A waterborne outbreak of multiple diarrhoeagenic Escherichia coli infections associated with drinking water at a school camp

Accepted Manuscript Title: A waterborne outbreak of multiple diarrhoeagenic Escherichia coli infections associated with drinking water at a school camp Authors: Jungsun Park, Jin Seok Kim, Soojin Kim, Eunkyung Shin, Kyung-Hwan Oh, Yonghoon Kim, Cheon Hyeon Kim, Min Ah Hwang, Chan Mun Jin, Kyoungin Na, Jin Lee, Enhi Cho, Byung-Hak Kang, Hyo-Sun Kwak, Won Keun Seong, Junyoung Kim PII: DOI: Reference:

S1201-9712(17)30246-1 https://doi.org/10.1016/j.ijid.2017.09.021 IJID 3044

To appear in:

International Journal of Infectious Diseases

Received date: Revised date: Accepted date:

20-7-2017 15-9-2017 20-9-2017

Please cite this article as: Park Jungsun, Kim Jin Seok, Kim Soojin, Shin Eunkyung, Oh Kyung-Hwan, Kim Yonghoon, Kim Cheon Hyeon, Hwang Min Ah, Jin Chan Mun, Na Kyoungin, Lee Jin, Cho Enhi, Kang Byung-Hak, Kwak Hyo-Sun, Seong Won Keun, Kim Junyoung.A waterborne outbreak of multiple diarrhoeagenic Escherichia coli infections associated with drinking water at a school camp.International Journal of Infectious Diseases https://doi.org/10.1016/j.ijid.2017.09.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A waterborne outbreak of multiple diarrhoeagenic Escherichia coli infections associated with drinking water at a school camp.

Jungsun Park1, Jin Seok Kim1, Soojin Kim1, Eunkyung Shin1, Kyung-Hwan Oh1, Yonghoon Kim1, Cheon Hyeon Kim2, Min Ah Hwang2, Chan Mun Jin2, Kyoungin Na3, Jin Lee3, Enhi Cho3, Byung-Hak Kang1, Hyo-Sun Kwak1, Won Keun Seong1 and Junyoung Kim1

1

Division of Bacterial Diseases, Center for Laboratory Control of Infectious Diseases, Korea

Centers for Disease Control and Prevention, Chungcheongbuk-do, Republic of Korea 2

Division of Microorganisms, Jeollabukdo Institute of Health and Environment Research,

Jeollabuk-do, Republic of Korea 3

Division of Infectious Disease Control, Center for Infectious Disease Control, Korea Centers

for Disease Control and Prevention, Chungcheongbuk-do, Republic of Korea

#Corresponding author: Junyoung Kim Division of Bacterial Diseases, Center for Laboratory Control of Infectious Diseases, Korea Centers for Disease Control and Prevention, Heungdeok-gu, Cheongju-si, Chungcheongbukdo, 28159, Republic of Korea Tel.: +82-43-719-8116; Fax: +82-43-719-8109; E-mail: [email protected]

1

Highlights  In June 2015, an outbreak of acute gastroenteritis occurred in a school camp.  We isolated diarrhoeagenic E. coli strains from clinical and drinking water samples.  The camp used groundwater drawn from a private underground reservoir.  The outbreak investigation revealed some problems with the water supply system, such as the use of inappropriate filters in the water purifier and a defect in the pipeline between the reservoir and the chlorination device.  The management of drinking water quality in group facilities should be strengthened.

Abstract Background: In June 2015, a local public health laboratory was notified that students had developed gastroenteritis symptoms after participating in a camp. An outbreak investigation was conducted to determine the extent and cause of the outbreak. Method: We conducted a retrospective cohort study to determine the correlations between the illness and specific exposures at the school camp. All attendees were interviewed with a standard questionnaire that addressed clinical symptoms, food consumption and environmental exposures. Clinical specimens were cultured using standard microbiological methods for bacterial or viral pathogens. The genetic relationships of all isolates were determined using pulsed-field gel electrophoresis (PFGE). Results: We identified 188 patients with symptoms of diarrhoea, abdominal pain and nausea. Their completed questionnaires suggested that the consumption of drinking water was likely to be linked to this outbreak. Using microbiological methods, we isolated enterohemorrhagic Escherichia coli, enteropathogenic E. coli and enteroaggregative E. coli, and the isolates from both patient stools and environmental water samples displayed indistinguishable XbaI-PFGE 2

patterns. The water system in the camp used groundwater drawn from a private underground reservoir for cooking and drinking. The environmental investigation revealed some problems with the water supply system, such as the use of inappropriate filters in the water purifier and a defect in the pipeline between the reservoir and the chlorination device. Conclusions: This outbreak points to the importance of drinking water quality management in group facilities using underground water and emphasizes the need for periodic sanitation and inspection to prevent possible waterborne outbreaks.

Keywords: Outbreak; diarrhoeagenic Escherichia coli; drinking water; O103:H2

Background Waterborne diseases caused by the consumption of contaminated water can affect a large number of people in a short time (Gallay et al., 2006;Mellou et al., 2014). The World Health Organization(WHO) has reported that worldwide, two million deaths related to such diseases occur annually, primarily in individuals under five years of age; in addition, approximately 663 million people continue to lack access to improved drinking water sources (WHO/UNICEF, 2015).

Significant sources of water pollution include human sewage and animal waste poured into water distribution systems and surface water (Wallender et al., 2014). The resulting faecal contamination of drinking water is a known route for the waterborne transmission of enteric pathogens; thus, the removal of biological and chemical contaminants in drinking water prior to human consumption is an essential step for securing water safety (Braeye et al., 2015;Riera-Montes et al., 2011).

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Public water is regularly managed to ensure water quality. However, groundwater that has not undergone purification processes is sometimes used as drinking water because groundwater is believed to be relatively safe for human consumption (Sezen et al., 2015). The use of groundwater for drinking or cooking without adequate water treatment or sanitation can increase the risk of infection in individuals who consume it (Laine et al., 2011;Licence et al., 2001), particularly if this water is contaminated with pathogenic agents such as norovirus (Hoebe et al., 2004) and Entamoeba histolytica (Chen et al., 2001). Therefore, closer inspections of drinking water are needed to reduce the risk of potential outbreaks. Noncommunity places such as camps, restaurants and hotels have a variety of water distribution systems, both private and public, for drinking water. These water distribution systems are managed with multistep treatment systems or frequent monitoring. The sources of the drinking water in these distribution systems ranges from forest catchments to rivers (Pons et al., 2015). A number of factors can contribute to microbial contamination in drinking water, including low pressure conditions, low residual chlorine concentrations and pipe breaks that can result in the intrusion of pathogenic microorganisms (Ashbolt, 2015; Ercumen et al., 2014).

A number of waterborne gastroenteritis outbreaks have been caused by diarrhoeagenic Escherichia coli (DEC) (Swerdlow et al., 1992), which has been detected in diverse ecological niches ranging from mammalian intestines to various aquatic environments, such as surface water and groundwater (Coleman et al., 2013). DEC can be divided into five groups based on virulence properties: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC) and enteroinvasive E. coli (EIEC) (Lienemann et al., 2011). Depending on the type of infection, DEC can cause a wide spectrum of human diseases ranging from mild diarrhoea to severe

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haemorrhagic colitis (Sinclair et al., 2017).

In general, an outbreak is likely to be attributed to a single E. coli clone as an aetiological agent; however, multiple DEC strains are occasionally involved in a waterborne outbreak (Lienemann et al., 2011;McCall et al., 2010). In this report, we describe the epidemiological and microbiological investigations of an outbreak involving multiple DEC strains that was associated with contaminated underground water at a school camp in Korea in 2015.

Methods Background On June 8, 2015, the Jeollabuk-do Public Health Laboratory (JPHL) was notified that a group of middle school students (from school A in Gwangju Metropolitan City) had developed diarrhoea and vomiting after attending a school camp located in Jeollabuk-do from June 3 to June 5. The JPHL found that two additional schools (school B in Yeosu, Jeollanam-do Province and school C in Sejong Metropolitan Autonomous City) attended the same camp and that the camp attendees had acute gastrointestinal symptoms. Thus, the JPHL launched an investigation to assess the extent of the outbreak, determine the aetiological agent and implement control measures.

Epidemiological investigation An suspected case in this outbreak was defined as a person who attended the school camp from June 3 to June 5 and who had one or more symptoms, including diarrhoea (three or more watery stools per day), abdominal pain, vomiting, nausea or chills. A laboratory-confirmed case was a suspected case with a culture-confirmed pathogenic E. coli infection. We 5

conducted a retrospective cohort study to determine the correlations between illness and specific exposures at the school camp. To collect information, all attendees, including camp staff, students and teachers from the three schools, were interviewed using a standard questionnaire that addressed clinical symptoms, food consumption and environmental exposures while attending the camp and the time of symptom onset. The number of newly infected patients was continuously monitored during the period of investigation, and additional hygiene education was provided to all camp participants to control foodborne or waterborne outbreaks. This outbreak investigation was part of an urgent response to a public health emergency and did not require institutional review board approval. Microbiological analysis Three local public health laboratories collected clinical specimens from rectal swabs of students and teachers in schools A, B and C and from camp staff. All samples were cultured using standard microbiological methods for bacterial or viral pathogens (Shin et al., 2015). Isolated DEC strains were pathotyped using an 8-plex PCR assay for the stx1, stx2, st, lt, eaeA, bfpA, aggR and ipaH genes (KogeneBiotech, Seoul, Korea) in accordance with the manufacturer’s instructions. The isolates were submitted to the Korea Centers for Disease Control and Prevention (KCDC) for confirmation of their identification and further characterization. They were serotyped via agglutination using E. coli antisera for the O and H antigens (Denka Seiken, Tokyo, Japan) and analysed using pulsed-field gel electrophoresis (PFGE) after XbaI digestion in accordance with the PulseNet International protocol. Genetic similarities between PFGE patterns were determined using BioNumerics v7.5 software (Applied Maths, Sint-Martens-Latem, Belgium) with the Dice coefficient and 1.5% band tolerance and via the unweighted pair-group method using arithmetic averages (UPGMA).

The antimicrobial susceptibilities and minimum inhibitory concentrations (MICs) of the

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EPEC and EHEC isolates were determined using the broth microdilution method with customized Sensititre panels (Trek Diagnostic Systems, OH, USA) in accordance with guidelines established by the Clinical and Laboratory Standards Institute (CLSI). The tested antimicrobial agents were ampicillin, ampicillin/sulbactam, amoxicillin/clavulanic acid, cephalothin, cefoxitin, ceftriaxone, cefotaxime, nalidixic acid, ciprofloxacin, tetracycline, chloramphenicol, imipenem, streptomycin, gentamicin, amikacin and trimethoprim/sulfamethoxazole.

Environmental investigation The JPHL inspected the camp facilities (the cafeteria and the water supply system), interviewed the catering staff and reviewed food safety records. Environmental specimens were initially collected from food items served from June 3 to June 5, and four samples were obtained from cooking utensils. Additional samples were collected from water; 16 samples were from drinking water from water purifiers throughout the camp cafeteria, four samples were from the upper and lower regions of a valley near a camp, and two samples were from washbasins in the toilet. All samples were tested as described above for bacterial and viral identification, and the residual free chlorine concentrations in the drinking water samples were measured.

Statistical analysis Risk factors were compared using χ2 tests. Univariate analysis was performed to calculate the adjusted relative risks (RRs) with 95% confidence intervals (CIs) for all food items. A P value < 0.05 was considered statistically significant, and all statistical analyses were performed using Microsoft Excel (Microsoft, USA).

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Results Epidemiological investigation An initial interview was completed for 475 people who attended a school camp from June 3 to June 5. The outbreak affected 188 out of 609 people (attack rate 30.9%). The epidemic curve indicated that the first six cases of gastroenteritis were reported on June 4 and that additional cases were discovered by June 12, with a peak incidence of 89 cases on June 5 (the third day of the camp; Figure 1). The patients’ common symptoms were diarrhoea (100%), abdominal pain (72.9%), nausea (36.7%), chills (15.4%), fever (9.6%) and vomiting (6.9%). The mean incubation period was 54.5 h, and incubation periods ranged from 10 h to 120 h.

Based on the completed questionnaires, kimchi (spicy pickled cabbage), bulgogi (marinated pork) and drinking water served during the period were thought to be the food items potentially associated with illness (Table 1). The relative risks of the kimchi served for lunch on June 3 and the bulgogi served for dinner on June 4 were 2.33 (95% CI 1.80-3.01) and 2.25 (95% CI 1.14-4.43), respectively. The drinking water served at dinner on June 3, lunch on June 4 and breakfast on June 5 had relative risks and 95% CIs of 2.90 (1.01-8.28), 2.38 (1.085.27) and 2.35 (1.19-4.65), respectively.

Microbiological analysis A total of 235 rectal swabs collected from students, staff, and food employees with gastroenteritis were subjected to bacteriological and virological assessments, and 82 (34.9%) samples were positive for DEC. The most frequently isolated type of DEC was EPEC (58.5%, 48/82), followed by EHEC (26.8%, 22/82) and EAEC (14.7%, 12/82). All EHEC isolates were serotyped as O103:H2, whereas the EPEC and EAEC specimens were O untypeable

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(ONT). Among the 55 environmental samples, we isolated 14 EHEC O103:H2 strains from drinking water obtained from the camp cafeteria and four EPEC ONT strains (two from water and two from a washbasin). No microorganisms were found in samples taken from preserved foods or cooking utensils. The genetic relatedness of the clinical and environmental strains was analysed via PFGE. All but one strain of the EHEC isolates exhibited an identical EHHX01.161 PFGE pattern (Figure 2). Nine different PFGE patterns were found among the EPEC isolates: EPCX01.229 in 33 isolates, including four environmental strains (two from drinking water and two from a washbasin), and EPCX01.228 in 9 human isolates. Antimicrobial susceptibility testing showed that all the EHEC and EPEC isolates were susceptible to the tested antimicrobial agents, with the exceptions of one EHEC isolate that was resistant to cefotaxime (MIC, 16 µg/ml) and 37 EPEC isolates clustered based on PFGE patterns that exhibited a MIC value of >128 µg/ml for nalidixic acid.

Environmental investigation The noncommunity water system in the camp used groundwater drawn from a private underground reservoir for cooking and drinking. An inspection revealed critical flaws in the camp’s water distribution system: 1) The outbreak investigation found that a pipeline connected to an auto-chlorination device had been pulled out of the water storage tank. Before the outbreak, the chlorination level of water from this camp had been being regularly tested and maintained according to the camp’s own management process. Therefore, we assumed that the pipeline connecting the tank and the drinking water supply might have been disconnected shortly before this outbreak, resulting in an increased possibility of contamination of the water distribution system; 2) residual free chlorine was not detected in the water samples because the residual chlorine levels of the water were lower than the 9

recommend level of 0.2 ppm (Ercumen et al., 2014); and 3) the water purifier in the camp cafeteria was equipped with filtering cartridges for the removal of fine particles (such as sand, silt and clay) rather than bacteria or viruses.

Discussion Surveillance for diarrhoeal diseases in 2012-2015 in Korea (http://www.is.cdc.go.kr) indicated that approximately 25.6% of cases of acute bacterial diarrhoea were attributable to pathogenic E. coli. Among these cases, school outbreaks accounted for 66 of the 120 (55%) reported cases of E. coli outbreaks (KCDC, 2013; KCDC, 2014; KCDC, 2015; KCDC, 2016). In humans, symptoms of DEC infection are associated with gastrointestinal disease and can range from mild diarrhoea to severe abdominal pain and/or cramping, depending on the pathogenic type of E. coli involved (Dutta et al., 2013). Particularly, EHEC infection can cause bloody diarrhoea and potentially life-threatening complications, such as haemolytic uremic syndrome (Brooks et al., 2004). In Korea, EHEC infection was designated a nationally notifiable disease and was associated with 12 foodborne and waterborne outbreaks from 2012 to 2015.

In June 2015, a waterborne outbreak of E. coli infections associated with contaminated drinking water occurred at a school camp; in this outbreak, 188 patients (30.9%) from three schools experienced acute gastrointestinal symptoms. We hypothesized that the main cause of this outbreak was drinking water that had not been properly disinfected or filtered prior to consumption. This assumption was supported by an epidemiological investigation indicating that acute gastroenteritis in the patients was linked to the drinking water supplied by the camp during the outbreak period and by the shape of the epidemic curve, which was consistent with a continuous-source outbreak. Additionally, the EHEC and EPEC strains isolated from clinical

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faecal specimens and water samples obtained from water purifiers and water basins, respectively, displayed indistinguishable PFGE banding patterns.

A number of waterborne outbreaks have been associated with the following potential water system-related causative factors: inappropriate cross-connections between the reservoir and drinking water pipelines, failure of the disinfection system, and leaks in the underground storage tank (Franklin et al., 2009;Gallay et al., 2006). In this study, water distribution at the camp was regarded as the most likely source of the outbreak. We found that the filtration provided by a water purifier at a camp cafeteria was insufficient to remove contaminants during the outbreak period. Furthermore, the cross-connection between the reservoir and the chlorine sterilization pipelines in the camp’s water supply system had recently broken. This issue was known to be recent because a periodic water inspection conducted before the outbreak indicated that the camp had safe drinking water. The sewage at the camp was managed in two stages: the first treatment was a disinfection process (chlorination) to remove pathogenic microorganisms that cause disease. The second treatment was a filtration system that used a filter capable of removing dissolved particles such as dust, sand, parasites, bacteria and viruses. The drinking water went through a purification process that used appropriate treatment and filtration to remove contaminants. Typically, the purification process for drinking water is controlled by a public system or local government; however, in this case, the camp managed the purification process for drinking water their own. Given that E. coli is used as a bacterial indicator of faecal contamination in water (Saxena et al., 2015), the isolation of DEC strains from this outbreak indicated that the camp’s drinking water might have been mixed with faeces. Eventually, contaminated water caused disease among camp attendees. Unfortunately, however, potential sources of groundwater contamination were not identified in our study.

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As follow-up actions in response to the described outbreak, the cafeteria’s filter was replaced with a reverse osmosis membrane capable of removing viruses and bacteria during the purification process. Chlorination was reinitiated following the cleaning and disinfection of the water distribution system, including underground storage tanks and pipelines.

Waterborne outbreaks associated with contaminated drinking water remain a public health concern around the world and are continuously monitored in several countries. Because outbreaks in schools, camps and group facilities that use groundwater are frequently reported in Korea, the systematic surveillance and management of drinking water quality with respect to waterborne diseases should be strengthened to prevent and control waterborne outbreaks.

Conflict of interest The authors report no potential conflicts of interest in relation to this paper.

Acknowledgements This work was supported by a grant from the Korea Centers for Disease Control and Prevention (4847-311-210).

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References Ashbolt NJ. Microbial Contamination of Drinking Water and Human Health from Community Water Systems. Curr Environ Health Rep 2015;2:95-106. Braeye T, K DES, Wollants E, van Ranst M, Verhaegen J. A large community outbreak of gastroenteritis associated with consumption of drinking water contaminated by river water, Belgium, 2010. Epidemiol Infect 2015;143:711-9. Brooks JT, Bergmire-Sweat D, Kennedy M, Hendricks K, Garcia M, Marengo L, et al. Outbreak of Shiga toxin-producing Escherichia coli O111:H8 infections among attendees of a high school cheerleading camp. Clin Infect Dis 2004;38:190-8. Chen KT, Chen CJ, Chiu JP. A school waterborne outbreak involving both Shigella sonnei and Entamoeba histolytica. J Environ Health 2001;64:9-13, 26. Coleman BL, Louie M, Salvadori MI, McEwen SA, Neumann N, Sibley K, et al. Contamination of Canadian private drinking water sources with antimicrobial resistant Escherichia coli. Water Res 2013;47:3026-36. Dutta S, Guin S, Ghosh S, Pazhani GP, Rajendran K, Bhattacharya MK, et al. Trends in the prevalence of diarrheagenic Escherichia coli among hospitalized diarrheal patients in Kolkata, India. PLoS One 2013;8:e56068. Ercumen A, Gruber JS, Colford JM, Jr. Water distribution system deficiencies and gastrointestinal illness: a systematic review and meta-analysis. Environ Health Perspect 2014;122:651-60. Franklin LJ, Fielding JE, Gregory J, Gullan L, Lightfoot D, Poznanski SY, et al. An outbreak of Salmonella Typhimurium 9 at a school camp linked to contamination of rainwater tanks. Epidemiol Infect 2009;137:434-40. Gallay A, De Valk H, Cournot M, Ladeuil B, Hemery C, Castor C, et al. A large multi-pathogen waterborne community outbreak linked to faecal contamination of a groundwater

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system, France, 2000. Clin Microbiol Infect 2006;12:561-70. Hoebe CJ, Vennema H, de Roda Husman AM, van Duynhoven YT. Norovirus outbreak among primary schoolchildren who had played in a recreational water fountain. J Infect Dis 2004;189:699-705. KCDC. Epidemiological Investigation of Infectious Diseases in Korea Annual Report 2012 2013. Available at: http://cdc.go.kr/CDC/cms/content/87/24787_view.html. [Accessed 28 August 2017.] KCDC. Epidemiological Investigation of Infectious Diseases in Korea Annual Report 2013 2014. Available at: http://cdc.go.kr/CDC/cms/content/02/26002_view.html. [Accessed 28 August 2017.] KCDC. Epidemiological Investigation of Infectious Diseases in Korea Annual Report 2014 2015. Available at: http://cdc.go.kr/CDC/cms/content/89/67789_view.html. [Accessed 28 August 2017.] KCDC. Epidemiological Investigation of Infectious Diseases in Korea Annual Report 2015 2016. Available at: http://cdc.go.kr/CDC/cms/content/77/71877_view.html. [Accessed 28 August 2017.] Laine J, Huovinen E, Virtanen MJ, Snellman M, Lumio J, Ruutu P, et al. An extensive gastroenteritis outbreak after drinking-water contamination by sewage effluent, Finland. Epidemiol Infect 2011;139:1105-13. Licence K, Oates KR, Synge BA, Reid TM. An outbreak of E. coli O157 infection with evidence of spread from animals to man through contamination of a private water supply. Epidemiol Infect 2001;126:135-8. Lienemann T, Pitkanen T, Antikainen J, Molsa E, Miettinen I, Haukka K, et al. Shiga toxinproducing Escherichia coli O100:H(-): stx2e in drinking water contaminated by waste water in Finland. Curr Microbiol 2011;62:1239-44.

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McCall BJ, Slinko VG, Smith HV, Heel K, Culleton TH, Kelk VR, et al. An outbreak of shiga toxin-producing Escherichia coli infection associated with a school camp. Communicable Diseases Intelligence Quarterly Report 2010;34:54-6. Mellou K, Katsioulis A, Potamiti-Komi M, Pournaras S, Kyritsi M, Katsiaflaka A, et al. A large waterborne gastroenteritis outbreak in central Greece, March 2012: challenges for the investigation and management. Epidemiol Infect 2014;142:40-50. Pons W, Young I, Truong J, Jones-Bitton A, McEwen S, Pintar K, et al. A Systematic Review of Waterborne Disease Outbreaks Associated with Small Non-Community Drinking Water Systems in Canada and the United States. PLoS One 2015;10:e0141646. Riera-Montes M, Brus Sjolander K, Allestam G, Hallin E, Hedlund KO, Lofdahl M. Waterborne norovirus outbreak in a municipal drinking-water supply in Sweden. Epidemiol Infect 2011;139:1928-35. Saxena T, Kaushik P, Krishna Mohan M. Prevalence of E. coli O157:H7 in water sources: an overview on associated diseases, outbreaks and detection methods. Diagn Microbiol Infect Dis 2015;82:249-64. Sezen F, Aval E, Agkurt T, Yilmaz S, Temel F, Gulesen R, et al. A large multi-pathogen gastroenteritis outbreak caused by drinking contaminated water from antique neighbourhood fountains, Erzurum city, Turkey, December 2012. Epidemiol Infect 2015;143:704-10. Shin J, Oh SS, Oh KH, Park JH, Jang EJ, Chung GT, et al. An outbreak of foodborne illness caused by enteroaggregative Escherichia coli in a high school in South Korea. Jpn J Infect Dis 2015;68:514-9. Sinclair C, Jenkins C, Warburton F, Adak GK, Harris JP. Investigation of a national outbreak of STEC Escherichia coli O157 using online consumer panel control methods: Great Britain, October 2014. Epidemiol Infect 2017;145:864-71.

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Swerdlow DL, Woodruff BA, Brady RC, Griffin PM, Tippen S, Donnell HD, Jr., et al. A waterborne outbreak in Missouri of Escherichia coli O157:H7 associated with bloody diarrhea and death. Ann Intern Med 1992;117:812-9. Wallender EK, Ailes EC, Yoder JS, Roberts VA, Brunkard JM. Contributing factors to disease outbreaks associated with untreated groundwater. Groundwater 2014;52:886-97. WHO/UNICEF. Progress on sanitation and drinking water 2015 update and MDG assessment 2015. Available at: http://www.who.int/water_sanitation_health/monitoring/jmp-2015update/en/. [Accessed 11 July 2017.]

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Figure legends Figure 1. Cases of gastroenteritis by onset of illness during a camp outbreak, 2015

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Figure 2. Dendrogram of the XbaI-PFGE patterns of (a) EHEC and (b) EPEC strains isolated from clinical and environmental samples. This dendrogram was constructed with BioNumerics v5.1 software (Applied-Maths, Belgium) by utilizing the unweighted-pair group method with arithmetic means and a Dice coefficient (1.5% optimization and 1.5% position tolerance).

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Table. Univariate analysis of the relative risk (RR) for gastroenteritis during a camp outbreak, June 3-5, 2015. AR, attack rate; RR, relative risk; CI, confidence interval. Exposed Date

6/3

Lunch

Dinner

6/4

Breakfast

Lunch

Dinner

6/5

Breakfast

Food item

Unexposed

Reliability AR (%)

P-value

Min

Max

3

43

0.8389

1.07

0.73

1.59

45

17

38

0.8389

1.07

0.73

1.59

30

8

26

0.2141

1.50

0.82

2.74

39

37

15

41

0.9479

0.98

0.65

1.47

127

33

81

54

67

0.0000

0.51

0.41

0.62

241

134

55

226

54

24

0.0000

2.33

1.80

3.01

417

175

42

20

4

20

0.0857

2.10

0.87

5.08

Rice

455

186

40

11

2

18

0.2281

2.25

0.64

7.91

Pork cutlet

336

179

53

10

4

40

0.6120

1.33

0.62

2.86

Cream soup

331

147

44

125

34

27

0.0012

1.63

1.20

2.23

Corn salad

347

141

40

108

29

27

0.4674

1.13

0.85

1.49

Pickled radish

383

156

40

70

23

33

0.2687

1.24

0.87

1.77

Kimchi

307

139

45

158

49

31

0.0041

1.46

1.12

1.90

Purified water

375

163

43

20

3

15

0.0226

2.90

1.01

8.28

Yogurt

332

174

52

23

6

26

0.0260

2.01

1.00

4.03

Rice

459

186

40

8

2

25

0.6003

1.62

0.49

5.41

Bean sprout soup

279

114

40

184

72

39

0.7836

1.04

0.83

1.31

Braised kimchi

387

163

42

73

21

29

0.0449

1.46

1.00

2.14

Stir-fried sausage

422

174

41

39

11

28

0.1564

1.46

0.87

2.44

Seaweed

411

167

40

51

17

33

0.3938

1.22

0.81

1.83

Purified water

423

169

40

18

6

33

0.7518

1.20

0.62

2.33

Kimchi

240

105

43

225

82

36

0.1308

1.20

0.96

1.50

Multi-grain rice

455

185

40

9

3

33

0.9200

1.22

0.48

3.09

Soybean soup

296

122

41

165

62

37

0.5054

1.10

0.86

1.39

Bulgogi

420

174

41

38

7

18

0.0092

2.25

1.14

4.43

Topokki

423

172

40

32

8

25

0.1189

1.63

0.88

3.00

Seasoned vegetables

212

90

42

244

89

36

0.2272

1.16

0.93

1.46

Purified water

415

190

45

26

5

19

0.0146

2.38

1.08

5.27

Kimchi

231

105

45

232

80

34

0.0206

1.32

1.05

1.65

Bibimbap

363

130

36

100

42

42

0.3092

0.85

0.65

1.12

Rice

422

165

39

30

11

37

0.9440

1.07

0.66

1.73

Purified water

410

166

40

31

7

23

0.0754

1.79

0.92

3.48

Fried tofu soup

373

124

33

80

53

66

0.0000

0.50

0.41

0.62

Rice

442

176

39

23

12

52

0.3374

0.76

0.51

1.15

Seaweed soup

380

149

39

82

36

44

0.5079

0.89

0.68

1.18

Stir-fried fish cake

363

148

41

96

34

35

0.4029

1.15

0.86

1.55

Stir-fried sausage

405

162

40

54

19

35

0.5948

1.14

0.78

1.66

Soy sauce braised pork

390

153

39

77

30

39

0.9335

1.01

0.74

1.37

Total Cases

AR (%)

Multi-grain rice

462

185

40

7

Fish cake soup

419

170

40

Braised spicy chicken

421

168

39

Sweet and sour pork

419

166

Seasoned vegetables

376

Kimchi Purified water

20

Total Cases

RR

0.95

Kimchi

225

97

43

237

86

36

0.1604

1.19

0.95

1.49

Purified water

401

165

41

40

7

17

0.0059

2.35

1.19

4.65

21