. Analysis of the resistance profile of the mutant revealed a loss of ␤-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 ␤-lactams was due to loss of the ␤-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.Staphylococcus aureus is one of the most common pathogens and is a trigger of community-acquired and nosocomial disease. The drugs of choice against methicillin-resistant S. aureus (MRSA) strains are the glycopeptide antibiotics vancomycin and teicoplanin. Since 1996, emergence of MRSA strains resistant to vancomycin has been reported in many countries (4-6, 13, 16, 21, 30, 36, 41, 45, 53). According to the NCCLS, strains for which the MIC of vancomycin is less than or equal to 4 g/ml are considered susceptible whereas those for which the MIC is greater than or equal to 32 g/ml are considered resistant. Strains for which the MICs are 8 to 16 g/ml are intermediately resistant. Detailed characterization of clinical isolates and in vitro-selected vancomycin-resistant mutants showed that the resistance phenotype of at least some vancomycin intermediately resistant S. aureus (VISA) strains is caused by activation of cell wall biosynthesis and a significant increase in free D-alanyl-D-alanine termini in the cell wall, which represent false target sites for vancomycin. Resistance was shown to go along with thickening of the cell wall, changes in the composition of the peptidoglycan, and the expression of penicillin-binding proteins (PBPs) (7,8,18,31,37,44,(46)(47)(48). Some authors have suggested that the genes involved in glycopeptide resistance are probably under the control of a comprehensive regulatory system (8, 33). However, the reported changes in cell wall biochemistry are not restricted to glycopeptide-resistant strains and the magnitude of the changes does not always correlate with the resistance level (7,39).In this study, we characterize two VISA isolates and one revertant strain. SA137/93A was identified by the screening of 457 clinical isolates of MRSA from the strain collection of the Reference Center for Staphylococci of the University of Bonn (4). A spontaneous ␤-lactam-susceptible mutant of SA137/93A with an increased vancomycin MIC was named SA137/93G. SA137/93G1 is a vancomycin-susceptible revertant of strain SA137/93G. We examined the three strains with regard to the previously reported characteristics of glycopeptide-resist...

The emergence of intermediate vancomycin resistance, mainly in methicillin-resistant Staphylococcus aureus strains, has become a great concern. Thorough characterization of clinical and laboratory vancomycinintermediately resistant S. aureus (VISA) strains identified multiple, resistance-associated changes most probably due to stepwise mutations. We hypothesized that an elevated mutation frequency as found, e.g., in mutator strains defective in DNA mismatch repair could allow rapid acquisition of adaptive mutations in the presence of vancomycin. We therefore subjected S. aureus RN4220 and its isogenic mutator strain, the mutS-knockout mutant RN4220⌬mutS, to a stepwise vancomycin selection procedure. Vancomycin resistance evolved much more quickly in the mutator background than in the wild type (5 versus 19 passages, respectively). In addition, a higher resistance level could be reached (MIC, 32 versus 4 g/ml, respectively). The susceptibility to other antibiotics with the exception of teicoplanin remained unchanged. Concomitantly with increasing vancomycin resistance, a loss of phage typeability and differences in growth behavior as well as an improved ability to regrow at high vancomycin concentrations were observed. In conclusion, an elevated mutation rate in S. aureus led to the rapid development of vancomycin resistance, indicating that a high mutation frequency could be one of the factors that favor the emergence of vancomycin resistance in S. aureus.Staphylococcus aureus causes severe community-acquired and nosocomial disease. The glycopeptide antibiotic vancomycin has long been reserved for treatment of infections with methicillin-resistant S. aureus (MRSA). However, over the last 10 years, vancomycin-intermediately resistant S. aureus strains, mainly MRSA strains, have emerged in many countries (5,11,14,15,25,35). According to the National Committee for Clinical Laboratory Standards (NCCLS), strains for which the MIC of vancomycin is Յ4 g/ml in Mueller-Hinton (MH) medium are considered susceptible whereas strains for which the MIC is Ͼ16 g/ml are considered resistant to vancomycin. For most clinical isolates that have been reported so far, MICs of vancomycin are 8 g/ml, and thus these isolates are considered intermediately resistant. Nevertheless, these strains are of clinical importance since treatment failure has been reported elsewhere (24,35,43). Many strains display a heterogeneous phenotype, i.e., although the overall MICs for them are Յ4 g/ml, subpopulations resistant to higher vancomycin concentrations are present at a frequency of at least 10 Ϫ6 (24). Higher MICs up to 100 g/ml have been obtained for strains that were selected by stepwise passage on vancomycin agar in the laboratory (7, 13, 40).The low-level resistance of clinical S. aureus isolates is not associated with one of the van gene clusters which mediate the high-level resistance in enterococci (21, 46). Up to now, the mechanism leading to vancomycin resistance in staphylococci has not been fully understood. The thorough characterizati...