VanA gene cluster in a Vancomycin-resistant clinical isolate of Bacillus circulans

We report on the cloning and sequencing of the vanA gene cluster present in the glycopeptide-resistant clinical isolate Bacillus circulans VR0709 (R. Fontana, M. Ligozzi, C. Pedrotti, E. M. Padovani, and G. Cornaglia, Eur. J. Clin. Microbiol. Infect. Dis. 16:473-474, 1997). The presence of a vanA- related gene in VR0709 was demonstrated in a PCR assay which permitted the specific amplification of an internal segment of vanA. Southern blotting suggested that the vanA gene was located in the chromosome in a 7.6-kb EcoRI fragment. DNA sequence analysis revealed the presence of all seven genes of the vanA cluster (vanR, vanS, vanH, vanA, vanX, vanY, and vanZ). The degree of identity between homologous proteins encoded by Tn1546 and the chromosome of B. circulans VR0709 ranged from 87 to 95%. Neither PCR nor Southern blotting with specific primers and probes, respectively, showed the presence of open reading frames (ORFs) 1 and 2 which encode the transposase and the resolvase of Tn1546, respectively, the transposon found to carry the vanA gene cluster in enterococci. Determination of the sequences of the flanking regions of the van gene cluster of B. circulans revealed perfect inverted repeats of 10 bp which delineated a 9.2-kb region containing the van gene cluster and an ORF which encoded a putative protein (178 residues) which displayed a low level of identity (28%) to the resolvase of Tn1546. These results suggest that glycopeptide resistance in B. circulans VR0709 is associated with the acquisition of a vanA gene cluster which shows a high degree of homology with that of enterococci. In B. circulans, however, the cluster is not carried by Tn1546 and is borne by the chromosome.