Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing.

PCR-ribotying, a typing method based on polymorphism in the 16S-23S intergenic spacer region, has been recently used to investigate outbreaks due to Clostridium difficile. However, this method generates bands of high and close molecular masses which are difficult to separate on agarose gel electrophoresis. To improve reading of banding patterns of PCR-ribotyping applied to C. difficile, a partial sequencing of the rRNA genes (16S and 23S) and intergenic spacer region has been performed, then a new set of primers located closer to the intergenic spacer region has been defined. The new PCR gave reproducible patterns of bands easy to separate on agarose gel electrophoresis. Each of the 10 serogroups and 11 subgroups of serogroup A produced a different pattern. This typing method has evidenced major qualities such as easiness, rapidity and reproducibility. However, its discriminatory power has to be evaluated to validate its importance as a typing tool for C. difficile.