Choi Hyemin, Lee Dong Gun
College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea.
Biochim Biophys Acta. 2012 Dec;1820(12):1831-8. doi: 10.1016/j.bbagen.2012.08.012. Epub 2012 Aug 16.
Pleurocidin, a 25-mer antimicrobial peptide (AMP), is known to exert bactericidal activity. However, the synergistic activity and mechanism(s) of pleurocidin in combination with conventional antibiotics, and the antibiofilm effect of the peptide are poorly understood.
The interaction between pleurocidin and antibiotics was evaluated using checkerboard assay. To study the mechanism(s) involved in their synergism, we detected hydroxyl radical formation using 3'-(p-hydroxyphenyl) fluorescein, measured the NAD(+)/NADH ratio by NAD(+) cycling assay, observed change in bacterial viability with the hydroxyl radical scavenger thiourea, and investigated cytoplasmic membrane damage using propidium iodide. Also, the antibiofilm effect of pleurocidin was examined with the tissue culture plate method.
All combinations of pleurocidin and antibiotics showed synergistic interaction against bacterial strains (fractional inhibitory concentration index (FICI)≤0.5) except for Enterococcus faecium treated with a combination of the peptide and ampicillin (FICI=0.75). We identified that pleurocidin alone and in combinations with antibiotics induced formation of hydroxyl radicals. The oxidative stress was caused by a transient NADH depletion and the addition of thiourea prevented bacterial death, especially in the case of the combined treatment of pleurocidin and ampicillin showing synergisms. The combination of pleurocidin and erythromycin increased permeability of bacterial cytoplasmic membrane. Additionally, pleurocidin exhibited a potent inhibitory effect on preformed biofilm of bacterial organisms. In conclusion, pleurocidin synergized with antibiotics through hydroxyl radical formation and membrane-active mechanism, and exerted antibiofilm activity.
The synergistic effect between pleurocidin and antibiotics suggests the AMP is a potential therapeutic agent and adjuvant for antimicrobial chemotherapy.
杀肺菌素是一种由25个氨基酸组成的抗菌肽（AMP），已知具有杀菌活性。然而，对于杀肺菌素与传统抗生素联合使用时的协同活性和作用机制，以及该肽的抗生物膜作用，人们了解甚少。
采用棋盘法评估杀肺菌素与抗生素之间的相互作用。为研究其协同作用所涉及的机制，我们使用3'-（对羟基苯基）荧光素检测羟基自由基的形成，通过NAD⁺循环分析法测定NAD⁺/NADH比值，用羟基自由基清除剂硫脲观察细菌活力的变化，并使用碘化丙啶研究细胞质膜损伤情况。此外，采用组织培养板法检测杀肺菌素的抗生物膜作用。
除用杀肺菌素与氨苄西林联合处理的粪肠球菌（FICI = 0.75）外，杀肺菌素与抗生素的所有组合对细菌菌株均显示出协同相互作用（分数抑制浓度指数（FICI）≤0.5）。我们发现，单独使用杀肺菌素以及与抗生素联合使用均会诱导羟基自由基的形成。氧化应激是由NADH的短暂消耗引起的，添加硫脲可防止细菌死亡，尤其是在杀肺菌素与氨苄西林联合治疗显示协同作用的情况下。杀肺菌素与红霉素的组合增加了细菌细胞质膜的通透性。此外，杀肺菌素对细菌生物被膜具有强大的抑制作用。总之，杀肺菌素通过形成羟基自由基和膜活性机制与抗生素协同作用，并发挥抗生物膜活性。
杀肺菌素与抗生素之间的协同作用表明该抗菌肽是抗菌化疗的潜在治疗剂和佐剂。
