Genetic basis for vancomycin-enhanced cephalosporin susceptibility in vancomycin-resistant enterococci revealed using counterselection with dominant-negative thymidylate synthase. Antimicrob Agents Chemother 2014;58(3):1556-64
Date
12/25/2013Pubmed ID
24366749Pubmed Central ID
PMC3957902DOI
10.1128/AAC.02001-13Scopus ID
2-s2.0-84896854268 (requires institutional sign-in at Scopus site) 19 CitationsAbstract
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.
Author List
Kristich CJ, Djorić D, Little JLAuthors
Dusanka Djoric Research Scientist I in the Microbiology and Immunology department at Medical College of WisconsinChristopher J. Kristich PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Anti-Bacterial AgentsCephalosporin Resistance
Cephalosporins
Directed Molecular Evolution
Drug Delivery Systems
Enterococcus faecalis
Microbial Sensitivity Tests
Penicillin-Binding Proteins
Reverse Transcriptase Polymerase Chain Reaction
Thymidylate Synthase
Vancomycin Resistance
