title: Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci.
abstract: Enterococci are naturally tolerant to typically bactericidal cell wall-active antibiotics, meaning that their growth is inhibited but they are not killed even when exposed to a high concentration of the drug. The molecular reasons for this extraordinary tolerance are still incompletely understood. Previous work showed that resistance to killing collapsed specifically in mutants affected in superoxide dismutase (Sod) activity, arguing that bactericidal antibiotic treatment led to induction of a superoxide burst. In the present work, we show that loss of antibiotic tolerance in ΔsodA mutants of pathogenic enterococci is dependent on the energy source present during antibiotic treatment. Hexoses induce greater killing than the pentose ribose, and no killing was observed with glycerol as the energy source. These results point to glycolytic reactions as crucial for antibiotic-mediated killing of ΔsodA mutants. A transposon mutant library was constructed in Enterococcus faecalis ΔsodA mutants and screened for restored tolerance of vancomycin. Partially restored tolerance was observed in mutants with transposon integrations into intergenic regions upstream of regulators implicated in arginine catabolism. In these mutants, the arginine deiminase operon was highly upregulated. A model for the action of cell wall-active antibiotics in tolerant and nontolerant bacteria is proposed.
  
  
  OBJECTIVE
  Antibiotic tolerance is a serious clinical concern, since tolerant bacteria have considerably increased abilities to resist killing by bactericidal drugs. Using enterococci as models for highly antibiotic-tolerant pathogens, we showed that tolerance of these bacteria is linked to their superoxide dismutase (Sod), arguing that bactericidal antibiotics induce generation of reactive oxygen species inside cells. Wild-type strains are tolerant because they detoxify these deleterious molecules by the activity of Sod, whereas Sod-deficient strains are killed. This study showed that killing depends on the energy source present during treatment and that an increase in arginine catabolism partially restored tolerance of the Sod mutants. These results are used to propose a mode-of-action model of cell wall-active antibiotics in tolerant and nontolerant bacteria.
authors.affiliation: Université de Caen Basse-Normandie, EA4655 U2RM-Stress et Virulence, Caen, France Université de Bejaia, FSNV, Laboratoire d'Ecologie Microbienne, Bejaia, Algeria.
authors.forename: Rabia
authors.initials: R
authors.lastname: Ladjouzi
authors.affiliation: Université de Caen Basse-Normandie, EA4655 U2RM-Stress et Virulence, Caen, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Bizzini
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Willem
authors.initials: W
authors.lastname: van Schaik
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Xinglin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Université de Caen Basse-Normandie, EA4655 U2RM-Stress et Virulence, Caen, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Rincé
authors.affiliation: Université de Caen Basse-Normandie, EA4655 U2RM-Stress et Virulence, Caen, France.
authors.forename: Abdellah
authors.initials: A
authors.lastname: Benachour
authors.affiliation: Université de Caen Basse-Normandie, EA4655 U2RM-Stress et Virulence, Caen, France axel.hartke@unicaen.fr.
authors.forename: Axel
authors.initials: A
authors.lastname: Hartke
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001120:Arginine / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D004251:DNA Transposable Elements; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D013293:Enterococcus faecalis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D009154:Mutation; D010406:Penicillins / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D014640:Vancomycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D010406:Penicillins; D014640:Vancomycin; D001120:Arginine; D013482:Superoxide Dismutase