In this study, we examined the prevalence of human fecal waste in Milwaukee's harbor. Previous attempts to identify fecal pollution sources and assess risk from pollution events have led to the development of human-associated fecal pollution assays, many of which target the order Bacteroidales
, a group of fecal anaerobes (21
). However, the exact specificity and applicability of these assays in varied environments remain unknown (48
); thus, we sought to identify another human fecal indicator that could complement existing genetic markers. Relying upon multiple taxa to create a human-specific indicator signature should improve source specificity and provide more consistent results among environments given likely differences in decay rates for various types of organisms.
The introduction of next-generation sequencing technology has allowed the undertaking of large microbial community sequencing projects (2
). These projects now provide a resource from which we can examine the specificity of tens of thousands of microbial phylotypes to a specific environmental habitat(s). In a previous microbial community study of WWTP influent, McLellan et al. (29
) suggested that the Lachnospiraceae
family would be an ideal bacterial group for fecal source tracking because of its abundance in WWTP influent samples. Closer examination of the microbial community data here revealed that a single phylotype (Lachno2), which is closely related to the genus Blautia
(), was especially abundant in both human fecal samples and Milwaukee sewage samples but was not present in cattle fecal samples, a common fecal pollution source in the harbor (). The identification of database sequences and clone sequences from our own libraries containing Lachno2 further confirmed that it belonged to a phylogenetically narrow group and would therefore be an excellent candidate for a host-associated fecal indicator.
Lachno2 was consistently present, but its relative abundance varied largely from human to human, which was in contrast to the small relative abundance variation among the sewage samples (). Previous large sequencing efforts related to the human microbiome have indicated immense variation in the fecal communities among humans (38
), which has prompted some researchers to suggest that a core fecal signature will be difficult to identify (52
). The sewage influent samples in this study spanned a 3-year period and represented annual, seasonal, and geographic (two WWTP service areas) variation in metropolitan Milwaukee's human population. The smaller variation in the sewage influent samples, which represent a composite of up to 1.1 million human fecal communities, suggests that the identification of core microbes in human fecal content may be identified through examination of WWTP influent samples. Future studies that examine a larger number of human fecal communities in addition to WWTP influent samples from a large geographic range could provide the needed insight to tease apart the core human fecal community.
We developed a qPCR assay for the Lachno2 phylotype and made use of the pyrosequencing data sets to examine in silico
the specificity of this assay as well as previously defined total and human Bacteroidales
qPCR assays. This process revealed that our assay likely targets more taxa than those associated with the unique Lachno2 phylotype, but the range of phylotypes remained phylogenetically narrow (98.9% of sequences targeted were RDP classified as Blautia
), were associated with humans and not our comparison host, cattle (), and were almost exclusively human associated in public 16S rRNA gene databases. Our in silico
analysis also revealed high person-to-person variability in the relative abundance of the total and human Bacteroidales
assays in human fecal samples (). Interestingly, the human Bacteroidales
assay targeted sequences that were a significantly (P
≤ 0.001) larger part of the community in the human fecal samples from the study of Dethlefsen et al. (11
) than from the study of Turnbaugh et al. (52
), while the Lachno2 marker exhibited the inverse of this relationship (P
≤ 0.001) (). The complementary nature of these two marker assays suggests that using them in tandem or as part of a larger profile may provide a more consistent measure of human fecal contamination than using either on its own.
Fecal pollution of urban waterways is a major contributor to waterborne illnesses in the United States and remains a widespread problem for both coastal freshwater and marine ecosystems (3
). While simply detecting fecal pollution provides evidence of health risks, identifying the pollution source is ultimately the information needed for effective remediation efforts to take place that will significantly reduce risk. In Milwaukee, WI, major fecal pollution events in nearshore waters occur each year during combined and sanitary sewer overflows. During these periods, untreated sewage is discharged to Milwaukee's rivers and subsequently the harbor and Lake Michigan, thereby providing us an opportunity to use conventional and alternative fecal indicators to compare known human fecal contamination events with other times in which human fecal pollution should not be present.
In this study, we found that fecal pollution is chronic in Milwaukee's harbor (). This persistent input into the harbor suggests multiple delivery routes outside of reported sewer overflows. In urban environments, it has been demonstrated that human fecal pollution may enter receiving bodies from stormwater runoff/outfall discharge, leaking sanitary pipes, and illicit sanitary sewer connections (40
). Potential routes of unrecognized sewage inputs have been documented in the Milwaukee area (41
), and our identification of significantly increased human fecal indicators following rain events provides further evidence that stormwater may be an important source of human-derived fecal pollution.
As human fecal content is a likely source for human health risks from fecal and/or sewage pollution, we focused on the contribution of human fecal pollution to the “total” fecal pollution in the harbor. Using the ratio of human Bacteroidales
to total Bacteroidales
spp. as a proxy for relative human fecal contribution to total fecal pollution (41
), we observed that CSO periods in the harbor had ratios similar to what was seen for sewage, which suggests that untreated sewage is the main source of fecal pollution during these periods. During the non-CSO periods, although the human Bacteroidales
component of fecal contamination remained high, the ratio dropped to roughly half of what is typically found in sewage, which suggests that both human and nonhuman sources contribute to the chronic fecal pollution in the harbor. Likewise, a comparison of the Lachno2 and enterococcal qPCR assays revealed an abundance ratio shift from a low to high Lachno2/enterococcus ratio when switching from non-CSO to CSO periods (C). As the enterococcal qPCR assay targets human and multiple animal fecal sources, these data further provide evidence of a decreased human fecal contribution to total fecal pollution in the harbor during non-CSO events. Nonhuman fecal pollution in urban environments can occur from multiple primary sources, including birds, domestic pets, and urban wildlife (19
). Further efforts in Milwaukee's urban environment are needed to identify and quantify all of the contributing sources.
Other studies have shown that the human Bacteroidales
assay may detect bacteria from nonhuman mammal fecal material, although this cross-reactivity appears fairly minor (48
). Excluding cattle, it is unknown whether the Lachno2 assay has cross-reactivity for bacteria from nonhuman animal fecal material. However, a tight correlation was observed between the human Bacteroidales
and Lachno2 assays in the harbor (C). It is unlikely that this tight correlation would exist unless the markers had detected the same source microbial community and this source was a major polluter of the harbor. It is also unlikely that these two bacterial indicators, as members of different phyla, have an identical host distribution; thus, we suggest that their correlation in the harbor water is strong evidence that the indicators specifically identify human sewage in our system. Because there may be no single genetic marker that is exclusive to a single source, a community approach (characterizing a suite of markers) may be a very effective alternative (29
). This is especially true in light of the large variability among human fecal communities (52
). The tight correlation of the Lachno2 and human Bacteroidales
assays suggests that these two markers are an excellent starting point for development of this source-specific community approach to human fecal pollution detection in surface waters.
Although human fecal indicators have revealed the presence of human fecal pollution in surface waters, a strong link between these indicators and human pathogen presence has yet to be established (58
). Our examination of adenovirus, a group commonly used as an index for the presence of human viruses in water (31
), showed that a linear abundance relationship is not present between the human indicators and virus abundance in sewage; therefore, we did not expect to observe a direct correlation in harbor water. Viral titers in sewage are known to fluctuate with season (43
). We also observed large fluctuations in adenovirus abundance in sewage across a year, which contrasted the relative stability over time of the bacterial human fecal markers (A). Further, different ecological forcings upon the bacteria and viruses once in the harbor waters may cause different retention times for each group and thus also affect a potential linear relationship. Our results did, however, suggest that it is likely to find human adenovirus in the harbor when the bacterial human fecal indicator abundance is high (B). In fact, a logistic regression model revealed that a 10-fold increase in human indicator abundance in the harbor results in a 154% increase in the odds of observing adenovirus; thus, it may be feasible to use a bacterial human fecal signature to assess pathogen risk. The abundance of these bacterial indicators, which is 2 to 4 orders of magnitude greater then the abundance of human adenovirus in sewage, provides increased sensitivity for detecting human fecal pollution in freshwaters and makes these markers particularly suitable for tracking sewage contamination. These results also add credence to the hypothesis that tracking multiple indicators and/or human pathogens may be required to adequately assess human health risk from human fecal contamination of surface waters.