Cell wall thickening is a common feature of vancomycin resistance in Staphylococcus aureus

Abstract

We have previously shown that a thickened cell wall is responsible for the vancomycin resistance of vancomycin-resistant Staphylococcus aureus (VRSA) (equivalent to vancomycin-intermediate S. aureus and glycopeptide-intermediate S. aureus) strain Mu50 (L. Cui, H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu, Antimicrob. Agents Chemother. 44:2276-2285, 2000). However, the mechanism of vancomycin resistance in other VRSA strains remained unclear. In this study, 16 clinical VRSA strains from seven countries were subjected to serial daily passage in drug-free medium. After 10 to 84 days of passage in the nonselective medium, passage-derived strains with decreased MICs of vancomycin (MIC, <4 mg/liter) were obtained. However, all of the passage-derived strains except one (15 of 16) still possessed subpopulations that were resistant to vancomycin as judged by population analysis, and vancomycin-resistant mutant strains were selected from the passage-derived strains by one-step vancomycin selection with a frequency of 4.25 x 10(-6) to 1.64 x 10(-3). The data indicated that vancomycin-resistant cells are frequently generated from the passage-derived strains even after vancomycin selective pressure is lifted. Cell wall thicknesses and MICs of glycopeptides (vancomycin and teicoplanin) and beta-lactams (imipenem and oxacillin) were determined for a total of 48 strains, including 15 sets of three strains: the clinical VRSA strain, the passage-derived strain, and the vancomycin-resistant mutant strain obtained from the passage-derived strain. No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (r = 0.679; P < 0.001) and between MICs of imipenem and oxacillin (r = 0.787; P < 0.001) were recognized. Moreover, all of the VRSA strains had significantly thickened cell walls, which became thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains. The data showed that cell wall thickness had high correlation with the MICs of the two glycopeptides (correlation coefficients, 0.908 for vancomycin and 0.655 for teicoplanin) but not with those of the beta-lactam antibiotics tested. These results together with coupled changes of cell wall thickness and vancomycin MICs in 16 isogenic sets of strains indicate that thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for vancomycin resistance in S. aureus.

FIG. 1.

Analysis of vancomycin-resistant subpopulations of VRSA strains and their passage-derived strains. The population curves of 16 VRSA strains and their passage-derived strains are compared with those of Mu50 and Mu3. All of the passage-derived strains except BR2-P25 retain subpopulations of cells capable of growth in 4 mg of vancomycin/liter despite their susceptible vancomycin MIC (2 or 3 mg/liter).

FIG. 2.

PFGE banding patterns of SmaI-digested chromosomal DNAs of VRSA strains and their passage-derived strains. All except three passage-derived strains (NJ-P50, AMC11094-P84, and BR3-P60) had PFGE patterns identical to those of the parent strains. Lanes MW, low-range PFGE marker (Bio-Rad Laboratories). The sizes of the fragments are in kilobases.

FIG. 3.

Transmission electron microscopy of representative VRSA strains, their passage-derived strains, and vancomycin-resistant mutant strains. Magnification, ×30,000. The values given under each panel are the means and SDs of the cell wall thickness of the cells in nanometers. Note that the cell walls of passage-derived strains (with suffix P) were much thinner than those of the parent VRSA strains and vancomycin-resistant mutant strains (suffix PR).

FIG. 4.

Regression analysis between cell wall thickness and MICs of glycopeptide and beta-lactam antibiotics. Circles, VRSA strains; squares, passage-derived strains; triangles, vancomycin-resistant mutant strains; diamonds, other S. aureus strains. A significant correlation was seen between vancomycin MIC and cell wall thickness, followed by that between teicoplanin MIC and cell wall thickness. No correlation was seen between beta-lactam antibiotic MICs and cell wall thickness.

FIG. 5.

Proposed cycle of vancomycin resistance expression in the MRSA population. Hetero-VRSA strains prevail in the hospital, and some of these strains become VRSA as a result of prolonged exposure of the strains to vancomycin. Once vancomycin pressure is lifted, VRSA gradually goes back to hetero-VRSA status, from which, however, VRSA is regenerated at a high frequency of 1 in 10³ to 1 in 10⁶. Some in vitro-selected hetero-VRSA strains are unstable (K. Hiramatsu, unpublished observation) and return to VSSA status within 2 weeks in drug-free culture. However, some hetero-VRSA clinical strains, represented by Mu3, are extremely stable, and constitute a great risk factor for the emergence of VRSA and vancomycin therapeutic failure once they are allowed to prevail in the hospital. Both beta-lactam and vancomycin serve as selective pressure in the generation of hetero-VRSA from VSSA (17).