Inhibiting Mycobacterium abscessus Cell Wall Synthesis: Using a Novel Diazabicyclooctane β-Lactamase Inhibitor To Augment β-Lactam Action.

Mycobacterium abscessus ( Mab ) infections are a growing menace to the health of many patients, especially those suffering from structural lung disease and cystic fibrosis. With multidrug resistance a common feature and a growing understanding of peptidoglycan synthesis in Mab , it is advantageous to identify potent β-lactam and β-lactamase inhibitor combinations that can effectively disrupt cell wall synthesis. To improve existing therapeutic regimens to address serious Mab infections, we evaluated the ability of durlobactam (DUR), a novel diazobicyclooctane β-lactamase inhibitor to restore in vitro susceptibilities in combination with β-lactams and provide a biochemical rationale for the activity of this compound. In cell-based assays, susceptibility of Mab subsp. abscessus isolates to amoxicillin (AMOX), imipenem (IMI), and cefuroxime (CXM) was significantly enhanced with the addition of DUR. The triple drug combinations of CXM-DUR-AMOX and IMI-DUR-AMOX were most potent, with MIC ranges of ≤0.06 to 1 μg/mL and an MIC 50 /MIC 90 of ≤0.06/0.25 μg/mL, respectively. We propose a model by which this enhancement may occur, DUR potently inhibited the β-lactamase Bla Mab with a relative Michaelis constant ( K i app ) of 4 × 10 -3 ± 0.8 × 10 -3  μM and acylation rate ( k 2 / K ) of 1 × 10 7 M -1 s -1 . Timed mass spectrometry captured stable formation of carbamoyl-enzyme complexes between DUR and Ldt Mab2-4 and Mab d,d-carboxypeptidase, potentially contributing to the intrinsic activity of DUR. Molecular modeling showed unique and favorable interactions of DUR as a Bla Mab inhibitor. Similarly, modeling showed how DUR might form stable Michaelis-Menten complexes with Ldt Mab2-4 and Mab d,d-carboxypeptidase. The ability of DUR combined with amoxicillin or cefuroxime and imipenem to inactivate multiple targets such as d,d-carboxypeptidase and Ldt Mab2,4 supports new therapeutic approaches using β-lactams in eradicating Mab . IMPORTANCE Durlobactam (DUR) is a potent inhibitor of Bla Mab and provides protection of amoxicillin and imipenem against hydrolysis. DUR has intrinsic activity and forms stable acyl-enzyme complexes with Ldt Mab2 and Ldt Mab4 . The ability of DUR to protect amoxicillin and imipenem against Bla Mab and its intrinsic activity along with the dual β-lactam target redundancy can explain the rationale behind the potent activity of this combination.