Peptide-antibiotic Combination Strategy for Combating Multiple Drug-resistant Bacteria

Han, Wenxu

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Peptide-antibiotic Combination Strategy for Combating Multiple Drug-resistant Bacteria

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The development of antibiotic resistance is a severe worldwide problem. What's even more concerning is that the development of new antibiotics is a challenging, time-consuming, expensive, and often unsuccessful process. To improve the effectiveness of antibiotics, antibiotic combinations are used in clinical as possible treatment options. However, the antibiotic combinations may promote bacteria to develop more severe resistance. Antimicrobial peptides (AMPs), like LL37, exhibit a broad spectrum of antibacterial effects, making them promising and potential antimicrobial agents. LL37 also plays important roles in the human immune system and is less likely to promote resistance. Thus, we first proposed a novel combination strategy by combining LL37 with antibiotics to combat two clinical Pseudomonas aeruginosa (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, which appears to explain why it was easier to restore the effectiveness of the antibiotic against the bacteria. 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 hypothesized to have contributed to the increase in outer membrane permeability. We also 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 decreased, suggesting LL37 can reduce the resistance through increase the outer membrane permeability of P. aeruginosa strains. While LL37 shows promise as an antimicrobial agent, the potential cytotoxicity at high concentrations and manufacturing cost may limit its clinical applications. To enhance the antibacterial properties of LL37 while mitigating some of its drawbacks, the N-terminal region and less structured fragments were cut from LL37, resulting in two more structured LL37 fragments: FK16 and FK13. To evaluate the potential of LL37 fragments in both antibacterial efficacy and combination strategy, we combined LL37, FK16, and FK13 with antibiotics to combat Methicillin-resistant Staphylococcus aureus (MRSA) strains in vitro. Our results showed that FK16 and FK13 have more synergistic inhibitory effects to MRSA strains when combined with penicillin G and ampicillin. In addition, AMPs exhibited strong membrane permeabilizing properties, and membrane permeabilizing effects can provide a possible explanation for the improved antibacterial effects of antibiotics. We further studied the electrostatic interactions between AMPs and bacteria and demonstrated the connection between membrane permeabilization and charge neutralization. Finally, we treated MRSA strains with AMPs and characterized the MICs of penicillin G and ampicillin. AMP exposure and subsequent membrane permeabilization provide a possible pathway to re-sensitize drug-resistant bacteria to traditional antibiotics. Re-sensitization may help preserve the effectiveness of traditional antibiotics, thus providing a potential new combination strategy for fighting MRSA infections. To study the influence of Mg2+ and Ca2+ ions and gain a deeper understanding of the AMP-antibiotic combination strategy, LL37, FK16, and FK13 were combined with polymyxins and vancomycin to combat three clinical Escherichia coli (E. coli) strains in vitro. More synergistic combinations were found when AMPs combined with polymyxin B and colistin. All three AMPs displayed strong outer membrane permeabilizing properties, and cationic AMPs demonstrated the capability to bind to negatively charged lipopolysaccharide (LPS) molecules on the outer membrane. We also found that the outer membrane permeabilizing properties are correlated with the net positive charge quantity in cationic antimicrobials. We found pretreatment of E. coli strains with AMPs can reduce the resistance of E. coli strains to vancomycin. We also studied the cytoplasmic membrane permeabilization capabilities of three AMPs. This is crucial because it determines the ability of AMPs to induce bacterial lysis and fatality. Finally, we evaluated the cytotoxicity of AMPs on human dermal fibroblasts, and FK13 exhibited the lowest toxicity, possibly because FK13 lacks hydrophobic residues. The results from this study provide a novel AMP-antibiotic combination strategy to combat multiple bacteria, offering insight into the interactions between AMPs and the bacterial membrane. Additionally, our study sheds light on the reasons behind synergistic combinations and demonstrates a possible strategy to broaden the antibacterial spectrum of antibiotics and reduce bacterial resistance.

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