Gogry Firdoos Ahmad, Siddiqui Mohammad Tahir, Sultan Insha, Husain Fohad Mabood, Al-Kheraif Abdulaziz A, Ali Asghar, Haq Qazi Mohd Rizwanul
Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India.
Department of Food Science and Nutrition, College of Food and Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia.
Pharmaceutics. 2022 Jan 27;14(2):295. doi: 10.3390/pharmaceutics14020295.
Colistin, a polycationic antimicrobial peptide, is one of the last-resort antibiotics for treating infections caused by carbapenem-resistant Gram-negative bacteria. The antibacterial activity of colistin occurs through electrostatic interaction between the polycationic peptide group of colistin and the negatively charged phosphate groups of lipid A membrane. This study investigated the interaction of colistin with the outer membrane and surface constituents of resistant and susceptible strains of and harboring 1 resistance gene. Bacterial membrane and lipopolysaccharide used in this study were isolated from susceptible as well as colistin-resistant strains of and . Interaction of colistin with the bacterial surface was studied by deoxycholate and lysozyme sensitivity test, N-phenyl-1-naphthylamine (NPN) uptake assay, Atomic force microscopy (AFM), Zeta potential measurements and H NMR. The binding affinity of colistin was found to be lower with outer membrane from resistant strains in comparison with the susceptible strains. Colistin exposure enhances the outer membrane permeability of the susceptible strains to deoxycholate and lysozyme. However, on the other hand, colistin dose of 256 µg/mL did not permeabilize the outer membrane of resistant bacteria. The NPN permeability in resistant strains was greater in comparison with susceptible strains. Atomic force microscopy images depicted smooth, featherless and deformed membranes in treated susceptible cells. Contrary to the above, resistant treated cells displayed surface roughness topography even at 256 µg/mL colistin concentration. Surface charge alterations were confirmed by Zeta potential measurements as a function of the growth phase. Mid-logarithmic phase susceptible strains showed a greater negative charge than resistant strains upon exposure to colistin. However, there was no statistical variation in the Zeta potential measurements between resistant and susceptible strains at the stationary phase. NMR analysis revealed line broadening in susceptible strains with increasing colistin: LPS aggregates mass ratio. Moreover, resistant strains did not show line broadening for the outer membrane, even at the highest mass ratio. The findings of this study suggest that the resistant strains of and can block the electrostatic contact between the cationic peptide and anionic lipid A component that drives the first phase of colistin action, thereby preventing hydrophobically driven second-tier action of colistin on the outer lipopolysaccharide layer.
黏菌素是一种聚阳离子抗菌肽，是治疗由耐碳青霉烯革兰氏阴性菌引起的感染的最后手段之一。黏菌素的抗菌活性通过黏菌素的聚阳离子肽基团与脂多糖膜带负电荷的磷酸基团之间的静电相互作用产生。本研究调查了黏菌素与携带1个耐药基因的肺炎克雷伯菌和鲍曼不动杆菌耐药及敏感菌株的外膜和表面成分之间的相互作用。本研究中使用的细菌膜和脂多糖分别从肺炎克雷伯菌和鲍曼不动杆菌的敏感菌株以及黏菌素耐药菌株中分离得到。通过脱氧胆酸盐和溶菌酶敏感性试验、N-苯基-1-萘胺（NPN）摄取试验、原子力显微镜（AFM）、zeta电位测量和核磁共振氢谱（1H NMR）研究了黏菌素与细菌表面的相互作用。结果发现，与敏感菌株相比，黏菌素与耐药菌株外膜的结合亲和力较低。黏菌素处理可增强敏感菌株外膜对脱氧胆酸盐和溶菌酶的通透性。然而，另一方面，256μg/mL的黏菌素剂量并未使耐药菌的外膜通透性增加。与敏感菌株相比，耐药菌株中的NPN通透性更高。原子力显微镜图像显示，经处理的敏感细胞中的膜光滑、无羽毛且变形。与此相反，即使在256μg/mL的黏菌素浓度下，经处理的耐药细胞仍呈现表面粗糙的形貌。通过zeta电位测量证实了表面电荷变化是生长阶段的函数。对数中期的敏感菌株在暴露于黏菌素后显示出比耐药菌株更大的负电荷。然而，在稳定期，耐药菌株和敏感菌株之间的zeta电位测量没有统计学差异。核磁共振分析显示，随着黏菌素与脂多糖聚集体质量比的增加，敏感菌株中的谱线变宽。此外，即使在最高质量比下，耐药菌株的外膜也没有显示出谱线变宽。本研究结果表明，肺炎克雷伯菌和鲍曼不动杆菌的耐药菌株可以阻断阳离子肽与阴离子脂多糖成分之间的静电接触，而这种静电接触驱动了黏菌素作用的第一阶段，从而阻止了黏菌素在外脂多糖层上由疏水作用驱动的第二阶段作用。
