Isolation,Identification And Biological Characteristics Analysis Of Lactococcus Petauri From Siberian Sturgeon

China is the largest sturgeon farming country in the world.The rich sub-cold water resources in the southwest have promoted the rapid development of the regional sturgeon farming industry.Hybrid sturgeon bred with Siberian sturgeon(Acipenser baerii)as the female parent is the main farming species in the current sturgeon industry.However,the problem of bacterial infection in A.baerii broodstock after laparotomy in summer and spring has become a clinical bottleneck that threatens the broodstock reserves of A.baerii.Bacterial diseases are one of the important infectious diseases in sturgeon farming.Multi-bacterial co-infection is the focus of clinical disease research.However,the role of non-lethal strain in the development of co-infection was rarely reported.In this study,the pathogenic bacteria were isolated from the bacterial septicemia of A.baerii broodstock by microbiology and pathology experiments.Further sequencing to obtain the complete genome sequence of the non-pathogenic lactococcus sp.JLlg08PX.Then the genome component and gene annotation were analyzed.Phylogenetic tree based on a full-length sequence of 16S ribosomal RNA(16S r RNA)gene,average nucleotide identity(ANI)and digital DNA-DNA hybridization(d DDH)analysis were used to determine the species-level classification of Lactococcus sp.JLlg08PX and reclassification of five L.garvieae strains.To enrich the biological characteristics of lactococcus sp.JLlg08PX,growth curve determination,morphological observation and culture condition tolerance analysis were carried out by bacterial culture experiments.Bacterial biochemical properties were analyzed by VITEK?2 gram-positive(GP)identification card,and the gastrointestinal resistance characteristics were analyzed by acid and bile salt tolerance tests.In addition,the bacteriostatic properties of the strain were analyzed by the in vitro antibacterial experiment of strain cell-free supernatant(CFS)and the in vivo Aeromonas spp.load experiments of co-infection.The main findings are as follows:1.Discovery of non-pathogenic Lactococcus sp.JLlg08PXTwo kinds of bacteria,JLAHX and JLlg X were isolated and purified from the pericardial cavity,liver,mesonephros and spleen of the five dying A.baerii broodstock after laparotomy.The JLAHX hasβ-hemolytic activity and carried virulence genes of hemolysin(hly A)and cytotoxic enterotoxin(alt),while the JLlg X has no hemolytic activity.16S r RNA gene universal primer amplification sequence alignment and phylogenetic tree analysis verified that the JLAHX belongs to Aeromonas hydrophila and the JLlg X belongs to genus Lactococcus,but the latter one could not be classified at the species level.Interestingly,the animal regression infection showed that the cumulative mortality of hybrid sturgeon(A.baerii♀×A.schrenckii♂)was 0%in the Lactococcus sp.group.The cumulative mortality rate of hybrid sturgeon infected with A.hydrophila ranged from 77.78%to 88.89%.However,the value was decreased to 33.33%after co-infection with Lactococcus sp.and A.hydrophila.Meanwhile,histopathological observation showed that the necrosis of hepatocytes and the damage of valvular intestinal mucosa caused by A.hydrophila infection were alleviated in the co-infection group.In the pathogenicity test of Lactococcus sp.JLlg08PX was further infected with A.baerii by intraperitoneal injection with 1.0×10~8 CFU/fish,oral gavage with 1.0×10~8 CFU/fish and immersion with 5.74×10~7 CFU/fish.No death and clinical symptoms were observed within 14 d.The detection rate of Lactococcus sp.JLlg08PX was only 16.67%at 14 d past intraperitoneal infection.These results indicated that A.hydrophila was the pathogen of bacterial septicemia in A.baerii broodstock,while Lactococcus sp.was a non-pathogenic strain,and the co-infection of Lactococcus sp.could alleviate the histopathological damage and morbidity mortality of sturgeon caused by A.hydrophila.2.Complete genome sequencing and analysis of Lactococcus sp.JLlg08PXThe genome of Lactococcus sp.JLlg08PX consists of a 2,113,250 bp chromosome and a 45,868 bp plasmid,with a total genome length of 2,159,118 bp and a GC content of 38.01%.2,132 coding genes,59 r RNAs,16 t RNAs and 35 s RNAs were predicted in the Lactococcus sp.JLlg08PX genome,and the prediction information of tandem repeat,interspersed repeat,genome island and prophage in the genome was obtained.Meanwhile,1790,1520,1160,114 and 449 genes were annotated in COG,GO,KEGG,CARD and PHI databases,respectively.It was worth noting that the ANI values of Lactococcus sp.JLlg08PX with L.petauri strains 159469,LZys1 and CF11 were greater than 96%(98.55%,98.32%and98.15%,respectively)and the d DDH values were greater than 70%(86.00%,84.30%and83.80%,respectively).The results showed that Lactococcus sp.JLlg08PX belongs to L.petauri.In addition,the ANI and d DDH values of L.garvieae strains ERR1600742-bin.28,FDAARGOS1002,FDAARGOS1062,FDAARGOS1063 and MGYG-HGUT-00230 with L.petauri JLlg08PX were greater than 96%and 70%,respectively.Therefore,five L.garvieae strains were can be re-identified as L.petauri based on the complete genome sequence of L.petauri JLlg08PX.3.Biological characteristics analysis of L.petauri JLlg08PXWhen L.petauri JLlg08PX was inoculated in TSB culture at 28℃and 180 r/min,it entered the logarithmic phase from 2 h and stationary phase from 6 h.L.petauri JLlg08PX grew on BHIA and MRSA medium as translucent,neat edge,circular bulge shape,moist and milky white colonies.The bacteria were ovoid with an average size of 1.00±0.13μm×0.63±0.03μm and distributed single or in chains.L.petauri JLlg08PX grew at 18～45℃,p H 7～11 and 2～6%Na Cl(w/v).Moreover,the survival rate of L.petauri JLlg08PX reached56.65%±11.10%in p H 4 medium for 3 h,and it could also tolerate 0.2%,0.3%and 0.4%bile salt.In addition,in vitro bacteriostatic experiments showed CFS of L.petauri JLlg08PX could inhibit the growth of A.hydrophila,A.veronii and Yersinia ruckeri except Streptococcus agalactiae.In summary,a non-pathogenic L.petauri JLlg08PX was obtained from A.baerii broodstock.The pathogenicity and biological characteristics analysis of the L.petauri JLlg08PX enriched the basic data of L.petauri and provided new insight for the impact of non-lethal strains on the development of the disease in co-infection.