Blautia and Prevotella sequences distinguish human and animal fecal pollution in Brazil surface waters

Abstract

Summary Untreated sewage discharges and limited agricultural manure management practices contribute to fecal pollution in rural Brazilian waterways. Few microbial source tracking studies have tested host-specific indicators in underdeveloped regions and have mostly focused on Bacteroidales. Sequencing of sewage, human feces, and animal feces with Illumina HiSeq revealed Prevotellaceae as the most abundant family in humans, with Lachnospiraceae and Ruminococcaceae also comprising a large proportion of the microbiome. These same families were also dominant in animals. Bacteroides, the most commonly utilized human-specific marker in the US, was present in very low abundance. We used oligotyping to identify Prevotella and Blautia sequences that can distinguish human fecal contamination. Thirty-five of 61 Blautia oligotypes and 13 of 108 Prevotella oligotypes in humans were specific to or highly abundant (i.e. host-preferred) compared to pig, dog, horse, and cow sources. Certain human Prevotella and Blautia oligotypes increased more than an order of magnitude along a polluted river transect in rural Brazil, but traditional fecal indicator levels followed a steady or even decreasing trend. While both Prevotella and Blautia oligotypes distinguished human and animal fecal pollution in Brazil surface waters, Blautia appears to contain more discriminatory and globally applicable markers for tracking sources of fecal pollution.

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

“…We previously sequenced DNA extracted from 53 fecal samples from animals (cats, chickens, cows, deer, dogs, swine) and two primary influent sewage samples and reported on the fine-scale population structure within the genus Blautia (35). The environmental samples and additional sewage samples analyzed in this study were also sequenced previously, and methods for their collection and processing are reported elsewhere (18,36).…”

“…The percent abundance of an oligotype in sewage was expressed relative to the abundance only in the fecal portion of sewage rather than the total number of reads (31). Environmental samples with known human fecal or sewage contamination were obtained from a Brazilian river with direct human fecal inputs from a small village, stormwater with high copy numbers of human fecal indicators Lachno2 and HF183, and surface water samples taken from Lake Michigan after a combined sewer overflow (CSO) event (18,36,41). Stormwater samples with very low copy numbers or results below the detection limit from the Lachno2 and HF183 assays and Lake Michigan water collected during the baseflow were used as comparison samples with low levels of contamination.…”

“…Differences in diet, geography, and many other factors can shape the fecal bacterial community in human populations (51)(52)(53). Of particular note for MST purposes is the relatively lower Bacteroidaceae:Prevotellaceae ratios in fecal samples from people with a non-Western diet (18,53) and the higher abundances of Lachnospiraceae in those who eat a more plant-based diet (54). The currently used genetic markers may therefore be less effective in regions of the world where populations lack high proportions of Bacteroidaceae in their fecal communities (16,18).…”

“…Of particular note for MST purposes is the relatively lower Bacteroidaceae:Prevotellaceae ratios in fecal samples from people with a non-Western diet (18,53) and the higher abundances of Lachnospiraceae in those who eat a more plant-based diet (54). The currently used genetic markers may therefore be less effective in regions of the world where populations lack high proportions of Bacteroidaceae in their fecal communities (16,18). We observed a shift from a Bacteroides-dominated signature to a Lachnospiraceae-dominated signature in U.S. versus Brazilian sewage and water samples, but the majority of our candidate indicators were present in Brazilian sewage, even though the ratios differed from those in U.S. sewage.…”

“…Bacteroidales markers for humans, ruminants, pigs, and other animals have been used extensively with much success (9,12,15). However, additional markers are needed to support the results of these assays, to provide alternative indicators in regions where Bacteroidales are a minor component of human fecal pollution, and to resolve contributions from multiple hosts in environments with complex pollution sources (16,18). Only a few studies have proposed alternatives to Bacteroidales markers for the identification of mammalian fecal sources (7,(19)(20)(21), and even fewer studies have compared the performance of marker genes from the Bacteroidales with that of marker genes from other major taxonomic groups (6,19,22).…”