Novel Mutation Sites in the Development of Vancomycin- Intermediate Resistance in .

Increased use of vancomycin has led to the emergence of vancomycin-intermediate (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 for 60 days. MICs were determined by the agar dilution and -test methods. Vancomycin was then removed to assess the stability of VISA strains and mutations. Following 60 days of vancomycin treatment , 29/42 VISA strains were generated. The complete sequences of , and were compared with those in the parental strains. Seven missense mutations including four novel mutations (L466S in , R232K in , I594M in , and A111T in ) 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 , and are more important than and in VISA development.