Role of penicillin-binding protein 4 in expression of vancomycin resistance among clinical isolates of oxacillin-resistant Staphylococcus aureus

It has been reported that penicillin-binding protein 4 (PBP4) activity decr eases when a vancomycin-susceptible Staphylococcus aureus isolate is passag ed in vitro to vancomycin resistance. We analyzed the PBP profiles of four vancomycin intermediately susceptible S. aureus (VISA) clinical isolates an d found that PBP4 was undetectable in three isolates (HIP 5827, HIP 5836, a nd HIP 6297) and markedly reduced in a fourth (Mu50). PBP4 was readily visi ble in five vancomycin-susceptible, oxacillin-resistant S. aureus (ORSA) is olates. The nucleotide sequences of the pbp4 structural gene and flanking s equences did not different between the VISA and vancomycin-susceptible isol ates. Overproduction of PBP4 on a high-copy-number plasmid in the VISA isol ates produced a two- to threefold decrease in vancomycin MICs. Inactivation of pbp4 by allelic replacement mutagenesis in three vancomycin-susceptible ORSA strains (COL, RN450M, and N315) led to a decrease in vancomycin susce ptibility, an increase in highly vancomycin-resistant subpopulations, and d ecreased cell wall cross-linking by high-performance liquid chromatography analysis. Complementation of the COL mutant with plasmid-encoded pbp4 resto red the vancomycin MIC and increased cell wall cross-linking. These data su ggest that alterations in PBP4 expression are at least partially responsibl e for the VISA phenotype.