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
2019-10-22
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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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