Coexistence of Multiple VNTR sub-typing Profiles of Clostridium Difficle Strains within Faecal Samples

Hannah Jarivs, HPA

Jarvis, H.E, Hardy KJ, Hawkey PM

Health Protection Agency, Heartlands Hospital, Birmingham; Dept of Infection and Immunity, Birmingham University, Birmingham

Background: Approximately 66% of all C. difficile isolates typed routinely at HPA Birmingham are PCR-Ribotype 027. The commonness of this profile limits the epidemiological utility of PCR-Ribotyping.

Aim: To investigate the usefulness of variable number tandem repeat (VNTR) loci from two published MLVA analysis methods which are able to sub-divide C. difficile isolates of PCR-Ribotype 027.

Method: Faeces samples were inoculated onto C. difficile selective agar and incubated anaerobicly. Five C. difficile colonies were picked from each of these initial culture plates and PCR-Ribotyped. Where the ribotype was 027, six VNTR loci were amplified. Tandem repeat numbers at each locus were estimated by PCR product size on agarose gel electrophoresis.

Results and Conclusion: Of 26 specimens tested, all were found to contain only one PCR-Ribotype within the specimen. However, in 4 out of 10 VNTR typed specimens, at least two distinct VNTR repeat number profiles were detected. Tandem repeat number variation at three of the loci appears to be as variable within one faecal specimen as between isolates from different specimens. This observation may limit the application of the published VNTR typing methods for determining epidemiological links between patients with or outbreaks of C. difficile associated disease.