Characterization of broad-spectrum Mycobacterium abscessus class A β-lactamase.

Objectives: Imipenem and cefoxitin are used to treat Mycobacterium abscessus infections and have moderate activity against this fast-growing mycobacterium (MIC50 of 16 and 32 mg/L, respectively). M. abscessus is highly resistant to most other beta-lactams, although the underlying mechanisms have not been explored. Here, we characterized M. abscessus class A beta-lactamase (Bla(Mab)) and investigated its role in beta-lactam resistance. Methods: Hydrolysis kinetic parameters of purified Bla(Mab) were determined by spectrophotometry for various beta-lactams and compared with those of related BlaC from Mycobacterium tuberculosis. MICs of beta-lactams were determined for M. abscessus CIP104536 and for Escherichia coli producing Bla(Mab) and BlaC. Results: Bla(Mab) had a broad hydrolysis spectrum, similar to that of BlaC, but with overall higher catalytic efficiencies, except for cefoxitin. As expected from its in vivo efficacy, cefoxitin was very slowly hydrolysed by Bla(Mab) (k(cat)/K-m = 6.7 M-1 s(-1)). Bla(Mab) hydrolysed imipenem more efficiently (k(cat)/K-m = 3.0x10(4) M-1 s(-1)), indicating that the in vivo activity of this drug might be improved by combination with beta-lactamase inhibitor. beta-Lactamase inhibitors clavulanate, tazobactam and sulbactam did not inhibit Bla(Mab). This enzyme efficiently hydrolysed clavulanate, in contrast to BlaC, which is irreversibly acylated by this inhibitor. Bla(Mab) and BlaC were functional in E. coli and the resistance profiles mediated by these enzymes were in agreement with the kinetic parameters. Conclusions: M. abscessus produces a clavulanate-insensitive broad-spectrum beta-lactamase that limits the in vivo efficacy of beta-lactams.