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. 1992 Jul;30(7):1823–1828. doi: 10.1128/jcm.30.7.1823-1828.1992

Use of nonradioactive DNA hybridization for identification of enterotoxigenic Escherichia coli harboring genes for colonization factor antigen I, coli surface antigen 4, or putative colonization factor O166.

H Sommerfelt 1, H M Grewal 1, W Gaastra 1, A M Svennerholm 1, M K Bhan 1
PMCID: PMC265388  PMID: 1629340

Abstract

We developed an accurate nonradioactive colony hybridization assay (NCHA) using a digoxigenin-labeled polynucleotide probe and an antidigoxigenin alkaline phosphatase conjugate for the identification of enterotoxigenic Escherichia coli (ETEC) harboring genes for colonization factor antigen I (CFA/I), coli surface antigen 4 (CS4), or putative colonization factor O166 (PCFO166). In this 2-day assay, visual registration of color intensity could be used to distinguish between CFA/I-positive strains and strains with the genetic potential to express CS4 or PCFO166. A rapid NCHA was developed by which the results could be read visually 7 h and 45 min after inoculation of the bacteria. In the rapid NCHA, densitometry verified the visual discrimination between four groups of E. coli; ETEC with the CFA/I gene, ETEC with the CS4 gene, ETEC with the PCFO166 gene, and E. coli strains that lack such genes. As a confirmatory test, plasmids from ETEC with the CFA/I, CS4, or PCFO166 gene were differentiated by their characteristic restriction fragment patterns in nonradioactive Southern blot hybridization.

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Selected References

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  1. Bhan M. K., Raj P., Levine M. M., Kaper J. B., Bhandari N., Srivastava R., Kumar R., Sazawal S. Enteroaggregative Escherichia coli associated with persistent diarrhea in a cohort of rural children in India. J Infect Dis. 1989 Jun;159(6):1061–1064. doi: 10.1093/infdis/159.6.1061. [DOI] [PubMed] [Google Scholar]
  2. Binsztein N., Jouve M. J., Viboud G. I., López Moral L., Rivas M., Orskov I., Ahrén C., Svennerholm A. M. Colonization factors of enterotoxigenic Escherichia coli isolated from children with diarrhea in Argentina. J Clin Microbiol. 1991 Sep;29(9):1893–1898. doi: 10.1128/jcm.29.9.1893-1898.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black R. E., Lopez de Romaña G., Brown K. H., Bravo N., Bazalar O. G., Kanashiro H. C. Incidence and etiology of infantile diarrhea and major routes of transmission in Huascar, Peru. Am J Epidemiol. 1989 Apr;129(4):785–799. doi: 10.1093/oxfordjournals.aje.a115193. [DOI] [PubMed] [Google Scholar]
  4. Casey J., Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. doi: 10.1093/nar/4.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Changchawalit S., Echeverria P., Taylor D. N., Leksomboon U., Tirapat C., Eampokalap B., Rowe B. Colonization factors associated with enterotoxigenic Escherichia coli isolated in Thailand. Infect Immun. 1984 Aug;45(2):525–527. doi: 10.1128/iai.45.2.525-527.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clemens J. D., Sack D. A., Harris J. R., Chakraborty J., Neogy P. K., Stanton B., Huda N., Khan M. U., Kay B. A., Khan M. R. Cross-protection by B subunit-whole cell cholera vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial. J Infect Dis. 1988 Aug;158(2):372–377. doi: 10.1093/infdis/158.2.372. [DOI] [PubMed] [Google Scholar]
  7. Cravioto A., Scotland S. M., Rowe B. Hemagglutination activity and colonization factor antigens I and II in enterotoxigenic and non-enterotoxigenic strains of Escherichia coli isolated from humans. Infect Immun. 1982 Apr;36(1):189–197. doi: 10.1128/iai.36.1.189-197.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Gaastra W., de Graaf F. K. Host-specific fimbrial adhesins of noninvasive enterotoxigenic Escherichia coli strains. Microbiol Rev. 1982 Jun;46(2):129–161. doi: 10.1128/mr.46.2.129-161.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gothefors L., Ahrén C., Stoll B., Barua D. K., Orskov F., Salek M. A., Svennerholm A. M. Presence of colonization factor antigens on fresh isolates of fecal Escherichia coli: a prospective study. J Infect Dis. 1985 Dec;152(6):1128–1133. doi: 10.1093/infdis/152.6.1128. [DOI] [PubMed] [Google Scholar]
  11. Grewal H. M., Sommerfelt H., Gaastra W., Svennerholm A. M., Bhan M. K., Hamers A. M., Kumar R., Wiklund G., Bjorvatn B. Detection of colonization factor antigen I-positive enterotoxigenic Escherichia coli with a cloned polynucleotide probe. J Clin Microbiol. 1990 Oct;28(10):2264–2268. doi: 10.1128/jcm.28.10.2264-2268.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heery D. M., Gannon F., Powell R. A simple method for subcloning DNA fragments from gel slices. Trends Genet. 1990 Jun;6(6):173–173. doi: 10.1016/0168-9525(90)90158-3. [DOI] [PubMed] [Google Scholar]
  13. Hibberd M. L., McConnell M. M., Field A. M., Rowe B. The fimbriae of human enterotoxigenic Escherichia coli strain 334 are related to CS5 fimbriae. J Gen Microbiol. 1990 Dec;136(12):2449–2456. doi: 10.1099/00221287-136-12-2449. [DOI] [PubMed] [Google Scholar]
  14. Honda T., Arita M., Miwatani T. Characterization of new hydrophobic pili of human enterotoxigenic Escherichia coli: a possible new colonization factor. Infect Immun. 1984 Mar;43(3):959–965. doi: 10.1128/iai.43.3.959-965.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Honda T., Wetprasit N., Arita M., Miwatani T. Production and characterization of monoclonal antibodies to a pilus colonization factor (colonization factor antigen III) of human enterotoxigenic Escherichia coli. Infect Immun. 1989 Nov;57(11):3452–3457. doi: 10.1128/iai.57.11.3452-3457.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Klemm P. Fimbrial adhesions of Escherichia coli. Rev Infect Dis. 1985 May-Jun;7(3):321–340. doi: 10.1093/clinids/7.3.321. [DOI] [PubMed] [Google Scholar]
  17. Lopez-Vidal Y., Klemm P., Svennerholm A. M. Monoclonal antibodies against different epitopes on colonization factor antigen I of enterotoxin-producing Escherichia coli. J Clin Microbiol. 1988 Oct;26(10):1967–1972. doi: 10.1128/jcm.26.10.1967-1972.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lopez-Vidal Y., Svennerholm A. M. Monoclonal antibodies against the different subcomponents of colonization factor antigen II of enterotoxigenic Escherichia coli. J Clin Microbiol. 1990 Sep;28(9):1906–1912. doi: 10.1128/jcm.28.9.1906-1912.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McConnell M. M., Chart H., Field A. M., Hibberd M., Rowe B. Characterization of a putative colonization factor (PCFO166) of enterotoxigenic Escherichia coli of serogroup O166. J Gen Microbiol. 1989 May;135(5):1135–1144. doi: 10.1099/00221287-135-5-1135. [DOI] [PubMed] [Google Scholar]
  20. McConnell M. M., Hibberd M., Field A. M., Chart H., Rowe B. Characterization of a new putative colonization factor (CS17) from a human enterotoxigenic Escherichia coli of serotype O114:H21 which produces only heat-labile enterotoxin. J Infect Dis. 1990 Feb;161(2):343–347. doi: 10.1093/infdis/161.2.343. [DOI] [PubMed] [Google Scholar]
  21. McConnell M. M., Thomas L. V., Willshaw G. A., Smith H. R., Rowe B. Genetic control and properties of coli surface antigens of colonization factor antigen IV (PCF8775) of enterotoxigenic Escherichia coli. Infect Immun. 1988 Aug;56(8):1974–1980. doi: 10.1128/iai.56.8.1974-1980.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mooi F. R., de Graaf F. K. Molecular biology of fimbriae of enterotoxigenic Escherichia coli. Curr Top Microbiol Immunol. 1985;118:119–138. doi: 10.1007/978-3-642-70586-1_7. [DOI] [PubMed] [Google Scholar]
  23. Moseley S. L., Echeverria P., Seriwatana J., Tirapat C., Chaicumpa W., Sakuldaipeara T., Falkow S. Identification of enterotoxigenic Escherichia coli by colony hybridization using three enterotoxin gene probes. J Infect Dis. 1982 Jun;145(6):863–869. doi: 10.1093/infdis/145.6.863. [DOI] [PubMed] [Google Scholar]
  24. Nishibuchi M., Arita M., Honda T., Miwatani T. Evaluation of a nonisotopically labeled oligonucleotide probe to detect the heat-stable enterotoxin gene of Escherichia coli by the DNA colony hybridization test. J Clin Microbiol. 1988 Apr;26(4):784–786. doi: 10.1128/jcm.26.4.784-786.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sommerfelt H., Grewal H. M., Bhan M. K. Simplified and accurate nonradioactive polynucleotide gene probe assay for identification of enterotoxigenic Escherichia coli. J Clin Microbiol. 1990 Jan;28(1):49–54. doi: 10.1128/jcm.28.1.49-54.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sommerfelt H., Grewal H. M., Gaastra W., Bhan M. K., Svennerholm A. M., Kalland K. H., Asphaug V., Aasland R., Bjorvatn B. Presence of cfaD-homologous sequences and expression of coli surface antigen 4 on enterotoxigenic Escherichia coli; relevance for diagnostic procedures. Microb Pathog. 1991 Oct;11(4):297–304. doi: 10.1016/0882-4010(91)90034-8. [DOI] [PubMed] [Google Scholar]
  27. Sommerfelt H., Kalland K. H., Raj P., Moseley S. L., Bhan M. K., Bjorvatn B. Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli. J Clin Microbiol. 1988 Nov;26(11):2275–2278. doi: 10.1128/jcm.26.11.2275-2278.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  29. Svennerholm A. M., Holmgren J., Sack D. A. Development of oral vaccines against enterotoxinogenic Escherichia coli diarrhoea. Vaccine. 1989 Jun;7(3):196–198. doi: 10.1016/0264-410x(89)90228-4. [DOI] [PubMed] [Google Scholar]
  30. Svennerholm A. M., Vidal Y. L., Holmgren J., McConnell M. M., Rowe B. Role of PCF8775 antigen and its coli surface subcomponents for colonization, disease, and protective immunogenicity of enterotoxigenic Escherichia coli in rabbits. Infect Immun. 1988 Feb;56(2):523–528. doi: 10.1128/iai.56.2.523-528.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tacket C. O., Maneval D. R., Levine M. M. Purification, morphology, and genetics of a new fimbrial putative colonization factor of enterotoxigenic Escherichia coli O159:H4. Infect Immun. 1987 May;55(5):1063–1069. doi: 10.1128/iai.55.5.1063-1069.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thomas L. V., McConnell M. M., Rowe B., Field A. M. The possession of three novel coli surface antigens by enterotoxigenic Escherichia coli strains positive for the putative colonization factor PCF8775. J Gen Microbiol. 1985 Sep;131(9):2319–2326. doi: 10.1099/00221287-131-9-2319. [DOI] [PubMed] [Google Scholar]
  33. Thomas L. V., Rowe B. The occurrence of colonisation factors (CFA/I, CFA/II and E8775) in enterotoxigenic Escherichia coli from various countries in South East Asia. Med Microbiol Immunol. 1982;171(2):85–90. doi: 10.1007/BF02124915. [DOI] [PubMed] [Google Scholar]

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