Isolation And Identification Of Phage PT2 And Its Combined Application With Phage PZY-Ah In Treatment Of Aeromonas Hydrophila

Aeromonas hydrophila（A.hydrophila）is a common pathogenic bacterium of human and aquatic organisms.It exists widely in aquatic environment and food,and causes gastrointestinal bleeding of freshwater fishes,or even serious septicemia in fishes.This bacterium not only infected fish but also caused gastroenteritis,peritonitis and bacteremia in humans.Antibiotics as a simple and inexpensive drug,were widely used in aquaculture to control A.hydrophila infection.Due to the excessive use of antibiotics,bacteria have developed resistance to antibiotics,which was difficult to achieve the desired therapeutic effect.Therefore,it was urgently to find an antibiotic substitutes to solve the huge economic losses caused by A.hydrophila in aquaculture industry.Bacteriophage is a kind of virus that can specifically kill its host bacteria.It was widely distributed in any environment and was one of the most abundant organisms on earth.Compared with conventional antibiotics and other chemical drugs,phage therapy has the following advantages:strong specificity for host bacteria,short development cycle and easy preservation,and no drug residues in animals.However,the disadvantages of single phage are that the host range is narrow and phage-resistant strains showed during treatment.Step-By-Step（SBS）method isolates bacteriophages using wild-type bacteria and continuous bacteriophage-resistant bacteria as hosts.Simply,the primary phage and wild-type bacteria were co-incubated for more than 24h to produce bacteriophage-resistant second-generation bacteria,and then the second-generation phage was isolated.By combining the second-generation phage and the primary phage to form a mixed phage preparation,better therapeutic effect was achieved for the primary host bacteria.The main advantages of this mixed phages in clinical medicine,compared with single phage therapy,mixed phage could effectively expand the host range and showed better bactericidal effect which reduce the mutation rate of bacteria.In addition,this method is more targeted to host bacteria and resistant bacteria than conventional phage cocktails.In conclusion,A.hydrophila TPS was co-cultured with bacteriophage PZY-Ah,and isolated phage-resistant A.hydrophila TPS-2 and its bacteriophage PT2.Finally,a mixture of two bacteriophages was used to treat A.hydrophila disease of crucian carp.The biological characteristics of TPS-2 and its phage were analyzed and compared.The results showed that compared with TPS,the growth rate of TPS-2 was slow,but the ability of forming biofilm was enhanced,and the sensitivity of TPS-2 to cefoperazone and kanamycin was increased.Phage PT2 is a short-tailed phage family（Podoviridae）,and its optimal MOI=1.The incubation period was 40 min,and the maximum effective price was more than 10⁸PFU/m L when it reached the plateau.It has relatively stable activity at the temperature from 4℃to 50℃and p H from 4 to 9.Long term storage in 4°C refrigerator.There was no overlap between PT2 and PZY-Ah in the host range except the common host TPS.Furthermore,the whole genome of PT2 was sequenced and showed that the PT2was 41404 bp with double stranded linear DNA（G+C content is 50.86%）encoding44 predictive open reading frames（ORFs）.Phylogenetic and collinearity analysis showed that the phage PT2 and PZY-Ah belonged to the same branch of phylogenetic tree.And the peak heights of the two are close in the same collinear module.The difference of host range between PT2 and PZY-Ah was caused by the different amino acid sequence of the tail fiber protein.Two phages did not contain lysogenicity,virulence factors and pathogenicity integration genes,so they were safe at the gene level and had great potential for combined treatment of A.hydrophila infection.A.hydrophila TPS was used as host bacteria to test the therapeutic effect of single phage and mixed phages（PT2+PZY-Ah）on A.hydrophila.The results showed that mixed phages could improve the therapeutic effect in vitro and in vivo.In vitro treatment showed that mixed phages could rapidly kill A.hydrophila TPS while reducing the mutation rate from 5.2%×10^-4to 1.1%×10^-4 of A.hydrophila TPS,and had a better effect on biofilm eradication with the clearance rate up to 65.3%.The results of in vivo phage treatment showed that the survival rate of crucian carp treated with mixed phages was significantly higher than that treated with single phage,the Aeromonas bacterial load in the intestinal contents of crucian carp decreased rapidly to 2.6 lg,and the mutation rate in the bacteria decreased from 25.5%to 4.6%.In conclusion,based SBS mixed phage（PT2+PZY-Ah）therapy can provide a more effective and targeted treatment for A.hydrophila infection,providing a new method to solve the harm of A.hydrophila to aquaculture industry.