Molecular analysis of novel tetracycline resistance genes and the elements involved in their transfer between diverse gut bacteria
A previously undescribed tetracycline resistance gene, tet(W), was isolated by our group from the rumen anaerobe Butyrivibrio fibrisolvens. tet(W) is less than 65% homologous to tet(M) and in the B. fibrisolvens strain 1.230, is carried on a 50 kb mobile chromosomal element, TnB1230, which is unrelated to known conjugative transposons. This thesis has identified a gene >99% homologous to tet(W) from two groups of human faecal bacteria, a low % G+C Gram Positive human colonic bacteria, Strain K10 and Bifidobacterium longum. In each isolate tet(W) was identified by Southern blot on differently sized chromosomal fragments which are unrelated to TnB1230. Sequence analysis of 13 kb of TnB1230 from a cosmoid library clone identified several open reading frames with identity to transfer functions encoded by conjugative elements from important human pathogens. The DNA% G+C of tet(W) is considerably higher than the flanking orf's in TnB1230 and also the Butyrivibrio host itself, suggesting that it may have originated in a higher % G+C bacterium. The tetracycline resistance (TcR) was transmissible from Strain K10 to the rumen anaerobe B. fibrisolvens 2221R by filter mating, however tet(W) was not detected in the transconjugants by PCR with the tet(W) specific primers. Instead a second novel TcR gene designated tet(32) was shown to be responsible for transmissible resistance in K10. This novel ribosome protection gene has also been sequenced and characterised and is most related to tet(O), expresses a higher level of TcR than other ribosome protection proteins and is abundant in the ovine rumen, human and porcine gut.