Morphological and genetic differences in two isogenic Staphylococcus aureus strains with decreased susceptibilities to vancomycin

Abstract

Many VISA (vancomycin intermediately resistant Staphylococcus aureus) strains are characterized by increased cell wall biosynthesis and decreased cross-linking of the peptide side chains, leading to accumulation of free D-alanyl-D-alanine termini in the peptidoglycan, which act as false target sites for vancomycin. A spontaneous mutant of methicillin-resistant VISA strain SA137/93A (vancomycin MIC [E-test], 8 micro g/ml), called SA137/93G, showed increased resistance to vancomycin (MIC [E-test], 12 micro g/ml). Analysis of the resistance profile of the mutant revealed a loss of beta-lactam resistance with a concomitant increase in resistance to glycopeptides. In both strains, cell wall thickness was 1.4-fold greater than that of control isolates. However, cross-linking of the cell wall was drastically lower in SA137/93A than in SA137/93G. The sensitivity of strain SA137/93G to beta-lactams was due to loss of the beta-lactamase plasmid and a deletion that comprises 32.5 kb of the methicillin resistance cassette SCCmec, as well as 65.4 kb of chromosomal DNA. A spontaneous mutant of SA137/93G with higher sensitivity to vancomycin displayed a cell wall profile similar, in some respects, to that of an fmhB mutant. Results described here and elsewhere show that the only feature common to all VISA strains is a thickened cell wall, which may play a central role in the vancomycin resistance mechanism.

FIG. 1.

Population analysis of VISA isolates SA137/93A (▴) and SA137/93G (×) and control strains NCTC 8325 (♦), SA1450/94 (▪), and Mu50 (•). The points on the abscissa refer to zero CFU values.

FIG. 2.

PFGE of the strains employed in this study. Lanes: 1, NCTC 8325; 2, SA137/93G; 3, SA1450/94; 4, SA137/93A.

FIG. 3.

(a) The ca. 100-kb chromosomal fragment that is present in MRSA strains SA1450/94 and SA137/93A and that was deleted in methicillin-susceptible S. aureus SA137/93G. (b) Nucleotide sequence present in SA137/93G. The 5′ end comprises the indirect repeat of IS431, which is directly attached to the 3′ end of ORF SA0132.

FIG. 4.

Electron micrographs of ultrathin sections of VISA strains SA137/93A (a) and SA137/93G (b), susceptible revertant SA137/93G1 (c), and control strain NCTC 8325 (d). Magnification, ×63,000.

FIG. 5.

PBP patterns of control strains NCTC 8325 (lane 5) and SA1450/94 (lane 6) and VISA isolates SA137/93A (lanes 1 and 2) and SA137/93G (lanes 3 and 4) cultured in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of vancomycin. About 125 μg of separated membrane proteins was loaded onto the gel. The PBPs were labeled with biotinylated ampicillin and detected by Western blotting. The numbers on the left correspond to PBP1 to -4.

FIG. 6.

Uptake of vancomycin from medium by VISA cells. Vancomycin at 30 μg/ml was added to a growing culture (OD₆₀₀, 0.4), and binding of vancomycin to the cells was monitored by determining the concentration of vancomycin in the supernatant. a, SA137/93A; b, SA137/93G.