DNA microarray based PCR ribotyping of Clostridium difficile

This study presents a DNA microarray based assay for fast and simple PCR-ribotyping of Clostridium difficile. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR) which is also the template for conventional and seq-PCR ribotyping. Probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well characterized C. difficile isolates representing 48 seq-PCR-ribotypes. The reference hybridization patterns calculated by arithmetic mean were compared among themselves by similarity matrix analysis. The investigated 48 seq-PCR-ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human pathogenic ribotypes 001, 014/020, 027 and 078/126 could be discriminated by the microarray. Clostridium difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652 and 660) and 014/020 (014, 020 and 449), respectively, showed similar hybridization patterns confirming their genetic relatedness which was previously reported. A panel of 50 C. difficile field isolates were tested by seq-PCR-ribotyping and the DNA microarray based assay in parallel. Taking into account that the current version of the microarray cannot discriminate some closely related seq-PCR-ribotypes, all isolates were typed correctly. Moreover, seq-PCR-ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes confirming the performance and expansion potential of the microarray.