The mechanism of glycopeptide resistance in Staphylococcus aureus is not known with certainty. Because the target of vancomycin is the D-Ala-D-Ala terminus of the stem peptide of the peptidoglycan precursor, by subjecting muropeptides to reversed-phase high-performance liquid chromatography, we investigated peptidoglycan obtained from glycopeptide-intermediate S. aureus (GISA) isolates for changes in composition and evaluated whether any peptidoglycan structural change was a consistent feature of clinical GISA isolates. GISA isolates Mu50 and Mu3 from Japan had the large glutamate-containing monomeric peak demonstrated previously, although strain H1, a vancomycin-susceptible MRSA isolate from Japan that was clonally related to Mu3 and Mu50, and a femC mutant that we studied, did also. For the U.S. GISA isolates, strain NJ had a large monomeric peak with a retention time identical to that described for the glutamate-containing monomer in strains H1, Mu3, and Mu50. However, a much smaller corresponding peak was seen in GISA MI, and this peak was absent from both GISA PC and a recent GISA isolate obtained from an adult patient in Illinois (strain IL). These data suggest that a uniform alteration in peptidoglycan composition cannot be discerned among the GISA isolates and indicate that a single genetic or biochemical change is unlikely to account for the glycopeptide resistance phenotype in the clinical GISA isolates observed to date. Furthermore, a large monomeric glutamate-containing peak is not sufficient to confer the resistance phenotype.

The glycopeptide antimicrobials vancomycin and teicoplanin inhibit the transglycosylation and transpeptidation reactions that are required for synthesis of peptidoglycan by binding to the D-alanyl-D-alanine (D-Ala-D-Ala) termini of peptidoglycan pentapeptide precursors (reviewed in reference 6). Despite the identification of several properties that differ between susceptible staphylococcal isolates and those with decreased susceptibility or resistance to glycopeptides (8,13,27,31,35,40), the mechanism mediating glycopeptide resistance in any staphylococcal isolate remains unknown.To investigate the vancomycin resistance mechanism in Staphylococcus aureus, we (13, 31) and others (7,20,27,44) have produced isolates with decreased susceptibility (resistance) to vancomycin and teicoplanin by serial incubation of susceptible clinical isolates in the presence of increasing concentrations of vancomycin. In one such glycopeptide-resistant isolate, 523k, derived from the susceptible clinical isolate 523, the MICs of both vancomycin and teicoplanin increased above the susceptibility breakpoints and no change occurred in the susceptibility of any other antimicrobial tested (13). Moreover, the resistance phenotype was stable in the absence of glycopeptides (13).By nucleotide and peptide sequencing, we recently identified a 37-kDa cytoplasmic protein, provisionally called Ddh, that is constitutively overproduced by isolate 523k (13, 30). Ddh is related (30) to the family of bacterial D-2-hydroxyacid dehydrogenases, which includes NAD ϩ -linked D-lactate dehydrogenases (D-nLDH by the nomenclature of Garvie [18]) and the VanH dehydrogenase (2), which is essential for acquired high-level vancomycin resistance in Enterococcus faecium and Enterococcus faecalis (1). The role of VanH in vancomycin resistance in enterococci which harbor the vanH gene is to synthesize D-lactate, which becomes the carboxyl-terminal residue of the stem peptide portion of peptidoglycan precursors (11). The presence of D-lactate instead of the usual D-Ala residue at the terminal position decreases the affinity of the precursor for vancomycin 1,000-fold (11).There are a few bacterial species that have low-level intrinsic glycopeptide resistance by producing a small proportion of peptidoglycan precursors terminating in D-lactate or serine, in addition to the wild-type precursor, which terminates in D-Ala (9,22). In some of these bacteria, such as Lactobacillus casei, the lactate produced for metabolic purposes is presumably funneled into the production of the D-lactate-terminating precursors. Similarly, in a vancomycin-resistant Staphylococcus haemolyticus isolate, although the bulk of the total pool of peptidoglycan precursors terminate in D-Ala, a small fraction terminate in D-lactate (8).The observation that increased cytoplasmic D-nLDH activity corresponded to increased production of the 37-kDa protein in S. aureus isolate 523k (30) strongly suggested that this protein is the S. aureus D-nLDH described previously (19) and provided a possible explanatio...

We previously reported the isolation of a laboratory-derived Staphylococcus aureus mutant, 523k, that has constitutive low-level resistance to vancomycin (MIC = 5 micrograms/ml) and teicoplanin (MIC = 5 micrograms/ml) and elaborates a ca. 39-kDa cytoplasmic protein that was not detected in the parent strain 523 (MIC = 1 micrograms/ml). We have now detected the protein in strain 523 by immunoblotting with antiserum raised against the protein. Consistent with our initial observations, densitometric analysis of the immunoblots revealed an increased production of the protein in 523k compared with that of the susceptible parent 523. The 5' region of the gene encoding the protein of interest was identified by nucleotide sequencing a PCR product amplified from the genome of 523k with degenerate primers designed to encode the amino acid sequence of proteolytic peptides obtained from the protein. The remainder of the gene was identified by library screening, PCR, and nucleotide sequencing. The gene encodes a 36.7-kDa protein with homology to a family of bacterial NAD+-dependent, D-specific 2-hydroxyacid dehydrogenases which includes both D-lactate dehydrogenase and the enterococcal vancomycin resistance protein VanH and is therefore designated ddh. Increased production of the product of ddh, Ddh, was associated with increased D-lactate dehydrogenase activity in 523k, a finding which suggested that Ddh is likely to be the D-lactate dehydrogenase previously identified in S. aureus. The increased D-lactate dehydrogenase activity in strain 523k and the structural similarities among Ddh, D-lactate dehydrogenase, and VanH suggest that overproduction of Ddh might play a role in vancomycin resistance in this strain.