Genomic analysis of Shiga toxin-producing Escherichia coli from patients and asymptomatic food handlers in Japan

Autoři: Hiroaki Baba aff001;  Hajime Kanamori aff001;  Hayami Kudo aff002;  Yasutoshi Kuroki aff002;  Seiya Higashi aff002;  Kentaro Oka aff002;  Motomichi Takahashi aff002;  Makiko Yoshida aff001;  Kengo Oshima aff001;  Tetsuji Aoyagi aff001;  Koichi Tokuda aff001;  Mitsuo Kaku aff001
Působiště autorů: Department of Infectious Diseases, Internal Medicine, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan aff001;  Miyarisan Pharmaceutical Co., Ltd., Saitama-shi, Saitama, Japan aff002
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225340


Shiga toxin-producing Escherichia coli (STEC) can cause severe gastrointestinal disease and colonization among food handlers. In Japan, STEC infection is a notifiable disease, and food handlers are required to undergo routine stool examination for STEC. However, the molecular epidemiology of STEC is not entirely known. We investigated the genomic characteristics of STEC from patients and asymptomatic food handlers in Miyagi Prefecture, Japan. Whole-genome sequencing (WGS) was performed on 65 STEC isolates obtained from 38 patients and 27 food handlers by public health surveillance in Miyagi Prefecture between April 2016 and March 2017. Isolates of O157:H7 ST11 and O26:H11 ST21 were predominant (n = 19, 29%, respectively). Non-O157 isolates accounted for 69% (n = 45) of all isolates. Among 48 isolates with serotypes found in the patients (serotype O157:H7 and 5 non-O157 serotypes, O26:H11, O103:H2, O103:H8, O121:H19 and O145:H28), adhesion genes eae, tir, and espB, and type III secretion system genes espA, espJ, nleA, nleB, and nleC were detected in 41 to 47 isolates (85–98%), whereas isolates with other serotypes found only in food handlers were negative for all of these genes. Non-O157 isolates were especially prevalent among patients younger than 5 years old. Shiga-toxin gene stx1a, adhesion gene efa1, secretion system genes espF and cif, and fimbrial gene lpfA were significantly more frequent among non-O157 isolates from patients than among O157 isolates from patients. The most prevalent resistance genes among our STEC isolates were aminoglycoside resistance genes, followed by sulfamethoxazole/trimethoprim resistance genes. WGS revealed that 20 isolates were divided into 9 indistinguishable core genomes (<5 SNPs), demonstrating clonal expansion of these STEC strains in our region, including an O26:H11 strain with stx1a+stx2a. Non-O157 STEC with multiple virulence genes were prevalent among both patients and food handlers in our region of Japan, highlighting the importance of monitoring the genomic characteristics of STEC.

Klíčová slova:

Age groups – Antimicrobial resistance – Escherichia coli – Japan – Phylogenetic analysis – Secretion systems – Virulence factors – Hemolytic-uremic syndrome


1. Amezquita-Lopez BA, Soto-Beltran M, Lee BG, Yambao JC, Quinones B. Isolation, genotyping and antimicrobial resistance of Shiga toxin-producing Escherichia coli. J Microbiol Immunol Infect. 2018;51: 425–434. doi: 10.1016/j.jmii.2017.07.004 28778595

2. Valilis E, Ramsey A, Sidiq S, DuPont HL. Non-O157 Shiga toxin-producing Escherichia coli-A poorly appreciated enteric pathogen: Systematic review. Int J infect Dis. 2018;76: 82–87. doi: 10.1016/j.ijid.2018.09.002 30223088

3. Terajima J, Iyoda S, Ohnishi M, Watanabe H. Shiga Toxin (Verotoxin)-Producing Escherichia coli in Japan. Microbiol Spectr. 2014;2(5).

4. Harada T, Hirai Y, Itou T, Hayashida M, Seto K, Taguchi M, et al. Laboratory investigation of an Escherichia coli O157:H7 strain possessing a vtx2c gene with an IS1203 variant insertion sequence isolated from an asymptomatic food handler in Japan. Diagn Microbiol Infect Dis. 2013;77: 176–178. doi: 10.1016/j.diagmicrobio.2013.06.012 23891550

5. Scheutz F, Teel LD, Beutin L, Pierard D, Buvens G, Karch H, et al. Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol. 2012;50: 2951–2963. doi: 10.1128/JCM.00860-12 22760050

6. Day M, Doumith M, Jenkins C, Dallman TJ, Hopkins KL, Elson R, et al. Antimicrobial resistance in Shiga toxin-producing Escherichia coli serogroups O157 and O26 isolated from human cases of diarrhoeal disease in England, 2015. J Antimicrob Chemother. 2017;72: 145–152. doi: 10.1093/jac/dkw371 27678285

7. Haugum K, Johansen J, Gabrielsen C, Brandal LT, Bergh K, Ussery DW, et al. Comparative genomics to delineate pathogenic potential in non-O157 Shiga toxin-producing Escherichia coli (STEC) from patients with and without haemolytic uremic syndrome (HUS) in Norway. PLoS ONE. 2014;9(10):e111788. doi: 10.1371/journal.pone.0111788 25360710

8. Ferdous M, Friedrich AW, Grundmann H, de Boer RF, Croughs PD, Islam MA, et al. Molecular characterization and phylogeny of Shiga toxin-producing Escherichia coli isolates obtained from two Dutch regions using whole genome sequencing. Clin Microbiol Infect. 2016;22(7):642.e1–9.

9. World Health Organization. World health report. 50 Facts: Global health situation and trends 1955–2025. 2019. Available from:

10. Nishijima S, Suda W, Oshima K, Kim SW, Hirose Y, Morita H, et al. The gut microbiome of healthy Japanese and its microbial and functional uniqueness. DNA Res. 2016;23: 125–133. doi: 10.1093/dnares/dsw002 26951067

11. Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics. 2014;30: 2114–2120. doi: 10.1093/bioinformatics/btu170 24695404

12. Kajitani R, Toshimoto K, Noguchi H, Toyoda A, Ogura Y, Okuno M, et al. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res. 2014;24: 1384–1395. doi: 10.1101/gr.170720.113 24755901

13. Joensen KG, Tetzschner AM, Iguchi A, Aarestrup FM, Scheutz F. Rapid and easy in silico serotyping of Escherichia coli isolates by use of whole-genome sequencing data. J Clin Microbiol. 2015;53: 2410–2426. doi: 10.1128/JCM.00008-15 25972421

14. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, et al. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol. 2012;50:1355–1361. doi: 10.1128/JCM.06094-11 22238442

15. Joensen KG, Scheutz F, Lund O, Hasman H, Kaas RS, Nielsen EM, et al. Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli. J Clin Microbiol. 2014;52: 1501–1510. doi: 10.1128/JCM.03617-13 24574290

16. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67: 2640–2644. doi: 10.1093/jac/dks261 22782487

17. Treangen TJ, Ondov BD, Koren S, Phillippy AM. The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biol. 2014;15: 524. doi: 10.1186/s13059-014-0524-x 25410596

18. Dallman TJ, Byrne L, Ashton PM, Cowley LA, Perry NT, Adak G, et al. Whole-genome sequencing for national surveillance of Shiga toxin-producing Escherichia coli O157. Clin Infect Dis. 2015;61: 305–312. doi: 10.1093/cid/civ318 25888672

19. Stamatakis A. Using RAxML to infer phylogenies. Curr Protoc Bioinformatics. 2015;51: 6.14.1–14.

20. DebRoy C, Fratamico PM, Yan X, Baranzoni G, Liu Y, Needleman DS, et al. Comparison of O-antigen gene clusters of all O-serogroups of Escherichia coli and proposal for adopting a new nomenclature for O-typing. PLoS One, 2016;11: e0147434. doi: 10.1371/journal.pone.0147434 26824864

21. Byrne L, Vanstone GL, Perry NT, Launders N, Adak GK, Godbole G, et al. Epidemiology and microbiology of Shiga toxin-producing Escherichia coli other than serogroup O157 in England, 2009–2013. J Med Microbiol. 2014;63: 1181–1188. doi: 10.1099/jmm.0.075895-0 24928216

22. Matussek A, Jernberg C, Einemo IM, Monecke S, Ehricht R, Engelmann I, et al. Genetic makeup of Shiga toxin-producing Escherichia coli in relation to clinical symptoms and duration of shedding: a microarray analysis of isolates from Swedish children. Eur J Clin Microbiol Infect Dis. 2017;36: 1433–1441. doi: 10.1007/s10096-017-2950-7 28421309

23. Kanayama A, Yahata Y, Arima Y, Takahashi T, Saitoh T, Kanou K, et al. Enterohemorrhagic Escherichia coli outbreaks related to childcare facilities in Japan, 2010–2013. BMC Infect Dis. 2015;15: 539. doi: 10.1186/s12879-015-1259-3 26589805

24. Karmali MA, Mascarenhas M, Shen S, Ziebell K, Johnson S, Reid-Smith R, et al. Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease. J Clin Microbiol. 2003;41: 4930–4940. doi: 10.1128/JCM.41.11.4930-4940.2003 14605120

25. McWilliams BD, Torres AG. Enterohemorrhagic Escherichia coli adhesins. Microbiol Spectr. 2014;2(3).

26. Cepeda-Molero M, Berger CN, Walsham ADS, Ellis SJ, Wemyss-Holden S, Schuller S, et al. Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors. PLoS Pathog. 2017;13(10):e1006706. doi: 10.1371/journal.ppat.1006706 29084270

27. Steyert SR, Sahl JW, Fraser CM, Teel LD, Scheutz F, Rasko DA. Comparative genomics and stx phage characterization of LEE-negative Shiga toxin-producing Escherichia coli. Front Cell Infect Microbiol. 2012;2: 133. doi: 10.3389/fcimb.2012.00133 23162798

28. Iguchi A, Shirai H, Seto K, Ooka T, Ogura Y, Hayashi T, et al. Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli. PLoS ONE. 2011;6(8):e23250. doi: 10.1371/journal.pone.0023250 21876740

29. Hosoi Y, Asai T, Koike R, Tsuyuki M, Sugiura K. Sales of veterinary antimicrobial agents for therapeutic use in food-producing animal species in Japan between 2005 and 2010. Rev Sci Tech. 2014;33: 1007–1015. doi: 10.20506/rst.33.3.2337 25812223

30. Kobayashi H, Kanazaki M, Ogawa T, Iyoda S, Hara-Kudo Y. Changing prevalence of O-serogroups and antimicrobial susceptibility among STEC strains isolated from healthy dairy cows over a decade in Japan between 1998 and 2007. J Vet Med Sci. 2009;71: 363–366. doi: 10.1292/jvms.71.363 19346709

31. Agger M, Scheutz F, Villumsen S, Molbak K, Petersen AM. Antibiotic treatment of verocytotoxin-producing Escherichia coli (VTEC) infection: a systematic review and a proposal. J Antimicrob Chemother. 2015;70: 2440–2446. doi: 10.1093/jac/dkv162 26093376

32. Rabinowitz PM, Kock R, Kachani M, Kunkel R, Thomas J, Gilbert J, et al. Toward proof of concept of a one health approach to disease prediction and control. Emerg Infect Dis. 2013;19(12).

33. Soborg B, Lassen SG, Muller L, Jensen T, Ethelberg S, Molbak K, et al. A verocytotoxin-producing E. coli outbreak with a surprisingly high risk of haemolytic uraemic syndrome, Denmark, September-October 2012. Euro Surveill. 2013;18(2).

34. Lavina M, Gaggero C, Moreno F. Microcin H47, a chromosome-encoded microcin antibiotic of Escherichia coli. J Bacteriol. 1990;172: 6585–6588. doi: 10.1128/jb.172.11.6585-6588.1990 2228975

35. Ishijima N, Lee KI, Kuwahara T, Nakayama-Imaohji H, Yoneda S, Iguchi A, et al. Identification of a new virulent clade in enterohemorrhagic Escherichia coli O26:H11/H- sequence type 29. Sci Rep. 2017;7:43136. doi: 10.1038/srep43136 28230102

36. Bielaszewska M, Mellmann A, Bletz S, Zhang W, Kock R, Kossow A, et al. Enterohemorrhagic Escherichia coli O26:H11/H-: a new virulent clone emerges in Europe. Clin Infect Dis. 2013;56: 1373–1381. doi: 10.1093/cid/cit055 23378282

37. National Institute of Infectious Diseases. Enterohemorrhagic Escherichia coli (EHEC) infection, as of March 2019, Japan. Infect Agents Surveill Rep. 2019;40: 71–72.

38. Rusconi B, Sanjar F, Koenig SS, Mammel MK, Tarr PI, Eppinger M. Whole genome sequencing for genomics-guided investigations of Escherichia coli O157:H7 outbreaks. Front Microbiol. 2016;7:985. doi: 10.3389/fmicb.2016.00985 27446025

Článek vyšel v časopise


2019 Číslo 11