Zhang Huanhuan, Feng Wei, Sun Fengjun, Xu Te, Qian Yan
Department of Pharmacy, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China; Department of Pharmacy, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China.
Department of Pharmacy, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China.
J Glob Antimicrob Resist. 2025 Jun;43:143-154. doi: 10.1016/j.jgar.2025.04.009. Epub 2025 Apr 29.
This study aimed to investigate the effects of polymyxin B (PMB) in combination with other antibiotics on delaying resistance in Klebsiella pneumoniae and to explore the mechanisms underlying PMB-induced resistance.
In vitro continuous induction experiments were performed to observe changes in drug susceptibility with PMB alone vs. in combination. RNA-seq, quantitative reverse transcription PCR, and proteomic analyses were utilized to evaluate differential gene and protein expression between induced-resistant strains and those exhibiting delayed resistance. Then, gene knockout experiments were performed to validate the functional roles of relevant genes.
These findings indicated that PMB alone could induce resistance within 1-2 d, whereas the combination with amikacin (AMK) or tigecycline delayed the onset of resistance by 6 d. RNA-seq, quantitative reverse transcription PCR, and proteomic analyses revealed significant upregulation of nlpE, two-component systems, and AcrAB-TolC efflux pump-associated genes in PMB-induced resistant strains, whereas these genes were downregulated in the delayed resistant strains of PMB combined with AMK. Deletion and complementation experiments demonstrated that the expression levels of two-component systems and efflux pump-related genes were downregulated in nlpE knockout strains. Furthermore, PMB induction experiments revealed a significant upregulation of PmrA, PhoP, PhoQ, PagP, and AcrB proteins associated with cationic antimicrobial peptide pathways in the wild-type and nlpE complemented strains, whereas no differential change was observed in the nlpE knockout strain.
nlpE contributes to PMB resistance by modulating the AcrAB-TolC efflux pump and the PhoP/Q and PmrA/B two-component systems. The combined use of PMB with AMK effectively delays the development of resistance in K. pneumoniae through the regulation of nlpE and its associated signalling pathways.
本研究旨在探讨多粘菌素B（PMB）与其他抗生素联合使用对延缓肺炎克雷伯菌耐药性的影响，并探索PMB诱导耐药的机制。
进行体外连续诱导实验，观察单独使用PMB与联合使用时药物敏感性的变化。利用RNA测序、定量逆转录PCR和蛋白质组学分析评估诱导耐药菌株与表现出延缓耐药性的菌株之间基因和蛋白质表达的差异。然后，进行基因敲除实验以验证相关基因的功能作用。
这些发现表明，单独使用PMB可在1 - 2天内诱导耐药，而与阿米卡星（AMK）或替加环素联合使用可将耐药发生时间推迟6天。RNA测序、定量逆转录PCR和蛋白质组学分析显示，在PMB诱导的耐药菌株中，nlpE、双组分系统和AcrAB - TolC外排泵相关基因显著上调，而在PMB与AMK联合的延缓耐药菌株中这些基因下调。缺失和互补实验表明，在nlpE敲除菌株中双组分系统和外排泵相关基因的表达水平下调。此外，PMB诱导实验显示，在野生型和nlpE互补菌株中，与阳离子抗菌肽途径相关的PmrA、PhoP、PhoQ、PagP和AcrB蛋白显著上调，而在nlpE敲除菌株中未观察到差异变化。
nlpE通过调节AcrAB - TolC外排泵以及PhoP/Q和PmrA/B双组分系统来促进对PMB的耐药性。PMB与AMK联合使用通过调节nlpE及其相关信号通路有效延缓肺炎克雷伯菌耐药性的发展。
