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  • Lemaire et al. Page 1 of 31

    AAC 1192-06 Revised version

    Intraphagocytic Methicillin-Resistant Staphylococcus aureus are susceptible to

    Meropenem and Cloxacillin: role of acid pH.

    Sandrine Lemaire,1 Franoise Van Bambeke,1 Marie-Paule Mingeot-Leclercq,1 Youri

    Glupczynski,2 and Paul M. Tulkens.1

    1 Unit de Pharmacologie cellulaire et molculaire, Universit catholique de Louvain,

    Brussels; 2 Laboratoire de Microbiologie, Cliniques universitaires de Mont-Godinne,

    Yvoir; Belgium

    Running title: Susceptibility of intraphagocytic MRSA to d-lactams

    Corresponding author: Paul M. Tulkens, Unit de pharmacologie cellulaire et

    molculaire, Universit catholique de Louvain, UCL 73.70 Avenue E. Mounier 73, B-

    1200 Brussels, Belgium. Phone: 32-2-762.21.36-764.73.71; Fac-simile: 32-2-

    7647373;

    electronic mail address: tulkens@facm.ucl.ac.be

    Word count of abstract: 249

    Page count of text: 13

    Number of inserts (figures/tables): 7 (6 figures; 1 Table)

    Number of references: 23

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    Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.01192-06 AAC Accepts, published online ahead of print on 16 February 2007

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  • Lemaire et al. Page 2 of 31

    ABSTRACT

    Early studies showed that methicillin-resistant S. aureus (MRSA) are

    susceptible to d-lactams when exposed to pH ~ 5.5 in broth. Because S. aureus

    survives in phagolysosomes of macrophages where pH may be acidic, we have

    examined the susceptibility of MRSA ATCC 33591 phagocytized by human THP-1

    macrophages to meropenem (MEM) and cloxacillin (CLX). Using a

    pharmacodynamic model assessing key pharmacological (EC50, Emax) and

    microbiological (static concentration) descriptors of antibiotic activity, we show that

    intraphagocytic MRSA are as sensitive to MEM and CLX as methicillin susceptible

    S. aureus (MSSA ATCC 25923). This observation was replicated in broth if pH was

    brought to 5.5, and was confirmed with clinical strains. Electron microscopy showed

    that both MRSA and MSSA localized and multiplied in membrane-bounded structures

    (phagolysosomes) in the absence of d-lactams. Incubation of the infected

    macrophages with ammonium chloride (to raise the phagolysosomal pH) made

    MRSA insensitive to MEM and CLX. No difference was seen in mec, mecA, mecI,

    mecR1, femA, and femB expression (reversed-transcription PCR), and in PB2a

    content (immunodetection) in MRSA grown in broth at pH 5.5 vs. 7.4. [14C]-benzyl-

    penicillin binding to cell walls prepared from a non d-lactamase producing MRSA

    (clinical isolate) was 2 times lower than for MSSA ATCC 25923 at pH 7.4, but

    increased to similar values for both strains at pH 5.5. These data suggests that the

    restoration of susceptibility of intraphagocytic of MRSA to MEM and CLX is due to

    the acid pH prevailing in phagolysosomes, and is mediated by an enhanced binding

    to PBP's.

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  • Lemaire et al. Page 3 of 31

    Keywords: MRSA, MSSA, susceptibility, acid pH, meropenem, cloxacillin, mec,

    penicillin-binding protein, PBP-2a, macrophages, intracellular

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  • Lemaire et al. Page 4 of 31

    INTRODUCTION

    Staphylococcus aureus causes a wide range of severe and often life-

    threatening infections, such as endocarditis, osteomyelitis and complicated skin and

    skin structure infections. Often considered as an extracellular organism, S. aureus is

    capable of surviving within phagocytic and non-phagocytic cells (4,11), which is

    probably an important determinant in the recurrent and relapsing character of these

    infections. There is substantial evidence that the intracellular milieu may modulate

    both the pharmacological properties of the antibiotics and the response of the

    bacteria (20). Yet, this factor is not taken into account in the assessment of bacterial

    susceptibility to drugs in routine clinical microbiology. In the course of a study on the

    activity of d-lactams against S. aureus phagocytozed by human THP-1 macrophages

    (10), we noted that methicillin-resistant (MRSA) and methicillin-sensitive (MSSA)

    S. aureus showed similar susceptibilities to meropenem (Lemaire et al.,

    Carbapenems are Active against Intracellular MRSA. In Program and Abstracts of

    the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy,

    Washington DC, Dec. 16-Dec. 19, 2005, Abstract A-1832) suggesting a restoration of

    susceptibility of MRSA to d-lactams in the intracellular milieu. We present here a

    detailed account of this finding and extend it to cloxacillin, taken as typical

    d-lactamase-resistant penicillin. Our studies suggest that the restoration of the

    susceptibility to d-lactams is due to the acid pH prevailing in the vacuoles where

    S. aureus sojourns and thrives.

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  • Lemaire et al. Page 5 of 31

    MATERIALS AND METHODS

    Bacterial strains and growth conditions. All key experiments were

    performed with one methicillin susceptible strain of S. aureus (MSSA ATCC 25923;

    American Type Culture Collection (ATCC), Manassas, VA), and one methicillin-

    resistant d-lactamase producing strain (MRSA ATCC 33591). Additional surveys

    were made with recent Belgian MRSA isolates (3 hospital-acquired [obtained during

    this study by Y.G.] and 3 community-acquired (2 obtained during this study by Y.G.,

    and one obtained from the USA [NRS 192; Network on Antimicrobial Resistance in

    Staphylococcus aureus (NARSA) at Focus Technologies, Inc., Herndon, VA]).

    For benzyl[14C]penicillin binding studies, we used a non-penicillinase

    producing MRSA (Strain 459 (clinical isolate); checked for absence of production of

    penicillinase with the BBLTM DryslideTM Nitrocefin reagent (Becton, Dickinson and

    Co., Cockeysville, MD) and for the presence of mecA by PCR amplification (see

    below)). All bacteria were grown in Mueller-Hinton broth supplemented with 2 %

    NaCl (w/v) for the MRSA strains.

    Cell cultures. All experiments were performed with THP-1 cells (ATCC TIB-

    202), a human myelomonocytic cell line displaying macrophage-like activity, exactly

    as previously described (10).

    Susceptibility testing and 24 h dose response curves in broth. Bacteria in

    exponential phase of growth were harvested and resuspended at a final density of

    106 CFU/mL in broth adjusted to pH 7.4, 6.0 or 5.5. MICs determinations and 24-h

    dose-response curves studies were performed as previously described (10). We

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  • Lemaire et al. Page 6 of 31

    checked that the final pH of the broths at the end of the 24 h incubation period was

    close to the original one at all antibiotic concentrations equal to the MIC or above.

    Cell infection, assessment of intracellular activities of antibiotics and

    morphological studies. Infection of THP-1 cells, assessment of intracellular

    activity, and electron microscopy studies were performed as described earlier for

    S. aureus ATCC 25923 (10), except that the concentration of gentamicin added to

    the culture medium of controls (no d-lactam added) to prevent the extracellular

    growth of bacteria was reduced to 0.5 x MIC to better minimize its influence on the

    intracellular bacterial growth (see (1) for a full description of the gentamicin

    concentration effects). As previously reported (1), small colony variants were only

    very infrequently observed under our conditions of experiments.

    Reverse-Transcription-PCR studies. RT-PCR was used for semi-

    quantitative detection of mecA, mecI, mecR1, femA, and femB, using 16S rRNA as

    housekeeping gene, and following a previously established procedure (9) and

    published primers (7,14,21), but with the following modifications. Bacteria were

    grown in broth adjusted to pH 7.4 or 5.5, collected in mid-exponential phase of

    growth (O.D.(600 nm) ~ 0.3), and lysed with lysostaphin (100 mg/L) and lysozyme

    (3 g/L). Total RNA was extracted with Rneasy Mini kit (Qiagen, Hilden, Germany),

    the contaminating DNA eliminated with RQ1 Rnase-free Dnase I (Promega

    Corporation, Madison, WI, USA), and RNA further extracted with Rneasy mini

    columns. PCR were carried out in a Gene CyclerTM Thermal Cycler (Biorad

    Laboratories, Hercules, CA) with 30 cycles to ensu