Research And Analysis Of Bacterial Sepsis And Antibacterial Peptide NK-lysin In Pseudocrossocheilus Bamaensis

Pseudocrossocheilus bamaensis belongs to Cypriniformes,Cyprinidae,and Pseudocrossocheilus.It is mainly distributed in the Red Water River system in Guangxi and is a valuable indigenous economy fish in Guangxi.Affected by factors such as overfishing and the deterioration of natural habitats,the wild resources of P.bamaensis gradually dried up.Due to the high requirements for artificial breeding environment of P.bamaensis,especially when the water temperature is high,bacterial diseases mainly caused by septicemia are prone to outbreak,which greatly restricts the large-scale breeding of P.bamaensis.At present,the prevention and treatment of bacterial diseases in fish are mainly based on the application of conventional chemical drugs such as antibiotics.However,the research on the theory and practice of fish antibiotics is still relatively weak,and the method of administration is lacking.Misuse of antibiotics not only makes it difficult to effectively control the prevalence and outbreak of farmed fish diseases,but also causes the resistance of bacteria to increase continuously.Therefore,it is of great significance to carry out research on the prevention and treatment of P.bamaensis diseases and new antibacterial drugs such as antibacterial peptides for the protection of germplasm resources and healthy breeding.1.Isolation,identification and pathogenicity analysis of A.hydrophila from P.bamaensisIn this study,a dominant strain of bacteria（named BM178）was isolated and purified from the diseased P.bamaensis.Comprehensive the comprehensive morphological characteristics,physical and chemical characteristics and sequence analysis results,it was identified as A.hydrophila.After artificial infection in the abdominal cavity,the pectoral fin,ventral fin,and anal fin base of the P.bamaensis bleed,and the anus is red and swollen.The necropsy shows spotted bleeding of the visceral mass,liver congestion,splenomegaly,and intestinal congestion.The symptoms are basically the same as the natural symptoms.The BM178 strain has median lethal dose(LD₅₀)of 1.26×10² CFU/g for healthy P.bamaensis.In addition,the detection rate of six common virulence genes in this strain reached 100.0%,and the virulence genotype was hly⁺aer⁺act⁺alt⁺ahp⁺ahal⁺.Drug sensitivity results showed that the strain was highly sensitive to seven drugs including ceftriaxone,cefoperazone,gentamicin,doxycycline,norfloxacin,compound sulfamethoxazole,and florfenicol.However it has developed resistance to penicillin G.The above results indicate that A.hydrophila is the main pathogenic bacteria leading to the violent death of P.bamaensis,and has strong pathogenicity.During the breeding process,doxycycline,compound sulfamethoxazole and florfenicol can be used for prevention and treatment.2.Analysis of sequence and expression characteristics of antibacterial peptide NK-lysin gene（PbNKL）in P.bamaensisThe PbNKL gene has a total length of 523 bp,which is similar to the carp Nkl6 gene structure,and is composed of three exons and two introns.The ORF spans 339 bp and encodes 112 amino acids.The mature peptide of PbNKL is a hydrophilic cationic peptide,without signal peptide,containing a conserved Saposin B domain and 6 highly conserved cysteine.It belongs to the typical SAPLIP family members.Homology and phylogenetic analysis showed that PbNKL and carp Nkl6 had a high homology of 85.0%,and they clustered with carp Nkl6 in the phylogenetic tree.q PCR analysis showed that in healthy fish,PbNKL is expressed differently in different tissues,mainly in gills,head kidney,spleen and trunk kidney.After A.hydrophila infection,the expression of PbNKL in liver and head kidney increased significantly（0～6 h）（P<0.05）;while the expression in spleen was significantly reduced（0～3 h）（P<0.05）;In the gills,the expression of PbNKL showed a tendency to increase first,then decrease,and then increase again within 0～72 h after infection by A.hydrophila,indicating that PbNKL may play a role in the immune response of fish to pathogenic infection.3.Analysis of antibacterial activity of NKL26 derived peptides and preliminary study of antibacterial mechanismBased on multi-sequence alignment,the conserved region of fish-derived NK-lysin peptides was analyzed,and the sequence information of PbNKL derived peptide（NKL26）from P.bamaensis was successfully obtained,and the high-purity peptide（98.9%）was synthesized chemically.MIC and MBC results show that NKL26 has a broad-spectrum antibacterial activity,especially a strong inhibitory effect on Gram-positive bacteria.The analysis of dose-effect and time-effect curves showed that the bactericidal effect of NKL26 on A.hydrophila and Streptococcus agalactiae was positively correlated with the concentration and duration of action.Further gel electrophoresis results showed that NKL26 can enter the A.hydrophila by destroying the target cell membrane and interact with bacterial genomic DNA.In summary,A.hydrophila BM178 is the main pathogenic bacteria causing septicemia in P.bamaensis and has strong pathogenicity.It is recommended to use drugs such as doxycycline to prevent and treat it.The expression pattern of PbNKL in different tissues is different when the P.bamaensis responds to BM178 strain infection,and the derived peptide NKL26 has extensive antibacterial activity,indicating that the P.bamaensis NK-lysin plays a important role in the immune response.