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ABSTRACTEnterococcus faecalisis a low-GC Gram-positive bacterium, a normal resident of the gastrointestinal (GI) tract, and an important hospital-acquired pathogen. An important risk factor for hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporins, antibiotics that impair cell wall biosynthesis by inhibiting peptidoglycan cross-linking. Enterococci are intrinsically resistant to cephalosporins; however, environmental factors that modulate cephalosporin resistance have not been described. While searching for the genetic determinants of cephalosporin resistance in E. faecalis, we unexpectedly discovered that oxidative stress, whether from external sources or derived from endogenous metabolism, drives enhanced intrinsic resistance to cephalosporins. A particular source of oxidative stress, H2O2, activates signaling through the CroR-CroS two-component signaling system, a known determinant of cephalosporin resistance in E. faecalis. We find that CroR-CroS is required for adaptation to H2O2stress and that H2O2potentiates the activities of cephalosporins against E. faecaliswhen the CroR-CroS signaling system is nonfunctional. Rather than directly detecting H2O2, our data suggest that the CroR-CroS system responds to cell envelope damage caused by H2O2exposure in order to promote cell envelope repair and enhanced cephalosporin resistance.

ABSTRACTAntibiotic-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 vanresistance genes, as a mutant in which the vangenes 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 VanYBis both necessary and sufficient to dramatically sensitize E. faecalisto cephalosporins.

N1-(2,6-dichlorobenzyl)-1,4-(1H2)dihydronicotinamide and N1-(2,6-dichlorobenzyl)-1,4-(1H,2H)dihydronicotinamide were oxidized with nitrosobenzene (NOB), 2,6-dichloroindophenol (DCPIP), 2,4,6-trinitrobenzenesulfonic acid (TNBS), 10-methyl-acridinium ion (MAI), and 10-methyl-9-phenyl-acridinium ion (MPAI). In addition, 2-propanol-h8and 2-propanol-d8were oxidized by NAD+in the presence of yeast alcohol dehydrogenase. Investigation of the temperature dependence of the kinetic isotope effect indicated that the redox reactions with MAI and MPAI have very similar thermodynamic characteristics and proceed via a linear transition state. Redox reactions with NOB, DCPIP, and TNBS have entirely different thermodynamic characteristics, and probably proceed via a different transition state. Thermodynamic characteristics of enzymatic reaction are very similar to model redox reactions with MAI and MPAI.

Enterococcus faecalisand Enterococcus faeciumare commensals of the gastrointestinal tract of most terrestrial organisms, including humans, and are major causes of health care-associated infections. Such infections are difficult or impossible to treat, as the enterococcal strains responsible are often resistant to multiple antibiotics. One intrinsic resistance trait that is conserved among E. faecalisand E. faeciumis cephalosporin resistance, and prior exposure to cephalosporins is one of the most well-known risk factors for acquisition of an enterococcal infection.