Impact of relebactam-mediated inhibition of Mycobacterium abscessus BlaMab ß-lactamase on the in vitro and intracellular efficacy of imipenem.

OBJECTIVES: Imipenem is one of the recommended ß-lactams for the treatment of Mycobacterium abscessus pulmonary infections in spite of the production of BlaMab ß-lactamase. Avibactam, a second-generation ß-lactamase inhibitor, was previously shown to inactivate BlaMab, but its partner drug, ceftazidime, is devoid of any antibacterial activity against M. abscessus. Here, we investigate whether relebactam, a novel second-generation inhibitor developed in combination with imipenem, improves the activity of this carbapenem against M. abscessus. METHODS: The impact of BlaMab inhibition by relebactam was evaluated by determining MICs, time-kill curves and M. abscessus intracellular proliferation in human macrophages. Kinetic parameters for the inhibition of BlaMab by relebactam were determined by spectrophotometry using nitrocefin as the substrate. The data were compared with those obtained with avibactam. RESULTS: Combination of relebactam (4 mg/L) with ß-lactams led to >128- and 2-fold decreases in the MICs of amoxicillin (from >4096 to 32 mg/L) and imipenem (from 8 to 4 mg/L). In vitro, M. abscessus was not killed by the imipenem/relebactam combination. In contrast, relebactam increased the intracellular activity of imipenem, leading to 88% killing. Relebactam and avibactam similarly potentiated the antibacterial activities of ß-lactams although BlaMab was inactivated 150-fold less effectively by relebactam than by avibactam. CONCLUSIONS: Inhibition of BlaMab by relebactam improves the efficacy of imipenem against M. abscessus in macrophages, indicating that the imipenem/relebactam combination should be clinically considered for the treatment of infections due to M. abscessus.

In Vitro and Intracellular Activity of Imipenem Combined with Tedizolid, Rifabutin, and Avibactam against Mycobacterium abscessus.

Mycobacterium abscessus infections are difficult to treat because of their resistance to many antibiotics. In vitro, tedizolid combined with imipenem displayed a moderate synergistic effect (fractional inhibitory concentration index, 0.41) but no bactericidal activity. Intracellularly, tedizolid 2 µg/ml (half of the MIC), corresponding to the peak serum concentration, increased the efficacy of imipenem at 8 and 32 µg/ml. Addition of avibactam and rifabutin, alone or in combination, improved the activity of the imipenem-tedizolid combination.

In Vitro and Intracellular Activity of Imipenem Combined with Rifabutin and Avibactam against Mycobacterium abscessus.

Repurposing drugs may be useful as an add-on in the treatment of Mycobacterium abscessus pulmonary infections, which are particularly difficult to cure. M. abscessus naturally produces a ß-lactamase, BlaMAb, which is inhibited by avibactam. The recommended regimens include imipenem, which is hydrolyzed by BlaMAb and used without any ß-lactamase inhibitor. Here, we determine whether the addition of rifabutin improves the activity of imipenem alone or in combination with avibactam against M. abscessus CIP104536. Rifabutin at 16 µg/ml was only bacteriostatic (MIC of 4 µg/ml) and was moderately synergistic in combination with imipenem (fractional inhibitory concentration [FIC] index of 0.38). Addition of rifabutin (16 µg/ml) moderately increased killing by a low (8 µg/ml) but not by a high (32 µg/ml) concentration of imipenem. Addition of avibactam (4 µg/ml) did not further increase killing by the former combination. In infected macrophages, rifabutin (16 µg/ml) increased the activity of imipenem at 8 and 32 µg/ml, achieving 3- and 100-fold reductions in the numbers of intracellular bacteria, respectively. Avibactam (16 µg/ml) improved killing by imipenem at 8 µg/ml. A 5-fold killing was obtained for a triple combination comprising avibactam (16 µg/ml) and therapeutically achievable doses of imipenem (8 µg/ml) and rifabutin (1 µg/ml). These results indicate that the imipenem-rifabutin combination should be further considered for the treatment of M. abscessus pulmonary infections in cystic fibrosis patients and that addition of a ß-lactamase inhibitor might improve its efficacy. Mechanistically, the impact of BlaMAb inhibition by avibactam on antibiotic activity was assessed by comparing CIP104536 and a ß-lactamase-deficient derivative.

Synthesis of Avibactam Derivatives and Activity on ß-Lactamases and Peptidoglycan Biosynthesis Enzymes of Mycobacteria.

There is a renewed interest for ß-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus because their ß-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction is reported. The amoxicillin-DBO combinations were active, indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 µg mL-1 for both species). Mechanistically, ß-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, the latter compounds were investigated as inhibitors of l,d-transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria.

Evolution towards extremely high ß-lactam resistance in Mycobacterium abscessus outbreak strains.

Treatment of Mycobacterium abscessus pulmonary disease requires multiple antibiotics including intravenous ß-lactams (e.g., imipenem, meropenem). M. abscessus produces a ß-lactamase (BlaMab) that inactivates ß-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in M. abscessus and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected M. abscessus subsp. massiliense isolates from the index case of a CF center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC >512 µg/ml). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the ß-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in ß-lactamase activity and increased bla Mab mRNA level. Concurrent increase in transcription of preceding ppiA gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin mspA coincided with the first increase in MIC (from 8 to 32 µg/ml). A frameshift mutation in msp2 responsible for the rough colony morphology, and a SNP in ATP-dependent helicase hrpA co-occurred with the second increase in MIC (from 32 to 256 µg/ml). Increased BlaMab expression and enzymatic activity may have been due to altered regulation of the ppiA-bla Mab operon by the mutated HrpA alone, or in combination with other genes described above. This work supports using carbapenem/ß-lactamase inhibitor combinations for treating M. abscessus, particularly imipenem resistant strains.