Use of Barcoded Pyrosequencing and Shared OTUs To Determine Sources of Fecal Bacteria in Watersheds

Abstract

While many current microbial source tracking (MST) methods rely on the use of specific molecular marker genes to identify sources of fecal contamination, these methods often fail to determine all point and nonpoint contributors of fecal inputs into waterways. In this study, we developed a new library-dependent MST method that uses pyrosequencing-derived shared operational taxonomy units (OTUs) to define sources of fecal contamination in waterways. A total 56,841 pyrosequencing reads of 16S rDNA obtained from the feces of humans and animals were evaluated and used to compare fecal microbial diversity in three freshwater samples obtained from the Yeongsan river basin in Jeonnam Province, South Korea. Sites included an urbanized agricultural area (Y1) (Escherichia coli counts ≥ 1600 CFU/100 mL), an open area (Y2) with no major industrial activities (940 CFU/100 mL), and a typical agricultural area (Y3) (≥ 1600 CFU/100 mL). Data analyses indicated that the majority of bacteria in the feces of humans and domesticated animals were comprised of members of the phyla Bacteroidetes or Firmicutes, whereas the majority of bacteria in wild goose feces and freshwater samples were classified to the phylum Proteobacteria. Analysis of OTUs shared between the fecal and environmental samples suggested that the potential sources of the fecal contamination at the sites were of human and swine origin. Quantification of fecal contamination was also examined by comparing the density of pyrosequencing reads in each fecal sample within shared OTUs. Taken together, our results indicated that analysis of shared OTUs derived from barcoded pyrosequencing reads provide the necessary resolution and discrimination to be useful as a next generation platform for microbial source tracking studies.

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

“…However, human and other animal gut microbiota contain an array of other taxonomic groups that may serve as novel indicators of fecal pollution along with Bacteroidales. Unno and colleagues [136] reported the development of a new library-dependent method using pyrosequencing-derived shared operational taxonomic units (OTUs) to identify the sources of fecal pollution in waterways in South Korea. Their results indicated that the majority of bacteria in the feces of humans and domesticated animals belonged to the phyla Bacteroidetes and Firmicutes, whereas the predominant bacteria in the feces of geese and in freshwater samples belonged to Proteobacteria.…”

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

“…Currently, there is little information available on data acceptance criteria for fecal source identification technologies. The lack of data acceptance criteria is, in part, due to an active method development research community where new technologies are introduced on a regular basis.Fecal source identification technologies are diverse and range from bacterial community approaches (19)(20)(21)(22) to canine scent detection (23), making it difficult to choose the most suitable technology. In this study, the human-associated HF183/BacR287 (24) and HumM2 (25) quantitative real-time PCR (qPCR) methods were selected for data acceptance criteria development.…”

“…Fecal source identification technologies are diverse and range from bacterial community approaches (19)(20)(21)(22) to canine scent detection (23), making it difficult to choose the most suitable technology. In this study, the human-associated HF183/BacR287 (24) and HumM2 (25) quantitative real-time PCR (qPCR) methods were selected for data acceptance criteria development.…”

Application of Phylogenetic Microarray Analysis to Discriminate Sources of Fecal Pollution

“…These results are consistent with previous surveys of other mammalian gut microbial communities. 12,13,21 In contrast to mammals, avian feces contained far less taxa in the Clostridia and Bacteroidales and instead were dominated by Gammaproteobacteria and Bacilli (Figure 2).…”