Molecular typing of C. difficile clinical isolates in an outbreak evaluation

Introduction: Clostridium difficile is a Gram-positive spore-forming anaerobic bacillus. C. difficile is one of the major responsible of hospital acquired infections, since its spores are highly resistant to heat and common detergents treatment.The aim of this study was to exclude a nosocomial infection in an Intensive Care Unit at Bambino Gesù Children Hospital by using a molecular typing method (rep-PCR). Methods: 9 C. difficile strains were collected within 2 weeks from 9 faecal samples, belonging to 4 patients hospitalized in the intensive care unit. Identification of C. difficile was obtained by phenotypic method and confirmed by genotypic methods. Susceptibility of isolates was evaluated by using E-test. Molecular typing of these strains was obtained by semi-automatic rep-PCR. Results: All antibiotic susceptibility tests resulted overlapping. Genotypic analysis by rep-PCR showed that isolates belonging to the same patient had a similarity index > 96%, whereas, by comparing strains from different patients, a similarity index < 87% was observed. Conclusions: Molecular fingerprinting of 9 C. difficile isolates arranged the strains in 4 different groups, corresponding to 4 patients.The rep- PCR with the semi-automated Diversilab platform was able to ruled out the presence of a C. difficile nosocomial outbreak in a paediatric Intensive Care Unit. Finally, this system can be used as a “first line” tool, nevertheless, confirmation by “gold standard” fingerprinting methods is necessary. However, even though the system may seem easy to use, it is still not reproducible and criteria for interpretation of analytical results must be fully assessed by trained personnel in the practice of microbial fingerprinting.