Abstractβ-lactam antibiotics are ineffective against Mycobacterium tuberculosis, being rapidly hydrolyzed by the chromosomally encoded blaC gene product. The carbapenem class of β-lactams are very poor substrates for BlaC, allowing us to determine the three-dimensional structure of the covalent BlaCmeropenem covalent complex at 1.8 angstrom resolution. When meropenem was combined with the β-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.

Members of the beta-lactam class of antibiotics, which inhibit the bacterial d,d-transpeptidases involved in cell wall biosynthesis, have never been used systematically in the treatment of Mycobacterium tuberculosis infections because of this organism's resistance to beta-lactams. The critical resistance factor is the constitutive production of a chromosomally encoded, Ambler class A beta-lactamase, BlaC in M. tuberculosis. We show that BlaC is an extended spectrum beta-lactamase (ESBL) with high levels of penicillinase and cephalosporinase activity as well as measurable activity with carbapenems, including imipenem and meropenem. We have characterized the enzyme's inhibition by three FDA-approved beta-lactamase inhibitors: sulbactam, tazobactam, and clavulanate. Sulbactam inhibits the enzyme competitively and reversibly with respect to nitrocefin. Tazobactam inhibits the enzyme in a time-dependent manner, but the activity of the enzyme reappears due to the slow hydrolysis of the covalently acylated enzyme. In contrast, clavulanate reacts with the enzyme quickly to form hydrolytically stable, inactive forms of the enzyme that have been characterized by mass spectrometry. Clavulanate has potential to be used in combination with approved beta-lactam antibiotics to treat multi-drug resistant (MDR) and extremely drug resistant (XDR) strains of M. tuberculosis.

More than 60 years after the introduction of penicillin, the class of ␤-lactam antibiotics remains the most commonly used to treat severe infections despite the emergence of various resistance mechanisms, including drug detoxification by production of ␤-lactamases (1), decreased affinity of the target (2), and decreased permeability (3). The success story of ␤-lactams involves the development of several generations of ␤-lactams to defeat the resistance mechanisms and their association with inhibitors of ␤-lactamases (4, 5). All ␤-lactams have a common mechanism of action, as they share structural similarity with bacterial cell wall precursors. These drugs act as suicide substrates of the DD-transpeptidase catalytic domain of the penicillin-binding proteins (PBPs) 2 responsible for the last cross-linking step of cell wall assembly (Fig. 1A) (2, 6, 7).Among the DD-transpeptidases of Enterococcus faecium, the low affinity PBP5 is unessential for growth but responsible for intrinsic low level resistance to ampicillin (8, 9), the first-line drug used at high dosage to treat severe enterococcal infections. In clinical isolates, higher levels of resistance to ampicillin are commonly associated with multiple amino acid substitutions in the transpeptidase domain of PBP5 (10, 11). We have shown previously that even higher levels of resistance to ampicillin can be achieved by a novel mechanism in the absence of PBP5 (12 (Fig. 1B). In the presence of ampicillin, the novel L-Lys 3 3D-iAsn-L-Lys 3 cross-links were exclusively found in the peptidoglycan of E. faecium M512, indicating that the DD-transpeptidase activity of the PBPs did not participate in the formation of the cross-links. Selection of E. faecium M512 from E. faecium D344S in five consecutive steps on increasing concentrations of ampicillin led to the gradual activation of the LD-transpeptidation pathway because the proportion of L-Lys 3 3D-iAsn-L-Lys 3 was 3.1% for D344S and increased at each selection step (13). Activation of the resistance pathway was associated with the production of a DD-carboxypeptidase that generated precursors containing a tetrapeptide stem in the cytoplasm of E. faecium M512 (13). Based on these observations, we have speculated that precursors containing a tetrapeptide stem are essential for the formation of the L-Lys 3 3D-iAsn-L-Lys 3 cross-links. In the present work, we have identified in E. faecium an LD-transpeptidase as the key enzyme of this alternate transpeptidation pathway and have shown that its substrate does not bear structural similarity to ␤-lactams. The bypass mechanism could invalidate the ␤-lactams as the major class of antibiotics. EXPERIMENTAL PROCEDURES Purification of the LD-Transpeptidase and N-terminal Sequencing-The LD-transpeptidase from E. faecium (Ldt fm ) was partially purified in four chromatographic steps using a radioactive exchange assay (13) to detect active fractions. Briefly, E. faecium M512 (12) was grown to an A 650 of 0.7 in 24 liters of brain heart infusion broth (Difco), harvested by centrifugati...