Abstract
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 tRNAAla 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.
Highlights
In recent years, Clostridium difficile infection has become a major problem in hospital environments worldwide
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
PCR ribotyping is based on fragment length variations in the 16S–23S ISR, the detailed mechanism underlying the formation of the variations in C. difficile is currently unknown
Summary
Clostridium difficile infection has become a major problem in hospital environments worldwide. Disease due to C. difficile is associated with a wide range of clinical manifestations ranging from mild diarrhoea, through moderately severe illness with watery diarrhoea, to life-threatening and sometimes fatal pseudomembranous colitis, which can be accompanied by toxic megacolon or perforation of the bowel Several typing methods, such as PFGE, repetitive extragenic palindromic PCR, restriction endonuclease analysis and PCR ribotyping, have been developed for C. difficile; they are hampered by problems concerning interlaboratory exchangeability, reproducibility and comparability (Killgore et al, 2008). C. difficile PCR ribotyping, the most widely used method in Europe, exploits differences in the length of the 16S–23S rRNA intergenic spacer region (16S–23S ISR) (Bidet et al, 1999; Stubbs et al, 1999). To elucidate the mechanisms underlying the length variations of 16S–23S ISR sequences in C. difficile, we analysed six C. difficile isolates of six PCR ribotypes
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