Acinetobacter, Aeromonas and Trichococcus populations dominate the microbial community within urban sewer infrastructure.

We evaluated the population structure and temporal dynamics of the dominant community members within sewage influent from two wastewater treatment plants (WWTPs) in Milwaukee, WI. We generated > 1.1 M bacterial pyrotag sequences from the V6 hypervariable region of 16S rRNA genes from 38 influent samples and two samples taken upstream in the sanitary sewer system. Only a small fraction of pyrotags from influent samples (∼ 15%) matched sequences from human faecal samples. The faecal components of the sewage samples included enriched pyrotag populations from Lactococcus and Enterobacteriaceae relative to their fractional representation in human faecal samples. In contrast to the large number of distinct pyrotags that represent faecal bacteria such as Lachnospiraceae and Bacteroides, only one or two unique V6 sequences represented Acinetobacter, Aeromonas and Trichococcus, which collectively account for nearly 35% of the total sewage community. Two dominant Acinetobacter V6 pyrotags (designated Acineto tag 1 and Acineto tag 2) fluctuated inversely with a seasonal pattern over a 3-year period, suggesting two distinct Acinetobacter populations respond differently to ecological forcings in the system. A single nucleotide change in the V6 pyrotags accounted for the difference in these populations and corresponded to two phylogenetically distinct clades based on full-length sequences. Analysis of wavelet functions, derived from a mathematical model of temporal fluctuations, demonstrated that other abundant sewer associated populations including Trichococcus and Aeromonas had temporal patterns similar to either Acineto tag 1 or Acineto tag 2. Populations with related temporal fluctuations were found to significantly correlate with the same WWTP variables (5-day BOD, flow, ammonia, total phosphorous and suspended solids). These findings illustrate that small differences in V6 sequences can represent phylogenetically and ecologically distinct taxa. This work provides insight into microbial community composition and dynamics within the defined environment of urban sewer infrastructure.

Water use and acute diarrhoeal illness in children in a United States metropolitan area.

We examined the association between water exposures and acute diarrhoeal illness (ADI) in children under non-outbreak conditions in a major US metropolitan area. We used a nested case-control study of children seen in an urban/suburban emergency department. Cases were those seen for a complaint of diarrhoea, while controls were age-matched children with a non-gastrointestinal complaint. Parents of subjects completed a validated water-use survey. Stratum-specific adjusted odds ratios (aOR) were calculated for the three main water effects: water source [surface vs. ground (well)], drinking-water type (tap vs. bottled), and use of water filters. Of 2472 subjects, 45% drank mostly or only bottled water. Well-water use was associated with increased odds of ADI compared to surface water [aOR 1·38, 95% confidence interval (CI) 1·01-1·87]. Use of bottled water did not affect the odds of ADI in well-water users, but increased the odds of ADI for surface-water users (aOR 1·27, 95% CI 1·02-1·57). We conclude that well-water use and bottled-water use are associated with increased odds of ADI in children.

Reliability of mCP method for identification of Clostridium perfringens from faecal polluted aquatic environments.

AIMS: The purpose of the work was to evaluate the mCP method to correctly identify and enumerate Clostridium perfringens that are present in surface waters impacted by a mixture of faecal pollution sources. METHODS: Clostridium perfringens were enumerated and isolated from sewage influent, surface water and suspended sediments using the mCP method. Molecular characterization of isolates was performed using species-specific PCR, along with full-length sequencing of the 16S rRNA gene for a subset of isolates. RESULTS: The environmental isolates were presumptively identified as C. perfringens based on utilization of sucrose, inability to ferment cellobiose and a positive action for acid phosphatase activity. All isolates (n = 126) were classified as C. perfringens based on positive results with species-specific PCR with a subset confirmed as C. perfringens based on the 16S rRNA gene identity. CONCLUSIONS: The molecular results indicated all of the presumptive positive isolates were C. perfringens regardless of the source, e.g. sewage influent or environmental water samples. Sequencing revealed that C. perfringens obtained from sewage and the aquatic environment were nearly identical (c. 99.5% similarity). SIGNIFICANCE AND IMPACT OF THE STUDY: From this study we conclude that the mCP method is a robust approach to enumerate and isolate C. perfringens from aquatic environments that receive diverse sources of faecal pollution.

Diversity and population structure of sewage-derived microorganisms in wastewater treatment plant influent.

The release of untreated sewage introduces non-indigenous microbial populations of uncertain composition into surface waters. We used massively parallel 454 pyrosequencing of hypervariable regions in rRNA genes to profile microbial communities from eight untreated sewage influent samples of two wastewater treatment plants (WWTPs) in metropolitan Milwaukee. The sewage profiles included a discernible human faecal signature made up of several taxonomic groups including multiple Bifidobacteriaceae, Coriobacteriaceae, Bacteroidaceae, Lachnospiraceae and Ruminococcaceae genera. The faecal signature made up a small fraction of the taxa present in sewage but the relative abundance of these sequence tags mirrored the population structures of human faecal samples. These genera were much more prevalent in the sewage influent than standard indicators species. High-abundance sequences from taxonomic groups within the Beta- and Gammaproteobacteria dominated the sewage samples but occurred at very low levels in faecal and surface water samples, suggesting that these organisms proliferate within the sewer system. Samples from Jones Island (JI--servicing residential plus a combined sewer system) and South Shore (SS--servicing a residential area) WWTPs had very consistent community profiles, with greater similarity between WWTPs on a given collection day than the same plant collected on different days. Rainfall increased influent flows at SS and JI WWTPs, and this corresponded to greater diversity in the community at both plants. Overall, the sewer system appears to be a defined environment with both infiltration of rainwater and stormwater inputs modulating community composition. Microbial sewage communities represent a combination of inputs from human faecal microbes and enrichment of specific microbes from the environment to form a unique population structure.

The potential for beach sand to serve as a reservoir for Escherichia coli and the physical influences on cell die-off.

AIMS: The Escherichia coli burden at a Great Lakes urban beach was evaluated during the summer months to determine if sand served as a reservoir for E. coli, and if there was evidence of cell replication in situ. Field and laboratory studies investigated the effects of moisture, temperature and UV on E. coli densities in the sand. METHODS AND RESULTS: Sand samples (n = 481) were collected across three distinct transects of the beach, the top, a middle streamline, and the berm, over 15 sample days. The highest levels were found in the middle streamline, which was affected by stormwater discharge from nearby outfalls and roosting gulls; daily geometric mean levels of these seven sites ranged from 6700 to 40,900 CFU per 100 g of sand. Escherichia coli levels were greatest in samples with moisture levels between 15% and 19%, and were significantly higher than 0-4 and 20-24% ranges (P < 0.05). Pre- and post-rain samples at the beach demonstrated an increase in E. coli levels nearly 100-fold within 30 min, suggesting sand washout as a major mechanism for loading of E. coli into the beach waters. Rep PCR analysis of 160 isolates obtained from eight sites demonstrated that 21% of the isolates fell into one of the six clonal patterns, suggesting that bacteria may be able to replicate and possibly colonize beach sand. Sand field plots inoculated with E. coli cells containing pGFPuv that expresses GFP (green fluorescent protein) as a marker showed an initial two- to 100-fold increase at 24 h, depending on the temperature condition. The sand appeared to provide considerable protection from UV exposure as no significant difference was seen in cell densities within the first 2-4 cm of sand between exposed and unexposed plots (P < 0.05). CONCLUSIONS: Beach sand may act as a reservoir for E. coli. Replication of cells appears to be one possible contributing factor to the persistently high levels, as indicated by both field studies and laboratory studies, and warrants further investigation. Moisture content of sand may also be a determinant of cell persistence in the sand environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli is used as an indicator organism for faecal pollution at most Great Lakes coastal beaches; therefore, a better understanding of how E. coli might survive, or possibly replicate, in the environment would improve interpretation of beach monitoring results.

Evidence for localized bacterial loading as the cause of chronic beach closings in a freshwater marina.

We conducted a comprehensive regional spatial assessment of bacterial water quality in order to determine the points of entry of fecal pollution into a swimming beach area on Lake Michigan that historically has had numerous water quality advisories for elevated levels of Escherichia coli (E. coli). Intensive, consecutive-day water samples were collected during dry and rainy conditions across multiple shoreline and offshore sites, and E. coli levels were enumerated from these samples. For both dry and rainy days, shoreline sites demonstrated significantly higher E. coli levels than offshore regions. We found that offshore (10-150m from shore) E. coli levels did not exceed 235CFU/100ml in more than 5% of the samples collected for 19 surveys (n=209). In contrast, samples taken at the beach area exceeded 235CFU/100ml in 66% of the samples collected for 43 shoreline surveys (n=675). Locally high E. coli levels coincided with bird presence and stormwater at the swimming beach located within the marina, and were unrelated to E. coli levels in connecting harbor waters. We conclude that beach water quality may be impacted by local, persistent contamination, which may confound routine beach monitoring and prevent the detection of regional pollution from other sources.

Clonal populations of thermotolerant Enterobacteriaceae in recreational water and their potential interference with fecal Escherichia coli counts.

Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for Escherichia coli enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of beta-glucuronidase activity revealed that these clonal isolates were Klebsiella, Citrobacter, and Enterobacter spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be E. coli, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. beta-Glucuronidase activity was critical in distinguishing E. coli from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that E. coli is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.