Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis

Abstract

A tetracycline resistance (Tc') determinant from Closfridium &Bile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SucI-Hind111 fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. &mile strains and to Bacillus subtilis at a frequency of lo-' per donor cell. The element could be returned from B. subtilis to C. &@cik at a frequency of per donor cell. This is the first demonstration of C. &Wile acting as a recipient in intergeneric crosses. DNA from C. dzBcile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtiZis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transpomn Tn916. The Him11 restriction maps of the two elements Mered and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.

Antimicrobial Resistance, Toxinotype, and Genotypic Profiling of Clostridium difficile Isolates of Swine Origin

“…One recent study found all isolates of swine origin to be sensitive to metronidazole and vancomycin, 98% were sensitive to chloramphenicol, and 90% were sensitive to tetracycline (20). Previous studies have reported that the majority of C. difficile strains with tetracycline resistance have the tetM gene, which is carried on a conjugative transposon (19). The tetW gene has the second largest host range of the tetracycline resistance genes, second only to tetM.…”

Presence of the Listeria tetracycline resistance gene tet(S) in Enterococcus faecalis

“…The tet(M) gene is common among enterococci and streptococci, where it is part of broad-host-range conjugative transposons and is thus usually associated with int-Tn, the gene encoding the integrase of these elements (23,24). In contrast, tet(O) is rarely encountered in these genera, where it is carried by conjugative plasmids (33,35,37) (19). Lack of association of tet(M) with int-Tn could be due to deletions in conjugative transposons or to the existence of genetic support(s) other than Tn916-related elements for this resistance determinant in gram-positive cocci.…”

“…(29,31). Originally discovered in Streptococcus agalactiae (4), tet(M) has been subsequently detected in a large variety of aerobic and anaerobic gram-positive and gram-negative bacterial genera, including Enterococcus (2, 27, 37), Streptococcus (27,37), Peptostreptococcus (26,27), Staphylococcus (3), Listeria (9,25), Clostridium (11,19), Neisseria (16), Kingella (16), Gardnerella (27), Haemophilus (26), and Bacteroides (1), and in cell wall-less organisms such as Mycoplasma and Ureaplasma spp. (26).…”

Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630Δerm) and demonstration that the conjugative transposon Tn916ΔE enters the genome of this strain at multiple sites

“…Towards this end we have investigated the behaviour of the tetracycline-resistance-encoding conjugative transposon Tn916 and its erythromycin-resistant derivative Tn916˜E (Rubens & Heggen, 1988;Mullany et al, 1991) and the endogenous tetracycline-resistance-encoding conjugative transposon Tn5397 in C. difficile (for a recent review on the properties of these elements see Mullany, 2002, and accompanying articles). All these elements enter the genome of C. difficile CD37 at highly preferred sites (Mullany et al, 1990;Mullany et al, 1991;Wang et al, 2000a). Tn5398 is a transferable genetic element (Mullany et al, 1995); however, it does not contain the genes required for self-conjugation, therefore it is likely to be a mobilizable element (Farrow et al, 2001).…”