小核糖核酸调控的外排泵表达影响肺炎克雷伯氏菌临床分离株对替加环素的耐药性和异抗性。

IF 6.1 1区 生物学 Q1 MICROBIOLOGY
Yuqiao Han , Yilin Xiong , Mengyao Wang , Jia Wang , Tao Song , Jing Yu , Jia Hu , Zinan Zhao , Ming Li , Ying Li , Yang Chen
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引用次数: 0

摘要

替加环素和新近获得美国食品和药物管理局批准的四环素类药物(包括克拉维酸和奥马他环素)被视为治疗耐多药肠杆菌属的最后手段。然而,肺炎克雷伯菌对替加环素的耐药性有所增加,尤其是异抗性的内在机制尚不清楚。本研究旨在阐明临床肺炎克雷伯菌分离株对替加环素耐药和异耐药的机制。本研究共收集了 153 株临床肺炎克雷伯菌分离株,采用肉汤微量稀释法和群体分析图谱法分别鉴定出 15 株对替加环素耐药的分离株和 3 株对替加环素异耐药的分离株。提取了肺炎克氏菌 ATCC13883 和实验室诱导的耐替加环菌株的总 RNA,并在 Illumina 平台上进行了测序。利用定量实时 PCR 分析并验证了肺炎克雷伯菌临床分离株中的差异表达基因和调控小 RNA(sRNA)。RNA 测序结果显示,耐替加环素菌株中的 mdtABC 外排泵基因显著上调。在临床肺炎克雷伯菌分离株中观察到了 mdtABC 的过表达,这增加了替加环素的最低抑菌浓度(MICs),并参与了替加环素的异抗性。sRNA 测序分析表明,候选 sRNA-120 直接与 mdtABC 操作子相互作用,并在耐替加环素菌株中被下调。我们生成了一株 sRNA-120 缺失突变菌株和一株肺炎双球菌互补菌株。缺失 sRNA-120 的菌株显示 mdtA、mdtB 和 mdtC 的 mRNA 水平升高,对替加环素的 MICs 也升高。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。为了验证 sRNA-120 与 mdtABC 之间的相互作用,我们进行了 RNA 反义纯化和平行反应监测质谱分析。总之,我们的研究强调了通过 sRNA-120 对 mdtABC 的转录后抑制可能提供了另一层外排泵基因表达控制,这对临床肺炎克雷伯菌分离株的耐药性和异抗性非常重要。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Small RNA-regulated expression of efflux pump affects tigecycline resistance and heteroresistance in clinical isolates of Klebsiella pneumoniae

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.

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来源期刊
Microbiological research
Microbiological research 生物-微生物学
CiteScore
10.90
自引率
6.00%
发文量
249
审稿时长
29 days
期刊介绍: Microbiological Research is devoted to publishing reports on prokaryotic and eukaryotic microorganisms such as yeasts, fungi, bacteria, archaea, and protozoa. Research on interactions between pathogenic microorganisms and their environment or hosts are also covered.
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