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Ten clinical isolates and the type strain (H37Rv) of Mycobacterium tuberculosis were shown to produce an intracellular beta-lactamase. Crude enzyme preparations were extracted from acetone cell powders by grinding with zirconium beads in 0.133 M glycine with 1.0% Triton X-100. The enzymes had identical patterns on isoelectric focusing, with two major bands at isoelectric points of 4.9 and 5.1. The beta-lactamase was highly susceptible to the new beta-lactamase inhibitor BRL 42715, with an I50 of 0.0001 microgram/ml. The enzyme was also susceptible to clavulanic acid with an I50 (0.05 microgram/ml), which was similar to the value for the common bacterial beta-lactamase TEM-1 (0.01 microgram/ml). The latter result is consistent with previous MIC studies with M. tuberculosis, which have shown synergy between clavulanic acid and amoxicillin. BRL 42715 and clavulanic acid were more active than sulbactam, tazobactam, and cloxacillin. These studies support the potential value of penicillin/clavulanic acid and penicillin/BRL 42715 combinations in the treatment of tuberculosis.

The beta-lactamases from the 3 biovariants of M. fortuitum were compared on the basis of substrate profiles, susceptibility to enzyme inhibitors, and inducibility in the presence of selected beta-lactams. Despite differences in the distribution of beta-lactamase bands observed when enzymes from different isolates were subjected to isoelectric focusing, substrate profiles for the 3 biovariants were similar. All demonstrated a comparable broad spectrum hydrolytic activity for both cephalosporins and penicillins. The MIC for amoxicillin were reduced 4- to 16-fold when combined with the beta-lactamase inhibitor clavulanic acid, but not to a clinically susceptible range. The degree of reduction in MIC for amoxicillin correlated well with the susceptibility of enzyme to inhibition by clavulanic acid as determined in an in vitro assay. Although all M. fortuitum strains produce beta-lactamase under routine growth conditions, 90% of strains demonstrated an increase in the amount of this enzyme when cultured in the presence of selected beta-lactams as potential inducers. Quantitative assays and isoelectric focusing further indicated that this apparent induction of beta-lactamase is a simple enhancement of the same enzyme(s) produced in the absence of a known inducer. This is the first demonstration of any inducibility among mycobacterial beta-lactamases and suggests that synthesis of these enzymes in M. fortuitum is under some form of regulatory control. These results indicate that the beta-lactamases have a role in resistance of M. fortuitum to the beta-lactams. Other factors, such as permeability and penicillin-binding proteins, were not evaluated.

The uninduced culture supernatants and cell extracts from 58 strains of the 3 biovariants (biovar) of Mycobacterium fortuitum were all positive for beta-lactamase with the chromogenic cephalosporin substrate. By analytical isoelectric focusing (IEF), 29 of 30 strains of biovar fortuitum exhibited an identical beta-lactamase pattern with 1 major band. In contrast, the beta-lactamases of biovar peregrinum and the unnamed third biovar were heterogeneous, with multiple bands and a variety of patterns. The pH range of isoelectric points for the beta-lactamases was relatively narrow, however, with most bands appearing between pH 4.3 and 5.2. Although additional genetic studies are required, these enzymes appear to be chromosomal, as they are present in all strains including some without detectable plasmids. Repeat isolates from the same patient obtained up to six months apart always had the same beta-lactamase pattern by IEF. Of the third biovar complex, 30% are cefoxitin resistant with minimal inhibitory concentrations greater than 32 micrograms/ml. All 9 cefoxitin-resistant isolates tested had the same unique beta-lactamase pattern by IEF, although this enzyme failed to hydrolyze cefoxitin while hydrolyzing cephalothin and benzylpenicillin. Thus, despite the association of cefoxitin-resistance with a single enzyme pattern, the role of this beta-lactamase in resistance is not known.