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Characterisation of clinical Clostridium difficile isolates by rep-PCR, PFGE and PCR ribotyping

Abstract number: P1731

Frye S., Smith C., Reece K., Wise M., Krishnaswami N., Foussadier A., Embry J., Abedi S., Ross T., Healy M.

Objectives:Clostridium difficile is a major cause of nosocomial infections. In recent years, a highly virulent strain (NAP1/ribotype 027) that causes more serious disease and increased mortality has emerged in both Europe and North America. Tracking the source and spread of these infections through strain-typing will help to contain hospital associated infections. Several molecular typing methods are used as tools to strain-type C. difficile, including PFGE and PCR ribotyping. An automated repetitive sequence-based PCR system, the DiversiLab System, has also been used in strain typing of C. difficile. This study compares rep-PCR, PCR ribotyping and PFGE methods for strain typing.

Methods: A total of 84 C. difficile consisting of clinical isolates from a healthcare facility in the UK (17 isolates) and a healthcare facility in the US (67 isolates) were previously characterised by PCR ribotyping. The sample set consisted of 25 different PCR ribotypes including multiple ribotype 027. Each isolate was cultured and genomic DNA was extracted using the UltraCleanTM Microbial DNA Isolation Kit. For rep-PCR, DNA was amplified using the Clostridium Kit for DNA Fingerprinting. The amplified product fragments were separated using microfluidics lab-on-a-chip technology and analyzed using web-based data analysis software. PFGE was carried out using the CHEF-DRTMII (Bio-Rad Laboratories) electrophoresis system following a previously described method.

Results: Rep-PCR and PCR ribotyping fingerprints were generated for every sample. PFGE fingerprints were generated for over 95% of isolates. Rep-PCR, PFGE and PCR ribotyping clustering showed a high concordance; however, rep-PCR and PFGE showed a higher level of discrimination by divisions within some ribotype clusters. Additionally, rep-PCR and PFGE provided multiple fingerprints for ribotype 027.

Conclusions: Rep-PCR and PCR ribotyping had a more rapid turnaround time and were more robust because DNA degradation was less of a concern as compared to PFGE. However, both PFGE and rep-PCR provided a higher level of discrimination than PCR ribotyping including for isolates of ribotype 027.

Session Details

Date: 16/05/2009
Time: 00:00-00:00
Session name: 19th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Helsinki, Finland, 16 - 19 May 2009
Presentation type:
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