Detection Of Avian Pathogenic Escherichia Coli Biofilm Formation And Study On Its Forming Gene

Escherichia coli is a normal intestinal parasite in human and animal. However, some strains of Escherichia coli have evoluted that become one of the most common pathogens in the medicine and veterinary surgeon clinically. Colibaciilosis often happens together with various diseases. With the development of poultry industry, situation of the disease is more and more serious and has caused huge losses. Antibiotic has played an important role in the control of APEC, but abuse of antimicrobials causes the appearance of multi-drug resistant strain, which makes the prevention and treatment of chicken colibacillosis more difficult. There is significantly increasing tendency of multi-resistance to bacteria. Escherichia coli resistance to antibiotics has become the focus of society's attention.The special structure of biofilm is the fundamental factor of drug resistance of bacterial biofilm.Biofilms play a key role in the pathogenesis of many bacterial infections. Bacteria in a biofilm are inherently protected from their surrounding environment and often exhibit increased resistance to host defense and antimicrobial agents, making most infections difficult or impossible to eradicate. Plasmid-containing avian E. coli even may cause extraintestinal infections or serve as sources of virulence or resistance genes for human pathogens.A total of 47 avian pathogenic Escherichia coli(APEC) were isolated and identified from 58 samples which were collected from a poultry yard in Dehui JILIN province. 3 APEC of kanamycin-senstive were screened from 47 strains by the K-B round scrips method. Other 11 kinds of antibacterial drugs were determined too. The result of susceptiblility tests showed that the medicine resistance of the isolated strains was rather strong, The resistance rate of Tetracycline is highest, up to 97.87%. The resistance rate of Ampicillin,Piperacillin,Doxycycline is more than 90%. All of bacteria from 58 samples were the tendency of multi-resistance to bacteria. Among them 19.15% of strains were resistant to less than seven antibiotic drugs; 12.77% were resistant to eight antibiotic drugs; 10.64%were resistant to nine antibiotic drugs; 19.15% were resistant to ten antibiotic drugs; 23.40% were resistant to eleven antibiotic drugs; 14.89% were resistant to twelve antibiotic drugs. APEC-8 was chosen that was the strongest biofilm formation producter in vitro by a quantitative method and a qualitative method. The results showed that the biofilm was well developed after 48 hours cultivation at 37℃with U-bottom 96-well polystyrene microplate (USA Corning Costar) as substratum and 5%TSB as medium. A kanamycin-resistant strain of E.coliβ2155 (pUT-miniTn5-Km2) as the donor was conjugated to a kanamycin-sensitive strain of APEC-8 as a recipient. Transconjugants were passed by three time to get 269 mutants and identified by PCR methods. 4 defective-biofilm mutants were chosen from 193 mutants which were kanamycin-resistant by PCR. Genome walking by PCR get the flanking fragements of the transposon. Sequence determination and analysis revealed that rfaG, flhE, OmpR, ycfM genes associated APEC biofilm formation.