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Objectives: Mycobacterium tuberculosis and Mycobacterium abscessus produce broad-spectrum class A β-lactamases, BlaC and Bla Mab , which are inhibited by clavulanate and avibactam, respectively. BlaC differs from Bla Mab at Ambler position 132 in the conserved motif SDN (SDG versus SDN, respectively). Here, we investigated whether this polymorphism could account for the inhibition specificity of β-lactamases from slowly and rapidly growing mycobacteria., Methods: Enzyme kinetics were determined to assess the impact of the substitutions G 132 N in BlaC and N 132 G in Bla Mab on β-lactamase inhibition by clavulanate and avibactam. The stability of acylenzymes was evaluated by MS. The impact of the substitutions on the antibacterial activity of drug combinations was determined based on production of the β-lactamases in Escherichia coli ., Results: The substitution G 132 N increased 140-fold the efficacy of BlaC inhibition by avibactam and abolished clavulanate inhibition due to acylenzyme hydrolysis. Bla Mab efficiently hydrolysed clavulanate, but the substitution N 132 G led to a 5600-fold reduction in the hydrolysis rate constant k cat due to stabilization of Bla Mab -clavulanate covalent adducts. The N 132 G substitution also led to a 610-fold reduction in the efficacy of Bla Mab carbamylation by avibactam. Testing resistance to the amoxicillin/clavulanate and amoxicillin/avibactam combinations revealed that modifications in the catalytic properties of the β-lactamases resulted in opposite shifts from susceptibility to resistance and vice versa., Conclusions: G 132 N and N 132 G had opposite effects on the inhibition of BlaC and Bla Mab , indicating that these substitutions might lead to acquisition of resistance to either of the β-lactamase inhibitors, but not to both of them., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The production of β-lactamases Bla(Mab) and BlaC contributes to β-lactam resistance in Mycobacterium abscessus and Mycobacterium tuberculosis, respectively. Ceftaroline was efficiently hydrolyzed by these enzymes. Inhibition of M. tuberculosis BlaC by clavulanate decreased the ceftaroline MIC from ≥ 256 to 16 to 64 μg/ml, but these values are clinically irrelevant. In contrast, the ceftaroline-avibactam combination should be evaluated against M. abscessus since it inhibited growth at lower and potentially achievable drug concentrations., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Objectives: Two β-lactams, cefoxitin and imipenem, are part of the reference treatment for pulmonary infections with Mycobacterium abscessus. M. abscessus has recently been shown to produce a broad-spectrum β-lactamase, BlaMab, indicating that the combination of β-lactams with a BlaMab inhibitor may improve treatment efficacy. The objectives of this study were to evaluate the impact of BlaMab production on the efficacy of β-lactams in vitro and to assess the benefit of BlaMab inhibition on the activity of β-lactams intracellularly and in an animal model., Methods: We analysed the mechanism and kinetics of BlaMab inactivation by avibactam, a non-β-lactam β-lactamase inhibitor currently in Phase III of development, in combination with ceftazidime for the treatment of serious infections due to Gram-negative bacteria. We then deleted the gene encoding BlaMab to assess the extent of BlaMab inhibition by avibactam based on a comparison of the impact of chemical and genetic inactivation. Finally, the efficacy of amoxicillin in combination with avibactam was evaluated in cultured human macrophages and in a zebrafish model of M. abscessus infection., Results: We showed that avibactam efficiently inactivated BlaMab via the reversible formation of a covalent adduct. An inhibition of BlaMab by avibactam was observed in both infected macrophages and zebrafish., Conclusions: Our data identify avibactam as the first efficient inhibitor of BlaMab and strongly suggest that β-lactamase inhibition should be evaluated to provide improved therapeutic options for M. abscessus infections., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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 β-lactams, although the underlying mechanisms have not been explored. Here, we characterized M. abscessus class A β-lactamase (Bla(Mab)) and investigated its role in β-lactam resistance., Methods: Hydrolysis kinetic parameters of purified Bla(Mab) were determined by spectrophotometry for various β-lactams and compared with those of related BlaC from Mycobacterium tuberculosis. MICs of β-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) = 3.0 × 10(4) M(-1) s(-1)), indicating that the in vivo activity of this drug might be improved by combination with a β-lactamase inhibitor. β-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 β-lactamase that limits the in vivo efficacy of β-lactams.

The in vitro activity of cefoxitin and imipenem was compared for 43 strains of the Mycobacterium abscessus complex, mostly isolated from cystic fibrosis patients. The MICs of imipenem were lower than those of cefoxitin, although the number of imipenem-resistant strains was higher according to the CLSI breakpoints. Strain comparisons indicated that the MICs of cefoxitin were significantly higher for Mycobacterium bolletii than for M. abscessus. The MICs of both β-lactams were higher for the rough morphotype than for the smooth morphotype. The clinical impact of the in vitro difference between the activity of imipenem and that of cefoxitin remains to be determined. [ABSTRACT FROM AUTHOR]