Sequencing And Analysis Of Plasmid Sequence Of Avian Pasteurella Multocida

Pasteurellosis is a poultry contagious disease caused by Pasteurella multocida(Pm),whose characteristic performances including septicemia,inflammatory hemorrhaged diarrhea and high mortality.The abuse and high dose of antibiotics result in more drug resistance to Pm,and poor effect in clinical prevention and treatment.In order to study the relationship between Pasteurella multocida plasmid and drug resistance,two strains of Pseudomonas aeruginosa(FC6605 and FC6607)were identified by bacterical culture,capsular identified,drug sensitivity assay,extraction of plasmid and agarose gel electrophoresis.Then constructed positive recombinant plasmid and software Seqman was used to connect sequences obtained from recombinant plasmids.A pair of primers were designed for PCR according to the sequences of FC6605-3 and FC6607-3.The sequence of PCR products was compared with the sequence published on NCBI using the sequence alignment tool like DNAMAN and DNASTAR.The results showed that both strains FC6605 and FC6607 were capsule A,and strain FC6605 carrying 5 different length plasmids,and FC6607 carriying 7 different length plasmids.According to the drug sensitivity test of 24 different tablets,the strain FC6605 was only sensitive to florfenicol,and the strain FC6607 was sensitive to amoxicillin,florfenicol and doxycycline.Both strains were Multidrug resistant Pasteurella multocida.The full length of Plasmid FC6605-3 sequences was 3086bp,which contained 23 open reading frame(ORF).And the full length of plasmid FC6607-2 sequences was 3086bp,which contained 25 open reading frame(ORF).The results showed that there were 80 gene sequences or genomes homologous to the nucleotide sequence of plasmid FC6605-3,among which 10(95%?100%)had higher homology.The average G+C content was 43.51%with even distribution among the whole sequences.There were 80 gene sequences or genomes homologous to the nucleotide sequences of plasmid FC6607-2,among which 9(96%?100%)had higher homology,the average G+C content was 43.51%with even distribution among the whole sequences.The results of BLAST showed that 23 different sized open reading frame-encoded amino acids were found in plasmid FC6605-3,Seven of these ORFs were highly similar(100%)with trimethoprim resistant dihydrofolate reductase DfrA from different bacteria two ORF and aminoglycoside adenosine transferase similarity of 100%,two ORF-encoded amino acid and dynamic protein similarity of 100%,one ORF-encoded amino acids and transposase similarity of 50%,one ORF-encoded amino acid has a similarity of 68%to the transcriptional regulator,one ORF-encoded amino acids and dihydropterin synthase reductase similarity of 100%,five ORF-encoded amino acids and the hypothetical protein of different bacteria has similar(59%?95%),four ORF-encoded amino acids did not find amino acids similar to any bacteria.In the amino acids encoded by the 25 open reading frames of plasmid FC6607-2,seven of these ORFs had a high similarity(80%?100%)with trimethoprim resistant dihydrofolate reductase DfrA from different bacteria,two ORF and aminoglycoside adenosine transferase similarity of 100%,three ORF-encoded amino acids and dynamic protein similarity of 96 to 100%,one ORF-encoded amino acid has a similarity of 68%to the transcriptional regulator,one ORF-encoded amino acids and dihydropterin synthase reductase similarity of 100%,five ORF-encoded amino acids and the hypothetical protein of different bacteria has similar(59%?95%),five ORF-encoded amino acids did not find amino acids similar to any bacteria.Through the amino acid comparison of ORF,it was found that the resistance genes of trimethoprim resistant dihydrofolate reductase DfrA and aminoglycoside adenosine transferase were found on plasmids FC6605-3 and FC6607-3,The results of further susceptibility test showed that FC6605 and FC6607 had different resistance to the antibiotics,revealing the presence of antibiotic resistant genes in the plasmid of Pasteurella multocida.