| We analyzed the penicillin-binding protein profiles of four clinical vancomycin intermediate resistant S. aureus (VISA) isolates and found that PBP4 activity as measured by ability to bind labelled penicillin, was undetectable in three isolates (HIP 5827, HIP 5836 and HIP 6297) and markedly reduced in a fourth isolate (Mu50). In addition, overproduction of PBP4 from a high-copy-number plasmid that was transformed into the VISA isolates produced a two- to three-fold decrease in the vancomycin MIC values of these strains. Finally, inactivation of the pbp4 gene by allelic replacement mutagenesis in three vancomycin susceptible ORSA strains (COL, RN450M, and N315) led to a decrease in susceptibility to vancomycin, an increase in the highly vancomycin-resistant sub-populations, and decreased cell wall cross-linking, as evidenced by high-performance liquid chromatography analysis. Possible abnormalities of PBP4 in VISA verses vancomycin-susceptible S. aureus isolates were investigated and the results were as follows. First, the nucleotide sequences of the pbp4 structural gene and flanking regions did not differ significantly between VISA and vancomycin-susceptible isolates. Second, PBP4 expression was studied using a pbp4-beta-galactosidase fusion and found to be growth-phase regulated in both VISA and non-VISA isolates with the peak PBP4 expression level of vancomycin susceptible RN450M 20% higher than that of the VISA isolate HIP5827. However, immunoreactive PBP4 was present in all of the VISA isolates examined and in the same quantity as in non-VISA isolates. The initial observation that PBP4 activity was absent in VISA isolates was found to be due to the inactivation of labelled penicillin by beta-lactamase. Inactivation of beta-lactamase by clavulanic acid restored the PBP4 band to activity gels. (Abstract shortened by UMI.)... |
