Mu50 (NRS1) [1] and MI (NRS3) [47] represent high-level VISA, whereas the majority of clinical VISA strains are low-level VISA (Table 1). As illustrated in Fig. 4B, introduction of graR(N197S) raised vancomycin resistance of hVISA strain Mu3 to the level of a low-level VISA, and subsequent introduction of rpoB(H481Y) converted it into high-level VISA indistinguishable from Mu50 [34]. Subsequently, the VISA phenotype of Mu50 was successfully Galunisertib reconstituted in VSSA strain N315ΔIP by sequentially introducing four mutations, i.e. vraS(S329L),

msrR(E146K), graR(N197S) and rpoB(H481Y) (Katayama Y, unpublished data) ( Fig. 4B). However, we were not convinced with the idea that combination of graR and rpoB mutations was the only way for Mu3 to become high-level VISA. To obtain a more comprehensive view on the genetic events underlying hVISA-to-VISA conversion, we established 45 high-level VISA strains by selecting Mu3 and its related hVISA strains with 6 mg/L vancomycin and determined their whole genome sequences [38]. VISA is generated by spontaneous mutation from hVISA at a frequency

of ≥10−6[3], [48] and [49]. Since the usual appearance rate for a spontaneous mutation is ca. 10−8–10−9, such a high incidence of emergence of VISA from hVISA indicates the presence of great numbers of alternative mutations. The whole-genome sequences of 45 VISA-converted mutant strains revealed a surprising result that each converted strain had one to four mutations, but no two strains shared the same mutation [38]. Table 2 shows the Buparlisib molecular weight list of non-synonymous single mutation found in 32 of the 45 VISA-converted strains. Each strain carried a unique mutation in 1 of the 20 genes. Therefore, those genes listed in Table 2 were considered to have a direct contribution to the hVISA-to-VISA phenotypic conversion as a single determinant. By far the most

frequently affected genes were rpoB and rpoC in six strains, cmk in another six strains, followed by tarO in three strains [38]. They affected various cellular processes and metabolic pathways of the cell, and five of them were reported previously in association with raised vancomycin resistance, including Reverse transcriptase SAHV_1209 encoding PP2C phosphatase [50], pbp4 [51], rpoB [34], [39] and [52], rpoC [53] and walK [28], [29], [30] and [54]. Reduction in PBP4 activity decreases PG cross-linkages and thus increases the number of false targets of vancomycin. PP2C phosphatase and the walKR TCRS are considered to be associated with cell wall metabolism and the control of autolysis [50] and [55]. Accelerated cell wall synthesis and decreased autolysis are two alternative ways to thicken cell wall PG layers. In this regard, it is also noteworthy that the orf SAHV_1760 encoding putative autolysin is among the singly mutated gene list ( Table 2). As shown in Table 1, rpoB mutation is frequently found in clinical VISA strains.