This further contributed to the increase of free d-alanyl-d-alanine residues in the PG layers and increased consumption of vancomycin. Furthermore, by another curious coincidence, the non-amidated muropeptide happened to have a greater binding affinity to vancomycin than the amidated muropeptide [16]. These observations correlated well with the experimental data that Mu50 consumes ≥2.8 times more vancomycin in its cell wall than VSSA strains [16]. Fig. 2B illustrates that VISA strain Mu50

with a vancomycin MIC of 8 mg/L can grow in learn more medium containing as high as 30 mg/L vancomycin [13]. When the concentration of vancomycin was monitored by bioassay, a significant drop in the concentration was observed from 30 mg/L to ca. 16–17 mg/L within a few minutes of addition of the cells to the medium. This is due to the rapid adsorption of vancomycin to the cell wall PG of Mu50. The

vancomycin concentration gradually decreased before the cells started to re-grow. It is noted that prior to the growth initiation of Mu50 cells, the concentration of vancomycin dropped to <10 mg/L. This decline of vancomycin concentration is due to the sequestration Epigenetics Compound Library of the drug by the false targets in PG of Mu50. The vancomycin MIC of the re-grown Mu50 cells was the same as that of the inoculum. This clearly demonstrates the ‘inoculum effect’ in S. aureus susceptibility to vancomycin [13]. When VISA cells are exposed to vancomycin, their PG layers adsorb a huge number of vancomycin molecules as described above. As a result, bound vancomycin molecules obliterate the PG mesh structure and prevent further passage of vancomycin molecules from outside of the cell. This ‘clogging effect’ is clearly observed in Fig. 3. Fig. 3A shows the time-dependent change in vancomycin concentration of the medium inoculated with ca. 5 × 108 cells with thick and thin PG layers. In less than 5 min, 15 mg/L vancomycin was adsorbed Oxalosuccinic acid by both cells, after which no more decrease was observed with cells with thin PG layers. Complete saturation of

vancomycin binding targets in PG is achieved. In contrast, cells with thick PG continued adsorbing vancomycin from the culture medium, but the rate of decrease in vancomycin concentration was blunted after 5 min and gradually reached saturation (denoted by an arrow in Fig. 3A). The thick cell wall finally adsorbed 25 mg/L vancomycin (Fig. 3A). Slowing of the decrease in vancomycin concentration was lost by brief treatment of the cells with lysostaphin, which breaks the PG mesh and allows vancomycin to penetrate the entire PG layer without hindrance (Fig. 3B). The more critical sequel of clogging is observed in Fig. 3A, in which the timing of saturation of thick PG layers is delayed by 20–40 min, which is almost comparable with the doubling time of S. aureus. This signifies that S. aureus cells would continue producing PG during the time delay and provide new PG layers from beneath the older PG layers.