinfection (chlorination vs UV) or season. Results of this study provide evidence that qPCR may not be suitable for monitoring efficacy of wastewater disinfection on the inactivation of bacterial pathogens.

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese.

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland-a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

Distribution of genetic marker concentrations for fecal indicator bacteria in sewage and animal feces.

Very little is known about the density and distribution of fecal indicator bacteria (FIB) genetic markers measured by quantitative real-time PCR (qPCR) in fecal pollution sources. Before qPCR-based FIB technologies can be applied to waste management and public health risk applications, it is vital to characterize the concentrations of these genetic markers in pollution sources (i.e., untreated wastewater and animal feces). We report the distribution of rRNA genetic markers for several general FIB groups, including Clostridium spp., Escherichia coli, enterococci, and Bacteroidales, as determined by qPCR on reference collections consisting of 54 primary influent sewage samples collected from treatment facilities across the United States and fecal samples representing 20 different animal species. Based on raw sewage sample collection data, individual FIB genetic markers exhibited a remarkable similarity in concentration estimates from locations across the United States ranging from Hawaii to Florida. However, there was no significant correlation between genetic markers for most FIB combinations (P > 0.05). In addition, large differences (up to 5 log(10) copies) in the abundance of FIB genetic markers were observed between animal species, emphasizing the importance of indicator microorganism selection and animal source contribution for future FIB applications.

Correlation between quantitative PCR and culture-based methods for measuring Enterococcus spp. over various temporal scales at three California marine beaches.

Several studies have examined how fecal indicator bacteria (FIB) measurements compare between quantitative PCR (qPCR) and the culture methods it is intended to replace. Here, we extend those studies by examining the stability of that relationship within a beach, as affected by time of day and seasonal variations in source. Enterococcus spp. were quantified at three southern California beaches in the morning and afternoon using two qPCR assays, membrane filtration, and defined-substrate testing. While qPCR and culture-based measurements were consistently and significantly correlated, strength of the correlation varied both among and within beaches. Correlations were higher in the morning (0.45 < ρ < 0.74 [P < 0.002]) than in the afternoon (0.18 < ρ < 0.45 [P < 0.021]) and higher when the fecal contamination was concentrated (0.38 < ρ < 0.83 [P < 0.001]) than when it was diffuse (0.19 < ρ < 0.34 [P < 0.003]). The ratios of culture-based and qPCR results (CFU or most probable number [MPN] per calibrator cell equivalents [CCE]) also varied spatially and temporally. Ratios ranged between 0.04 and 0.85 CFU or MPN per CCE and were lowest at the beach affected by diffuse pollution. Patterns in the ratios over the course of the day were dissimilar across beaches, increasing with time at one beach and decreasing at another. The spatial and temporal variability we observed indicate that the empirical relationship between culture-based and qPCR results is not universal, even within a beach.

Fecal indicators in sand, sand contact, and risk of enteric illness among beachgoers.

BACKGROUND: Beach sand can harbor fecal indicator organisms and pathogens, but enteric illness risk associated with sand contact remains unclear. METHODS: In 2007, visitors at 2 recreational marine beaches were asked on the day of their visit about sand contact. Ten to 12 days later, participants answered questions about health symptoms since the visit. F+ coliphage, Enterococcus, Bacteroidales, fecal Bacteroides, and Clostridium spp. in wet sand were measured using culture and molecular methods. RESULTS: We analyzed 144 wet sand samples and completed 4999 interviews. Adjusted odds ratios (aORs) were computed, comparing those in the highest tertile of fecal indicator exposure with those who reported no sand contact. Among those digging in sand compared with those not digging in sand, a molecular measure of Enterococcus spp. (calibrator cell equivalents/g) in sand was positively associated with gastrointestinal (GI) illness (aOR = 2.0 [95% confidence interval (CI) = 1.2-3.2]) and diarrhea (2.4 [1.4-4.2]). Among those buried in sand, point estimates were greater for GI illness (3.3 [1.3-7.9]) and diarrhea (4.9 [1.8-13]). Positive associations were also observed for culture-based Enterococcus (colony-forming units/g) with GI illness (aOR digging = 1.7 [1.1-2.7]) and diarrhea (2.1 [1.3-3.4]). Associations were not found among nonswimmers with sand exposure. CONCLUSIONS: We observed a positive relationship between sand-contact activities and enteric illness as a function of concentrations of fecal microbial pollution in beach sand.

Comparison of Enterococcus quantitative polymerase chain reaction analysis results from fresh and marine waters on two real-time instruments.

The U.S. Environmental Protection Agency will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for implementation of this and other qPCR methods is whether the results are comparable on different PCR instruments. In this study, quantitative estimates of Enterococcus densities from marine and freshwater samples were determined by the qPCR method from cycle threshold (Ct) measurements obtained on Applied Biosystems StepOnePlus and Cepheid SmartCycler instruments. Three variations of a comparative Ct model, differing in their sources of calibration data, were used in the estimations. Both traditional and Bayesian statistical modeling approaches were examined in the instrument comparisons. The traditional analysis of variance (ANOVA) approach indicated no significant differences (p>0.05) between mean density estimates from the instruments in two of the three model variations. The Bayesian approach indicated that the 95% Bayesian credible intervals of density estimates from the instruments overlapped in all models; however, the uncertainty of the estimates varied depending on the model. These results support the interchangeable use of the two instruments in the method and also illustrate the importance of defining the source of calibration data used in the comparative Ct model.

Dramatic improvements in beach water quality following gull removal.

Gulls are often cited as important contributors of fecal contamination to surface waters, and some recreational beaches have used gull control measures to improve microbial water quality. In this study, gulls were chased from a Lake Michigan beach using specially trained dogs, and water quality improvements were quantified. Fecal indicator bacteria and potentially pathogenic bacteria were measured before and during gull control using culture methods and quantitative polymerase chain reaction (qPCR). Harassment by dogs was an effective method of gull control: average daily gull populations fell from 665 before to 17 during intervention; and a significant reduction in the density of a gull-associated marker was observed (p < 0.001). Enterococcus spp. and Escherichia coli densities were also significantly reduced during gull control (p < 0.001 and p = 0.012, respectively for culture methods; p = 0.012 and p = 0.034, respectively for qPCR). Linear regression results indicate that a 50% reduction in gulls was associated with a 38% and 29% decrease in Enterococcus spp. and E. coli densities, respectively. Potentially human pathogenic bacteria were detected on 64% of days prior to gull control and absent during gull intervention, a significant reduction (p = 0.005). This study demonstrates that gull removal can be a highly successful beach remedial action to improve microbial water quality.

Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator bacteria.

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.

Performance of NIST SRM® 2917 with 13 recreational water quality monitoring qPCR assays.

Fecal pollution remains a significant challenge for recreational water quality management worldwide. In response, there is a growing interest in the use of real-time quantitative PCR (qPCR) methods to achieve same-day notification of recreational water quality and associated public health risk as well as to characterize fecal pollution sources for targeted mitigation. However, successful widespread implementation of these technologies requires the development of and access to a high-quality standard control material. Here, we report a single laboratory qPCR performance assessment of the National Institute of Standards and Technology Standard Reference Material 2917 (NIST SRM® 2917), a linearized plasmid DNA construct that functions with 13 recreational water quality qPCR assays. Performance experiments indicate the generation of standard curves with amplification efficiencies ranging from 0.95 ± 0.006 to 0.99 ± 0.008 and coefficient of determination values (R2) ≥ 0.980. Regardless of qPCR assay, variability in repeated measurements at each dilution level were very low (quantification threshold standard deviations ≤ 0.657) and exhibited a heteroscedastic trend characteristic of qPCR standard curves. The influence of a yeast carrier tRNA added to the standard control material buffer was also investigated. Findings demonstrated that NIST SRM® 2917 functions with all qPCR methods and suggests that the future use of this control material by scientists and water quality managers should help reduce variability in concentration estimates and make results more consistent between laboratories.

Differential decay of human faecal Bacteroides in marine and freshwater.

Genetic markers from Bacteroides and other faecal bacteria are being tested for inclusion in regulations to quantify aquatic faecal contamination and estimate public health risk. For the method to be used quantitatively across environments, persistence and decay of markers must be understood. We measured concentrations of contaminant molecular markers targeting Enterococcus and Bacteroides spp. in marine and freshwater microcosms spiked with human sewage and exposed to either sunlight or dark treatments. We used Bayesian statistics with a delayed Chick-Watson model to estimate kinetic parameters for target decay. DNA- and RNA-based targets decayed at approximately the same rate. Molecular markers persisted (could be detected) longer in marine water. Sunlight increased the decay rates of cultured indicators more than those of molecular markers; sunlight also limited persistence of molecular markers. Within each treatment, Bacteroides markers had similar decay profiles, but some Bacteroides markers significantly differed in decay rates. The role of extracellular DNA in persistence appeared unimportant in the microcosms. Because conditions were controlled, microcosms allowed the effects of specific environmental variables on marker persistence and decay to be measured. While marker decay profiles in more complex environments would be expected to vary from those observed here, the differences we measured suggest that water matrix is an important factor affecting quantitative source tracking and microbial risk assessment applications.

Large-scale comparison of E. coli levels determined by culture and a qPCR method (EPA Draft Method C) in Michigan towards the implementation of rapid, multi-site beach testing.

Fecal pollution remains a challenge for water quality managers at Great Lakes and inland recreational beaches. The fecal indicator of choice at these beaches is typically Escherichia coli (E. coli), determined by culture-based methods that require over 18 h to obtain results. Researchers at the United States Environmental Protection Agency (EPA) have developed a rapid E. coli qPCR methodology (EPA Draft Method C) that can provide same-day results for improving public health protection with demonstrated sensitivity, specificity, and data acceptance criteria. However, limited information is currently available to compare the occurrence of E. coli determined by cultivation and by EPA Draft Method C (Method C). This study provides a large-scale data collection effort to compare the occurrence of E. coli determined by these alternative methods at more than 100 Michigan recreational beach and other sites using the complete set of quantitative data pairings and selected subsets of the data and sites meeting various eligibility requirements. Simple linear regression analyses of composite (pooled) data indicated a correlation between results of the E. coli monitoring approaches for each of the multi-site datasets as evidenced by Pearson R-squared values ranging from 0.452 to 0.641. Theoretical Method C threshold values, expressed as mean log10 target gene copies per reaction, that corresponded to an established E. coli culture method water quality standard of 300 MPN or CFU /100 mL varied only from 1.817 to 1.908 for the different datasets using this model. Different modeling and derivation approaches that incorporated within and between-site variability in the estimates also gave Method C threshold values in this range but only when relatively well-correlated datasets were used to minimize the error. A hypothetical exercise to evaluate the frequency of water impairments based on theoretical qPCR thresholds corresponding to the E. coli water quality standard for culture methods suggested that the methods may provide the same beach notification outcomes over 90% of the time with Method C results differing from culture method results that indicated acceptable and unacceptable water quality at overall rates of 1.9% and 6.6%, respectively. Results from this study provide useful information about the relationships between E. coli determined by culture and qPCR methods across many diverse freshwater sites and should facilitate efforts to implement qPCR-based E. coli detection for rapid recreational water quality monitoring on a large scale in the State of Michigan.

Performance assessment PCR-based assays targeting bacteroidales genetic markers of bovine fecal pollution.

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.

Performance of PCR-based assays targeting Bacteroidales genetic markers of human fecal pollution in sewage and fecal samples.

There are numerous PCR-based assays available to characterize human fecal pollution in ambient waters. Each assay employs distinct oligonucleotides and many target different genes and microorganisms leading to potential variations in assay performance. Performance comparisons utilizing feces and raw sewage samples are needed to determine which assays are best suited for expensive and time-consuming field validation, fate, transport, and epidemiology studies. We report the assessment of five end-point PCR and 10 real-time quantitative PCR (qPCR) assays that target genes from presumptive Bacteroidales microorganisms reported to be associated with human feces. Each assay was tested against a reference collection of 54 primary influent sewage samples collected from different geographical locations across the United States and 174 fecal DNA extracts from 23 different animal sources. Experiments indicate that human-associated genetic markers are distributed across a broad range of human populations but show substantial differences in specificity for human feces suggesting that particular assays may be more suitable than others depending on the abundance of genetic marker required for detection and the animal sources impacting a particular watershed or beach of interest.