New antimicrobial peptide-antibiotic combination strategy for Pseudomonas aeruginosa inactivation.

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.