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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1992 May;30(5):1189–1193. doi: 10.1128/jcm.30.5.1189-1193.1992

Rapid and specific detection of the pap, afa, and sfa adhesin-encoding operons in uropathogenic Escherichia coli strains by polymerase chain reaction.

C Le Bouguenec 1, M Archambaud 1, A Labigne 1
PMCID: PMC265248  PMID: 1349900

Abstract

Adhesin-encoding operons (pap, sfa/foc, and afa) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. A quick and sensitive assay to identify these operons was developed by using the polymerase chain reaction (PCR). Three pairs of 25-mer primers were defined from the sequences of the DNA fragments used as probes in hybridization studies to identify each of the three operons, and the six primers were used together in a single reaction of amplification. To validate the PCR approach for detection of adhesin-encoding operons among clinical isolates, we investigated a collection of 97 E. coli isolates with the following characteristics: all isolates originated from the urine of patients with pyelonephritis, and the adhesin responsible for specific binding of the isolates to uroepithelial cells was previously characterized by phenotypic assays, as well as genotypic tests based on hybridization. There was a perfect correlation between the results obtained with the PCR approach and those previously obtained by using DNA probes. These results indicate that the PCR method, which is highly specific and easier to perform than the hybridization method, is a powerful genotypic assay for detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as for epidemiological studies.

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Selected References

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  1. Archambaud M., Courcoux P., Labigne-Roussel A. Detection by molecular hybridization of pap, afa, and sfa adherence systems in Escherichia coli strains associated with urinary and enteral infections. Ann Inst Pasteur Microbiol. 1988 Sep-Oct;139(5):575–588. doi: 10.1016/0769-2609(88)90156-1. [DOI] [PubMed] [Google Scholar]
  2. Archambaud M., Courcoux P., Ouin V., Chabanon G., Labigne-Roussel A. Phenotypic and genotypic assays for the detection and identification of adhesins from pyelonephritic Escherichia coli. Ann Inst Pasteur Microbiol. 1988 Sep-Oct;139(5):557–573. doi: 10.1016/0769-2609(88)90155-x. [DOI] [PubMed] [Google Scholar]
  3. Bilge S. S., Clausen C. R., Lau W., Moseley S. L. Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells. J Bacteriol. 1989 Aug;171(8):4281–4289. doi: 10.1128/jb.171.8.4281-4289.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Buchanan K., Falkow S., Hull R. A., Hull S. I. Frequency among Enterobacteriaceae of the DNA sequences encoding type 1 pili. J Bacteriol. 1985 May;162(2):799–803. doi: 10.1128/jb.162.2.799-803.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dowling K. J., Roberts J. A., Kaack M. B. P-fimbriated Escherichia coli urinary tract infection: a clinical correlation. South Med J. 1987 Dec;80(12):1533–1536. doi: 10.1097/00007611-198712000-00012. [DOI] [PubMed] [Google Scholar]
  8. Edén C. S., Hanson L. A., Jodal U., Lindberg U., Akerlund A. S. Variable adherence to normal human urinary-tract epithelial cells of Escherichia coli strains associated with various forms of urinary-tract infection. Lancet. 1976 Sep 4;1(7984):490–492. [PubMed] [Google Scholar]
  9. Fowler J. E., Jr, Stamey T. A. Studies of introital colonization in women with recurrent urinary infections. VII. The role of bacterial adherence. J Urol. 1977 Apr;117(4):472–476. doi: 10.1016/s0022-5347(17)58501-8. [DOI] [PubMed] [Google Scholar]
  10. Frankel G., Giron J. A., Valmassoi J., Schoolnik G. K. Multi-gene amplification: simultaneous detection of three virulence genes in diarrhoeal stool. Mol Microbiol. 1989 Dec;3(12):1729–1734. doi: 10.1111/j.1365-2958.1989.tb00158.x. [DOI] [PubMed] [Google Scholar]
  11. Gander R. M., Thomas V. L., Forland M. Mannose-resistant hemagglutination and P receptor recognition of uropathogenic Escherichia coli isolated from adult patients. J Infect Dis. 1985 Mar;151(3):508–513. doi: 10.1093/infdis/151.3.508. [DOI] [PubMed] [Google Scholar]
  12. Grüneberg R. N. Antibiotic sensitivities of urinary pathogens, 1971-82. J Antimicrob Chemother. 1984 Jul;14(1):17–23. doi: 10.1093/jac/14.1.17. [DOI] [PubMed] [Google Scholar]
  13. Göransson M., Uhlin B. E. Environmental temperature regulates transcription of a virulence pili operon in E. coli. EMBO J. 1984 Dec 1;3(12):2885–2888. doi: 10.1002/j.1460-2075.1984.tb02225.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hacker J., Schmidt G., Hughes C., Knapp S., Marget M., Goebel W. Cloning and characterization of genes involved in production of mannose-resistant, neuraminidase-susceptible (X) fimbriae from a uropathogenic O6:K15:H31 Escherichia coli strain. Infect Immun. 1985 Feb;47(2):434–440. doi: 10.1128/iai.47.2.434-440.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hull R. A., Gill R. E., Hsu P., Minshew B. H., Falkow S. Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect Immun. 1981 Sep;33(3):933–938. doi: 10.1128/iai.33.3.933-938.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnson J. R., Stamm W. E. Urinary tract infections in women: diagnosis and treatment. Ann Intern Med. 1989 Dec 1;111(11):906–917. doi: 10.7326/0003-4819-111-11-906. [DOI] [PubMed] [Google Scholar]
  17. Johnson J. R. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev. 1991 Jan;4(1):80–128. doi: 10.1128/cmr.4.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Källenius G., Möllby R., Svenson S. B., Helin I., Hultberg H., Cedergren B., Winberg J. Occurrence of P-fimbriated Escherichia coli in urinary tract infections. Lancet. 1981 Dec 19;2(8260-61):1369–1372. doi: 10.1016/s0140-6736(81)92797-5. [DOI] [PubMed] [Google Scholar]
  19. Labigne-Roussel A. F., Lark D., Schoolnik G., Falkow S. Cloning and expression of an afimbrial adhesin (AFA-I) responsible for P blood group-independent, mannose-resistant hemagglutination from a pyelonephritic Escherichia coli strain. Infect Immun. 1984 Oct;46(1):251–259. doi: 10.1128/iai.46.1.251-259.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Labigne-Roussel A., Falkow S. Distribution and degree of heterogeneity of the afimbrial-adhesin-encoding operon (afa) among uropathogenic Escherichia coli isolates. Infect Immun. 1988 Mar;56(3):640–648. doi: 10.1128/iai.56.3.640-648.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lomberg H., Hellström M., Jodal U., Leffler H., Lincoln K., Svanborg Edén C. Virulence-associated traits in Escherichia coli causing first and recurrent episodes of urinary tract infection in children with or without vesicoureteral reflux. J Infect Dis. 1984 Oct;150(4):561–569. doi: 10.1093/infdis/150.4.561. [DOI] [PubMed] [Google Scholar]
  22. Low D., Robinson E. N., Jr, McGee Z. A., Falkow S. The frequency of expression of pyelonephritis-associated pili is under regulatory control. Mol Microbiol. 1987 Nov;1(3):335–346. doi: 10.1111/j.1365-2958.1987.tb01940.x. [DOI] [PubMed] [Google Scholar]
  23. Lund B., Marklund B. I., Strömberg N., Lindberg F., Karlsson K. A., Normark S. Uropathogenic Escherichia coli can express serologically identical pili of different receptor binding specificities. Mol Microbiol. 1988 Mar;2(2):255–263. doi: 10.1111/j.1365-2958.1988.tb00027.x. [DOI] [PubMed] [Google Scholar]
  24. Meyrier A., Condamin M. C., Fernet M., Labigne-Roussel A., Simon P., Callard P., Rainfray M., Soilleux M., Groc A. Frequency of development of early cortical scarring in acute primary pyelonephritis. Kidney Int. 1989 Feb;35(2):696–703. doi: 10.1038/ki.1989.41. [DOI] [PubMed] [Google Scholar]
  25. Norgren M., Båga M., Tennent J. M., Normark S. Nucleotide sequence, regulation and functional analysis of the papC gene required for cell surface localization of Pap pili of uropathogenic Escherichia coli. Mol Microbiol. 1987 Sep;1(2):169–178. doi: 10.1111/j.1365-2958.1987.tb00509.x. [DOI] [PubMed] [Google Scholar]
  26. Norgren M., Normark S., Lark D., O'Hanley P., Schoolnik G., Falkow S., Svanborg-Edén C., Båga M., Uhlin B. E. Mutations in E coli cistrons affecting adhesion to human cells do not abolish Pap pili fiber formation. EMBO J. 1984 May;3(5):1159–1165. doi: 10.1002/j.1460-2075.1984.tb01945.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. O'Hanley P., Lark D., Falkow S., Schoolnik G. Molecular basis of Escherichia coli colonization of the upper urinary tract in BALB/c mice. Gal-Gal pili immunization prevents Escherichia coli pyelonephritis in the BALB/c mouse model of human pyelonephritis. J Clin Invest. 1985 Feb;75(2):347–360. doi: 10.1172/JCI111707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. O'Hanley P., Low D., Romero I., Lark D., Vosti K., Falkow S., Schoolnik G. Gal-Gal binding and hemolysin phenotypes and genotypes associated with uropathogenic Escherichia coli. N Engl J Med. 1985 Aug 15;313(7):414–420. doi: 10.1056/NEJM198508153130704. [DOI] [PubMed] [Google Scholar]
  29. Ott M., Schmoll T., Goebel W., Van Die I., Hacker J. Comparison of the genetic determinant coding for the S-fimbrial adhesin (sfa) of Escherichia coli to other chromosomally encoded fimbrial determinants. Infect Immun. 1987 Aug;55(8):1940–1943. doi: 10.1128/iai.55.8.1940-1943.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pere A., Nowicki B., Saxén H., Siitonen A., Korhonen T. K. Expression of P, type-1, and type-1C fimbriae of Escherichia coli in the urine of patients with acute urinary tract infection. J Infect Dis. 1987 Oct;156(4):567–574. doi: 10.1093/infdis/156.4.567. [DOI] [PubMed] [Google Scholar]
  31. Rhen M., Väisänen-Rhen V., Saraste M., Korhonen T. K. Organization of genes expressing the blood-group-M-specific hemagglutinin of Escherichia coli: identification and nucleotide sequence of the M-agglutinin subunit gene. Gene. 1986;49(3):351–360. doi: 10.1016/0378-1119(86)90371-9. [DOI] [PubMed] [Google Scholar]
  32. Riegman N., Kusters R., Van Veggel H., Bergmans H., Van Bergen en Henegouwen P., Hacker J., Van Die I. F1C fimbriae of a uropathogenic Escherichia coli strain: genetic and functional organization of the foc gene cluster and identification of minor subunits. J Bacteriol. 1990 Feb;172(2):1114–1120. doi: 10.1128/jb.172.2.1114-1120.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schmoll T., Morschhäuser J., Ott M., Ludwig B., van Die I., Hacker J. Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesion determinant: nucleotide sequence of the genes sfa B, C, D, E, F. Microb Pathog. 1990 Nov;9(5):331–343. doi: 10.1016/0882-4010(90)90067-z. [DOI] [PubMed] [Google Scholar]
  36. Swanson T. N., Bilge S. S., Nowicki B., Moseley S. L. Molecular structure of the Dr adhesin: nucleotide sequence and mapping of receptor-binding domain by use of fusion constructs. Infect Immun. 1991 Jan;59(1):261–268. doi: 10.1128/iai.59.1.261-268.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Väisänen-Rhen V., Elo J., Väisänen E., Siitonen A., Orskov I., Orskov F., Svenson S. B., Mäkelä P. H., Korhonen T. K. P-fimbriated clones among uropathogenic Escherichia coli strains. Infect Immun. 1984 Jan;43(1):149–155. doi: 10.1128/iai.43.1.149-155.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Väisänen V., Elo J., Tallgren L. G., Siitonen A., Mäkelä P. H., Svanborg-Edén C., Källenius G., Svenson S. B., Hultberg H., Korhonen T. Mannose-resistant haemagglutination and P antigen recognition are characteristic of Escherichia coli causing primary pyelonephritis. Lancet. 1981 Dec 19;2(8260-61):1366–1369. doi: 10.1016/s0140-6736(81)92796-3. [DOI] [PubMed] [Google Scholar]
  39. Westerlund B., Kuusela P., Risteli J., Risteli L., Vartio T., Rauvala H., Virkola R., Korhonen T. K. The O75X adhesin of uropathogenic Escherichia coli is a type IV collagen-binding protein. Mol Microbiol. 1989 Mar;3(3):329–337. doi: 10.1111/j.1365-2958.1989.tb00178.x. [DOI] [PubMed] [Google Scholar]
  40. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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