Potentiation of p-lactam antibiotics and β-lactam/β-lactamase inhibitor combinations against MDR and XDR Pseudomonas aeruginosa using non-ribosomal tobramycin-cyclam conjugates

Objectives: To develop a multifunctional adjuvant molecule that can rescue beta-lactam antibiotics and beta-lactam/beta-lactamase inhibitor combinations from resistance in carbapenem-resistant Pseudomonas aeruginosa clinical isolates. Methods: Preparation of adjuvant was guided by structure-activity relationships, following standard protocols. Susceptibility and chequerboard studies were assessed using serial 2-fold dilution assays. Toxicity was evaluated against porcine erythrocytes, human embryonic kidney (HEK293) cells and liver carcinoma (HepG2) cells via MTS assay. Preliminary in vivo efficacy was evaluated using a Galleria mellonella infection model. Results: Conjugation of tobramycin and cyclam abrogates the ribosomal effects of tobramycin but confers a potent adjuvant property that restores full antibiotic activity of meropenem and aztreonam against carbapenem-resistant P. aeruginosa. Therapeutic levels of susceptibility, as determined by CLSI susceptibility breakpoints, were attained in several MDR clinical isolates, and time-kill assays revealed a synergistic dose-dependent pharmacodynamic relationship. A triple combination of the adjuvant with ceftazidime/avibactam (approved), aztreonam/avibactam (Phase III) and meropenem/avibactam enhances the efficacies of beta-lactam/beta-lactamase inhibitors against recalcitrant strains, suggesting rapid access of the combination to their periplasmic targets. The newly developed adjuvants, and their combinations, were non-haemolytic and non-cytotoxic, and preliminary in vivo evaluation in G. mellonella suggests therapeutic potential for the double and triple combinations. Conclusions: Non-ribosomal tobramycin-cyclam conjugate mitigates the effect of OprD/OprF porin loss in P. aeruginosa and potentiates beta-lactam/beta-lactamase inhibitors against carbapenem-resistant clinical isolates, highlighting the complexity of resistance to beta-lactam antibiotics. Our strategy presents an avenue to further preserve the therapeutic utility of beta-lactam antibiotics.