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

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

Classifying spa Types in Complexes Improves Interpretation of Typing Results for Methicillin-Resistant Staphylococcus aureus

A total of 382 isolates of methicillin-resistant Staphylococcus aureus originating from three Austrian regions and one adjacent Italian region (Vienna, Lower Austria, North Tyrol, and South Tyrol) were typed by DNA sequence analysis of the variable repeat region of the protein A gene (spa typing). The strain collection consisted of arbitrarily chosen isolates originating from clinical specimens taken in the years 2003 to 2005 at 17 hospitals. The most common spa types found were t001 (28.8% of all isolates), t190 (27.0%), t008 (14.1%), and t041 (11.3%). The 42 remaining spa types accounted for ≤2.4% each. The dominating spa types varied between the different regions. As short sequence DNA repeat units are unstable entities, the 46 spa types were classified into seven spa complexes with respect to short sequence repeat unit composition and organization. Such classification into complexes can provide additional information for the hospital epidemiologist, empowering one to differentiate the introduction of a new strain from mere variation of endemic spa types