生物被膜态和游离态临床耐碳青霉烯类鲍曼不动杆菌转录组的差异分析  

Transcriptome analysis of planktonic and biofilm forms of clinical carbapenem-resistant Acinetobacter baumannii

作　　者：刘巍巍 国果 吴兆颖 毛成菊 李忠旬 贾利娜 尚小丽 彭建[2,3] 吴建伟 LIU Weiwei;GUO Guo;WU Zhaoying;MAO Chengju;LI Zhongxun;JIA Lina;SHANG Xiaoli;PENG Jian;WU Jianwei(Department of Human Parasitology,School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Modern Pathogen Biology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;School of Biology&Engineering,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院人体寄生虫学教研室,贵州贵阳550025 [2]贵州医科大学基础医学院现代病原生物学特色重点实验室,贵州贵阳550025 [3]贵州医科大学生物工程学院,贵州贵阳550025

出　　处：《贵州医科大学学报》2021年第11期1249-1257,共9页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81660347);国家科技支撑计划(2011BAC06B12);贵州省科技厅科技支撑计划[黔科合支撑(2019)2823]。

摘　　要：目的了解临床分离的耐碳青霉烯类鲍曼不动杆菌(CRAB)生物被膜态和游离态的转录组差异,探究生物被膜形成的机制。方法从住院病人痰液或血液样本分离CRAB 4、55、78及117菌株,挑取单菌落分别培养生物被膜态菌(A组)和游离态菌(B组),采用Trizol法提取2组细菌核糖核酸(RNA)样本进行转录组测序;使用DESeq软件进行差异表达基因(DEGs)分析,通过GOSeq R软件进行基因本体(GO)富集分析,通过KOBAS软件进行京都基因与基因组百科全书(KEGG)通路富集分析;选择8个DEGs,利用实时荧光定量PCR(qRT-PCR)验证转录组测序分析结果。结果RNA样品完整无污染满足测序要求,测序数据错误率不超过0.03%;A与B组相比鉴定到407个差异基因,其中212条基因为上调表达,195条基因为下调表达,涉及铁的摄取和转运、菌毛合成的调控、生化代谢酶类及转录调节因子等;GO分析显示,GO条目主要富集于分子功能类别和生物过程类别;KEGG分析显示,DEGs富集到硫代谢、ATP结合盒(ABC)转运系统、万古霉素耐药、群体感应及阳离子抗菌肽耐药等多条通路;选取的8个差异基因通过qRT-PCR进行验证,结果与转录组结果趋势一致。结论临床分离的CRAB菌株,其生物被膜态和游离态存在基因表达差异,生物被膜的形成涉及多基因和多信号通路参与。Objective To understand the difference between clinical carbapenem-resistant Acinetobacter baumannii(CRAB)transcriptomes in biofilm-form and planktonic bacteria,and investigate the mechanism of biofilm formation.Methods Clinical isolates of CRAB 4,55,78,and 117 were collected from sputum or blood samples of hospitalized patients.A single colony was separately chosen to culture biofilm bacteria(A group)and planktonic bacteria(B group),and RNA samples of both groups were extracted by Trizol for transcriptome sequencing.DESeq software was used for screening differentially expressed genes(DEGs).GOSeq R software was used for performing Gene Ontology(GO)function annotation and KOBAS software was used for Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Eight DEGs were chosen and amplified by quantitative real-time PCR(qRT-PCR)to verify the results of transcriptome sequencing analysis.Results The RNA sample was intact and contamination-free,in line with the sequencing requirements.The error rate of sequencing data did not exceed 0.03%.Comparing with the B group,407 DEGs were identified in the A group.Of which,212 genes were up-regulated and 195 genes down-regulated,involving iron intake and transport,regulation of fimbriae synthesis,biochemical metabolic enzymes,transcription regulators,etc.GO analysis showed that GO items were mainly enriched in molecular function and biological process.KEGG analysis showed that DEGs were enriched in multiple pathways such as sulfur metabolism,ABC transport system,vancomycin resistance,quorum sensing and cationic antimicrobial peptide resistance.The qRT-PCR results of the eight DEGs were consistent with the results of transcriptome sequencing.Conclusion Four hundred and seven DEGs are identified in the biofilms of the clinical CRAB isolates compared with the planktonic ones.The biofilm formation of clinical CRAB isolates involved multiple genes and regulation of multiple signal pathways,which will be the target of drug.

关 键 词：细菌 耐碳青霉烯类鲍曼不动杆菌 生物被膜 游离菌 转录组 差异表达基因 

分 类 号：R384.2[医药卫生—医学寄生虫学]