Ⅰ类整合子在多重耐药大肠埃希菌中的作用探讨

发布时间：2018-04-14 10:29

  本文选题：大肠埃希菌 + 多重耐药　； 参考：《泸州医学院》2010年硕士论文

【摘要】：目的：了解本地区多重耐药大肠埃希菌(Escherichia coli)中Ⅰ类整合子的流行分布情况,并分析Ⅰ类整合子与多重耐药之间可能存在的关系,探讨十二烷基硫酸钠(Sodium dodecyl sulfate, SDS)对Ⅰ类整合子的作用影响。方法：1.纸片扩散法(Kerby-Bauer法,K-B法)：采用NCCLS推荐的该方法测定71株大肠埃希菌对三类4种常用抗菌药物——庆大霉素(G)、左氧氟沙星(L)、头孢噻肟(CTX)、头孢他啶(CAZ)的耐药性。大肠埃希菌标准菌株ATCC 25922进行质控。2.PCR技术：采用Biospin细菌基因组DNA试剂盒提取大肠埃希菌DNA,并用PCR方法检测Ⅰ类整合子3个不同部位的基因。根据整合酶1(IntI1)基因、可变区两侧5’端和3’端保守序列及3’保守端qacE△1-sul1基因(Ⅰ类整合子遗传标记)序列设计三对引物,以提取的大肠埃希菌DNA为PCR反应模板进行基因检测。扩增产物进行琼脂糖凝胶电泳、紫外线检测仪观察结果及送检做基因测序。3.SDS处理后PCR法测定Ⅰ类整合子基因：以高温(41℃)、高浓度(60μg/ml)SDS对挑选出的Ⅰ类整合子阳性的多重耐药大肠埃希菌进行处理,并用PCR法对处理后的大肠埃希菌行Ⅰ类整合子基因检测,与处理前结果进行比较。4.SDS处理前后细菌最低抑菌浓度(MIC)测定:SDS处理方法同前,将处理前与处理后的大肠埃希菌分别置于含不同浓度的抗生素培养基内生长,得到其MIC值,从而分析SDS对细菌耐药性的影响。结果：1.71株大肠埃希菌的耐药情况：(1)对各种常用抗生素的耐药分布：66.20%耐庆大霉素(47株)、63.38%耐左氧氟沙星(45株)、59.15%耐头孢噻肟(42株)、18.31%耐头孢他定(13株)；(2)其耐药模式分布：以多重耐药菌株(23株,32.39%)和双耐菌株(25株,35.21%)为主,其次是单耐菌株(16株,22.54%),全敏感菌株(7株,9.86%)最少。其中多耐模式的表型以G+L+CTX(14株,19.72%)为主。2.PCR法检测大肠埃希菌中Ⅰ类整合子的存在情况：(1)71株大肠埃希菌中Ⅰ类整合子的总检出率为88.73%。各耐药模式菌株的Ⅰ类整合子的检出率有差异(P0.05),两两比较示多耐菌株与全敏菌株、双耐菌株与全敏菌株的检出率有明显差异；(2)PCR法对三种基因检出率分别为：整合酶基因74.65%、整合子遗传标记基因78.87%、整合子可变区基因21.13%。3.Ⅰ类整合子与耐药的相关分析：(1)与4种抗生素耐药的关系：整合子阳性株与阴性株对庆大霉素、左氧氟沙星、头孢噻肟耐药与敏感的分布情况有明显的差异(P0.05)；(2)与耐药模式的关系：多耐菌株与双耐菌株的Ⅰ类整合子基因的阳性率最高(均为100.00%),其次为单耐菌株(13株,81.25%),全敏菌株的基因阳性率最低(2株,28.57%),且其差异有统计学差异(P0.05),两两比较发现多耐菌株与全敏菌株、双耐菌株与全敏菌株有统计学差异(P0.05)。 4.SDS的干扰作用：(1)经SDS处理后,多重耐药大肠埃希菌中的Ⅰ类整合子基因阳性率有不同程度的降低(100.00%降为30.43%),有统计学差异；(2)SDS处理前后,多重耐药大肠埃希菌的对庆大霉素、头孢噻肟的MIC值均有明显差异(P0.05)。结论：1.本地区多重耐药大肠埃希菌普遍存在；Ⅰ类整合子的阳性率高,是多重耐药的重要原因之一；2.多重耐药大肠埃希菌整合酶基因、遗传标记基因的检出率高于可变区基因；3.SDS对多重耐药大肠埃希菌的Ⅰ类整合子基因阳性率与MIC值均有不同程度的影响。
[Abstract]:Objective: to understand the region in multidrug-resistant Escherichia coli (Escherichia coli) popular in the distribution of integron, and analyze the possible relationship between integrons and multiple drug resistance, twelve of sodium dodecyl sulfate (Sodium dodecyl, sulfate, SDS) of class I integron. Methods: 1. pieces of paper diffusion method (Kerby-Bauer method, K-B method): 71 strains of Escherichia coli were determined in three categories of 4 kinds of commonly used antimicrobial agents - gentamicin by the method recommended by NCCLS (G), levofloxacin (L), cefotaxime (CTX), ceftazidime (CAZ). The drug resistance of Escherichia coli strains ATCC 25922 quality control.2.PCR Technology: using Biospin bacterial genomic DNA extraction kit of Escherichia coli DNA, and using PCR method for detection of integron in 3 different parts of the gene. According to the 1 integrase (IntI1) gene and the variable region on both sides of the 5 'and 3' ends. Keep the sequence and the 3 'end of qacE Delta 1-sul1 gene conservative (class I integron) three primer sequences, Escherichia coli DNA extraction for PCR reaction template gene was detected by agarose gel electrophoresis. The amplification products, UV detector observations and submission to do gene sequencing after treatment with.3.SDS PCR method for the determination of integron gene: with high temperature (41 DEG C), high concentration (60 g/ml) SDS positive on the selected integron in multidrug-resistant Escherichia coli for processing, and use the PCR method to the treatment of Escherichia coli for integron gene detection, and the results were compared before treatment.4.SDS before and after the treatment, the minimum inhibitory concentration (MIC) determination: SDS processing method with treatment before and after the treatment of Escherichia coli were treated with different concentrations of the antibiotic growth medium, the MIC value, and SDS analysis of bacteria 鑰愯嵂鎬х殑褰卞搷.缁撴灉锛，

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