1例复发类鼻疽的病原学鉴定及生物学特性研究  

Etiological identification and biological characteristics of recurrent melioidosis:report of 1 case

作　　者：杨文波 张纳安 饶承龙 夏瑀培 章美娟 李骁 闫晶敏 陈海 李倩[1] 毛旭虎[1] YANG Wenbo;ZHANG Na’an;RAO Chenglong;XIA Yupei;ZHANG Meijuan;LI Xiao;YAN Jingmin;CHEN Hai;LI Qian;MAO Xuhu(Department of Clinical Microbiology and Immunology,Faculty of Pharmacy and Laboratory Medicine,Army Medical University(Third Military Medical University),Chongqing,400038;College of Life Science and Technology Huazhong Agricultural University,Wuhan,Hubei Province,430070;Department of Clinical Laboratory Sanya People’s Hospital,Sanya,Hainan Province,572000,China)

机构地区：[1]陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室,重庆400038 [2]华中农业大学生命科学技术学院,湖北430070 [3]三亚市人民医院检验科,海南572000

出　　处：《第三军医大学学报》2021年第22期2441-2448,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81971907)。

摘　　要：目的通过对1例复发类鼻疽进行系统病原学鉴定及生物学特性研究,分析和探讨类鼻疽菌长期滞留机体可能的适应宿主机制。方法对临床收集的来源于同一个患者时隔5年分离到的两株类鼻疽菌,进行微生物学鉴定、全基因组测序,并使用相关软件对测序数据进行多位点序列分型(multilocus sequence typing,MLST)、同源比对进化树、单核苷酸多态性(single nucleotide polymorphism,SNP)以及插入缺失(insertion-deletion,Indel)分析;进一步在感染肺上皮A549细胞模型中比较感染不同时间细胞内细菌载量变化;双向电泳-质谱技术分析两株细菌在蛋白水平的表达变化。结果分离到的两株类鼻疽菌,全基因组均约7.12 Mb,COG功能注释编码蛋白大致约5 000个;MLST分型均为ST58型;同源比对进化树分析显示它们位于同一分支且与其他分离自中国的类鼻疽菌亲缘关系较近;SNP/Indel分析显示复发株有114个SNP位点、55个Indel位点;与原发株相比,双向电泳-质谱结果表明复发株共有68个蛋白的表达水平发生了显著的变化;同时复发株感染A549后细胞内细菌载量明显增加(P<0.05)。结论以上复发菌株的基因组学、蛋白组学以及胞内感染增殖能力的系统研究提示类鼻疽菌可能在机体长期的选择压力下微进化,通过对细菌基因转录表达、分泌系统、耐药以及能量代谢等方面的调控更加适应其在宿主体内的生存而实现长期滞留。Objective To analyze and explore the possible adaptive mechanisms that Burkholderia pseudomallei(B.pseudomallei) could long-term reside in the host cells based on the systematic etiological identification and biological characteristics of a patient with recurrent melioidosis.Methods The 2 B.pseudomallei strains isolated from the same patient at an interval of 5 years were performed for microbiological identification and genome sequencing,and the sequencing data were further analyzed by multilocus sequence typing(MLST),phylogenetic tree analysis of homologous alignment,single nucleotide polymorphism(SNP) analysis and insertion-deletion(Indel) analysis, respectively.Furthermore, the intracellular survival of the 2 clinical isolates of B.pseudomallei were examined at different times by counting the viable bacteria in the A549 cells.Two-dimensional electrophoresis(2-DE) and mass spectrometry was employed to analyze the protein expression of 2 strains of bacteria.Results The 2 clinical isolates had a whole genome of about 7.12 Mb,and up about 5 000 Clusters of Orthologous Groups(COG) annotations,and was identified as sequence type(ST) 58 by MLST analysis.Phylogenetic analysis of homologous alignment showed that they were located in the same branch and were closely related to other B.pseudomallei isolates from China.SNP/Indel analysis indicated that there were 114 SNP sites and 55 Indel sites in the recurrent strain.The results of 2-DE displayed that the expression levels of 68 proteins in the recurrent strain were significantly changed compared with the primary strain.The intracellular bacterial load of recurrent strain was significantly increased compared to the primary strain in A549 cells(P < 0.05).Conclusion Based on a series of studies with genomics,proteomics and ability of intracellular survival of the 2 clinical isolates of B.pseudomallei,the bacterium may evolve slightly under the long-term selective pressure of the organism,and adapt to the host environment for its own survival through the regulation of bact

关 键 词：类鼻疽菌 复发 全基因组 差异蛋白 

分 类 号：R372[医药卫生—病原生物学] R378.99[医药卫生—基础医学]