Journal of Great Lakes Research 38 (2012) 129–133

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Journal of Great Lakes Research

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Escherichia coli toxin and attachment genes in sand at Great Lakesrecreational beaches

Leah Bauer, Elizabeth Alm ⁎

Institute for Great Lakes Research, Department of Biology, Central Michigan University, Mount Pleasant, MI, 48859, USA

⁎ Corresponding author at: Microbiology, 157 Brooks HUSA. Tel.: +1 989 774 2503.

E-mail address: alm1ew@cmich.edu (E. Alm).

0380-1330/$ – see front matter © 2011 International Adoi:10.1016/j.jglr.2011.10.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 July 2011Accepted 13 October 2011Available online 1 November 2011

Communicated by Joseph Makarewicz

Keywords:Escherichia coliFecal indicator bacteriaBeach sandeaestxbfp

The United States Environmental Protection Agency recommends density thresholds for the fecal indicatororganism Escherichia coli in order to ensure the safety of recreational waters. A number of studies publishedover the past ten years indicate that E. coli is encountered frequently in sand at recreational beaches. While amajority of the sand-associated E. coli may be commensal or environmental strains, the potential for patho-genic strains of E. coli to be present exists. Therefore, the aim of this study was to assess the presence ofattachment and virulence genes associated with enteropathogenic and enterohemorrhagic strains of E. coli(EPEC and EHEC) in populations of E. coli recovered from swash zone sand from seven recreational beachesalong Lake Huron and Lake St. Clair in eastern Michigan, USA. Genes coding for attachment proteins in EPECand EHEC were very prevalent in sand E. coli, but genes coding for toxin genes were uncommon. The paucityof genes associated with E. coli toxins suggests that the EPEC and EHEC pathotypes are not common in sand;however, the high prevalence of genes associated with attachment in E. coli pathotypes suggests that thesegenes are being retained within the beach sand E. coli population.

© 2011 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Introduction

Enteropathogenic E. coli (EPEC) are among the most commoncauses of infantile diarrhea in developing countries, and enterohemor-rhagic E. coli (EHEC) have been implicated in outbreaks of food andwater-borne illnesses that have resulted in cases of hemorrhagic coli-tis and hemolytic uremic syndrome. The pathogenic nature of theseorganisms is characterized by virulence genes that confer the abilityto attach and to colonize a host, and in the case of EHEC, secrete toxins.The attachment genes, eae and bfp, encode intimin and bundle-forming pilus, respectively, and both are employed by EPEC, whileintimin alone is associated with EHEC (Donnenberg and Whittam,2001). EHEC pathogenicity is also characterized by the ability to secreteshiga toxins 1 and 2,which are coded by the stx1 and stx2 genes, respec-tively (O'Brien et al., 1984).

Intimin is an adhesion protein that allows pathogenic E. coli to bindto the intestinal epithelium of the host via a self-translocated inti-min receptor, tir (Donnenberg and Whittam, 2001). Attachmentvia intimin is characterized by the formation of attaching and effac-ing lesions and actin pedestal formation. Bundle-forming pilus isthought to aid in attachment of EPEC both to host intestinal cellsas well as to other E. coli, forming microcolonies (Cleary et al., 2004).Both eae and bfp are located on mobile genetic elements: eae on thelocus of enterocyte effacement (LEE) pathogenicity island and bfp on

all, Mount Pleasant, MI 48859,

ssociation for Great Lakes Research.

the EPEC adherence factor (EAF) plasmid. The shiga toxin genesencode two closely related shiga toxins that are similar to toxinssecreted by Shigella. These genes are carried by lysogenic bacterio-phages (Donnenberg and Whittam, 2001; O'Brien et al., 1984).

The possible presence of such genes in microorganisms living inenvironments secondary to the host intestinal system may poseimmediate risks to human health. During 2005–2006, a total of 78water-borne disease outbreaks affected 4,412 persons in the UnitedStates, and the most common etiologic agents of gastrointestinaloutbreaks were Shigella and E. coli O157:H7 (Yoder et al., 2008).Shigella carries the stx1 gene, and E. coli O157:H7 is an EHEC thattypically carries the eae, stx1, and stx2 genes. Previous studies haveidentified associations between contact with beach sand and entericillness (Bonilla et al., 2007; Heaney et al., 2009), and in 2003 our labrecovered an E. coli 0157:H7 isolate from sand at a beach along LakeHuron in Michigan, USA (unpublished).

E. coli has been shown to survive and persist at high densities inbeach sand at freshwater recreational beaches (Alm et al., 2003, 2006;Francy and Gifford, 2002; Haack et al., 2003; Whitman and Nevers,2003). In fact, it has been shown that E. coli can survive inmany second-ary environments (Byappanahalli et al., 2003; Carrillo et al., 1985; Ishiiet al., 2006; Whitman et al., 2005). Survival and persistence in the sec-ondary environment could provide an opportunity for horizontal genetransfer (HGT) of virulence and attachment genes between microor-ganisms outside of the mammalian host.

Despite the fact that many of the E. coli present in the beach sandenvironment are likely part of a naturalized population or transientcommensals, it is possible that organisms possessing these virulence

Published by Elsevier B.V. All rights reserved.

130 L. Bauer, E. Alm / Journal of Great Lakes Research 38 (2012) 129–133

genes are present. The 2011 outbreak of hemolytic uremic syndromeassociated with E. coli O104:H4 illustrates that E. coli acquire newgenetic material readily (Scheutz et al., 2011), and the potentialfor HGT events between E. coli possessing virulence genes and natu-ralized or transient E. coli may pose additional health risks. Thisstudy was designed to assess the presence of genes associatedwith pathogenic E. coli in beach sand at freshwater recreational bea-ches on Lake St. Clair and Lake Huron in eastern Michigan, USA.

Materials and methods

Field collection

During June, July, and August of 2005 and 2006, sand cores (20 cmdeep) were aseptically collected from the swash zone at seven recre-ational beaches on Lake Huron and Lake St. Clair in eastern Michigan,USA (Fig. 1). These cores were subsequently divided into four sections(5 cm thick) that were placed in individual Whirl-Pak bags andlabeled according to section depth (1–5 cm, 6–10 cm, 11–15 cm,and 16–20 cm). Samples were transported back to the lab on iceand stored at 4 °C until processing.

Sample processing

Sand filtration was performed for each depth separately to avoidcross-contamination. Five grams of sand was transferred to a sterile,100 mL Erlenmeyer flask. Following the addition of 100 mL of deio-nized water, the sand and water were swirled vigorously in theflask for approximately 1 min. The sand slurry was then poured intoa vacuum filtration tower lined with a Millipore® membrane (Milli-pore, Massachusetts, USA). This wash process was repeated a totalof three times, pooling washes on the membrane filter. Using sterile

LakeHuron

SaginawBay

12

3

4

1. Tawas Point State Park2. Tawas City Park3. Bay City State Recreation Area4. Conger-Lighthouse Beach5. Metro Beach Metropark6. Memorial Park7. Blossom Heath Beach

Legend

Fig. 1. Map of beach sand

forceps, the membrane was transferred to a Petri dish containingModified membrane Thermotolerant E. coli (mTEC) Agar (Difco™,Becton, Dickinson Co., New Jersey, USA), incubated for 2 h at 35±2 °C,and then transferred to a 44.5±0.5 °Cwater bath to incubate overnight.After incubation, DNA was extracted from the entire filter membraneusing BIO 101© Systems FastDNA® SPIN® Kit for Soil (BIO 101, Inc.,California, USA) according to themanufacturer's protocol. The extractedDNA was used as the template in PCR analysis.

PCR analysis

Four-gene multiplex PCR amplification was performed to detectthe presence of three pathogen genes eae, stx1, stx2, and mdh, thegene for malate dehydrogenase, an internal positive control for E. colitemplate DNA quality. A separate single PCR reaction was used fordetection of bfp. The four-gene multiplex reaction was performedaccording to a protocol adapted from Lacher et al. (2004). A total ofeight primers were used; primer sequences for each gene are shownin Table 1.

A master-mix was prepared using the following ingredients:nuclease-free H2O, 10× buffer (1× final concentration; Buffer B,Thermo Fisher Scientific, Massachusetts, USA), MgCl2 (25 mM), dNTPmix (2 mM each), mdh-F41, mdh-R875, stx1A-F122, stx1A-R813 (10 μMeach), and eae-F626 eae-R1166, stx2A-F481, and stx2A-R864 (5 μMeach). Approximately 50 ng/μL of template DNA was added to each50 μL reaction. Following a hot start at 94 °C, 1 μL of Taq polymerase(diluted 1:1 with nuclease-free H2O) was added, and the thermal cy-cling profile was run as follows: 1 cycle, 10 min at 94 °C; 40 cycles of20 s at 94 °C, 10 s at 52 °C, and 45 s at 72 °C; 1 cycle, 5 min at 72 °C(Bio-Rad iCycler, Bio-Rad Laboratories, Inc., California, USA).

The separate PCR amplification of bfp was also a 50 μL reaction(Vidal et al., 2004). Master-mix components included nuclease-free

N

40 km

Lake St. Clair

5

6

7

sampling locations.

Table 1Primers used in the multiplex PCR assay to detect mdh, eae, stx1, and stx2, and the cor-responding amplicon sizes.

Targetgene

Primer Primer sequence Amplicon(bp)

mdh mdh-F41 5′-AGG CGC TTG CAC TAC TGT TA-3′mdh-R875 5′-AGC GCG TTC TGT TCA AAT G-3′ 835

eae eae-F626 5′-ATT ATG GAA CGG CAG AGG TTA AT-3′eae-R1166 5′-ATC CCC ATC GTC ACC AGA GG-3′ 541

stx1 stx1A-F122 5′-CAT TCG CTC TGC AAT AGG TA-3′stx1A-R813 5′-AAC TCG CGA TGC ATG ATG A-3′ 691

stx2 stx2A-F481 5′-TAT CTG GCG TTA ATG GAG TT-3′stx2A-R864 5′-CCT GTC GCC AST TAT CTG AC-3′ 384

131L. Bauer, E. Alm / Journal of Great Lakes Research 38 (2012) 129–133

H2O, 10× buffer with MgCl2 (1× final concentration; Buffer A, ThermoFisher Scientific), dNTP mix (2 mM each dNTP), and two primers(20 μM each). Primer sequences for bfp were: bfp-F-23, 5′-GGA AGTCCA ATT CAT GGG GGT AT-3′, and bfp-R-23, 5′-GGA ATC AGA CGCAGA CTG GTA GT-3′, producing a 300 bp amplicon. Approximately50 ng/μL of template DNA was added followed by 1 μL Taq polymerase(diluted 1:1 with nuclease-free H2O) after a hot start at 94 °C. The ther-mal cycling profile comprised 35 cycles, each cycle consisting of 1.5 minat 94 °C, 1.5 min at 64 °C, and 1.5 min at 72 °C.

The positive control for the four-gene multiplex PCR was a wholecell lysate of an E. coli isolate collected from water from CongerLighthouse Beach, Lake Huron, in 2003 that had been previouslyidentified as an E. coli O157:H7 and positive for all four genes ofinterest. The positive control for the bfp PCR was a whole cell lysateof EPEC1 Isolate E2348/69, obtained from the STEC collection ofThomas S. Whittam at Michigan State University. Two negative con-trols were included for each PCR: a whole cell lysate of E. coli ATCC25922 (negative for all pathogen genes, but positive for mdh) and ano DNA, nuclease-free H2O control.

Five microliters of PCR product was combined with 5 μL 2× DNAsample loading buffer and loaded onto a 1.5% agarose gel containingethidium bromide. A 100 bp marker (Promega) was also loaded ontothe gel, and the gels were electrophoresed at 90 V for 1 h. Gels werevisualized using an Eagle EyeUVTransilluminator (Stratagene, California,USA).

Statistical analysis

The proportion of positives for each gene in addition to the pro-portion of samples having both eae and bfp was compared betweenLake Huron and Lake St. Clair beaches using Fisher's exact test of pro-portions. NCSS 2007 statistical software (NCSS LLC, Utah, USA) wasused for the analysis, and p-valuesb0.05 were considered significant.

Table 2Summary of E. coli attachment and toxin genes detected by PCR in swash zone sand from s

Beach Lake No. of samples analyzed

Tawas City Park Huron 20Tawas Point State Park Huron 20Bay City State Recreation Area Saginaw Bay Huron 2Conger-Lighthouse Beach Huron 20Lake Huron totals 62Metro Beach Metropark St. Clair 32Memorial Park St. Clair 16Blossom Heath St. Clair 11Lake St. Clair totals 59Totals 121

a mdh was included as an internal positive control for E. coli template DNA quality.b Percent positive of total number analyzed.c Significantly different by Fisher's exact test of proportions (pb0.05).d Significantly different by Fisher's exact test of proportions (pb0.05).

Results

DNA extracted from a total of 121 incubated modified mTEC filterswere analyzed by PCR; 62 of the samples were collected at the fourLake Huron beaches and 59 of the samples were collected at threeLake St. Clair beaches. Table 2 summarizes the genes detected ateach of the seven beaches. The genes coding for the attachment pro-teins of E. coli pathotypes were quite prevalent; eae was detected in78% of total membrane DNA samples, while 34% were positive forbfp. When comparing Lake Huron beaches to Lake St. Clair beaches,beaches along Lake St. Clair had higher percentages of samples posi-tive for bfp (46% vs. 23%, p=0.008) in addition to the highest percent-age of samples in which both genes were detected (37% vs. 19%,p=0.042). The eae gene was equally prevalent at the sampled bea-ches along Lake Huron and Lake St. Clair (81% vs. 74%, p=0.388).Both genes were detected in incubated modified mTEC filters recov-ered from all sand depths (1–5 cm, 6–10 cm, 11–15 cm, 16–20 cm).The gene eae was detected at all beaches, while bfp was detected insamples from six of the seven beaches, although bfp detection variedby sampling date.

The toxin gene stx1 was not detected in any of the samples, andstx2 was found in only two (1.7%) samples. The stx2-positive sampleswere collected from the same Lake Huron beach on the same sam-pling date from the 1 to 5 cm and 6 to 10 cm depths.

Discussion

The low prevalence of stx2 and the absence of stx1 in our samplesare consistent with the findings of several other studies. Analysis ofE. coli isolated from Great Lakes sand, water, and green algal mats,and marine coastal sediments by membrane filtration followed byincubation at 44.5 °C yielded negative results with respect to detec-tion of stx1 and stx2 (Hamelin et al., 2007; Ishii et al., 2007; Lauberet al., 2003; Luna et al., 2010). In contrast, using similar incubationconditions, Duris et al. (2009) detected stx1 in 11.9% and stx2 in53.7% of inland water samples.

The ubiquity of eae in our study was consistent with the 95% eae-positivity reported for samples from river watersheds in southwesternand southeastern Michigan and northern Indiana (Duris et al., 2009).However, two of the threewatersheds sampled in that study are locatedin areas where land-use is primarily agricultural, and the high preva-lence of eae may have been due, in part, to agricultural runoff. Reasonsfor the high frequency of eae detected in our study are unclear, as sim-ilar studies of water, sand, and sediment from the Great Lakes and othercoastal areas reported much lower eae positivity.

Ishii et al. (2007) detected eae in only 0.85% of sand and water sam-ples froma Lake Superior beach inMinnesota, while Lauber et al. (2003)

even Michigan beaches.

mdha eae stx1 stx2 bfp eae+bfp

20 (100%)b 17 (85%) 0 2 (10%) 4 (20%) 4 (20%)20 (100%) 14 (70%) 0 0 4 (20%) 2 (10%)2 (100%) 2 (100%) 0 0 0 020 (100%) 13 (65%) 0 0 6 (30%) 6 (30%)62 (100%) 46 (74%) 0 2 (3%) 14 (23%)c 12 (19%)d

32 (100%) 28 (88%) 0 0 14 (44%) 10 (31%)16 (100%) 11 (69%) 0 0 8 (50%) 8 (50%)11 (100%) 9 (82%) 0 0 5 (45%) 4 (36%)59 (100%) 48 (81%) 0 0 27(46%)c 22 (37%)d

121 (100%) 94 (78%) 0 2 (2%) 41 (34%) 34 (28%)

132 L. Bauer, E. Alm / Journal of Great Lakes Research 38 (2012) 129–133

reported 0.84% of their samples from a Lake Erie beach were eae-positive. Only a single E. coli strain of 308 total strains isolated fromLake Ontario waterwas reported to be positive for a set of genes includ-ing eae (Hamelin et al., 2006), and the highest percentage of eae-positive isolates reported for water from the Lake St. Clair and DetroitRiver areawas4% (Hamelin et al., 2007). Furthermore, a study ofmarinesediments sampled from the Adriatic Sea found eae in only 9.5% of sam-ples (Luna et al., 2010).

The assessment of the presence of virulence gene sets characteris-tic of E. coli pathogens in water from Lake Ontario also included bfp,which was not detected (Hamelin et al., 2006). To our knowledge,our study is the first to evaluate the presence of bfp in sand, and wedetected a high frequency of the gene. Given that possession of theEAF plasmid is considered typical of EPEC of human origin (Trabulsiet al., 2002) and the presence of bfp is thought to be one of the hall-marks of human pathogenic strains (Ishii et al., 2007), the detectionof bfp indicates the potential that E. coli of human origin had beenpresent in the beach sand environment (Kobayashi et al., 2002,2003; Souza et al., 2002).

The low frequency of eae reported in previous studies compared toour results may be due to differing sampling methods and detectiontechniques. Some of these studies sampled only water, while someincluded swash zone, foreshore, and backshore sand in addition towater, making it difficult to directly compare the results to those ofour study, which examined swash zone sand only. Luna et al. (2010)collected offshore sediments from the Adriatic Sea, and differences inthe marine versus freshwater environments may have contributed tothe much lower frequency of eae detection compared to our study. Itis possible that the attachment proteins coded by eae and bfp aremore important for persistence in the swash zone habitat of the beach.The high prevalence of these genes in E. coli isolated from the swashzone, relative to habitats sampled in other studies, may indicate a sub-population of E. coli particularly suited for this habitat.

All of the aforementioned studies, with the exception of thoseby Duris et al. (2009) and Luna et al. (2010), used a detection meth-odology other than PCR. Hamelin et al. (2006, 2007) employed DNAmicroarrays in both of their studies, Ishii et al. (2007) used DNAfingerprinting, and Lauber et al. (2003) performed DNA–DNA hybridi-zation followed by PCR confirmation of positives. Duris et al. (2009) alsoutilized a multiplex PCR analysis but with primers targeting differentsequences than those included in our multiplex assay, and the eaeprimer sequences used by Luna et al. (2010) also differed fromours. The fact that the PCR technique of Duris et al. (2009) was sim-ilar to ours and yielded similar results suggests that perhaps differ-ences in detection techniques are responsible for the discrepancyin findings between our study and previous work. However, thehigher prevalence of stx1 and stx2 reported by Duris et al. (2009)suggests otherwise.

Although the prevalence of stx2was low and stx1was not detectedat all in our study, these genes may have been present in levels thatevaded detection by our PCR method. Duris and coworkers (2009)reported a theoretical detection limit of one cell per 100 mL basedon deductions from previous studies reporting detection limits deter-mined experimentally. As we utilized a PCR protocol previously opti-mized for environmental samples, we did not perform a formal analysisof our detection limit, and thus a direct comparison with their theoret-ical limit is not possible. However, our culture methods were similar,and using the same line of reasoning, we could suggest the same theo-retical detection limit for our analysis. Detection limit is related toseveral variables including the volume of water that is filtered, theefficiency of target recovery, and the presence of PCR inhibitors(Loge et al., 2002), and extraction of bacteria from sand may presenta variety of factors not yet characterized that influence PCR detec-tion. To our knowledge, no study has assessed detection limits formolecular assays using samples isolated from sand. Future studiesare necessary to identify the detection limits of assays for samples

isolated from sand and to compare these values with those alreadydetermined for environmental water samples.

The EHEC are generally not thermotolerant and the EHEC strainE. coli O157:H7 grows poorly if at all at temperatures above 44 °C(Doyle and Schoeni, 1984; Raghubeer and Matches, 1990). The incu-bation of mTEC plates at 44.5 °C in this study might have preventedthe growth of non-thermotolerant E. coli, resulting in an underesti-mation of the prevalence of stx1 and stx2 genes. Prevalence of stx1and stx2 may also be low if they are not required in the extraintestinalenvironment and are therefore under negative regulation under envi-ronmental conditions (House et al., 2009). It is also possible that theE. coli strains persisting in the beach sand environment are evolvingsuch that they are not retaining the stx1 and stx2 genes. Additionalresearch is necessary to identify factors influencing the abundanceof E. coli possessing eae at Lake Huron and Lake St. Clair beaches,and to determine whether the scarcity of stx1 and stx2 is a functionof adaption to the beach sand environment or a result of inadequatesampling and/or analysis methods.

If the attachment proteins they encode are being expressed, theprevalence of E. coli carrying eae and bfp at these beaches may poseimmediate risks to human health. The existence of these genes onmobile genetic elements also suggests that horizontal gene transferevents in E. coli could occur in sand. Given that the possession ofappropriate combinations of virulence genes belonging to knownpathotypes can render E. coli strains pathogenic (Kaper et al., 2004),our findings support the potential that recreational beach sand containsE. coli pathotypes of intestinal origin, specifically EPEC and EHEC. Fur-thermore, the beach sand environment may serve as a reservoir foremerging pathogens.

Conclusions

The presence in recreational beach sand of attachment and viru-lence genes associated with E. coli pathotypes suggests that horizon-tal gene transfer events could occur in this secondary environment.This could have public health implications in that beach sand maybe serving as a reservoir of pathogenicity genes that could contributeto the emergence of novel pathogens. Furthermore, expression ofthe proteins encoded by these genes could pose immediate risks tobeachgoers. Additional studies are warranted to quantify the pres-ence of these pathogen genes and to determine whether their corre-sponding proteins are being expressed. Future research should alsobe conducted to verify the occurrence of horizontal gene transferamong E. coli residing in beach sand and to determine why eae isso abundant at beaches along Lake Huron and Lake St. Clair.

Acknowledgements

This publication is a result of work sponsored by the Michigan SeaGrant College Program, R/PSC-5, under: NA05OAR4171045 fromNational Sea Grant, NOAA, U.S. Department of Commerce, with fundsfrom the State of Michigan. The authors would like to thank DavidSadler, Luiza Assis, and Krista Reitenga for their assistance in the fieldand laboratory.

References

Alm, E.W., Spain, A., Burke, J., 2003. Fecal indicator bacteria are abundant in wet sand atfreshwater beaches. Water Res. 37, 3978–3982.

Alm, E.W., Burke, J., Hagan, E., 2006. Persistence and potential growth of the fecalindicator bacteria, Escherichia coli, in shoreline sand at Lake Huron. J. GreatLakes Res. 32 (2), 401–405.

Bonilla, T.D., Nowosielski, K., Cuvelier, M., Hartz, A., Green, M., Esiobu, N., McCorquodale,D.S., Fleisher, J.M., Rogerson, A., 2007. Prevalence and distribution of fecal indica-tor organisms in South Florida beach sand and preliminary assessment of healtheffects associated with beach sand exposure. Mar. Pollut. Bull. 54 (9), 1472–1482.

Byappanahalli, M.N., Shively, D.A., Nevers, M.B., Sadowsky, M.J., Whitman, R.L., 2003.Growth and survival of Escherichia coli and enterococci populations in the macro-alga Cladophora (Chlorophyta). FEMS Microbiol. Ecol. 46, 203–211.

133L. Bauer, E. Alm / Journal of Great Lakes Research 38 (2012) 129–133

Carrillo, M., Estrada, E., Hazen, T.C., 1985. Survival and enumeration of the fecal indica-tors Bifidobacterium adolescentis and Escherichia coli in a tropical rain forest water-shed. Appl. Environ. Microbiol. 50, 468–476.

Cleary, J., Li-Ching, L., Shaw, R.K., Straatman-Iwanowska, A., Donnenberg, M.S., Frankel,G., Knutton, S., 2004. Enteropathogenic Escherichia coli (EPEC) adhesion to intesti-nal epithelial cells: role of bundle forming pili (BFP), EspA filaments and intimin.Microbiol. 150, 527–538.

Donnenberg, M.S., Whittam, T.S., 2001. Pathogenesis and evolution of virulence inenteropathogenic and enterohemorrhagic Escherichia coli. J. Clin. Invest. 107 (5),539–548.

Doyle, M.P., Schoeni, J.L., 1984. Survival and growth characteristics of Escherichia coliassociated with hemorrhagic colitis. Appl. Environ. Microbiol. 48, 855–856.

Duris, J.W., Haack, S.K., Fogarty, L.R., 2009. Gene and antigen markers of Shiga-toxinproducing E. coli from Michigan and Indiana river water: occurrence and relationto recreational water quality criteria. J. Environ. Qual. 38, 1878–1886.

Francy, D.S., Gifford, A.M., 2002. E. coli in the swash zone of four Ohio bathing beaches.U.S. Geological Survey Fact Sheet FS-132-02. 4 pp.

Haack, S.K., Fogarty, L.R., Wright, C., 2003. Escherichia coli and enterococci at beaches inthe Grand Traverse Bay, Lake Michigan: sources, characteristics, and environmen-tal pathways. Environ. Sci. Technol. 37 (15), 3275–3282.

Hamelin, K., Bruant, G., El-Shaarawi, A., Hill, S., Edge, T.A., Bekal, S., Fairbrother, J.M.,Harel, J., Maynard, C., Masson, L., Brousseau, R., 2006. A virulence and antimicrobialresistance DNA microarray detects a high frequency of virulence genes in Escheri-chia coli isolates from Great Lakes recreational waters. Appl. Environ. Microbiol. 72(6), 4200–4206.

Hamelin, K., Bruant, G., El-Shaarawi, A., Hill, S., Edge, T.A., Fairbrother, J.M., Harel, J.,Maynard, C., Masson, L., Brousseau, R., 2007. Occurrence of virulence and antimi-crobial resistance genes in Escherichia coli isolates from different aquatic ecosys-tems within the St. Clair River and Detroit River areas. Appl. Environ. Microbiol.73 (2), 477–484.

Heaney, C.D., Sams, E., Wing, S., Marshall, S., Brenner, K., Dufour, A.P., Wade, T.J., 2009.Contact with beach sand among beachgoers and risk of illness. Am. J. Epidemiol.170 (2), 164–172.

House, B., Kus, J.V., Prayitno, N., Mair, R., Que, L., Chingcuanco, F., Gannon, V., Cvitkovitch,D.G., Barnett Foster, D., 2009. Acid-stress-induced changes in enterohaemorrhagicEscherichia coli O157: H7 virulence. Microbiol. 155 (9), 2907–2918.

Ishii, S., Ksoll, W.B., Hicks, R.E., Sadowsky, M.J., 2006. Presence and growth of natural-ized Escherichia coli in temperate soils from Lake Superior watersheds. Appl. Envi-ron. Microbiol. 72, 612–621.

Ishii, S., Hansen, D.L., Hicks, R.E., Sadowsky, M.J., 2007. Beach sand and sediments aretemporal sinks and sources of Escherichia coli in Lake Superior. Environ. Sci. Tech-nol. 41, 2203–2209.

Kaper, J.B., Nataro, J.P., Mobley, H.L.T., 2004. Pathogenic Escherichia coli. Nat. Rev.Microbiol. 2, 123–140.

Kobayashi, H., Pohjanvirta, T., Pelkonen, S., 2002. Prevalence and characteristics ofintimin- and shiga toxin-producing Escherichia coli from gulls, pigeons, andbroilers in Finland. J. Vet. Med. Sci. 64 (11), 1071–1073.

Kobayashi, H.,Miura, A., Hayashi, H., Ogawa, T., Endô, T., Hata, E., Eguchi,M., Yamamoto, K.,2003. Prevalence and characteristics of eae-positive Escherichia coli from healthycattle in Japan. Appl. Environ. Microbiol. 69 (9), 5690–5692.

Lacher, D.W., Walk, S.T., Wick, L.M., Whittam, T.S., 2004. Multiplex PCR detection ofintimin and Shiga toxin genes. NIAID STEC Center/National Food Safety andToxicology Center, Michigan State University. http://www.shigatox.net/stec/cgi-bin/stx-eae.

Lauber, C.L., Glatzer, L., Sinsabaugh, R.L., 2003. Prevalence of pathogenic Escherichia coliin recreational waters. J. Great Lakes Res. 29 (2), 301–306.

Loge, F.J., Thompson, D.E., Call, D.R., 2002. PCR detection of specific pathogens in water:a risk-based analysis. Environ. Sci. Technol. 36, 2754–2759.

Luna, G.M., Vignaroli, C., Rinaldi, C., Pusceddu, A., Nicoletti, L., Gabellini, M., Danovaro,R., Biavasco, F., 2010. Extraintestinal Escherichia coli carrying virulence genes incoastal marine sediments. Appl. Environ. Microbiol. 76 (17), 5659–5668.

O'Brien, A.D., Newland, J.W., Miller, S.F., Holmes, R.K., Smith, H.W., Formal, S.B., 1984.Shiga-like toxin-converting phages from Escherichia coli strains that cause hemor-rhagic colitis or infantile diarrhea. Science 226, 694–696.

Raghubeer, E.V., Matches, J.R., 1990. Temperature range for growth of Escherichia coliserotype O157:H7 and selected coliforms in E. coli medium. J. Clin. Microbiol. 28,803–805.

Scheutz, F., Møller Nielsen, E., Frimodt-Møller, J., Boisen, N., Morabito, S., Tozzoli, R.,Nataro, J.P., Caprioli, A., 2011. Characteristics of the enteroaggregative shiga toxin/verotoxin producing Escherichia coliO104:H4 strain causing the outbreak of haemoly-tic uraemic syndrome in Germany, May to June 2011. Eurosurveillance 16 (24), 1–6.

Souza, V., Castillo, A., Equiarte, L., 2002. The evolutionary ecology of Escherichia coli.Am. Sci. 90 (4), 332–342.

Trabulsi, L.R., Keller, R., Tardelli Gomes, T.A., 2002. Typical and atypical enteropatho-genic Escherichia coli. CDC EID 8 (5), 508–513.

Vidal, R., Vidal, M., Lagos, R., Levine, M., Prado, V., 2004. Multiplex PCR for diagnosis ofenteric infections associated with diarrheagenic Escherichia coli. J. Clin. Microbiol.42 (4), 1787–1789.

Whitman, R.L., Nevers, M.B., 2003. Foreshore sand as a source of Escherichia coli in near-shore water of a Lake Michigan beach. Appl. Environ. Microbiol. 69 (9), 5555–5562.

Whitman, R.L., Byers, S.E., Shively, D.A., Ferguson, D.M., Byappanahalli, M., 2005. Occur-rence and growth characteristics of Escherichia coli and enterococci within theaccumulated fluid of the northern pitcher plant (Sarracenia purpurea L.). Can. J.Microbiol. 51, 1027–1037.

Yoder, J.S., Hlavsa, M.C., Craun, G.F., Hill, V., Roberts, V., Yu, P.A., Hicks, L.A., Alexander,N.T., Calderon, R.L., Roy, S.L., Beach, M.J., 2008. Surveillance for waterborne diseaseand outbreaks associated with recreational water use and other aquatic facility-associated health events—United States, 2005–2006. MMWR 57 (SS09), 1–29.