本文選題：大腸埃希菌 + 多重耐藥　； 參考：《瀘州醫(yī)學(xué)院》2010年碩士論文

【摘要】：目的：了解本地區(qū)多重耐藥大腸埃希菌(Escherichia coli)中Ⅰ類整合子的流行分布情況,并分析Ⅰ類整合子與多重耐藥之間可能存在的關(guān)系,探討十二烷基硫酸鈉(Sodium dodecyl sulfate, SDS)對Ⅰ類整合子的作用影響。方法：1.紙片擴散法(Kerby-Bauer法,K-B法)：采用NCCLS推薦的該方法測定71株大腸埃希菌對三類4種常用抗菌藥物——慶大霉素(G)、左氧氟沙星(L)、頭孢噻肟(CTX)、頭孢他啶(CAZ)的耐藥性。大腸埃希菌標(biāo)準菌株ATCC 25922進行質(zhì)控。2.PCR技術(shù)：采用Biospin細菌基因組DNA試劑盒提取大腸埃希菌DNA,并用PCR方法檢測Ⅰ類整合子3個不同部位的基因。根據(jù)整合酶1(IntI1)基因、可變區(qū)兩側(cè)5’端和3’端保守序列及3’保守端qacE△1-sul1基因(Ⅰ類整合子遺傳標(biāo)記)序列設(shè)計三對引物,以提取的大腸埃希菌DNA為PCR反應(yīng)模板進行基因檢測。擴增產(chǎn)物進行瓊脂糖凝膠電泳、紫外線檢測儀觀察結(jié)果及送檢做基因測序。3.SDS處理后PCR法測定Ⅰ類整合子基因：以高溫(41℃)、高濃度(60μg/ml)SDS對挑選出的Ⅰ類整合子陽性的多重耐藥大腸埃希菌進行處理,并用PCR法對處理后的大腸埃希菌行Ⅰ類整合子基因檢測,與處理前結(jié)果進行比較。4.SDS處理前后細菌最低抑菌濃度(MIC)測定:SDS處理方法同前,將處理前與處理后的大腸埃希菌分別置于含不同濃度的抗生素培養(yǎng)基內(nèi)生長,得到其MIC值,從而分析SDS對細菌耐藥性的影響。結(jié)果：1.71株大腸埃希菌的耐藥情況：(1)對各種常用抗生素的耐藥分布：66.20%耐慶大霉素(47株)、63.38%耐左氧氟沙星(45株)、59.15%耐頭孢噻肟(42株)、18.31%耐頭孢他定(13株)；(2)其耐藥模式分布：以多重耐藥菌株(23株,32.39%)和雙耐菌株(25株,35.21%)為主,其次是單耐菌株(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%)為主。2.PCR法檢測大腸埃希菌中Ⅰ類整合子的存在情況：(1)71株大腸埃希菌中Ⅰ類整合子的總檢出率為88.73%。各耐藥模式菌株的Ⅰ類整合子的檢出率有差異(P0.05),兩兩比較示多耐菌株與全敏菌株、雙耐菌株與全敏菌株的檢出率有明顯差異；(2)PCR法對三種基因檢出率分別為：整合酶基因74.65%、整合子遺傳標(biāo)記基因78.87%、整合子可變區(qū)基因21.13%。3.Ⅰ類整合子與耐藥的相關(guān)分析：(1)與4種抗生素耐藥的關(guān)系：整合子陽性株與陰性株對慶大霉素、左氧氟沙星、頭孢噻肟耐藥與敏感的分布情況有明顯的差異(P0.05)；(2)與耐藥模式的關(guān)系：多耐菌株與雙耐菌株的Ⅰ類整合子基因的陽性率最高(均為100.00%),其次為單耐菌株(13株,81.25%),全敏菌株的基因陽性率最低(2株,28.57%),且其差異有統(tǒng)計學(xué)差異(P0.05),兩兩比較發(fā)現(xiàn)多耐菌株與全敏菌株、雙耐菌株與全敏菌株有統(tǒng)計學(xué)差異(P0.05)。 4.SDS的干擾作用：(1)經(jīng)SDS處理后,多重耐藥大腸埃希菌中的Ⅰ類整合子基因陽性率有不同程度的降低(100.00%降為30.43%),有統(tǒng)計學(xué)差異；(2)SDS處理前后,多重耐藥大腸埃希菌的對慶大霉素、頭孢噻肟的MIC值均有明顯差異(P0.05)。結(jié)論：1.本地區(qū)多重耐藥大腸埃希菌普遍存在；Ⅰ類整合子的陽性率高,是多重耐藥的重要原因之一；2.多重耐藥大腸埃希菌整合酶基因、遺傳標(biāo)記基因的檢出率高于可變區(qū)基因；3.SDS對多重耐藥大腸埃希菌的Ⅰ類整合子基因陽性率與MIC值均有不同程度的影響。
[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.緇撴灉錛，

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