Pseudomonas aeruginosa is a Gram-negative pathogen that frequently causes life-threatening infections in immunocompromised patients. We previously showed that subinhibitory concentrations of short synthetic peptides permeabilize P. aeruginosa and enhance the lethal action of co-administered antibiotics. Long-term permeabilization caused by exposure of multidrug-resistant P. aeruginosa strains to peptide P4–9 was investigated by measuring the uptake of several antibiotics and fluorescent probes and by using confocal imaging and atomic force microscopy. We demonstrated that P4–9, a 13-amino acid peptide, induces a growth delay (i.e. post-antibiotic effect) of 1.3 h on a multidrug-resistant P. aeruginosa clinical isolate. Remarkably, when an independently P4–9-treated culture was allowed to grow in the absence of the peptide, cells remained sensitive to subinhibitory concentrations of antibiotics such as ceftazidime, fosfomycin and erythromycin for at least 2 h. We designated this persistent sensitization to antibiotics occurring in the absence of the sensitizing agent as Post-Antibiotic Effect associated Permeabilization (PAEP). Using atomic force microscopy, we showed that exposure to P4–9 induces profound alterations on the bacterial surface and that treated cells need at least 2 h of growth to repair those lesions. During PAEP, P. aeruginosa mutants overexpressing either the efflux pump MexAB-OprM system or the AmpC β-lactamase were rendered sensitive to antibiotics that are known substrates of those mechanisms of resistance. Finally, we showed for the first time that the descendants of bacteria surviving exposure to a membrane disturbing peptide retain a significant level of permeability to hydrophobic compounds, including propidium iodide, even after 20 h of growth in the absence of the peptide. The phenomenon of long-term sensitization to antibiotics shown here may have important therapeutic implications for a combined peptide-antibiotic treatment because the peptide would not need to be present to exert its antibiotic enhancing activity as long as the target organism retains sensitization to the antibiotic.

中文翻译：

---

合成肽可使多重耐药铜绿假单胞菌对抗生素敏感两个多小时，并使其包膜通透二十个小时。


铜绿假单胞菌是一种革兰氏阴性病原体，经常引起免疫功能低下患者危及生命的感染。我们之前表明，亚抑制浓度的短合成肽可透化铜绿假单胞菌并增强共同施用的抗生素的致死作用。通过测量几种抗生素和荧光探针的摄取以及使用共焦成像和原子力显微镜，研究了由多重耐药铜绿假单胞菌菌株暴露于肽 P4-9 引起的长期透化。我们证明，P4-9（一种 13 个氨基酸的肽）可诱导多重耐药铜绿假单胞菌临床分离株生长延迟（即抗生素后效应）1.3 小时。值得注意的是，当独立 P4-9 处理的培养物在没有肽的情况下生长时，细胞对亚抑制浓度的抗生素（如头孢他啶、磷霉素和红霉素）保持敏感至少 2 小时。我们将这种在没有致敏剂的情况下发生的对抗生素的持续致敏称为抗生素后效应相关透化（PAEP）。使用原子力显微镜，我们发现暴露于 P4-9 会引起细菌表面的深刻改变，并且处理过的细胞需要至少 2 小时的生长才能修复这些病变。在 PAEP 期间，过度表达外排泵 MexAB-OprM 系统或 AmpC β-内酰胺酶的铜绿假单胞菌突变体对抗生素敏感，而抗生素是这些耐药机制的已知底物。 最后，我们首次证明，暴露于膜干扰肽后幸存的细菌后代即使在没有肽的情况下生长 20 小时后，仍对疏水性化合物（包括碘化丙啶）保持显着水平的渗透性。这里显示的对抗生素长期敏感的现象可能对肽-抗生素联合治疗具有重要的治疗意义，因为只要目标生物体保持对抗生素的敏感，就不需要存在肽来发挥其抗生素增强活性。