Detection of a genetic linkage between genes coding for resistance to tetracycline and erythromycin in Clostridium difficile

Elements carrying more than one antibiotic resistance gene have never been found in Clostridium difficile, one of the major causes of nosocomial diarrheic diseases. In this study, C. difficile isolates were investigated for a possible genetic linkage between tet(M) and erm(B), the most frequent genes found in strains resistant to tetracycline and erythromycin. In the majority of C. difficile strains, tet(M) is carried by Tn5397. However, tet(M) genes carried by Tn916-like elements have been found in recent clinical isolates. As far as erythromycin resistance is concerned, the only completely characterized transposon harboring an erm(B) gene in C. difficile is Tn5398, even if ErmB determinants probably carried by other elements have been identified. Among the 100 C difficile isolates screened in this study, 27 were positive for tet(M) and erm(B). Twenty five of these strains were positive for tndX, used as marker for Tn5397, whereas two were positive for int, used as marker for Tn916-like elements. The latter isolates showed two tet(M) genes: one was carried by a Tn916-like element, able to transfer to a recipient C difficile strain, whereas the second was genetically linked to an erm(B) in a composite element probably unable to conjugate. Molecular analysis of C difficile cd1911 tet(M)-erm(B) DNA sequence demonstrated that this region has arisen by recombination of DNA fragments from different plasmids and transposons. This is the first demonstration that C difficile is able to accumulate and maintain antibiotic resistance genes, as observed in other pathogens.