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.

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.

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.

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.

Multi-laboratory survey of qPCR enterococci analysis method performance in U.S. coastal and inland surface waters.

Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta-delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta-delta tended to bias recoveries from apparent partially inhibitory samples on the high side which could help in avoiding potential underestimates of enterococci--an important consideration in a public health context. Control assay and delta-delta recovery results were largely consistent across the range of habitats sampled, and among laboratories. The methodological option that best balanced acceptable estimated target gene recoveries with method sensitivity and avoidance of underestimated enterococci densities was Method 1609 without extract dilution and using the delta-delta calculation method. The applicability of this method can be extended by the analysis of diluted extracts to sites where interference is indicated but, particularly in these instances, should be confirmed by augmenting the control assays with analyses for target gene recoveries from spiked target organisms.

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.

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

A method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for detecting interferences in RNA recovery and analysis, was developed for the direct, culture-independent detection of genetic markers from FRNA coliphage genogroups I, II & IV in water samples. Results were obtained from an initial evaluation of the performance of this method in analyses of waste water, ambient surface water and stormwater drain and outfall samples from predominantly urban locations. The evaluation also included a comparison of the occurrence of the FRNA genetic markers with genetic markers from general and human-related bacterial fecal indicators determined by current or pending EPA-validated qPCR methods. Strong associations were observed between the occurrence of the putatively human related FRNA genogroup II marker and the densities of the bacterial markers in the stormwater drain and outfall samples. However fewer samples were positive for FRNA coliphage compared to either the general bacterial fecal indicator or the human-related bacterial fecal indicator markers particularly for ambient water samples. Together, these methods show promise as complementary tools for the identification of contaminated storm water drainage systems as well as the determination of human and non-human sources of contamination.

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.

Water quality, weather and environmental factors associated with fecal indicator organism density in beach sand at two recreational marine beaches.

Recent studies showing an association between fecal indicator organisms (FIOs) in sand and gastrointestinal (GI) illness among beachgoers with sand contact have important public health implications because of the large numbers of people who recreate at beaches and engage in sand contact activities. Yet, factors that influence fecal pollution in beach sand remain unclear. During the 2007 National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study, sand samples were collected at three locations (60 m apart) on weekend days (Sat, Sun) and holidays between June and September at two marine beaches - Fairhope Beach, AL and Goddard Beach, RI - with nearby publicly-owned treatment works (POTWs) outfalls. F(+) coliphage, enterococci, Bacteroidales, fecal Bacteroides spp., and Clostridium spp. were measured in sand using culture and qPCR-based calibrator-cell equivalent methods. Water samples were also collected on the same days, times and transects as the 144 sand samples and were assayed using the same FIO measurements. Weather and environmental data were collected at the time of sample collection. Mean FIO concentrations in sand varied over time, but not space. Enterococci CFU and CCE densities in sand were not correlated, although other FIOs in sand were. The strongest correlation between FIO density in sand and water was fecal Bacteroides CCE, followed by enterococci CFU, Clostridium spp. CCE, and Bacteroidales CCE. Overall, the factors associated with FIO concentrations in sand were related to the sand-water interface (i.e., sand-wetting) and included daily average densities of FIOs in water, rainfall, and wave height. Targeted monitoring that focuses on daily trends of sand FIO variability, combined with information about specific water quality, weather, and environmental factors may inform beach monitoring and management decisions to reduce microbial burdens in beach sand. The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.

Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods.

The U.S. EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 recreational water quality criteria (RWQC). The CCE quantification unit stems from the calibration model used to estimate enterococci densities in recreational beach waters in the EPA National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study and directly informed the derivation of the RWQC recommendations. Recent studies have demonstrated that CCE estimates from the method can vary when using different cultured Enterococcus cell preparations in calibrator samples. These differences have been attributed to differences in the quantities of targeted gene copies (target sequences) that are recovered per nominal calibrator cell by DNA extraction. Standardization of results from the calibration model will require the estimation of target sequence recoveries from the calibrator and water samples. In addition, comparisons of water sample results with the RWQC values will require a knowledge of target sequence recoveries from the NEEAR study calibrator samples. In this study recoveries of target sequences and the mean target sequence/cell ratio for the NEEAR study calibrator samples were retrospectively estimated with a corroborated standard curve. A modification of the calibration model was then used to estimate enterococci target sequence quantities in water samples from eight midwestern U.S. rivers. CCE estimates were obtained by dividing these target sequence quantities by the mean NEEAR study target sequence/cell ratio. This target sequence-based quantification approach resulted in a high degree of agreement in beach action decisions (determinations of whether bacterial fecal indicator densities are above or below RWQC-recommended values) from CCE results of the qPCR method and from culture dependent enumeration of both enterococci and Eschericia coli in the corresponding water samples.

Comparison of Enterococcus quantitative polymerase chain reaction analysis results from Midwest U.S. river samples using EPA Method 1611 and Method 1609 PCR reagents.

Enterococci target sequence density estimates from analyses of diluted river water DNA extracts by EPA Methods 1611 and 1609 and estimates with lower detection limits from undiluted DNA extracts by Method 1609 were indistinguishable. These methods should be equally suitable for comparison with U.S. EPA 2012 Recreational Water Quality Criteria values.

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.

Human fecal source identification with real-time quantitative PCR.

Waterborne diseases represent a significant public health risk worldwide and can originate from contact with water contaminated with human fecal material. We describe a real-time quantitative PCR (qPCR) method that targets a genetic marker of the human-associated Bacteroides dorei for identification of human fecal pollution in ambient water samples. The following protocol includes water sample collection, filtration, DNA isolation with a sample processing control, qPCR amplification with an internal amplification control, and quality control data analysis.

Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods.

Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an option for recreational water quality testing in the United States (USEPA, 2011. EPA-OW-2011-0466, FRL-9609-3, Notice of Availability of Draft Recreational Water Quality Criteria and Request for Scientific Views). However, transition of qPCR from a research tool to routine water quality testing requires information on how various method variations affect target enumeration. Here we compared qPCR performance and enumeration of enterococci in spiked and environmental water samples using three qPCR platforms (Applied Biosystem StepOnePlus™, the BioRad iQ™5 and the Cepheid SmartCycler(®) II), two reference materials (lyophilized cells and frozen cells on filters) and two comparative CT quantification models (ΔCT and ΔΔCT). Reference materials exerted the biggest influence, consistently affecting results by approximately 0.5 log(10) unit. Platform had the smallest effect, generally exerting <0.1 log(10) unit difference in final results. Quantification model led to small differences (0.04-0.2 log(10) unit) in this study with relatively uninhibited samples, but has the potential to cause as much as 8-fold (0.9 log(10) unit) difference in potentially inhibitory samples. Our findings indicate the need for a certified and centralized source of reference materials and additional studies to assess applicability of the quantification models in analyses of PCR inhibitory samples.

Influences of sample interference and interference controls on quantification of enterococci fecal indicator bacteria in surface water samples by the qPCR method.

A quantitative polymerase chain reaction (qPCR) method for the detection of enterococci fecal indicator bacteria has been shown to be generally applicable for the analysis of temperate fresh (Great Lakes) and marine coastal waters and for providing risk-based determinations of water quality at recreational beaches. In this study we further examined the applicability of the method for analyses of diverse inland waters as well as tropical marine waters from Puerto Rico based on the frequencies of samples showing presumptive PCR interference. Interference was assessed by salmon DNA sample processing control (SPC) and internal amplification control (IAC) assay analysis results and pre-established acceptance criteria of <3.0 and <1.5 cycle threshold (Ct) offsets from control samples, respectively. SPC assay results were accepted in analyses of 93% of the inland water samples whereas the criterion was met at frequencies of 60% and 97% in analyses of samples from Puerto Rico in two different years of sampling. The functionality of the control assays and their acceptance criteria was assessed on the basis of relative recovery estimates of spiked enterococci target organisms extracted in the presence of water sample filters and sample-free control filters and was supported by observations that recovery estimates from the water sample and control filters were substantially different for samples that failed these criteria. Through the combined use of the SPC and IAC assays, two presumptive types of interference were identified. One type, observed in the tropical marine water samples, appeared to primarily affect the availability of the DNA templates for detection. The second type, observed in river water samples, appeared to primarily affect PCR amplification efficiency. In the presence of DNA template interference, adjustments from SPC assay results by the ΔΔCt comparative Ct calculation method decreased the variability of spiked enterococci recovery estimates and increased the similarity with control filters as compared to unadjusted recovery estimates obtained by the ΔCt calculation method. Use of a higher salmon DNA concentration in the extraction buffer also reduced this type of interference. The effects of amplification interference were largely reversed by dilution of the DNA extracts and even more effectively by the use of an alternative, commercial PCR reagent, designed for the analysis of environmental samples.

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.

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.