Comparison of Sewage and Animal Fecal Microbiomes by Using Oligotyping Reveals Potential Human Fecal Indicators in Multiple Taxonomic Groups

Abstract

dMost DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales, but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCRbased assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the family Lachnospiraceae to provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts. Microbial source tracking (MST) is used to determine sources of fecal pollution in surface waters and recreational beaches with the goal of minimizing the risk to human health (1-3). Transmission of bacterial, viral, and zoonotic diseases occurs through feces-contaminated water (4), and identification of the type of host inputs (e.g., sewage, wildlife, agricultural) can provide a more accurate assessment of the risks to human health and better direct management actions to reduce likely sources of pollution (5). Identification of fecal sources is based on the assumption that some microorganisms exhibit host-specific distribution patterns (6). The majority of microbial population studies performed to identify members that specifically or preferentially associate with particular animal hosts have used the 16S rRNA gene as a marker (7-9).Bacteria within the order Bacteroidales have been the major focus of molecular MST efforts (10-16), as they have many qualities that make them an effective indicator. These organisms are abundant in the gastrointestinal tract of many animals, some species exhibit an association with particular hosts, and many have persistence and survival chara...

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

“…A recent study compared the V6 region of the 16S rRNA gene in fecal samples from six animals to sewage [137]. The authors focused on 10 abundant genera and used oligotyping, which can distinguish ecologically distinct organisms whose rRNA sequences differ from each other by few nucleotides [138].…”

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.…”

Microbial communities as biosensors for monitoring urban environments

“…Oligotyping, a computational method that makes use of highly variable nucleotide positions in 16S sequences, can distinguish closely related organisms that would normally be classified into one taxon (Eren et al, 2013). Oligotyping has led to the identification of human faecal indicators from multiple taxonomic groups (Koskey et al, 2014;Fisher et al, 2015). Human-specific taxa were found not only in populations of Bacteroidetes, but also in the bacterial phylum Firmicutes, including oligotypes under the genera Blautia, Coprococcus, Dorea, Faecalibacterium and Roseburia (Fisher et al, 2015).…”

“…Oligotyping has led to the identification of human faecal indicators from multiple taxonomic groups (Koskey et al, 2014;Fisher et al, 2015). Human-specific taxa were found not only in populations of Bacteroidetes, but also in the bacterial phylum Firmicutes, including oligotypes under the genera Blautia, Coprococcus, Dorea, Faecalibacterium and Roseburia (Fisher et al, 2015). In addition, community profiling of pipes transmitting sewage from households to wastewater treatment facilities has shown that Arcobacter species are abundant and prevalent, suggesting that using community composition in pollution source tracking has the potential to detect signatures from urban water infrastructure .…”