Enterococci are dangerous opportunistic pathogens with the potential to cause life-threatening infections due in part to their intrinsic resistance to cephalosporin antibiotics. Elucidating the molecular mechanisms that provide this resistance is critical for the development of strategies to both prevent and treat enterococcal infections.
Enterococcus faecalis is 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, H 2 O 2 , 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 H 2 O 2 stress and that H 2 O 2 potentiates the activities of cephalosporins against E. faecalis when the CroR-CroS signaling system is nonfunctional. Rather than directly detecting H 2 O 2 , our data suggest that the CroR-CroS system responds to cell envelope damage caused by H 2 O 2 exposure in order to promote cell envelope repair and enhanced cephalosporin resistance. Enterococci are ubiquitous inhabitants of the gastrointestinal tract in healthy animals, including humans. However, antibiotic-resistant enterococci are also major causes of hospital-acquired infections (1, 2) and therefore represent a serious public health problem. One well-known risk factor for the acquisition of enterococcal hospital-acquired infections is prior therapy with broad-spectrum cephalosporins (3), antibiotics that belong to the -lactam family and interfere with cell wall biosynthesis by inhibiting the penicillin-binding proteins (PBPs) that cross-link peptidoglycan. Enterococci exhibit intrinsic resistance to cephalosporins, enabling them to proliferate and achieve abnormally high densities in the gastrointestinal (GI) tract of patients during cephalosporin therapy (4), thereby promoting dissemination to other sites, where they cause infection (5). This intrinsic cephalosporin resistance is a trait shared by essentially all isolates of Enterococcus faecalis; however, our understanding of the genetic and biochemical basis underlying cephalosporin resistance in enterococci remains incomplete. One important well-characterized factor that is required for cephalosporin resistance is a specialized low-affinity PBP (Pbp5) that does not get inactivated by cephalosporins and can therefore perform cross-linking of peptidoglycan to permit growth in the presence of the antibiotic (6, 7). Although required, Pbp5 is not sufficient for resistance, as mutations in other loci render E. faecalis susceptible to cephalosporin by mechanisms that have not been fully worked out (8-12).Among the additional determinants required for intrinsi...
Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired resistance to commonly used antibiotics.
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.
Enterococcus faecalis and Enterococcus faecium are 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. faecalis and E. faecium is cephalosporin resistance, and prior exposure to cephalosporins is one of the most well-known risk factors for acquisition of an enterococcal infection. Cephalosporins inhibit peptidoglycan biosynthesis by acylating the active-site serine of penicillin-binding proteins (PBPs) to prevent the PBPs from catalyzing cross-linking during peptidoglycan synthesis. For decades, a specific PBP (known as Pbp4 or Pbp5) that exhibits low reactivity toward cephalosporins has been thought to be the primary PBP required for cephalosporin resistance. We analyzed other PBPs and report that in both E. faecalis and E. faecium, a second PBP, PbpA(2b), is also required for resistance; notably, the cephalosporin ceftriaxone exhibits a lethal effect on the ΔpbpA mutant. Strikingly, PbpA(2b) exhibits low intrinsic reactivity with cephalosporins in vivo and in vitro. Unlike the Δpbp5 mutant, the ΔpbpA mutant exhibits a variety of phenotypic defects in growth kinetics, cell wall integrity, and cellular morphology, indicating that PbpA(2b) and Pbp5(4) are not functionally redundant and that PbpA(2b) plays a more central role in peptidoglycan synthesis. Collectively, our results shift the current understanding of enterococcal cephalosporin resistance and suggest a model in which PbpA(2b) and Pbp5(4) cooperate to coordinately mediate peptidoglycan cross-linking in the presence of cephalosporins.
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