Abstract：
Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates.
摘要：
替加环素和新的食品和药物管理局批准的四环素，包括eravacycline和omadacycline,被认为是耐多药肠杆菌的最后手段。然而,肺炎克雷伯菌对替加环素的耐药性有所增加，特别是异质抗性的潜在机制尚不清楚。本研究旨在阐明临床肺炎克雷伯菌对替加环素耐药和异质耐药的潜在机制。共收集临床肺炎克雷伯菌153株，并使用肉汤微量稀释和群体分析概况方法鉴定了15株替加环素抗性和3株替加环素异源抗性分离株，分别。在Illumina平台上提取来自肺炎克雷伯菌ATCC13883和实验室诱导的替加环素抗性菌株的总RNA并测序。使用定量实时PCR在肺炎克雷伯菌的临床分离株中分析和验证差异表达基因和调节小RNA(sRNA)。RNA测序结果显示，mdtABC外排泵基因在替加环素耐药菌株中显著上调。在临床肺炎克雷伯菌分离物中观察到mdtABC的过表达，增加了替加环素的最低抑制浓度（MIC），并参与了替加环素的异质耐药。sRNA的测序分析表明，候选sRNA-120直接与mdtABC操纵子相互作用，并在替加环素耐药菌株中下调。我们产生了一个sRNA-120缺失突变菌株和一个互补的肺炎克雷伯菌菌株。sRNA-120缺失菌株显示mdtA的mRNA水平增加，mdtB,和mdtC以及替加环素的MIC增加。sRNA-120的互补菌株恢复了这些基因的mRNA水平和对替加环素的敏感性。进行RNA反义纯化和平行反应监测质谱以验证sRNA-120与mdtABC之间的相互作用。总的来说，我们的研究强调，通过sRNA-120的mdtABC的转录后抑制可能提供了一个额外的外排泵基因表达控制层，这对临床肺炎克雷伯菌分离株的耐药性和耐药性很重要。
