ABSTRACT
The susceptibility of clinical isolates of Mycobacterium avium to rifampicin (RIF) was examined. All 32 clinical isolates tested, including 18 from Japan, 13 from Poland and 1 from Thailand, were resistant to RIF (minimum inhibitory concentrations (MICs) > or =32 microg/mL for 17 isolates and 2-16 microg/mL for 15 isolates), whereas the type strain of M. avium ATCC 25291 was susceptible to RIF (MIC < or = 0.03 microg/mL). Mutations in nucleotides 1276-1356 of the rpoB gene, termed the 81 bp core region, are associated with RIF resistance in Mycobacterium tuberculosis. No mutations were found in this region in any of the M. avium clinical isolates tested. However, mutation of G-->A to give a Gly544-->Asp substitution was identified within the rpoB gene downstream of the 81 bp region in all clinical isolates. A RIF-resistant strain (ATCC 25291 Rif(r); MIC> or =32 microg/mL) obtained by culturing the type strain in RIF-containing broth possessed a mutation C-->T to give a His445-->Tyr substitution within the 81 bp region. When the rpoB gene of the ATCC 25291 Rif(r) strain and of a clinical isolate were inserted into Mycobacterium smegmatis, organisms with the ATCC 25291 Rif(r) sequence, but not those with the clinical isolate sequence, showed resistance to RIF. These results suggest that mutations of the 81 bp region of rpoB, as well as factors other than rpoB mutation, confer RIF resistance in M. avium.
Subject(s)
Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Mutation , Mycobacterium avium/drug effects , Rifampin/pharmacology , Amino Acid Sequence , Bacterial Proteins/drug effects , Base Sequence , DNA-Directed RNA Polymerases , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Mycobacterium avium/genetics , Mycobacterium avium/isolation & purification , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Transformation, BacterialABSTRACT
Macrolide 2'-phosphotransferase (MPH(2')) catalyzes the transfer of the gamma-phosphate of ATP to the 2'-hydroxyl group of macrolide antibiotics. In this study, H198 and H205, conserved in the ATP-binding region motif 1 in the putative amino acid sequence of MPH(2')II, were replaced by Ala to investigate their role. H205 was also subsequently replaced by Asn. H198A and H205N mutant enzymes retained more than 50% of the specific activity of the original enzyme to substrate oleandomycin. On the other hand, the specific activity of the H205A mutant enzyme was reduced to less than 1% of that of the wild enzyme. The results suggested that H205 is crucial for maintaining the catalytic activity of MPH(2')II, and Asn can substitute for His at this position.
