Ⅰ型整合子与E.coli多重耐药性的相关性研究
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摘要
大肠杆菌是医学和兽医临床上最常见的病原菌之一，给人类的健康和畜牧业的发展造成了严重的损害。抗菌药在控制大肠杆菌感染方面发挥了重要作用，但随着抗菌药物的广泛应用，导致大肠杆菌耐药株尤其是多重耐药菌株不断出现，大肠杆菌的耐药问题已成为影响人类健康和养殖业发展的重要因素。细菌耐药机制—整合子(integron)系统得到研究者们的广泛注意，并取得了很大的进展。细菌通过整合子系统，通过整合酶的作用，捕获外来的耐药基因，并在位于整合子上游启动子的作用下得到表达，使细菌具有耐药及多重耐药性，造成细菌多重耐药性的传播。在整合子介导细菌的耐药机制中，第一类整合子起着非常重要的作用。大部分的耐药基因水平传播都是由Ⅰ类整合子介导的。因此调查研究大肠杆菌耐药性、扩增Ⅰ型整合子基因检测Ⅰ型整合子分子流行病学情况对从基因水平上探讨细菌耐药性转移的分子机制具有重要的理论和实践意义。
我们从黑龙江省地区3个猪场内分离出20株耐药大肠杆菌，分析大肠杆菌对16种抗生素的耐药特点。采用PCR方法检测Ⅰ型整合子—基因盒系统，并对所得基因测序和序列分析。研究养殖场大肠杆菌Ⅰ型整合子分子流行病学特点。
16种抗菌药物的药敏试验结果表明：20株大肠杆菌均表现多重耐药，对链霉素、庆大霉素、壮观霉素、氯霉素、痢特灵、磺胺甲基异恶唑、复方新诺明的耐药率在本次试验中已经达到了100％，对新霉素、环丙沙星耐药率超过90％，对诺氟沙星、阿莫西林、卡那霉素耐药率也较高，头孢氨苄利阿米卡星相对其它抗菌药物耐药率较低，对多粘菌素B的耐药率则为0。在这些耐药菌株中，均为多重耐药菌株，耐药谱集中在11～15耐，其中14耐，12耐菌株居多。克隆整合子基因片断，大小约为2000bp，经测序鉴定PCR扩增产物大小为1991bp，结果经Genebank检索同源性分析如下：整合子内部插入磺胺类耐药基因二氢叶酸还原酶基因dhfrⅫ和氨基糖苷类耐药基因核苷转移酶基因aadA2。猪场大肠杆菌中Ⅰ型整合子流行较普遍，检出率为60％。
结果表明所分离大肠杆菌对抗菌药存在严重的耐药性，耐药谱不断增加，其耐药性亦不断发生变迁。采用PCR方法成功克Ⅰ型整合子基因片断，并采用整合子PCR方法检测大肠杆菌Ⅰ型整合子流行病学特点，本研究结果为大肠杆菌耐药性监测提供了一种实用、快速、准确的检测方法，也为进一步从基因水平探讨大肠杆菌多重耐药性的产生与传播奠定了理论基础。
Escherichia coli (E.Coli) is one of the most common pathogens in the medicine and veterinary surgeon clinically, which endanger the poultry industry and humans. Antimicrobial agents play a very important role in controlling diseases caused by Escherichia coli. Along with the widely use of antibiotics, antibiotic resistance to bacteria is getting worse. The problem of bacterial resistance is getting worse. There is a significantly increasing tendency of multi-resistance to bacteria.The resistance of Escherichiacoli has raised several concerns related to human health and the development of poultry industry. The mechanism of bacterial resistance integron system base on the distribution and characterization of class I integron among Escherichiacoli isolated from animals. The integron of bacterial is DNA system which carry gene cassette encoding antibiotic resistance, and also is the importance molecular foundation of bacteria to capture and disseminate the resistance gene class I integron one of the most important mechaism of bacterial resistance .Examined for bacterial sensitivity to common antimicrobials and detect the integrase gene by PCR,suitable for mechaism of bacteria and clinical studies.
In this paper, the sensitivities of 20 resistant E.coli isolated from pigs to 16 antibiotics were assessed with a controlled disk diffusion test;the distribution and tenency of resistance were studied and the resistance characteristic were analyzed.The presence and genetic content of class I integron were examined with specific primers of class 1 integron by PCR and sequencing..
The results of the sensitivity test of 20 resistant-E.coli isolates to 16 kinds antibiotics showed that all isolate presented a multiple antibitic resistance, the resistant ratio of E.coli isolates to phytomycin, cidomycin, actinospectacin, amphemycin, furaxone, cotrimoxazole and SMZ-TMP were 100%, the resistant rate of E.coli to fradiomycin and ciprofloxacin were exceeded 90%, the resistance radio to ampligram and amikacin was relative lower, the resistent style of Escherichiacoli to antibiotics was concentrate in 11-15 kinds .Two primers were designed to amplify the gene of class I integron, the transformants were identified by PCR test , and then sequenced. 13 were positive of for Escherichia coli class I integron dihydrofolate reductase (dhfrXIl) and putative protein genes, complete cds, and streptomycin/spectinomycin 3' adenyltransferase (aadA2) pseudogene .The rates of class I integron in E.coli isolated from pig was about 60%.
The result shows that the resistance rates of Escherichiacoli is increasing, there is a significantly increasing tendency of multi-resistance ,the problem of bacterial resistance is getting worse .The presence and genetic content of class I integron were examined with specific primers of class I integron by PCR.
我们从黑龙江省地区3个猪场内分离出20株耐药大肠杆菌，分析大肠杆菌对16种抗生素的耐药特点。采用PCR方法检测Ⅰ型整合子—基因盒系统，并对所得基因测序和序列分析。研究养殖场大肠杆菌Ⅰ型整合子分子流行病学特点。
16种抗菌药物的药敏试验结果表明：20株大肠杆菌均表现多重耐药，对链霉素、庆大霉素、壮观霉素、氯霉素、痢特灵、磺胺甲基异恶唑、复方新诺明的耐药率在本次试验中已经达到了100％，对新霉素、环丙沙星耐药率超过90％，对诺氟沙星、阿莫西林、卡那霉素耐药率也较高，头孢氨苄利阿米卡星相对其它抗菌药物耐药率较低，对多粘菌素B的耐药率则为0。在这些耐药菌株中，均为多重耐药菌株，耐药谱集中在11～15耐，其中14耐，12耐菌株居多。克隆整合子基因片断，大小约为2000bp，经测序鉴定PCR扩增产物大小为1991bp，结果经Genebank检索同源性分析如下：整合子内部插入磺胺类耐药基因二氢叶酸还原酶基因dhfrⅫ和氨基糖苷类耐药基因核苷转移酶基因aadA2。猪场大肠杆菌中Ⅰ型整合子流行较普遍，检出率为60％。
结果表明所分离大肠杆菌对抗菌药存在严重的耐药性，耐药谱不断增加，其耐药性亦不断发生变迁。采用PCR方法成功克Ⅰ型整合子基因片断，并采用整合子PCR方法检测大肠杆菌Ⅰ型整合子流行病学特点，本研究结果为大肠杆菌耐药性监测提供了一种实用、快速、准确的检测方法，也为进一步从基因水平探讨大肠杆菌多重耐药性的产生与传播奠定了理论基础。
Escherichia coli (E.Coli) is one of the most common pathogens in the medicine and veterinary surgeon clinically, which endanger the poultry industry and humans. Antimicrobial agents play a very important role in controlling diseases caused by Escherichia coli. Along with the widely use of antibiotics, antibiotic resistance to bacteria is getting worse. The problem of bacterial resistance is getting worse. There is a significantly increasing tendency of multi-resistance to bacteria.The resistance of Escherichiacoli has raised several concerns related to human health and the development of poultry industry. The mechanism of bacterial resistance integron system base on the distribution and characterization of class I integron among Escherichiacoli isolated from animals. The integron of bacterial is DNA system which carry gene cassette encoding antibiotic resistance, and also is the importance molecular foundation of bacteria to capture and disseminate the resistance gene class I integron one of the most important mechaism of bacterial resistance .Examined for bacterial sensitivity to common antimicrobials and detect the integrase gene by PCR,suitable for mechaism of bacteria and clinical studies.
In this paper, the sensitivities of 20 resistant E.coli isolated from pigs to 16 antibiotics were assessed with a controlled disk diffusion test;the distribution and tenency of resistance were studied and the resistance characteristic were analyzed.The presence and genetic content of class I integron were examined with specific primers of class 1 integron by PCR and sequencing..
The results of the sensitivity test of 20 resistant-E.coli isolates to 16 kinds antibiotics showed that all isolate presented a multiple antibitic resistance, the resistant ratio of E.coli isolates to phytomycin, cidomycin, actinospectacin, amphemycin, furaxone, cotrimoxazole and SMZ-TMP were 100%, the resistant rate of E.coli to fradiomycin and ciprofloxacin were exceeded 90%, the resistance radio to ampligram and amikacin was relative lower, the resistent style of Escherichiacoli to antibiotics was concentrate in 11-15 kinds .Two primers were designed to amplify the gene of class I integron, the transformants were identified by PCR test , and then sequenced. 13 were positive of for Escherichia coli class I integron dihydrofolate reductase (dhfrXIl) and putative protein genes, complete cds, and streptomycin/spectinomycin 3' adenyltransferase (aadA2) pseudogene .The rates of class I integron in E.coli isolated from pig was about 60%.
The result shows that the resistance rates of Escherichiacoli is increasing, there is a significantly increasing tendency of multi-resistance ,the problem of bacterial resistance is getting worse .The presence and genetic content of class I integron were examined with specific primers of class I integron by PCR.
引文
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