Characterization of Clostridium difficile isolates using capillary gel electrophoresis-based PCR ribotyping

Abstract

We have developed a Clostridium difficile PCR ribotyping method based on capillary gel electrophoresis and have compared it with conventional PCR ribotyping. A total of 146 C. difficile isolates were studied: five isolates were reference strains (PCR ribotypes 001, 014, 017, 027 and 053); 141 were clinical isolates comprising 39 Austrian PCR ribotypes collected in the period 2006-2007 at 25 Austrian healthcare facilities. Capillary gel electrophoresis yielded up to 11 fragments per isolate and 47 ribotype patterns. All but one of the five PCR ribotypes of reference strains were clearly reflected in the chromatograms of capillary-based typing. Capillary gel electrophoresis divided 24 isolates belonging to PCR ribotype type 014 into seven subgroups, whereas subtyping the same isolates using multiple-locus variable-number tandem-repeat analysis yielded three unrelated subgroups, without obvious correlation to sr subgroups. Using a web-based software program (http://webribo.ages.at), we were able to correctly identify these 014 isolates by simply allocating the seven subgroup patterns to one ribotype, i.e. to PCR ribotype 014. We consider capillary gel electrophoresis-based PCR ribotyping to be a way of overcoming the problems associated with inter-laboratory comparisons of typing results, while at the same time substantially diminishing the hands-on time for PCR ribotyping.

Fig. 1.

Ribotype patterns obtained by classic agarose gel-based electrophoresis (on the left), compared with those obtained using the capillary gel electrophoresis-based method (column labelled seq-PCR ribotyping). PCR ribotypes resulting from capillary gel electrophoresis received the prefix ‘sr’. Control reference strains are boxed.

Fig. 2.

MLVA and capillary gel electrophoresis-based PCR ribotyping of 24 isolates belonging to ribotype 014. Capillary gel electrophoresis-based ribotyping yielded seven different 014 subgroups with assigned names sr014/0–sr014/6 (right), whereas MLVA subtyping of the same isolates yielded three unrelated subgroups without obvious correlation to sr subgroups (left).