Cloning of the Staphylococcus aureus ddh gene encoding NAD+-dependent D-lactate dehydrogenase and insertional inactivation in a glycopeptide-resistant isolate

The mechanism of low-level glycopeptide resistance among staphylococci is not known. A cytoplasmic protein, provisionally called Ddh (W. M. Milewski, S. Boyle-Vavra, B. Moreira, C. C. Ebert, and R. S. Daum, Antimicrob. Agents Chemother. 40:166-172, 1996), and the RNA transcript that contains the ddh gene, which encodes Ddh, are present in increased amounts in a vancomycin-resistant isolate, 523k, compared with the susceptible parent isolate, 523. Sequence analysis had previously revealed that Ddh is related to NAD+-dependent D-lactate dehydrogenase (D-nLDH) and VanH. This latter protein is essential for high-level glycopeptide resistance in Enterococcus faecium and Enterococcus faecalis by synthesizing the D-lactate needed for biosynthesis of D-lactate-terminating peptidoglycan precursors with low affinity for vancomycin. We now provide the direct evidence that the ddh gene product is Staphylococcus aureus D-nLDH and hereafter refer to the protein as D-nLDH. However, overproduction of this protein in isolate 523k did not result in production of D-lactate-containing peptidoglycan precursors, and susceptibility testing of ddh mutants of 523k demonstrated that S. aureus D-nLDH is not necessary for glycopeptide resistance in this isolate. We conclude that the mechanism of glycopeptide resistance in this isolate is distinct from that in enterococci.