[A NOVEL APPROACH TO GENOTYPING OF HOSPITAL ISOLATES OF CLOSTRIDIUM DIFFICILE].

AIM: Development of a novel approach in genotyping of Clostridium difficile and its testing on the example of 140 hospital isolates. MATERIALS AND METHODS: The approach is based on an idea of double digest and selective label (DDSL), used previously during genotyping of other bacterial pathogens. Selection of optimal enzymes for restriction of MluI and Mph1103I was carried out, condition of DDSL reaction execution were optimized. RESULTS: Genotyping of C. difficile hospital isolates was carried out, index of strain discrimination was calculated, conclusions regarding possibilities of the method in elucidation of spread pathways and identification of infection sources were made. CONCLUSION: The developed method of genotyping has a number of advantages over the existing method and can be used to'address issues in epidemiology of infections caused by C. difficile.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.