Low permeability across the outer membrane is a major reason why most antibiotics are ineffective against Gram-negative bacteria. Agents that permeabilize the outer membrane are typically toxic at their effective concentrations. Here, we report the development of a broad-spectrum homodimeric tobramycin adjuvant that is nontoxic and more potent than the gold standard permeabilizing agent, polymyxin B nonapeptide. In pilot studies, the adjuvant confers potent bactericidal activity on novobiocin against Gram-negative bacteria, including carbapenem-resistant and colistin-resistant strains bearing plasmid-borne mcr-1 genes. Resistance development to the combination was significantly reduced, relative to novobiocin alone, and there was no induction of cross-resistance to other antibiotics, including the gyrase-acting fluoroquinolones. Tobramycin homodimer may allow the use of lower doses of novobiocin, overcoming its twin problem of efficacy and toxicity.

Objectives To develop a multifunctional adjuvant molecule that can rescue β-lactam antibiotics and β-lactam/β-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 β-lactam/β-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 β-lactam/β-lactamase inhibitors against carbapenem-resistant clinical isolates, highlighting the complexity of resistance to β-lactam antibiotics. Our strategy presents an avenue to further preserve the therapeutic utility of β-lactam antibiotics.

The lack of therapeutic options to treat infections caused by multidrug-resistant (MDR) pathogens, especially Gram-negative bacteria, is apparent. Therefore, it is imperative to develop new strategies to address the problem of antimicrobial resistance. Repurposing non-antibiotic commercial drugs for antimicrobial therapy presents a viable option. We screened six anticancer drugs for their potential use in antimicrobial therapy. Here, we provide in vitro evidence that suggests feasibility to repurpose the anticancer drug mitomycin C against MDR Gram-negative bacteria. We also demonstrated that mitomycin C, etoposide and doxorubicin were affected by drug efflux in Pseudomonas aeruginosa. In combination with a tobramycin-ciprofloxacin antibiotic hybrid (TOB-CIP), the antibacterial activity of mitomycin C was enhanced against MDR clinical isolates of P. aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. In fact, 4 µg/mL (3 µM) TOB-CIP reduced the minimum inhibitory concentration of mitomycin C to ≤1 µg/mL against MDR Gram-negative bacteria, except A. baumannii. We showed that synergy was inherent to TOB-CIP and that neither tobramycin nor ciprofloxacin individually synergized with mitomycin C. Our finding supports identifying adjuvant partners for mitomycin C, such as TOB-CIP, to enhance suitability for antimicrobial therapy.

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