天然β-内酰胺酶抑制剂的筛选研究

发布时间：2018-01-29 18:19

  本文关键词： β-内酰胺酶 耐药性 耐药基因 大肠杆菌 中药单体　出处：《贵州大学》2016年硕士论文　论文类型：学位论文

【摘要】：β-内酰胺酶是病原菌对β-内酰胺类药物耐药的主要机制,尤其是超广谱β-内酰胺酶的产生导致该类抗菌药物在临床上治疗感染的疗效下降甚至失效,目前研发β-内酰胺酶抑制剂是解决这一耐药机制的一种有效途径。本研究首先采用微量肉汤稀释法测定采自贵州不同地区规模化养猪场的482株大肠杆菌对9种β-内酰胺类抗菌药物的敏感性,其次采用PCR法和凝胶电泳法检测482株大肠杆菌所携带的表达超广谱β-内酰胺酶(Extended-Spectrumβ-Lactamases,ESBL)的四种耐药基因情况,再次采用紫外分光光度法测定15种中药单体对β-内酰胺酶的抑制作用;最后采用微量肉汤稀释法和平板计数法测有效中药单体对产ESBL大肠杆菌的增敏作用,试验结果如下:1、482株猪源大肠杆菌对9种抗菌药物表现出不同程度的耐药,其中氨苄西林的耐药率最高,为86.72%;氨曲南的耐药率最低,为38.38%;五重、四重和六重耐药菌株数相对较多,分别为124株、105株和95株;各个地区大肠杆菌菌株耐药情况存在一定的差异。2、482株猪源大肠杆菌中,TEM,OXA-1,CTX-M-1和SHV基因的总检出率分别为59.13%,46.89%,32.57%和41.70%;其中20株同时检测出四种目的基因,90株同时检测出三种基因,175株同时检测出两种基因,166株仅检测出TEM基因,31株大肠杆菌未检测出这四种目的基因。3、15种中药单体中,有七种能抑制β-内酰胺酶,且其抑制作用随其浓度的增大逐渐增强。在浓度为10μg/mL时,芦荟大黄素、苦参碱和香紫苏醇与克拉维酸相比对β-内酰胺酶的抑制作用无显著性差异(P0.05);在浓度为30μg/mL时,木犀草素和香紫苏醇与克拉维酸相比无显著性差异(P0.05);在浓度为70μg/mL时,芦荟大黄素与克拉维酸相比无显著性差异(P0.05);在浓度为90μg/mL时,芦荟大黄素对β-内酰胺酶的抑制效果最好。4、七个中药单体分别联合头孢他啶、头孢噻肟、氨苄西林、阿莫西林和头孢噻呋五种抗菌药物,对产ESBL大肠杆菌均有不同程度的增敏作用,其中增敏作用效果较好的为芦荟大黄素和大蒜素,效果一般的为木犀草素、香紫苏醇和苦参碱,效果最差的槲皮素和五味子甲素。除了大蒜素以外,其余六种中药单体增敏试验结果与其抑制β-内酰胺酶活性试验结果一致,说明其增敏机制可能是抑制β-内酰胺酶使抗菌药物发挥作用。
[Abstract]:尾-lactamase is the main mechanism of bacterial resistance to 尾-lactams, especially the production of extended-spectrum 尾-lactamases, which leads to the decline or even invalidation of the clinical efficacy of these antibiotics in the treatment of infections. At present, the development of 尾 -lactamases inhibitor is an effective way to solve this drug resistance mechanism. In this study, 482 strains of Escherichia coli collected from large scale pig farms in different regions of Guizhou were determined by broth dilution method. Sensitivity of 9 尾-lactam antibiotics. Secondly, Extended-Spectrum 尾 -Lactamases carried by 482 strains of Escherichia coli were detected by PCR and gel electrophoresis. The inhibitory effect of 15 traditional Chinese medicine monomers on 尾 -lactamases was determined by UV spectrophotometry. Finally, the sensitizing effect of effective traditional Chinese medicine monomers on ESBL Escherichia coli was determined by broth dilution method and plate counting method. The results are as follows: 1. 482 strains of porcine Escherichia coli showed different degrees of resistance to 9 antimicrobial agents, of which ampicillin resistance rate was the highest (86.72%). The resistance rate of aztreonam was the lowest, 38.38%. The number of five, four and six resistant strains was relatively high, 105 and 95 strains, respectively. The resistance of Escherichia coli strains in different regions was different. The total detection rates of CTX-M-1 and SHV genes were 59.13% and 46.89%, respectively. Among them, 20 strains simultaneously detected four target genes, 90 strains simultaneously detected three genes, 175 strains simultaneously detected two genes, 166 strains detected only TEM gene. Of the 15 Chinese medicine monomers, 7 of the 31 strains of E. coli could inhibit 尾 -lactamases. The inhibitory effect of aloe emodin was increased with the increase of the concentration of aloe emodin at the concentration of 10 渭 g / mL. The inhibitory effects of matrine and perilla alcohol on 尾 -lactamases were not significantly different from those of clavulanic acid (P 0.05). At the concentration of 30 渭 g / mL, luteolin and perilla alcohol had no significant difference compared with clavulanic acid. At the concentration of 70 渭 g / mL, there was no significant difference between aloe emodin and clavulanic acid (P 0.05). At the concentration of 90 渭 g / mL, aloe emodin had the best inhibitory effect on 尾 -lactamases. Seven traditional Chinese medicine monomers combined with ceftazidime, cefotaxime and ampicillin respectively. Amoxicillin and cefotaxime had different sensitizing effects on ESBL producing Escherichia coli, among which aloe emodin and allicin were better sensitizers. Luteolin, perilla perilla and matrine, quercetin and Schisandrin were the most effective, with the exception of allicin. The results of the other six Chinese medicine monomer sensitizing tests were consistent with the results of the inhibition of 尾 -lactamase activity, which suggested that the mechanism of the sensitizing effect of 尾 -lactamases might be to inhibit the effect of 尾 -lactamases on antimicrobial agents.
【学位授予单位】：贵州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R978.1
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本文编号：1474054