title: Genetic basis for vancomycin-enhanced cephalosporin susceptibility in vancomycin-resistant enterococci revealed using counterselection with dominant-negative thymidylate synthase.
abstract: 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 VanYB is both necessary and sufficient to dramatically sensitize E. faecalis to cephalosporins.
authors.affiliation: Department of Microbiology and Molecular Genetics, Center for Infectious Disease Research, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Kristich
authors.forename: Dusanka
authors.initials: D
authors.lastname: Djorić
authors.forename: Jaime L
authors.initials: JL
authors.lastname: Little
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018550:Cephalosporin Resistance / Q000235:genetics; D002511:Cephalosporins / Q000494:pharmacology*; D019020:Directed Molecular Evolution / Q000379:methods; D016503:Drug Delivery Systems; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests; D046915:Penicillin-Binding Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013940:Thymidylate Synthase / Q000235:genetics*; D020713:Vancomycin Resistance / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D046915:Penicillin-Binding Proteins; D013940:Thymidylate Synthase