Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1995 Aug;33(8):2016–2021. doi: 10.1128/jcm.33.8.2016-2021.1995

PCR ribotyping and arbitrarily primed PCR for typing strains of Clostridium difficile from a Polish maternity hospital.

G Martirosian 1, S Kuipers 1, H Verbrugh 1, A van Belkum 1, F Meisel-Mikolajczyk 1
PMCID: PMC228327  PMID: 7559940

Abstract

Detection of the source of Clostridium difficile strains is of importance for the control of the nosocomial spread of this microorganism. For this purpose, vaginal and rectal swabs from 183 mothers, duplicate fecal samples (taken on days 1 and 4 after birth) from 183 neonates, and 94 environmental samples were cultured for C. difficile. The microorganism was never detected in the meconium obtained on day 1 after birth. On the other hand, an incidence of 17% C. difficile positivity was noted in the fecal samples obtained on day 4 after birth. Forty-two percent of the 31 colonized neonates had been delivered with complications. The bacteria were never encountered in the rectal swabs of the mothers, and C. difficile was identified in only one vaginal swab. In contrast, 13% of the environmental samples were positive for C. difficile. No major difference was encountered between patient and environmental isolates with respect to toxigenicity (58 to 65% toxigenic isolates). All strains were subsequently typed by PCR amplification of the 16S-23S ribosomal intergenic spacer regions and by arbitrarily primed PCR (AP-PCR) with different primers and combinations thereof. All environmental isolates and 11 of 31 neonatal strains were of a single type. The vaginal strain was unique, and among the maternity ward- and neonate-related isolates, only two additional AP-PCR types were identified. When a collection of C. difficile strains from patients hospitalized in other institutions and suffering from antibiotic-associated diarrhea or pseudomembranous colitis was analyzed in a similar manner, it appeared that the strain from the maternity ward was unique.(ABSTRACT TRUNCATED AT 250 WORDS)

Full Text

The Full Text of this article is available as a PDF (454.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barbut F., Mario N., Delmée M., Gozian J., Petit J. C. Genomic fingerprinting of Clostridium difficile isolates by using a random amplified polymorphic DNA (RAPD) assay. FEMS Microbiol Lett. 1993 Dec 1;114(2):161–166. doi: 10.1111/j.1574-6968.1993.tb06567.x. [DOI] [PubMed] [Google Scholar]
  2. Bartlett J. G. Clostridium difficile: clinical considerations. Rev Infect Dis. 1990 Jan-Feb;12 (Suppl 2):S243–S251. doi: 10.1093/clinids/12.supplement_2.s243. [DOI] [PubMed] [Google Scholar]
  3. Bartlett J. G. Clostridium difficile: history of its role as an enteric pathogen and the current state of knowledge about the organism. Clin Infect Dis. 1994 May;18 (Suppl 4):S265–S272. doi: 10.1093/clinids/18.supplement_4.s265. [DOI] [PubMed] [Google Scholar]
  4. Boom R., Sol C. J., Salimans M. M., Jansen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. doi: 10.1128/jcm.28.3.495-503.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borriello S. P. 12th C. L. Oakley lecture. Pathogenesis of Clostridium difficile infection of the gut. J Med Microbiol. 1990 Dec;33(4):207–215. doi: 10.1099/00222615-33-4-207. [DOI] [PubMed] [Google Scholar]
  6. Borriello S. P., Ketley J. M., Mitchell T. J., Barclay F. E., Welch A. R., Price A. B., Stephen J. Clostridium difficile--a spectrum of virulence and analysis of putative virulence determinants in the hamster model of antibiotic-associated colitis. J Med Microbiol. 1987 Aug;24(1):53–64. doi: 10.1099/00222615-24-1-53. [DOI] [PubMed] [Google Scholar]
  7. Cartmill T. D., Panigrahi H., Worsley M. A., McCann D. C., Nice C. N., Keith E. Management and control of a large outbreak of diarrhoea due to Clostridium difficile. J Hosp Infect. 1994 May;27(1):1–15. doi: 10.1016/0195-6701(94)90063-9. [DOI] [PubMed] [Google Scholar]
  8. Cartwright C. P., Stock F., Beekmann S. E., Williams E. C., Gill V. J. PCR amplification of rRNA intergenic spacer regions as a method for epidemiologic typing of Clostridium difficile. J Clin Microbiol. 1995 Jan;33(1):184–187. doi: 10.1128/jcm.33.1.184-187.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clabots C. R., Johnson S., Bettin K. M., Mathie P. A., Mulligan M. E., Schaberg D. R., Peterson L. R., Gerding D. N. Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems. J Clin Microbiol. 1993 Jul;31(7):1870–1875. doi: 10.1128/jcm.31.7.1870-1875.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clabots C. R., Johnson S., Olson M. M., Peterson L. R., Gerding D. N. Acquisition of Clostridium difficile by hospitalized patients: evidence for colonized new admissions as a source of infection. J Infect Dis. 1992 Sep;166(3):561–567. doi: 10.1093/infdis/166.3.561. [DOI] [PubMed] [Google Scholar]
  11. Clabots C. R., Peterson L. R., Gerding D. N. Characterization of a nosocomial Clostridium difficile outbreak by using plasmid profile typing and clindamycin susceptibility testing. J Infect Dis. 1988 Oct;158(4):731–736. doi: 10.1093/infdis/158.4.731. [DOI] [PubMed] [Google Scholar]
  12. Cooperstock M. S., Steffen E., Yolken R., Onderdonk A. Clostridium difficile in normal infants and sudden infant death syndrome: an association with infant formula feeding. Pediatrics. 1982 Jul;70(1):91–95. [PubMed] [Google Scholar]
  13. Gürtler V., Mayall B. C. Genotyping of Clostridium difficile isolates. J Clin Microbiol. 1994 Dec;32(12):3095–3095. doi: 10.1128/jcm.32.12.3095-.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gürtler V. Typing of Clostridium difficile strains by PCR-amplification of variable length 16S-23S rDNA spacer regions. J Gen Microbiol. 1993 Dec;139(12):3089–3097. doi: 10.1099/00221287-139-12-3089. [DOI] [PubMed] [Google Scholar]
  15. Heard S. R., O'Farrell S., Holland D., Crook S., Barnett M. J., Tabaqchali S. The epidemiology of Clostridium difficile with use of a typing scheme: nosocomial acquisition and cross-infection among immunocompromised patients. J Infect Dis. 1986 Jan;153(1):159–162. doi: 10.1093/infdis/153.1.159. [DOI] [PubMed] [Google Scholar]
  16. Holst E., Helin I., Mårdh P. A. Recovery of Clostridium difficile from children. Scand J Infect Dis. 1981;13(1):41–45. doi: 10.1080/00365548.1981.11690365. [DOI] [PubMed] [Google Scholar]
  17. Johnson S., Clabots C. R., Linn F. V., Olson M. M., Peterson L. R., Gerding D. N. Nosocomial Clostridium difficile colonisation and disease. Lancet. 1990 Jul 14;336(8707):97–100. doi: 10.1016/0140-6736(90)91605-a. [DOI] [PubMed] [Google Scholar]
  18. Kaatz G. W., Gitlin S. D., Schaberg D. R., Wilson K. H., Kauffman C. A., Seo S. M., Fekety R. Acquisition of Clostridium difficile from the hospital environment. Am J Epidemiol. 1988 Jun;127(6):1289–1294. doi: 10.1093/oxfordjournals.aje.a114921. [DOI] [PubMed] [Google Scholar]
  19. Killgore G. E., Kato H. Use of arbitrary primer PCR to type Clostridium difficile and comparison of results with those by immunoblot typing. J Clin Microbiol. 1994 Jun;32(6):1591–1593. doi: 10.1128/jcm.32.6.1591-1593.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim K., Pickering L. K., DuPont H. L., Sullivan N., Wilkins T. In vitro and in vivo neutralizing activity of human colostrum and milk against purified toxins A and B of Clostridium difficile. J Infect Dis. 1984 Jul;150(1):57–62. doi: 10.1093/infdis/150.1.57. [DOI] [PubMed] [Google Scholar]
  21. Kristjánsson M., Samore M. H., Gerding D. N., DeGirolami P. C., Bettin K. M., Karchmer A. W., Arbeit R. D. Comparison of restriction endonuclease analysis, ribotyping, and pulsed-field gel electrophoresis for molecular differentiation of Clostridium difficile strains. J Clin Microbiol. 1994 Aug;32(8):1963–1969. doi: 10.1128/jcm.32.8.1963-1969.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Larson H. E., Barclay F. E., Honour P., Hill I. D. Epidemiology of Clostridium difficile in infants. J Infect Dis. 1982 Dec;146(6):727–733. doi: 10.1093/infdis/146.6.727. [DOI] [PubMed] [Google Scholar]
  23. Martirosian G., Meisel-Mikolajczyk F., Stańczak J., Mierzejewski J., Flis D. Identyfikacja metoda PCR toksynotwórczych szczepów Clostridium difficile wyhodowanych z przewodu pokarmowego psów. Med Dosw Mikrobiol. 1994;46(3):201–206. [PubMed] [Google Scholar]
  24. Maslow J. N., Mulligan M. E., Arbeit R. D. Molecular epidemiology: application of contemporary techniques to the typing of microorganisms. Clin Infect Dis. 1993 Aug;17(2):153–164. doi: 10.1093/clinids/17.2.153. [DOI] [PubMed] [Google Scholar]
  25. McMillin D. E., Muldrow L. L. Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers. FEMS Microbiol Lett. 1992 Apr 1;71(1):5–9. doi: 10.1016/0378-1097(92)90532-s. [DOI] [PubMed] [Google Scholar]
  26. Mitchell T. J., Ketley J. M., Haslam S. C., Stephen J., Burdon D. W., Candy D. C., Daniel R. Effect of toxin A and B of Clostridium difficile on rabbit ileum and colon. Gut. 1986 Jan;27(1):78–85. doi: 10.1136/gut.27.1.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rothman S. W. Technique for measuring 50% end points in cytotoxicity assays for Clostridium difficile toxins. J Clin Pathol. 1986 Jun;39(6):672–676. doi: 10.1136/jcp.39.6.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Samore M. H., Bettin K. M., DeGirolami P. C., Clabots C. R., Gerding D. N., Karchmer A. W. Wide diversity of Clostridium difficile types at a tertiary referral hospital. J Infect Dis. 1994 Sep;170(3):615–621. doi: 10.1093/infdis/170.3.615. [DOI] [PubMed] [Google Scholar]
  29. Silva J., Jr Clostridium difficile nosocomial infections--still lethal and persistent. Infect Control Hosp Epidemiol. 1994 Jun;15(6):368–370. doi: 10.1086/646933. [DOI] [PubMed] [Google Scholar]
  30. Silva J., Jr, Tang Y. J., Gumerlock P. H. Genotyping of Clostridium difficile isolates. J Infect Dis. 1994 Mar;169(3):661–664. doi: 10.1093/infdis/169.3.661. [DOI] [PubMed] [Google Scholar]
  31. Stark P. L., Lee A., Parsonage B. D. Colonization of the large bowel by Clostridium difficile in healthy infants: quantitative study. Infect Immun. 1982 Mar;35(3):895–899. doi: 10.1128/iai.35.3.895-899.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tabaqchali S., O'Farrell S., Nash J. Q., Wilks M. Vaginal carriage and neonatal acquisition of Clostridium difficile. J Med Microbiol. 1984 Aug;18(1):47–53. doi: 10.1099/00222615-18-1-47. [DOI] [PubMed] [Google Scholar]
  33. Triadafilopoulos G., Pothoulakis C., O'Brien M. J., LaMont J. T. Differential effects of Clostridium difficile toxins A and B on rabbit ileum. Gastroenterology. 1987 Aug;93(2):273–279. doi: 10.1016/0016-5085(87)91014-6. [DOI] [PubMed] [Google Scholar]
  34. Versalovic J., Koeuth T., Lupski J. R. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1991 Dec 25;19(24):6823–6831. doi: 10.1093/nar/19.24.6823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Welsh J., McClelland M. Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers. Nucleic Acids Res. 1991 Oct 11;19(19):5275–5279. doi: 10.1093/nar/19.19.5275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilks M., Tabaqchali S. Typing of Clostridium difficile by polymerase chain reaction with an arbitrary primer. J Hosp Infect. 1994 Nov;28(3):231–234. doi: 10.1016/0195-6701(94)90105-8. [DOI] [PubMed] [Google Scholar]
  37. Williams J. G., Kubelik A. R., Livak K. J., Rafalski J. A., Tingey S. V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990 Nov 25;18(22):6531–6535. doi: 10.1093/nar/18.22.6531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. van Belkum A., Bax R., Peerbooms P., Goessens W. H., van Leeuwen N., Quint W. G. Comparison of phage typing and DNA fingerprinting by polymerase chain reaction for discrimination of methicillin-resistant Staphylococcus aureus strains. J Clin Microbiol. 1993 Apr;31(4):798–803. doi: 10.1128/jcm.31.4.798-803.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. van Belkum A. DNA fingerprinting of medically important microorganisms by use of PCR. Clin Microbiol Rev. 1994 Apr;7(2):174–184. doi: 10.1128/cmr.7.2.174. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES