P-775. Unlocking Synergistic Potency: Dual Action of β-Lactam and β-Lactamase Inhibitor in Combating Mycobacterium abscessus through β-Lactamase Inhibition and Multi-Target Receptor Inactivation Open Access

Mycobacterium abscessus (Mab) poses significant clinical challenges, leading to chronic pulmonary disease in immunocompromised patients. Current treatment involve amikacin (AMK), which is toxic, highlighting the imperative for safer options. This study continues our evaluation of the synergistic effects of a β-lactam and β-lactamase inhibitor (BL/BLI) against Mab and to elucidate the underlying mechanism.

The inhibition constant (Ki,app)

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The binding affinities and chemical interactions between target receptors (LDT1-5, DDC, PBP B, and PBP-lipo) and BL/BLI were determined via kinetics, mass spectrometry, DSF, flow cytometry and microscopy. The synergistic effects of BL/BLI were evaluated in time-kill studies, conducted over 10 days, using ATCC 19977 producing Bla_Mab.

Time-kill curves of monotherapy or double β-lactams (A, D, and G) and their combinations with avibactam (B, E, and H) or durlobactam+sulbactam (C, F, and I).

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Imipenem (IPM) showed high binding affinities for both LDTs and PBPs, with an extremely low inhibition constant (K_i,app; Tab. 1). Cephalosporins exhibited moderate binding affinity for both LDTs and PBPs, whereas amoxicillin (AMX) selectively targeted PBPs. The inactivation of Bla_Mab and LDTs/PBPs by durlobactam (DUR) exhibited greater efficacy compared to avibactam (AVI), aligning with their respective bactericidal effects. DUR showed a 75-fold lower K_i,app for Bla_Mab in comparison to AVI. The K_i,app of DUR for PBP B, PBP-lipo, and LDT2 fell below the clinical concentration, whereas those of AVI did not. Single β-lactam treatment resulted in minimal killing (∼1 log₁₀ reduction), but adding AVI significantly enhanced killing by inhibition of β-lactamase (∼4 log₁₀ reduction for IPM+AVI and ∼2 log₁₀ reduction for others; Fig. 1). DUR alone showed 2 log₁₀ reduction, and when combined with IPM or dual BL, it achieved near-eradication, surpassing the SOC (AMK + Clarithromycin + IPM or cefoxitin). Inhibition of PBP-lipo by sulopenem, IPM, DUR, and AMX changed the cell morphology to filaments. In accordance with the binding affinity to LDTs and PBPs, alterations in melting temperature were detected.

IPM + DUR + SUL showed the most killing of Mab with limited regrowth. This outcome can be attributed to β-lactamase inhibition and the inactivation of multiple targets, not only LDTs but also PBPs. This combination strategy holds promise for enhanced efficacy of Mab treatment, necessitating further study in clinical trials.

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