Genotypic characterisation of binary-toxin-producing Clostridium difficile strains
Abstract number: P524
Marín M., Martín A., Alcalá L., Pélaez T., Domínguez E., Sánchez-Somolinos M., Insa R., Catalán P., Bouza E.
Background:Clostridium difficile (CD) is an important cause of communitary and nosocomial diarrhoea. In recent years, epidemic strains belonging to the 027 and 078 ribotypes have emerged. Both ribotypes are characterised by binary toxin production and deletions of tcdC gene (negative regulator of toxin production), the latter possibly related to increased toxin production.
Objective: To genotype the binary-toxin-positive CD strains (bin+) circulating in our hospital during the year 2007.
Methods: CD strains were cultured and identified by conventional microbiological methods. Toxins A and B were detected in isolates using an immunochromatographic method (ImmunoCard, Meridian Bioscience). DNA was obtained from pure cultures using Chelex resin (Instagene matrix, BioRad). The tcdA gene (toxin A), tcdB gene (toxin B), and binary-toxin genes cdtA and cdtB were detected by PCR following methods previously described (Kato, 1991; Wolfhagen, 1994; and Stubbs, 2000, respectively). Bin + isolates were characterised by PCR-ribotyping (Bidet et al. 2000). Phylogenetic analysis of ribotyping profiles was conducted using Bionumerics software 5.0. The tcdC gene was amplified by PCR using the method described by Spigaglia and Mastrantonio (2002), sequenced by the Big Dye Terminator method, and detected in an AbiPrism 3100 automatic DNA sequencer (Applied Biosystems Inc.). Sequences were aligned using BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html).
Results: Seven hundred and forty three CD toxigenic strains were isolated from patients with CD associated diarrhoea during 2007. Eighty-eight isolates were bin + (62 patients) and all were also tox A+B+. The isolates were from 8 different ribotypes. Most CD isolates belonged to ribotype 078 (63 isolates from 45 patients). Only 6 isolates (2 patients) belonged to ribotype 027. The analysis of the tcdC gene revealed deletions of 18 bp in 10 isolates (2 different ribotypes), 36 bp in 5 isolates (2 different ribotypes), 39 bp in 69 isolates (2 different ribotypes), and 54 bp in 6 isolates (only 1 ribotype).
Conclusions: Twelve percent of our CD toxigenic isolates had binary-toxin genes. Ribotype 078 is a frequent cause of diarrhoea in our patients, representing 8% of toxigenic CD isolated at our institution during 2007. In our study, epidemic strains of ribotype 027 were detected in only 2 patients. All the bin + CD detected had deletions in the tcdC gene.
|Session name:||19th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Helsinki, Finland, 16 - 19 May 2009|
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