Ethambutol and meropenem/clavulanate synergy promotes enhanced extracellular and intracellular killing of Mycobacterium tuberculosis

Increasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy. However, additional research on the interaction of these drugs with conventional anti-TB agents is still warranted. Since the complex cell envelope of Mycobacterium tuberculosis (Mtb) may pose an additional obstacle to the diffusion of beta-lactams, an improved activity upon combination with drugs that inhibit the synthesis of outer cell wall elements is particularly relevant. In this context, we aimed to determine potential synergies between beta-lactams and the antimycobacterial drugs ethambutol and isoniazid. This was followed by experiments that aimed to confirm if the increased antimicrobial effects remained within the intracellular milieu and if they promoted heightened immune responses. Results of checkerboard assays with H37Rv and eight clinical isolates, including four drug-resistant Mtb strains, exposed that only the treatments containing ethambutol and beta-lactams achieved synergistic effects, while the standard ethambutol and isoniazid association failed to produce synergy in any of the tested isolates. In Mtb-infected THP-1 macrophages, combinations of ethambutol with increasing meropenem concentrations consistently displayed superior killing activities over the individual antibiotics. Flow cytometry with BODIPY FL vancomycin, which binds directly to the peptidoglycan, confirmed an increased exposure of this layer after co-treatment. This was reinforced by the high IL-1β secretion levels found in infected macrophages after incubation with concentrations of meropenem above 5 mg/L, which indicated an exposure of the host innate response sensors to pathogen-associated molecular patterns in the PG. Our findings show that the proposed impaired access of beta-lactams to periplasmic transpeptidases is counteracted by concomitant administration with ethambutol. The efficiency of this combination may be attributed to the synchronized inhibition of arabinogalactan and peptidoglycan synthesis, two key cell wall components. Given that beta-lactams exhibit a time-dependent bactericidal activity, a more effective pathogen recognition and killing prompted by this association may be highly beneficial to optimize TB regimens containing carbapenems.