Can Penicillins and Other (cid:98) -Lactam Antibiotics Be Used To Treat Tuberculosis?

An increase in the number of tuberculosis cases caused by multiple-drug-resistant strains of Mycobacterium tuberculosis has stimulated search for new antituberculous agents. Beta-lactam antibiotics, traditionally regarded as ineffective against tuberculosis, merit consideration. Four major penicillin-binding proteins (PBPs) with approximate molecular sizes of 94, 82, 52, and 37 kDa were detected by ﬂuorography of [ 3 H]penicillin-radiolabeled membrane proteins prepared from M. tuberculosis H37Ra. The presence of membrane-associated (cid:98) -lactamase precluded the use of membranes for assaying the binding afﬁnities of beta-lactam antibiotics. Therefore, ampicillin afﬁnity chromatography was used to purify these four PBPs from crude membranes in order to assay the binding afﬁnities of beta-lactam antibiotics. Ampicillin, amoxicillin, and imipenem, beta-lactam antibiotics previously reported to be active in vitro against M. tuberculosis , bound to M. tuberculosis PBPs at therapeutically achievable concentrations. Binding of the 94-, 82-, and 52-kDa PBPs, but not the 37-kDa PBP, was associated with antibacterial activity, suggesting that these PBPs are the critical targets. Studies of mycobacterial cell wall permeability, which was assayed with a panel of reference cephalosporins and penicillins with different charge positivities, indicated that the rate of penetration of beta-lactam antibiotics to the target PBPs could not account for resistance. Resistance could be reversed with the (cid:98) -lactamase inhibitors clavulanate or sulbactam or could be circumvented by the use of a (cid:98) -lactamase-stable drug, imipenem, indicating that mycobacterial (cid:98) -lactamase, probably in conjunction with slow penetration, is a major deter-minant of M. tuberculosis resistance to beta-lactam antibiotics. These ﬁndings conﬁrm in vitro data that M. tuberculosis is susceptible to some beta-lactam antibiotics. Further evaluation of these drugs for the treatment of tuberculosis in animal models and in clinical trials is warranted. centrifugation. PBP binding assay. Ampicillin afﬁnity-puriﬁed PBP samples of approximately 5 (cid:109) g of protein were incubated with antibiotics at ﬁnal concentrations of 0, 0.1, 0.5, 1, 5, 10, 20, 50, and 100 (cid:109) g/ml at 37 (cid:56) C for 20 min, and then 20 (cid:109) g (ﬁnal concentrations) of [ 3 H]benzylpenicillin (30 Ci/mmol; a gift from Merck Sharpe & Dohme) per ml was added for 20 Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide, 0.26% bisacrylam-* Staphylococcus 209P determined by the broth dilution method in Mueller-Hinton broth on successive days for both the frozen aliquot and the 37 (cid:56) C aliquot. The ratios of these two MICs was determined, and the half-life was calculated from the slope of the regression curve obtained by plotting the log 2 ratio versus the number of days of incubation at 37 (cid:56) C. Assay of mycobacterial cell wall permeability to beta-lactam antibiotics. Cell wall permeability was determined by assaying the entry of beta-lactam via spontaneous diffusion based on the rate of hydrolysis by periplasmic M.