The transcriptomes of vancomycin intermediate-resistance Staphylococcus aureus (VISA) clinical isolates HIP5827 and Mu50 (MIC ‫؍‬ 8 g/ml) were compared to those of highly vancomycin-resistant S. aureus (VRSA; MIC ‫؍‬ 32 g/ml) passage derivatives by microarray. There were 35 genes with increased transcription and 16 genes with decreased transcription in common between the two VRSAs compared to those of their VISA parents. Of the 35 genes with increased transcription, 15 involved purine biosynthesis or transport, and the regulator (purR) of the major purine biosynthetic operon (purE-purD) was mutant. We hypothesize that increased energy (ATP) is required to generate the thicker cell walls that characterize resistant mutants.Vancomycin is the treatment of choice for serious infections caused by oxacillin-resistant Staphylococcus aureus, and some isolates are reported that are susceptible only to this antibiotic (8, 12). Oxacillin-resistant S. aureus isolates with reduced susceptibility to vancomycin (vancomycin intermediate-resistance S. aureus [VISA] isolates; the vancomycin MIC increases from 1 to 8 g/ml) have been recovered from patients receiving prolonged courses of vancomycin (1-3). A number of abnormalities have been noted among VISA isolates, including reduced rate of growth, decreased cell wall cross-linking (6, 16), increased cell wall thickness (4, 10), decreased autolysis (17), changes in penicillin binding proteins (6, 9, 15), and alterations in glutamate amidation of the peptidoglycan stem peptide (5). No unifying molecular hypothesis has been proposed that explains the VISA phenotype, but the current hypothesis is that the thickened, poorly cross-linked cell wall provides more peripheral targets for vancomycin, trapping the antibiotic before it can reach its site of lethal action at the cell membrane (17). It is also assumed that because of the long clinical vancomycin exposure times required to generate resistance, multiple genes and, probably, multiple metabolic pathways have been altered.We sought to assess genomic changes in gene transcription (the transcriptome) by DNA microarray and took a number of approaches different from those taken by others in trying to understand genomic changes associated with the VISA phenotype. First, we used clinical VISA isolates as parents rather than vancomycin-susceptible laboratory strains. There is evidence that there is something unique about VISA isolates that allows them to become resistant to vancomycin more easily than other S. aureus isolates (13). Second, we sought to amplify the phenotype by continued exposure of VISA isolates to vancomycin in vitro, further increasing the vancomycin MIC. We hypothesized that in this way, we would create isogenic strain sets that exaggerate the changes present in clinical VISA isolates. Third, we compared stable mutants of VISA strains for which vancomycin MICs were increased and we did not grow strains in the presence of the antibiotic, removing any direct effect of the antibiotic on transcription.Generation of mutant...

It has been reported that penicillin-binding protein 4 (PBP4) activity decreases when a vancomycinsusceptible Staphylococcus aureus isolate is passaged in vitro to vancomycin resistance. We analyzed the PBP profiles of four vancomycin intermediately susceptible S. aureus (VISA) clinical isolates and found that PBP4 was undetectable in three isolates (HIP 5827, HIP 5836, and HIP 6297) and markedly reduced in a fourth (Mu50). PBP4 was readily visible in five vancomycin-susceptible, oxacillin-resistant S. aureus (ORSA) isolates. The nucleotide sequences of the pbp4 structural gene and flanking sequences did not different between the VISA and vancomycin-susceptible isolates. Overproduction of PBP4 on a high-copy-number plasmid in the VISA isolates produced a two-to threefold decrease in vancomycin MICs. Inactivation of pbp4 by allelic replacement mutagenesis in three vancomycin-susceptible ORSA strains (COL, RN450M, and N315) led to a decrease in vancomycin susceptibility, an increase in highly vancomycin-resistant subpopulations, and decreased cell wall cross-linking by high-performance liquid chromatography analysis. Complementation of the COL mutant with plasmid-encoded pbp4 restored the vancomycin MIC and increased cell wall cross-linking. These data suggest that alterations in PBP4 expression are at least partially responsible for the VISA phenotype.Vancomycin is currently the treatment of choice for serious infections caused by oxacillin-resistant Staphylococcus aureus (ORSA). ORSA isolates recovered from patients with serious infections in both Japan (11) and the United States (23) have recently been described that have reduced susceptibility to vancomycin and other glycopeptides (vancomycin intermediately susceptible S. aureus [VISA]). In addition to having reduced vancomycin susceptibility, these isolates are also resistant to other antimicrobials, leaving few options for effective antimicrobial therapy.The mechanism of decreased vancomycin susceptibility is poorly understood and still largely speculative (4,7,16). VISA isolates demonstrate decreased autolysis, slower growth rates, and thicker cell walls in comparison to vancomycin-susceptible S. aureus (7). In a vancomycin-resistant S. aureus strain derived by in vitro passage, the cell walls of the mutant have a decrease in peptidoglycan cross-bridges and an increase in monomeric muropeptides carrying intact carboxyl-terminal D-alanyl-D-alanine residues (19,20), the targets of vancomycin, as stem peptide termini. The passage mutant has also been shown to have markedly decreased or absent PBP4, as assessed by radiolabeled penicillin binding (19). PBP4 is a low-molecular-weight (LMW) PBP hypothesized to be involved in secondary cell wall remodeling (9, 10, 26). It has transpeptidase activity and appears also to act as a D,D-carboxypeptidase, cleaving terminal D-alanine residues from un-cross-linked muropeptides (12). It has been proposed that VISA strains, with their thicker cell walls, provide an increased number of vancomycin targets at the cell wall peri...

Staphylococci that acquire the mecA gene are usually resistant to ␤-lactam antibiotics (methicillin or oxacillin resistance). mecA encodes a penicillin-binding protein (PBP 2a) that has a reduced affinity for ␤-lactams. In some isolates with methicillin or oxacillin resistance, only a small proportion (<0.1%) of the population expresses resistance to >10 g of oxacillin per ml (heterotypic resistance [HeR]), while in other isolates most of the population expresses resistance (homotypic resistance [HoR]). In the present study, growth of Staphylococcus aureus or Staphylococcus epidermidis strains with HeR in concentrations of oxacillin (0.3 to 0.7 g/ml) that produced a fall or a lag in optical density converted the strains from the HeR to the HoR phenotype. The conversion from the HeR to the HoR phenotype appeared to be due to the selection of a highly resistant mutant population, as determined by fluctuation analysis and the failure of populations with HoR to revert to HeR after 60 generations of growth in antibiotic-free media. The frequencies of conversion were as high as 10 ؊3 to 10 ؊2 . Conversion to HoR required an intact mecA gene and an increase in the level of mecA transcription since no highly resistant subpopulation could be selected after growth in oxacillin when mecA transcription was constitutively repressed or when mecA had been inactivated. In addition, in both S. epidermidis and S. aureus the level of resistance to vancomycin, which also acts directly on the staphylococcal cell wall, was greater among convertants with HoR than their isogenic parents. The conversion of a population from HeR to HoR involves the selection of a mutation(s) that occurs at a high frequency and most likely requires abundant PBP 2a.