刘巍巍;国果;吴兆颖;毛成菊;李忠旬;贾利娜;尚小丽;彭建;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 Medical Sciences,Guizhou Medical University;Key Laboratory of Modern Pathogen Biology,School of Basic Medical Sciences,Guizhou Medical University;School of Biology & Engineering,Guizhou Medical University;

• 1:贵州医科大学基础医学院人体寄生虫学教研室
• 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
• 3:贵州医科大学生物工程学院

摘要(Abstract)：

目的了解不同生物被膜形成能力的临床分离耐碳青霉烯类鲍曼不动杆菌菌株(CRAB)基因表达的差异。方法选取8株临床分离CRAB,将生物被膜形成能力强的菌株4、55、78、117号作为强生物被膜(BF)组,将生物被膜形成能力弱的菌株13、177、191、196号作为弱生物被膜(WBF)组,培养两组菌株形成生物被膜,分别提取RNA进行文库构建和转录组测序(RNA-Seq),以鲍曼不动杆菌AB030(NZ_CP009257.1)的基因组作为参考基因组对测序数据进行分析,鉴定差异表达基因(DEGs),并进行基因本体(GO)功能注释和京都基因与基因组百科全书(KEGG)通路富集分析,选取8个DEGs利用实时荧光定量PCR(qRT-PCR)验证转录组测序结果。结果 RNA样品完整无污染,测序数据错误率为0.03%;BF组与WBF组相比,鉴定到171个DEGs,其中106条基因为上调表达,65条基因为下调表达;这些基因主要涉及DNA结合、膜蛋白、菌毛调控、ATP的合成和分解等;GO分析显示较多基因被注释到生物过程部分,KEGG分析显示DEGs富集到多种物质代谢途径;选取的8个DEGs利用qRT-PCR进行验证,结果与转录组测序结果趋势一致。结论不同生物被膜形成能力的临床CRAB的基因表达存在差异,多基因和多条信号通路影响生物被膜的形成。
Objective To understand the transcriptome variation in gene expression of clinically carbapenem-resistant Acinetobacter baumannii with different biofilm formation ability.Methods Eight clinically isolated carbapenem-resistant Acinetobacter baumannii were selected,and the strains 4,55,78,and 117 with strong biofilm formation ability were grouped into a group and named as strong biofilm(BF) Group,the strains 13,177,191,and 196 with weak biofilm formation ability were divided into another group and named the Weak Biofilm(WBF) group.Two groups of strains were cultivated to form biofilm,and ribonucleic acid(RNA) samples were extracted,library construction and transcriptome sequencing were performed.Use the genome of Acinetobacter baumannii AB030(NZ_CP009257.1) as the reference genome to analyze the sequencing data,identify differentially expressed genes(DEGs),and perform Gene Ontology(GO) functional annotations and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis.To validate the results of RNA-Seq,eight DEGs were selected for Quantitative Real-time PCR(qRT-PCR).Results The RNA sample is intact and pollution-free,and the error rate of sequencing data does not exceed 0.03 %.After transcriptome sequencing,compared with the WBF group,171 DEGs were identified in the BF group,of which 106 genes were up-regulated and 65 genes were down-regulated.These genes mainly involved in DNA binding,membrane protein,fimbriae regulation,ATP synthesis and decomposition,etc.GO analysis showed that more genes were annotated to the biological process part.KEGG analysis showed that DEGs were enriched in a variety of material metabolism pathways,such as pyruvate metabolism,methane metabolism,etc.,as well as signal pathways such as biosynthesis of antibiotics and vancomycin resistance.Eight DEGs were selected and analysised by qRT-PCR,and the results were consistent with the results of RNA-Seq.Conclusion There are differences in the gene expression of clinical CRABs with different biofilm formation ability,and multiple genes and multiple signal pathways affect the formation of biofilms.

关键词(KeyWords)： 细菌;耐碳青霉烯类鲍曼不动杆菌;生物被膜形成能力;菌毛;外膜蛋白;信号通路;差异表达基因
bacteria;carbapenem-resistant Acinetobacter baumannii(CRAB);biofilm formation;fimbria;outer membrane protein;pathway;differentially expressed genes(DEGs)

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基因（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]

作者(Authors): 刘巍巍;国果;吴兆颖;毛成菊;李忠旬;贾利娜;尚小丽;彭建;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 Medical Sciences,Guizhou Medical University;Key Laboratory of Modern Pathogen Biology,School of Basic Medical Sciences,Guizhou Medical University;School of Biology & Engineering,Guizhou Medical University;

DOI: 10.19367/j.cnki.2096-8388.2021.11.001

参考文献(References)：

• [1] EZE E C, CHENIA H Y, ELZOWALATY M E.Acinetobacter baumanniibiofilms:effects of physicochemical factors, virulence, antibiotic resistance determinants, gene regulation, and future antimicrobial treatments[J]. Infection and Drug Resistance, 2018, 11:2277-2299.
• [2] DONLAN R M, COSTERTON J W. Biofilms:survival mechanisms of clinically relevant microorganisms[J].Clinical Microbiology Reviews, 2002, 15(2):167-193.
• [3] SONG F, KOO H, REN D. Effects of material properties on bacterial adhesion and biofilm formation[J]. Journal of Dental Research, 2015, 94(8):1027-1034.
• [4] DAROUICHE RO. Treatment of infections associated with surgical implants[J]. The New England Journal of Medicine, 2004, 350(14):1422-1429.
• [5] RAJENDRAN R, MAY A, SHERRY L, et al. Integrating Candida albicansmetabolism with biofilm heterogeneity by transcriptome mapping[J]. Scientific Report, 2016,6:35436.
• [6] RAJENDRAN R, SHERRY L, NILE C J, et al. Biofilm formation is a risk factor for mortality in patients with Candida albicansbloodstream infection-Scotland, 2012-2013[J]. Clinical Microbiology and Infection, 2016,22(1):87-93.
• [7] BARDBARI A M, ARABESTANI M R, KARAMI M, et al. Correlation between ability of biofilm formation with their responsible genes and MDR patterns in clinical and environmentalAcinetobacter baumanniiisolates[J]. Microbial Pathogenesis, 2017, 108:122-128.
• [8] ZEIGHAMI H, VALADKHANI F, SHAPOURI R, et al.Virulence characteristics of multidrug resistant biofilm formingAcinetobacter baumanniiisolated from intensive care unit patients[J]. BMC Infectious Diseases, 2019,19(1):629.
• [9] RAMIREZ M S, BONOMO R A, TOLMASKY M E. Carbapenemases:transforming into a yet more dangerous menace[J]. Biomolecules, 2020, 10(5):720.
• [10]戚丽华．鲍曼不动杆菌耐药特性及其与生物膜的相关性研究[D]．北京：中国人民解放军军事医学科学院，2016.
• [11]PARKHOMCHUK D, BORODINA T, AMSTISLAVSKIY V, et al. Transcriptome analysis by strand-specific sequencing of complementary DNA[J]. Nucleic Acids Research, 2009, 37(18):e123-e123.
• [12] LANGMEAD B, SALZBERG S L. Fast gapped-read alignment with Bowtie 2[J]. Nature Methods, 2012, 9(4):357-359.
• [13]ANDERS S, HUBER W. Differential expression analysis for sequence count date[J]. Genome biology,2010,11(10):106.
• [14]IACONO M, VILLA L, FORTINI D, et al. Whole-genome pyrosequencing of an epidemic multidrug-resistant Acinetobacter baumanniistrain belonging to the European clone II group[J]. Antimicrobial Agents and Chemotherapy, 2008, 52(7):2616-2625.
• [15]田德英，倪明，余冰，等．新型mucA基因突变的铜绿假单胞菌生物被膜的形成和藻酸盐相关基因表达的研究[J]．中华微生物学和免疫学杂志，2006(1):14-17.
• [16]尹珍，黄文祥，杨均均，等．耐碳青霉烯类鲍曼不动杆菌流行克隆株生物被膜形成能力分析[J]．中国感染与化疗杂志，2018,18(2):184-189.
• [17]DAFOPOULOU K, VOURLI S, TSAKRIS A, et al. An update on polymyxin susceptibility testing methods for Acinetobacter baumannii[J]. Expert Review of Anti-Infective Therapy, 2019, 17(9):699-713.
• [18]李艳．抗菌多肽对细菌生物被膜作用的研究进展[J]．临床医药文献电子杂志，2018, 5(53):191-192,194.
• [19] AZIZI O, SHAKIBAIE M R, MODARRESI F, et al.Molecular Detection of Class-D OXA Carbapenemase Genes in Biofilm and Non-Biofilm Forming Clinical Isolates ofAcinetobacter baumannii[J]. Jundishapur Journal of Microbiology, 2015, 8(1):e21042.
• [20]裘鹏，郭玉茹，张泽辉，等．基于转录组学技术分析细菌耐药机制的研究进展[J]．中国家禽，2019,41(4):45-49.
• [21]VEERACHAMY S, YARLAGADDA T, MANIVASAGAM G, et al. Bacterial adherence and biofilm formation on medical implants:a review[J]. Proc Inst Mech Eng H,2014, 228(10):1083-1099.
• [22]MCCONNELL M J, ACTIS L, PACHóN J.Acinetobacter baumannii:human infections, factors contributing to pathogenesis and animal models[J]. FEMS Microbiology Reviews, 2013, 37(2):130-155.
• [23]GADDY J A, TOMARAS A P, ACTIS L A. TheAcinetobacter baumannii19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells[J]. Infection and Immunity, 2009, 77(8):3150-3160.
• [24]LEE C-R, LEE J H, PARK M, et al. Biology ofAcinetobacter baumanniipathogenesis, antibiotic resistance mechanisms, and prospective treatment options[J]. Frontiers in Cellular and Infection Microbiology, 2017, 7:55.
• [25] CHOPRA S, RAMKISSOON K, ANDERSON D C. A systematic quantitative proteomic examination of multidrug resistance inAcinetobacter baumannii[J]. Journal of Proteomics, 2013, 84:17-39.

文章评论(Comment)：