1887

Microbiology Society logo

Volume 139, Issue 12

Typing of strains by PCR-amplification of variable length 16S-23S rDNA spacer regions Free

Abstract

To develop a rapid and accurate method of typing large numbers of clinical isolates of four regions of the rRNA operon [A, 15–1407 and B, 907–1407 (16S–16S); C, 1392–507 and D, 907–507 (16S–23S)] were enzymically amplified from 24 strains. When region A was hybridized to III-digested genomic DNA isolated from strains, all of the variable length restriction fragments hybridized. When region B was hybridized to dIII-digested genomic DNA isolated from strains, a set of variable length restriction fragments (Group II) hybridized predominantly. When region C was separated by agarose gel electrophoresis, a series of products ranging in size from approximately 800–1300 bp was obtained. When regions C and D were digested with dIII, a constant region of 430 bp was found in both products and in all strains. From the above experiments it was concluded that the variable length Group II restriction fragments and the variable length region C amplification products were due to variable length 16S-23S spacer regions between alleles of the one strain. When region C amplification products were separated by denaturing PAGE, 16 variable length rRNA alleles A-P) were demonstrated from 24 strains ranging in size from 852–1210 bp. After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes. The product C ribotypes and band sizes were stable after 14 single colony passages on horse blood agar plates and stable since ribotype G was isolated twice from one patient and ribotype E was isolated three times from another patient (all on separate occasions). The ribotyping method described here has clear advantages over existing typing methods; it has universal applicability, it is objective and is moderately rapid.

  • Received:
  • Accepted:
  • Published Online:
© Society for General Microbiology, 1993
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-12-3089
1993-12-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/12/mic-139-12-3089.html?itemId=/content/journal/micro/10.1099/00221287-139-12-3089&mimeType=html&fmt=ahah

References

  1. Altwegg M., Hickmann-Brenner F.W., Farmer J.J.III 1989; Ribosomal RNA gene restriction patterns provide increased sen-sitivity for typing Salmonella typhi strains. Journal of Infectious Diseases 160:145–149
     [Google Scholar]
  2. Bartlett J.G., Chang T.W., Gurwith M., Gorbach S.L., Onderdonk A.B. 1978; Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. New England Journal of Medicine 298:531–534
     [Google Scholar]
  3. Bingen E.H, Denamur E., Lambert-Zechvosky N.Y., Bourdois A., Mariani-Kurkdjian P., Cezard J.-P., Navarro J., Elion J. 1991; DNA restriction fragment length polymorphism differen-tiates crossed from independent infections in nosocomial Xanthomonas maltophilia bacteremia. Journal of Clinical Microbiology 29:1348–1350
     [Google Scholar]
  4. Boondeekhun H.S., Gürtler V., Odd M.L., Wilson V.A., Mayall B.C. 1993; Detection of the Clostridium difficile enterotoxin gene in clinical specimens by the polymerase chain reaction. Journal of Medical Microbiology 38:384–387
     [Google Scholar]
  5. Bowman R.A., O̓Neill G.L., Riley T.V. 1991; Nonradioactive restriction fragment length polymorphism (RFLP) typing of Clostridium difficile. FEMS Microbiology Letters 63:269–272
     [Google Scholar]
  6. Brosius J., Palmer M.L., Kennedy P.J., Noller H.F. 1978; Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 72:4801–4805
     [Google Scholar]
  7. Brosius J., Dull T.J., Sleeter D.D., Noller H.F. 1981; Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. Journal of Molecular Biology 148:107–127
     [Google Scholar]
  8. Burgin A.B., Parodos K., Lane D.J., Pace N.R. 1990; The excision of intervening sequences from Salmonella 23S ribosomal RNA. Cell 60:405–414
     [Google Scholar]
  9. Cato E.P., George W.L., Finegold S.M. 1986; Genus Clostridium Prazmowski 1880, 23AL. In berger’s manual of systematic bacteriology 2 pp. 1141–1200 Sneath P.H.A., Mair N.S., Sharpe M.E., Holt J.G. Edited by Baltimore: Williams & Wilkins.;
     [Google Scholar]
  10. Clabots C., Lee S., Gerding D., Mulligan M., Kwok R., Schaberg D., Fekety R., Peterson L. 1988a; Clostridium difficile plasmid isolation as an epidemiologic tool. European Journal of Clinical Microbiology and Infectious Diseases 7:312–315
     [Google Scholar]
  11. Clabots C.R., Peterson L.R., Gerding D.N. 1988b; Characterization of a nosocomial Clostridium difficile outbreak by using plasmid profile typing and clindamycin susceptibility testing. Journal of Infectious Diseases 158:731–736
     [Google Scholar]
  12. De Buyser M.-L., Morvan A., Aubert S., Dilasser F., El Sohl N. 1992; Evaluation of a ribosomal RNA gene probe for the identification of species and subspecies within the genus Staphylococcus. Journal of General Microbiology 138:889–899
     [Google Scholar]
  13. Delmee M., Avesani V. 1988; Correlation between serogroup and susceptibility to chloramphenicol, clindamycin, erythromycin, rifampicin and tetracycline among 308 isolates of Clostridium difficile. Journal of Antimicrobial Chemotherapy 22:325–331
     [Google Scholar]
  14. Fournier M.J., Ozeki H. 1985; Structure and organization of the transfer ribonucleic acid genes of Eschericia coli K-12. Microbiological Reviews 49:379–397
     [Google Scholar]
  15. Garnier T., Canard B., Cole S.T. 1991; Cloning, mapping, and molecular characterization of the rRNA operons of Clostridium perfringens. Journal of Bacteriology 173:5431–5438
     [Google Scholar]
  16. George R.H.S., Symonds J.M., Dimock F., Brown J.D., Arabi Y., Shinagawa N., Keighley M.R.B., Alexander-Williams J., Burdon D.W. 1978; Identification of Clostridium difficile as a cause of pseudomembranous colitis. British Medical Journal 1:695
     [Google Scholar]
  17. Gürtler V., Wilson V.A., Mayall B.C. 1991; Classification of medically important Clostridia using restriction endonuclease site differences of PCR-amplified 16S rDNA. Journal of General Microbiology 137:2673–2679
     [Google Scholar]
  18. Guttell R.R., Fox G.E. 1988; A compilation of large sub-unit RNA sequences presented in a structural format. Nucleic Acids Research 16:r175–r269
     [Google Scholar]
  19. Harrison T.G., Saunders N.A., Haththotuwa A., Doshi N., Taylor N.G. 1992; Further evidence that genotypically closely related strains of Legionella pneumophila can express different serogroup specific antigens. Journal of Medical Microbiology 37:155–161
     [Google Scholar]
  20. Heard S.R., Rasburn B., Matthews R.C., Tabaqchali S. 1986; Immunoblotting to demonstrate antigenic and immunogenic differences among nine standard strains of Clostridium difficile. Journal of Clinical Microbiology 24:384–387
     [Google Scholar]
  21. Hsu D., Pan M.-J., Zee Y.C., Le Febvre R.B. 1990; Unique ribosome structure of Leptospira interrogans is composed of four rRNA components. Journal of Bacteriology 172:3478–3480
     [Google Scholar]
  22. Hsu D., Zee Y.C., Ingraham J., Shih L.-M. 1992; Diversity of cleavage patterns of Salmonella 23S rRNA. Journal of General Microbiology 138:199–203
     [Google Scholar]
  23. Johnson S., Clabots C.R., Linn F.V., Olson M.M., Peterson L.R., Gerding D.N. 1990; Nosocomial Clostridium difficile colonisation and disease. Lancet 336:97–100
     [Google Scholar]
  24. Lipuma J.J., Stull T.L., Dasen S.E., Pidcock K.A., Kaye D., Korzeniowski O.M. 1989; DNA polymorphisms among Escherichia coli isolated from bacteriuric women. Journal of Infectious Diseases 159:526–532
     [Google Scholar]
  25. Loughney K., Lund E., Dahlberg J.E. 1982; tRNA genes are found between the 16S and 23S rRNA genes in Bacillus subtilis. Nucleic Acids Research 10:1607–1624
     [Google Scholar]
  26. Mahony D.E., Clow J., Atkinson L., Vakharia N., Schlech W.F. 1991; Development and application of a multiple typing system for Clostridium difficile. Applied and Environmental Microbiology 57:1873–1879
     [Google Scholar]
  27. Mcfarland L.V., Mulligan M.E., Kwok R.Y., Stamm W.E. 1989; Nosocomial acquisition of Clostridium difficile infection. New England Journal of Medicine 320:204–210
     [Google Scholar]
  28. Muldrow L.L., Archibold E.R., Nunez M.O., Sheehy R.J. 1982; Survey of the extrachromosomal gene pool of Clostridium difficile. Journal of Clinical Microbiology 16:637–640
     [Google Scholar]
  29. Mulligan M.E., Peterson L.R., Kwok R.Y., Clabots C.R., Gerding D.N. 1988; Immunoblots and plasmid fingerprints compared with serotyping and polyacrylamide gel electrophoresis for typing Clostridium difficile. Journal of Clinical Microbiology 26:41–46
     [Google Scholar]
  30. Neefs J.-M., Van De Peer Y., Hendriks L., De Wachter R. 1990; Compilation of small ribosomal sub-unit RNA sequences. Nucleic Acids Research 18:r2237–r2317
     [Google Scholar]
  31. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.;
     [Google Scholar]
  32. Sell T.L., Schaberg D.R., Fekety F.R. 1983; Bacteriophage and bacteriocin typing scheme for Clostridium difficile. Journal of Clinical Microbiology 17:1148–1152
     [Google Scholar]
  33. Sutter V.L., Citron D.M., Edelstein M.A.C., Finegold S.M. 1985 Wadsworth Anaerobic Bacteriology Manual, 4th edn.. Belmont, California: Star Publishing Company.;
     [Google Scholar]
  34. Swofford D.L. 1985; Phylogenetic analysis using parsimony (PAUP) version 2·4. University of Illinois.:
     [Google Scholar]
  35. Tabaqchali S. 1990; Epidemiologic markers of Clostridium difficile. Reviews of Infectious Diseases 12: suppl. 2 192–199
     [Google Scholar]
  36. Tabaqchali S., Holland D., O’farrell S., Silman R. 1984; Typing scheme for Clostridium difficile: its application in clinical and epidemiological studies. Lancet 1:935–938
     [Google Scholar]
  37. Vaneechoutte M., Rossau R., De Vos P., Gillis M., Janssens D., Paepe N., De Rouck A., Fiers T., Clayes G., Kersters K. 1992; Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA). FEMS Microbiology Letters 93:227–234
     [Google Scholar]
  38. Vold B.S. 1985; Structure and organization of genes for transfer ribonucleic acid in Bacillus subtilis. Microbiological Reviews 49:71–80
     [Google Scholar]
  39. Wren B.W., Tabaqchali S. 1987; Restriction endonuclease DNA analysis of Clostridium difficile. Journal of Clinical Microbiology 25:2402–2404
     [Google Scholar]
  40. Wust J., Sullivan N.M., Hardegger U., Wilkins T.D. 1982; Investigation of an outbreak of antibiotic-associated colitis by various typing methods. Journal of Clinical Microbiology 16:1096–1101
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-12-3089
Loading
Typing of Clostridium difficile strains by PCR-amplification of variable length 16S-23S rDNA spacer regions
Microbiology 139, 3089 (1993); https://doi.org/10.1099/00221287-139-12-3089
/content/journal/micro/10.1099/00221287-139-12-3089
/content/journal/micro/10.1099/00221287-139-12-3089
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error