A fixed number of cells were inoculated on each of the agar plates containing various concentrations of vancomycin (abscissa). The plates were incubated Z-VAD-FMK chemical structure at 37 °C for 48 h. Then the number of grown colonies were counted and plotted on the semi-logarithmic graph. Note that the precursor strain Mu3 with MIC 2 mg/L is distinct from VSSA strain ΔIP (MIC 1 mg/L). Whereas the growth of ΔIP is completely depressed by 2 mg/L of vancomycin, the minor proportions of cells of Mu3 grew up to 12 mg/L of vancomycin though the 99.999% of the entire cell population is depressed with 3 mg/L of vancomycin. This clearly showed

that Mu3 is composed of heterogeneous cell subpopulations with different levels of vancomycin resistance. Within the subpopulations grown on the agar plates containing 4 mg/L or greater concentrations of vancomycin, we identified VISA converted strains. http://www.selleckchem.com/products/nutlin-3a.html To distinguish Mu3 from VSSA, we classified it as a heterogeneously vancomycin-resistant S. aureus (hVRSA; now is called hVISA) [52]. VISA is generated by accumulation of several spontaneous mutations [33] and [34]. The VISA phenotype of Mu50, for instance, can be reconstituted in VSSA strain ΔIP by sequentially

introducing four mutations in the genes vraS, msrR, rpoB and graR (Katayama, Y. in preparation). The first couple of two mutations in vraS and msrR converted ΔIP into PAK5 a hVISA strain with a similar PA pattern with that of Mu3, then rpoB mutation converted the hVISA into VISA with vancomycin MIC 4 mg/L. Addition of graR mutation further increased vancomycin MIC to 8 mg/L. vraS is the sensor histidine kinase of two-component regulatory

systems (TCRS), which is known to up-regulate the genes in the cell-wall synthesis pathway in response to the exposure to cell-wall-acting antibiotics [35] and [36]. graR is a response regulator of another TCRS which is involved in resistance to cationic antimicrobial peptides (CAMP) [37], [38] and [39]. msrR is considered to be involved in the production of wall-teichoic acid (WTA) [40] and [41]. The RNA polymerase (RNAP) core enzyme is composed of five subunits as represented by α2ββ′ω. Remarkably, as many as 64% of VISA clinical strains possessed more than one mutation in rpoB gene encoding the β subunit of the RNAP core enzyme [42]. When introduced individually into vancomycin-susceptible S. aureus strain ΔIP, the above four mutations either increased vancomycin MIC slightly, (i.e, within the susceptible range), or changed the susceptible patterns of PA curves to those of hVISA. Besides the four genes described above, great number of different mutations and their combinations were found to raise vancomycin resistance of S. aureus. A single mutation incorporated in any of the 20 genes in diverse metabolic pathways was found to raise vancomycin resistance [33].