猪源化脓隐秘杆菌的分离鉴定及氨基糖苷类药物耐药株转录谱分析

发布时间：2018-02-25 09:28

本文关键词： 猪化脓隐秘杆菌毒力基因耐药机制转录组　出处：《吉林农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：化脓隐秘杆菌(Trueperella pyogenes)是一种重要的条件致病菌,能够引起猪、牛、羊和其他动物的化脓性感染,由于缺少预防手段或误诊给养殖业带来巨大的经济损失。由于滥用抗生素导致该菌的耐药率逐渐升高,而在国内还没有关于化脓隐秘杆菌的耐药机制的研究。本文目的在于研究我国吉林省地区分离到的化脓隐秘杆菌的耐药机制及检测其主要毒力基因。通过PCR扩增16S rRNA基因的方法,我们共分离得到8株化脓隐秘杆菌。药敏结果显示多数分离株对氨基糖苷类药物耐药,因此我们从靶位突变、整合子和耐药基因这三个方面入手研究其耐药机制,并对所鉴定的的氨基糖苷类敏感株与耐药株进行转录谱分析,从mRNA水平对耐药机制进行解析。以期为新型防治技术以及新型药物与耐药抑制剂的研发提供一定的数据支持。1.化脓隐秘杆菌的分离鉴定及主要毒力基因检测为了了解吉林省地区猪源化脓隐秘杆菌的毒力基因携带情况,通过细菌形态观察,生化试验及16S rRNA基因序列测定方法对分离菌株进行鉴定,共获得8株猪源化脓隐秘杆菌。应用PCR方法对分离菌株的毒力基因进行检测。结果表明,不同地区分离菌株神经氨酸酶H(nanH)基因及神经氨酸酶P(nanP)基因的携带情况略有不同,分离菌株都携带溶血素(plo)基因,而全部分离菌株都缺失胶原结合蛋白(cbpA)基因。2.化脓隐秘杆菌对氨基糖苷类药物的耐药机制的初步研究通过平板稀释法测定8株化脓隐秘杆菌对5种氨基糖苷类药物的MIC值,经判定共6株耐药菌,2株敏感菌。为了研究化脓隐秘杆菌耐药机制,我们对靶位突变、整合子和耐药基因进行检测。耐药基因PCR检测结果均为阴性,化脓隐秘杆菌两个16S核糖体RNA的测序结果与参考株序列进行比对分析,结果显示耐药株与敏感株都不存在突变。但多数分离菌株携带Ⅰ类整合子,且全部携带基因盒,基因盒中包括aadA1,aadA2,aadA5,aacA4与aac(6')-Ib 5种诱导氨基糖苷类药物耐药的基因。3.氨基糖苷类药物敏感株与耐药株转录谱分析基于转录组测序技术(RNA Sequencin,RNA-seq),对氨基糖苷类药物耐药株进行转录谱分析,以深入了解化脓隐秘杆菌对氨基糖苷类药物耐药的机制。结果显示,化脓隐秘杆菌氨基糖苷类耐药菌与敏感菌相比,存在245个差异表达基因。对差异表达基因进行了功能注释和相关代谢通路的绘制,其中细菌趋化性代谢通路中rbsB基因的显著上调,该基因参与调控细菌生物膜的形成,这可能是导致化脓隐秘杆菌对氨基糖苷类抗生素耐药的重要因素。
[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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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