Department of Biochemistry, University of Missouri, Columbia, Missouri, USA
Experiment Station Chemical Laboratories, University of Missouri, Columbia, Missouri, USA.
Antimicrob Agents Chemother. 2018 Jul 27;62(8). doi: 10.1128/AAC.02349-17. Print 2018 Aug.
Multidrug-resistant bacterial infections are being increasingly treated in clinics with polymyxins, a class of antibiotics associated with adverse effects on the kidney, nervous system, or airways of a significant proportion of human and animal patients. Although many of the resistant pathogens display enhanced virulence, the hazard of cytotoxic interactions between polymyxin antibiotics and bacterial virulence factors (VFs) has not been assessed, to date. We report here the testing of paired combinations of four VF phenazine toxins, pyocyanin (PYO), 1-hydroxyphenazine (1-HP), phenazine-1-carboxylic acid (PCA), and phenazine-1-carboxamide (PCN), and two commonly prescribed polymyxin drugs, colistin-colistimethate sodium (CMS) and polymyxin B, in three human airway cell lines, BEAS-2B, HBE-1, and CFT-1. Cytotoxicities of individual antibiotics, individual toxins, and their combinations were evaluated by the simultaneous measurement of mitochondrial metabolic, total transcriptional/translational, and Nrf2 stress response regulator activities in treated cells. Two phenazines, PYO and 1-HP, were cytotoxic at clinically relevant concentrations (100 to 150 μM) and prompted a significant increase in oxidative stress-induced transcriptional activity in surviving cells. The polymyxin antibiotics arrested cell proliferation at clinically achievable (<1 mM) concentrations as well, with CMS displaying surprisingly high cytotoxicity (50% effective dose [ED] = 180 μM) in BEAS-2B cells. The dose-response curves were probed by a median-effect analysis, which established a synergistically enhanced cytotoxicity of the PYO-CMS combination in all three airway cell lines; a particularly strong effect on BEAS-2B cells was observed, with a combination index (CI) of 0.27 at the ED PCA, PCN, and 1-HP potentiated CMS cytotoxicity to a smaller extent. The cytotoxicity of CMS could be reduced with 10 mM -acetyl-cysteine. Iron chelators, while ineffective against the polymyxins, could rescue all three bronchial epithelial cell lines treated with lethal PYO or CMS-PYO doses. These findings suggest that further evaluations of CMS safety are needed, along with a search for means to moderate potentially cytotoxic interactions.
多药耐药菌感染在临床上越来越多地用多粘菌素类抗生素治疗，这类抗生素与相当一部分人和动物患者的肾脏、神经系统或气道的不良反应有关。尽管许多耐药病原体表现出增强的毒力，但迄今为止，尚未评估多粘菌素类抗生素与细菌毒力因子 (VF) 之间细胞毒性相互作用的危险。我们在这里报告了对四种 VF 吩嗪毒素、绿脓菌素 (PYO)、1-羟基吩嗪 (1-HP)、吩嗪-1-羧酸 (PCA) 和吩嗪-1-甲酰胺 (PCN) 以及两种常用的多粘菌素药物黏菌素-黏菌素甲磺酸钠 (CMS) 和多粘菌素 B 的配对组合的测试，在三种人呼吸道细胞系 BEAS-2B、HBE-1 和 CFT-1 中进行。通过同时测量处理细胞中的线粒体代谢、总转录/翻译和 Nrf2 应激反应调节剂活性，评估了单独抗生素、单独毒素及其组合的细胞毒性。两种吩嗪，PYO 和 1-HP，在临床相关浓度 (100 至 150 μM) 下具有细胞毒性，并促使存活细胞中氧化应激诱导的转录活性显著增加。黏菌素类抗生素也以临床可达到的浓度 (<1 mM) 阻止细胞增殖，CMS 在 BEAS-2B 细胞中显示出惊人的高细胞毒性 (50%有效剂量 [ED] = 180 μM)。通过中值效应分析探测剂量反应曲线，该分析在所有三种气道细胞系中建立了 PYO-CMS 组合的协同增强的细胞毒性；在 BEAS-2B 细胞中观察到特别强的作用，组合指数 (CI) 在 ED PCA、PCN 和 1-HP 处为 0.27，CMS 细胞毒性增强较小。10 mM N-乙酰半胱氨酸可降低 CMS 的细胞毒性。铁螯合剂虽然对多粘菌素类无效，但可挽救用致死剂量 PYO 或 CMS-PYO 处理的所有三种支气管上皮细胞系。这些发现表明需要进一步评估 CMS 的安全性，并寻找减轻潜在细胞毒性相互作用的方法。
