CLONING OF THE STAPHYLOCOCCUS-AUREUS DDH GENE ENCODING NAD(-DEPENDENTD-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, Ant imicrob. Agents Chemother. 40:166-172, 1996), and the RNA transcript t hat contains the ddh gene, which encodes Ddh, are present in increased amounts in a vancomycin-resistant isolate, 523k, compared with the su sceptible parent isolate, 523. Sequence analysis had previously reveal ed that Ddh is related to NAD(+)-dependent D-lactate dehydrogenase (D- nLDH) and VanH. This latter protein is essential for high-level glycop eptide resistance in Enterococcus faecium and Enterococcus faecalis by synthesizing the D-lactate needed for biosynthesis of D-lactate-termi nating peptidoglycan precursors with low affinity for vancomycin. We n ow provide the direct evidence that the ddh gene product is Staphyloco ccus aureus D-nLDH and hereafter refer to the protein as D-nLDH. Howev er, overproduction of this protein in isolate 523k did not result in p roduction of D-lactate-containing peptidoglycan precursors, and suscep tibility 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.