Typing of Clostridium difficile strains with an automated repetitive PCR system in comparison to PCR ribotyping
Abstract number: O147
Pasanen T., Kotila S., Mero S., Antikainen J., Tarkka E., Eerola E., Jalava J., Virolainen-Julkunen A., Könönen E., Vaara M., Tissari P.
Objectives: An accurate and rapid method is needed for typing of toxigenic Clostridium difficile. A commercial automated repetitive PCR system (rep-PCR; DiversiLab®, Biomérieux Inc., St Louis, USA) utilises amplification and subsequent automated electrophoretic separation of the repetitive extragenic palindromic sequences of C. difficile. Our aim was to evaluate the performance of this rep-PCR method for genotyping of C. difficile isolates and to compare it to PCR ribotyping. In addition, the correlation between the rep-PCR and the virulence gene profiles of C. difficile strains was studied.
Methods: A total of 195 toxin-positive C. difficile isolates were studied. We included consecutive isolates from two laboratories in Finland, containing also strains of the hypervirulent C. difficile ribotype 027. In addition, selected C. difficile strains with >18 bp deletions in their tcdC genes were analyzed.
The DNA was extracted and the rep-PCR performed according to the manufacturer's instructions. The amplification products of rep-PCR were detected and analyzed using the DiversiLab system. Further analysis was performed with the web-based software accompanying the system. The usefulness of the library construction option of the DiversLab system for isolate comparison was tested. The virulence genes (tcdA, tcdB, cdtA, cdtB and tcdC) were analyzed by conventional PCR and the whole gene sequencing of tcdC was performed from isolates with deletions >18 bp. PCR ribotyping was performed using the protocol of the Anaerobe Reference Unit in Cardiff, UK.
Results: The correlation between the rep-PCR profile and the ribotype was excellent. All major ribotype groups were clustered in their own rep-PCR groups. Interestingly, subgroups could be found with rep-PCR within two most prevalent ribotypes 001 and 027.
The automated rep-PCR proved to be reproducible; the results from separate DNA isolations and PCR-runs/microfluid electrophoresis as well as the results performed by different individuals of laboratory personnel were comparable. The rep-PCR profiles and PCR ribotypes correlated also with the virulence gene profiles.
Conclusion: This automated rep-PCR represents an effective and reproducible method for the genetic characterisation of C. difficile strains in clinical laboratories with molecular biology facilities. The constructed C. difficile library allows comparing the relatedness of C. difficile strains and their fingerprints over time.
|Session name:||19th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Helsinki, Finland, 16 - 19 May 2009|
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