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

Abstract

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

Current Status of Marker Genes of Bacteroides and Related Taxa for Identifying Sewage Pollution in Environmental Waters

“…Since its development, 2966 individual non-human fecal samples have been screened for the presence of the HF183 marker, of which 2807 were PCR negative, yielding an overall host specificity value of 94.6% (Supplementary Table S11). Several studies have reported 100% host specificity of the HF183 marker for non-human fecal samples [51,[66][67][68][69][70]. In contrast, the occasional presence of the HF183 markers in non-human fecal samples has also been reported [52,57,61,71,72], particularly in dog, deer, and chicken feces.…”

Survival of Host-Associated Bacteroidales Cells and Their Relationship with Enterococcus spp., Campylobacter jejuni, Salmonella enterica Serovar Typhimurium, and Adenovirus in Freshwater Microcosms as Measured by Propidium Monoazide-Quantitative PCR

“…Significant concentrations of dissolved DNA are found in marine water, freshwater, and sediments, potentially complicating the interpretation of information from qPCR data due to the relatively long persistence of target DNA (23). The PMA-qPCR method could avoid the issue of persistent DNA and provide a relatively fast, simple, and inexpensive means to discriminate intact/dead cells in mixed populations (2,22,42). However, some limitations of PMA-qPCR have been reported, such as the interference of higher particle concentration (2) and highly complex chemical and biological matrices in environmental samples.…”

“…Furthermore, FIB are inadequate to identify the source of fecal pollution because they are observed in both warm-and cold-blooded animal feces (30,35). Microbial source tracking (MST) can discriminate between human and nonhuman fecal contamination such as cow, dog, and pig in water using microbiological or chemical traits of source identifiers (19,22). Bacteroidales have been proposed as an alternative fecal indicator as well as source identifier because they are abundant in the gastrointestinal tract and genetic markers based on 16S rRNA have host-associated distributions (7,13,14,17,18,25,30,32).…”

Unsuitability of Quantitative Bacteroidales 16S rRNA Gene Assays for Discerning Fecal Contamination of Drinking Water

“…The Bacteroidales counts were high compared to the number of E. coli that are occasionally observed in fecally contaminated drinking water (17a) but low compared to numbers observed in surface water (4,20,22). Water from the extraction wells and raw water used for unchlorinated drinking water production were analyzed, and Bacteroidales species were detected in 10 out of 15 samples (Table 1).…”