Genetic Basis for Vancomycin-Enhanced Cephalosporin Susceptibility in Vancomycin-Resistant Enterococci Revealed Using Counterselection with Dominant-Negative Thymidylate Synthase

Antibiotic-resistant enterococci are major causes of hospital-acquired infections. All enterococci are intrinsically resistant to most cephalosporins, antibiotics in the beta-lactam family that impair peptidoglycan synthesis by inactivating the transpeptidases responsible for cross-linking. In addition, clinical isolates of enterococci often possess acquired resistance to vancomycin, a glycopeptide antibiotic that impairs peptidoglycan biosynthesis by a mechanism distinct from that of the beta-lactams, namely, by binding to the D-Ala-D-Ala termini found in peptidoglycan precursors to prevent their utilization by biosynthetic transglycosylases. Antimicrobial synergism between vancomycin and beta-lactams against vancomycin-resistant enterococci was originally described decades ago, but the genetic basis for synergy has remained unknown. Because a complete understanding of the mechanism underlying synergy between vancomycin and beta-lactams might suggest new targets or strategies for therapeutic intervention against antibiotic-resistant enterococci, we explored the genetic basis for synergy between vancomycin and cephalosporins in Enterococcus faecalis. To do so, we developed a counterselection strategy based on a dominant-negative mutant of thymidylate synthase and implemented this approach to create a panel of mutants in vancomycin-resistant E. faecalis. Our results confirm that vancomycin promotes synergy by inducing expression of the van resistance genes, as a mutant in which the van genes are expressed in the absence of vancomycin exhibits susceptibility to cephalosporins. Further, we show that peptidoglycan precursors substituted with D-Ala-D-Lac are not required for vancomycin-enhanced cephalosporin sensitivity. Instead, production of the D, D-carboxypeptidase VanY(B) is both necessary and sufficient to dramatically sensitize E. faecalis to cephalosporins.