Abstract
Increased use of vancomycin has led to the emergence of vancomycin-intermediate Staphylococcus aureus (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 in vitro for 60 days. MICs were determined by the agar dilution and E-test methods. Vancomycin was then removed to assess the stability of VISA strains and mutations. Following 60 days of vancomycin treatment in vitro, 29/42 VISA strains were generated. The complete sequences of rpoB, vraS, graR, graS, walK, and walR were compared with those in the parental strains. Seven missense mutations including four novel mutations (L466S in rpoB, R232K in graS, I594M in walk, and A111T in walR) 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 rpoB, graS, walk, and walR are more important than vraS and graR in VISA development.
Highlights
Multiple antibiotic resistant Staphylococcus aureus continues to be one of the most common pathogens of both hospital-associated and community-associated infections worldwide (Klevens et al, 2007; Popovich et al, 2007; Hidron et al, 2008; Kallen et al, 2010)
Our results suggest that four novel mutation sites are important for vancomycin-intermediate Staphylococcus aureus (VISA) development: L466S in rpoB, R232K in graS, I594M in walK, and A111T in walR
Twenty-nine VISA strains were generated within 60 days, while 13 strains remained vancomycin susceptible (MIC < 4 μg/mL)
Summary
Multiple antibiotic resistant Staphylococcus aureus continues to be one of the most common pathogens of both hospital-associated and community-associated infections worldwide (Klevens et al, 2007; Popovich et al, 2007; Hidron et al, 2008; Kallen et al, 2010). The vraS I5N mutation was found to confer heterogeneous vancomycin resistance when introduced into a vancomycin-susceptible MRSA strain (Katayama et al, 2009). The H481Y mutation in rpoB has been confirmed by allelic replacement experiments to increase vancomycin resistance during development of the VISA phenotype in the Mu3 (hVISA) strain (Matsuo et al, 2011). The majority of previous reports on the genetic mechanism of VISA development have focused on clinical MRSA strains (Doddangoudar et al, 2012). 39 methicillin-susceptible S. aureus and 3 MRSA strains were treated with increasing concentrations of vancomycin in vitro to investigate the genetic mechanism underlying development of vancomycin resistance. The genes (rpoB, vraS, graSR, and walRK) important for development of vancomycin non-susceptibility were analyzed after 60 days of vancomycin treatment and compared with those in the parental strains. Our results suggest that four novel mutation sites are important for VISA development: L466S in rpoB, R232K in graS, I594M in walK, and A111T in walR
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