Outbreak of CTX-M-15-producing enterotoxigenic Escherichia coli O159:H20 in the

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1 AAC Accepted Manuscript Posted Online 26 June 2017 Antimicrob. Agents Chemother. doi: /aac Copyright 2017 American Society for Microbiology. All Rights Reserved. 1 2 Outbreak of CTX-M-15-producing enterotoxigenic Escherichia coli O159:H20 in the Republic of Korea, Jin Seok Kim 1, Jungsun Park 1, Eunkyung Shin 1, Soojin Kim 1, Sung Suck Oh 2, Hyo-Jin Yang 3, Dae-Won Kim 3, Kyung-Hwan Oh 1, Yonghoon Kim 1, Min Kim 2, Mun Ju Kwon 2, Kyoungin Na 4, Jin Lee 4, En-hi Cho 4, Byung- Hak Kang 1, Hyo-Sun Kwak 1, Won Keun Seong 1 and Junyoung Kim 1# 1 Division of Enteric Diseases, Center for Infectious Diseases, National Institute of Health, Chungcheongbuk-do, Republic of Korea 2 Incheon Research Institute of Public Health and Environment, Incheon Metropolitan City, Republic of Korea 3 Division of Biosafety Evaluation and Control, National Institute of Health, Chungcheongbuk-do, Republic of Korea 4 Division of Infectious Disease Surveillance, Korea Centers for Disease Control and Prevention, Chungcheongbuk-do, Republic of Korea Running Title: Outbreak of CTX-M-15-producing ETEC in Korea # Corresponding author: Junyoung Kim Division of Enteric Diseases, Center for Infectious Diseases, National Institute of Health, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, 28159, Republic of Korea Tel: ; Fax: ; jun49@hanmail.net 1

2 Abstract We investigated an outbreak of enterotoxigenic Escherichia coli (ETEC) O159:H20 associated with the consumption of a tossed noodle dish in a high school in Thirty-three ETEC strains isolated from both clinical and food samples were genetically indistinguishable. The outbreak strains were resistant to third-generation cephalosporins and harbored a bla CTX- M-15 gene on a 97-kb self-transferable IncK plasmid. This is the first outbreak caused by CTX- M-15-producing ETEC strains. Main Text Enterotoxigenic Escherichia coli (ETEC) is a major etiological agent of acute gastroenteritis in developing countries, where an estimated 2.5 million cases of infection and 700,000 deaths in children younger than 5 years of age are attributable to ETEC each year (1). In developed countries, ETEC is frequently implicated as a cause of diarrhea among travelers visiting endemic areas and of foodborne outbreaks (1, 2). The main pathogenic characteristic of ETEC strains is two classes of enterotoxins, namely, heat-stable toxin (ST) and heat-labile toxin (LT), which elevate intracellular levels of cyclic GMP and cyclic AMP, respectively, resulting in secretory diarrhea (3). Most cases of ETECinduced acute diarrhea are self-limiting; however, because prolonged diarrhea can result in severe dehydration, which is a high risk factor for mortality, antimicrobials are sometimes considered for the treatment of ETEC infections such as traveler s diarrhea (1). However, the emergence of drug resistance to available antimicrobials complicates treatment and leads to public health concerns (1, 4, 5). Here, we describe the first outbreak caused by cefotaximeresistant ETEC O159:H20 strains at a high school in Incheon, Korea. 2

3 In June 2016, the Incheon public health department was notified of acute diarrheal illness in a high school (with the first reported diarrheal episode occurring on June 23); subsequently, the department initiated an outbreak investigation to identify patients and sources of infection and determine the extent of the outbreak. In total, 653 persons who had eaten meals served at the school from June 20 to June 23 were interviewed regarding their symptoms of illness and food consumption. A case-patient was defined as an individual with at least three loose stools per day or two watery stools accompanied by other gastrointestinal symptoms. On June 27, we collected 51 rectal swabs (38 from case-patients and 13 from food handlers) and 113 environmental samples including 106 preserved food products consumed during the outbreak, 4 cooking utensils, and 3 drinking water samples. All samples were screened for enteric pathogens using routine microbiological protocols (6). Bacterial identification was performed using the Vitek 2 system (biomérieux, Marcy-l Etoile, France), and presumptively identified diarrheagenic E. coli strains were then confirmed based on toxin-specific PCRs (esta and eltb for ETEC and eaea for enteropathogenic E. coli [EPEC]) (7). All isolated strains were sent to the Korea National Institute of Health for further analysis, where serotyping for O and H antigens (Denka Seiken, Tokyo, Japan), multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed (8). Antimicrobial susceptibility was tested via the broth microdilution method using a customized KRCDCF panel with 16 antimicrobials and a commercial ESB1F plate (Sensititre, TREK Diagnostic Systems, OH, USA) according to CLSI guidelines (9); for cefotaxime-resistant strains, extended-spectrum beta-lactamase (ESBL) genes were analyzed using multiplex PCR and sequencing (10). From one selected isolate, the plasmid harboring the bla gene was transferred into the azide-resistant E. coli strain J53 by transconjugation and was 3

4 characterized by PCR-based Inc/rep typing (PBRT) (11, 12). The number and size of the plasmid in the donor and transconjugant strains were verified by PFGE with S1-nuclease digestion of whole-cell DNA (S1-PFGE) (13). The genetic environment of the bla CTX-M-15 gene was determined using PCR, sequencing and the primer walking method (10) From clinical specimens, we isolated 35 pathogenic E. coli strains, including 33 ETEC strains (esta only; STh) and 2 EPEC strains (eaea); one ETEC (STh) strain was also identified from a food item served on June 21, tofu-konjac noodles tossed with mixed vegetables. All ETEC strains, except for one strain from a food handler, were serotyped as O159:H20 and classified into ST2040 by MLST ( Consistently, PFGE results indicated that thirty-three ETEC O159 strains had an identical XbaI macrorestriction pattern (ETCX01.177, as assigned by Korea PulseNet) (Fig. 1). The outbreak strains exhibited the same antimicrobial resistance to third-generation cephalosporins (cefotaxime MIC, 16 μg/ml), and their ESBL phenotypes were confirmed by the synergy test with clavulanic acid (Table 1). PCR and sequencing analysis indicated that these strains harbor the bla CTX-M-15 gene located on an approximately 90-kb IncK plasmid (estimated by S1-PFGE profiling), which was successfully transferred from one representative strain (ET ) into E. coli J53. Analysis of the regions flanking bla CTX-M-15 revealed that an orf477 sequence was found downstream but that none of the transposable elements frequently associated with the bla CTX- M were observed in the upstream region. Instead, sequencing by primer walking revealed a truncated bla TEM gene and putative promoter sequences between a right inverted repeat of an ISEcp1 transposable element and the bla gene (Fig. 2). This genetic structure (Δbla TEM - 4

5 bla CTX-M-15 -orf477) has not previously been described in the literature, but the analyzed sequences were similar to those of an E. coli plasmid isolated from a sewage treatment plant in India (14). Interestingly, a plasmid of Shigella sonnei isolated from a diarrheal patient in Incheon in 2015 shared the same plasmid replicon type (IncK) and genetic environment of bla CTX-M-15 (J. Kim, submitted for publication) To determine the complete structure of pet , plasmid DNA was extracted using the Qiagen plasmid Midi kit (Qiagen, CA, USA), and then sequenced using the Illumina Miseq platform (Illumina, CA, USA). The sequencing reads were assembled as described previously (12), and annotations were performed using Rapid Annotation using Subsystem Technology (RAST; Plasmid pet is 97,940 bp in size, with an average GC content of 51.0% and harbors 128 predicted open reading frames (ORFs). Based on comparative analysis of IncK plasmids, pet contains an IncK replicon detected in the PBRT scheme (11) and conserved plasmid backbone of IncK responsible for plasmid replication, stability and partitioning (12, 15, 16). However, this plasmid was untypeable with new sets of primers for IncK sequence typing (12), and showed 46.6 and 47.5% overall nucleotide identity with the plasmid pct (IncK1, GenBank accession no. NC_014477) and pdv10 (IncK2, GenBank accession no. KR905390), respectively. Furthermore, conjugal transfer system (tra locus) of IncI1 plasmids was found in pet These results indicated that pet differs from the recently described IncK1 and IncK2 plasmids (12, 16), and may be a variant of the IncK plasmid. In this study, we investigated a school outbreak of gastroenteritis due to the ingestion of food contaminated with ETEC O159:H20 strains. Unfortunately, the inspection of companies that supplied food materials to school was not carried out during outbreak, thus we were 5

6 unable to identify the potential sources of food contamination. ETEC can attach firmly to the surface of salad leaves using their flagella and survive environmental conditions for several months (17). As observed in the large E. coli O104:H4 outbreaks associated with contaminated sprouts in Europe in 2011 (17), raw vegetables in the tossed noodle dish in this study may have acted as favorable vehicles for pathogenic E. coli transmission; this possibility emphasizes the importance of sanitation and hand hygiene in the food-processing workplace. ETEC serotype O159 was reported to be a common serotype in Japan, where it was linked to 12 outbreaks from 1966 to 2009 in Tokyo (18). Similarly, in Korea, this serotype has been identified in sporadic cases and in three outbreaks since 2014; however, these prior ETEC serotype O159 pathogens exhibited different PFGE patterns from the pattern observed in this outbreak strain and were all susceptible to third-generation cephalosporins (J. Kim, unpublished data). To our knowledge, the outbreak described here is the first instance of an outbreak of ETEC O159:H20 that produces CTX-M-15 ESBL. Third-generation cephalosporins, which have been used for the empirical treatment of infections with various enteric pathogens, are not the current first-line treatment for ETEC infections or traveler s diarrhea. However, cefotaxime resistance is rapidly increasing among ETEC strains and could be horizontally transferred by a self-transferable plasmid. Therefore, it is necessary to monitor antimicrobial susceptibilities for pathogenic E. coli strains, including ETEC Nucleotide sequence accession number The DNA sequence of pet , which was examined in this paper, has been deposited in GenBank under accession no. KY and MF

7 Acknowledgments This work was supported by a grant from the Korea Centers for Disease Control and Prevention [ ] Conflict of interest We have no conflicts of interest to declare. Downloaded from on April 9, 2018 by guest 7

8 References 1. Qadri F, Svennerholm AM, Faruque AS, and Sack RB Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev 18: Dalton CB, Mintz ED, Wells JG, Bopp CA, and Tauxe RV Outbreaks of enterotoxigenic Escherichia coli infection in American adults: a clinical and epidemiologic profile. Epidemiol Infect 123: Kaper JB, Nataro JP, and Mobley HL Pathogenic Escherichia coli. Nat Rev Microbiol 2: Oh KH, Kim DW, Jung SM, and Cho SH Molecular characterization of enterotoxigenic Escherichia coli strains isolated from diarrheal patients in Korea during PLoS One 9:e Begum YA, Talukder KA, Azmi IJ, Shahnaij M, Sheikh A, Sharmin S, Svennerholm AM, and Qadri F Resistance pattern and molecular characterization of enterotoxigenic Escherichia coli (ETEC) strains isolated in Bangladesh. PLoS One 11:e Shin J, Oh SS, Oh KH, Park JH, Jang EJ, Chung GT, Yoo CK, Bae GR, and Cho SH An outbreak of foodborne illness caused by enteroaggregative Escherichia coli in a high school in South Korea. Jpn J Infect Dis 68: Oh KH, Kim SB, Park MS, and Cho SH Development of a one-step PCR assay with nine primer pairs for the detection of five diarrheagenic Escherichia coli types. J Microbiol Biotechnol 24: Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, and Barrett TJ Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. 8

9 Foodborne Pathog Dis 3: Clinical and Laboratory Standards Institute Performance standards for antimicrobial susceptibility testing; Twenty-fourth informational supplement (M100- S24). Clinical and Laboratory Standards Institute, Wayne, PA. 10. Kim JS, Kim MJ, Kim SJ, Shin E, Oh KH, Kim SG, Chung GT, Yoo CK, Seo KW, and Kim J First description of CTX-M-3 extended-spectrum beta-lactamase in an outbreak strain of Shiga toxin-producing Escherichia coli O103:H2. Int J Antimicrob Agents 47: Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, and Threlfall EJ Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 63: Rozwandowicz M, Brouwer MS, Zomer AL, Bossers A, Harders F, Mevius DJ, Wagenaar JA, and Hordijk J Plasmids of distinct IncK lineages show compatible phenotypes. Antimicrob Agents Chemother 61:e Barton BM, Harding GP, and Zuccarelli AJ A general method for detecting and sizing large plasmids. Anal Biochem 226: Akiba M, Sekizuka T, Yamashita A, Kuroda M, Fujii Y, Murata M, Lee K, Joshua DI, Balakrishna K, Bairy I, Subramanian K, Krishnan P, Munuswamy N, Sinha RK, Iwata T, Kusumoto M, and Guruge KS Distribution and relationships of antimicrobial resistance determinants among extended-spectrumcephalosporin-resistant or carbapenem-resistant Escherichia coli isolates from rivers and sewage treatment plants in India. Antimicrob Agents Chemother 60: Hansen KH, Bortolaia V, Nielsen CA, Nielsen JB, Schonning K, Agerso Y, and Guardabassi L Host-specific patterns of genetic diversity among IncI1-Iγ and IncK plasmids encoding CMY-2 β-lactamase in Escherichia coli isolates from humans, 9

10 poultry meat, poultry, and dogs in Denmark. Appl Environ Microbiol 82: Seiffert SN, Carattoli A, Schwendener S, Collaud A, Endimiani A, and Perreten V Plasmids carrying bla CMY-2/4 in Escherichia coli from poultry, poultry meat, and humans belong to a novel IncK subgroup designated IncK2. Front Microbiol 8: Shaw RK, Berger CN, Pallen MJ, Sjoling A, and Frankel G Flagella mediate attachment of enterotoxigenic Escherichia coli to fresh salad leaves. Environ Microbiol Rep 3: Konishi N, Obata H, Monma C, Nakama A, Kai A, and Tsuji T Bacteriological and epidemiological characteristics of enterotoxigenic Escherichia coli isolated in Tokyo, Japan, between 1966 and J Clin Microbiol 49: Downloaded from on April 9, 2018 by guest 10

11 Table 1 Antimicrobial susceptibility profiles of enterotoxigenic Escherichia coli ET , the representative strain of a school outbreak in Incheon, and its transconjugant strain ET TC Antimicrobial agents enterotoxigenic E. coli (ET ) MIC, μg/ml E. coli J53 E. coli J53 (ET TC) Ampicillin >64 >64 >64 Ampicillin-sulbactam Amoxicillin-clavulanate Piperacillin-Tazobactam <4 <4 <4 Cephalothin >64 16 >64 Cefepime 2 <1 <1 Cefoxitin Cefpodoxime > >32 Ceftazidime 8 < Ceftriaxone 32 < Cefotaxime 32 < Cefotaxime-clavulanate <0.12 <0.12 <0.12 Imipenem <0.5 <0.5 <0.5 Meropenem <1 <1 <1 Gentamicin <1 <1 <1 Amikacin <4 <4 <4 Streptomycin Tetracycline <2 <2 <2 Nalidixic acid Ciprofloxacin Trimethoprim-sulfamethoxazole <1 <1 <1 Chloramphenicol

12 Figure legends Figure 1. Dendrogram of XbaI-PFGE patterns of 34 ETEC isolates in this foodborne outbreak This dendrogram was constructed with BioNumerics v5.1 software (Applied-Maths, Belgium) by utilizing the unweighted-pair group method with arithmetic means (UPGMA) and a Dice coefficient (1.5% optimization and 1.5% position tolerance). Figure 2. Schematic representation of the genetic structure surrounding bla CTX-M-15 on plasmid pet The hatched box and the arrow indicate the inverted repeat (IR) of the ISEcp1 element and the transcriptional start site for the bla CTX-M-15 gene, respectively. Downloaded from on April 9, 2018 by guest 12

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