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Ecological and Technical Mechanisms for Cross-Reaction of Human Fecal Indicators with Animal Hosts.

ABSTRACT: Quantitative PCR (qPCR) assays for human/sewage marker genes have demonstrated sporadic positive results in animal feces despite their high specificities to sewage and human feces. It is unclear whether these positive reactions are caused by true occurrences of microorganisms containing the marker gene (i.e., indicator organisms) or nonspecific amplification (false positive). The distribution patterns of human/sewage indicator organisms in animals have not been explored in depth, which is crucial for evaluating a marker gene's true- or false-positive reactions. Here, we analyzed V6 region 16S rRNA gene sequences from 257 animal fecal samples and tested a subset of 184 using qPCR for human/sewage marker genes. Overall, specificities of human/sewage marker genes within sequencing data were 99.6% (BacV6-21), 96.9% (Lachno3), and 96.1% (HF183, indexed by its inferred V6 sequence). Occurrence of some true cross-reactions was associated with atypical compositions of organisms within the genera Blautia or Bacteroides For human/sewage marker qPCR assays, specificities were 96.7% (HF183/Bac287R), 96.2% (BacV6-21), 95.6% (human Bacteroides [HB]), and 94.0% (Lachno3). Select assays duplexed with either Escherichia coli or Enterococcus spp. were also validated. Most of the positive qPCR results in animals were low level and, on average, 2 orders of magnitude lower than the copy numbers of E. coli and Enterococcus spp. The lower specificity in qPCR assays compared to sequencing data was mainly caused by amplification of sequences highly similar to the marker gene and not the occurrence of the exact marker sequence in animal fecal samples.IMPORTANCE Identifying human sources of fecal pollution is critical to remediate sanitation concerns. Large financial investments are required to address these concerns; therefore, a high level of confidence in testing results is needed. Human fecal marker genes validated in this study showed high specificity in both sequencing data and qPCR results. Human marker sequences were rarely found in individual animals, and in most cases, the animals had atypical microbial communities. Sequencing also revealed the presence of closely related organisms that could account for nonspecific amplification in certain assays. Both the true cross-reactions and the nonspecific amplification had low signals well below E. coli or Enterococcus levels and likely would not impact the assay's ability to reliably detect human fecal pollution. No animal source had multiple human/sewage marker genes present; therefore, using a combination of marker genes would increase the confidence of human fecal pollution detection.

SUBMITTER: Feng S

PROVIDER: S-EPMC7028960 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Ecological and Technical Mechanisms for Cross-Reaction of Human Fecal Indicators with Animal Hosts.

Feng Shuchen S Ahmed Warish W McLellan Sandra L SL

Applied and environmental microbiology 20200218 5

Quantitative PCR (qPCR) assays for human/sewage marker genes have demonstrated sporadic positive results in animal feces despite their high specificities to sewage and human feces. It is unclear whether these positive reactions are caused by true occurrences of microorganisms containing the marker gene (i.e., indicator organisms) or nonspecific amplification (false positive). The distribution patterns of human/sewage indicator organisms in animals have not been explored in depth, which is crucia ...[more]

PMID: 31862726

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Project description:The model prokaryote Escherichia coli can exist as a either a commensal or a pathogen in the gut of diverse mammalian hosts. These associations, coupled with its ease of cultivation and genetic variability, have made E. coli a popular indicator organism for tracking the origin of fecal water contamination. Source tracking accuracy is predicated on the assumption that E. coli isolates recovered from contaminated water present a genetic signature characteristic of the host from which they originated. In this study, we compared the accuracy with which E. coli isolated from humans, bear, cattle and deer could be identified by standard fingerprinting methods used for library-based microbial source tracking (repetitive element PCR and pulsed-field gel electrophoresis) in relation to microarray-based analysis of genome content. Our results show that patterns of gene presence or absence were more useful for distinguishing E. coli isolates from different sources than traditional fingerprinting methods, particularly in the case of human strains. Host-associated differences in genome composition included the presence or absence of mobile IS1 elements as well as genes encoding the ferric dicitrate iron transporter (fec), E. coli common pilus (ECP), type 1 fimbriae and the CRISPR associated cas proteins. Many of these differences occurred in regions of the E. coli chromosome previously shown to be M-bM-^@M-^\hot spotsM-bM-^@M-^] for the integration of horizontally-acquired DNA. PCR primers designed to amplify the IS1 and fec loci confirmed array results and demonstrated the ease with which gene presence/absence data can be converted into a diagnostic assay. The data presented here suggest that, despite the high level of genetic diversity observed among isolates by PFGE, human-derived strains may constitute a distinct ecotype distinguished by multiple potential library-independent source tracking markers. Twelve isolates of E. coli ( 3 from bear, 3 from cattle, 3 from deer and 3 from humans) were isolated from feces and/or raw sewage. Genome content for each strain was assessed in duplicate using comparative genome hybridization with E. coli K12 MG1655 as the reference for a total of 24 arrays.

Dataset Information

Ecological and Technical Mechanisms for Cross-Reaction of Human Fecal Indicators with Animal Hosts.

Publications

Ecological and Technical Mechanisms for Cross-Reaction of Human Fecal Indicators with Animal Hosts.

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