Mechanisms behind variation in the Clostridium difficile 16S–23S rRNA intergenic spacer region

Clostridium difficile infection is an increasing problem in hospitals worldwide, mainly due to the recent emergence of a hypervirulent C. difficile strain. C. difficile PCR ribotyping, based on size variation of the 16S–23S rRNA intergenic spacer region (16S–23S ISR), is widely used in Europe for molecular epidemiological investigation. The mechanism underlying the 16S–23S ISR size variations in the genome of C. difficile is currently not completely understood. To elucidate this mechanism, isolates of six different PCR ribotypes were analysed by cloning and sequencing the 16S–23S ISR. A direct repeat, IB, of 9 bp was detected up to five times in the 16S–23S ISR in all 47 clones investigated. Thirty-five clones displayed differences either by ribotype or by nucleotide sequence. The sequences of the 16S–23S ISR of C. difficile showed a uniformly organized structure, composed of a tRNA(Ala) gene and spacers of 33 and 53 bp separated by the 9 bp direct repeat IB. The results of the study support the hypothesis that this composition is responsible for the length variations seen in the 16S–23S ISR, and indicate that these length variations result from slipped-strand mispairing and intra- and possibly interchromosomal homologous recombination.