临床分离替加环素不敏感鲍曼不动杆菌的基因组和转录组学研究
发布时间：2018-08-26 13:40
【摘要】：替加环素(tigecycline,Tgc)是FDA批准临床使用的第一个甘氨酰类抗生素,主要用于复杂腹腔感染、皮肤感染和社区获得性肺炎的治疗,和黏菌素一起被称为人类抵抗细菌感染的最后"防线"。但是,近年来,有关替加环素耐药菌株发现的报道越来越多,给临床治疗效果和感染控制带来了挑战。替加环素2007年获美国FDA批准,2012年底正式在国内应用于临床,本课题对本三甲医院2012年底使用替加环素以来,临床分离报告替加环素耐药的鲍曼不动杆菌进行研究。鲍曼不动杆菌具有很强的耐药基因获得能力,可以在干燥、辐射、消毒剂等极端条件下生存,是导致院内感染的重要条件致病菌。根据卫计委全国细菌耐药监测网Mohnarin数据显示,临床多重药耐药(MDR)鲍曼不动杆菌近5年分离率均高于50%,而且泛耐药(XDR)和全耐药(PDR)的鲍曼不动杆菌也逐年增多,并且已经开始对替加环素产生耐药性。本研究利用高通量测序技术和分子生物学手段,从分子流行病学、比较基因组学和比较转录组学角度,系统研究了临床分离的鲍曼不动杆菌替加环素耐药性发展的基因组学基础和转录调控机制,并探讨了在替加环素耐药鲍曼不动杆菌中毒力因子和耐药基因的分布和关系。通过本研究,系统阐明了鲍曼不动杆菌对替加环素耐药的形成机制,为耐药控制和院感防控提供了一定的理论基础。在第一部分研究中,对本医院替加环素使用一年半以来的临床实验室分离报告替加环素耐药的鲍曼不动杆菌,使用微量肉汤稀释法测定替加环素的体外最低抑菌浓度(MIC),确定了 56株替加环素不敏感株,进行样本类型、科室分布、疾病、和抗生素使用等分析,揭示替加环素不敏感鲍曼不动杆菌临床分布特征;分子型别(MLST)分析揭示了其分子流行特征。第二部分研究以第一部分研究为基础,选择替加环素不敏感鲍曼不动杆菌3株以及分离自相同病人的不同替加环素耐药程度菌株3株,共6株鲍曼不动杆菌,进行了高通量基因组测序和比较基因组学分析,构建遗传进化树,比较基因组序列、结构和功能基因的差异,研究发现了1株替加环素耐药菌可能是由同病人分离的另外2株替加环素耐药性较低的菌株之间同源重组形成的,并且约9kbp的基因组重组区域包含了 6个基因,其中4个是外排泵、转录调控因子或膜蛋白。对这3株菌在不同替加环素浓度(0μg/mL,0.5μg/mL和1μg/mL)条件下基因表达情况进行链特异性转录组测序和比较转录组学分析,发现了在梯度增加的替加环素浓度下基因转录水平的变化,并确定了所有显著差异表达的基因。这些基因的转录水平变化进一步通过半定量qRT-PCR进行了实验验证。基因组水平上包括6个基因的变异可能导致了相应基因功能变化,结合转录水平的表达调控分析讨论了其可能对替加环素耐药产生的影响及途径,差异转录组展示了基因调控可能对替加环素耐药的较大影响并且确定了起显著调控作用的基因。同时,本研究对所有替加环素耐药的鲍曼不动杆菌19株进行了比较基因组学研究,发现了19株鲍曼不动杆耐药菌在进化关系上比较接近,耐药基因和毒力因子均具有多态性,这些耐药菌之间以及与GeneBank已有耐药菌全基因组相比较,可以通过marker基因进行不同耐药基因或毒力因子型的区分。
[Abstract]:Tigecycline (Tgc) is the first glycyl antibiotic approved by the FDA for clinical use. It is mainly used in the treatment of complex abdominal infections, skin infections and community-acquired pneumonia. Together with mucin, tigecycline is known as the last line of defense against bacterial infections in humans. Tigacycline was approved by FDA in 2007 and formally used in clinical practice in China at the end of 2012. Since the end of 2012, Tigacycline-resistant Acinetobacter baumannii has been reported in clinical isolates from our tertiary hospital. Strong ability to acquire resistant genes that can survive in extreme conditions such as dryness, radiation, disinfectants and so on is an important conditional pathogen causing nosocomial infections. Acinetobacter baumannii resistant to tigacycline is also increasing year by year, and has begun to develop resistance to tigacycline. This study systematically studied the development of tigacycline resistance in clinical isolates of Acinetobacter baumannii from molecular epidemiology, comparative genomics and comparative transcriptome perspectives using high-throughput sequencing and molecular biology techniques. The genomics basis and transcriptional regulation mechanism of Tigacycline-resistant Acinetobacter baumannii were studied. The distribution and relationship of virulence factors and drug-resistant genes in Tigacycline-resistant Acinetobacter baumannii were discussed. Through this study, the mechanism of Tigacycline-resistant Acinetobacter baumannii was systematically elucidated. In part of the study, Acinetobacter baumannii, which was reported to be resistant to tigacycline, was isolated from the clinical laboratory of our hospital for one and a half years. Minimum inhibitory concentration (MIC) of tigacycline in vitro was determined by broth dilution method. Fifty-six tigacycline-insensitive strains were identified for sample type, Department distribution, disease, and resistance. Analysis of biotin use revealed the clinical distribution of tigacycline-insensitive Acinetobacter baumannii, and molecular typing (MLST) revealed its molecular epidemiological characteristics. Three strains and six strains of Acinetobacter baumannii were analyzed by high throughput genome sequencing and comparative genomics. A genetic evolutionary tree was constructed to compare the differences of genome sequence, structure and function genes. It was found that one strain of tigacycline-resistant bacteria might be the same as the other two strains with lower tigacycline resistance isolated from the same patient. The recombinant region consisted of six genes, four of which were efflux pumps, transcription regulators or membrane proteins. The gene expression profiles of the three strains under different tigacycline concentrations (0 ug/mL, 0.5 ug/mL and 1 ug/mL) were sequenced by chain-specific transcriptome analysis and comparative transcriptome analysis. Changes in gene transcription levels at gradient tigacycline concentrations were further validated by semi-quantitative qRT-PCR. Variations in six genes at the genome level may result in changes in gene function associated with transcriptional water Differential transcriptomes showed that gene regulation may have a greater effect on tigacycline resistance and identified genes that play a significant role in the regulation. In addition, 19 tigacycline-resistant Acinetobacter baumannii strains were compared. Genomic analysis revealed that 19 strains of Baumann fixed-rod resistant bacteria were similar in evolutionary relationship, and the drug-resistant genes and virulence factors were polymorphic. Compared with the genome of the existing drug-resistant bacteria in GeneBank, marker gene could be used to distinguish different drug-resistant genes or virulence factors.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R446.5
本文编号：2205029
[Abstract]:Tigecycline (Tgc) is the first glycyl antibiotic approved by the FDA for clinical use. It is mainly used in the treatment of complex abdominal infections, skin infections and community-acquired pneumonia. Together with mucin, tigecycline is known as the last line of defense against bacterial infections in humans. Tigacycline was approved by FDA in 2007 and formally used in clinical practice in China at the end of 2012. Since the end of 2012, Tigacycline-resistant Acinetobacter baumannii has been reported in clinical isolates from our tertiary hospital. Strong ability to acquire resistant genes that can survive in extreme conditions such as dryness, radiation, disinfectants and so on is an important conditional pathogen causing nosocomial infections. Acinetobacter baumannii resistant to tigacycline is also increasing year by year, and has begun to develop resistance to tigacycline. This study systematically studied the development of tigacycline resistance in clinical isolates of Acinetobacter baumannii from molecular epidemiology, comparative genomics and comparative transcriptome perspectives using high-throughput sequencing and molecular biology techniques. The genomics basis and transcriptional regulation mechanism of Tigacycline-resistant Acinetobacter baumannii were studied. The distribution and relationship of virulence factors and drug-resistant genes in Tigacycline-resistant Acinetobacter baumannii were discussed. Through this study, the mechanism of Tigacycline-resistant Acinetobacter baumannii was systematically elucidated. In part of the study, Acinetobacter baumannii, which was reported to be resistant to tigacycline, was isolated from the clinical laboratory of our hospital for one and a half years. Minimum inhibitory concentration (MIC) of tigacycline in vitro was determined by broth dilution method. Fifty-six tigacycline-insensitive strains were identified for sample type, Department distribution, disease, and resistance. Analysis of biotin use revealed the clinical distribution of tigacycline-insensitive Acinetobacter baumannii, and molecular typing (MLST) revealed its molecular epidemiological characteristics. Three strains and six strains of Acinetobacter baumannii were analyzed by high throughput genome sequencing and comparative genomics. A genetic evolutionary tree was constructed to compare the differences of genome sequence, structure and function genes. It was found that one strain of tigacycline-resistant bacteria might be the same as the other two strains with lower tigacycline resistance isolated from the same patient. The recombinant region consisted of six genes, four of which were efflux pumps, transcription regulators or membrane proteins. The gene expression profiles of the three strains under different tigacycline concentrations (0 ug/mL, 0.5 ug/mL and 1 ug/mL) were sequenced by chain-specific transcriptome analysis and comparative transcriptome analysis. Changes in gene transcription levels at gradient tigacycline concentrations were further validated by semi-quantitative qRT-PCR. Variations in six genes at the genome level may result in changes in gene function associated with transcriptional water Differential transcriptomes showed that gene regulation may have a greater effect on tigacycline resistance and identified genes that play a significant role in the regulation. In addition, 19 tigacycline-resistant Acinetobacter baumannii strains were compared. Genomic analysis revealed that 19 strains of Baumann fixed-rod resistant bacteria were similar in evolutionary relationship, and the drug-resistant genes and virulence factors were polymorphic. Compared with the genome of the existing drug-resistant bacteria in GeneBank, marker gene could be used to distinguish different drug-resistant genes or virulence factors.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R446.5
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