Evidence of Genetic Fecal Marker Interactions between Water Column and Periphyton in Artificial Streams.

Periphyton is a complex mixture of algae, microbes, inorganic sediment, and organic matter that is attached to submerged surfaces in most flowing freshwater systems. This natural community is known to absorb pollutants from the water column, resulting in improved water quality. However, the role of periphyton in the fate and transport of genetic fecal markers suspended in the water column remains unclear. As application of genetic-based methodologies continues to increase in freshwater settings, it is important to identify any interactions that could potentially confound water quality interpretations. A 16 week indoor mesocosm study was conducted to simultaneously measure genetic fecal markers in the water column and in the associated periphyton when subject to wastewater source loading. Treated wastewater effluent was pumped directly from a treatment facility adjacent to the experimental stream facility. Inflow and outflow surface water grabs were paired with the collection of periphyton samples taken from the mesocosm substrates on a weekly basis. Samples were analyzed with three genetic fecal indicator quantitative real-time polymerase chain reaction assays targeting Escherichia coli (EC23S857), enterococci (Entero1), and Bacteroidales (GenBac3), as well as, two human host-associated fecal pollution markers (HF183 and HumM2). In addition, periphyton dry mass was measured. During wastewater effluent loading, genetic markers were detected in periphyton at frequencies up to 100% (EC23S857, Entero1, and GenBac3), 59.4% (HF183), and 21.9% (HumM2) confirming sequestration from the water column. Mean net-flux shifts in water column inflow and outflow genetic indicator concentrations further supported interactions between the periphyton and water column. In addition, positive correlations were observed between periphyton dry mass and genetic marker concentrations ranging from r = 0.693 (Entero1) to r = 0.911 (GenBac3). Overall, findings support the notion that genetic markers suspended in the water column can be trapped by periphyton, further suggesting that the benthic environment in flowing freshwater systems may be an important factor to consider for water quality management with molecular methods.

Factors affecting the presence of human-associated and fecal indicator real-time quantitative PCR genetic markers in urban-impacted recreational beaches.

Urban runoff can carry a variety of pollutants into recreational beaches, often including bacterial pathogens and indicators of fecal contamination. To develop complete recreational criteria and risk assessments, it is necessary to understand conditions under which human contamination could be present at beaches solely impacted by urban runoff. Accurately estimating risk requires understanding sources, concentrations, and transport mechanisms of microbial contaminants in these environments. By applying microbial source tracking methods and empirical modeling, we assessed the presence and level of human contamination at urban runoff impacted recreational beaches. We also identified environmental parameters and pollution sources that can influence the concentration and transport of culturable and molecular fecal indicator bacteria (FIB) in systems impacted solely by urban runoff. Water samples and physico-chemical parameters were collected from shoreline locations from three South Carolina (SC) beaches (five locations per beach) and two Florida (FL) beaches (three locations per beach). Each SC beach was directly impacted by swashes or tidal creeks receiving stormwater runoff from the urbanized area and therefore were designated as swash drain associated (SDA) beaches, while FL beaches were designated as non-swash drain associated (NSDA). Sampling in swash drains (SD; three sites per SD) directly impacting each SC beach was also conducted. Results indicate that although culturable (enterococci) and real-time quantitative polymerase chain reaction (qPCR) (EC23S857, Entero1, and GenBac3) FIB concentrations were, on average, higher at SD locations, SDA beaches did not have consistently higher molecular FIB signals compared to NSDA beaches. Both human-associated markers (HF183 and HumM2) were concomitantly found only at SDA beaches. Bacteroidales species-specific qPCR markers (BsteriF1 and BuniF2) identified differences in the Bacteroidales community, depending on beach type. The marker for general Bacteroidales was most abundant at SD locations and exhibited a high correlation with both culturable and other molecular markers. Combining molecular information with predictive modeling allowed us to identify both alongshore movement of currents and SD outflow as significant influences on the concentration of molecular and culturable indicators in the bathing zone. Data also suggests that combining methodologies is a useful and cost effective approach to help understand transport dynamics of fecal contamination and identify potential sources of contamination at marine beaches.

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.

Age-related shifts in the density and distribution of genetic marker water quality indicators in cow and calf feces.

Calves make up about 16% of the current bovine population in the United States and can excrete high levels of human pathogens in their feces. We describe the density and distribution of genetic markers from 9 PCR- and real-time quantitative PCR-based assays, including CF128, CF193, CowM2, CowM3, GenBac3, Entero1, EC23S857, CampF2, and ttr-6, commonly used to help assess ambient surface water quality. Each assay was tested against a collection of 381 individual bovine fecal samples representing 31 mother and calf pairings collected over a 10-month time period from time of birth through weaning. Genetic markers reported to be associated with ruminant and/or bovine fecal pollution were virtually undetected in calves for up to 115 days from birth, suggesting that physiological changes in calf ruminant function impact host-associated genetic marker shedding. In addition, general fecal indicator markers for Bacteroidales, Escherichia coli, and Enterococcus spp. exhibited three separate trends across time, indicating that these bacteria respond differently to age-related physiological and dietary changes during calf development. The results of this study suggest that currently available PCR-based water quality indicator technologies can under- or overestimate fecal pollution originating from calves and identify a need for novel calf-associated source identification methods.

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study.

Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods.

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study.

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.

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.

Decay of bacterial pathogens, fecal indicators, and real-time quantitative PCR genetic markers in manure-amended soils.

This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green fluorescent protein-expressing Escherichia coli O157:H7/pZs and red fluorescent protein-expressing Salmonella enterica serovar Typhimurium/pDs were added to laboratory-scale manure-amended soil microcosms with moisture contents of 60% or 80% field capacity and incubated at temperatures of -20°C, 10°C, or 25°C for 120 days. A two-stage first-order decay model was used to determine stage 1 and stage 2 first-order decay rate coefficients and transition times for each organism and qPCR genetic marker in each treatment. Genetic markers for FIB (Enterococcus spp., E. coli, and Bacteroidales) exhibited decay rate coefficients similar to that of E. coli O157:H7/pZs but not of S. enterica serovar Typhimurium/pDs and persisted at detectable levels longer than both pathogens. Concentrations of these two bacterial pathogens, their counterpart qPCR genetic markers (stx1 and ttrRSBCA, respectively), and FIB genetic markers were also correlated (r = 0.528 to 0.745). This suggests that these qPCR genetic markers may be reliable conservative surrogates for monitoring fecal pollution from manure-amended land. Host-associated qPCR genetic markers for microbial source tracking decayed rapidly to nondetectable concentrations, long before FIB, Salmonella enterica serovar Typhimurium/pDs, and E. coli O157:H7/pZs. Although good indicators of point source or recent nonpoint source fecal contamination events, these host-associated qPCR genetic markers may not be reliable indicators of nonpoint source fecal contamination events that occur weeks following manure application on land.

Combining land use information and small stream sampling with PCR-based methods for better characterization of diffuse sources of human fecal pollution.

Diffuse sources of human fecal pollution allow for the direct discharge of waste into receiving waters with minimal or no treatment. Traditional culture-based methods are commonly used to characterize fecal pollution in ambient waters, however these methods do not discern between human and other animal sources of fecal pollution making it difficult to identify diffuse pollution sources. Human-associated quantitative real-time PCR (qPCR) methods in combination with low-order headwatershed sampling, precipitation information, and high-resolution geographic information system land use data can be useful for identifying diffuse source of human fecal pollution in receiving waters. To test this assertion, this study monitored nine headwatersheds over a two-year period potentially impacted by faulty septic systems and leaky sanitary sewer lines. Human fecal pollution was measured using three different human-associated qPCR methods and a positive significant correlation was seen between abundance of human-associated genetic markers and septic systems following wet weather events. In contrast, a negative correlation was observed with sanitary sewer line densities suggesting septic systems are the predominant diffuse source of human fecal pollution in the study area. These results demonstrate the advantages of combining water sampling, climate information, land-use computer-based modeling, and molecular biology disciplines to better characterize diffuse sources of human fecal pollution in environmental waters.

Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR.

Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4×10(4) target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>10(3)copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R(2)≥0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters.

Genetic and functional analyses of the mob operon on conjugative transposon CTn341 from Bacteroides spp.

Bacteroides are Gram-negative anaerobes indigenous to the intestinal tract of humans, and they are important opportunistic pathogens. Mobile genetic elements, such as conjugative transposons (CTns), have contributed to an increase in antibiotic resistance in these organisms. CTns are self-transmissible elements that belong to the superfamily of integrative and conjugative elements (ICEs). CTn341 is 52 kb; it encodes tetracycline resistance and its transfer is induced by tetracycline. The mobilization region of CTn341 was shown to be comprised of a three-gene operon, mobABC, and the transfer origin, oriT. The three genes code for a nicking accessory protein, a relaxase, and a VirD4-like coupling protein, respectively. The Mob proteins were predicted to mediate the formation of the relaxosome complex, nick DNA at the oriT, and shuttle the DNA/protein complex to the mating-pore apparatus. The results of mutational studies indicated that the three genes are required for maximal transfer of CTn341. Mob gene transcription was induced by tetracycline, and this regulation was mediated through the two-component regulatory system, RteAB. The oriT region of CTn341 was located within 100 bp of mobA, and a putative Bacteroides consensus nicking site was observed within this region. Mutation of the putative nick site resulted in a loss of transfer. This study demonstrated a role of the mobilization region for transfer of Bacteroides CTns and that tetracycline induction occurs for the mob gene operon, as for the tra gene operon(s), as shown previously.