New variants of the tet(M) gene in Clostridium difficile clinical isolates harbouring Tn916-like elements

Objectives: To detect Tn916-Iike elements in Clostridium difficile clinical isolates from different time periods and to analyse the genetic structure of these elements, in particular the tet(M) region. Methods: Ninety C. difficile clinical isolates were examined by PCR assays for tet(M) and int, which are markers for the Tn916family of elements. Positive isolates were typed by PCR-ribotyping, and tetracycline MIC values were evaluated by Etest. The genetic organization of the Tn916 elements was investigated by PCR mapping and hybridization assays. The tet(M) region of eight selected C. difficile isolates was sequenced. Results: Nineteen isolates were tet(M)/int positive and the majority (12/19) belonged to PCR-ribotype R, previously found to be predominant in clinical strains of more recent isolation. Eleven isolates were tetracycline resistant, three inducibly resistant and five susceptible. Fifty-eight per cent of the C. difficile isolates harboured one Tn916 element and 42% harboured two. Most isolates showed elements with a genetic organization very similar to that of Enterococcus faecalis DS16 Tn916. Sequence analysis highlighted variations in the leader peptide and six tet(M) variants were identified, five of which have never been described before. Conclusions: C. difficile isolates harbouring Tn916-like elements have mainly been isolated since 1997, suggesting a recent circulation of these elements among C. difficile strains in Italian hospitals. Molecular analysis of these Tn916-Iike elements showed that they may have different genetic structures and carry new tet(M) alleles.