沙門(mén)菌體內(nèi)耐藥誘導(dǎo)模型的建立及體外誘導(dǎo)耐藥菌RNA-seq分析
發(fā)布時(shí)間：2018-07-16 15:18
【摘要】：沙門(mén)菌是一類(lèi)常見(jiàn)的人畜共患病原菌,絕大多數(shù)對(duì)人和動(dòng)物致病,可造成人和動(dòng)物的傷寒、副傷寒、敗血癥和食物中毒等疾病,是公共衛(wèi)生的重要威脅。隨著氟喹諾酮類(lèi)藥物(fluoroquinolones,FQs)的普遍使用,沙門(mén)菌對(duì)FQs的耐藥水平不斷升高,耐藥問(wèn)題日益嚴(yán)重。目前通常使用FQs對(duì)敏感菌進(jìn)行體外誘導(dǎo)來(lái)研究耐藥機(jī)制,但是體外誘導(dǎo)并不能確切反映沙門(mén)菌在體內(nèi)時(shí)的耐藥性產(chǎn)生機(jī)理,因?yàn)閯?dòng)物體內(nèi)是復(fù)雜的生物環(huán)境,機(jī)體的代謝、免疫機(jī)制等都可能會(huì)對(duì)細(xì)菌的耐藥機(jī)理產(chǎn)生影響。本研究以秀麗隱桿線蟲(chóng)N2野生型為宿主,將鼠傷寒沙門(mén)菌ATCC13311定植于線蟲(chóng)體內(nèi),然后使用梯度濃度遞增法提高培養(yǎng)液中的環(huán)丙沙星濃度,誘導(dǎo)在線蟲(chóng)體內(nèi)的沙門(mén)菌產(chǎn)生耐藥性,同時(shí)進(jìn)行體外誘導(dǎo)耐藥實(shí)驗(yàn)。分別擴(kuò)增體內(nèi)誘導(dǎo)和體外誘導(dǎo)耐藥菌的gyrA、gyrB、parC和parE基因的氟喹諾酮耐藥決定區(qū)(Quinolone resistance determining regions,QRDR)以及外排泵抑制子基因(acrR、marR、ramR和soxR),對(duì)PCR擴(kuò)增產(chǎn)物進(jìn)行測(cè)序和分析。然后分別構(gòu)建鼠傷寒沙門(mén)菌ATCC13311和體外誘導(dǎo)耐藥株的轉(zhuǎn)錄組測(cè)序文庫(kù)并進(jìn)行Illumina RNA-seq雙向測(cè)序,對(duì)ATCC13311和體外誘導(dǎo)耐藥菌的轉(zhuǎn)錄組測(cè)序結(jié)果進(jìn)行數(shù)據(jù)分析。最后采用熒光定量PCR技術(shù)對(duì)轉(zhuǎn)錄組測(cè)序結(jié)果進(jìn)行驗(yàn)證。結(jié)果表明,鼠傷寒沙門(mén)菌ATCC13311可以穩(wěn)定定植于線蟲(chóng)體內(nèi),在線蟲(chóng)消化道內(nèi)集中分布于咽部和腸道,經(jīng)過(guò)環(huán)丙沙星誘導(dǎo)后可產(chǎn)生穩(wěn)定耐藥菌株。經(jīng)過(guò)誘導(dǎo)后得到環(huán)丙沙星MIC為4μg/mL的耐藥菌TN4,體外誘導(dǎo)實(shí)驗(yàn)獲到了環(huán)丙沙星MIC為4μg/mL的耐藥菌TW4。TN4的gyrA基因發(fā)生了Asp87Asn突變。體外誘導(dǎo)耐藥菌TW4的gyrA基因發(fā)生了Ser83Phe和Asp87Val突變,ramR基因出現(xiàn)了20bp的缺失。對(duì)ATCC13311和體外誘導(dǎo)耐藥菌TW4進(jìn)行RNA-seq測(cè)序后獲得了4.46 G有效數(shù)據(jù),通過(guò)de novo拼接,獲得了311條unigene,平均長(zhǎng)度為15587 bp。拼接所得到的全序列長(zhǎng)度為4847532 bp,經(jīng)預(yù)測(cè)4998條基因中有4902條得到GO注釋。采用COG功能將注釋基因劃分為25類(lèi),共有3735個(gè)基因得到注釋。將TW4與ATCC13311相比較,有3434個(gè)基因表達(dá)量顯著上升,32個(gè)基因表達(dá)量顯著下降,7條代謝途徑表達(dá)差異顯著。對(duì)表達(dá)量改變明顯的基因進(jìn)行統(tǒng)計(jì),發(fā)現(xiàn)22個(gè)與耐藥直接相關(guān)或者與藥物轉(zhuǎn)運(yùn)系統(tǒng)相關(guān)基因表達(dá)量顯著上升。熒光定量PCR結(jié)果顯示acrB、acrD和soxS的基因表達(dá)量與RNA-seq結(jié)果趨勢(shì)一致,證實(shí)RNA-seq可靠。本研究建立了鼠傷寒沙門(mén)菌-秀麗隱桿線蟲(chóng)體內(nèi)耐藥性誘導(dǎo)模型,并比較了體內(nèi)誘導(dǎo)耐藥菌和體外誘導(dǎo)耐藥菌的基因突變差異。利用轉(zhuǎn)錄組測(cè)序技術(shù)(RNA-seq)對(duì)鼠傷寒沙門(mén)菌ATCC13311和其誘導(dǎo)耐藥株TW4進(jìn)行研究,揭示了鼠傷寒沙門(mén)菌耐藥性誘導(dǎo)前后差異表達(dá)的基因和顯著變化的代謝途徑,為深入研究細(xì)菌耐藥機(jī)制及與細(xì)菌代謝途徑之間的聯(lián)系提供重要依據(jù),為進(jìn)一步研究生物體內(nèi)的細(xì)菌耐藥機(jī)理奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.61
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