Isolation,purification And Preliminary Application Of Phages Against Aeromonas Hydrophila ZYAH131 Strain

Aeromonas hydrophila is a common opportunistic pathogen in the aquatic environment,with a wide range of infecting hosts,almost all major freshwater aquaculture species can be infected.Grass carp infection can cause enteritis,sepsis and other diseases,causing serious losses to my country’s aquaculture industry.In recent years,its drug resistance problem has brought new challenges to the prevention and control of the bacteria.Phage can specifically lyse bacteria,and has the advantages of fast reproduction,strong specificity,and low pollution,and has the potential to become an alternative to antibiotics.Characterization,genome sequencing,and preliminary application studies are carried out.The details are as follows:The Aeromonas hydrophila grass carp isolate ZYAH131 was subjected to multi-locus sequence analysis（MLST）,and ZYAH131 was determined to be ST517based on the six housekeeping gene sequences of gyr B,gro L,glt A,met G,pps A and rec A.Taking ZYAH131 as the host strain,158 samples were collected from nine provinces,municipalities and autonomous regions including Harbin,Heilongjiang,Hefei,Anhui,Wuhan,Hubei,Chengdu,Sichuan,Guiyang,Zhejiang,Guangzhou,Guangxi,and Hulunbuir,Inner Mongolia.For phage screening,a total of phages were isolated and obtained.24 strains,named ZPAH127-ZPAH150.The results of the lysis curve showed that all 24 phages of Aeromonas hydrophila could inhibit the growth of Aeromonas hydrophila ZYAH131 within a certain period of time,and had good lysis activity.Among them,phages ZPAH132 and ZPAH140 had the longest antibacterial time and lysis activity.the strongest.Bacteriophages ZPAH132 and ZPAH140 were both lytic phages,and they appeared round translucent plaques with regular size on double-layer agar.Transmission electron microscope observation showed that phages ZPAH132 and ZPAH140 both have tails and icosahedral heads,belonging to the order of caudate phages.Phages ZPAH132 and ZPAH140 have good temperature tolerance and can maintain stable titers in the temperature range of 30℃-60℃.ZPAH132 has a stable titer between p H 4-10,while ZPAH140 has a wider p H tolerance range of p H 4-11.Under the optimal multiplicity of infection（MOI=0.001）,the adsorption rate of phage ZPAH132 for 15 min was 98.85%.The growth curve results showed that the incubation time of phage ZPAH132 was 25 min,the growth time was 50 min,and the titer in the stationary phase was 10¹⁰PFU/m L.At the optimal multiplicity of infection（MOI=0.1）,the adsorption rate of phage ZPAH140 for10 min was 90.77%.The growth curve results showed that the incubation time of ZPAH140 was 25 min,the growth time was 25 min,and the titer in the plateau phase was10⁹PFU/m L.Both of the above two phages showed the characteristics of short incubation period,high titer in the plateau phase and strong lysis ability.The results of whole genome sequencing showed that the genome size of ZPAH132 was 53.340 kb,the G/C content was 52.3%,and there were 69 open reading frames（ORF）;the genome size of ZPAH140 was 52.412 kb,and the G/C content was 46.9%.There were a total of 64 open reading frames（ORFs）,and neither virulence genes nor antibiotic resistance genes were found in the above genomes.In the grass carp control test,ZPAH132 can provide 50%（prevention）and 62.5%（treatment）protection against the infection of Aeromonas hydrophila ZYAH131 strain,while ZPAH140 can provide 50%（prevention）and 50%（treatment）.protective effect.In conclusion,the phage ZPAH132 and ZPAH140 have strong lytic ability and low potential risk,and have a certain protective effect on the infection of Aeromonas hydrophila.The results of this study provide a scientific basis for the development of new biological control strategies for Aeromonas hydrophila.