Yuqiao Han , Yilin Xiong , Mengyao Wang , Jia Wang , Tao Song , Jing Yu , Jia Hu , Zinan Zhao , Ming Li , Ying Li , Yang Chen
{"title":"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。","authors":"Yuqiao Han , Yilin Xiong , Mengyao Wang , Jia Wang , Tao Song , Jing Yu , Jia Hu , Zinan Zhao , Ming Li , Ying Li , Yang Chen","doi":"10.1016/j.micres.2024.127825","DOIUrl":null,"url":null,"abstract":"<div><p>Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales<em>.</em> However, tigecycline resistance in <em>Klebsiella pneumoniae</em> has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical <em>K. pneumoniae</em> isolates. A total of 153 clinical <em>K. pneumoniae</em> 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 <em>K. pneumoniae</em> 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 <em>K. pneumoniae</em> using quantitative real-time PCR. RNA sequencing results showed that <em>mdtABC</em> efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of <em>mdtABC</em> was observed in a clinical <em>K. pneumoniae</em> 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 <em>mdtABC</em> operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of <em>K. pneumoniae.</em> The sRNA-120 deletion strain displayed increased mRNA levels of <em>mdtA, mdtB,</em> and <em>mdtC</em> 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 <em>mdtABC</em>. Collectively, our study highlights that the post-transcriptional repression of <em>mdtABC</em> through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical <em>K. pneumoniae</em> isolates.</p></div>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":null,"pages":null},"PeriodicalIF":6.1000,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Small RNA-regulated expression of efflux pump affects tigecycline resistance and heteroresistance in clinical isolates of Klebsiella pneumoniae\",\"authors\":\"Yuqiao Han , Yilin Xiong , Mengyao Wang , Jia Wang , Tao Song , Jing Yu , Jia Hu , Zinan Zhao , Ming Li , Ying Li , Yang Chen\",\"doi\":\"10.1016/j.micres.2024.127825\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales<em>.</em> However, tigecycline resistance in <em>Klebsiella pneumoniae</em> has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical <em>K. pneumoniae</em> isolates. A total of 153 clinical <em>K. pneumoniae</em> 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 <em>K. pneumoniae</em> 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 <em>K. pneumoniae</em> using quantitative real-time PCR. RNA sequencing results showed that <em>mdtABC</em> efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of <em>mdtABC</em> was observed in a clinical <em>K. pneumoniae</em> 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 <em>mdtABC</em> operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of <em>K. pneumoniae.</em> The sRNA-120 deletion strain displayed increased mRNA levels of <em>mdtA, mdtB,</em> and <em>mdtC</em> 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 <em>mdtABC</em>. Collectively, our study highlights that the post-transcriptional repression of <em>mdtABC</em> through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical <em>K. pneumoniae</em> isolates.</p></div>\",\"PeriodicalId\":18564,\"journal\":{\"name\":\"Microbiological research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":6.1000,\"publicationDate\":\"2024-07-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Microbiological research\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S094450132400226X\",\"RegionNum\":1,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Microbiological research","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S094450132400226X","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
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.
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.