Molecular Characteristics Of Plasmid And Its Resistance Genes In Pasteurella Mulotcida From Duck

Pasteurella multocida is a pathogen that is responsible for causing hemorrhagic septicemia or infectious pneumonia in livestock and poultry.Fowl cholera caused by the disease causes huge impact on the poultry industry.As the rational use of antimicrobial agents,the multi-resistant situation of Pasteurella multocida is more and more serious.Currently,reports about drug resistance mechanism of duck-origin Pasteurella multocida which plasmid mediated are rarely.The study is based on whole genome sequence and plasmid sequence of a duck source Pasteurella multocida strain RCAD0259,the sequence analysis showed that the strain is ST129 model.Altogether contains seven resistance genes,including five on the plasmid which carries 4 resistant genes.Plasmid p RCADGH-2 also contains a complete type IV secretion system.This is the first time found on Pasteurella multocida plasmid type IV secretion system.This study based on plasmid elimination methods reported in literature,SDS-high temperature subculture and electroporation method which possess the higher rate of elimination were chosen to eliminate plasmid.Measured by the preliminary experiment selected 42?,0.3%SDS conditions of plasmid elimination,but after many attempts,the highest subculture to 100 generations still failed to extract plasmid eliminates strain.In addition,according to the previous electroporation conditions of Pasteurella multocida,2.0kV to 4.0kV voltage conditions,pulse 1 to 3times,respectively,were chosen.By repeated experiments,plasmid elimination was failed.May be caused by the large plasmid which resulted in increased the difficulty of the plasmid elimination or did not choose the suitable method to eliminate.E.coli J53Az^r(sodium azide resistant strain Lac-strain)was chosen as the receptors of transfer conjugation,though many times test,the zygote E.coli J53-0259,which plasmid is stable,was obtained and transfer frequency range is in 2×10^-6to7×10^-6.PCR amplification results confirmed that the four drug resistant genes which the plasmid p RCADGH-2 carried is transferred to E.coli J53-0259 along with plasmid p RCADGH-2,and drug resistance gene arr-2,bla _VEB-18can successfully mediate the resistance of the strain to rifampicin,penicillin and cephalosporin.To explore the reason that drug resistant genes aad B and cmlA could not mediate mechanism of the strain,expression plasmid p ET32a was chosen to construct the express positive strains E.coli J53-cmlA,E.coli J53-aad B.After determination of MIC and RT-PCR validation,it proved aad B gene can mediated the aminoglycoside drug resistance of the strains,and cmlA gene could not mediate the resistant mechanism of the strains to chloramphenicol.The MIC determination of twenty Pasteurella multocida isolates which laboratory existing and drug-resistant genetic epidemiology investigation of the seven drug resistant genes which existing in Pasteurella multocida RCAD0259 show that the resistance genes aad B,arr-2,sul2 and dfr A widely exists(?65%)in Pasteurella multocida,and the gene arr-2 which 75%of Pasteurella multocida strains carry had not successfully mediated rifampicin resistant of strains.