本文關鍵詞： β-內(nèi)酰胺酶 耐藥性 耐藥基因 大腸桿菌 中藥單體　出處：《貴州大學》2016年碩士論文　論文類型：學位論文

【摘要】：β-內(nèi)酰胺酶是病原菌對β-內(nèi)酰胺類藥物耐藥的主要機制,尤其是超廣譜β-內(nèi)酰胺酶的產(chǎn)生導致該類抗菌藥物在臨床上治療感染的療效下降甚至失效,目前研發(fā)β-內(nèi)酰胺酶抑制劑是解決這一耐藥機制的一種有效途徑。本研究首先采用微量肉湯稀釋法測定采自貴州不同地區(qū)規(guī)�；B(yǎng)豬場的482株大腸桿菌對9種β-內(nèi)酰胺類抗菌藥物的敏感性,其次采用PCR法和凝膠電泳法檢測482株大腸桿菌所攜帶的表達超廣譜β-內(nèi)酰胺酶(Extended-Spectrumβ-Lactamases,ESBL)的四種耐藥基因情況,再次采用紫外分光光度法測定15種中藥單體對β-內(nèi)酰胺酶的抑制作用;最后采用微量肉湯稀釋法和平板計數(shù)法測有效中藥單體對產(chǎn)ESBL大腸桿菌的增敏作用,試驗結果如下:1、482株豬源大腸桿菌對9種抗菌藥物表現(xiàn)出不同程度的耐藥,其中氨芐西林的耐藥率最高,為86.72%;氨曲南的耐藥率最低,為38.38%;五重、四重和六重耐藥菌株數(shù)相對較多,分別為124株、105株和95株;各個地區(qū)大腸桿菌菌株耐藥情況存在一定的差異。2、482株豬源大腸桿菌中,TEM,OXA-1,CTX-M-1和SHV基因的總檢出率分別為59.13%,46.89%,32.57%和41.70%;其中20株同時檢測出四種目的基因,90株同時檢測出三種基因,175株同時檢測出兩種基因,166株僅檢測出TEM基因,31株大腸桿菌未檢測出這四種目的基因。3、15種中藥單體中,有七種能抑制β-內(nèi)酰胺酶,且其抑制作用隨其濃度的增大逐漸增強。在濃度為10μg/mL時,蘆薈大黃素、苦參堿和香紫蘇醇與克拉維酸相比對β-內(nèi)酰胺酶的抑制作用無顯著性差異(P0.05);在濃度為30μg/mL時,木犀草素和香紫蘇醇與克拉維酸相比無顯著性差異(P0.05);在濃度為70μg/mL時,蘆薈大黃素與克拉維酸相比無顯著性差異(P0.05);在濃度為90μg/mL時,蘆薈大黃素對β-內(nèi)酰胺酶的抑制效果最好。4、七個中藥單體分別聯(lián)合頭孢他啶、頭孢噻肟、氨芐西林、阿莫西林和頭孢噻呋五種抗菌藥物,對產(chǎn)ESBL大腸桿菌均有不同程度的增敏作用,其中增敏作用效果較好的為蘆薈大黃素和大蒜素,效果一般的為木犀草素、香紫蘇醇和苦參堿,效果最差的槲皮素和五味子甲素。除了大蒜素以外,其余六種中藥單體增敏試驗結果與其抑制β-內(nèi)酰胺酶活性試驗結果一致,說明其增敏機制可能是抑制β-內(nèi)酰胺酶使抗菌藥物發(fā)揮作用。
[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.
【學位授予單位】：貴州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R978.1
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本文編號：1474054