The use of PCR ribotyping for molecular typing of clinically significant Clostridium difficile Romanian isolates

Clostridium difficile infections (CDI) became a significant infectious pathology in Romania in 2010-2011, when first major outbreaks were reported. The increase of case numbers made molecular typing of C. difficile strains necessary for epidemiological purposes. In this study, 96 C. difficile strains, isolated from CDI patients, were identified using PCR ribotyping. The method was selected considering the context of efforts being made to standardise C. difficile typing in Europe, using PCR ribotyping as the method of choice, with a type nomenclature for cross-country comparison in CDI surveillance, but also taking into account technical accessibility. The study results showed a majority of isolates (n=63) with the toxigenic profile A+/B+/CDT+, most of them (n=61) identified as hyper virulent ribotype 027. Eighteen more ribotypes were identified, in lower proportions. Fourteen corresponded to ribotypes from the Brazier nomenclature: 002, 012, 014, 015, 017, 018, 031, 046, 053, 085, 087, 106, 115 and 140. Ribotype 014 was the most representative (7 isolates), followed by 002 (4 isolates). Three isolates were non-toxigenic: ribotypes 031, 085 and 140. Other four ribotypes remained unidentified and were named U.01, U.02, U.03 and U.04. Two isolates clustering as ribotype U.02 were A+/B+/CDT+. By selecting this typing method, a prevalent, highly epidemic ribotype and 18 other ribotypes were detected, providing information about the C. difficile type diversity of the analysed group of strains and proving its usefulness as an instrument in epidemiological studies.