Temporal Assessment of the Impact of Exposure to Cow Feces in Two Watersheds by Multiple Host-Specific PCR Assays

Exposure to feces in two watersheds with different management histories was assessed by tracking cattle feces bacterial populations using multiple host-specific PCR assays. In addition, environmental factors affecting the occurrence of these markers were identified. Each assay was performed using DNA extracts from water and sediment samples collected from a watershed directly impacted by cattle fecal pollution (WS1) and from a watershed impacted only through runoff (WS2). In WS1, the ruminant-specific Bacteroidales 16S rRNA gene marker CF128F was detected in 65% of the water samples, while the non-16S rRNA gene markers Bac1, Bac2, and Bac5 were found in 32 to 37% of the water samples. In contrast, all source-specific markers were detected in less than 6% of the water samples from WS2. Binary logistic regressions (BLRs) revealed that the occurrence of Bac32F and CF128F was significantly correlated with season as a temporal factor and watershed as a site factor. BLRs also indicated that the dynamics of fecal-source-tracking markers correlated with the density of a traditional fecal indicator (P < 0.001). Overall, our results suggest that a combination of 16S rRNA gene and non-16S rRNA gene markers provides a higher level of confidence for tracking unknown sources of fecal pollution in environmental samples. This study also provided practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.     

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.     

Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers

PCR-based analysis of Bacteroidales 16S rRNA genes has emerged as a promising tool to identify sources of fecal water pollution. In this study, three TaqMan real-time PCR assays (BacGeneral, BacHuman, and BacBovine) were developed and evaluated for their ability to quantitatively detect general (total), human-specific, and bovine-specific Bacteroidales 16S rRNA genetic markers. The detection sensitivity was determined to be 6.5 copies of 16S rRNA gene for the BacGeneral and BacHuman assays and 10 copies for the BacBovine assay. The assays were capable of detecting approximately one to two cells per PCR. When tested with 70 fecal samples from various sources (human, cattle, pig, deer, dog, cat, goose, gull, horse, and raccoon), the three assays positively identified the target markers in all samples without any false-negative results. The BacHuman and BacBovine assays exhibited false-positive reactions with non-target samples in a few cases. However, the level of the false-positive reactions was about 50 times smaller than that of the true-positive ones, and therefore, these cross-reactions were unlikely to cause misidentifications of the fecal pollution sources. Microbial source-tracking capability was tested at two freshwater streams of which water quality was influenced by human and cattle feces, respectively. The assays accurately detected the presence of the corresponding host-specific markers upon fecal pollution and the persistence of the markers in downstream areas. The assays are expected to reliably determine human and bovine fecal pollution sources in environmental water samples.     

Chicken- and duck-associated Bacteroides–Prevotella genetic markers for detecting fecal contamination in environmental water

Bacteroides–Prevotella group is one of the most promising targets for detecting fecal contamination in water environments, principally due to its host-specific distributions and high concentrations in feces of warm-blooded animals. We developed real-time PCR assays for quantifying chicken/duck-, chicken-, and duck-associated Bacteroides–Prevotella 16S rRNA genetic markers (Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac). A reference collection of DNA extracts from 143 individual fecal samples and wastewater treatment plant influent was tested by the newly established markers. The quantification limits of Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac markers in environmental water were 54, 57, and 12 copies/reaction, respectively. It was possible to detect possible fecal contaminations from wild ducks in environmental water with the constructed genetic marker assays, even though the density of total coliforms in the identical water samples was below the detection limit. Chicken/Duck-Bac marker was amplified from feces of wild duck and chicken with the positive ratio of 96 and 61 %, respectively, and no cross-reaction was observed for the other animal feces. Chicken-Bac marker was detected from 70 % of chicken feces, while detected from 39 % of cow feces, 8.3 % of pig feces, and 12 % of swan feces. Duck-Bac marker was detected from 85 % of wild duck feces and cross-reacted with 31 % of cow feces. These levels of detection specificity are common in avian-associated genetic markers previously proposed, which implies that there is a practical limitation in the independent application of avian-associated Bacteroides–Prevotella 16S rRNA genetic markers and a combination with other fecal contamination markers is preferable for detecting fecal contamination in water environments.     

Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific “Bacteroidales” Populations

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 “Bacteroidales” 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution.The size of swine farming operations has increased significantly during the last few decades as a result of the high demand for pork products. In fact, pork is now considered the most popular meat worldwide (15). In the United States, the number of large confined swine animal units increased by 3 orders of magnitude from 1982 to 1997 (18), making the swine industry among the top three producers of domesticated animal feces. A direct consequence of this trend is the increase in swine fecal waste, which in turn has raised environmental concerns. When introduced to water, swine fecal waste can present a risk to human health because this waste can harbor a variety of human pathogens (5, 13, 15, 21, 36). The diversity and relatively high frequency of human pathogens in swine feces make swine important reservoirs of zoonotic pathogens. Moreover, the marked increase in the number of large operations has resulted in increased manure production and application in small geographic areas, creating an imbalance between the assimilative capacity of manure-treated farmland and the amount of manure nutrients produced on each farm. This imbalance is evidenced by the 20% increase (from 1982 to 1997) in nitrogen and phosphorus produced in swine operations, thus potentially contributing to the detrimental eutrophication of aquatic ecosystems (18). Swine manure spills and leaks are commonplace in the top hog production states, such as Iowa and North Carolina, due to failure or overflow of manure storage, uncontrolled runoff from open feedlots, improper manure application on cropland, deliberate pumping of manure onto the ground, and intentional breaches in storage lagoons (28, 37).Recently, swine-associated PCR-based methods targeting members of the “Bacteroidales” order (i.e., Prevotella species) and methanogen populations (12, 29, 35) have been proposed to discriminate swine fecal pollution events from other potential fecal contributions (i.e., human, bovine, and wildlife) to environmental waters. Nevertheless, the value of these assays in reliably detecting fecal pollution sources in watershed-based studies has not been thoroughly investigated. The main goals of this study were to determine host specificity, frequency of detection, and detection limits of currently available swine-associated PCR-based, microbial source tracking assays. To achieve these objectives, assays were tested against swine and nontarget fecal samples, samples from swine manure pits and swine waste lagoons, and water samples presumed to be impacted by swine fecal sources. Furthermore, we investigated the phylogenetic diversity of Bacteroidales 16S rRNA gene sequences derived from some of the aforementioned samples to resolve the level of specificity, relative abundance, and environmental occurrence of Bacteroidales-specific 16S rRNA gene sequences.     

Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 × 10⁶ copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.     

Coherence among Different Microbial Source Tracking Markers in a Small Agricultural Stream with or without Livestock Exclusion Practices

Over 1,400 water samples were collected biweekly over 6 years from an intermittent stream protected and unprotected from pasturing cattle. The samples were monitored for host-specific Bacteroidales markers, Cryptosporidium species/genotypes, viruses and coliphages associated with humans or animals, and bacterial zoonotic pathogens. Ruminant Bacteroidales markers did not increase within the restricted cattle access reach of the stream, whereas the ruminant Bacteroidales marker increased significantly in the unrestricted cattle access reach. Human Bacteroidales markers significantly increased downstream of homes where septic issues were documented. Wildlife Bacteroidales markers were detected downstream of the cattle exclusion practice where stream and riparian habitat was protected, but detections decreased after the unrestricted pasture, where the stream and riparian zone was unprotected from livestock. Detection of a large number of human viruses was shown to increase downstream of homes, and similar trends were observed for the human Bacteroidales marker. There was considerable interplay among biomarkers with stream flow, season, and the cattle exclusion practices. There were no to very weak associations with Bacteroidales markers and bacterial, viral, and parasitic pathogens. Overall, discrete sample-by-sample coherence among the different microbial source tracking markers that expressed a similar microbial source was minimal, but spatial trends were physically meaningful in terms of land use (e.g., beneficial management practice) effects on sources of fecal pollution.     

Competitive Metagenomic DNA Hybridization Identifies Host-Specific Microbial Genetic Markers in Cow Fecal Samples

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales-like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities.     

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.     

Basin-Wide Analysis of the Dynamics of Fecal Contamination and Fecal Source Identification in Tillamook Bay, Oregon

The objectives of this study were to elucidate spatial and temporal dynamics in source-specific Bacteroidales 16S rRNA genetic marker data across a watershed; to compare these dynamics to fecal indicator counts, general measurements of water quality, and climatic forces; and to identify geographic areas of intense exposure to specific sources of contamination. Samples were collected during a 2-year period in the Tillamook basin in Oregon at 30 sites along five river tributaries and in Tillamook Bay. We performed Bacteroidales PCR assays with general, ruminant-source-specific, and human-source-specific primers to identify fecal sources. We determined the Escherichia coli most probable number, temperature, turbidity, and 5-day precipitation. Climate and water quality data collectively supported a rainfall runoff pattern for microbial source input that mirrored the annual precipitation cycle. Fecal sources were statistically linked more closely to ruminants than to humans; there was a 40% greater probability of detecting a ruminant source marker than a human source marker across the basin. On a sample site basis, the addition of fecal source tracking data provided new information linking elevated fecal indicator bacterial loads to specific point and nonpoint sources of fecal pollution in the basin. Inconsistencies in E. coli and host-specific marker trends suggested that the factors that control the quantity of fecal indicators in the water column are different than the factors that influence the presence of Bacteroidales markers at specific times of the year. This may be important if fecal indicator counts are used as a criterion for source loading potential in receiving waters.     

Use of a genetically-engineered Escherichia coli strain as a sample process control for quantification of the host-specific bacterial genetic markers

Quantitative PCR (qPCR) assays targeting the host-specific Bacteroides-Prevotella 16S rRNA genetic markers have been proposed as one of the promising approaches to identify the source of fecal contamination in environmental waters. One of the concerns of qPCR assays to environmental samples is the reliability of quantified values, since DNA extraction followed by qPCR assays are usually performed without appropriate sample process control (SPC) and internal amplification controls (IACs). To check the errors in sample processing and improve the reliability of qPCR results, it is essential to evaluate the DNA recovery efficiency and PCR amplification efficiency of the target genetic markers and correct the measurement results. In this study, we constructed a genetically-engineered Escherichia coli K12 strain (designated as strain MG1655 Δlac::kan) as sample process control and evaluated the applicability to environmental water samples. The recovery efficiency of the SPC strain MG1655 Δlac::kan was similar to that of Bacteroides fragilis JCM 11019, when DNA were extracted from water samples spiked with the two bacteria. Furthermore, the SPC was included in the qPCR assays with propidium monoazide (PMA) treatment, which can exclude the genetic markers from dead cells. No significant DNA loss was observed in the PMA treatment. The inclusion of both the SPC (strain MG1655 Δlac::kan) and IAC in qPCR assays with PMA treatment gave the assurance of reliable results of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental water samples.     

Genotypic and Phenotypic Characterization of Escherichia coli Isolates from Feces,Hands, and Soils in Rural Bangladesh via the Colilert Quanti-Tray System

The increased awareness of the role of environmental matrices in enteric disease transmission has resulted in the need for rapid, field-based methods for fecal indicator bacteria and pathogen detection. Evidence of the specificity of β-glucuronidase-based assays for detection of Escherichia coli from environmental matrices relevant to enteric pathogen transmission in developing countries, such as hands, soils, and surfaces, is limited. In this study, we quantify the false-positive rate of a β-glucuronidase-based E. coli detection assay (Colilert) for two environmental reservoirs in Bangladeshi households (hands and soils) and three fecal composite sources (cattle, chicken, and humans). We investigate whether or not the isolation source of E. coli influences phenotypic and genotypic characteristics. Phenotypic characteristics include results of biochemical assays provided by the API-20E test; genotypic characteristics include the Clermont phylogroup and the presence of enteric and/or environmental indicator genes sfmH, rfaI, and fucK. Our findings demonstrate no statistically significant difference in the false-positive rate of Colilert for environmental compared to enteric samples. E. coli isolates from all source types are genetically diverse, representing six of the seven phylogroups, and there is no difference in relative frequency of phylogroups between enteric and environmental samples. We conclude that Colilert, and likely other β-glucuronidase-based assays, is appropriate for detection of E. coli on hands and in soils with low false-positive rates. Furthermore, E. coli isolated from hands and soils in Bangladeshi households are diverse and indistinguishable from cattle, chicken, and human fecal isolates, using traditional biochemical assays and phylogrouping.     

Molecular Diversity of Bacteroidales in Fecal and Environmental Samples and Swine-Associated Subpopulations

Several swine-specific microbial source tracking methods are based on PCR assays targeting Bacteroidales 16S rRNA gene sequences. The limited application of these assays can be explained by the poor understanding of their molecular diversity in fecal sources and environmental waters. In order to address this, we studied the diversity of 9,340 partial (>600 bp in length) Bacteroidales 16S rRNA gene sequences from 13 fecal sources and nine feces-contaminated watersheds. The compositions of major Bacteroidales populations were analyzed to determine which host and environmental sequences were contributing to each group. This information allowed us to identify populations which were both exclusive to swine fecal sources and detected in swine-contaminated waters. Phylogenetic and diversity analyses revealed that some markers previously believed to be highly specific to swine populations are shared by multiple hosts, potentially explaining the cross-amplification signals obtained with nontargeted hosts. These data suggest that while many Bacteroidales populations are cosmopolitan, others exhibit a preferential host distribution and may be able to survive different environmental conditions. This study further demonstrates the importance of elucidating the diversity patterns of targeted bacterial groups to develop more inclusive fecal source tracking applications.     

Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.     

Identification of Nonpoint Sources of Fecal Pollution in Coastal Waters by Using Host-Specific 16S Ribosomal DNA Genetic Markers from Fecal Anaerobes

We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 × 10⁻⁵ to 2.8 × 10⁻⁷ g (dry weight) of feces/liter and 6.8 × 10⁻⁷ g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.     

Identification of sources of Escherichia coli in South Carolina estuaries using antibiotic resistance analysis

Fecal pollution from nonhuman (pets, livestock or wildlife) and human sources is often one of the major factors associated with urbanization that contribute to the degradation of water quality. Methods to differentiate animal from human sources of fecal coliform contamination could assist resource managers in developing strategies to protect shellfish harvesting areas and recreational waters. In this study, surface water samples were collected from both a developed and an undeveloped watershed in coastal South Carolina. Influent and effluent samples from several wastewater treatment plants (WWTPs) in the same area were also collected. Most Probable Numbers (MPNs) of fecal coliforms were determined for all samples. Escherichia coli isolates were analyzed for antibiotic resistance (AR) to 10 antibiotics. Then, AR indices (no. of resistant/total no. of antibiotics tested), were calculated for each isolate and site. Results indicated that MPNs from the WWTP samples were significantly higher than those from the developed watershed which were significantly higher than those from the undeveloped watershed (p<0.0001). The AR analyses suggested that there was a trend toward increased antibiotic resistance in samples for the urbanized Broad Creek (BC) watershed. In the Okatee River (OR), E. coli isolates from three sites (20%) showed resistance to a single antibiotic (penicillin) but in BC, isolates from seven sites (47%) were resistant to multiple antibiotics, and the predominant resistance pattern was chlortetracycline-oxytetracycline-tetracycline. Raw sewage isolates from most WWTPs contained E. coli that exhibited resistance to multiple antibiotics. Cluster analysis indicated that all resistant OR sites had antibiotic resistant isolates that matched AR patterns found in isolates from WWTPs. Similarly, six of the seven sites in BC had AR patterns that matched with resistance patterns in WWTPs. These results suggest that AR testing may be a useful tool for differentiating E. coli from human and wildlife sources. Further testing of bacterial isolates from known animal sources is necessary to better assess the utility of this approach.     

Geographical Variation in Ribotype Profiles of Escherichia coli Isolates from Humans, Swine, Poultry, Beef, and Dairy Cattle in Florida

Waters impacted by fecal pollution can exact high risks to human health and can result in financial losses due to closures of water systems used for recreation and for harvesting seafood. Identifying the sources of fecal pollution in water is paramount in assessing the potential human health risks involved as well as in assessing necessary remedial action. Recently, various researchers have used the ribotyping method to identify sources of bacterial indicators (Escherichia coli and enterococci) in environmental waters. While these studies have identified genotypic differences between human- and animal-derived indicators that are capable of differentiating organisms isolated from humans and various animal hosts, most have focused on organisms collected from a confined geographic area and have not addressed the question of whether these ribotype profiles are watershed specific or if they can be applied universally to organisms from other geographic locations. In this study, E. coli isolates were obtained from humans, beef cattle, dairy cattle, swine, and poultry from locations in northern, central, and southern Florida and were subjected to ribotyping analysis. The intent was to determine (i) if ribotype profiles are capable of discriminating the source of E. coli at the host species level and (ii) if the resulting fingerprints are uniform over an extended geographic area or if they can be applied only to a specific watershed. Our research indicated that, using a single restriction enzyme (HindIII), the ribotyping procedure is not capable of differentiating E. coli isolates from the different animal species sampled in this study. Results indicate, however, that this procedure can still be used effectively to differentiate E. coli as being either human or animal derived when applied to organisms isolated from a large geographic region.     

Bifidobacteria in Feces and Environmental Waters

Bifidobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-specific 16S rRNA gene PCR assays were used to study the presence of bifidobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-specific assays showed that Bifidobacterium adolescentis, B. bifidum, B. dentium, and B. catenulatum had the broadest host distribution (11.9 to 17.4%), whereas B. breve, B. infantis, and B. longum were detected in fewer than 3% of all fecal samples. Phylogenetic analysis of 356 bifidobacterial clones obtained from different animal feces showed that ca. 67% of all of the sequences clustered with cultured bifidobacteria, while the rest formed a supercluster with low sequence identity (i.e., <94%) to previously described Bifidobacterium spp. The B. pseudolongum subcluster (>97% similarity) contained 53 fecal sequences from seven different animal hosts, suggesting the cosmopolitan distribution of members of this clade. In contrast, two clades containing B. thermophilum and B. boum clustered exclusively with 37 and 18 pig fecal clones, respectively, suggesting host specificity. Using species-specific assays, bifidobacteria were detected in only two of the surface water DNA extracts, although other fecal anaerobic bacteria were detected in these waters. Overall, the results suggest that the use of bifidobacterial species as potential markers to monitor human fecal pollution in natural waters may be questionable.     

Competitive metagenomic DNA hybridization identifies host-specific microbial genetic markers in cow fecal samples

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales-like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities.