Synthetic polypeptide adjuvant-antibiotic combination eradicates polymicrobial assemblies, dormant bacteria and bacterial biofilm.

In this work, we explore the potency of poly(D-peptide) (D-PP) in combination with diverse classes of antibiotics to address multidrug-resistant (MDR) Gram-negative superbugs, including their mature biofilms, polymicrobial assemblies, and dormant subpopulations, which are among complex challenging targets. The polypeptide adjuvant displayed significant antibiotic potentiation, resulting in a moderate to rapid bactericidal activity against the Gram-negative pathogens in their planktonic stage. The adjuvant and its combination restored susceptibility in dormant bacterial subpopulations. In polymicrobial settings, these combinations were able to reduce both Gram-negative and Gram-positive bacterial species, unlike individual antibiotic exposure. Importantly, one of the lead combinations (D-PP-rifampicin) resulted in moderate biofilm eradication of P. aeruginosa. Transcriptomic analysis demonstrated the differentially expressed genes (DEGs) involved in membrane integrity, stress response, and transport systems. Together with our previous mechanistic findings, this further confirmed that bacterial membrane perturbation is caused by the adjuvant. Such perturbation facilitated antibiotic accumulation within microbial cells. Furthermore, polypeptide reduces the bacterial resistance development towards doxycycline and rifampicin, unlike polymyxin B. Taken together, these findings exhibit that membrane-active polypeptide adjuvants can enhance antibiotic efficacy across diverse bacterial states and environments, providing a promising strategy to combat persistent and multidrug-resistant infections.