采用MALDI-TOF MS和全基因组测序鉴定和分析动弯杆菌属及其亲缘关系  

Classification and phylogenetic analysis of the genus Mobiluncus based on matrix-assisted laser desorption/ionization time.of-flight mass spectrometry （MALDI-TOF MS） and whole genome sequencing

作　　者：张雪英 叶红[1] 李爱云[1] 叶光勇[1] 冯南[1] 马裕[1] 朱宇宁[1] 沈应博 张嵘[3] Zhang Xueying;Ye Hong;Li Aiyun;Ye Guangyong;Feng Nan;Ma Yu;Zhu Yuning;Shen Yingbo;Zhang Rong(Department of Clinical Laboratory, Women＇s Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China;College of Veterinary Medicine, China Agricultural University, Beijing 100193, Chin;Department of Clinical Laboratory, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China)

机构地区：[1]浙江大学医学院附属妇产科医院检验科,杭州310016 [2]中国农业大学动物医学院,北京100193 [3]浙江大学医学院附属第二医院检验科,杭州310009

出　　处：《中华微生物学和免疫学杂志》2018年第6期446-450,共5页Chinese Journal of Microbiology and Immunology

基　　金：浙江省自然科学基金资助项目（LY18H040001）

摘　　要：目的用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）技术结合16S rRNA基因测序鉴定动弯杆菌，并通过全基因组测序分析动弯杆菌的耐药基因及该菌属种间亲缘关系。方法将65例细菌性阴道病（bacterial vaginosis，BV）患者的阴道分泌物标本厌氧培养获得25株动弯杆菌，采用MALDI-TOF MS及16S rRNA基因测序鉴定细菌种属，并提取细菌全基因组DNA进行二代测序，测序后得到的原始数据根据SPAdes软件进行拼接组装，并与ARGD耐药基因库比对得到这些细菌含有的获得性耐药基因，最后通过Harvest软件对所有菌株进行核心基因组多样性比较，构建进化树。结果采用MALDI-TOF MS鉴定动弯杆菌属仅能获得柯氏动弯杆菌的鉴定结果；采用16S rRNA基因测序能将动弯杆菌属菌株区分为柯氏动弯杆菌和羞怯动弯杆菌；全基因组分析显示动弯杆菌主要含有四环素耐药基因tet（o）及大环内酯类耐药基因erm（x），其中tet（o）基因的检出率为84.7%，erm（x）基因的检出率为61.5%；动弯杆菌属两个种内亲缘关系相近，种间亲缘关系甚远。结论本实验结果表明目前MALDI-TOF MS不能鉴定出动弯杆菌属中的羞怯动弯杆菌；动弯杆菌对四环素类及大环内酯类抗生素具有潜在的耐药性；动弯杆菌在种内进化缓慢，暂无新种出现的趋势。ObjectiveTo identify Mobiluncus species with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF MS） and 16S rRNA gene sequencing and to analyze antibiotic resistance genes and phylogenetic relationships among Mobiluncus species with whole genome sequencing.MethodsTwenty-five Mobiluncus strains were isolated after anaerobic culture of 65 vaginal secretion samples of patients with bacterial vaginosis （BV） and identified to species level by MALDI-TOF MS and 16S rRNA gene sequencing. The whole genome DNA of each strain was extracted for next-generation sequencing. SPAdes was used to assembly genomes. Resistance genes were searched in ARGD database. The core genome of all isolates was analyzed by Harvest to construct phylogenetic tree.ResultsMALDI-TOF MS could only identify Mobiluncus curtisii, while 16S rRNA gene sequencing could identify both Mobiluncus mulieris and Mobiluncus curtisii. Results of the whole genome sequencing showed that tetracycline resistance gene tet（o） and macrolides resistance gene erm（x） were the two predominant acquired resistance genes of Mobiluncus with a positive rate of 84.7% and 61.5% respectively. Intra-species relationships of the two Mobiluncus species were close, but a distant phylogenetic relationship was found between the two species.ConclusionThis study shows that MALDI-TOF MS can′t be used to identify Mobiluncus mulieris at present. Mobiluncus strains have potential resistance to tetracycline and macrolides. Intra-species evolution of Mobiluncus is slow, which indicates that there is no growing trend towards new species for the time being.

关 键 词：动弯杆菌 细菌性阴道病(BV) MALDI—TOF MS 全基因组测序 耐药基因 

分 类 号：R446.5[医药卫生—诊断学]