A new Open Access study published in the American Society for Microbiology journal mBio reports that a team of scientists from the University of Wisconsin School of Freshwater Sciences and the the Marine Biological Laboratory at Woods Hole, Massachusetts; has found that by using molecular characterizations of the gut microbiome from individual human stool samples, they’ve been able to have identify community and demographics patterns in subject populations correlating to age, disease, diet, and other human characteristics.
However, the paper, entitled “Sewage Reflects the Microbiomes of Human Populations“ (24 February 2015 mBio vol. 6 no. 2 e02574-14 doi: 10.1128/mBio.02574-14), coauthored by Ryan J. Newton, Sandra L. McLellan, and Deborah K. Dila of the University of Wisconsin School of Freshwater Sciences, Milwaukee, Wisconsin; and Joseph H. Vineis, Hilary G. Morrison, A. Murat Eren, and Mitchell L. Sogin of the Josephine Bay Paul Center of the Marine Biological Laboratory at Woods Hole, Massachusetts; edited by Gary B. Huffnagle of the University of Michigan Medical School
notes that resources for marker gene studies of microbiome trends among human populations scale with the number of individuals sampled from each population.
As an alternative strategy for sampling populations, the researchers examined whether sewage accurately reflects the microbial community of a mixture of stool samples, using oligotyping of high-throughput 16S rRNA gene sequence data to compare the bacterial distribution in a stool data set to a sewage influent data set from 71 U.S. cities.
On average, they found that only 15 percent of sewage sample sequence reads were attributable to human fecal origin, but sewage recaptured most (97 percent) human fecal oligotypes — the most common oligotypes in stool matching the most common and abundant in sewage.
Comparisons among municipal sewage communities revealed ubiquitous and abundant occurrence of 27 human fecal oligotypes, representing an apparent core set of microbiome organisms in U.S. populations. Fecal community variability among U.S. populations was found to be significantly lower than among individuals — clustered into three primary community structures distinguished by oligotypes from either: Bacteroidaceae, Prevotellaceae, or Lachnospiraceae/Ruminococcaceae. The coauthors say these distribution patterns reflect human population variation and predict whether samples represented lean or obese populations with 81 to 89 percent accuracy, and that their findings demonstrate that sewage represents the fecal microbial community of human populations and captures population-level traits of the human microbiome.
These findings signify that gut microbiota serve important functions in healthy humans, and that numerous projects have aimed to define a healthy gut microbiome and its association with general health outcomes. However, they observe that financial considerations and privacy concerns impose limits on numbers of individuals who can be screened.
On the other hand, by analyzing sewage from 71 cities, the investigation team have been able to demonstrate that geographically distributed U.S. populations share a small set of bacteria whose members represent various common community states within U.S. adults. The respective cities were found to be differentiated by their sewage bacterial communities, and the community structures were good predictors of a city’s estimated level of obesity for example. The researchers say their approach demonstrates how sewage can serve as a means and medium for sampling fecal microbiota characteristics from millions of people and demonstrates potential to reveal microbiome patterns associated with human demographics.
The scientists observe that the human fecal microbial community serves as a proxy for the human gut community, which exhibits considerable diversity and variability among individuals, and that human microbiome data sets reveal that most human gut communities share specific functional gene profiles rather than a single core set of microbial species. These functional similarities coupled with taxonomic variability indicate niche overlap among taxa, which in the gut microbiome of healthy individuals reflects taxonomically distinct sets of cooccurring taxa or enterotypes that contain similar functional gene profiles.
The study coauthors note that despite inter-individual taxonomic variability, studies that include multiple samples have identified correlations between functional gene composition and taxonomic composition and relationships between human characteristics and the gut microbial composition. For example, the gut community from an individual is more similar to itself through time than to samples collected from other individuals. Marked shifts in gut microbial communities are also reported for the very young and very old, in healthy versus diseased states, across different diet regimes and in culturally isolated human population. They observe that coherence of the gut microbial community within individuals and among individuals with specific characteristics suggests that gut communities maintain relatively stable equilibrium states. Consequently, they reason that if gut community composition tracks human characteristics, then identifying community members or community states that differ across human population boundaries could lead to an improved understanding of how these communities influence human health.
Sampling individuals has proven an effective approach for identifying gut microbial community patterns associated with human health states, but a large variation among gut microbiomes and the expense of sequencing libraries from many individuals limit the efficacy of microbial community comparisons from human populations over different demographic scales, e.g., city, country, or continent. The coauthors hypothesize that comparison of untreated sewage samples might provide a means to assess the human fecal microbiome, and by proxy the gut microbiome, within and among human populations.
They note that sewage influent accurately reflects a composite human stool bacterial community, and that the 15 most abundant bacterial families, which on average accounted for 98 percent of reads in the human stool data set, represented 26 percent of the sequence reads in a sewage sample. Another finding was that low representation of human fecal bacteria in sewage concurs with previous reports that 80 to 90% of bacterial sequences in sewage originate from non-human-fecal sources. Since sewage contains many organisms of nonfecal origin, the research team’s analysis focused on sequences from bacterial families that each represented fewer than three percent of total reads in the human stool data set: Bacteroidaceae, Ruminococcaceae, Lachnospiraceae, Porphyromonadaceae, Rikenellaceae, and Prevotellaceae. Five of these represented the most abundant of the human fecal matter-associated families in sewage, with the sixth, Rikenellaceae, contributing less than Veillonellaceae. Normalization of the sewage data set to the 15 most abundant families in the human stool data set showed comparable community compositions at the family level, and an overrepresentation of Lachnospiraceae and Prevotellaceae.
Homogeneity In The Fecal Microbial Composition Of Human Populations
The researchers found that sewage samples from different U.S. cities displayed very similar human fecal oligotype compositions within and among sample periods, with the oligotype composition of the pooled stool data set strikingly similar to individual sewage samples. For example, on average, two sewage samples captured 90 percent of measured fecal oligotype diversity in the sewage data set, whereas the same diversity levels required data from 71 individual human stool samples.
Core Fecal Microorganisms In The United States
The scientists found that no oligotype occurred in all human stool samples, with the two most prevalent oligotypes detected in 129/137 stool samples. In contrast, 17 oligotypes were present in all 207 sewage samples and 10 others were present in 205 (99 percent) of sewage samples. These 27 oligotypes also represented the most abundant amplicon sequences in the sewage samples, and the 27 most common and abundant oligotypes represent core gut microbiota among human populations in the United States. In the human stool data set, one of the core oligotypes represented the most abundant oligotype in 117 of the 137 samples.
Drivers Of Sewage Community Differences Among Cities
The researchers report finding human fecal oligotype composition in sewage reflected increased representation of oligotypes from one family group over the others. Of 51 treatment plant sites that had data for all three sample collections, 21 exhibited the same enrichment patterns, a result which exceeds random expectations. Cities with more than one treatment plant exhibited a higher level of consistency for paired sample comparisons between plants, and principal coordinate analyses (PCoAs) did not indicate significant (P 0.01) relationships between the geographic location of treatment plants, the plant chemical/physical measurements, or the population size served and the human fecal oligotype composition.
In contrast to the human fecal oligotypes, the nonfecal oligotype data set exhibited greater variation among cities and sample periods, with strong geographic and seasonal trends corresponding to air temperature and latitude differences. A constrained ordination for the city temperature profile parameter further illustrated that nonfecal oligotype composition was more strongly related to a city’s yearly temperature profile than to the human fecal oligotype composition. This relationship in the nonfecal community appeared as a significant divide between the northern and southern U.S. cities.
Human Demographics Represented In Sewage Microbial Communities
Although no measured factors explained a high percentage of the human fecal community variation among cities, the percent obesity in a city’s population had explanatory power. A random forest classification algorithm demonstrated that human fecal oligotype composition in sewage predicted whether a sample derived from a lean or obese population with 81 to 89 percent accuracy. The researchers say this relationship was driven partly by an increase in the relative abundance of Bacteroidaceae oligotypes in samples from the most obese city populations.
The scientists note that large populations with highly variable phenotypic characters (e.g., human weight and flower color) will include a greater number of variants with more even distributions than small populations. A character variant common among individuals in populations will be abundant in population-level assessments and highly prevalent among populations. However, unlike weight or flower color, where a single variant represents each individual at a given moment in time, microbiomes encompass hundreds to thousands of different kinds of microorganisms or operational taxonomic units (OTUs) that collectively define the character variant of an individual.
In support of this concept, the scientists found that (i) the population-level (sewage) samples recaptured the majority (97 percent) of oligotypes from individual stool samples, (ii) a pooled data set of human stool and sewage samples exhibited highly similar oligotype distribution patterns, (iii) sewage samples had higher richness and diversity than stool samples, and (iv) oligotypes that were more prevalent among individuals were more prevalent and more abundant in sewage. From these observations they infer that sewage influent represents the composite fecal microbiomes of many individuals and provides a metric to assess the relationship of these population-level microbial distributions with large-scale patterns in human demographics.
By sampling sewage, the researchers found that U.S. populations have a much less variable fecal bacterial community composition than that of individuals, and that this community composition convergence among populations suggests a finite level of composition variability — at least among U.S. populations — and that this variability can be overcome with large sample sizes to make meaningful inferences regarding the gut microbiome. From the sewage sampling, they were able to identify a set of core bacteria both common to and abundant among U.S. populations. They say that although no single species dominates the fecal microbial communities among individuals, their results demonstrate consistent differential abundance in human populations for some bacterial taxa over others.
The study coauthors also observe that sewage sampling described distinct community compositions among U.S. populations, and that although not a majority, the level of community consistency signifies that human populations at the citywide scale can have characteristic microbial community compositions, and although they did not identify the ultimate causes of bacterial community composition differences among U.S. cities, their single measure of lifestyle differences for individuals in these cities (obesity percent) explained a significant, albeit small, proportion of the community variation.
These lifestyle differences can reproducibly alter the human gut microbiome, and microbial community composition is a known indicator of obesity with up to 90 percent predictive accuracy for individuals. The researchers observed that the obesity signal in an individual’s gut microbial community composition scaled up, with nearly equivalent predictive capabilities (81 to 89 percent accuracy), to the level of human populations in cities, and that these community composition relationships to the population obesity gradient were driven in large part by increased representation of Bacteroides spp. and decreased representation of Faecalibacterium spp. in more obese populations. They note that Bacteroides spp. have been found to increase in abundance in humans consuming a high-animal-fat diet, and are associated with low-diversity proinflammatory gut communities, while Faecalibacterium spp. are more prevalent in high-diversity anti-inflammatory gut communities.
The scientists deduce that given the relatively minor difference in population obesity percentage (as low as 9 percent) between city populations considered lean and obese, the observed correlations between obesity and the microbial community in sewage might reflect other, more pronounced lifestyle differences in these cities, including the influence of diet on gut microbial communities.
In summary, the research team concludes that after filtering out overprinting sewer-associated taxa, sewage serves as a composite proxy for population-level human fecal microbiota, and that comparative sewage analysis provides a unique opportunity to explore relationships between human fecal communities and lifestyle or demographic differences in human populations.