title: Intracellular forms of menadione-dependent small-colony variants of methicillin-resistant Staphylococcus aureus are hypersusceptible to β-lactams in a THP-1 cell model due to cooperation between vacuolar acidic pH and oxidant species.
abstract: OBJECTIVE
  Phagocytosed methicillin-resistant Staphylococcus aureus (MRSA) are susceptible to β-lactams because of an acid-induced conformational change of penicillin-binding protein (PBP) 2a within phagolysosomes. We have examined whether this mechanism applies to menD and hemB small-colony variants (SCVs) of the COL MRSA strain, using cloxacillin, meropenem, doripenem, and vancomycin as comparator.
  
  
  METHODS
  Intracellularly, the change in cfu from post-phagocytosis inoculum was measured after 24 h of incubation with antibiotics combined or not with N-acetylcysteine (NAC; oxidant species scavenger); the relative potency (C(s)) was calculated from the Hill equation of concentration-response curves. Extracellularly, the effect of a pre-incubation with H(2)O(2) was determined on MICs and killing at pH 7.4 and 5.5.
  
  
  RESULTS
  Intracellularly, the β-lactam C(s) was similar for the COL strain and the hemB mutant and not modified or slightly decreased (2- to 16-fold) by NAC. In contrast, the C(s) was 100- to 900-fold lower for the menD mutant, but similar to that for the COL strain when NAC was present. Extracellularly, β-lactam MICs were markedly reduced at pH 5.5 for the parental strain and the haemin-supplemented hemB mutant, with limited additional effect of pre-incubation with H(2)O(2). In contrast, MICs remained elevated at pH 5.5 for the menD mutant (supplemented with menadione sodium bisulphite or not), but were 7-10 dilutions lower after pre-incubation with H(2)O(2). Vancomycin MICs were unaltered in all conditions, with no marked effect of NAC on C(s).
  
  
  CONCLUSIONS
  Cooperation between acidic pH and oxidant species confers high potency to β-lactams against intracellular forms of menD SCVs of MRSA.
authors.affiliation: Pharmacologie cellulaire et moléculaire & Louvain Drug Research Institute,  Université catholique de Louvain, Brussels,  Belgium.
authors.forename: Laetitia G
authors.initials: LG
authors.lastname: Garcia
authors.forename: Sandrine
authors.initials: S
authors.lastname: Lemaire
authors.forename: Barbara C
authors.initials: BC
authors.lastname: Kahl
authors.forename: Karsten
authors.initials: K
authors.lastname: Becker
authors.forename: Richard A
authors.initials: RA
authors.lastname: Proctor
authors.forename: Paul M
authors.initials: PM
authors.lastname: Tulkens
authors.forename: Françoise
authors.initials: F
authors.lastname: Van Bambeke
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002264:Carboxylic Acids / Q000494:pharmacology*; D002460:Cell Line; D015169:Colony Count, Microbial; D006805:Human Experimentation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects* / Q000254:growth & development* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D009000:Monocytes / Q000276:immunology / Q000382:microbiology*; D016877:Oxidants / Q000494:pharmacology*; D010587:Phagocytosis; D014617:Vacuoles / Q000737:chemistry / Q000382:microbiology; D024483:Vitamin K 3 / Q000378:metabolism*; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D002264:Carboxylic Acids; D016877:Oxidants; D047090:beta-Lactams; D024483:Vitamin K 3