1642 - A Novel β-lactamase Inhibitor (Durlobactam, DUR) and β-Lactams Enhance Susceptibility Against Multidrug-Resistant (MDR) Mycobacterium abscessus (Mab)

Background: Mab is a MDR nontuberculous mycobacterium that causes lung infections in patients with structural lung disease. Mab harbors a chromosomally encoded class A β-lactamase, Bla_Mab, able to hydrolyze penicillins, cephalosporins and carbapenems. L,D- and D,D-transpeptidases (L,D TP and D,D TP) shape peptidoglycan (PG) synthesis and contribute to cell wall structure. Select combinations of β-lactams that inhibit L,D TP and D,D TPs and Bla_Mab are desirable as they can potentially improve treatment outcomes. DUR is a novel DBO β-lactamase inhibitor (BLI) with broad-spectrum activity against Ambler class A, C, and D β-lactamases (Fig.). Here, we investigated the mechanism of action and efficacy of DUR alone and combined with select β-lactams in restoring susceptibility of Mab to β-lactam antibiotics

Methods: Minimum inhibitory concentrations (MICs) of cefuroxime (CEF), imipenem (IMI) and amoxicillin (Amox) with or without DUR were determined using microdilution. Approximately 5 x 10⁵ colony-forming units per milliliter were inoculated into Middlebrook 7H9 Broth supplemented with 10% (vol/vol) oleic albumin dextrose catalase and 0.05% (vol/vol) Tween 80. When more than 2 drugs were combined, Amox was added at fixed concentration of 8 µg/ml to serial dilutions of CEF-DUR or IMI-DUR. Mab isolates were incubated with test agents at 30°C for 48 h, and MIC was defined as lowest antibiotic concentration that prevented visible bacterial growth. Reaction intermediates in the inactivation pathway of Bla_Mab, L,D-TP and D,D-TPs with DUR

Results: One hundred clinically derived and previously characterized isolates were tested in these assays. MIC₅₀ and MIC₉₀ of DUR alone was 4 and 8 µg/ml, demonstrating intrinsic activity. Combinations of DUR-IMI or DUR-CEF plus 8 µg/mL Amox lowered MIC₅₀ to < 0.06 µg/ml in all 100 clinical isolates (Table). Mass spectrometry analyses of Bla_Mab, L,D-TP and D,D-TPs _{Mab (2,4)} inactivated by DUR showed formation of stable adducts of DUR to Bla_Mab, L,D-TP and D,D-TPs (Fig.)Chemical composition of durlobactam (DUR) and mass spectrometry of BlaMab, L,D TP and D,D TPs incubated with DUR
MIC50 and MIC90 of 100 Mab clinical strains against DUR alone and in combination with Amox, CEF and IMI


Conclusion: We demonstrate that a novel DBO BLI, DUR, is an active agent with potent intrinsic activity against Bla_Mab and Mab L,D-TPs and D,D-TPs. We hypothesize that DUR improves b-lactam activity by protecting against the hydrolytic activity of Bla_Mab and by targeting multiple steps in PG synthesis