Alterations of cell wall structure and metabolism accompany reduced susceptibility to vancomycin in an isogenic series of clinical isolates of Staphylococcus aureus

Abstract

A series of isogenic methicillin-resistant Staphylococcus aureus isolates recovered from a bacteremic patient were shown to acquire gradually increasing levels of resistance to vancomycin during chemotherapy with the drug (K. Sieradzki, T. Leski, L. Borio, J. Dick, and A. Tomasz, J. Clin. Microbiol. 41:1687-1693, 2003). We compared properties of the earliest (parental) vancomycin-susceptible isolate, JH1 (MIC, 1 microg/ml), to two late (progeny) isolates, JH9 and JH14 (vancomycin MIC, 8 microg/ml). The resistant isolates produced abnormally thick cell walls and poorly separated cells when grown in antibiotic-free medium. Chemical analysis of the resistant isolates showed decreased cross-linkage of the peptidoglycan and drastically reduced levels of PBP4 as determined by the fluorographic assay. Resistant isolates showed reduced rates of cell wall turnover and autolysis. In vitro hydrolysis of resistant cell walls by autolytic extracts prepared from either susceptible or resistant strains was also slow, and this abnormality could be traced to a quantitative (or qualitative) change in the wall teichoic acid component of resistant isolates. Some change in the structure and/or metabolism of teichoic acids appears to be an important component of the mechanism of decreased susceptibility to vancomycin in S. aureus.

FIG. 1.

Phase-contrast and thin-section micrographs of the JH1 (A and C) and JH9 (B and D) cells. Bacteria, grown in TSB, were harvested at the mid-exponential stage of growth. Bar = 1 μm.

FIG. 2.

Triton X-100-stimulated autolysis of the parental strain, JH1, and its progeny, strains JH2 through JH14, as well as strain JH15. Cultures were suspended in autolysis buffer to an initial OD of ≅1.0, and the rates of autolysis were monitored as decrease of OD in time.

FIG. 3.

Suppression of cell wall turnover in strains JH9 and JH14. Bacterial cells were prelabeled with radioactive N-acetylglucosamine and then transferred into isotope-free medium, and the rate of cell wall turnover was measured as described in Materials and Methods.

FIG. 4.

Bacteriolytic enzyme profiles on an SDS-7.5% polyacrylamide gel containing NCTC 8325 cell walls (0.1%) as a substrate (top panel). Autolytic enzyme extracts were prepared and subjected to electrophoresis (40 μg of proteins per line). After electrophoresis, the gels were renatured and, after overnight incubation at 37°C, bands with lytic activity were observed as clear zones in the opaque gel. The clear zones appeared as dark bands against a dark background. The same protein samples, resolved on gels that did not contain cell walls, were subsequently stained with Coomassie brilliant blue (bottom).

FIG. 5.

Solubilization of cell walls (CWs) by LiCl cell extracts. Crude autolytic extracts prepared from strains JH1, JH9, and JH14 were used to test the susceptibility of JH1 cell walls for autolytic degradation in vitro (upper panel). In a parallel set of experiments, isolated cell walls of JH1, JH9, and JH14 were subjected to degradation in vitro by autolytic extract prepared from strain JH1 (bottom).

FIG. 6.

Solubilization of peptidoglycan by LiCl JH1 cell extract. Purified peptidoglycan samples prepared from strains JH1, JH9, and JH14 were incubated with autolytic extract prepared from strain JH1, and the turbidity (OD₆₂₀) of the samples was monitored at intervals.

FIG. 7.

Solubilization by LiCl JH1 cell extract of JH1 peptidoglycan suspended in clear cell wall hydrolysates. Cell walls (CW) of JH9 and JH14 were digested overnight with muramidase and lysostaphin, incubated for 10 min at 100°C, and centrifuged, and clear hydrolysates were used as a medium for JH1 peptidoglycan hydrolysis.

FIG. 8.

Reduced rate of peptidoglycan synthesis for the strain JH9. Cultures of the parental strain, JH1, and its vancomycin-resistant derivative JH9 were grown in TSB. Optical densities were recorded, and the rate of incorporation of radioactive N-acetylglucosamine into the cell wall material during 5-min pulses was determined. The specific rates of incorporation (the counts per minute [CPM] of radioactive label associated with the cell wall divided by the OD) are plotted as a function of time.

FIG. 9.

Muropeptide profiles of the parental strain JH1 (A) and its vancomycin-resistant progeny, JH9 (B) and JH14 (C). Peptidoglycan was purified and digested with muramidase, and the muropeptides were separated by HPLC.

FIG. 10.

Membrane proteins (upper panel) and the PBP patterns (bottom) of the JH strains. The purified plasma membranes were incubated with (¹⁴C)benzylpenicillin and subjected to SDS-polyacrylamide gel electrophoresis, gels were stained with Coomassie brilliant blue, and the PBPs were finally detected by fluorography. MWM, molecular weight markers.