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.     

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.     

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).     

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 × 10⁴ 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 (>10³ 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 (R² ≥ 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.     

Quantification of host-specific <Emphasis Type="Italic">Bacteroides–Prevotella</Emphasis> 16S rRNA genetic markers for assessment of fecal pollution in freshwater

Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides–Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9–11 log₁₀ copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides–Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r ² = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50–800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments.     

Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Water quality monitoring techniques that target microorganisms in the order Bacteroidales are potential alternatives to conventional methods for detection of fecal indicator bacteria. Bacteroidales and members of the genus Bacteroides have been the focus of microbial source tracking (MST) investigations for discriminating sources of fecal pollution (e.g., human or cattle feces) in environmental waters. For accurate source apportionment to occur, one needs to understand both the abundance of Bacteroides in host feces and the survival of these host-associated microbial markers after deposition in the environment. Studies were undertaken to evaluate the abundance, persistence, and potential for growth of Bacteroidales originating from poultry litter under oxic and anoxic environmental conditions. Bacteroidales abundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml⁻¹ and 2 log gene copies g litter⁻¹ under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of the Bacteroidales organisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related to Bacteroides uniformis, B. ovatus, and B. vulgatus. These results suggest that MST studies using qPCR methods targeting Bacteroidales in watersheds that are affected by poultry litter should be interpreted cautiously. Growth of Bacteroidales originating from poultry litter in environmental waters may occur while Bacteroidales growth from other fecal sources declines, thus confounding the interpretation of MST results.     

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.     

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.     

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.     

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.     

Estimation of the Relative Abundance of Different Bacteroides and Prevotella Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.     

Diversity and abundance of single-stranded DNA viruses in human feces

In this study, we investigated the abundance and diversity of single-stranded DNA (ssDNA) viruses in fecal samples from five healthy individuals through a combination of serial filtration and CsCl gradient ultracentrifugation. Virus abundance ranged from 10⁸ to 10⁹ per gram of feces, and virus-to-bacterium ratios were much lower (less than 0.1) than those observed in aquatic environments (5 to 10). Viral DNA was extracted and randomly amplified using phi29 polymerase and analyzed through high-throughput 454 pyrosequencing. Among 400,133 sequences, an average of 86.2% viromes were previously uncharacterized in public databases. Among previously known viruses, double-stranded DNA podophages (52 to 74%), siphophages (11 to 30%), myophages (1 to 4%), and ssDNA microphages (3 to 9%) were major constituents of human fecal viromes. A phylogenetic analysis of 24 large contigs of microphages based on conserved capsid protein sequences revealed five distinct newly discovered evolutionary microphage groups that were distantly related to previously known microphages. Moreover, putative capsid protein sequences of five contigs were closely related to prophage-like sequences in the genomes of three Bacteroides and three Prevotella strains, suggesting that Bacteroides and Prevotella are the sources of infecting microphages in their hosts.     

Fecal Microbiota Composition and Frailty

The relationship between fecal microbiota composition and frailty in the elderly was studied. Fecal samples from volunteers with high frailty scores showed a significant reduction in the number of lactobacilli (26-fold). At much higher population levels, both the Bacteroides/Prevotella (threefold) and the Faecalibacterium prausnitzii (fourfold) groups showed a significant reduction in percentage of total number of hybridizable bacteria in the elderly with high frailty scores. In contrast to this, the number of Enterobacteriaceae was significantly higher (sevenfold) in samples from very frail volunteers.     

Method for Isolation of Bacteroides Bacteriophage Host Strains Suitable for Tracking Sources of Fecal Pollution in Water

Bacteriophages infecting Bacteroides are potentially a good tool for fecal source tracking, but different Bacteroides host strains are needed for different geographic areas. A feasible method for isolating Bacteroides host strains for phages present in human fecal material is described. Useful strains were identified for application in Spain and the United Kingdom. One strain, GA-17, identified as Bacteroides thetaiotaomicron, was tested in several locations in Europe with excellent performance in Southern Europe.     

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.     

Use of Bacteroidales Microbial Source Tracking To Monitor Fecal Contamination in Fresh Produce Production

In recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of food-borne illnesses. Most pathogens associated with fresh produce are enteric (fecal) in origin, and contamination can occur anywhere along the farm-to-fork chain. Microbial source tracking (MST) is a tool developed in the environmental microbiology field to identify and quantify the dominant source(s) of fecal contamination. This study investigated the utility of an MST method based on Bacteroidales 16S rRNA gene sequences as a means of identifying potential fecal contamination, and its source, in the fresh produce production environment. The method was applied to rinses of fresh produce, source and irrigation waters, and harvester hand rinses collected over the course of 1 year from nine farms (growing tomatoes, jalapeño peppers, and cantaloupe) in Northern Mexico. Of 174 samples, 39% were positive for a universal Bacteroidales marker (AllBac), including 66% of samples from cantaloupe farms (3.6 log₁₀ genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log₁₀ GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log₁₀ GEC/100 ml). Of 68 AllBac-positive samples, 46% were positive for one of three human-specific markers, and none were positive for a bovine-specific marker. There was no statistically significant correlation between Bacteroidales and generic Escherichia coli across all samples. This study provides evidence that Bacteroidales markers may serve as alternative indicators for fecal contamination in fresh produce production, allowing for determination of both general contamination and that derived from the human host.     

Evaluation of new gyrB-based real-time PCR system for the detection of B. fragilis as an indicator of human-specific fecal contamination

A rapid and specific gyrB-based real-time PCR system has been developed for detecting Bacteroides fragilis as a human-specific marker of fecal contamination. Its specificity and sensitivity was evaluated by comparison with other 16S rRNA gene-based primers using closely related Bacteroides and Prevotella. Many studies have used 16S rRNA gene-based method targeting Bacteroides because this genus is relatively abundant in human feces and is useful for microbial source tracking. However, 16S rRNA gene-based primers are evolutionarily too conserved among taxa to discriminate between human-specific species of Bacteroides and other closely related genera, such as Prevotella. Recently, one of the housekeeping genes, gyrB, has been used as an alternative target in multilocus sequence analysis (MLSA) to provide greater phylogenetic resolution. In this study, a new B. fragilis-specific primer set (Bf904F/Bf958R) was designed by alignments of 322 gyrB genes and was compared with the performance of the 16S rRNA gene-based primers in the presence of B. fragilis, Bacteroides ovatus and Prevotella melaninogenica. Amplicons were sequenced and a phylogenetic tree was constructed to confirm the specificity of the primers to B. fragilis. The gyrB-based primers successfully discriminated B. fragilis from B. ovatus and P. melaninogenica. Real-time PCR results showed that the gyrB primer set had a comparable sensitivity in the detection of B. fragilis when compared with the 16S rRNA primer set. The host-specificity of our gyrB-based primer set was validated with human, pig, cow, and dog fecal samples. The gyrB primer system had superior human-specificity. The gyrB-based system can rapidly detect human-specific fecal source and can be used for improved source tracking of human contamination.     

Analysis of the Gull Fecal Microbial Community Reveals the Dominance of Catellicoccus marimammalium in Relation to Culturable Enterococci

Gulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB. Enterococcaceae and Enterobacteriaceae were the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealed Catellicoccus marimammalium as the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcus indoxyl-β-d-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified as Enterococcus spp., 1.2% were identified as Streptococcus spp., and none were identified as C. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions of C. marimammalium 16S rRNA gene sequences (>50-fold) relative to typical mEI culturable Enterococcus spp. C. marimammalium therefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment.     

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.     

Dysbiosis Signature of Fecal Microbiota in Colorectal Cancer Patients

The human gut microbiota is a complex system that is essential to the health of the host. Increasing evidence suggests that the gut microbiota may play an important role in the pathogenesis of colorectal cancer (CRC). In this study, we used pyrosequencing of the 16S rRNA gene V3 region to characterize the fecal microbiota of 19 patients with CRC and 20 healthy control subjects. The results revealed striking differences in fecal microbial population patterns between these two groups. Partial least-squares discriminant analysis showed that 17 phylotypes closely related to Bacteroides were enriched in the gut microbiota of CRC patients, whereas nine operational taxonomic units, represented by the butyrate-producing genera Faecalibacterium and Roseburia, were significantly less abundant. A positive correlation was observed between the abundance of Bacteroides species and CRC disease status (R?=?0.462, P?=?0.046?<?0.5). In addition, 16 genera were significantly more abundant in CRC samples than in controls, including potentially pathogenic Fusobacterium and Campylobacter species at genus level. The dysbiosis of fecal microbiota, characterized by the enrichment of potential pathogens and the decrease in butyrate-producing members, may therefore represent a specific microbial signature of CRC. A greater understanding of the dynamics of the fecal microbiota may assist in the development of novel fecal microbiome-related diagnostic tools for CRC.