Two plus One: Combination Therapy Tri-systems Involving Two Membrane-Disrupting Antimicrobial Macromolecules and Antibiotics.

The escalating issue of multidrug-resistant (MDR) bacteria indicates the urgent need for new and effective strategies to combat this global health challenge. Here, we describe a new combinatorial approach that can be put forward for experimental therapy application against MDR bacteria. Specifically, we have developed a tri-system that includes the coadministration of two different membrane-disrupting-type antimicrobial agents─a synthetic antimicrobial polymer P and an antimicrobial peptide (AMP) colistin methanesulfonate (Col)─in conjunction with an antibiotic [doxycycline (Dox), rifampicin (Rif), or azithromycin (Azi)]. Traditionally, the administration of membrane-disrupting antimicrobial agents causes toxicity, but, in comparison, we demonstrated synergy and biocompatibility using this combinatorial approach. Checkerboard assays showed the occurrence of synergistic interactions in Col-Dox-P, Col-Rif-P, and Col-Azi-P tri-systems against wild-type and MDR Pseudomonas aeruginosa, with the Col-Dox-P system being the most effective. The ability to synergize thus enables the use of a lower dosage in combinations compared to the standalone agents. The tri-systems not only demonstrated bacteriostatic activity but were also bactericidal. For example, the Col-Dox-P system (at 8, 4, and 8 µg mL-1, respectively) and the Col-Rif-P system (at 4, 8, and 16 µg mL-1, respectively) were able to kill >99.999% of planktonic P. aeruginosa cells within 3 h of treatment. More importantly, an improvement of the therapeutic/selectivity index was achieved via combination therapy. Taking the Col-Dox-P system as an example, its biocompatibility with murine embryonic fibroblast cells was found to be comparable to that of polymer P alone despite the synergistic enhancement in antimicrobial activity of the combination. This resulted in a significant increase in selectivity by 16-fold for the Col-Dox-P combination system compared to P alone. Furthermore, the broad applicability of this tri-system strategy was demonstrated via the successful application of the AMP melittin in place of Col or P. Overall, this study sheds new insights on the application of membrane-disrupting antimicrobial agents in combination therapy and their potential for safer clinical use. Additionally, the information gathered in this study could inform the development of future combination therapy systems involving the simultaneous employment of multiple AMPs with antibiotics.