辽宁地区奶牛乳房炎源大肠杆菌毒力与耐药相关性研究

发布时间：2019-06-26 20:38

【摘要】：大肠杆菌可引发多种动物疾病,对畜牧业及人类健康构成极大威胁。大肠杆菌耐药现象已十分普遍,给大肠杆菌感染性疾病的有效防治带来较大困难。细菌耐药可以使其致病性发生变化,有关大肠杆菌耐药对自身毒力影响的研究已引起学者们的广泛关注。本研究系统分析了分离自辽宁地区患临床型乳房炎奶牛的大肠杆菌耐药性对其毒力的影响,以期为临床科学防治奶牛乳房炎提供指导。本研究应用PCR技术,对79株分离自辽宁地区的患临床型乳房炎奶牛的大肠杆菌进行毒力基因的检测,分析了辽宁地区奶牛乳房炎源大肠杆菌种系发育分型情况及毒力基因流行特点。结果显示,79株大肠杆菌中,种系发育A型占59.5%,B1型占27.9%,B2型和D型各占6.3%。对分离自患临床型乳房炎奶牛的大肠杆菌进行了 22种毒力基因的筛查,共检出10种毒力基因,分别为fimH(89.9%)、ECs3703(88.6%)、ompC(100%)、ompF(73.4%)、colV(5.1%)、irp2(2.5%)、fyuA(2.5%)、eaeA(1.3%)、ler(1.3%)和iucD(1.3%)。大肠杆菌分离株中A型大肠杆菌数量最多,所含毒力基因种类亦最多。采用实时荧光定量PCR技术测定4种不同耐药表型的大肠杆菌毒力基因ompF和ECs3703mRNA表达量。结果表明,ompF基因mRNA表达量随大肠杆菌耐抗菌药物种类的增多而下降,耐2类抗菌药组与敏感组相比,ompF基因mRNA表达量的差异显著,P0.05;耐4类抗菌药组、耐6类抗菌药组与敏感组相比,ompF基因mRNA表达量的差异极显著,P0.01。ECs3703基因mRNA表达量随大肠杆菌耐抗菌药物种类的增多变化不明显;但是,与敏感组相比,耐6类抗菌药物的大肠杆菌ECs3703基因mRNA表达量下降显著,P0.05。采用小鼠致病性试验对4种不同耐药表型的大肠杆菌的致病性进行比较,分析不同耐药表型对毒力的影响。攻毒后,敏感组、耐2类抗菌药物组、耐4类抗菌药物组、耐6类抗菌药物组小鼠死亡率的均值分别为72.22%、61.11%、33.33%、16.67%。随着奶牛乳房炎源大肠杆菌耐药种类的增加,被接种的小鼠死亡率逐渐下降;且接种耐4类抗菌药物大肠杆菌和耐6类抗菌药物大肠杆菌小鼠的致死率,明显低于接种对6类抗菌药物均敏感大肠杆菌小鼠的致死率。研究表明,对于四环素类、β-内酰胺类、氟喹诺酮类、氨基糖苷类、酰胺醇类、大环内酯类6类抗生素,大肠杆菌随着自身耐药种类的增多,毒力减弱。本研究分析了辽宁地区奶牛临床型乳房炎源大肠杆菌毒力因子的流行情况,及大肠杆菌耐药性对其致病性的影响,将为该地区奶牛乳房炎的有效防控提供理论依据。
[Abstract]:Escherichia coli can cause a variety of animal diseases, which poses a great threat to animal husbandry and human health. The drug resistance of Escherichia coli has been very common, which brings great difficulties to the effective prevention and treatment of Escherichia coli infectious diseases. Bacterial drug resistance can change its pathogenicity, and the study on the effect of Escherichia coli resistance on its toxicity has attracted extensive attention of scholars. In this study, the effect of Escherichia coli resistance on the virulence of dairy cows with clinical mastitis isolated from Liaoning was systematically analyzed in order to provide guidance for clinical scientific prevention and treatment of mastitis in dairy cows. In this study, PCR technique was used to detect the virulence genes of 79 strains of Escherichia coli isolated from Liaoning dairy cows with clinical mastitis, and the developmental typing and epidemic characteristics of virulence genes of Escherichia coli from dairy cows from Liaoning area were analyzed. The results showed that among 79 strains of Escherichia coli, type A was 59.5%, type B1 was 27.9%, type B2 and type D were 6.3% respectively. 22 virulence genes were screened from Escherichia coli isolated from clinical mastitis cows. 10 virulence genes were detected as fimH (89.9%), ECs3703 (88.6%), ompC (100%), ompF (73.4%), colV (5.1%), irp2 (2.5%), fyuA (2.5%), eaeA (1.3%), ler (1.3%) and iucD (1.3%). The number of type A Escherichia coli isolated from Escherichia coli was the largest, and the kinds of virulence genes were the most. The expression of virulence genes ompF and ECs3703mRNA in E. coli with different drug resistance phenotypes was detected by real-time fluorescence quantitative PCR. The results showed that the expression of ompF gene mRNA decreased with the increase of antibiotic resistance in E. coli. The expression of ompF gene mRNA in resistant group was significantly different from that in sensitive group (P0.05), and the expression of ompF gene mRNA in resistant group was significantly different from that in sensitive group. The expression of P0.01.ECs3703 gene mRNA did not change significantly with the increase of antibiotic resistance in E. coli. However, compared with the sensitive group, the expression of ECs3703 gene mRNA in Escherichia coli resistant to 6 kinds of antibiotics decreased significantly (P 0.05). The pathogenicity of E. coli with different drug resistance phenotypes was compared by mouse pathogenicity test, and the effects of different drug resistance phenotypes on virulence were analyzed. After challenge, the average mortality of mice in sensitive group, resistant to two kinds of antibiotics group, resistant to 4 types of antibiotics group and resistant to 6 types of antibiotics group was 72.22%, 61.11%, 33.33% and 16.67%, respectively. the average mortality of mice was 72.22%, 61.11%, 33.33% and 16.67%, respectively. With the increase of resistant types of Escherichia coli from dairy cows, the mortality of inoculated mice decreased gradually, and the fatality rate of mice inoculated with Escherichia coli resistant to four kinds of antibiotics and resistant to 6 kinds of antibiotics was significantly lower than that of mice inoculated with Escherichia coli sensitive to 6 kinds of antibiotics. The results showed that for tetracycline, 尾-lactam, fluoroquinolone, aminoglycosides, amides and macrolides, Escherichia coli became less virulent with the increase of drug resistance. In this study, the prevalence of virulence factors of Escherichia coli from clinical mastitis in dairy cows in Liaoning Province and the effect of Escherichia coli drug resistance on its pathogenicity were analyzed, which will provide a theoretical basis for the effective prevention and control of mastitis in dairy cows in Liaoning Province.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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