Complete Reconstitution of Vancomycin-Intermediate Resistance of Mu50 in a Vancomycin-Susceptible Staphylococcus aureus Strain (CBX257). unidentified

Three mutations in vraSR, graRS, and rpoB genes contribute to the VISA phenotype of vancomycin-intermediate Staphylococcus (S.) aureus (VISA) strain Mu50 (MIC=8 mg/L). A mutation in a two-component regulatory system (TCRS) vraSR raises vancomycin resistance to the level of heterogeneously vancomycin-intermediate S. aureus (hVISA). Two more mutations in TCRS graRS and in rpoB encoding ? subunit of RNA polymerase confer on hVISA strain Mu3 VISA phenotype. As the final step of our study, we tried to reconstitute the VISA phenotype of Mu50 by introducing the three mutations into a na?ve S. aureus strain N315??IP (DIP) (MIC=1). Unexpectedly, the procedure did not confer the VISA phenotype on ?IP. By re-reviewing the whole genome sequence of Mu3, we noticed a mutation msrR (E146K). The gene msrR, encoding methionine sulfoxide reductase regulator, has been known to raise vancomycin resistance when introduced in N315 (the parent strain of DIP) on a multicopy plasmid. Additional introduction of the msrR mutation conferred the VISA-level vancomycin resistance on DIP. Surprisingly, however, the constructed strain did not show the cell-wall thickening characteristic to VISA strains. Further study showed that the cell-wall thickening of the construct was inducible by vancomycin. Finally, the introduction of another mutated gene SA2102(A297V) of Mu50 into the construct converted the inducible cell-wall thickening into a constitutive one. Thus consecutive introduction of the five mutations completed the transfer of the VISA phenotype of Mu50 into the na?ve S. aureus strain. Biological significance of the msrR and SA2102 mutations is discussed.