Novel antimicrobials or new treatment strategies are urgently needed to treat Pseudomonas aeruginosa (P. aeruginosa) related infections and especially to address the problem of antibiotic resistance. We propose a novel strategy that combines the human antimicrobial peptide (AMP) LL37 with different antibiotics to find synergistic AMP-antibiotic combinations against P. aeruginosa strains in vitro. Our results showed that LL37 exhibited synergistic inhibitory and bactericidal effects against P. aeruginosa strains PAO1 and PA103 when combined with the antibiotics vancomycin, azithromycin, polymyxin B, and colistin. In addition, LL37 caused strong outer membrane permeabilization, as demonstrated through measurement of an increased uptake of the fluorescent probe N-phenyl-1-naphthylamine. The membrane permeabilization effects appear to explain why it was easier to rescue the effectiveness of the antibiotic toward the bacteria because the outer membrane of P. aeruginosa exhibits barrier function for antibiotics. Furthermore, the change in the zeta potential was measured for P. aeruginosa strains with the addition of LL37. Zeta potentials for P. aeruginosa strains PAO1 and PA103 were −40.9 and −10.9 mV, respectively. With the addition of LL37, negative zeta potentials were gradually neutralized. We found that positively charged LL37 can interact with and neutralize the negatively charged bacterial outer membrane through electrostatic interactions, and the process of neutralization is believed to have contributed to the increase in outer membrane permeability. Finally, to further illustrate the relationship between outer membrane permeabilization and the uptake of antibiotics, we used LL37 to make the outer membrane of P. aeruginosa strains more permeable, and minimum inhibitory concentrations (MICs) for several antibiotics (colistin, gentamicin, polymyxin B, vancomycin, and azithromycin) were measured. The MICs decreased were twofold to fourfold, in general. For example, the MICs of azithromycin and vancomycin decreased more than fourfold when against P. aeruginosa strain PAO1, which were the greatest decrease of any of the antibiotics tested in this experiment. As for PA103, the MIC of polymyxin B2 decreased fourfold, which was the strongest decrease seen for any of the antibiotics tested in this experiment. The increased uptake of antibiotics not only demonstrates the barrier role of the outer membrane but also validates the mechanism of synergistic effects that we have proposed. These results indicate the great potential of an LL37-antibiotic combination strategy and provide possible explanations for the mechanisms behind this synergy.

中文翻译：

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新的抗菌肽-抗生素联合策略用于铜绿假单胞菌的灭活

迫切需要新型抗菌药物或新的治疗策略来治疗铜绿假单胞菌( P. aeruginosa ) 相关感染，尤其是解决抗生素耐药性问题。我们提出了一种新策略，将人类抗菌肽 (AMP) LL37 与不同的抗生素相结合，以在体外找到针对铜绿假单胞菌菌株的协同 AMP-抗生素组合。我们的结果表明，LL37 对铜绿假单胞菌表现出协同抑制和杀菌作用菌株 PAO1 和 PA103 与抗生素万古霉素、阿奇霉素、多粘菌素 B 和粘菌素联合使用时。此外，LL37 引起强烈的外膜透化，如通过测量荧光探针N -苯基-1-萘胺的增加摄取所证明的那样。膜透化作用似乎可以解释为什么更容易挽救抗生素对细菌的有效性，因为铜绿假单胞菌的外膜对抗生素具有屏障功能。此外，还测量了添加 LL37 的铜绿假单胞菌菌株的 zeta 电位变化。铜绿假单胞菌的 Zeta 电位菌株 PAO1 和 PA103 分别为 -40.9 和 -10.9 mV。随着 LL37 的加入，负 zeta 电位逐渐被中和。我们发现带正电荷的 LL37 可以通过静电相互作用与带负电荷的细菌外膜相互作用并中和，中和过程被认为有助于外膜通透性的增加。最后，为了进一步说明外膜透化与抗生素摄取之间的关系，我们使用 LL37 制作铜绿假单胞菌的外膜菌株的渗透性更强，并测量了几种抗生素（粘菌素、庆大霉素、多粘菌素 B、万古霉素和阿奇霉素）的最低抑菌浓度 (MIC)。一般来说，MIC 降低了两倍到四倍。例如，阿奇霉素和万古霉素对铜绿假单胞菌的 MIC 降低超过四倍菌株 PAO1，这是本实验中测试的任何抗生素中降幅最大的。至于 PA103，多粘菌素 B2 的 MIC 降低了四倍，这是本实验中测试的所有抗生素中降幅最大的。抗生素摄入量的增加不仅证明了外膜的屏障作用，而且验证了我们提出的协同作用机制。这些结果表明 LL37-抗生素组合策略的巨大潜力，并为这种协同作用背后的机制提供了可能的解释。