Inhibition of the ß-Lactamase BlaMab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against Mycobacterium abscessus.

Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a ß-lactam (cefoxitin or imipenem). The triple combination is used without any ß-lactamase inhibitor, even though Mabscessus produces the broad-spectrum ß-lactamase BlaMab We determine whether inhibition of BlaMab by avibactam improves the activity of imipenem against M. abscessus The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of BlaMab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 µg/ml), amikacin (32 µg/ml), and avibactam (4 µg/ml) was bactericidal in broth (<0.1% survival), with 3.2- and 4.3-log10 reductions in the number of CFU being achieved at 72 h when imipenem was used at 8 and 32 µg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 µg/ml) and high (32 µg/ml) dosages of imipenem, respectively. In vivo inhibition of BlaMab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding BlaMab was induced (20-fold) in the infected macrophages. Inhibition of BlaMab by avibactam improved the efficacy of imipenem against M. abscessusin vitro, in macrophages, and in zebrafish embryos, indicating that this ß-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of BlaMab, since the production of the ß-lactamase is inducible in macrophages.

Bactericidal and intracellular activity of ß-lactams against Mycobacterium abscessus.

OBJECTIVES: Cefoxitin and imipenem are the sole recommended ß-lactams for the treatment of Mycobacterium abscessus pulmonary infections. Here, we investigated whether one of these drugs displays superiority in terms of killing and intracellular activity. We have also evaluated whether the use of a ß-lactamase inhibitor could improve their activity. METHODS: The impact of the ß-lactamase BlaMab on the activity of ß-lactams was assessed by comparing M. abscessus CIP104536 and its ß-lactamase-deficient ΔblaMab derivative, as well as by using the ß-lactamase inhibitor avibactam. The activity of cefoxitin, imipenem, amoxicillin and ceftaroline, alone and in various combinations including amikacin, was compared based on determination of time-kill curves and of intracellular proliferation in human macrophages. RESULTS: Imipenem was superior to cefoxitin in both the time-kill and macrophage assays. Production of BlaMab limited the activity of imipenem. The combination of imipenem and amikacin was bactericidal against the ΔblaMab mutant. Deletion of blaMab extended the spectrum of ß-lactams active against M. abscessus to include amoxicillin and ceftaroline. In the absence of BlaMab, amoxicillin was as active as imipenem. These drugs were more active than ceftaroline and cefoxitin was the least active. Avibactam increased the intracellular activity of ceftaroline, but inhibition of BlaMab was only partial, as previously reported for amoxicillin. CONCLUSIONS: Evaluation of the killing and intracellular activities of ß-lactams indicates that imipenem is superior to cefoxitin at clinically achievable drug concentrations. Inhibition of BlaMab could improve the efficacy of imipenem and extend the spectrum of drugs potentially useful to treat pulmonary infections.

Impact of ß-lactamase inhibition on the activity of ceftaroline against Mycobacterium tuberculosis and Mycobacterium abscessus.

The production of ß-lactamases Bla(Mab) and BlaC contributes to ß-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.

ß-Lactamase inhibition by avibactam in Mycobacterium abscessus.

OBJECTIVES: Two ß-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 ß-lactamase, BlaMab, indicating that the combination of ß-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 ß-lactams in vitro and to assess the benefit of BlaMab inhibition on the activity of ß-lactams intracellularly and in an animal model. METHODS: We analysed the mechanism and kinetics of BlaMab inactivation by avibactam, a non-ß-lactam ß-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 ß-lactamase inhibition should be evaluated to provide improved therapeutic options for M. abscessus infections.