Metagenome-based comparisons of decay rates and host-specificity of fecal microbial communities for improved microbial source tracking

Abstract

Fecal material in the environment is a primary source of pathogens that cause waterborne diseases and affect over a billion people worldwide. Microbial source tracking (MST) assays based on single genes (e.g., 16S rRNA) do not always provide the resolution needed to attribute fecal contamination sources. In this work, we used dialysis bag mesocosms simulating a freshwater habitat that were spiked separately with cow, pig, or human feces to monitor the decay of host-specific fecal signals over time with metagenomics, traditional qPCR, and culture-based methods. Sequencing of the host fecal communities used as inocula recovered 79 non-redundant metagenome-assembled genomes (MAGs) whose abundance patterns showed that the majority of the fecal community signal was not detectable in the mesocosm metagenomes after four days. Several MAGs showed high host specificity, and thus are promising candidates for biomarkers for their respective host type. Traditional qPCR methods varied in their correlation with MAG decay kinetics. Notably, the human-specific Bacteroides assay, HF183/BFDRev, consistently under-estimated fecal pollution due to not being present in all hosts and/or primer mismatches. This work provides new insights on the persistence and decay kinetics of host-specific gut microbes in the environment and identifies several MAGs as putative biomarkers for improved MST.

Advancing source tracking: systematic review and source-specific genome database curation of fecally shed prokaryotes

“…The human gut microbiome represents the best studied fecal source with work summarizing our understanding of its extant species diversity estimating it is probably comprised of about 3,000 reoccurring (i.e., excluding singletons) species globally 20 . Work examining human feces at the metagenomic level previously reported sequence diversity values less than those we found for our wastewater samples but greater than those of the dog and cat fecal slurries 34,35 .…”

Advancing Source Tracking: Systematic Review and Source-Specific Genome Database Curation of Fecally Shed Prokaryotes

“…20 Surveys of human feces at the metagenomic level previously reported sequence diversity values less than those we found for our wastewater samples but greater than those of the dog and cat fecal slurries. 38,39 Unsurprisingly, this suggests there are a substantial number of species missing from the databases of many of the sources we examined. Indeed, no source saw read capture rates exceed 60% in our noncompetitive read mapping exercises (Figure 3).…”

Novel municipal sewage-associated bacterial genomes and their potential in source tracking

“…Mesocosm sampling, DNA extraction and subsequent qPCR analysis occurred as described previously in Suttner et al (35). Briefly, water samples were passed through 0.45 μm poly-carbonate (PC) membranes and stored at -80 °C in 2 mL screw cap bead tubes until processed (within 1-3 months).…”

“…Template DNAs were run undiluted or diluted 5-fold (to remove the effect of PCR inhibitors) depending on the expected marker concentration and quality of each sample. Further details on qPCR reaction set up and standard plasmids for absolute quantification are provided in Suttner et al (35) and reiterated within the supplement here (Supplement Table S1). To test for extraneous DNA and potential contamination from sample handling, 50 mL of sterile PBS was also filtered onto PC membranes and processed following the same DNA extraction at every sampling time point as described above.…”