To investigate the roles of RamA, RarA, and SoxS regulators in tigecycline resistance in Klebsiella pneumoniae, carbapenem-resistant (C248-ST11-bla_KPC-2 and C182-ST11-bla_NDM-1), colistin-resistant (HK1-ST562-mcr-1), and tigecycline-resistant (KP17) strains with single or double regulon gene knockouts were evolved in vitro to generate tigecycline-resistant mutants. CRISPR-Cas9 was used to create KP17 mutants lacking rarA, ramA, lon, or their combinations. The mutant prevention concentration (MPC) of tigecycline and mutation frequency of regulon mutants were assessed. Phenotypic differences between the mutants and parents were assessed using growth curves, in vitro competition growth, serum bactericidal activity, biofilm formation, and hydrogen peroxide resistance tests. Genetic and transcriptomic variations were analyzed using whole-genome and RNA sequencing. Adaptive compensatory mechanisms of RamA, RarA, and SoxS in tigecycline resistance of K. pneumoniae were investigated. Acquired high-level tigecycline resistance in carbapenem- and colistin-resistant strains incurred fitness costs and reduced virulence. Colistin-resistant strains rapidly evolved high-level tigecycline resistance, with minimum inhibitory concentration of up to 256 mg/L. The RamRA-AcrAB/OqxAB pathway was pivotal for tigecycline resistance in carbapenem- and colistin-resistant strains. Lon was not related to tigecycline resistance but appeared to be linked to oxidative stress. Although knocking out the key regulon genes RamA and/or RarA did not impede tigecycline resistance development, these knockouts influenced mutation frequencies and MPCs, with RarA knockout increasing the number of mutation sites. RarA and SoxS served as compensatory regulons in the absence of RamA, or in double knockouts. These findings improved our understanding of the mechanisms underlying tigecycline resistance in K. pneumoniae.

Keywords: Klebsiella pneumoniae; RamA; RarA; SoxS; Tigecycline resistance.

Copyright © 2025. Published by Elsevier Ltd.

为了探究RamA、RarA和SoxS调节因子在肺炎克雷伯菌（Klebsiella pneumoniae）中替加环素耐药性中的作用，我们在体外进化了携带单个或双倍数调控基因敲除的碳青霉烯类耐药株(C248-ST11-bla_KPC-2和C182-ST11-bla_NDM-1)、多粘菌素耐药株(HK1-ST562-mcr-1)以及替加环素耐药株(KP17)，以生成替加环素耐药突变体。使用CRISPR-Cas9技术创建了KP17中缺少rarA、ramA、lon或其组合的突变体。评估了突变体和亲本菌株在替加环素中的突变预防浓度（MPC）以及调控基因突变体的突变频率。使用生长曲线、体内竞争生长、血清杀菌活性、生物膜形成和过氧化氢抗性测试评估了各突变体与亲本菌株之间的表型差异。利用全基因组测序和RNA测序分析遗传和转录组变异。研究了RamA、RarA和SoxS在肺炎克雷伯菌替加环素耐药适应性补偿机制中的作用。碳青霉烯类和多粘菌素耐药菌株获得高水平的替加环素耐药性会带来适应度代价并降低毒力。多粘菌素耐药菌株能够迅速进化出高达256 mg/L最小抑菌浓度的高水平替加环素耐药性。对于碳青霉烯类和多粘菌素耐药菌株而言，RamRA-AcrAB/OqxAB途径在替加环素耐药中至关重要。Lon不与替加环素耐药相关，但似乎与氧化应激有关联。虽然敲除关键调控基因RamA和/或RarA不会阻碍替加环素耐药性的发育，但这些敲除影响了突变频率和MPCs，其中RarA的敲除增加了突变位点的数量。在没有RamA的情况下，RarA和SoxS作为补偿性调控因子发挥作用，在双敲除中也是如此。这些发现提高了我们对肺炎克雷伯菌替加环素耐药机制的理解。

关键词：肺炎克雷伯菌；RamA；RarA；SoxS；替加环素耐药性。