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
species.

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
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.

PMID: 23002174  [PubMed - in process]