The molecular basis of glycopeptide Resistence in two Clinical Isolates: Bacillus Lentus RSA1208 and Paenibacillus Thiaminolyticus RSA 1221

Abstract

The molecular mechanisms of glycopeptide resistance in two Gram-positive clinical isolates, Bacillus lentus RSA1208 and Paenibacillus thiaminolyticus RSA1221 were investigated. The glycopeptide resistance genotypes were determined by PCR. If van genes were detected, recombinant DNA techniques and sequencing were used to determine the gene sequence. The location of the resistance determinant was investigated using Southern hybridization techniques. To determine the 5’ and 3’ ends flanking the resistance operon, sub-genomic libraries were constructed. Transmission electron microscopy was used to assess possible structural changes of the B. lentus RSA1208 cell wall. B. lentus RSA1208 exhibits inducible, high-level resistance to both glycopeptides, but does not possess any known van resistance genes. Electron micrographs showed a visible increase in cell wall thickness in B. lentus RSA1208 grown in vancomycin compared to the isolate grown in vancomycin-free media. However, it remains to be confirmed as to whether this resistance is solely responsible for the high-level resistance phenotype. P. thiaminolyticus RSA1221 exhibits constitutive, high-level resistance to vancomycin only. It was found to possess a chromosomally-borne, vanA gene cassette. The vanA gene showed the highest amino acid identity to the vanA-like D-ala: D-lac gene found in P. thiaminolyticus PT-2B1 and Enterococcus faecium BM4147. All five genes of the vanA gene cluster (vanR, vanS, vanH, vanX, vanY) were amplified and sequenced. No vanZ gene was detected. The vanA operon in P. thiaminolyticus RSA1221 was found not to be associated with any known mobile elements. The observed constitutive expression of resistance maybe due to a two amino acid insertion in the VanSBpt1221 protein. v