Gradual alterations in cell wall structure and metabolism in vancomycin-resistant mutants of Staphylococcus aureus

Abstract

In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL (MIC of methicillin, 800 microg/ml; MIC of vancomycin, 1.5 microg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to beta-lactam antibiotics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective.

FIG. 1

Phenotypic expression of resistance to vancomycin (left) and methicillin (right) in a series of vancomycin-resistant step mutants. Bacterial cultures were grown in TSB to stationary phase and plated at several dilutions on TSA containing various concentrations of vancomycin or methicillin, as described for the method of population analysis (13). Asterisks indicate colonies that served as sources for higher-level step mutants.

FIG. 2

Slow growth rate of mutant VM50. A mixture of mutant VM50 and parental strain COL was plated on TSA and incubated for 72 h at 37°C. The large colonies represent the parental strain; the small colonies represent the mutant.

FIG. 3

Levels of mecA transcription in the parental strain COL (lane 1) and the vancomycin-resistant step mutants VM3 through VM50 (lanes 2 to 6). RNA was extracted from mid-log-phase cultures and resolved by electrophoresis on agarose-formaldehyde gels, and mecA RNA was located after hybridization with a ³²P-labeled mecA DNA probe as described in Materials and Methods.

FIG. 4

PBP patterns of parental strain COL (lane 1) and its vancomycin-resistant derivatives VM3 (lane 2), VM6 (lane 3), VM12 (lane 4), VM25 (lane 5), and VM50 (lane 6). Purified plasma membranes were incubated with [³H]benzylpenicillin and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then fluorography as described in Materials and Methods.

FIG. 5

HPLC elution profiles of muropeptides isolated from the parental strain COL and the vancomycin-resistant step mutants VM3 through VM50. Peptidoglycan was purified and digested with muramidase, and the muropeptides were separated by HPLC as described in Materials and Methods.

FIG. 6

Inhibitory effect of β-lactams on the phenotypic expression of vancomycin resistance in strain VM50 (A) and massive reduction of vancomycin resistance in strain VM50 with inactivated PBP2 (B). (A) Cultures of VM50 grown in TSB overnight were plated at different cell concentrations on TSA containing various concentrations of vancomycin and one-fourth the MIC of β-lactam antibiotics with selective affinities for PBP1, PBP2, PBP3, and PBP4. (B) The pbp2 gene in strain VM50 was inactivated by allele replacement with strain RU130 (7) as described in Materials and Methods. Open squares represent strain VM50 with inactivated PBP2 (VM-RU130); solid circles represent VM50 with the intact pbp2 gene. Colonies were counted, and data were plotted to provide population analysis profiles as described before (13).