Role of penicillin-binding protein 4 in expression of vancomycin resistance among clinical isolates of oxacillin-resistant Staphylococcus aureus - PubMed (original) (raw)

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

J E Finan et al. Antimicrob Agents Chemother. 2001 Nov.

Abstract

It has been reported that penicillin-binding protein 4 (PBP4) activity decreases when a vancomycin-susceptible Staphylococcus aureus isolate is passaged in vitro to vancomycin resistance. We analyzed the PBP profiles of four vancomycin intermediately susceptible S. aureus (VISA) clinical isolates and found that PBP4 was undetectable in three isolates (HIP 5827, HIP 5836, and HIP 6297) and markedly reduced in a fourth (Mu50). PBP4 was readily visible in five vancomycin-susceptible, oxacillin-resistant S. aureus (ORSA) isolates. The nucleotide sequences of the pbp4 structural gene and flanking sequences did not different between the VISA and vancomycin-susceptible isolates. Overproduction of PBP4 on a high-copy-number plasmid in the VISA isolates 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 susceptibility, an increase in highly vancomycin-resistant subpopulations, and decreased cell wall cross-linking by high-performance liquid chromatography analysis. Complementation of the COL mutant with plasmid-encoded pbp4 restored the vancomycin MIC and increased cell wall cross-linking. These data suggest that alterations in PBP4 expression are at least partially responsible for the VISA phenotype.

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Figures

FIG. 1

FIG. 1

PBP profiles in the presence and absence of vancomycin. Lanes: 1, RN450M without vancomycin; 2, HIP5827 with vancomycin; 3, HIP5827 without vancomycin; 4, HIP5836 with vancomycin; 5, HIP5836 without vancomycin; 6, Mu50 with vancomycin; 7, Mu50 without vancomycin; 8, Mu3 with vancomycin; 9, Mu3 without vancomycin.

FIG. 2

FIG. 2

PBP profiles of control strains RN4220 (lane 1), RN4220/pJF3 (lane 2), and RN450M (lane 3) and VISA isolates Mu3 (lane 4), Mu3/pJF3 (lane 5), Mu50 (lane 6), Mu50/pJF3 (lane 7), HIP6297 (lane 8), HIP6297/pJF3 (lane 9), HIP 5836 (lane 10), HIP5836/pJF3 (lane 11), HIP5827 (lane 12), and HIP5827/pJF3 (lane 13).

FIG. 3

FIG. 3

EOP curves for HIP5836 and HIP5827. Shown are numbers of_S. aureus_ bacteria (in log10 CFU per milliliter) remaining on plates containing various concentrations of vancomycin. Each parental VISA isolate is represented as a solid square. Each parental VISA isolate containing the cloning vector alone (pSK265) is represented as a solid circle. Each VISA isolate expressing_pbp4_ on high-copy plasmid pJF3 is represented as a solid diamond.

FIG. 4

FIG. 4

EOP curves for pbp4 knockouts of COL and N315. Shown on the y axis are the numbers of_S. aureus_ bacteria (in log10 CFU per milliliter) remaining on the plates containing various concentrations of vancomycin. Parent strains N315 and COL are represented by solid squares, and their isogenic pbp4 knockouts are represented by solid circles.

FIG. 5

FIG. 5

Analysis of peptidoglycan from S. aureus COL (A), COL with pbp4 inactivated following allelic mutagenesis (B), and COL with pbp4_inactivated complemented with pJF3 (C) by reverse-phase HPLC. COL with_pbp4 inactivated demonstrates a lower degree of muropeptide cross-linking, as evidenced by the lower number of oligomeric muropeptides at the end of the chromatogram. Complementation of the inactivated pbp4 gene with pJF3 leads to a higher degree of cross-linking, as evidenced by the increase in trimers and oligomers in panel C. mAU, milliabsorption units.

FIG. 6

FIG. 6

Vancomycin gradient plate of COL and COLΔ_pbp4_ before and after overnight exposure to vancomycin at 1 μg/ml. Lanes: 1, COL with no exposure; 2, COL after overnight exposure to vancomycin; 3, COLΔ_pbp4_ with no exposure; 4, COLΔ_pbp4_ after overnight exposure to vancomycin.

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