【作者】 王建峰；

【导师】 俞云松；

【作者基本信息】 浙江大学 ， 内科学（传染病学）， 2016， 博士

【摘要】 不动杆菌属细菌是造成院内获得性感染的重要病原菌之一。近年来,不动杆菌属细菌,尤其是以耐碳青霉烯类抗生素为代表的多重耐药鲍曼不动杆菌分离率显著上升,给临床诊治带来极大挑战。多重耐药鲍曼不动杆菌的感染控制通常需要全方面的应对措施,包括风险因素识别、微生物的准确鉴定、耐药菌株的快速检测和识别,并实施感染控制和预防策略。由于不动杆菌属各菌种类型较多,且属内部分菌种间差异小,其菌种鉴定及分类长期以来一直不理想,而基于表型鉴定、16SrRNA基因测序等传统方法难以将其准确鉴定到种,在实际临床应用中一直缺乏快速、准确的鉴定方法。针对临床提出的现实问题,本研究选取2009年1月至2010年1月期间全国27个省市收集的2582株非重复不动杆菌属菌株,首先通过VITEC 2生化鉴定法和blaOxA-51-like基因进行菌种初步鉴定。其中385株blaOXA-51-like基因阴性菌和24株blaOxA-51-like基因阳性菌应用MALDI-TOF MS质谱进行二次鉴定,再通过16S rRNA基因与rpoB基因测序鉴定。由于公共数据库中暂缺相对完善与可靠的rpoB基因参考序列,本研究下载了全基因组序列截取rpoB基因作为参考。研究结果表明,目前临床广泛应用的生化鉴定、质谱都不足以准确鉴定不动杆菌属细菌,16SrRNA基因测序法也只能将不动杆菌属细菌准确鉴定到属。本研究设计的基于rpoB基因序列的鉴定方法克服了现有文献rpoB基因扩增片段过短、引物位置设计不一、相互之间无法比较、无鉴定标准等诸多缺陷,并根据现有数据提出rpoB基因序列相似度阈值为97%可作为界定不动杆菌属各菌种的分类新标准。另外,本研究通过对rpoB基因序列系统发生树分析,发现一簇由11株临床菌株构成的独立分支,极可能代表了一个不动杆菌属新菌种,我们依照惯例将其按不动杆菌属基因型命名为A.genomic species 33YU。细菌Ⅵ型分泌系统(T6SS)负责捕捉原核生物和真核生物细胞,将有毒效应分子定位到靶细胞,从而杀死靶细胞。T6SS细胞器在功能上类似于收缩噬菌体的尾部,由VgrG长钉聚体蛋白复合物穿透、攻击靶细胞。本研究通过敲除鲍曼不动杆菌ATCC17978、ATCC19606的vgrG基因明确T6SS元件在鲍曼不动杆菌生存能力、侵袭宿主细胞能力及调控相关耐药基因表达等方面的重要作用。研究结果表明。当敲除vgrG基因后,鲍曼不动杆菌的生长速度减慢,对细胞粘附能力降低,对小鼠的致死能力降低,但是生物被膜形成能力并无明显改变,提示vgrG基因在鲍曼不动杆菌的毒力形成中扮演重要角色,其毒力效应的改变并非通过生物被膜相关途径发挥效应。另外,vgrG基因的敲除对其耐药机制也存在影响,vgrG基因敲除株对哌拉西林、氨苄西林/舒巴坦、头孢哌酮/舒巴坦等药物产生耐药,对氯霉素和链霉素药物敏感性增加。实验结果提示了鲍曼不动杆菌对β内酰胺类药物和舒巴坦复合制剂的一种可能耐药机制,并解释了抗生素环境下细菌毒力降低的原因,即抗生素环境下细菌可能通过vgrG基因表达下调或沉默产生耐药,但是毒力也随之降低。本研究也进一步提示在将来开发以细菌毒力效应为标靶的药物时,需考虑其对抗菌药物耐药所带来的负面影响。综上所述,rpoB基因测序法的分辨率高于生化鉴定、质谱鉴定与16SrRNA基因测序法,是一种快速、准确的不动杆菌属菌种鉴定新方法。T6SS元件中的vgrG基因在鲍曼不动杆菌的毒力与耐药机制调控中扮演重要的角色,是未来新药物开发的潜在标靶。更多还原

【Abstract】 Acinetobacter spp.are important Gram-negative bacteria of hospital-acquired infections.In recent years,isolation rate of multi-drug resistant Acinetobacter baumannii(A.baumannii),especially Ab with resistance to carbapenems,increased significantly,which posed a great challenge to clinics.It needs all counter-measure for treatment and preventing A.baumannii infecton,including the risk factors identification,accurate identification of microorganisms,rapid detection drug-resistant strains,and implementation of infection control and prevention strategies.There are so many species in Acinetobacter spp.with only minor differences among different genus,which caused the identification and classification of them were unsatisfactory for a long time.It was difficult to accurately identify their species based on phenotypic identification,MALDI-TOF-MS and 16S rRNA gene sequencing.There was a lack of available rapid and accurate identification methods in clinics.In orde to solve the clinical identification problem,we collected 2582 non-repetitive strains of Acinetobacter spp.from 27 provinces in China between January 2009 and January 2010.All of these isolates were identified by amplifying blaOXA-51-like gene and VITEK 2 system.Then 385 blaOXA-51-like negative isolates and 24 blaOXA-51-like positive isolates were seclected for MALDI-TOF MS identification,16S rRNA and rpoB gene sequencing.Because there was a lack of adequate and reliable rpoB gene reference sequence in public databases,we downloaded all the Acinetobacter spp.genome and intercepted rpoB gene as reference.According to the results of this study,we showed that biochemical identification and MALDI-TOF MS can not identify Acinetobacter spp.accurately.By 16S rRNA sequencing,Acinetobacter spp.can only be identified accurately at the genus level.We redesigned the rpoB gene primers and resolved these problem shown in the previous literature,such as rpoB gene amplified fragment was too short,primers were designed in different location and can not be compared with each other and no availabe criteria for the classification.Our study results proved that Acinetobacter spp.can be identified accurately with our rpoB gene primers.Based on existing data,we suggested that the 97%similarity threshold of rpoB gene can be defined as an ideal species classification criteria.In addition,we found an independent branch composed of 11 clinical isolates by rpoB gene sequence phylogenetic tree analysis,which probably represented a new species.In the light of convention,they were named A.genomic species 33YU according to Acinetobacter genotype.The type VI secretion system(T6SS)is a large dynamic multicomponent macromolecular organelle.The T6SS organelle function was similar to the phage tail.It was a complex of VgrG spike proteins,which is referred to the polymer and began to penetrate,attack target cells.In this study,we showed T6SS element play an important role in the viability,biofilm formation,adhesion,invasion,and drug resistance by knockout vgrG gene of Acinetobacter baumannii ATCC 17978 and ATCC 19606.When the vgrG gene were knocked out,the Ab showed a lower growth rates,reduced cell adhesion ability and the impared lethality in mice.But there was no significant change in their biofilm formation ability,which suggested vgrG gene play an important role in Ab bacterial virulence,but the virulence regulation is not through the biofilm-related pathway.In addition,bacterial drug resistance mechanisms were affected.When vgrG gene was knocked out,the bacteria experienced an increase resistance to piperacillin,ampicillin/sulbactam,cefoperazone/sulbactam increased but decreased chloramphenicol and streptomycin.The experimental results showed a new resistance mechanism toβ-lactam drugs and sulbactam compound,it also partialy explained why the bacterial virulence was redued in antibiotic environment.When bacteria was in antibiotics environment,the vgrG gene expression was down-regulated or silenced for purpose of resistance,the virulence was also reduced.It needs to consider the adverse impact on bacterial resistance when drug targeted the bacterial virulence gene.In brief,rpoB gene identification can identify Acinetobacter spp.rapidly and accurately and it is superior to biochemical identification,MALDI-TOF-MS and 16S rRNA.This study also demonstrated the vgrG genes of T6SS played a very important role in drug resistance and virulence of Ab.It is a promising target for drug development in the future.更多还原