沙门菌体内耐药诱导模型的建立及体外诱导耐药菌RNA-seq分析
发布时间：2018-07-16 15:18
【摘要】：沙门菌是一类常见的人畜共患病原菌,绝大多数对人和动物致病,可造成人和动物的伤寒、副伤寒、败血症和食物中毒等疾病,是公共卫生的重要威胁。随着氟喹诺酮类药物(fluoroquinolones,FQs)的普遍使用,沙门菌对FQs的耐药水平不断升高,耐药问题日益严重。目前通常使用FQs对敏感菌进行体外诱导来研究耐药机制,但是体外诱导并不能确切反映沙门菌在体内时的耐药性产生机理,因为动物体内是复杂的生物环境,机体的代谢、免疫机制等都可能会对细菌的耐药机理产生影响。本研究以秀丽隐杆线虫N2野生型为宿主,将鼠伤寒沙门菌ATCC13311定植于线虫体内,然后使用梯度浓度递增法提高培养液中的环丙沙星浓度,诱导在线虫体内的沙门菌产生耐药性,同时进行体外诱导耐药实验。分别扩增体内诱导和体外诱导耐药菌的gyrA、gyrB、parC和parE基因的氟喹诺酮耐药决定区(Quinolone resistance determining regions,QRDR)以及外排泵抑制子基因(acrR、marR、ramR和soxR),对PCR扩增产物进行测序和分析。然后分别构建鼠伤寒沙门菌ATCC13311和体外诱导耐药株的转录组测序文库并进行Illumina RNA-seq双向测序,对ATCC13311和体外诱导耐药菌的转录组测序结果进行数据分析。最后采用荧光定量PCR技术对转录组测序结果进行验证。结果表明,鼠伤寒沙门菌ATCC13311可以稳定定植于线虫体内,在线虫消化道内集中分布于咽部和肠道,经过环丙沙星诱导后可产生稳定耐药菌株。经过诱导后得到环丙沙星MIC为4μg/mL的耐药菌TN4,体外诱导实验获到了环丙沙星MIC为4μg/mL的耐药菌TW4。TN4的gyrA基因发生了Asp87Asn突变。体外诱导耐药菌TW4的gyrA基因发生了Ser83Phe和Asp87Val突变,ramR基因出现了20bp的缺失。对ATCC13311和体外诱导耐药菌TW4进行RNA-seq测序后获得了4.46 G有效数据,通过de novo拼接,获得了311条unigene,平均长度为15587 bp。拼接所得到的全序列长度为4847532 bp,经预测4998条基因中有4902条得到GO注释。采用COG功能将注释基因划分为25类,共有3735个基因得到注释。将TW4与ATCC13311相比较,有3434个基因表达量显著上升,32个基因表达量显著下降,7条代谢途径表达差异显著。对表达量改变明显的基因进行统计,发现22个与耐药直接相关或者与药物转运系统相关基因表达量显著上升。荧光定量PCR结果显示acrB、acrD和soxS的基因表达量与RNA-seq结果趋势一致,证实RNA-seq可靠。本研究建立了鼠伤寒沙门菌-秀丽隐杆线虫体内耐药性诱导模型,并比较了体内诱导耐药菌和体外诱导耐药菌的基因突变差异。利用转录组测序技术(RNA-seq)对鼠伤寒沙门菌ATCC13311和其诱导耐药株TW4进行研究,揭示了鼠伤寒沙门菌耐药性诱导前后差异表达的基因和显著变化的代谢途径,为深入研究细菌耐药机制及与细菌代谢途径之间的联系提供重要依据,为进一步研究生物体内的细菌耐药机理奠定了基础。
[Abstract]:Salmonella is a kind of common zoonotic pathogens, most of which are pathogenic to human and animal, and can cause typhoid, paratyphoid, septicemia and food poisoning. It is an important threat to public health. With the widespread use of fluoroquinolones (FQs), the level of resistance of Salmonella to FQs is increasing, and the problem of drug resistance is becoming more and more serious. At present, FQs are usually used to induce sensitive bacteria in vitro to study the mechanism of drug resistance, but in vitro induction can not exactly reflect the mechanism of drug resistance of Salmonella in vivo, because the animal body is a complex biological environment, the body metabolism. The immune mechanism may affect the drug resistance mechanism of bacteria. In this study, the wild-type N _ 2 type of nematode was used as the host, and ATCC13311 of Salmonella typhimurium was planted in the nematode, then the concentration of ciprofloxacin in the culture medium was increased by gradient concentration increasing method, which induced the resistance of Salmonella in vivo. Drug resistance induction in vitro was also carried out. The quinolone resistance determining regions (QRDR) and the efflux pump suppressor genes (acrRr Marr Rimar R and soxR) of gyrAjrBnparC and pare genes were amplified, and the PCR amplification products were sequenced and analyzed. Then, the transcription group sequencing library of Salmonella typhimurium ATCC13311 and drug-resistance strain induced in vitro was constructed, and the bidirectional sequencing of Illumina RNA-seq was carried out. The results of transcription group sequencing of ATCC13311 and drug-resistant strains in vitro were analyzed. Finally, fluorescent quantitative PCR was used to verify the results of transcriptome sequencing. The results showed that Salmonella typhimurium ATCC13311 could be stably colonized in nematodes, and concentrated in pharynx and intestine in the digestive tract of online worms, and stable drug-resistant strains could be produced after induction of ciprofloxacin. After induction, 4 渭 g / mL ciprofloxacin resistant strain TN4 was obtained. In vitro, the gyrA gene of ciprofloxacin resistant strain TW4.TN4 was found to have Asp87Asn mutation. The gyrA gene of resistant strain TW4 was induced to have 20bp deletion in Ser83Phe and Asp87Val mutation in vitro. After RNA-seq sequencing of ATCC13311 and TW4, the effective data of 4.46G were obtained. By de novo splicing, 311 Unigenees were obtained, with an average length of 15587 BP. The length of the whole sequence was 4847532 BP, 4902 of the 4998 predicted genes were annotated by go. The annotated genes were classified into 25 classes by COG function, and a total of 3735 genes were annotated. Comparing TW4 with ATCC 13311, 3434 genes were significantly increased and 32 genes were significantly decreased. There were significant differences in the expression of 7 metabolic pathways between TW4 and ATCC 13311. The expression of 22 genes directly related to drug resistance or related to drug transport system was found to be significantly increased. The results of fluorescence quantitative PCR showed that the gene expression of acrBnacrD and soxS was consistent with the result of RNA-seq, which confirmed that RNA-seq was reliable. In this study, a drug resistance induction model of Salmonella typhimurium in vivo was established, and the difference of gene mutation between drug-resistant bacteria induced in vivo and drug-resistant bacteria in vitro was compared. Transcription sequencing technique (RNA-seq) was used to study Salmonella typhimurium ATCC13311 and its induced drug resistance strain TW4, which revealed the differentially expressed genes and the metabolic pathways of significant changes before and after induction of drug resistance of Salmonella typhimurium. It provides an important basis for the further study of the mechanism of bacterial drug resistance and the relationship between bacterial drug resistance and bacterial metabolic pathway, and lays a foundation for further study on the mechanism of bacterial drug resistance in organisms.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
[Abstract]:Salmonella is a kind of common zoonotic pathogens, most of which are pathogenic to human and animal, and can cause typhoid, paratyphoid, septicemia and food poisoning. It is an important threat to public health. With the widespread use of fluoroquinolones (FQs), the level of resistance of Salmonella to FQs is increasing, and the problem of drug resistance is becoming more and more serious. At present, FQs are usually used to induce sensitive bacteria in vitro to study the mechanism of drug resistance, but in vitro induction can not exactly reflect the mechanism of drug resistance of Salmonella in vivo, because the animal body is a complex biological environment, the body metabolism. The immune mechanism may affect the drug resistance mechanism of bacteria. In this study, the wild-type N _ 2 type of nematode was used as the host, and ATCC13311 of Salmonella typhimurium was planted in the nematode, then the concentration of ciprofloxacin in the culture medium was increased by gradient concentration increasing method, which induced the resistance of Salmonella in vivo. Drug resistance induction in vitro was also carried out. The quinolone resistance determining regions (QRDR) and the efflux pump suppressor genes (acrRr Marr Rimar R and soxR) of gyrAjrBnparC and pare genes were amplified, and the PCR amplification products were sequenced and analyzed. Then, the transcription group sequencing library of Salmonella typhimurium ATCC13311 and drug-resistance strain induced in vitro was constructed, and the bidirectional sequencing of Illumina RNA-seq was carried out. The results of transcription group sequencing of ATCC13311 and drug-resistant strains in vitro were analyzed. Finally, fluorescent quantitative PCR was used to verify the results of transcriptome sequencing. The results showed that Salmonella typhimurium ATCC13311 could be stably colonized in nematodes, and concentrated in pharynx and intestine in the digestive tract of online worms, and stable drug-resistant strains could be produced after induction of ciprofloxacin. After induction, 4 渭 g / mL ciprofloxacin resistant strain TN4 was obtained. In vitro, the gyrA gene of ciprofloxacin resistant strain TW4.TN4 was found to have Asp87Asn mutation. The gyrA gene of resistant strain TW4 was induced to have 20bp deletion in Ser83Phe and Asp87Val mutation in vitro. After RNA-seq sequencing of ATCC13311 and TW4, the effective data of 4.46G were obtained. By de novo splicing, 311 Unigenees were obtained, with an average length of 15587 BP. The length of the whole sequence was 4847532 BP, 4902 of the 4998 predicted genes were annotated by go. The annotated genes were classified into 25 classes by COG function, and a total of 3735 genes were annotated. Comparing TW4 with ATCC 13311, 3434 genes were significantly increased and 32 genes were significantly decreased. There were significant differences in the expression of 7 metabolic pathways between TW4 and ATCC 13311. The expression of 22 genes directly related to drug resistance or related to drug transport system was found to be significantly increased. The results of fluorescence quantitative PCR showed that the gene expression of acrBnacrD and soxS was consistent with the result of RNA-seq, which confirmed that RNA-seq was reliable. In this study, a drug resistance induction model of Salmonella typhimurium in vivo was established, and the difference of gene mutation between drug-resistant bacteria induced in vivo and drug-resistant bacteria in vitro was compared. Transcription sequencing technique (RNA-seq) was used to study Salmonella typhimurium ATCC13311 and its induced drug resistance strain TW4, which revealed the differentially expressed genes and the metabolic pathways of significant changes before and after induction of drug resistance of Salmonella typhimurium. It provides an important basis for the further study of the mechanism of bacterial drug resistance and the relationship between bacterial drug resistance and bacterial metabolic pathway, and lays a foundation for further study on the mechanism of bacterial drug resistance in organisms.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
【相似文献】
相关期刊论文 前10条
1 单玉兴,张海玉,张绍昆,刘一,徐莘香;兔骨髓间充质干细胞体外诱导软骨形成[J];吉林大学学报(医学版);2004年02期
2 刘敏;孔祥鑫;杨苹;陈玉斌;李校X;王会岩;;成纤维细胞生长因子-21对体外诱导的非酒精性脂肪肝细胞模型脂代谢的影响[J];吉林大学学报(医学版);2012年03期
3 杨自权,卫小春,郝一勇,李鹏翠,丁娟,焦强;体外诱导骨髓间质干细胞向软骨方向分化的研究[J];中华实验外科杂志;2004年04期
4 常卓;侯玲玲;周雅琼;彭洪尚;柯\~;;哺乳动物精子体外诱导的研究进展[J];中国生物工程杂志;2013年03期
5 贾延R，
本文编号：2126796
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2126796.html
