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.

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.

Genetic markers for rapid PCR-based identification of gull, Canada goose, duck, and chicken fecal contamination in water.

Avian feces contaminate waterways but contribute fewer human pathogens than human sources. Rapid identification and quantification of avian contamination would therefore be useful to prevent overestimation of human health risk. We used subtractive hybridization of PCR-amplified gull fecal 16S RNA genes to identify avian-specific fecal rRNA gene sequences. The subtracters were rRNA genes amplified from human, dog, cat, cow, and pig feces. Recovered sequences were related to Enterobacteriaceae (47%), Helicobacter (26%), Catellicoccus (11%), Fusobacterium (11%), and Campylobacter (5%). Three PCR assays, designated GFB, GFC, and GFD, were based on recovered sequence fragments. Quantitative PCR assays for GFC and GFD were developed using SYBR green. GFC detected down to 0.1 mg gull feces/100 ml (corresponding to 2 gull enterococci most probable number [MPN]/100 ml). GFD detected down to 0.1 mg chicken feces/100 ml (corresponding to 13 Escherichia coli MPN/100 ml). GFB and GFC were 97% and 94% specific to gulls, respectively. GFC cross-reacted with 35% of sheep samples but occurred at about 100,000 times lower concentrations in sheep. GFD was 100% avian specific and occurred in gulls, geese, chickens, and ducks. In the United States, Canada, and New Zealand, the three markers differed in their geographic distributions but were found across the range tested. These assays detected four important bird groups contributing to fecal contamination of waterways: gulls, geese, ducks, and chickens. Marker distributions across North America and in New Zealand suggest that they will have broad applicability in other parts of the world as well.

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.

Survival and persistence of human and ruminant-specific faecal Bacteroidales in freshwater microcosms.

Amplification of host-specific markers from Bacteroidales faecal anaerobes can rapidly identify the source of faecal pollution. It is necessary to understand persistence and survival of these markers and marker cells, both to interpret quantitative source-tracking data, and to use such data to predict pathogen occurrence. We measured marker persistence and cell survival of two human (HF134, HF183) and two ruminant (CF128, CF193) faecal Bacteroidales markers, compared with Escherichia coli and enterococci. Freshwater microcosms were inoculated with fresh cattle or human faeces and incubated at 13 degrees C in natural light or darkness. Marker persistence was measured by polymerase chain reaction (PCR) and quantitative PCR. Survival of marker cells was measured by real-time quantitative PCR. There was no difference in persistence between the two human-specific Bacteroidales DNA markers in the light and dark microcosms. Cell survival profiles of the two human markers were also similar; both were significantly affected by light. Ruminant markers persisted and survived longer than human markers (14 versus 6 days respectively). CF193 decreased more rapidly than CF128, and light significantly affected CF128 but not CF193. These results support use of host-specific faecal Bacteroidales markers as indicators of recent faecal pollution, but suggest that caution is needed in interpreting quantitative results to indicate proportional contribution of different sources, as individual markers differ in their survival, persistence and response to environmental variables. The survival and persistence profiles for Bacteroidales markers are consistent with survival profiles for several faecal pathogens.

Learning to Write Like a Scientist: A Writing-Intensive Course for Microbiology/Health Science Students.

Learning the tools and conventions of expert communication in the sciences provides multiple benefits to bioscience students, yet often these skills are not formally taught. To address this need, we designed a writing-intensive microbiology course on emerging infectious diseases to provide upper-division students with science-specific writing skills along with disciplinary course content. The course followed the guidelines of our university's Writing Intensive Curriculum (WIC) program. Students wrote a press release, a case study, a controversy/position paper, and a grant prospectus, and revised drafts after feedback. To assess the course, in 2015 and 2016 we administered pre-post surveys and collected writing samples for analysis. Students reported on their experience, training, skills, and knowledge before taking the course. They then rated the extent to which the assignments, lectures, in-class activities, and writing activities contributed to their attainment of the learning outcomes of the course. Students entering the class were inexperienced in tools of science writing and the specific genres covered by the class. Their confidence levels rose in both skills and knowledge. Feedback from instructors was cited as most helpful in the majority of the areas where students reported the most gains. The survey provided evidence that discipline-specific knowledge had been acquired through writing activities. Teaching science writing by allowing the students to write "fiction" (e.g., a case report about a fictional patient) was effective in maintaining a high level of interest, both in learning the conventions of the genre and in seeking out detailed information about emerging infectious diseases. Both the course structure and the specific assignments would be useful at other institutions to teach science writing.

Fecal source tracking, the indicator paradigm, and managing water quality.

Fecal source tracking is used because standard methods of measuring fecal contamination in water by enumerating fecal indicator bacteria (FIB) do not identify the sources of the contamination. This paper presents a critical review of source tracking with emphasis on the extent to which methods have been tested (especially in comparison with other methods and/or with blind samples), when methods are applicable, their shortcomings, and their usefulness in predicting public health risk or pathogen occurrence. In addition, the paper discusses the broader question of whether fecal source tracking and fecal indicator monitoring is the best approach to regulate water quality and protect human health. Many fecal source-tracking methods have only been tested against sewage or fecal samples or isolates in laboratory studies (proof of concept testing) and/or applied in field studies where the "real" answer is not known, so their comparative performance and accuracy cannot be assessed. For source tracking to be quantitative, stability of ratios between host-specific markers in the environment must be established. In addition, research is needed on the correlation between host-specific markers and pathogens, and survival of markers after waste treatments. As a result of the exclusive emphasis on FIB in legislation, monitoring has concentrated on FIB and lost sight of pathogens. A more rational approach to regulating water quality would start with available epidemiological data to identify pathogens of concern in a particular water body, and then use targeted pathogen monitoring coupled with targeted fecal source tracking to control them. Baseline monitoring of indicators would become just one tool among many.

Bayesian meta-analysis to synthesize decay rate constant estimates for common fecal indicator bacteria.

For decades, fecal indicator bacteria have been used as proxies to quantitatively estimate fecal loading into water bodies. Widely used cultured indicators (e.g. Escherichia coli and Enterococcus spp.) and more recently developed genetic markers are well studied, but their decay in the environment is still poorly understood. We used Hierarchical Bayesian Linear Modeling to conduct a series of meta-analyses using published decay rate constant estimates, to synthesize findings into pooled estimates and identify gaps in the data preventing reliable estimates. In addition to the meta-analysis assuming all estimates come from the same population, meta-regressions including covariates believed to contribute to decay were fit and used to provided synthesized estimates for specific combinations of significant variables. Additionally, statements regarding the significance of variables across studies were made using the 95% confidence interval for meta-regression coefficients. These models were used to construct a mean decay rate constant estimate as well as credible intervals for the mean and the distribution of all likely data points. While synthesized estimates for each targeted indicator bacteria were developed, the amount of data available varied widely for each target, as did the predictive power of the models as determined by testing with additional data not included in the modeling. Temperature was found to be significant for all selected indicators, while light was found to be significant only for culturable indicators. Results from the models must be interpreted with caution, as they are based only on the data available, which may not be representative of decay in other scenarios.

Microbial community dynamics based on 16S rRNA gene profiles in a Pacific Northwest estuary and its tributaries.

We analyzed bacterioplankton community structure in Tillamook Bay, Oregon and its tributaries to evaluate phylogenetic variability and its relation to changes in environmental conditions along an estuarine gradient. Using eubacterial primers, we amplified 16S rRNA genes from environmental DNA and analyzed the PCR products by length heterogeneity polymerase chain reaction (LH-PCR), which discriminates products based on naturally occurring length differences. Analysis of LH-PCR profiles by multivariate ordination methods revealed differences in community composition along the estuarine gradient that were correlated with changes in environmental variables. Microbial community differences were also detected among different rivers. Using partial 16S rRNA sequences, we identified members of dominant or unique gene fragment size classes distributed along the estuarine gradient. Gammaproteobacteria and Betaproteobacteria and members of the Bacteroidetes dominated in freshwater samples, while Alphaproteobacteria, Cyanobacteria and chloroplast genes dominated in marine samples. Changes in the microbial communities correlated most strongly with salinity and dissolved silicon, but were also strongly correlated with precipitation. We also identified specific gene fragments that were correlated with inorganic nutrients. Our data suggest that there is a significant and predictable change in microbial species composition along an estuarine gradient, shifting from a more complex community structure in freshwater habitats to a community more typical of open ocean samples in the marine-influenced sites. We also demonstrate the resolution and power of LH-PCR and multivariate analyses to provide a rapid assessment of major community shifts, and show how these shifts correlate with environmental variables.

Application of a rapid method for identifying fecal pollution sources in a multi-use estuary.

We demonstrate the application of a new PCR assay to detect and differentiate human and ruminant sources of fecal pollution in natural water samples. We tested samples collected from Tillamook Bay, Oregon, which has a long history of fecal pollution levels that exceed acceptable standards. The most likely sources are from dairy operations and ineffective sewage treatment. Using a suite of three PCR primer pairs specific for human or ruminant bacterial 16S ribosomal DNA markers, we detected at least one marker in 17 of 22 samples. In general, host-specific fecal markers were detected in areas that are heavily impacted by anthropogenic activities. Nine out of 11 sites classified as either urban or near a sewage point source were positive for the human marker while only five of these same sites were positive for ruminant markers. Conversely, 12 out of 21 sites classified as rural or agricultural use were positive for ruminant markers, while only six of these sites were positive for human pollution. This suite of host-specific genetic markers holds promise for identifying non-point source fecal pollution in coastal waters.

A comparative study of culture-independent, library-independent genotypic methods of fecal source tracking.

Culture-independent fecal source tracking methods have many potential advantages over library-dependent, isolate-culture methods, but they have been subjected to limited testing. The purpose of this study was to compare culture-independent, library-independent methods of fecal source tracking. Five laboratories analysed identical sets of aqueous samples that contained one or more of the following sources: sewage, human feces, dog feces, cattle feces and gull feces. Two investigators used methods based on PCR amplification of Bacteroidetes marker genes and both successfully discriminated between samples that did or did not contain human fecal material. One of these investigators was also able to identify the remaining sources, except for gull, with a low rate of false positives. A method based on E. coli toxin genes successfully identified samples containing sewage and cattle feces, but missed some samples with human feces because of low marker prevalence in individual human fecal samples. Researchers who used community terminal restriction fragment length polymorphism (T-RFLP) were limited by the amount of DNA recovered from samples, but they correctly identified human and cattle fecal contamination when sufficient DNA was obtained. Culture independent methods show considerable promise; further research is needed to develop markers for additional fecal sources and to understand the correlation of these source-tracking indicators to measures of human and environmental health.

Detection of Morganella morganii, a prolific histamine former, by the polymerase chain reaction assay with 16S rDNA-targeted primers.

A polymerase chain reaction (PCR) assay for the rapid and sensitive detection of the most prolific histamine former, Morganella morganii, was developed. 16S rDNA targeted PCR primers were designed, and the primer specificity and sensitivity of the PCR assay were evaluated. The 16S rDNA sequence (1,503 bp) for M. morganii showed 95% identity to those for enteric bacteria, i.e., Enterobacter spp., Klebsiella spp., Citrobacter spp., Hafnia alvei, Proteus spp., and Providencia spp. The unique primers for M. morganii were designed on the basis of the variable regions in the 16S rDNA sequence. The primers showed positive reactions with all M. morganii strains tested. However, PCR amplification was not detected when the primers were tested with other enteric or marine bacteria. When the sensitivity of the assay was evaluated, M. morganii was detected at levels ranging from 10(6) to 10(8) CFU/ml in albacore homogenate after the PCR amplification. The sensitivity of the assay was greatly improved with the enrichment of samples, and 9 CFU of M. morganii per ml of albacore homogenate was detected after 6 h of enrichment at 37 degrees C.

Enterococcus and Escherichia coli fecal source apportionment with microbial source tracking genetic markers--is it feasible?

Fecal pollution is measured in surface waters using culture-based measurements of enterococci and Escherichia coli bacteria. Source apportionment of these two fecal indicator bacteria is an urgent need for prioritizing remediation efforts and quantifying health risks associated with source-specific pathogens. There are a number of quantitative real-time PCR (QPCR) assays that estimate concentrations of source-associated genetic markers; however, their concentrations are not necessarily amenable to source apportionment because the markers may differ in prevalence across sources. Here we mathematically derive and test, under ideal conditions, a method that utilizes the ratios of fecal source-associated genetic markers and culture and molecular measurements of general fecal indicators to apportion enterococci and E. coli. The source contribution is approximately equal to the ratio of the source-associated and the general fecal indicator concentrations in a water sample divided by their ratio in the source material, so long as cross-reactivity is negligible. We illustrate the utility of the ratio method using samples consisting of mixtures of various fecal pollution sources. The results from the ratio method correlated well with the actual source apportionment in artificial samples. However, aging of contamination can confound source allocation predictions. In particular, culturable enterococci and E. coli, the organisms presently regulated in the United States and much of the world, decay at different rates compared to source-associated markers and as a result cannot be apportioned using this method. However, limited data suggest a similar decay rate between source-associated and QPCR-measured Enterococcus and E. coli genetic markers, indicating that apportionment may be possible for these organisms; however further work is needed to confirm.

Sensitive detection of sample interference in environmental qPCR.

Sample interference in environmental applications of quantitative PCR (qPCR) can prevent accurate estimations of molecular markers in the environment. We developed a spike-and-recovery approach using a mutant strain of Escherichia coli that contains a chromosomal insertion of a mutant GFP gene. The method was tested in water samples by separately reducing extraction efficiency or adding humic acids and ethanol, compounds that often contaminate environmental DNA extracts, and analyzing qPCR amplification of the spiked E. coli control and human fecal Bacteroides markers (HF183 and HF134). This approach, coupled with previously developed kinetic outlier detection (KOD) methods, allowed sensitive detection of PCR inhibition at much lower inhibitor concentrations than alternative approaches using Cq values or amplification efficiencies. Although HF183 was more sensitive to the effects of qPCR inhibitors than the E. coli control assay, KOD methods correctly identified inhibition of both control and HF183 assays in samples containing as little as 0.1 ng humic acids per reaction or 5% ethanol. Because sigmoidal modeling methods allow distinction of qPCR inhibition from poor DNA recovery, we were able to simultaneously identify qPCR-inhibited reactions and estimate recovery of nucleic acids in environmental samples using a single control assay. Since qPCR is currently used to estimate important water quality parameters that have serious economic and human health outcomes, these results are timely. While we demonstrate the methods in the context of water quality regulation, they will be useful in all areas of environmental research that use qPCR.

Detection of Bacteroidales fecal indicators and the zoonotic pathogens E. coli 0157:H7, salmonella, and campylobacter in river water.

Bacteroidales host-specific PCR offers a rapid method of diagnosing fecal pollution in water and identifying sources of input. To assess human health risks from exposure to fecal pathogens, however, Bacteroidales markers should be detectable when pathogens are present. To determine if Bacteroidales general, human-, ruminant-, and swine-specific markers correlate with certain fecal pathogens, we conducted a retrospective study on water samples for which the presence of E. coli O157:H7, Salmonella spp., and Campylobacter spp. had been determined. We found a positive relationship between detection of the Bacteroidales general fecal marker and presence of the pathogens. Detection of ruminant-specific markers predicted E. coli O157: H7 occurrence. There was a significant increase in the likelihood of detecting Salmonella when a ruminant marker was present, and Campylobacter spp. when human markers were present. For pathogens such as E. coli O157: H7 that are strongly associated with particular hosts, Bacteroidales host-specific markers can estimate the likelihood of pathogen occurrence, enabling more accurate health risk assessments.

Persistence and growth of fecal Bacteroidales assessed by bromodeoxyuridine immunocapture.

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.

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.

Development and applications of microbial ecogenomic indicators for monitoring water quality: report of a workshop assessing the state of the science, research needs and future directions.

This article brings forth recommendations from a workshop sponsored by the U.S. Environmental Protection Agency's Science to Achieve Results (STAR) and Environmental Monitoring and Assessment (EMAP) Programs and by the Council of State Governments, held during May 2002 in Kansas City, Kansas. The workshop assembled microbial ecologists and environmental scientists to determine what research and science is needed to bring existing molecular biological approaches and newer technologies arising from microbial genomic research into environmental monitoring and water quality assessments. Development of genomics and proteomics technologies for environmental science is a very new area having potential to improve environmental water quality assessments. The workshop participants noted that microbial ecologists are already using molecular biological methods well suited for monitoring and water quality assessments and anticipate that genomics-enabled technologies could be made available for monitoring within a decade. Recommendations arising from the workshop include needs for (i) identification of informative microbial gene sequences, (ii) improved understandings of linkages between indicator taxa, gene expression and environmental condition, (iii) technological advancements towards field application, and (iv) development of the appropriate databases.

Microplate subtractive hybridization to enrich for bacteroidales genetic markers for fecal source identification.

The ability to identify sources of fecal pollution plays a key role in the analysis of human health risk and the implementation of water resource management strategies. One approach to this problem involves the identification of bacterial lineages or gene sequences that are found exclusively in a particular host species or group. We used subtractive hybridization to enrich for target host-specific fecal Bacteroidales rRNA gene fragments that were different from those of very closely related reference (subtracter) host sources. Target host rRNA gene fragments were hybridized to subtracter rRNA gene fragments immobilized in a microplate well, and target sequences that did not hybridize were cloned and sequenced for PCR primer design. The use of microplates for DNA immobilization resulted in a one-step subtractive hybridization in which the products could be directly amplified with PCR. The new host-specific primers designed from subtracted target fragments differentiated among very closely related Bacteroidales rRNA gene sequences and distinguished between similar fecal sources, such as elk and cow or human and domestic pet (dog).

Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification.

The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.