Molecular characterization of strains of Clostridium difficile isolated from hospitalized geriatric patients

Introduction. Clostridium difficile (Cd) is a spore-forming, gram positive anaerobic bacillus. It can be found as commensal in the human intestinal flora. Cd is the etiologic agent of pseudomembranous colitis and the most common cause of nosocomial antibiotic-associated diarrhea. The pathogenic strains produce toxins (A and B) with cytotoxic effects on intestinal epithelium. Some strains producing binary toxin, cause more severe infections. So far it is not known the specific role of this toxin. The major risk factors for acquiring this infection are: advanced age (over 65), underlying disease, severity of illness, antibiotic exposure and prolonged hospitalization. The purpose of our work was to perform a molecular characterization of Cd strains isolated from patients hospitalized at ASP Pio Albergo Trivulzio. We also performed the analysis of sequential isolates in order to verify if the recurrent infections were due to relapse or reinfection. Methods. A total of 100 C.d. isolates were collected from 75 symptomatic patients from 6 different hospital wards. The strains were cultured on blood agar plates (CD agar – bioMérieux) in an anaerobic atmosphere for 48 h at 37°C. The identification of species was performed using Vitek automated system (bioMérieux). The molecular typing was performed by "ribotyping" according to the method described by Bidet et al., using the 16 S (5'-GTGCGGCTGGATCACCTCCT-3 ') and 23 S (5'-CCCTGCACCCTTAATAACTTGACC-3') sequences. Furthermore, all strains were subjected to PCR to detect the presence of the gene coding for binary toxin. Results. The analysis of patterns obtained by ribotyping showed 14 different ribotypes. None of the strains tested was found to produce binary toxin. Conclusions. The data obtained indicate that there is a considerable spread between the same ward in respect of the spread between different wards. For this phenomena we can assume that the spread has been fostered by the movements of patients in different rooms and that the environment is a possible source of infection. Most of the sequential samples belong to the same ribotype indicating that the recurrent infections may be due to relapse from treatment failure or reinfection with the same strain. The lack of toxin-producing strains binary allows for the time being, not to further increase the rate of mortality.