Abstract

Background: Antimicrobial combination therapies outperform traditional monotherapies by offering additive or synergetic effects, a broader spectrum of activity, enhanced efficacy against polymicrobial infections, and a reduced risk of resistance development and infection relapse. Methods: Herein, we have combined the chemotherapeutic antibiotic alkylating agent mitomycin C (MMC) with the wide-spectrum antiseptic octenidine hydrochloride (OCT). The non-specific mode of action via membrane disruption of the latter, together with the DNA-alkylating ability of the former, is here combined to produce bactericidal antibiotic-like mixtures against clinical isolates of Methicillin-resistant Staphylococcus aureus and uropathogenic Escherichia coli. The concentrations required for both antimicrobials to inhibit or eradicate bacterial biofilms as well as intracellular persisters are also determined here. The cytotoxicity of the selected combinations on eukaryotic cells was also evaluated in traditional 2D cultures and in 3D-fibroblast spheroids to mimic a physiologically relevant microenvironment present in topical infections. Finally, the ecotoxicity of the studied compounds was also evaluated through standardized bioassays using representative microorganisms commonly employed in environmental toxicity assessments such as Daphnia magna (D. magna) and Aliivibrio fischeri (A. fischeri). Results: Compared to monotherapy, these environmentally friendly antibiotic-like combinations at specific concentrations may achieve additive antimicrobial effects against planktonic, sessile, and intracellular pathogenic strains. However, the ecotoxicological response was bioindicator-dependent: the OCT–MMC combination reduced toxicity in A. fischeri, but not in D. magna, where toxicity was comparable to OCT alone, indicating OCT as the main driver. Thus, any reduction in ecotoxicity appears species-specific. Nonetheless, the use of lower doses may help minimize selective pressure for resistance. Conclusions: Collectively, these findings demonstrate that selected MMC–OCT combinations achieve antibacterial efficacy against planktonic, sessile, and intracellular pathogenic bacteria while preserving the viability of eukaryotic cells and exhibiting minimal toxicity toward standard ecotoxicological model organisms. Further studies are needed to guarantee the safe use of this combination at selected doses and to guarantee its non-systemic absorption after topical administration.

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