Meropenem-clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis

Abstract

beta-lactam antibiotics are ineffective against Mycobacterium tuberculosis, being rapidly hydrolyzed by the chromosomally encoded blaC gene product. The carbapenem class of beta-lactams are very poor substrates for BlaC, allowing us to determine the three-dimensional structure of the covalent BlaC-meropenem covalent complex at 1.8 angstrom resolution. When meropenem was combined with the beta-lactamase inhibitor clavulanate, potent activity against laboratory strains of M. tuberculosis was observed [minimum inhibitory concentration (MIC(meropenem)) less than 1 microgram per milliliter], and sterilization of aerobically grown cultures was observed within 14 days. In addition, this combination exhibited inhibitory activity against anaerobically grown cultures that mimic the "persistent" state and inhibited the growth of 13 extensively drug-resistant strains of M. tuberculosis at the same levels seen for drug-susceptible strains. Meropenem and clavulanate are Food and Drug Administration-approved drugs and could potentially be used to treat patients with currently untreatable disease.

Fig. 1

Kinetics of BlaC with meropenem. (A) Time courses of meropenem hydrolysis with various concentrations of BlaC. Enzyme concentrations are reported on the right. (B) Michaelis-Menten kinetics of BlaC with meropenem at single enzyme concentration (0.8 µM). (C) Mass spectra of enzyme-meropenem species. The 25+ charge state ions are shown. The mass reported for each peak was calculated as described in (20) from the two ions with m/z values of 1165.946 and 1167.702.

Fig. 2

(A) Overall structure of BlaC displayed in rainbow from N term (blue) to the C term (red), with the meropenem adduct displayed as a surface mesh. (B) F_o–F_c omit density (green) contoured at 4.0 σ surrounds the covalent meropenem adduct formed at the Ambler active-site residue Ser⁷⁰. Structure figures were produced using Pymol (www.pymol.org). (C) Proposed chemical mechanism for the BlaC-catalyzed reaction with meropenem.

Fig. 3

Killing curves of M. tuberculosis after exposure to β-lactams and clavulanate. (A) Aerobic growth using the microdilution method. Meropenem and clavulanate were added at 2 µg ml⁻¹ + 1 µg ml⁻¹ (■), 2 µg ml⁻¹ + 2 µg ml⁻¹ (□), 4 µg ml⁻¹ + 1 µg ml⁻¹ (▲), and 4 µg ml⁻¹ + 2 µg ml⁻¹ (△), respectively, for 5 consecutive days. (B) Meropenem is cidal for non-replicating anaerobic M. tuberculosis. Hypoxically adapted M. tuberculosis H37Rv was treated under anaerobic conditions with twofold dilutions of meropenem (0.19 to 12.5 µg ml⁻¹) in the presence or absence of 2.5 µg ml⁻¹ clavulanate. Isoniazid (0.16 to 1.0 µg ml⁻¹) and metronidazole (4.6 to 73 mM) served as negative and positive controls, respectively. Survival was determined by measurement of ATP amounts in surviving bacteria during aerobic outgrowth of 100-fold diluted cells at either 1 week (white bars) or 2 weeks (shaded bars) of treatment or by enumeration of CFUs (inset) after 2 weeks of compound exposure.