Characterization of mutations in the rpoB gene that confer rifampin resistance in Staphylococcus aureus

Abstract

Mutations in the rifampin resistance-determining (Rif) regions of the rpoB gene of Staphylococcus aureus mutants obtained during therapy or in vitro were analyzed by gene amplification and sequencing. Each of the resistant clinical isolates, including five nonrelated clones and two strains isolated from the same patient, and of the 10 in vitro mutants had a single base pair change that resulted in an amino acid substitution in the beta subunit of RNA polymerase. Eight mutational changes at seven positions were found in cluster I of the central Rif region. Certain substitutions (His481/Tyr and Asp471/Tyr [S. aureus coordinates]) were present in several mutants. Substitutions Gln468/Arg, His481/Tyr, and Arg484/His, which conferred high-level rifampin resistance, were identical or in the same codon as those described in other bacterial genera, whereas Asp550/Gly has not been reported previously. Substitutions at codon 477 conferred high- or low-level resistance, depending on the nature of the new amino acid. The levels of resistance of in vivo and one-step in vitro mutants carrying identical mutations were similar, suggesting that no other resistance mechanism was present in the clinical isolates. On the basis of these data and the population distribution of more than 4,000 clinical S. aureus isolates, we propose </=0.5 and >/=8 microg/ml as new breakpoints for the clinical categorization of this species relative to rifampin.

FIG. 1

Distribution of rifampin MICs for 4,644 S. aureus strains isolated at Henri Mondor Hospital between 1993 and 1996.

FIG. 2

Amino acid sequence comparison of the Rif regions of E. coli (Ec) and S. aureus (Sa). Clusters I, II, and III are indicated by dashed lines. Identical amino acids are indicated by asterisks, and gaps are indicated by dashes. Positions known to be involved in rifampin resistance in E. coli are indicated by downward-pointing arrows (mutations) or triangles (insertions) or are underlined (deletions). Mutations leading to low- and high-level rifampin resistance in S. aureus are indicated by single and double upward-pointing arrows, respectively.