1887

Abstract

The isolation of Shiga toxin-producing (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic . The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene , a microsphere suspension array targeting allelic variants of and a -based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the microsphere array and real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The -based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.

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2009-07-01
2020-04-10
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References

  1. Bastian S. N., Carle I., Grimont F. 1998; Comparison of 14 PCR systems for the detection and subtyping of stx genes in Shiga-toxin-producing Escherichia coli . Res Microbiol 149:457–472 [CrossRef]
    [Google Scholar]
  2. Belanger S. D., Boissinot M., Menard C., Picard F. J., Bergeron M. G. 2002; Rapid detection of Shiga toxin-producing bacteria in feces by multiplex PCR with molecular beacons on the smart cycler. J Clin Microbiol 40:1436–1440 [CrossRef]
    [Google Scholar]
  3. Beutin L., Zimmermann S., Gleier K. 2002; Evaluation of the VTEC-Screen “Seiken” test for detection of different types of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) in human stool samples. Diagn Microbiol Infect Dis 42:1–8 [CrossRef]
    [Google Scholar]
  4. Bischoff C., Luthy J., Altwegg M., Baggi F. 2005; Rapid detection of diarrheagenic E. coli by real-time PCR. J Microbiol Methods 61:335–341 [CrossRef]
    [Google Scholar]
  5. Boudailliez B., Berquin P., Mariani-Kurkdjian P., Ilef D., Cuvelier B., Capek I., Tribout B., Bingen E., Piussan C. 1997; Possible person-to-person transmission of Escherichia coli O111 – associated hemolytic uremic syndrome. Pediatr Nephrol 11:36–39 [CrossRef]
    [Google Scholar]
  6. Caprioli A., Luzzi I., Rosmini F., Resti C., Edefonti A., Perfumo F., Farina C., Goglio A., Gianviti A., Rizzoni G. 1994; Community-wide outbreak of hemolytic-uremic syndrome associated with non-O157 verocytotoxin-producing Escherichia coli . J Infect Dis 169:208–211 [CrossRef]
    [Google Scholar]
  7. Church D. L., Emshey D., Semeniuk H., Lloyd T., Pitout J. D. 2007; Evaluation of BBL CHROMagar O157 versus sorbitol-MacConkey medium for routine detection of Escherichia coli O157 in a centralized regional clinical microbiology laboratory. J Clin Microbiol 45:3098–3100 [CrossRef]
    [Google Scholar]
  8. Fey P. D., Wickert R. S., Rupp M. E., Safranek T. J., Hinrichs S. H. 2000; Prevalence of non-O157 : H7 shiga toxin-producing Escherichia coli in diarrheal stool samples from Nebraska. Emerg Infect Dis 6:530–533 [CrossRef]
    [Google Scholar]
  9. Gannon V. P., King R. K., Kim J. Y., Thomas E. J. 1992; Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Appl Environ Microbiol 58:3809–3815
    [Google Scholar]
  10. Gilmour M. W., Tracz D. M., Andrysiak A. K., Clark C. G., Tyson S., Severini A., Ng L. K. 2006; Use of the espZ gene encoded in the locus of enterocyte effacement for molecular typing of shiga toxin-producing Escherichia coli . J Clin Microbiol 44:449–458 [CrossRef]
    [Google Scholar]
  11. Gilmour M. W., Olson A. B., Andrysiak A. K., Ng L. K., Chui L. 2007a; Sequence-based typing of genetic targets encoded outside of the O-antigen gene cluster is indicative of Shiga toxin-producing Escherichia coli serogroup lineages. J Med Microbiol 56:620–628 [CrossRef]
    [Google Scholar]
  12. Gilmour M. W., Tabor H., Wang G., Clark C. G., Tracz D. M., Olson A. B., Mascarenhas M., Karmali M. A., Mailman T., Ng L. K. 2007b; Isolation and genetic characterization of a coinfection of non-O157 Shiga toxin-producing Escherichia coli . J Clin Microbiol 45:3771–3773 [CrossRef]
    [Google Scholar]
  13. Holland J. L., Louie L., Simor A. E., Louie M. 2000; PCR detection of Escherichia coli O157.  : H7 directly from stools: evaluation of commercial extraction methods for purifying fecal DNA. J Clin Microbiol 38:4108–4113
    [Google Scholar]
  14. Jelacic J. K., Damrow T., Chen G. S., Jelacic S., Bielaszewska M., Ciol M., Carvalho H. M., Melton-Celsa A. R., O'Brien A. D., Tarr P. I. 2003; Shiga toxin-producing Escherichia coli in Montana: bacterial genotypes and clinical profiles. J Infect Dis 188:719–729 [CrossRef]
    [Google Scholar]
  15. Johnson K. E., Thorpe C. M., Sears C. L. 2006; The emerging clinical importance of non-O157 Shiga toxin-producing Escherichia coli . Clin Infect Dis 43:1587–1595 [CrossRef]
    [Google Scholar]
  16. Karch H., Tarr P. I., Bielaszewska M. 2005; Enterohaemorrhagic Escherichia coli in human medicine. Int J Med Microbiol 295:405–418 [CrossRef]
    [Google Scholar]
  17. Karmali M. A., Petric M., Lim C., Fleming P. C., Arbus G. S., Lior H. 1985; The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli . J Infect Dis 151:775–782 [CrossRef]
    [Google Scholar]
  18. Lindstedt B. A., Brandal L. T., Aas L., Vardund T., Kapperud G. 2007; Study of polymorphic variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic Escherichia coli and Shigella isolates for use in a genotyping assay. J Microbiol Methods 69:197–205 [CrossRef]
    [Google Scholar]
  19. Lockary V. M., Hudson R. F., Ball C. L. 2007; Shiga toxin-producing Escherichia coli , Idaho. Emerg Infect Dis 13:1262–1264
    [Google Scholar]
  20. Louie M., Read S., Simor A. E., Holland J., Louie L., Ziebell K., Brunton J., Hii J. 1998; Application of multiplex PCR for detection of non-O157 verocytotoxin-producing Escherichia coli in bloody stools: identification of serogroups O26 and O111. J Clin Microbiol 36:3375–3377
    [Google Scholar]
  21. Manning S. D., Madera R. T., Schneider W., Dietrich S. E., Khalife W., Brown W., Whittam T. S., Somsel P., Rudrik J. T. 2007; Surveillance for Shiga toxin-producing Escherichia coli , Michigan, 2001–2005. Emerg Infect Dis 13:318–321 [CrossRef]
    [Google Scholar]
  22. McCarthy T. A., Barrett N. L., Hadler J. L., Salsbury B., Howard R. T., Dingman D. W., Brinkman C. D., Bibb W. F., Cartter M. L. 2001; Hemolytic-uremic syndrome and Escherichia coli O121 at a lake in Connecticut, 1999. Pediatrics 108:E59 [CrossRef]
    [Google Scholar]
  23. Miyajima Y., Takahashi M., Eguchi H., Honma M., Tanahashi S., Matui Y., Kobayashi G., Tanaka M., Higuchi T., Takeuchi Y. 2007; Outbreak of enterohemorrhagic Escherichia coli O26 in Niigata City, Japan. Jpn J Infect Dis 60:238–239
    [Google Scholar]
  24. Nataro J. P., Kaper J. B. 1998; Diarrheagenic Escherichia coli . Clin Microbiol Rev 11:142–201
    [Google Scholar]
  25. Nielsen E. M., Andersen M. T. 2003; Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5′ nuclease PCR assay. J Clin Microbiol 41:2884–2893 [CrossRef]
    [Google Scholar]
  26. Noller A. C., McEllistrem M. C., Stine O. C., Morris J. G. Jr, Boxrud D. J., Dixon B., Harrison L. H. 2003; Multilocus sequence typing reveals a lack of diversity among Escherichia coli O157.  : H7 isolates that are distinct by pulsed-field gel electrophoresis. J Clin Microbiol 41:675–679 [CrossRef]
    [Google Scholar]
  27. Park C. H., Kim H. J., Hixon D. L., Bubert A. 2003; Evaluation of the duopath verotoxin test for detection of shiga toxins in cultures of human stools. J Clin Microbiol 41:2650–2653 [CrossRef]
    [Google Scholar]
  28. Paton A. W., Paton J. C. 2002; Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx 1,stx 2, eae , ehxA ,and saa . J Clin Microbiol 40:271–274 [CrossRef]
    [Google Scholar]
  29. Perelle S., Dilasser F., Grout J., Fach P. 2007; Screening food raw materials for the presence of the world's most frequent clinical cases of Shiga toxin-encoding Escherichia coli O26, O103, O111, O145 and O157. Int J Food Microbiol 113:284–288 [CrossRef]
    [Google Scholar]
  30. Shen S., Mascarenhas M., Morgan R., Rahn K., Karmali M. A. 2005; Identification of four fimbria-encoding genomic islands that are highly specific for verocytotoxin-producing Escherichia coli serotype O157 strains. J Clin Microbiol 43:3840–3850 [CrossRef]
    [Google Scholar]
  31. Teel L. D., Daly J. A., Jerris R. C., Maul D., Svanas G., O'Brien A. D., Park C. H. 2007; Rapid detection of Shiga toxin-producing Escherichia coli by optical immunoassay. J Clin Microbiol 45:3377–3380 [CrossRef]
    [Google Scholar]
  32. Thompson L. H., Giercke S., Beaudoin C., Woodward D., Wylie J. L. 2005; Enhanced surveillance of non-O157 verotoxin-producing Escherichia coli in human stool samples from Manitoba. Can J Infect Dis Med Microbiol 16:329–334
    [Google Scholar]
  33. Toma C., Martinez Espinosa E., Song T., Miliwebsky E., Chinen I., Iyoda S., Iwanaga M., Rivas M. 2004; Distribution of putative adhesins in different seropathotypes of Shiga toxin-producing Escherichia coli . J Clin Microbiol 42:4937–4946 [CrossRef]
    [Google Scholar]
  34. Wang G., Clark C. G., Rodgers F. G. 2002; Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157: H7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR. J Clin Microbiol 40:3613–3619 [CrossRef]
    [Google Scholar]
  35. Ziebell K. A., Read S. C., Johnson R. P., Gyles C. L. 2002; Evaluation of PCR and PCR-RFLP protocols for identifying Shiga toxins. Res Microbiol 153:289–300 [CrossRef]
    [Google Scholar]
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