| Avian Pasteurella multocida is a pathogen that causes fowl cholera in poultry and wild birds,which seriously harm the poultry industry.In recent years,the multidrug resistance phenomenon of avian P.multocida has become increasingly serious because of the extensive and irrational use of antimicrobial agents in the poultry industry,which lead to a large number of sick poultry deaths and huge economic losses in the poultry industry as a result of bad antimicrobial treatment.Therefore,it is of great significance to study the drug resistance mechanism and to find new targets for drug action in avian P.multocida.Numerous previous studies indicated that the clinical isolates of avian P.multocida were multidrug resistant to antimicrobial agents such as aminoglycosides,tetracyclines,sulfonamides and fluoroquinolones,which was mainly caused by drug-resistant plasmids and drug target changes,but its exact resistance mechanism is not clear.In this study,the molecular resistance mechanism of avian P.multocida to streptomycin,ciprofloxacin and tetracycline was studied from the transcriptome level on the basis of resistant strains obtained by artificial induction in vitro.The main research contents and results shown as follows:Firstly,inducting the drug-resistant strains of avian P.multocida in vitro and detecting their characteristic.After the standard strain C48-1 of avian P.multocida was continuously cultured on TSA medium containing 1/2 MIC of corresponding antibiotics for25 generations,the highly resistant strain Str~R,moderate resistant strains Cip~R and Tet~R were successfully obtained.Compared with the standard strain,the growth rate,physiological and biochemical characteristics of the drug-resistant strains did not change significantly.The sensitivity of these drug-resistant strains to a variety of antimicrobial agents decreased or reached to resistance,and they still maintained a stable drug resistance even if these drug-resistant strains were continuously cultured on non-resistant medium for20 generations.These stable drug-resistant strains might be used for transcriptional sequencing.Secondly,the transcriptional sequencing and bioinformatics analysis of resistant strains.In order to compare the differences of gene expression before and after drug resistance,and investigate the function of differentially expressed genes and the metabolic pathways involved by analysis of GO,KEGG and ARDB databases,the total RNA was extracted from the standard strain and the drug-resistant strains and subjected to RNA-Seq sequencing.The results showed that compared with the standard strains,there were 625differentially expressed genes from Str~R strain,of which 584 were up-regulated and 41down-regulated,involved 11 types of molecular functions,19 biological processes and 84metabolic pathways,9 drug-resistance related genes mainly including aminoglycoside transferase and efflux pump retrieved by ARDB;There were 442 differentially expressed genes from Tet~R strain,of which 103 were up-regulated and 339 down-regulated,involved12 molecular functions,18 biological processes and 90 metabolic pathways,4drug-resistance related genes mainly including ribosomal protective protein and efflux pump retrieved by ARDB;Cip~R strains had 19 differentially expressed genes,of which 15were up-regulated and 4 down-regulated,involved 3 types of molecular functions,8biological processes and 6 metabolic pathways,and did not retrieve resistance-related genes.Finally,the differentially expressed genes were verified by fluorescence quantitative PCR.Ten differentially expressed genes were selected from the three groups of transcriptome data to verify.The melting curve,amplification curve and standard curve of each gene were analyzed by SYBR Green real-time quantitative PCR with 16S rRNA as internal reference gene and serial dilution standard strain cDNA as template.The results showed that there were the good reaction specificity with single peak of melting curve of each gene and the better reproducibility with the amplification curve.The correlation coefficient R~2 of the internal reference gene and the target genes were more than 0.99,and the amplification efficiency E value was 0.9-1.0,of which the difference between the E values was less than 5%,and the CT values were within 10-35.It was suitable for the2~-CtCt method to detect the mRNA expression level of the different genes.The relative expression of each gene was analyzed by the 2~-CtCt method of real-time fluorescence quantitative PCR with the appropriate dilution samples as the starting template concentration.The results showed that the expression trend of the three groups'differential genes was verified consistent with the transcriptome.In summary,three stable resistant strains(Tet~R,Str~R and Cip~R)were obtained in this study.A preliminary study suggested that the multidrug resistance of Tet~R and Str~R strains were mainly mediated by the synergistic effect overexpression of ABC transporter and ribosomal protective protein or aminoglycoside transferase expression.The multidrug resistance of the Cip~R strain was mainly mediated by the overexpression of the ABC transporter. |
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