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.

FORENSIC: an Online Platform for Fecal Source Identification

“…In this study, we demonstrated that the two most common gut-associated bacterial orders in mammals, i.e., Bacteroidales and Clostridiales ( 18 , 22 ), provide enough host-specific signal to perform accurate source identification. Similar observations were reported at the genus level ( 23 – 25 ), highlighting the fractal nature of specialization between gut microbiota and their hosts. Targeting taxonomically defined groups, rather than studying the whole bacterial community, may reduce the influence of large cross-phylum shifts due to diet or sequencing primer bias in the bacterial community ( 25 ) while providing relevant host-associated profiles.…”

“…Similar observations were reported at the genus level ( 23 – 25 ), highlighting the fractal nature of specialization between gut microbiota and their hosts. Targeting taxonomically defined groups, rather than studying the whole bacterial community, may reduce the influence of large cross-phylum shifts due to diet or sequencing primer bias in the bacterial community ( 25 ) while providing relevant host-associated profiles.…”

“…For the U.S. and Dominican Republic fecal samples, bacterial DNA was extracted from approximately 1 g of material using a QIAmp DNA stool minikit according to the manufacturer’s instructions (Qiagen, Valencia, CA) as described ( 25 ). About 200 mg of Australian stool samples was extracted using the QIAmp stool DNA kit, while about 500 mg of dry fecal sample for French samples was extracted using the FastDNA spin kit for soil (MP Biomedicals, Carlsbad, CA).…”

Superior performance of human wastewater-associated viral markers compared to bacterial markers in tropical environments

“…A similar trend showing higher specificity among viral markers than among bacterial markers has also been observed in Southeast Queensland, Australia (Ahmed et al, 2019a). This could be explained by the co-presence of certain gut bacterial genera in animals and humans (Feng et al, 2020).…”

“…Other than the intrinsic presence of marker sequence in nontargeted samples, low assay specificity could also be caused by nonspecific PCR amplification (Feng et al, 2020). In a previous study, the cross-reactivity of Lachno3 with animal feces was likely caused by nonspecific qPCR amplification, as supported by an absence of Lachno3 in the metagenomic analysis (Feng et al, 2018).…”