【摘要】：抗生素的使用不當導致細菌耐藥性的日益嚴重是臨床抗感染治療面臨的世界性難題。解決這一問題的方法之一是根據感染細菌的耐藥性有針對性地使用抗生素。目前對細菌耐藥性的檢測仍然依賴傳統(tǒng)的藥敏實驗,其最大的缺點是診斷周期長,臨床醫(yī)生無法在感染早期對患者合理用藥。通過基因芯片技術直接檢測細菌耐藥基因,可以快速、敏感地確定細菌的耐藥性,為合理使用抗生素提供依據。革蘭陰性桿菌是目前臨床常見的致病菌,其治療主要依賴β-內酰胺類抗生素。革蘭陰性桿菌耐受β-內酰胺類抗生素主要由細菌產生的β-內酰胺酶引起。β-內酰胺酶種類繁多,其中較為重要的包括AmpC 酶、ESBLs 等。AmpC 酶屬于Bush 分類中的I 類酶或Ambler C 類酶,其編碼基因是ampC。我們發(fā)現,不同種屬的細菌的ampC 基因序列顯著不同。 目的分析臨床革蘭陰性桿菌中ampC 耐藥結構基因的分布與耐藥狀況的關系;在此基礎上建立起利用多重PCR 及寡核苷酸芯片技術檢測ampC 耐藥結構基因的方法。 方法 1、標本菌株收集來源于第三軍醫(yī)大學第一附屬醫(yī)院及兒童醫(yī)院檢驗科2003 年10月至2004 年5 月臨床分離的菌株。 2、用PCR 檢測389 株革蘭陰性桿菌中ampC 基因的分布。 3、應用頭孢西丁三維實驗的方法檢測細菌產AmpC 酶的狀況。 4、應用microscan walkaway-4.0 鑒定細菌的耐藥性。 5、建立多重PCR 體系檢測7 種不同種屬細菌中的ampC 基因。 6、利用標記Cy3 的PCR 產物與寡核苷酸芯片雜交的技術建立檢測ampC 基因的方法。結果 1、在389 株革蘭陰性桿菌中,ampC 基因的陽性率為:51.7%。在不同種屬的細菌中,ampC 基因的分布不一致:陰溝腸桿菌34 株(79.1%)、埃希氏菌屬93 株(79.5%)、克雷伯氏菌屬14 株(1.22%)、假單胞菌屬41 株(61.2%)和不動桿菌屬14 株(50.0%)。 2、在201 株ampC 基因陽性的菌株中,有34 株細菌產AmpC 酶,ampC 基因表達的總陽性率為:16.9%。
[Abstract]:Improper use of antibiotics leads to the increasingly serious drug resistance of bacteria, which is a worldwide problem in clinical anti-infective therapy. One way to solve this problem is to target antibiotics based on the drug resistance of infected bacteria. At present, the detection of bacterial drug resistance still relies on the traditional drug sensitivity test, its biggest shortcoming is that the diagnosis cycle is long, the clinicians can not use reasonable drugs to the patients in the early stage of infection. Direct detection of bacterial drug resistance genes by gene chip technology can quickly and sensitively determine the drug resistance of bacteria and provide evidence for the rational use of antibiotics. Gram-negative bacilli are common pathogenic bacteria in clinic, and their treatment mainly depends on 尾-lactam antibiotics. Gram-negative bacilli tolerance to 尾 -lactam antibiotics is mainly caused by 尾 -lactamases produced by bacteria. There are many kinds of 尾 -lactamases, among which the more important ones are AmpC enzymes, such as ESBLs. AMPC enzymes belong to class I or Ambler C enzymes in Bush classification. The encoding gene is AMPC. We found that the ampC gene sequences of different species of bacteria were significantly different. Objective to analyze the relationship between the distribution of ampC resistance genes in clinical Gram-negative bacilli and the status of drug resistance, and to establish a method for the detection of multiplex PCR and oligonucleotide microarray for the detection of ampC resistance genes. Methods 1. The strains collected from the first affiliated Hospital of the third military Medical University and the Laboratory Department of Children's Hospital were isolated from October 2003 to May 2004. 2. PCR was used to detect the distribution of ampC gene in 389 Gram-negative bacilli. 3The method of cefxitin three-dimensional test was used to detect bacteria. The condition of producing AmpC enzyme. 4. Microscan walkaway-4.0 was used to identify the drug resistance of bacteria. 5. A multiplex PCR system was established to detect 7 different species. The method of detecting ampC gene was established by hybridization of PCR products labeled with Cy3 and oligonucleotide chip. Results 1. The positive rate of ampC gene in 389 Gram-negative bacilli was: 51.7%. The distribution of ampC gene was different among different species: Enterobacter cloacae 34 (79.1%), Escherichia coli 93 (79.5%), Klebsiella 14 (1.22%), Pseudomonas 41 (61.2%) and Acinetobacter 14 (50.0%). (2) among 201 ampC positive strains, The total positive rate of AmpC enzyme ampC gene expression in 34 strains of bacteria was: 16.9%.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

【引證文獻】

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1 何昕;多麗波;;AmpC β-內酰胺酶實驗室檢測方法及進展[J];中華醫(yī)院感染學雜志;2011年21期

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本文編號：2186088