Journal Article

Intracellular forms of menadione-dependent small-colony variants of methicillin-resistant <i>Staphylococcus aureus</i> are hypersusceptible to β-lactams in a THP-1 cell model due to cooperation between vacuolar acidic pH and oxidant species

Laetitia G. Garcia, Sandrine Lemaire, Barbara C. Kahl, Karsten Becker, Richard A. Proctor, Paul M. Tulkens and Françoise Van Bambeke

in Journal of Antimicrobial Chemotherapy

Published on behalf of British Society for Antimicrobial Chemotherapy

Volume 67, issue 12, pages 2873-2881
Published in print December 2012 | ISSN: 0305-7453
Published online September 2012 | e-ISSN: 1460-2091 | DOI: https://dx.doi.org/10.1093/jac/dks325
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

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Objectives

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 (Cs) was calculated from the Hill equation of concentration–response curves. Extracellularly, the effect of a pre-incubation with H2O2 was determined on MICs and killing at pH 7.4 and 5.5.

Results

Intracellularly, the β-lactam Cs 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 Cs 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 H2O2. 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 H2O2. Vancomycin MICs were unaltered in all conditions, with no marked effect of NAC on Cs.

Conclusions

Cooperation between acidic pH and oxidant species confers high potency to β-lactams against intracellular forms of menD SCVs of MRSA.

Keywords: cloxacillin; meropenem; doripenem; H2O2; haemin

Journal Article.  4692 words.  Illustrated.

Subjects: Medical Oncology ; Critical Care

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