Molecular characterizations of the gut microbiome from individual human stool samples have identified community patterns that correlate with age, disease, diet, and other human characteristics, but resources for marker gene studies that consider microbiome trends among human populations scale with the number of individuals sampled from each population. As an alternative strategy for sampling populations, we examined whether sewage accurately reflects the microbial community of a mixture of stool samples. We used 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, only 15% of sewage sample sequence reads were attributed to human fecal origin, but sewage recaptured most (97%) human fecal oligotypes. The most common oligotypes in stool matched the most common and abundant in sewage. After informatically separating sequences of human fecal origin, sewage samples exhibited ~3× greater diversity than stool samples. Comparisons among municipal sewage communities revealed the ubiquitous and abundant occurrence of 27 human fecal oligotypes, representing an apparent core set of organisms in U.S. populations. The fecal community variability among U.S. populations was significantly lower than among individuals. It clustered into three primary community structures distinguished by oligotypes from either: Bacteroidaceae, Prevotellaceae, or Lachnospiraceae/Ruminococcaceae. These distribution patterns reflected human population variation and predicted whether samples represented lean or obese populations with 81 to 89% accuracy. Our findings demonstrate that sewage represents the fecal microbial community of human populations and captures population-level traits of the human microbiome.

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modifled DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB+-dependent The locus known as mcrB was one of the first restriction systems to be discovered (33), by virtue of its action on special variants of T-even bacteriophage that incorporate 5-hydroxymethylcytosine (hm`C) into their DNA without further modification (see reference 50 for a review). This locus, formerly known as rglB (or r2,4) (48), was rediscovered because of difficulties encountered in cloning the genes for site-specific modification methylases associated with type II restriction-modification systems (7,26,40,49). In addition to hm5C-DNA, many but not all sequences methylated by site-specific cytosine modification methylases are restricted by the system in vivo, and the consensus recognition sequence 5'GmC was proposed (49). McrB is thus a sequence-specific, modification-requiring restriction system. We show here that the mcrB locus described above actually comprises two genes and that both are required for restriction of most the sequences previously characterized as sensitive. Thus, we will refer to the complete system as the McrBC system.The genes encoding the system are contained within the immigration control region of the Escherichia coli K-12 genome. Three restriction systems are encoded within 14 kilobases (kb) here (48). The well-studied hsdRMS locus (20, 31, 55) encodes the multisubunit type I system EcoK, which recognizes a seven-base sequence and cleaves the target when the sequence is not modified. The other two systems are the flanking loci mcrBC, described above, and mrr, which mediates site-specific restriction of adenine-modified DNA (22). The sequence organization of this region, judged by Southern blot analysis of chromosomal DNA, is highly variable in enteric bacteria (12), both in the hsd genes specifically and in the flanking sequences. Sequence analysis presented here is consistent with recent acquisition of the mcrBC genes by E. coli, possible accounting for some of the observed variability. * Corresponding author.At the molecular level, restriction systems consist of sequence-specific double-stranded endonucleases, usually accompanied by a sequence-specific modification methylase. So far, four classes of endonucleases have been described. The simplest are the type II enzymes, in which the endonuclease and protective methylase activities reside in separate enzymes. These endonucleases typically act as dimers of identical subunits and require only Mg2" for activity (38).One group of type II isoschizomers, typified by DpnI, recognizes a modified site (28), as McrBC appears to do. In contrast, type I and type III enzymes have separate specificity subunits that recognize the DNA site and require ATP in a...

BackgroundPast studies have demonstrated an association between waterborne disease and heavy precipitation, and climate change is predicted to increase the frequency of these types of intense storm events in some parts of the United States. In this study, we examined the linkage between rainfall and sewage contamination of urban waterways and quantified the amount of sewage released from a major urban area under different hydrologic conditions to identify conditions that increase human risk of exposure to sewage.Methods and findingsRain events and low-flow periods were intensively sampled to quantify loads of sewage based on two genetic markers for human-associated indicator bacteria (human Bacteroides and Lachnospiraceae). Samples were collected at a Lake Michigan estuary and at three river locations immediately upstream. Concentrations of indicators were analyzed using quantitative polymerase chain reaction (qPCR), and loads were calculated from streamflow data collected at each location. Human-associated indicators were found during periods of low flow, and loads increased one to two orders of magnitude during rain events from stormwater discharges contaminated with sewage. Combined sewer overflow (CSO) events increased concentrations and loads of human-associated indicators an order of magnitude greater than heavy rainfall events without CSO influence. Human-associated indicator yields (load per km2 of land per day) were related to the degree of urbanization in each watershed. Contamination in surface waters were at levels above the acceptable risk for recreational use. Further, evidence of sewage exfiltration from pipes threatens drinking water distribution systems and source water. While this study clearly demonstrates widespread sewage contamination released from urban areas, a limitation of this study is understanding human exposure and illness rates, which are dependent on multiple factors, and gaps in our knowledge of the ultimate health outcomes.ConclusionsWith the prediction of more intense rain events in certain regions due to climate change, sewer overflows and contamination from failing sewer infrastructure may increase, resulting in increases in waterborne pathogen burdens in waterways. These findings quantify hazards in exposure pathways from rain events and illustrate the additional stress that climate change may have on urban water systems. This information could be used to prioritize efforts to invest in failing sewer infrastructure and create appropriate goals to address the health concerns posed by sewage contamination from urban areas.

No abstract

Fecal contamination from sewage and agricultural runoff is a pervasive problem in Great Lakes watersheds. Most work examining fecal pollution loads relies on discrete samples of fecal indicators and modeling land use. In this study, we made empirical measurements of human and ruminant-associated fecal indicator bacteria and combined these with hydrological measurements in eight watersheds ranging from predominantly forested to highly urbanized. Flow composited river samples were collected over low-flow ( n = 89) and rainfall or snowmelt runoff events ( n = 130). Approximately 90% of samples had evidence of human fecal pollution, with highest loads from urban watersheds. Ruminant indicators were found in ∼60-100% of runoff-event samples in agricultural watersheds, with concentrations and loads related to cattle density. Rain depth, season, agricultural tile drainage, and human or cattle density explained variability in daily flux of human or ruminant indicators. Mapping host-associated indicator loads to watershed discharge points sheds light on the type, level, and possible health risk from fecal pollution entering the Great Lakes and can inform total maximum daily load implementation and other management practices to target specific fecal pollution sources.