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Laboratory investigation of an E. coli 0157:H7 outbreak associated with swimming in Battle Ground Lake, Vancouver, Washington. (Features).

Introduction

Escherichia coli 0157:H7 has emerged as a significant foodborne and waterborne pathogen. Over the past two decades, reports of waterborne E. coli 0157:H7 outbreaks attributed to drinking water or recreational contact have increased. The first reported isolation of E. coli 0157:H7 from water came from a freshwater sample taken from a water reservoir (without any connection to human illness) in the countryside surrounding the city of Philadelphia (McGowan, Wickersham, & Strockbin, 1989).

The first reported outbreak of E. coli 0157:H7 associated with recreational water occurred in the summer of 1991. The 1991 outbreak involved 21 cases of E. coli 0157:H7 and 59 cases of Shigella sonnei infections, all associated with swimming in Blue Lake near Portland, Oregon (Keene et al., 1994). Four more outbreaks of E. coli 0157:H7 have been epidemiologically linked to swimming in freshwater lakes (Ackman et al., 1997; Cransberg et al., 1996; Paunio et al., 1999; Warmer et al., 1996). In several waterborne outbreaks of E. coli 0157:H7, investigators were unable to isolate the pathogen from the suspected water (Ackman et al., 1997; Cransberg et al., 1996; Dev et al., 1984; Keene et al., 1994; Paunio et al., 1999; Swerdlow et al., 1992; Warmer et al., 1996). In August 1999, 36 case patients developed E. coil 0157:H7 infection. Most had a history of swimming in Battle Ground Lake (BGL) located in Battle Ground Lake State Park in Clark County, Washington (28 swimmers and 8 contacts of swimmers; 35 stool-cult ure confirmed, one serologically confirmed). The outbreak was detected through routine infectious-diseases surveillance by personnel of the Southwest Washington Health District. This paper reports the results of the environmental investigation into the E. coli O157:H7 outbreak linked to swimming in Battle Ground Lake.

Materials and Methods

Location and Sanitary Survey of Swimming Area

Battle Ground Lake is a 28-acre lake located in Battle Ground Lake State Park in Clark County, Washington. Sewage at the park is treated by a conventional community septic system.

Sampling

Eighty water and sediment samples were collected from the wading area of Battle Ground Lake and delivered on ice to the Southwest Washington Health District Public Health Laboratory or to the University of Washington. In addition, 28 fresh animal fecal samples (from cow coyote, deer, duck, and rabbit) were collected from the swimming-beach area and from around the lake. The samples were collected from September 6, 1999, through October 18, 1999. Samples were kept refrigerated until analysis, which was done within 48 hours of sample collection.

Fecal-Coliform Analysis

Fecal-coliform analysis of lake water samples was performed within six hours of collection by the multiple-tube fermentation and the membrane filtration methods as described in Standard Methods for the Examination of Water and Wastewater (Greenberg, Lenore, & Andrew, 1992).

Dye-Tracing

The septic tank serving the park was dosed with 5 pounds of fluorescein dye on September 2, 1999. Activated-charcoal packs were placed in the lake at seven locations within the swimming-beach area.

Detection of E. coli O157:H7

To increase the likelihood of detecting O157:H7 before performance of the polymerase chain reaction (PCR) procedure, the authors enriched the samples (both fecal and water samples) in modified trypticase soy broth (mTSB) for 24 hours. A 250-milliliter (250-mL) Erlenmeyer flask containing 90 mL of sterile mTSB--which in turn contained 100 microliters ([micro]L) of a 100-milligram-per-milliliter filter-sterilized vancomycin solution--was inoculated with 10 grams of feces or sediment and incubated overnight at 37[degrees]C with agitation. The authors then filtered 100 to 250 mL of water through a 0.45-micron (0.45-[micro]m) Gelman filter (Gelman Sciences, Inc., Ann Arbor, Michigan). The filters were put in 90 mL of vancomycin mTSB enrichment broth and incubated at 37[degrees]C overnight with agitation. After an overnight incubation of samples in mTSB enrichment broth, PCR was used to detect virulence factors associated with E. coli O157:H7. All enriched samples were plated on MacConkey sorbitol (Difco Laboratori es, Detroit, Michigan) with cefixime and tellurite (Dynal, Inc., Lake Success, New York) and were screened for growth of colonies characteristic of E. coli O157:H7 regardless of the PCR results.

To prepare the samples for PCR processing, 50 [micro]L of the enrichment broth was added to 400 [micro]L of lysis buffer consisting of 10-millimolars (mmol) Tris with 20 [micro]L per milliliter of achromopeptidase (Sigma, Saint Louis, Missouri). The lysates were incubated at 55[degrees]C for one hour, and a second incubation followed at 95[degrees]C for 10 minutes. Immediately after the incubations, the samples were placed on ice.

The lysate (10 [micro]L) was used in a PCR reaction containing 200 mmol of each dNTP (Promega Corp., Madison, Wisconsin), 1.5 units of Taq polymerase (Promega Corp.), 10 mM Tris-HCI, 50 mmol KCl, 0.1 percent Triton X-100, and 2 mmol [MgCl.sub.2]. Water was added to bring the total reaction volume to 50 [micro]L. The PCR reaction used was developed to detect three genes that are commonly associated with enterohemorrhagic E. coli: Stx (I and II), eaeA, and hlyA. A positive control, consisting of DNA from a previously characterized E. coli O157:H7 isolate, was used to ensure that the amplification reaction yielded desired products of appropriate sizes. A negative control, consisting of autoclaved deionized water, was used to ensure that the reaction cocktail, tips, tubes, and pipettes were not contaminated with DNA, which would have yielded an amplification product. The PCR primers and thermal cycle were used as described by Paton and Paton (1998) with a Perkin-Elmer 9600 thermocycler. In this study, the PCR pri mers were used in a multiplex format.

After the completion of the PCR reaction, loading dye was added to each reaction vial. Next, 25 [micro]L of each reaction was loaded into wells of a two percent agarose gel. The amplicons were resolved by agarose gel electrophoresis at 200 volts for 40 minutes. Following electrophoresis, the gel was stained for 15 minutes in ethidium bromide solution and destained for 10 minutes in distilled water. Banding patterns were visualized with an ultraviolet transilluminator.

Recovery of E. coli O157:H7

All sample enrichments were plated and screened for growth of colonies characteristic of E. coli O157:H7 regardless of the PCR results, Immediately following the preparation of the sample lysis buffer, a loop full of the enrichment broth was streaked onto a sorbitol MacConkey with an agar plate containing cefixime and tellurite (SMAC w/CT). Enrichment cultures that yielded amplicons corresponding to Stx (I and II), eaeA, and hly genes were pursued further for recovery of E. coli O157:H7. Colorless colonies were picked with sterile toothpicks, streaked onto trypticase soy agar grids, and grown overnight at 35[degrees]C. An average of 25 colorless colonies were picked per SMAC w/CT plate. The colorless colonies were then tested with the spot indole test. Colonies that had a positive reaction were tested with a latex agglutination test for E. coli O157:H7 (Oxoid Ltd., Basingstoke, Hampshire, England). Positive colonies were restreaked onto SMAC w/CT plates to ensure that they were pure colonies and then were str eaked onto trypticase soy agar for heavy growth.

Results

Sanitary Survey

On an average day during the summer months, Battle Ground Lake State Park is visited by an estimated 400 day users and 220 overnight campers. Sources for drinking water in the park and the swimming-beach area are water fountains that are fed from a well 85 to 120 feet deep. The well water is piped directly, without any treatment or disinfection. Visual inspection of the well head did not reveal a potential route of contamination. The toilets in the park are connected to a septic system. The drainfield is 1,200 yards away from the lake and the well, and is situated at a higher elevation

During the summer, approximately 10 to 20 horseback riders per day use dedicated park trails. The drainage from the horse trails does not appear to flow into the lake. There are two small dairy farms near the lake whose surface runoff has no direct drainage route to the lake for surface runoff. A resident population of a dozen ducks frequents the swimming-beach area. Other wildlife around the lake include deer, coyotes, rabbits, raccoons, and opossums.

Fecal-Coliform Analysis of Water Samples

Two water samples taken in the beginning of the outbreak investigation were analyzed for the presence of fecal coliform by the multiple-tube fermentation technique following Method 9221E (Greenberg et al., 1992). The samples yielded 18 and 93 most probable number (MPN) per 100 mL of water. Both results were well below the Washington State limit of 200 colony-forming units (CFU) per 100 mL of water for recreational waters (Washington Administrative Code, 2001).

Dye-Tracing

Dye-testing showed that the lake was not affected by the septic system. The activated-charcoal packs were removed on September 16, 1999 (two weeks later), and September 23, 1999 (three weeks later). The charcoal test packs were sent to the Ozark Underground Laboratory in Protem, Missouri, for analysis of fluorescein dye. No dye was detected. The minimum detection limit for this procedure is 15 micrograms ([micro]g) per liter.

Detection of E. coli O157:H7 Virulence Factors

A total of 108 environmental samples including water (51), soil (one), sand (eight), sediment (20), and animal fecal matter (28), were analyzed for the presence of E. coli O157:H7 virulence factors. Of the 108 samples tested, 59 (54.6 percent) tested positive for the presence of Stx (I and II) genes. Of the 59 Stx-positive samples, 32 (54.2 percent) tested positive only for Stx (I and II) genes, seven (11.9 percent) contained Stx and eaeA genes, 17 (28.9 percent) tested positive for Stx and hly genes, and three (5.1 percent) were positive for Stx, eaeA, and hly. Of the 108 environmental samples tested, one sample (0.9 percent) tested positive for eaeA gene only. Forty-eight of the 108 (44.4 percent) tested negative for Stx, eaeA, and hly genes. Results of the analysis are summarized in Table 1.

Virulence Factor and RFLP Analysis of the Recovered E. coli O157:H7

E. coli O157:H7 was recovered from the enrichment broths of a water sample and a duck fecal sample. The enrichment from the water sample had tested positive for Stx, eaeA, and hly genes, while the enrichment from the duck sample had tested positive for Stx and eaeA genes. The E. coli O157:H7 samples recovered from the water and the duck fecal sample off of SMAC w/CT were screened by the PCR reaction for evidence of the virulence genes. Both isolates yielded bands for Stx, eaeA, and hly genes, which were of the same size as the bands of the positive control. In addition to the environmental samples, the patient isolates were also tested by PCR and yielded bands of the same size as the positive control and the environmental isolates. All patient isolates were identical by pulsed-field gel electrophoresis/restriction fragment length polymorphism (PFGE/RFLP) typing. Comparison of the genetic fingerprints of the environmental isolates (water and duck feces) with those of the case patients from the outbreak showed that the environmental isolates were identical to most of the patient isolates (Figure 1). Columns 1 through 7 and 9 through 12 in Figure 1 show the genetic fingerprints of patient isolates. Columns 3 and 4 represent patients outside the outbreak cluster. Column 8 is a genetic fingerprinting of a duck isolate, and columns 13 and 14 are from water isolates.

Discussion

Although several outbreaks of E. coli O157:H7 linked to recreational waters have been reported (Ackman et al., 1997; Friedman et al., 1999; Kramer, Herwaldt, Craun, Calderon, & Juranek, 1994; Levy, Bens, Craun, Calderon, & Herwaldt, 1998), E. coli O157:H7 has never been isolated from implicated waters in those outbreaks. The Battle Ground Lake outbreak was detected by the Southwest Washington Health District as the result of an epidemiological investigation. The environmental investigation took place early during the course of the outbreak. A sanitary survey of Battle Ground Lake and Battle Ground Lake State Park identified several possible routes of contamination: the presence of a septic-tank system in the vicinity of the swimming beach, the untreated drinking-water source, wildlife in the area, and park visitors.

Visual inspection of the drainfield did not show any signs of seepage. This finding was confirmed by results from dye-testing of the septic system. The park drinking water is not chlorinated. It is, however, tested monthly for the presence of fecal coliforms. The test results have been consistently negative.

Early during the investigation, the authors decided against the use of the fecal-coliform assay for the recovery of E. coli O157:H7 because this organism grows poorly, or not at all, at 44.5[degress]C (Doyle & Schoeni, 1984). Instead, the investigation relied on an enrichment process followed by PCR screening for the virulence factors associated with this organism.

A large proportion (54.6 percent) of the samples produced amplicons for Stx genes. This proportion is much higher than reported levels in Stx-positive food and environmental samples, which range from 15 to 25 percent (Samadpour, Ongerth, & Liston, 1994; Samadpour, Ongerth, Liston, Tran, et al., 1994; Samadpour, 1995), but because the lake water was contaminated with E. coli 0157:H7, the finding of a higher percentage of E. coli O157:H7 virulence factors than previously reported was expected. Twenty-five percent of the samples (N = 27) contained two to three of the virulence genes associated with E. coli O157:H7 E. coli O157:H7 was recovered from two (7.4 percent) of the 27 samples. The environmental isolates had the same restriction fragment length polymorphism as the patient isolates from the outbreak.

After the closure of Battle Ground Lake to swimming, weekly water and sediment samples were analyzed for the presence of enterohemorraghic E. coli (EHEC), as determined by PCR detection of EHEC virulence factors. Three consecutive weekly samples following the BGL closure were positive for EHEC virulence factors; the next three sets of weekly samples taken from the lake all tested negative for the EHEC virulence genes.

Three of the four cow fecal samples and three of the 20 duck fecal samples tested positive for the presence of Stx genes. It is not unusual to find high carriage levels for Stx genes in cattle (Samadpour, Ongerth, & Liston, 1994; Samadpour, Ongerth, Liston, Tran, et al., 1994). The rest of the animal fecal samples (one coyote sample, two deer samples, and one rabbit sample) tested negative for the presence of Stx genes.

Although E. coli O157:H7 was recovered from a duck fecal sample, it is not clear if the resident population of ducks at the lake was the source of lake-water contamination. It is quite possible that the ducks at the lake were transiently infected by contaminated water. The initial source of contamination may have been an early human case or other animals that reside in the park or its vicinity. The fact that the lake water samples tested negative for E. coli O157:H7 a month after closure of the lake to swimming--while the ducks still resided at the park--may argue in favor of the theory that ducks were not the original source of the contamination. The presence of the ducks, however, may have helped sustain contamination levels over a period of time.

Despite numerous outbreaks of enteric diseases linked to swimming pools and water parks (Ackman et al., 1997; Cransberg et al., 1996; Friedman et al., 1999; Levy et al., 1998) with humans as the only conceivable source of contamination, the role of humans as sources of contamination of recreational waters is not fully appreciated. Humans can add a significant load of enteric pathogens to recreational waters. Fecal contamination of recreational waters is not limited only to accidental releases that are credited to toddlers; every person, regardless of age, adds a load of fecal coliforms (and other bacteria) to recreational waters upon immersing. The source is mostly the recto-genital flora and small quantities of fecal material that may be present in the anal area.

The fact that levels of fecal coliforms in the lake were well within the Washington State standard for recreational water of 200 CFU per 100 mL may argue against simplistic approaches such as continued reliance on indicators for determining the microbiological quality of water and food. Indeed, the last set of water samples from Battle Ground Lake, which tested negative for EHEC according to the PCR protocol, had fecal-coliform levels exceeding the Washington State standard.

In summary, the authors report the first isolation of E. coli O157:H7 from recreational waters implicated as a source of an E. coli O157:H7 outbreak. This study also constitutes the first reported isolation of the organism from duck feces. The levels of fecal coliforms at the time of the outbreak were below the regulatory limit of 200 CFU per 100 mL for recreational water in Washington State (Washington Administrative Code, 2001), which shows the limitations of using indicator organisms to determine the microbiological quality of water.

[FIGURE 1 OMITTED]
TABLE 1

PCR Analysis of Enriched Water, Sediment, and Animal-Fecal Sample for
the Presence of Virulence Factors Associated with E. coli O157:H7

Source Number Stx Stx, Stx, Stx, eaeA All E. coli
 of eaeA hly eaeA, Only Negative O157:H7
 Samples hly Recovered

Cow 4 3 -- -- -- -- 1 N
Coyote 1 -- -- -- -- -- 1 N
Deer 2 -- -- -- -- -- 2 N
Duck 20 3 2 2 -- -- 13 1
Rabbit 1 -- -- -- -- -- 1 N
Sand 8 3 -- 1 -- -- 4 N
Soil 1 -- -- -- -- -- 1 N
Sediment 20 9 -- 2 1 1 7 N
Water 51 14 5 12 2 -- 18 2
Total 108 32 7 17 3 1 48 3

N = not recovered.


REFERENCES

Ackman, D., Marks, S., Mack, P., Caldwell, M., Root, T., & Birkhead, G. (1997). Swimming-associated haemorrhagic colitis due to Escherichia coli O157:H7 infection: Evidence of prolonged contamination of a fresh water lake. Epidemiology of Infections, 119(1), 1-8.

Cransberg, K., van den Kerkhof, J.H., Banffer, J.R., Stijnen, C., Wernars, K., van de Kar, N.C., Nauta, J., & Wolff, ED. (1996). Four cases of hemolytic uremic syndrome--Source contaminated swimming water? Clinical Nephrology, 46(1), 45-49.

Dev, V.J., Main, M., & Gould, I. (1991). Waterborne outbreak of Escherichia coli O157 [letter to the editor]. Lancet, 337(8754), 1412.

Doyle, M.P., & Schoeni, J.L. (1984). Survival and growth characteristics of Escherichia coli associated with hemorrhagic colitis. Applied Environmental Microbiology, 48(4), 855-856.

Friedman, M.S., Roels, T., Koehler, J.E., Feldman, L., Bibb, W.F., & Blake, P. (1999). Escherichia coli O157:H7 outbreak associated with an improperly chlorinated swimming pool. Clinical Infectious Diseases, 29(2), 298-303.

Greenberg, E.A., Lenore, C.S., & Andrew, E.E. (1992). Standard methods for the examination of water and wastewater (18th ed.). Washington, DC: American Public Health Association; American Water Works Association; World Environmental Federation.

Keene, W.E., McAnulty, J.M., Hoesly, F.C., Williams, L.P., Jr., Hedberg, K., Oxman, G.L., Barrett, T.J., Pfaller, M.A., & Fleming, D.W. (1994). A swimming-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 and Shigella sonnei. New England Journal of Medicine, 331(9), 579-584.

Kramer, H.M., Herwaldt, L.B., Craun, F.G., Calderon, L.R., & Juranek, D.D. (1994). Surveillance for waterborne-disease outbreaks--United States 1993-1994. Morbidity and Mortal Weekly Report, CDC Control Surveillance Summaries, 45(1), 1-33.

Levy, D.A., Bens, M.S., Craun, G.F., Calderon, R.L., & Herwaldt, B.L. (1998). Surveillance for waterborne-disease outbreaks--United States, 1995-1996. Morbidity and Mortality Weekly Report, CDC Report Surveillance Summaries, 47(5), 1-34.

McGowan, K.L., Wickersham, E., & Strockbin, N.A. (1989). Escherichia coli O157:H7 from water [letter to the editor]. Lancet, 1(8644), 967-968.

Paton, W.A., & Paton, J.C. (1998). Detection and characterization of Shiga Toxigenic Escherichia coli by using multiplex PCR assays for [Stx.sub.1], [Stx.sub.2], eaeA, Enterohemorrhagic E. coli hlyA, [rfb.sub.0111], and [rfb.sub.0157]. Journal of Clinical Microbiology, 36(2), 598-602.

Paunio, M., Pebody, R., Keskimaki, M., Kokki, M., Ruutu, P., Oinonen, S., Vuotari, V., Siitonen, A., Lahti, E., & Leinikki, P. (1999). Swimming-associated outbreak of Escherichia coli O157:H7. Epidemiology of Infections, 122(1), 1-5.

Samadpour, M., Ongerth, J.E., & Liston, J. (1994). Development and evaluation of oligonucleotide DNA probes for detection and genotyping of Shiga-like toxin producing Escherichia coli. Journal of Food Protection, 57(5), 399-402.

Samadpour, M., Ongerth, J.E., Liston, J., Tran, N., Nguyen, D. Whittam, T.S., Wilson, R.A., & Tarr, P.I. (1994). Occurrence of Shiga-like toxin-producing Escherichia coli in retail fresh seafood, beef, lamb, pork, and poultry from grocery stores in Seattle, Washington. Applied Environmental Microbiology, 60(3), 1038-1040.

Samadpour, M. (1995). Molecular epidemiology of Escherichia coli O157:H7 by restriction fragment length polymorphism using Shiga-like toxin genes. Journal of Clinical Microbiology 33(8), 2150-2154.

Swerdlow, D.L., Woodruff, B.A., Brady, R.C., Griffin, P.M., Tippen, S., Donnel, H.D., Geldreich, E., Payne, B.J., Meyer, A., Wells, J.G., Greene, K.D., Bright, M., Bean, N.H., & Blake, P.A. (1992). A waterborne outbreak in Missouri of Escherichia coli O157:H7 associated with bloody diarrhea and death, Annals of Internal Medicine, 117(10), 812-819.

Warrner, M., Williams, K.K., Adam, B., Langkop, C., Ruden, R., Francis, B., & Haupt, T. (1996). Lake-associated outbreak of Escherichia coli O157:H7--Illinois, 1995. Morbidity and Mortality Weekly Report, CDC Report Surveillance Summaries, 45(21), 437-439.

Washington Administrative Code. (2001). [section] 173-201A-030 (2001).

RELATED ARTICLE: Practical Stuff!

* Several outbreaks of E. coli O157:H7 linked to recreational waters have been reported.

* Before this study, however, E. coli O157:H7 had never been isolated from implicated waters in an outbreak.

* The Battle Ground Lake outbreak was detected by the Southwest Washington Health District as the result of an epidemiological investigation.

* A sanitary survey identified several possible routes of contamination:

-- the presence of a septic-tank system in the vicinity of the swimming beach;

-- an untreated drinking-water source;

-- wildlife, including ducks, in the area; and

-- park visitors.

* The drainfield was exonerated by visual inspection and dye testing.

* The park drinking water is not chlorinated but is tested monthly for the presence of fecal coliforms.

* The test results have been consistently negative.

* The investigators decided against the use of the fecal-coliform assay for the recovery of E. coli O157:H7 since the organism grows poorly, or not at all, at 44.5[degrees]C.

* Instead, the investigation relied on an enrichment process followed by polymerase chain reaction (PCR) screening for the virulence factors associated with this organism (Stx, eaeA, and hlygenes).

* A large proportion of samples produced amplicons for Stx genes.

* The proportion was much higher than reported levels in Stx-positive food and environmental samples, which range from 15 to 25 percent.

* This finding was expected, however, because the lake water was contaminated with E. coli O157:H7

* The environmental isolates had the same restriction fragment length polymorphism as the patient isolates from the outbreak.

* Although E. coli O157:H7 was recovered from a duck fecal sample, it is not clear if the resident duck population was the source of lake-water contamination.

* It is possible that the ducks were transiently infected by contaminated water.

* The initial source of contamination may have been an early human case or other animals in the park or its vicinity.

* The presence of the ducks, however, may have helped sustain contamination levels over a period of time.

* Humans also can add a significant load of enteric pathogens to recreational waters.

* Fecal contamination is not due only to accidental releases credited to toddlers.

* Every person, regardless of age, adds a load of fecal coliforms (and other bacteria) to recreational waters upon immersing.

* The source is mostly the recto-genital flora and small quantities of fecal material that may be present in the anal area.

* It is important to note that levels of fecal coliforms in the lake were well within the Washington State standard of 200 CFU per 100 mL for recreational water.

* This fact may argue against continued reliance on indicators for determining the microbiological quality of water and food.

Corresponding Author: Mansour Samadpour, Ph.D., Assistant Professor, Department of Environmental Health, School of Public Health, University of Washington, Box 357234, Seattle, WA 98195. E-mail: <mansour@u.washington.edu>.
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Author:Newman, Tom
Publication:Journal of Environmental Health
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Geographic Code:1U9WA
Date:Jun 1, 2002
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