1. J Antimicrob Chemother. 2012 Dec;67(12):2873-81. doi: 10.1093/jac/dks325. Epub
2012 Sep 21.

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

Garcia LG, Lemaire S, Kahl BC, Becker K, Proctor RA, Tulkens PM, Van Bambeke F.

Pharmacologie cellulaire et moléculaire & Louvain Drug Research Institute,
Université catholique de Louvain, Brussels, Belgium.

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
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.

PMID: 23002174  [PubMed - in process]