Novel Mutation Sites in the Development of Vancomycin- Intermediate Resistance in Staphylococcus aureus

Abstract

Increased use of vancomycin has led to the emergence of vancomycin-intermediate <i>Staphylococcus aureus</i> (VISA). To investigate the mechanism of VISA development, 39 methicillin-susceptible strains and 3 MRSA strains were treated with vancomycin to induce non-susceptibility, and mutations in six genes were analyzed. All the strains were treated with vancomycin <i>in vitro</i> for 60 days. MICs were determined by the agar dilution and <i>E</i>-test methods. Vancomycin was then removed to assess the stability of VISA strains and mutations. Following 60 days of vancomycin treatment <i>in vitro</i>, 29/42 VISA strains were generated. The complete sequences of <i>rpoB, vraS, graR, graS, walK</i>, and <i>walR</i> were compared with those in the parental strains. Seven missense mutations including four novel mutations (L466S in <i>rpoB</i>, R232K in <i>graS</i>, I594M in <i>walk</i>, and A111T in <i>walR</i>) were detected frequently in strains with vancomycin MIC ≥ 12 μg/mL. Jonckheere-Terpstra trend test indicated these mutations might play an important role during VISA evolution. After the vancomycin treatment, strains were passaged to vancomycin-free medium for another 60 days, and the MICs of all strains decreased. Our results suggest that <i>rpoB, graS, walk</i>, and <i>walR</i> are more important than <i>vraS</i> and <i>graR</i> in VISA development.