Abstract
The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.
Full Text
The Full Text of this article is available as a PDF (796.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Barrett T. J., Kaper J. B., Jerse A. E., Wachsmuth I. K. Virulence factors in Shiga-like toxin-producing Escherichia coli isolated from humans and cattle. J Infect Dis. 1992 May;165(5):979–980. doi: 10.1093/infdis/165.5.979. [DOI] [PubMed] [Google Scholar]
- Barrett T. J., Lior H., Green J. H., Khakhria R., Wells J. G., Bell B. P., Greene K. D., Lewis J., Griffin P. M. Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing. J Clin Microbiol. 1994 Dec;32(12):3013–3017. doi: 10.1128/jcm.32.12.3013-3017.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Besser R. E., Lett S. M., Weber J. T., Doyle M. P., Barrett T. J., Wells J. G., Griffin P. M. An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157:H7 in fresh-pressed apple cider. JAMA. 1993 May 5;269(17):2217–2220. [PubMed] [Google Scholar]
- Beutin L., Geier D., Steinrück H., Zimmermann S., Scheutz F. Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. J Clin Microbiol. 1993 Sep;31(9):2483–2488. doi: 10.1128/jcm.31.9.2483-2488.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beutin L., Geier D., Zimmermann S., Karch H. Virulence markers of Shiga-like toxin-producing Escherichia coli strains originating from healthy domestic animals of different species. J Clin Microbiol. 1995 Mar;33(3):631–635. doi: 10.1128/jcm.33.3.631-635.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blanco M., Blanco J., Blanco J. E., González E. A., Gomes T. A., Zerbini L. F., Yano T., de Castro A. F. Genes coding for Shiga-like toxins in bovine verotoxin-producing Escherichia coli (VTEC) strains belonging to different O:K:H serotypes. Vet Microbiol. 1994 Nov;42(2-3):105–110. doi: 10.1016/0378-1135(94)90010-8. [DOI] [PubMed] [Google Scholar]
- Faith N. G., Shere J. A., Brosch R., Arnold K. W., Ansay S. E., Lee M. S., Luchansky J. B., Kaspar C. W. Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin. Appl Environ Microbiol. 1996 May;62(5):1519–1525. doi: 10.1128/aem.62.5.1519-1525.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldwater P. N., Bettelheim K. A. Hemolytic uremic syndrome due to shiga-like toxin producing Escherichia coli 048:H21 in South Australia. Emerg Infect Dis. 1995 Oct-Dec;1(4):132–133. doi: 10.3201/eid0104.950405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Griffin P. M., Tauxe R. V. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev. 1991;13:60–98. doi: 10.1093/oxfordjournals.epirev.a036079. [DOI] [PubMed] [Google Scholar]
- Grimm L. M., Goldoft M., Kobayashi J., Lewis J. H., Alfi D., Perdichizzi A. M., Tarr P. I., Ongerth J. E., Moseley S. L., Samadpour M. Molecular epidemiology of a fast-food restaurant-associated outbreak of Escherichia coli O157:H7 in Washington State. J Clin Microbiol. 1995 Aug;33(8):2155–2158. doi: 10.1128/jcm.33.8.2155-2158.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hancock D. D., Besser T. E., Kinsel M. L., Tarr P. I., Rice D. H., Paros M. G. The prevalence of Escherichia coli O157.H7 in dairy and beef cattle in Washington State. Epidemiol Infect. 1994 Oct;113(2):199–207. doi: 10.1017/s0950268800051633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hofmann S. L. Southwestern Internal Medicine Conference: Shiga-like toxins in hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. Am J Med Sci. 1993 Dec;306(6):398–406. doi: 10.1097/00000441-199312000-00009. [DOI] [PubMed] [Google Scholar]
- Hovde C. J., Calderwood S. B., Mekalanos J. J., Collier R. J. Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2568–2572. doi: 10.1073/pnas.85.8.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Imberechts H., De Greve H., Schlicker C., Bouchet H., Pohl P., Charlier G., Bertschinger H., Wild P., Vandekerckhove J., Van Damme J. Characterization of F107 fimbriae of Escherichia coli 107/86, which causes edema disease in pigs, and nucleotide sequence of the F107 major fimbrial subunit gene, fedA. Infect Immun. 1992 May;60(5):1963–1971. doi: 10.1128/iai.60.5.1963-1971.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Janke B. H., Francis D. H., Collins J. E., Libal M. C., Zeman D. H., Johnson D. D., Neiger R. D. Attaching and effacing Escherichia coli infection as a cause of diarrhea in young calves. J Am Vet Med Assoc. 1990 Mar 15;196(6):897–901. [PubMed] [Google Scholar]
- Jerse A. E., Gicquelais K. G., Kaper J. B. Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli. Infect Immun. 1991 Nov;59(11):3869–3875. doi: 10.1128/iai.59.11.3869-3875.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jerse A. E., Yu J., Tall B. D., Kaper J. B. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7839–7843. doi: 10.1073/pnas.87.20.7839. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karch H., Meyer T., Rüssmann H., Heesemann J. Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation. Infect Immun. 1992 Aug;60(8):3464–3467. doi: 10.1128/iai.60.8.3464-3467.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kudva I. T., Hatfield P. G., Hovde C. J. Effect of diet on the shedding of Escherichia coli O157:H7 in a sheep model. Appl Environ Microbiol. 1995 Apr;61(4):1363–1370. doi: 10.1128/aem.61.4.1363-1370.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kudva I. T., Hatfield P. G., Hovde C. J. Escherichia coli O157:H7 in microbial flora of sheep. J Clin Microbiol. 1996 Feb;34(2):431–433. doi: 10.1128/jcm.34.2.431-433.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McDaniel T. K., Jarvis K. G., Donnenberg M. S., Kaper J. B. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1664–1668. doi: 10.1073/pnas.92.5.1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meng J., Zhao S., Zhao T., Doyle M. P. Molecular characterisation of Escherichia coli O157:H7 isolates by pulsed-field gel electrophoresis and plasmid DNA analysis. J Med Microbiol. 1995 Apr;42(4):258–263. doi: 10.1099/00222615-42-4-258. [DOI] [PubMed] [Google Scholar]
- Montenegro M. A., Bülte M., Trumpf T., Aleksić S., Reuter G., Bulling E., Helmuth R. Detection and characterization of fecal verotoxin-producing Escherichia coli from healthy cattle. J Clin Microbiol. 1990 Jun;28(6):1417–1421. doi: 10.1128/jcm.28.6.1417-1421.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morris J. A., Wells G. A., Scott A. C., Sojka W. J. Colonisation of the small intestine of lambs by an enterotoxigenic Escherichia coli producing F41 fimbriae. Vet Rec. 1983 Nov 12;113(20):471–471. doi: 10.1136/vr.113.20.471. [DOI] [PubMed] [Google Scholar]
- O'Brien A. D., Holmes R. K. Shiga and Shiga-like toxins. Microbiol Rev. 1987 Jun;51(2):206–220. doi: 10.1128/mr.51.2.206-220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Perry M. B., Bundle D. R. Antigenic relationships of the lipopolysaccharides of Escherichia hermannii strains with those of Escherichia coli O157:H7, Brucella melitensis, and Brucella abortus. Infect Immun. 1990 May;58(5):1391–1395. doi: 10.1128/iai.58.5.1391-1395.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rasmussen M. A., Cray W. C., Jr, Casey T. A., Whipp S. C. Rumen contents as a reservoir of enterohemorrhagic Escherichia coli. FEMS Microbiol Lett. 1993 Nov 15;114(1):79–84. doi: 10.1016/0378-1097(93)90145-r. [DOI] [PubMed] [Google Scholar]
- Rice D. H., Hancock D. D., Besser T. E. Verotoxigenic E coli O157 colonisation of wild deer and range cattle. Vet Rec. 1995 Nov 11;137(20):524–524. doi: 10.1136/vr.137.20.524. [DOI] [PubMed] [Google Scholar]
- Rüssmann H., Kothe E., Schmidt H., Franke S., Harmsen D., Caprioli A., Karch H. Genotyping of Shiga-like toxin genes in non-O157 Escherichia coli strains associated with haemolytic uraemic syndrome. J Med Microbiol. 1995 Jun;42(6):404–410. doi: 10.1099/00222615-42-6-404. [DOI] [PubMed] [Google Scholar]
- Samadpour M. Molecular epidemiology of Escherichia coli O157:H7 by restriction fragment length polymorphism using Shiga-like toxin genes. J Clin Microbiol. 1995 Aug;33(8):2150–2154. doi: 10.1128/jcm.33.8.2150-2154.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanderson M. W., Gay J. M., Hancock D. D., Gay C. C., Fox L. K., Besser T. E. Sensitivity of bacteriologic culture for detection of Escherichia coli O157:H7 in bovine feces. J Clin Microbiol. 1995 Oct;33(10):2616–2619. doi: 10.1128/jcm.33.10.2616-2619.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shin S. J., Chang Y. F., Timour M., Lauderdale T. L., Lein D. H. Hybridization of clinical Escherichia coli isolates from calves and piglets in New York State with gene probes for enterotoxins (STaP, STb, LT), Shiga-like toxins (SLT-1, SLT-II) and adhesion factors (K88, K99, F41, 987P). Vet Microbiol. 1994 Jan;38(3):217–225. doi: 10.1016/0378-1135(94)90003-5. [DOI] [PubMed] [Google Scholar]
- Tesh V. L., O'Brien A. D. Adherence and colonization mechanisms of enteropathogenic and enterohemorrhagic Escherichia coli. Microb Pathog. 1992 Apr;12(4):245–254. doi: 10.1016/0882-4010(92)90043-n. [DOI] [PubMed] [Google Scholar]
- Tesh V. L., O'Brien A. D. The pathogenic mechanisms of Shiga toxin and the Shiga-like toxins. Mol Microbiol. 1991 Aug;5(8):1817–1822. doi: 10.1111/j.1365-2958.1991.tb00805.x. [DOI] [PubMed] [Google Scholar]
- Wang G., Zhao T., Doyle M. P. Fate of enterohemorrhagic Escherichia coli O157:H7 in bovine feces. Appl Environ Microbiol. 1996 Jul;62(7):2567–2570. doi: 10.1128/aem.62.7.2567-2570.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weinstein D. L., Jackson M. P., Perera L. P., Holmes R. K., O'Brien A. D. In vivo formation of hybrid toxins comprising Shiga toxin and the Shiga-like toxins and role of the B subunit in localization and cytotoxic activity. Infect Immun. 1989 Dec;57(12):3743–3750. doi: 10.1128/iai.57.12.3743-3750.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wells J. G., Shipman L. D., Greene K. D., Sowers E. G., Green J. H., Cameron D. N., Downes F. P., Martin M. L., Griffin P. M., Ostroff S. M. Isolation of Escherichia coli serotype O157:H7 and other Shiga-like-toxin-producing E. coli from dairy cattle. J Clin Microbiol. 1991 May;29(5):985–989. doi: 10.1128/jcm.29.5.985-989.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wieler L. H., Bauerfeind R., Baljer G. Characterization of Shiga-like toxin producing Escherichia coli (SLTEC) isolated from calves with and without diarrhoea. Zentralbl Bakteriol. 1992 Jan;276(2):243–253. doi: 10.1016/s0934-8840(11)80011-3. [DOI] [PubMed] [Google Scholar]
- Willshaw G. A., Scotland S. M., Smith H. R., Cheasty T., Thomas A., Rowe B. Hybridization of strains of Escherichia coli O157 with probes derived from the eaeA gene of enteropathogenic E. coli and the eaeA homolog from a Vero cytotoxin-producing strain of E. coli O157. J Clin Microbiol. 1994 Apr;32(4):897–902. doi: 10.1128/jcm.32.4.897-902.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yu J., Kaper J. B. Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol. 1992 Feb;6(3):411–417. doi: 10.1111/j.1365-2958.1992.tb01484.x. [DOI] [PubMed] [Google Scholar]
- Zhao T., Doyle M. P., Shere J., Garber L. Prevalence of enterohemorrhagic Escherichia coli O157:H7 in a survey of dairy herds. Appl Environ Microbiol. 1995 Apr;61(4):1290–1293. doi: 10.1128/aem.61.4.1290-1293.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Graaf F. K., Krenn B. E., Klaasen P. Organization and expression of genes involved in the biosynthesis of K99 fimbriae. Infect Immun. 1984 Feb;43(2):508–514. doi: 10.1128/iai.43.2.508-514.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]