Quantitative PCR Method for Sensitive Detection of Ruminant Fecal Pollution in Freshwater and Evaluation of This Method in Alpine Karstic Regions

174

159

The microbiological quality of coastal or river water can be affected by fecal contamination from human or animal sources. To discriminate pig fecal pollution from other pollution, a library-independent microbial source tracking method targeting Bacteroidales host-specific 16S rRNA gene markers by real-time PCR was designed. Two pig-specific Bacteroidales markers (Pig-1-Bac and Pig-2-Bac) were designed using 16S rRNA gene Bacteroidales clone libraries from pig feces and slurry. For these two pig markers, 98 to 100% sensitivity and 100% specificity were obtained when tested by TaqMan real-time PCR. A decrease in the concentrations of Pig-1-Bac and Pig-2-Bac markers was observed throughout the slurry treatment chain. The two newly designed pig-specific Bacteroidales markers, plus the human-specific (HF183) and ruminant-specific (BacR) Bacteroidales markers, were then applied to river water samples (n ‫؍‬ 24) representing 14 different sites from the French Daoulas River catchment (Brittany, France). Pig-1-Bac and Pig-2-Bac were quantified in 25% and 62.5%, respectively, of samples collected around pig farms, with concentrations ranging from 3.6 to 4.1 log 10 copies per 100 ml of water. They were detected in water samples collected downstream from pig farms but never detected near cattle farms. HF183 was quantified in 90% of water samples collected downstream near Daoulas town, with concentrations ranging between 3.6 and 4.4 log 10 copies per 100 ml of water, and BacR in all water samples collected around cattle farms, with concentrations ranging between 4.6 and 6.0 log 10 copies per 100 ml of water. The results of this study highlight that pig fecal contamination was not as frequent as human or bovine fecal contamination and that fecal pollution generally came from multiple origins. The two pig-specific

Section: Resultsmentioning

confidence: 99%

“…The detection of host-specific Bacteroidales markers is a promising library-independent method and has been used for identifying contamination from human and bovine origins (25,29,39,40,45). In this study, we selected Bacteroidales 16S rRNA gene markers and real-time PCR to focus on fecal contamination from pigs.…”

mentioning

confidence: 99%

Estimation of Pig Fecal Contamination in a River Catchment by Real-Time PCR Using Two Pig-Specific Bacteroidales 16S rRNA Genetic Markers

Mieszkin

Furet

Corthier

et al. 2009

174

159

“…The assays employed in this study targeted E. coli (55), Enterococcus spp. (Entero) (68), human-associated Lachnospiraceae (Lachno2) (26), human-associated Bacteroides (HB) (19,67,69), gull-associated Catellicoccus marimammalium (Gull2) (28,70), and ruminant-associated Bacteroidetes (BacR) (22). Standard curves were created using six serial 1:10 dilutions from 1.5 ϫ 10 6 to 15 copies per reaction of a linearized plasmid containing the target sequence.…”

Section: Methodsmentioning

confidence: 99%

“…Alternative indicator assays commonly target anaerobic fecal bacteria due to their high abundance in animal and human feces and their limited survival in the external environment (15)(16)(17). Bacteria within the order Bacteroidales are common targets for quantitative PCR (qPCR) assays and have been used for the detection of sewage (18)(19)(20)(21), ruminant (19,22), canine (19), and avian (23,24) sources of fecal pollution. Certain members within the order Clostridiales have also demonstrated host-associated patterns (25).…”

mentioning

confidence: 99%

Distribution and Differential Survival of Traditional and Alternative Indicators of Fecal Pollution at Freshwater Beaches

Cloutier

McLellan

2017

Alternative indicators have been developed that can be used to identify host sources of fecal pollution, yet little is known about how their distribution and fate compare to traditional indicators. Escherichia coli and enterococci were widely distributed at the six beaches studied and were detected in almost 95% of water samples (n ϭ 422) and 100% of sand samples (n ϭ 400). Berm sand contained the largest amount of E. coli (P Ͻ 0.01), whereas levels of enterococci were highest in the backshore (P Ͻ 0.01). E. coli and enterococci were the lowest in water, using a weight-to-volume comparison. The gull-associated Catellicoccus marimammalium (Gull2) marker was found in over 80% of water samples, regardless of E. coli levels, and in 25% of sand samples. Human-associated Bacteroides (HB) and Lachnospiraceae (Lachno2) were detected in only 2.4% of water samples collected under baseflow and post-rain conditions but produced a robust signal after a combined sewage overflow, despite low E. coli concentrations. Burdens of E. coli and enterococci in water and sand were disproportionately high in relation to alternative indicators when comparing environmental samples to source material. In microcosm studies, Gull2, HB, and Lachno2 quantitative PCR (qPCR) signals were reduced twice as quickly as those from E. coli and enterococci and approximately 20% faster than signals from culturable E. coli. High concentrations of alternative indicators in source material illustrated their high sensitivity for the identification of fecal sources; however, differential survival and the potential for long-term persistence of traditional fecal indicators complicate the use of alternative indicator data to account for the levels of E. coli and enterococci in environmental samples. IMPORTANCE E. coli and enterococci are general indicators of fecal pollution and may persist in beach sand, making their use problematic for many applications. This study demonstrates that gull fecal pollution is widespread at Great Lakes beaches, whereas human and ruminant contamination is evident only after major rain events. An exploration of sand as a reservoir for indicators found that E. coli was ubiquitous, while gull host markers were detected in only 25% of samples. In situ sand beach microcosms provided decay rate constants for E. coli and enterococci relative to alternative indicators, which establish comparative benchmarks that would be helpful to distinguish recent from past pollution. Overall, alternative indicators are useful for identifying sources and assessing potentially high health risk contamination events; however, beach managers should be cautious in attempting to directly link their detection to the levels of E. coli or enterococci.KEYWORDS beaches, water quality, human-associated indicators, gull-associated indicators, qPCR F ecal contamination of recreational waters can be a serious threat to public health. Due to the vast diversity of fecal-borne human pathogens, the USEPA has recommended the use of fecal indicator bacteria (FIB),...

“…At present, qualitative and quantitative analyses of Bacteroidales in feces-related samples are achieved mainly through cell cultivation and the use of conventional molecular tools (2,4,10,19,21,33). Both approaches, however, have their strengths and weaknesses.…”

Section: Approximately 10mentioning

confidence: 99%

Relative Abundance ofBacteroidesspp. in Stools and Wastewaters as Determined by Hierarchical Oligonucleotide Primer Extension

Hong

Liu

2008

A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), was used to determine the relative abundances of predominant Bacteroides spp. present in fecal microbiota and wastewaters. For this analysis, genomic DNA in feces of healthy human adults, bovines, and swine and in wastewaters was extracted and total bacterial 16S rRNA genes were PCR amplified and used as the DNA templates for HOPE. Nineteen oligonucleotide primers were designed to detect 14 Bacteroides spp. at different hierarchical levels (domain, order, cluster, and species) and were arranged into and used in six multiplex HOPE reaction mixtures. Results showed that species like B. vulgatus, B. thetaiotaomicron, B. caccae, B. uniformis, B. fragilis, B. eggerthii, and B. massiliensis could be individually detected in human feces at abundances corresponding to as little as 0.1% of PCR-amplified 16S rRNA genes. Minor species like B. pyogenes, B. salyersiae, and B. nordii were detected only collectively using a primer that targeted the B. fragilis subgroup (corresponding to ϳ0.2% of PCR-amplified 16S rRNA genes). Furthermore, Bac303-related targets (i.e., most Bacteroidales) were observed to account for 28 to 44% of PCR-amplified 16S rRNA genes from human fecal microbiota, and their abundances were higher than those detected in the bovine and swine fecal microbiota and in wastewaters by factors of five and two, respectively. These results were comparable to those obtained by quantitative PCR and to those reported previously from studies using whole-cell fluorescence hybridization and 16S rRNA clone library methods, supporting the conclusion that HOPE can be a sensitive, specific, and rapid method to determine the relative abundances of Bacteroides spp. predominant in fecal samples.