| Aeromonas hydrophila(A.hydrophila)is a typical opportunistic pathogen of fish,livestock,and humans.The bacterial sepsis caused by the bacteria every year hinders the healthy development of aquaculture industry and seriously threatens the veterinary public Health and safety.At present,our country is still using different chemical drugs and antibiotics to prevent and treat diseases caused by A.hydrophila.However,due to the uncontrolled use of antibiotics,the resistance of A.hydrophila is gradually increasing.In addition,excessive use of antibiotics will cause a large number of drug residues in fish,which will endanger the health of fish and even humans,and bring unprecedented challenges to the treatment of related bacterial diseases.According to related studies,A.hydrophila mostly exists in the environment in the form of Biofilm(BF).Bacterial BF can make it more capable of resisting bad environments and maintaining viability than bacteria in planktonic state,making the bacteria less vulnerable to antibiotics or conventional Disinfectant kills and can escape the defenses of the body’s immune system,playing an important role in the process of bacterial resistance and disease.Therefore,how to effectively prevent and remove the bacteria BF has become an urgent task and mission at this stage,which is of great significance to the prevention and treatment of diseases caused by A.hydrophila in the freshwater aquaculture industry in my country.Bacteriophage(phage)is a virus that completes self-growth and reproduction in bacteria and releases itself into the environment by lysing bacteria.Bacteriophages use bacteria as their natural hosts and can specifically kill the corresponding host bacteria.Therefore,it is of great significance to use bacteriophages to control the diseases caused by A.hydrophila.Studies have shown that bacteriophage derivative polysaccharide depolymerase(Depolymerase)has significant advantages in combating bacterial BF: 1.Phage polysaccharide depolymerase can effectively inhibit the formation of bacterial BF and destroy the formed bacterial BF,and can degrade bacteria Capsular polysaccharide;2.As a protein,polysaccharide depolymerase has good stability and is not easy to cause adverse reactions and bacterial mutations;3.As an enzyme preparation,polysaccharide depolymerase can be used alone to identify the serotype of capsular polysaccharide of capsular bacteria,mixed Use("cocktail therapy")to combat the complex biofilm environment and facilitate the research of bacterial resistance.Therefore,bacteriophages and the depolymerase secreted by them have important potential in dealing with bacterial infectious diseases.In this study,A.hydrophila Ah-152 was used as the host bacteria,and a strain of bacteriophage PZL-Ah152 was isolated from the sewage collected in Changchun City.The results of biological characteristics show that the phage can lyse 7 strains of Aeromonas hydrophila and 1 strain of Aeromonas velvet;the best MOI is 0.1;the incubation period is 20 min and the plateau period is 80 min;the p H value is 6,7,The most stable at 8;can maintain high activity at 30 ℃,40 ℃ and 50 ℃.According to transmission electron microscopy,the phage is classified into Caudovirales(Caudovirales)and Podoviridae(Podoviridae).In the double-layer plate experiment,it was found that the phage had an aperture around the plaque,and it was verified that the phage PZL-Ah152 can clear the formed biofilm.The genome-wide determination and analysis of PZL-Ah152 found that its full length is 40,828 bp,with 54 open reading frames(ORF),and G+C% is 51.75%,of which 45(83.33%)ORFs can be compared with the existing ones.The functions of known sequences have high similarity.The minimum size of the protein encoded by the 45 CDS is 6.26 k Da(ORF26)and the maximum is 149.96 k Da(ORF48).Among all 54 ORFs,49(90.7%)start codons are ATG,4(7.4%)start codons are GTG,and only 1 start codon is TTG;16(29.6%)One stop codon was TGA,33(61.1%)stop codons were TAA,and 5(9.3%)stop codons were TAG.Comparison analysis shows that PZL-Ah152 is similar to Aeromonas phage T7-Ah,Klebsiella phage v B＿Kpn P＿Sibilus,Dickeya phage v B＿Dso P＿JA10,Yersinia phage Yep-phi,Erwinia phage p Ep＿SNUABM＿h1,Viobrage JVP＿Vp＿SNUABM＿h1,Viobrage JV30 and Viiobrio phage phage p Ep＿SNUABM＿h1,Viiobrage J.The sexes are 95.76 %,79.03 %,78.85%,77.35%,74.81%,72.87%,71.82%,71.41%,71.35%,which are closely related to Aeromonas phage PZL-Ah1,but the PZL-Ah152 genome still has its own unique Part of the sequence of phage,such as 6529-6645 bp,7132-7290 bp,9361-9570 bp,15917-16279 bp,17798-18361 bp,18405-18563,22670-22861 bp,28018-29739 bp,is unique to the phage PZL-Ah152.In addition to the similarities between phage PZL-Ah152 and phages of different genus,we found that it also has 38% and 39%similarities with some genes of Fusobacterium(ORF32)and Escherichia coli(ORF33).It may be PZL-Ah152 has a competitive and co-evolution relationship with them.PZL-Ah152 was found in the double-layer plate experiment to produce an aperture around its plaque,indicating that the phage may encode and secrete depolymerase.The whole genome analysis of PZL-Ah152 speculated that ORF47(tail filament protein)might be the encoded depolymerase gene.The ORF was expressed and purified and named Dep47.Dep47 can be solublely expressed in BL21 competent cells.The depolymerase Dep47 is composed of 573 amino acids,the molecular weight is 62.4 KDa,and the PI is 5.5.The depolymerase Dep47 and Ralstonia solanacearum depolymerase(BCN10537.1)are on the same branch,and their relationship with other depolymerases is relatively compared.far.Dep47 can inhibit the biofilm formation of A.hydrophila and the growth of A.hydrophila,and its inhibitory ability is directly proportional to the concentration.According to the experimental results,we can know that Dep47 is a new depolymerase,which can be applied to the control of A.hydrophila in the biofilm state. |
