Isolation And Identification Of Lactic Acid Bacteria Against Aeromonas Hvdrophila And Screening Of Mutagenesis Strains

Aeromonas hydrophila can cause bacterial sepsis in many fishes,causing significant economic losses to farmers.Due to the unreasonable use of antibiotics and antibiotics,Aeromonas hydrophila has serious drug resistance.Therefore,screening probiotics that can replace antibiotics is of great significance for freshwater fish diseases caused by A.hydrophila.In this study,fishderived lactic acid bacteria were screened,and Atmospheric and Room Temperature Plasma mutagenesis was used for the first time to enhance the antibacterial effect of lactic acid bacteria against A.hydrophila,and related studies were carried out.The intestinal samples of fish were collected,enriched with Lactobacillus broth medium and screened with 1% calcium carbonate MRS medium.After morphological identification,16S rDNA identification,physiological and biochemical identification,one strain of L.lactis was obtained.The antibacterial experiment of L.lactis L-3 against A.hydrophila was carried out by Oxford cup method.It was found that L.lactis L-3 was an antibacterial lactic acid bacteria and could produce a 14 mm diameter inhibition zone against A.hydrophila.And the whole genome sequencing,the full length of its genome was 2.49 Mbp,GC content was 35.06%,The functional annotation of genome-coded proteins in KEGG,GO and COG databases was performed.The bacteriocin gene clusters were searched in BAGEL4 database,and the gene clusters of L-3 strain with six bacteriocins were analyzed.In order to enhance the bacteriostatic effect of L.lactis L-3,ARTP mutagenesis was carried out.The experimental conditions were as follows: Power 120 W,Gas 10 SLM,Space 2 mm.After treatment for different time,it was found that after 70 s mutagenesis,the fatality rate reached 90%,and the probability of obtaining high-yield mutagenesis strain was high under this condition.Through co-culture of the mutated strain and A.hydrophila,preliminary screening by turbidimetric method and re-screening by Oxford cup method,a mutant A13 with enhanced antibacterial activity was screened,and its antibacterial zone diameter was 18.6 mm.And it has genetic stability and antibacterial stability.The L.lactis L-3 and mutant A13 were subjected to transcriptome sequencing,the metabolic pathways of the significantly different genes were analyzed in the KEGG database.Differential genes are mainly enriched in purine metabolism,valine,leucine and isoleucine biosynthesis metabolism,and ABC transport system metabolic pathways.The antibacterial activity of mutant A13 was enhanced,which was preliminarily determined to be able to synthesize more purines,amino acids and ABC transporter proteins.Among them,leucine and isoleucine can be used as the substrates for bacteriocin synthesis,and can also induce bacteriocin production through quorum sensing mechanism.Moreover,the up-regulation of multiple proteins in the ABC transport system may be related to the extracellular transport of bacteriocin.In summary,a mutant strain was obtained by ARTP mutagenesis and A.hydrophila antibacterial screening for L.lactis isolated from the intestinal tract of fish in this study.Transcriptome sequencing showed that it can synthesize more purine and amino acids,which may be the potential reason for its enhanced antibacterial activity.This study provides strain resources and experimental basis for the development of new type lactic acid bacteria.