Resistance Transfer Study Of Riemerella Anatipestfer With Escherichia Coli

Riemerella anatipestfer infection and Escherichia coli infection are the most two serious infectious disease to poultry, and these two bacterial infection both can cause sepsis, fibrinous pericarditis, perihepatitis, gasbag inflammation and so on. In clinical, these two kinds of pathogens often appeared phenomenon of mixed infection, while mixed infection are prone to the transfer of resistance, making the drug-resistant spectrum widening and causing the failure of drug treatment, and finally causing serious economic losses to the poultry industry. Although the transfer of drug resistance about bacteria mixed infection has been a lot of research, how the resistance transfer between two different species of Riemerella anatipestfer and Escherichia coli has not been reported. In this study, RA P3 and E.coli 8099 as test bacterials trains for mixed subculture in vitro were selected from drug sensitive test, and analyzed the resistance strains of E.coli screened after mixed subculture.In this study,four strains of Riemerella anatipestfer were isolated and identified from suspected duck disease epidemic materials from Shandong province. The K-B(Kirby-Bauer) method is used for four strains of RA and two strains of E.coli standard strains to test drug susceptibility to 24 kinds of common antibiotics drug. The drug susceptibility results showed that four strains of RA are resistant to aminoglycoside drugs(gentamycin, tobramycin and kanamycin) and quinolone drugs(ciprofloxacin, ofloxacin, norfloxacin), while two strains of E.coli are sensitive to the six antibiotics. According to the specific data, RA P3 and E.coli standard strain 8099 were selected as the donor and acceptor of resistance transfer test respectively and used in mixed subculture. RA P3 and E.coli standard strain 8099 were measured the minimum inhibition concentration(MIC) to gentamycin, tobramycin, kanamycin, ciprofloxacin, ofloxacin and norfloxacin by the liquid tube double dilution method.The test strains RA P3 and E.coli 8099 we selected were used in mixed subculture. For the nutrition requirement of RA culture is high, we need to explore the mixed subculture conditions for these two kinds of bacteria. Final mixed subculture conditions: firstly RA and E.coli were cultured alone; the first generation, 10 ?L E.coli bacterium suspension(concentration is 1.5×103 cfu/mL) and 50 ?L RA bacterium suspension(concentration is 15×108 cfu/mL) were inoculated in 4 mL tryptone soy broth culture medium containing 4% serum, mixed culturing of 9 to 12 h; Starting from the second generation, 10 ?L generation on the mixed cultivating bacterial liquid(concentration is 1.5×105 cfu/mL) and 50 ?L RA bacterial liquid(concentration is 15×108 cfu/mL) were inculated in 4 mL TSB culture medium containing 4% serum, mixed culturing of 9 to 12 h.After mixed subculture, to screen resistance strains of E.coli, each generation of the mixed culture of bacteria liquid was coated on the macconkey resistance screening plate whose bacteriostasis concentration is two times higher than the E.coli standard strains of 8099, 0.25 ?g/mL of ciprofloxacin, 1 ?g/mL of ofloxacin, 1 ?g/mL of norfloxacin resistance plate are sterile growth, and 32 ?g/mL of kanamycin, 10 ?g/mL of gentamycin, 8 ?g/mL of tobramycin resistance plate has a single colony, respectively named E1, E2 and E3.To analyze drug resistance after mixed subculture, contrasting with before, the resistant strains of E.coli E1, E2 and E3 were analyzed the drug susceptibility and measured the minimum bacteriostasis concentration to the six drugs, and compared with the E.coli standard strains 8099, the drug susceptibility of drug-resistant strains E1 to gentamycin, tobramycin and kanamycin has significantly changes, the minimum bacteriostasis concentration was also significantly increased.To further study the level of transfer of drug resistance between RA and E.coli, we selected aminoglycoside resistant gene, aminoglycoside phosphotransferase gene aac6-? and aac3-?, aminoglycoside adenosine transferase gene aadA1, and aminoglycoside phosphotransferase gene aph3-? and designed primer, using polymerase chain reaction(PCR) to detect the resistant gene and determind sequence determination in RA P3 strains, standard strains of E.coli 8099 and drug-resistant strains of E.coli E1. Results show that aac6-?was detected in the RA P3 and drug-resistant strains E1, other genes are not detected, and no genes were detected in E.coli standard strain 8099.This study successfully mixed subcultured of Riemerella anatipestfer and Escherichia coli, at the same time analyzed the resistance after mixed subculture contrasting with before, and determined the resistance transfer in level between RA and E.coli. So the study laid the foundation of sensitive drug screening and development of new drug research.