Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

Abstract

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.

Phylogenetic Diversity and Molecular Detection of Bacteria in Gull Feces

“…In contrast to other gut systems, Bacteroidetes bacteria represent a small fraction of the bacteria in avian feces (e.g., as little as ϳ1% of the total community). This is an important finding to those developing markers to track avian fecal pollution in environmental waters, as it suggests that Bacteroidetes bacteria might not be practical targets for the development of avian-specific assays due to their low densities in the avian gut and considering that Bacteroidetes host-specific populations represent less that 1 to 10% of the total Bacteroidetes populations (4,20).…”

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

“…Texas water samples (100 ml) were collected, placed on ice for transportation to the laboratory, and filtered within 6 hours of sample collection. Samples were filtered through 0.22-m-pore-size filters as previously described (22). DNA was extracted using the QIAamp DNA minikit (Qiagen, Valencia, CA) and stored at Ϫ80°C until further analyses.…”

“…The performance of each swine-associated assay was determined in PCR mixtures containing known concentrations of fecal and water DNA extracts. Using this approach, it was possible to determine the detection limits of an assay against environmental extracts (22). PCR assays were performed using templates consisting of serial fecal DNA dilutions (1 ϫ 10 Ϫ8 to 1 ϫ 10 Ϫ16 g DNA) of composite fecal samples of swine from different age groups, lagoons, manure pits, and selected water samples yielding positive PCR results.…”

“…Samples were stored in ice coolers and transported to the laboratory within 4 hours of collection. Each sample was centrifuged at 3,600 ϫ g for 20 min at 4°C, after which the supernatants were filtered onto 0.22-m polycarbonate membranes (22). Filters and pellets were stored at Ϫ20°C until further processing.…”

Sustained Anthropogenic Impact in Carter Saltpeter Cave, Carter County, Tennessee and the Potential Effects on Manganese Cycling

“…Clone 4A08 shared 99% identity to environmental clones isolated from fecal contaminated watersheds (Lamendella et al, 2007;, and 97% identity to its closest cultured relative, Prevotella paludivivens, a species isolated from rice-plant residue (Ueki et al, 2007). Clone FB01 shared 99% identity to environmental clones isolated from equine-fecal contaminated water (Simpson et al, 2004).…”