Selection of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus by Imipenem

Vancomycin (VAN)-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates are considered to have emerged from VAN-susceptible S. aureus (VSSA) by spontaneous mutation during VAN exposure. We previously reported that laboratory mutant H14, obtained from VSSA strain ΔIP by exposure to imipenem (IPM), showed overexpression of the vraSR two-component system and a typical hVISA phenotype. In the present study, to elucidate the mechanism of VSSA conversion to hVISA, we further characterized strain H14 by determining its whole-genome sequence, morphology, cell wall synthetic activity, and gene expression. Genome sequencing revealed that H14 harbored a mutated vraS (designated vraS_H14) that caused an amino acid substitution (S₃₂₉→L). This mutation is different from the VraS mutation (N₅→I) identified in representative clinical hVISA strain Mu3. However, H14 exhibited a phenotype similar to that of Mu3, including heterogeneous resistance to VAN, enhanced cell wall synthetic activity, and vraSR overexpression. Replacement of the vraS gene of ΔIP with the mutated vraS_H14 gene confirmed that the S₃₂₉→L substitution was responsible for both the upregulation of vraSR and conversion to the hVISA phenotype. This conversion was also achieved by using the vraS gene of Mu3, which carries a mutation (N₅→I), but not with the native vraS gene of strain N315. Finally, we carried out a study to analyze the appearance of hVISA from VSSA by exposure of ΔIP to selective concentrations of VAN and beta-lactam antibiotics. A total of 8 and 5 hVISA isolates were detected among 50 isolates selected with VAN and IPM, respectively. Among the 13 hVISA mutants, mutation in vraSR was detected only in mutant strain H14, suggesting that additional mutational mechanisms can be responsible for evolution to the hVISA phenotype. We conclude that exposure not only to VAN but also to beta-lactams may select for reduced glycopeptide susceptibility in S. aureus.Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is a major cause of serious nosocomial infections, and the emergence of virulent MRSA strains in the community is of particular concern (6). Vancomycin (VAN) still serves as the main therapeutic agent for infections caused by multiresistant MRSA strains (17). However, MRSA strains with various degrees of reduced susceptibility to glycopeptides, vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) strains, have emerged among multidrug-resistant MRSA clinical strains (9, 16).Recently, we identified several genes that are overexpressed in VISA strain Mu50 and hVISA Mu3 compared to their levels of expression in their isogenic VAN-susceptible S. aureus (VSSA) strain, strain Mu50Ω (13); and among these, we found the overexpression of the vraSR two-component system (TCS), an upregulator of the S. aureus cell wall biosynthesis pathway (12, 13). We also demonstrated that the vraS gene is overexpressed in ΔIP-H14 (H14), a laboratory-derived hVISA strain obtained by selecting VSSA strain N315ΔIP (ΔIP) with 8 mg/liter of imipenem (IPM) (12) and showed that a single amino acid substitution in VraS was present in H14, Mu3, and Mu50 (10a).In the study described here, we further characterized hVISA strain H14, investigated the role of the vraS mutation on the phenotype of H14, and evaluated the rates of selection of hVISA from VSSA ΔIP following exposure to VAN and beta-lactam antibiotics.