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The Prevalence Of Florfenicol Resistance To Escherichia Coli Among Different Animals

Posted on:2011-07-25Degree:MasterType:Thesis
Country:ChinaCandidate:C C ZhengFull Text:PDF
GTID:2143360305969340Subject:Basic veterinary science
Abstract/Summary:PDF Full Text Request
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 classâ… integron.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.
Keywords/Search Tags:Escherichia coli, drug resistance, flor gene, prevalence
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