本文關(guān)鍵詞： 豬 化膿隱秘桿菌 毒力基因 耐藥機制 轉(zhuǎn)錄組　出處：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：化膿隱秘桿菌(Trueperella pyogenes)是一種重要的條件致病菌,能夠引起豬、牛、羊和其他動物的化膿性感染,由于缺少預(yù)防手段或誤診給養(yǎng)殖業(yè)帶來巨大的經(jīng)濟損失。由于濫用抗生素導(dǎo)致該菌的耐藥率逐漸升高,而在國內(nèi)還沒有關(guān)于化膿隱秘桿菌的耐藥機制的研究。本文目的在于研究我國吉林省地區(qū)分離到的化膿隱秘桿菌的耐藥機制及檢測其主要毒力基因。通過PCR擴增16S rRNA基因的方法,我們共分離得到8株化膿隱秘桿菌。藥敏結(jié)果顯示多數(shù)分離株對氨基糖苷類藥物耐藥,因此我們從靶位突變、整合子和耐藥基因這三個方面入手研究其耐藥機制,并對所鑒定的的氨基糖苷類敏感株與耐藥株進行轉(zhuǎn)錄譜分析,從mRNA水平對耐藥機制進行解析。以期為新型防治技術(shù)以及新型藥物與耐藥抑制劑的研發(fā)提供一定的數(shù)據(jù)支持。1.化膿隱秘桿菌的分離鑒定及主要毒力基因檢測為了了解吉林省地區(qū)豬源化膿隱秘桿菌的毒力基因攜帶情況,通過細菌形態(tài)觀察,生化試驗及16S rRNA基因序列測定方法對分離菌株進行鑒定,共獲得8株豬源化膿隱秘桿菌。應(yīng)用PCR方法對分離菌株的毒力基因進行檢測。結(jié)果表明,不同地區(qū)分離菌株神經(jīng)氨酸酶H(nanH)基因及神經(jīng)氨酸酶P(nanP)基因的攜帶情況略有不同,分離菌株都攜帶溶血素(plo)基因,而全部分離菌株都缺失膠原結(jié)合蛋白(cbpA)基因。2.化膿隱秘桿菌對氨基糖苷類藥物的耐藥機制的初步研究通過平板稀釋法測定8株化膿隱秘桿菌對5種氨基糖苷類藥物的MIC值,經(jīng)判定共6株耐藥菌,2株敏感菌。為了研究化膿隱秘桿菌耐藥機制,我們對靶位突變、整合子和耐藥基因進行檢測。耐藥基因PCR檢測結(jié)果均為陰性,化膿隱秘桿菌兩個16S核糖體RNA的測序結(jié)果與參考株序列進行比對分析,結(jié)果顯示耐藥株與敏感株都不存在突變。但多數(shù)分離菌株攜帶Ⅰ類整合子,且全部攜帶基因盒,基因盒中包括aadA1,aadA2,aadA5,aacA4與aac(6')-Ib 5種誘導(dǎo)氨基糖苷類藥物耐藥的基因。3.氨基糖苷類藥物敏感株與耐藥株轉(zhuǎn)錄譜分析基于轉(zhuǎn)錄組測序技術(shù)(RNA Sequencin,RNA-seq),對氨基糖苷類藥物耐藥株進行轉(zhuǎn)錄譜分析,以深入了解化膿隱秘桿菌對氨基糖苷類藥物耐藥的機制。結(jié)果顯示,化膿隱秘桿菌氨基糖苷類耐藥菌與敏感菌相比,存在245個差異表達基因。對差異表達基因進行了功能注釋和相關(guān)代謝通路的繪制,其中細菌趨化性代謝通路中rbsB基因的顯著上調(diào),該基因參與調(diào)控細菌生物膜的形成,這可能是導(dǎo)致化膿隱秘桿菌對氨基糖苷類抗生素耐藥的重要因素。
[Abstract]:Trueperella pyogenes is an important conditional pathogen that causes suppurative infections in pigs, cattle, sheep and other animals. The lack of preventive measures or misdiagnosis caused enormous economic losses to the aquaculture industry. The drug resistance rate of the bacterium gradually increased as a result of the abuse of antibiotics. The aim of this paper is to study the drug resistance mechanism and to detect the main virulence genes of Stereobacterium pyogenes isolated in Jilin province of China. The main virulence genes were amplified by PCR. The method of 16s rRNA gene, We isolated 8 strains of covert bacillus pyogenes. The results of drug sensitivity showed that most isolates were resistant to aminoglycosides, so we studied the drug resistance mechanism from three aspects: target mutation, integron and drug resistance gene. Transcriptional analysis of aminoglycoside sensitive and resistant strains was carried out. The mechanism of drug resistance was analyzed from the level of mRNA. The aim is to provide certain data support for the development of new prevention and treatment technology and new drugs and drug resistance inhibitors. 1. Isolation, identification and detection of main virulence genes of covert bacillus pyogenes. To understand the virulence gene carrying of porcine cryptomegalobacteria from swine in Jilin province, The isolated strains were identified by morphological observation, biochemical test and 16s rRNA gene sequencing, and 8 strains of Stereobacterium purpura were obtained. The virulence genes of the isolated strains were detected by PCR method. The carriers of neuraminidase HnH gene and neuraminidase PnanP gene were slightly different in different regions. The isolated strains all carried hemolysin plow gene. The resistance mechanism of Stereobacterium purpurans to aminoglycoside drugs was studied. The MIC values of 8 strains of C. pyogenosa to 5 aminoglycoside drugs were determined by plate dilution method. In order to study the mechanism of drug resistance, we detected target mutation, integron and drug resistance gene. The results of PCR analysis of drug resistance genes were negative. The sequencing results of two 16s ribosomal RNA were compared with those of reference strains. The results showed that there were no mutations in both resistant and sensitive strains, but most isolates carried class I integrons and all of them carried gene boxes. The gene cassette contains five genes, namely aadA1, aadA2, aadA5, aacA4 and aac(6')-Ib, which induce aminoglycoside resistance. The transcriptional analysis of aminoglycoside sensitive and resistant strains was carried out on the basis of transcriptome sequencing technique, RNA sequencin RNA-seqn, and the transcriptional spectrum of aminoglycoside resistant strains was analyzed. In order to understand the mechanism of drug resistance to aminoglycosides of covert bacillus pyogenes, the results showed that the aminoglycoside resistant bacteria of C. purpurus were more sensitive than those of sensitive bacteria. There were 245 differentially expressed genes. Functional annotation and related metabolic pathways were carried out for the differentially expressed genes, in which the rbsB gene was significantly up-regulated in the bacterial chemotactic metabolic pathway, which was involved in the regulation of bacterial biofilm formation. This may be an important factor leading to resistance to aminoglycoside antibiotics.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.61

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