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Beach Sand and Sediments are Temporal Sinks and Sources of Escherichia coli in Lake Superior S A T O S H I I S H I I , †,‡ D E N N I S L . H A N S E N , § RANDALL E. HICKS,§ AND M I C H A E L J . S A D O W S K Y * ,†,‡,| Department of Soil, Water, and Climate, Center for Microbial and Plant Genomics, and BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108, and Department of Biology, University of Minnesota-Duluth, Duluth, Minnesota 55812
The Duluth Boat Club (DBC) Beach, located in the Duluth-Superior harbor of Lake Superior, is frequently closed in summer due to high counts of Escherichia coli, an indicator of fecal contamination. However, the sources of bacteria contributing to beach closure are currently unknown. In this study, we investigated the potential sources of E. coli contaminating the DBC beach by using modified rep-PCR (HFERP) DNA fingerprinting. Over 3600 E. coli strains were obtained from 55 lake water, 25 sediment, and 135 sand samples taken from five transects at the DBC beach at 11 different times during the summer through fall months of 2004 and 2005. Potential sources of E. coli at this beach were determined by using a known-source DNA fingerprint library containing unique E. coli isolates from wildlife, waterfowl, and treated wastewater obtained near Duluth, MN. Amounts E. coli in the samples were enumerated by membrane filtration counting, and the presence of potentially pathogenic E. coli was determined by using multiplex PCR. E. coli counts in all samples increased during the summer and early fall (July to September). While E. coli in spring samples originated mainly from treated wastewater effluent, the percentage of E. coli from waterfowl increased from summer to fall. DNA fingerprint analyses indicated that some E. coli strains may be naturalized, and autochthonous members of the microbial community in the beach sand and sediments were examined. However, multiplex PCR results indicated that 71% of the time, a relatively high rate of correct classification as compared to the previous reports (14, 19). On average, 32% of the E. coli strains was identified to the source based on ID bootstrap analysis at P ) 0.90 (Figure S2, Supporting Information). The sources of the rest of the strains could not be identified at this probability level, even when using a more comprehensive Minnesota DNA fingerprint library (data not shown). The sources of the identified strains at the DBC beach changed monthly (Figure 2B1,B2). During the spring (April and May 2005), a relatively large number of E. coli strains was identified as originating from humans, while the contribution of E. coli from waterfowl increased from the summer to fall (June to October 2004 and 2005). When the percentage of E. coli identified as originating from humans was low in June 2005, E. coli strains originating from wildlife were higher than earlier in the spring. These observations were similar among many samples obtained in the 2-year study period but were most clearly shown in water, shoreline sand, and nearshore sand samples from 2005. There was no trend in source distribution of E. coli in upshore and far upshore sand samples in 2004 and 2005. Naturalized Populations. Diverse E. coli genotypes were revealed by analysis of the 3633 HFERP DNA fingerprints
from the beach isolates (data not presented), with relative similarity values ranging from 3.4 to 99.9%. On the basis of the criteria proposed by Ishii et al. (6), 106 naturalized E. coli strains were identified in water, sediment, and sand samples (see Materials and Methods). Most of the naturalized E. coli strains appeared in July through September 2005 (Figure S2, Supporting Information). The highest proportion (22%) of naturalized E. coli strains was seen in the August 2005 water samples, suggesting that these strains may contribute to high counts of E. coli in water. The naturalized E. coli were genetically distinct from all other strains obtained in these studies and had nearly identical genotypes with similarity coefficients g92% (Figure S3, Supporting Information). Pathogenic E. coli. Out of the 3557 E. coli strains examined, 3.3% (117 strains) were positive for hemolysin production, and only one of these (0.85%) was positive for the intimin gene, eaeA. None of the tested E. coli strains carried the enterohemolysin gene ehxA, indicating that only R-hemolysins were produced, and no strains contained PCR products consistent with Shiga-like toxin genes (stx1 or stx2). Thus, only one out of the 3557 strains could be classified as a potential human pathogen, an enteropathogenic E. coli (EPEC) (4).
Discussion Seasonal Change in the Concentration of E. coli. In this study, the population densities of E. coli in sand and sediment samples were expressed as CFU/g of oven-dried samples, as these values are not influenced by moisture content, whereas E. coli concentrations in water were expressed as CFU/100 mL of water (1). Moisture content was significantly greater in shoreline samples and the lowest in upshore sand samples (Figure S4, Supporting Information). When E. coli concentrations in sand and sediment samples were converted to CFU per interstitial water, the greatest numbers of E. coli were observed in nearshore and upshore sands, followed by shoreline sands and sediment (data not shown). These numbers were, on average, 63, 74, 1087, and 4982 times greater in sediment, shoreline, nearshore, and upshore sand samples, respectively, than the concentration of E. coli in lake water expressed as CFU/mL. It should be noted, however, that while the U.S. EPA assumes that CFU/cm3 of sand is roughly equivalent to CFU/mL of water, this relationship is more complicated than presumed (9), and CFU per unit of interstitial water is most likely a more accurate way to compare E. coli counts in sand and water. However, since sediment and sand moisture content can rapidly change, care should be taken when applying this expression. In this paper, we expressed counts as CFU/g of oven-dried sample since precipitation 24-48 h before sampling varied among sampling dates (data not shown but available at http:// climate.umn.edu/). The concentration of E. coli in sand at the DBC beach was very low, or below our detection limit (5 m from the shoreline) was also reported by Byappanahalli et al. (20). In contrast, E. coli counts in shoreline and nearshore sand samples were evenly distributed, suggesting that that wave action may have homogenized E. coli populations in these areas. Similarly, E. coli counts did not significantly vary according to the distance from shoreline in the May 2005 samples. While Whitman and Nevers (9) reported that E. coli concentrations decreased as the distance from the shoreline increased, they examined much longer distances than were used in our study. Diversity of E. coli Isolates from DBC Beach. HFERP DNA fingerprint analyses indicated that the E. coli isolated from the DBC beach were genotypically diverse. Byappanahalli and co-workers (20) also reported that E. coli ribotypes were diverse on the beaches they examined. Taken together, these results suggest that there may be limited selection for specific genotypes that are adapted to beach sand or continuous inputs of E. coli from several sources to these environments. Potential Sources. ID bootstrap analyses indicated that sources for E. coli isolates at the DBC beach changed seasonally and that the major contributors of E. coli on this beach were waterfowl and humans. In this study, the sources of only 32% of the isolates could be identified based on ID bootstrap analysis at P ) 0.90. The percentage of E. coli strains to which a source could be assigned was generally higher in the 2005 than in the 2004 samples (Figure S2, Supporting Information). This was likely due to the larger number of E. coli strains isolated in 2005 than in 2004. While there were some differences in source distribution between 2004 and 2005 sample dates, waterfowl appeared to be the major contributors of E. coli in summer months at this beach. In the spring of 2005, prior to waterfowl activity in the harbor area, the relative contribution of E. coli from treated wastewater to water, shoreline sand, and nearshore sand was greater than other source groups, suggesting that humans were the main contributors to beach contamination. McLellan (19) also reported that the majority of beach isolates they examined most likely originated from stormwater, and a lesser percentage was from gulls. Harwood and co-workers (5) reported that the contribution of human-derived E. coli was greater during spring and was maintained at a fairly constant level during the rest of the year. The lower percentage of E. coli identified as originating from humans in June, rather
TABLE 1. Assignment of E. coli Isolates to Source Groups in Duluth DNA Fingerprint Library by Jackknife Analysis percentage of E. coli isolates in assigned groupsa isolate group wildlife waterfowl humansb
wildlife
waterfowl
humansb
71.0a
3.0 80.1a 17.0
1.2 21.8 77.1a
19.4 9.7
a Values in bold indicate the percentages of E. coli isolates correctly assigned to source groups. b Treated wasterwater obtained from WLSSD was assumed to contain E. coli from human sources.
than in the spring, might be due to chlorination activity at the WLSSD treatment plant, which reduced effluent E. coli counts by 10-100-fold as compared to periods when no chlorine was used. Chlorination was performed intensively from late May to early June in 2005 (Tim Tuominen, WLSSD, personal communication). The relative contribution of E. coli from wildlife (mainly deer) to sand, sediment, and water was also greater in June than other months in 2005. This increase might be due to deer-derived fecal runoff by rain since precipitation was at its greatest in June in 2005 (data not shown but available at http://climate.umn.edu/). The contribution of waterfowl to E. coli loading at this beach increased from summer to fall in both years, probably due to the increased presence of terns, gulls, and geese in the Duluth area during these months. Canada geese arrive in the Duluth area in late April to May from southern states, and their breeding activities are at their peak in June and July. The greatest number (75) of Canada geese appeared on the beach in early July (Heidi Bauman and Melissa Rauner, MPCA, personal communication). We also often observed that geese rested and sometimes left fecal droppings on the beach during these months, most likely influencing E. coli levels at the beach (Figure S5, Supporting Information). There was no apparent seasonal trend in source distribution observed in the upshore and far upshore sand samples. This suggests that direct fecal deposition (e.g., from wildlife and waterfowl), rather than transportation by wave action, may contribute to E. coli counts at these positions. This hypothesis is also supported by patchy distribution of E. coli in upshore and far upshore sand discussed previously. Indigenous E. coli Populations. Naturalized E. coli strains (106) were isolated from multiple samples over several months during 2 years of this study, indicating that some E. coli strains were able to survive over winter in northern Minnesota. A similar observation was reported for E. coli obtained from temperate riverine soils (6). However, as previously reported for soils (6), our inability to identify the potential original sources for these naturalized E. coli strains might be due to an insufficient number or diversity of E. coli in our known-source DNA fingerprint libraries. While beach sand was not originally thought of as a habitat for E. coli, several studies have shown that E. coli can grow in sand (9, 22) and in other secondary habitats (6, 24, 25, 29). Potential Hazards to Humans. Our results indicated that 0.85% of the strains isolated from water, sand, and sediment at this beach site were potentially pathogenic. Similarly, Lauber et al. (34) reported that only 0.84% of the E. coli strains (four out of 472) they examined from a Lake Erie beach had the eaeA gene, and Boczek et al. (35) found only two eaeA positive E. coli out of 218 enterohemolysin-producing (hlyA positive) strains (0.92%) isolated from the effluent wastewater from treatment plants. In agreement with our results, the stx1 and stx2 genes were not detected in their isolates (34, 35). Interestingly, we also found that only three of 142 E. coli strains from Canada geese harbored the eaeA gene (Ishii, S., Meyer, K. P., and Sadowsky, M. J., unpublished data), supporting our current findings that the majority of waterVOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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fowl-derived E. coli are most likely not pathogenic. The low frequency of pathogenic E. coli detected at this beach site may be due to the method we used in this study. Our results also are in agreement with others that enterohemolysin production on the blood agar may not be a not reliable screen for STEC (35). More robust approaches may be needed to detect and quantify the limited number of pathogenic E. coli in the environment. Our results confirm that waterfowl in addition to humans can be a significant source of fecal indicator bacteria like E. coli at Great Lakes beaches. Although waterfowl have been reported to carry a limited number of pathogenic E. coli (36), which was also found our study, they may harbor other potential pathogens such as Salmonella and Campylobacter (37). The potential health risks associated with waterfowlborne bacteria found at beaches needs to be investigated in the future.
Acknowledgments We thank John Ferguson, Youngkwang Kim, Sarah Smith, Marisa Stanley, John Tomaszewski, and Ann Yang for technical assistance. We also thank Joe Stepun, Tim Tuominen, Mike Guite, Al Parrella, and Joe Mayasich at the WLSSD and Heidi Bauman and Melissa Rauner at the MPCA for help obtaining samples, for providing data, and for helpful discussions. This work was supported, in part, from grants from the Minnesota Sea Grant College Program, supported by the NOAA Office of Sea Grant, United States Department of Commerce, under Grant NA03-OAR4170048, the Western Lake Superior Sanitary District (R.E.H. and M.J.S.), and the University of Minnesota Agricultural Experiment Station (M.J.S.). The U.S. Government is authorized to reproduce and distribute reprints for government purposes, not withstanding any copyright notation that may appear hereon. This paper is journal reprint 533 of the Minnesota Sea Grant College Program.
Supporting Information Available Five figures of the concentration of E. coli at each transect, the moisture content in DBC beach sand samples, the percent of E. coli identified by ID bootstrap analysis, a dendrogram of HFREP DNA fingerprints, and a representation of how various sources contribute E. coli. This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review September 27, 2006. Revised manuscript received January 19, 2007. Accepted January 29, 2007. ES0623156
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