Biological Characteristics Of Phage VB＿AceP＿PAc Of A.caviae And Its Therapeutic Effect On Intestinal Infection In Mice

Aeromonas caviae（A.caviae）is a common conditioned pathogen of human-animal-aquatic animals,which can cause a variety of diseases in aquatic animals.The cases of gastroenteritis and even acute death caused by A.caviae strain are also gradually increasing,which seriously affects the development of aquaculture industry and public health security.At present,antibiotics are mainly used to treat diseases caused by A.caviae.However,the abuse of antibiotics not only causes serious drug residue problems,endangering human health,but also increases the difficulty of treating related diseases caused by drug-resistant A.caviae.Finding a new product that can replace antibiotics has become a current research hotspot.Bacteriophage is a kind of virus that can complete self-growth and reproduction in bacteria.It is widely distributed in any environment.Phages are highly specific to drug-resistant bacteria and can kill their target host bacteria with specific infection.Therefore,the application of phage to control the disease caused by A.caviae is of great significance.In addition,the isolation of phage is simple,the development cycle is short,and the cost is low.The dose required in the course of treatment is small,and it can quickly spread all over the body when entering the animal body,which has the advantage of small amount and high efficiency.In addition to killing pathogens,phages also have the function of removing biofilms.With various advantages,phage has great potential in the treatment of bacterial infection,especially drug-resistant bacterial infection.In conclusion,a lytic phage vB＿AceP＿PAc was isolated from wastewater of Changchun City using drug-resistant A.caviae AC-CY as host bacteria.Under transmission electron microscopy（TEM）,it was observed as a member of Phage cauda,and its optimum MOI was 1.One-step growth curve showed that the incubation period was 30 min and the stationary period was 80 min.It can maintain relatively stable activity in the range of pH 6-9 and temperature 4℃-50℃.Sensitive to ultraviolet light,and to ethanol,sodium hypochlorite chemical reagents are also sensitive;In addition,it also has a certain ability to clear the biofilm grown by host bacteria,which can be stored in a 4℃ refrigerator for a long timeWhole genome sequencing analysis of phage vB＿AceP＿PAc showed that its full length was 123,933 bp,which was linear double-stranded DNA with G+C content of35.73%,encoding 213 predictive open reading frames（ORFs）.The proteins encoded by ORFs ranged in size from 3.06 k Da to 131.39 k Da,among which 29 ORFs were functional proteins.The comparison analysis results on NCBI showed that there were only 9 phage strains（Aeromonas phage phi A047,Aeromonas phage phi A009,Aeromonas phage phi A019,Aeromonas Phage Phage）2L372X,Aeromonas phage2L372D,Aeromonas phage 4L372D,Aeromonas phage 4L372XY,Pseudaeromonas phage vB＿Ppe M＿KLEP7,Aeromonas phage phi A005)and vB＿AceP＿PAc have different degrees of similarity（92.03%-76.39%）,and the coverage rate is below 50%.Phylogenetic tree construction also showed that vB＿AceP＿PAc was a single phage,which was relatively distant from other phages,and may be a new phage.Its genome has multiple functional modules（structural component protein module,cracking function module and life activity function module）,does not contain lysogenicity module,and does not contain toxic force factors and drug resistance genes,so it is safe at the gene level and has therapeutic potential.The median lethal dose(LD₅₀)of A.caviae AC-CY in mice was 2.0×10⁷CFU/mouse.The phage treatment showed that mice in the phage treatment group survived30%better than those in the control group.The results of RT-qPCR showed that vB＿AceP＿PAc could reduce the expression of proinflammatory factors,and further pathological sections showed that vB＿AceP＿PAc could reduce the inflammatory diseases of tissues.In conclusion,vB＿AceP＿PAc can effectively treat intestinal infection caused by drug-resistant bacteria in mice,which lays a certain foundation for treating aeromonas infection in A.caviae.