Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene: Assessment of fecal pollution sources (original) (raw)
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FEMS Microbiology Ecology, 2000
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 humantargeted 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.
Journal of Applied Microbiology, 2010
Aims: The aims of this study were to evaluate the host-specific distribution of Bacteroidales 16S rRNA gene sequences from human-and animal-related effluents and faeces, and to define a ruminantspecific marker. Methods and Results: Bacteroidales 16S rRNA gene clone libraries were constructed from samples of effluent (sewage, bovine manure and pig slurry) and faeces (human, bovine, pig and wild bird), using PCR primers targeting order Bacteroidales. The phylogenetic analysis revealed six main distinct human-, bovine-, pig-and wild bird-specific clusters. From the bovine-specific cluster II, we designed a ruminant-specific marker, Rum-2-Bac, and this showed 97% sensitivity (n = 30) and 100% specificity (n = 40) when tested by TaqMan ® real-time PCR. Average concentrations of this marker in bovine and sheep faeces and in bovine manure were 8•2 ± 0•5, 8•4 ± 1•3 and 7 ± 0•5 log 10 copies per gram, respectively. It was also quantified in samples of runoff water impacted by bovine manure, with average concentrations of 5•1 ± 0•3 log 10 copies per millilitre water. Conclusions: Our results confirmed that some members of Bacteroidales isolated from effluents and faeces had host-specific distributions. Identification of a bovine-specific cluster made it possible to design a reliable ruminant-specific marker. Significance and Impact of the Study: The host-specific distribution of Bacteroidales sequences from effluents mirrored the host-specific distribution of sequences observed in individual faeces. This efficient new ruminant-specific Bacteroidales 16S rRNA marker represents a useful addition to the microbial source tracking toolbox.
Applied and Environmental Microbiology, 2006
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.
Applied and Environmental …, 2000
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.
Applied and …, 2008
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.
Applied and Environmental Microbiology, 2004
Assessment of health risk associated with fecal pollution requires a reliable fecal indicator and a rapid quantification method. We report the development of a Taq nuclease assay for enumeration of 16S rRNA genes of Bacteroidetes. Sensitivity and correlation with standard fecal indicators provide experimental evidence for application of the assay in monitoring fecal pollution.
The field of molecular fecal source tracking in the water environment has developed rapidly since the first PCR assays for general and host-‐specific Bacteroides 16s rRNA markers were published. Numerous host-‐specific molecular markers and PCR assays have been developed, adding greater specificity, sensitivity and quantitative methods to the array of options. The public demand for readying methods for transfer to the commercial lab, so that they may be used to generate data for public utilities, citizen action groups and regulatory agencies, has fueled the development of an entire new research community. These methods, however plentiful, have not found community agreement and there is no consensus concerning the appropriate implementation of molecular fecal source tracking in the field. Some issues plaguing the implementation include imperfect marker specificity, environmental variability, DNA extraction variability, PCR inhibition and high cost of molecular analysis. This thesis...
2013
Accurate identification of fecal contamination sources in an impaired watershed is crucial for developing best management practices. Microbial source tracking (MST) is commonly used to identify fecal pollution sources and includes both library-based and non-library-based approaches. The library-based approach often involves the development of a known source library from DNA fingerprints obtained from fecal samples of known host groups, allowing the DNA fingerprints from water samples to be compared against those in the known source library. The non-library based approach involves the direct detection of host-associated markers present in the water samples. In the first study, the library-based E. coli rep-PCR DNA fingerprinting method and Bacteroidales host-associated markers were used to identify fecal contamination sources in a mixed land-use watershed. DNA extracted from 64 water samples was analyzed using end-point and quantitative PCR. A total of 1,050 E. coli rep-PCR DNA fingerprints obtained from water samples were then compared against the known source library, which consisted of 945 unique E. coli DNA fingerprints from nine host groups. All of the water samples were positive for both general Bacteroidales markers and E. coli. The rep-PCR method detected human and cattle contamination in 94% and 75% of the water samples, respectively, while end-point PCR found human and cattle markers only in 27% and 28% of the samples. qPCR, on the other hand, detected human-associated markers in 64.0% of the samples. iii The second study evaluated human-and cattle-associated Bacteroidales genetic markers for their applicability in Alabama and identified the most suitable primer sets in qPCR assays for assessing fecal contamination in environmental samples. Four humanand seven cattle-associated genetic markers were tested and human-associated HF183 and cattle-associated CowM3 appeared to be the best human and cattle markers, respectively. DNA extracted from surface water samples was amplified with general Bacteroidales primers as well as human-and cattle-associated primers. The results indicated that general Bacteroidales genetic markers were positive for all samples, with the highest concentration being 1,180,500 gene copies/100 ml. Human-associated Bacteroidales markers were detected in 87% of the water samples, while only 8% of the water samples contained the cattle-associated Bacteroidales markers. The major disadvantage of PCR is its inability to discriminate between DNA from live and dead cells. Propidium monoazide (PMA) is a DNA intercalating agent that can be used to detect DNA from live cells. The third study focused on the survival of E. coli and live Bacteroidales in stream water and sediment microcosms. The general Bacteroidales markers were detectable up to 7 and 9 days in stream water and sediment microcosms, respectively, but human markers were detected only in the first 3 days in both microcosms. During the study period, a 3-log reduction of E. coli was observed, with 2,500 CFU/100 ml remaining in the water microcosm at the end of 14 days. However, E. coli survived in the sediment for more than 75 days. Experiments with PMA revealed that about 50% of the Bacteroidales gene copies amplified by qPCR were actually from dead cells or extracellular DNA.
Water research, 2007
We report the design and validation of new TaqMan((R)) assays for microbial source tracking based on the amplification of fecal 16S rRNA marker sequences from uncultured cells of the order Bacteroidales. The assays were developed for the detection and enumeration of non-point source input of fecal pollution to watersheds. The quantitative "universal"Bacteroidales assay BacUni-UCD detected all tested stool samples from human volunteers (18 out of 18), cat (7 out of 7), dog (8 out of 8), seagull (10/10), cow (8/8), horse (8/8), and wastewater effluent (14/14). The human assay BacHum-UCD discriminated fully between human and cow stool samples but did not detect all stool samples from human volunteers (12/18). In addition, there was 12.5% detection of dog stool (1/8), but no cross-reactivity with cat, horse, or seagull fecal samples. In contrast, all wastewater samples were positive for the BacHum-UCD marker, supporting its designation as 100% sensitive for mixed-human source ...