大肠杆菌氟苯尼考耐药性在不同动物间的传播研究
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摘要
本实验主要研究保定地区不同动物源(包括鸡源、猪源和牛源)大肠杆菌的传播情况,了解氟苯尼考耐药性在不同动物源大肠杆菌的变迁趋势、生物学特性和耐药谱,对大肠杆菌的氟苯尼考耐药性进行危险性评估,并从基因水平对大肠杆菌氟苯尼考耐药机制进行探讨,从而为大肠杆菌病的有效防治和新兽药开发提供参考。本研究主要分3部分:
1大肠杆菌的分离鉴定及耐药谱测定
自河北省保定市及其周边地区的鸡场、猪场和牛场分离出60株细菌,经生化实验鉴定出45株分离菌为大肠杆菌,其中有23株鸡源大肠杆菌,14株猪源大肠杆菌和8株牛源大肠杆菌。采用玻板凝集和试管凝集相结合的方法,对45株大肠杆菌进行了O血清型鉴定,鸡源大肠杆菌以O78、O35为优势血清型,猪源、牛源大肠杆菌以O101为优势血清型。按世界卫生组织(WHO)推荐的Kirby-Bauer菌液涂布法对45株大肠杆菌进行氟苯尼考、头孢喹肟、诺氟沙星等11种抗生素的药物敏感性试验,结果表明分离的大肠杆菌对各种抗生素呈现不同程度的耐受性,仅对头孢喹肟较敏感。其中氟苯尼考耐药菌检出率分别为62%,58%和38%。
2 IntⅠ基因、flor基因和cmlA基因的克隆
提取所分离的45株大肠杆菌的基因组DNA和质粒DNA,采用PCR扩增技术,以耐氟苯尼考大肠杆菌菌落、基因组DNA及质粒DNA为模板扩增整合酶基因,以耐氟苯尼考大肠杆菌菌落及质粒DNA为模板扩增flor基因及cmlA基因。结果从菌落及质粒DNA中均扩增出了IntⅠ基因(Ⅰ型整合子整合酶基因),且两者呈现良好的一致性;以菌落及质粒DNA为模板扩增出了flor基因和cmlA基因。从菌落和质粒DNA中扩增的同种基因碱基序列完全相同,flor基因与cmlA基因存在28.2%-28.4%同源性。
3不同动物源大肠杆菌耐药基因的测序及分析
对扩增的不同动物源大肠杆菌耐氟苯尼考基因flor进行测序和分析,根据测序结果绘制基因进化树图谱。发现来自同种属大肠杆菌的flor基因同源性为100%,猪源、牛源大肠杆菌flor基因同源性为100%,鸡源与猪源、牛源大肠杆菌的flor基因同源性高达99.8%。由flor基因编码的氨基酸序列与GenBank报道的flor基因存在3个氨基酸替代。通过连接、转化构建了基因工程菌,通过蓝白斑实验筛选阳性菌株。经酶切鉴定和质粒PCR确定阳性菌中转入了含耐药基因的重组质粒。比较阳性菌与阴性菌耐药性发现阳性菌对氟苯尼考耐受程度明显增强,对氯霉素的耐受性也有小幅增强,对其他抗生素均敏感。
The purpose of this study is to understand the transmission and prevelance of Escherichia coli from different regions of Baoding and acquire some information about the trend of transition, biological feature and spectrum of florfenicol resistance among different animals. Then the E.coli florfenicol resistance risk is to be evaluated and the mechanism of florfenicol resistance is to be discussed in the genetic level. Based on works we performed above, the prevention and treatment of Escherichia colibacillosis would be more effective and it may provide some references for the development of new veterinary pharmaceuticals. This study is divided into 3 parts and the details is reported as follows.
1. Identification of E.coli strains and assay of antibacterial spectrum.
60 bacterial strains were isolated from dead or sick animals showing typical symptoms of Escherichia colibacillosis in poultry, swine and bovine farms in several regions of Baoding. The 45 strains of E.coli were determined by biochemical tests which including 23,14, 8 strains isolated from poultry, swine and bovine, respectively. Slide and tube agglutination tests were used to determine the serotype of 45 strains and it showed that O78 and O35 were dominant strains of poultry, but in swine and bovine it were O101. The resistance test of 11 antibiotics, such as Florfenicol, Cefquinome, Norfloxacin and the like, were tested according to the Kirby-Bauer’s method recommended by the World Health Organization(WHO). The result revealled that drug resistance of variety of antibiotics was common, and most of them even had multidrug resistance. However, most of strains were sensitive to Cefquinome.The resistance proportion of 3 kinds of animals to Florfenicol were 62%, 58% and 38%, respectively.
2. Cloning of integrase gene, flor gene and cmlA gene of classⅠintegron.
The genomic DNA and plasmid from 45 isolates were extracted and the colony of E.coli with resistance to Florfenicol , genomic DNA and plasmid DNA were used as models to amplify the integrase gene. The colony of E.coli with resistance to Florfenicol and plasmid DNA were used as models to amplify flor gene and cmlA gene. The result turned out to be positive that integrase gene of class I integron from two models were amplified successfully, both of which had a good uniformity, as well as the flor gene and cmlA gene. The homogeneous base sequence amplified from the colony and plasmid DNA were identical. The flor gene and the cmlA gene had homology from 28.2% to 28.4%.
3. Sequence assay and analysis of resistance gene of E.coli from different animals.
The sequence assay and analysis upon amplified resistance gene of E.coli to Florfenicol from different animals were performed. Then the Gene Evolution Tree was made according to the result of sequence assay. It showed that the homology of the flor gene from homogeneous animal, the flor gene between swine and bovine, the flor gene between poultry and swine or bovine were 100%, 100% and 99.8%, respectively. There were three amino acid substitutions in the flor gene between the sequences coloned and reported by GenBank. Gene engineering bacteria were constructed by technology of linkage and transformation and positive bacterial strains were picked up by blue-white spotting test. The result of identification of restriction enzyme digestion and plasmid PCR showed that the ecombinant plasmid delivered resistance gene was transmitted into positive bacteria. The result of minimum Inhibition concentration test showed that positive bacteria had stronger resistance to florfenicol than negative ones. Also, the former had a little increased resistance to chloromycetin than the latter, however, it was sensitive to other antibiotics.
1大肠杆菌的分离鉴定及耐药谱测定
自河北省保定市及其周边地区的鸡场、猪场和牛场分离出60株细菌,经生化实验鉴定出45株分离菌为大肠杆菌,其中有23株鸡源大肠杆菌,14株猪源大肠杆菌和8株牛源大肠杆菌。采用玻板凝集和试管凝集相结合的方法,对45株大肠杆菌进行了O血清型鉴定,鸡源大肠杆菌以O78、O35为优势血清型,猪源、牛源大肠杆菌以O101为优势血清型。按世界卫生组织(WHO)推荐的Kirby-Bauer菌液涂布法对45株大肠杆菌进行氟苯尼考、头孢喹肟、诺氟沙星等11种抗生素的药物敏感性试验,结果表明分离的大肠杆菌对各种抗生素呈现不同程度的耐受性,仅对头孢喹肟较敏感。其中氟苯尼考耐药菌检出率分别为62%,58%和38%。
2 IntⅠ基因、flor基因和cmlA基因的克隆
提取所分离的45株大肠杆菌的基因组DNA和质粒DNA,采用PCR扩增技术,以耐氟苯尼考大肠杆菌菌落、基因组DNA及质粒DNA为模板扩增整合酶基因,以耐氟苯尼考大肠杆菌菌落及质粒DNA为模板扩增flor基因及cmlA基因。结果从菌落及质粒DNA中均扩增出了IntⅠ基因(Ⅰ型整合子整合酶基因),且两者呈现良好的一致性;以菌落及质粒DNA为模板扩增出了flor基因和cmlA基因。从菌落和质粒DNA中扩增的同种基因碱基序列完全相同,flor基因与cmlA基因存在28.2%-28.4%同源性。
3不同动物源大肠杆菌耐药基因的测序及分析
对扩增的不同动物源大肠杆菌耐氟苯尼考基因flor进行测序和分析,根据测序结果绘制基因进化树图谱。发现来自同种属大肠杆菌的flor基因同源性为100%,猪源、牛源大肠杆菌flor基因同源性为100%,鸡源与猪源、牛源大肠杆菌的flor基因同源性高达99.8%。由flor基因编码的氨基酸序列与GenBank报道的flor基因存在3个氨基酸替代。通过连接、转化构建了基因工程菌,通过蓝白斑实验筛选阳性菌株。经酶切鉴定和质粒PCR确定阳性菌中转入了含耐药基因的重组质粒。比较阳性菌与阴性菌耐药性发现阳性菌对氟苯尼考耐受程度明显增强,对氯霉素的耐受性也有小幅增强,对其他抗生素均敏感。
The purpose of this study is to understand the transmission and prevelance of Escherichia coli from different regions of Baoding and acquire some information about the trend of transition, biological feature and spectrum of florfenicol resistance among different animals. Then the E.coli florfenicol resistance risk is to be evaluated and the mechanism of florfenicol resistance is to be discussed in the genetic level. Based on works we performed above, the prevention and treatment of Escherichia colibacillosis would be more effective and it may provide some references for the development of new veterinary pharmaceuticals. This study is divided into 3 parts and the details is reported as follows.
1. Identification of E.coli strains and assay of antibacterial spectrum.
60 bacterial strains were isolated from dead or sick animals showing typical symptoms of Escherichia colibacillosis in poultry, swine and bovine farms in several regions of Baoding. The 45 strains of E.coli were determined by biochemical tests which including 23,14, 8 strains isolated from poultry, swine and bovine, respectively. Slide and tube agglutination tests were used to determine the serotype of 45 strains and it showed that O78 and O35 were dominant strains of poultry, but in swine and bovine it were O101. The resistance test of 11 antibiotics, such as Florfenicol, Cefquinome, Norfloxacin and the like, were tested according to the Kirby-Bauer’s method recommended by the World Health Organization(WHO). The result revealled that drug resistance of variety of antibiotics was common, and most of them even had multidrug resistance. However, most of strains were sensitive to Cefquinome.The resistance proportion of 3 kinds of animals to Florfenicol were 62%, 58% and 38%, respectively.
2. Cloning of integrase gene, flor gene and cmlA gene of classⅠintegron.
The genomic DNA and plasmid from 45 isolates were extracted and the colony of E.coli with resistance to Florfenicol , genomic DNA and plasmid DNA were used as models to amplify the integrase gene. The colony of E.coli with resistance to Florfenicol and plasmid DNA were used as models to amplify flor gene and cmlA gene. The result turned out to be positive that integrase gene of class I integron from two models were amplified successfully, both of which had a good uniformity, as well as the flor gene and cmlA gene. The homogeneous base sequence amplified from the colony and plasmid DNA were identical. The flor gene and the cmlA gene had homology from 28.2% to 28.4%.
3. Sequence assay and analysis of resistance gene of E.coli from different animals.
The sequence assay and analysis upon amplified resistance gene of E.coli to Florfenicol from different animals were performed. Then the Gene Evolution Tree was made according to the result of sequence assay. It showed that the homology of the flor gene from homogeneous animal, the flor gene between swine and bovine, the flor gene between poultry and swine or bovine were 100%, 100% and 99.8%, respectively. There were three amino acid substitutions in the flor gene between the sequences coloned and reported by GenBank. Gene engineering bacteria were constructed by technology of linkage and transformation and positive bacterial strains were picked up by blue-white spotting test. The result of identification of restriction enzyme digestion and plasmid PCR showed that the ecombinant plasmid delivered resistance gene was transmitted into positive bacteria. The result of minimum Inhibition concentration test showed that positive bacteria had stronger resistance to florfenicol than negative ones. Also, the former had a little increased resistance to chloromycetin than the latter, however, it was sensitive to other antibiotics.
引文
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