Screen And Functional Studies Of Antagonistic Bacteria And Antimicrobial Peptides Against Riemerella Anatipestifer

Riemerella anatipestifer(RA)is the main pathogen causing infectious serositis of ducklings.It is one of the most common bacterial pathogens endangering duck breeding industry in China.At present,antibiotics are mostly used to treat the diseases caused by the bacteria.However,due to genetic mutaions,bacterial drug resistance and other reasons,the effectiveness of antibiotic treatment is getting worse.Therefore,looking for new alternative control methods is one of the urgent problems to be solved in the aquaculture industry.In this study,microbial strains with inhibitory effect on R.anatipestifer were screened through the experiment of interspecific relationship of microorganisms.The phylogenetic and genomics analysis were performed to identity the taxonomy and predict the possible antibacterial substances and inhibitory mechanism.At the same time,other known antibacterial peptides were expressed and screened,so as to provide a certain basis and theoretical basis for the biological control of R.anatipestifer.The main research progress is as follows:(1)Through the drug sensitivity test with 11 common drugs,it was determined that R.anatipestifer RA-CC and RA-ZXP were resistant to a variety of antibiotics,among which aminoglycosides,tetracyclines and chloramphenicols were the most significant.Combined with the molecular amplification verification of known drug resistance genes,it is speculated that there is a certain correlation between the multi-drug resistance of RA and its drug resistance genes,which also provides a new basis and idea for the prevention and treatment of RA.(2)About 20 strains of antagonistic bacteria of R.anatipestifer were screened,isolated and purified from environmental samples and preserved strains.Among them,Streptomyces filamentosus MEY2 had the strongest inhibitory ability,followed by Paenibacillus polymyxa MEZ6 and MEY9.16S rDNA sequencing analysis showed that 10 strains were Enterococcal bacteria,and 5 strains had less than 98%homology with the known bacteria in NCBI database.Other co-inoculation experiments found that Paenibacillus polymyxa had the widest antibacterial spectrum and had inhibitory effect on most of the tested bacteria.(3)Effect of co-inoculation ratio on RA-CC inhibition of R.anatipestifer.Direct contact liquid co culture experiment showed that when the inoculation ratio of MEY2 and RA-CC was≤1:10,RA-CC was completely inhibited;When the inoculation ratio of MEZ6 and RA-CC was≤1:1000,RA-CC was quickly inhibited,and there was no RA-CC under microscope.Combined inoculation of MEY2,MEZ6 and probiotic Lacticaseibacillus rhamnosus MEY7 against a variety of pathogenic bacteria showed that the phenotype of combined inoculation was consistent with that of single inoculation of the dominant strain.(4)The synthetic genes of 11 major antimicrobial peptides in Paenibacillus polymyxa MEZ6 were heterologously expressed in Escherichia.coli.The pET32a expression vectors were constructed by enzyme digestion and enzyme linked system and transferred to BL21 strain for IPTG induced expression.Among them,9 proteins were successfully induced and 2 proteins were not detected.Through the co-inoculation experiment between the expression products and bacteria,it was found that there was no obvious inhibition zone in the co connection with bacteria.It is speculated that the single gene expression may be not function.However these constructions can provide the basis for the heterologous reconstruction of the complete synthesis pathways in future.(5)Expression and screening of other antimicrobial peptides.Twenty known broad-spectrum antimicrobial peptides were screened from the antimicrobial peptide database APD.Among them,the antimicrobial peptides AP03048 and AP02856 had a certain inhibitory effect on R.anatipestifer.However,fusion expression of these antimicrobial peptides did not have a significant inhibitory effect,indicating that the heterologous expression mode had an important impact on the activity of antimicrobial peptides.In summary,in this study microbial strains with antagonistic ability against R.anatipestifer were obtained through microbial isolation,purification and co-culture experiment.The possible key genes in the representative strains were predicted,and their heterologous expression together with some other known antimicrobial peptides were tested.All the results may provide new microbial and gene resources for the biological control of R.anatipestifer.