A Novel Pedioccocus Pentosaceus Screening And Its Effecting Disease-Resistance For Grass Carp

Probiotic as food additives continues to be an effective method to control the diseases caused by bacterial pathogens in freshwater fish aquaculture.In current study,a new strain of Pediococcus pentosaceus(SL001)was isolated from a soil sample collected at Sanya city,Hainan province.The genome and biological characterization of SL001 were studied.Its disease-resistance,growth and immunity for grass carp were also investigated.The main research contents and results are as follows:A lactic acid bacteria,named SL001,with broad-spectrum antibacterial activity against fish pathogens was isolated from soil sample at Sanya city in Hainan province.According to morphological characteristics,physiological&biochemical characterization as well as16S rRNA gene sequence,the strain was identificated towards a new P.pentosaceus strain.P.pentosaceus SL001 exhibited wide antimicrobial spectrum of against fish pathogens,including A.hydrophila,A.veronii,A.sobria,E.tarda,L.garvieae and P.shigelloide.In this study,it was found that the strain cells could withstand high heat(80°C for 15 min),antibotic(spectamycin etc.)and oxygen.In addition,SL001 was been evaluated that is safety to hepatic L8824 cells divided from liver of grass carp.The antagonistic active substance from the cell free supernatant of P.pentosaceus SL001 was not sensitive to temperature and protease.With culture time increasing,the concentration of lactic acid in the cell free supernatant was enhanced(up to 12.4mmol/L),as 0.3 mL cell free supernatant of lactic acid at a concentration of 6 mmol/L could completely inhibit the growth of A.hydrophila.The probiotic function of SL001 was studied using grass carp model.The grass carp was fed with SL001 supplemented diets at 1×10~9 CFU/g.Utilizing high-throughput sequencing technology based on Illumina MiSeq platform to sequence the intestinal microbiome of gut samples from SL001-fed grass carp,the results showed that Firmicutes,Proteobacteria and Fusobacteria was the dominant phyla in the experimental(PA)and control(DB)groups.In addition,the abundance of Pediococcus as potential probiotics in PA group was significantly higher than that in the control group,while the abundance of Aeromonas and Vibrio as potential pathogens in PA group was significantly lower than that of DB.The strain SL001 could stably colonize in gut of grass carp.After 30 days,the weight gain rate and specific growth rate of SL001-fed group increased when compared with to those of control group.It was found that more mucus-secreting goblet cells and longer intestinal villi could be seen in SL001-supplement group compared to non-supplement group by H&E staining of intestinal tissue.The genes involved in restraining muscle growth(MSTN-1 and MSTN-2)in SL001-fed group were significantly downregulated(P<0.05),whereas genes related to promoting muscle growth(IGF-1 and IGF-2)were notably upregulated(P<0.05).What's more,a significantly increased alkaline phosphatase(AKP)and acid phosphatase(ACP)activity was observed in SL001-fed groups compared with the control group after 30 days post-feeding.The immunoglobulin M(IgM)and complement 3(C3)showed higher expression in liver and spleen from grass carp with SL001 supplemented diets for 30 days,whereas the interleukin-8(IL-8)was significantly downregulated in both organs by qRT-PCR analysis.The above results indicated that SL001 strain could promote growth and enhance immunity of grass carp.Finally,the challenge test showed that SL001 could significantly reduce the mortality of grass carp after infection with A.hydrophila.The analysis of whole genome of P.pentosaceus strain SL001generated 1,842,476 bp longest contig(nuclear genome)and 76,699 bp shortest contig(plasmid genome),resulting in a genome assembly with total size of 1,919,175 bp,encoding 1,889 CDS and 2 CRISPR elements.According to COG,GO and KEGG functional annotation and RAST annotation,numerous genes involved in carbohydrates metabolism,protein and amino acid metabolism,nucleotide metabolism and membrane transport.The gene family was significantly different between P.pentosaceus SL001 and P.pentosaceus other strains through comparative genomic analysis.The gene clusters of secondary metabolite in SL001 were predicted by antiSMASH online software.Two secondary metabolite gene clusters were identified,among which cluster 1 had 40%similarity with coagulin biosynthesis gene cluster.It is speculated that cluster 1 may be a new member of the coagulin biosynthesis gene family.In summary,Pediococcus pentosaceus SL001 isolated from soil in Hainan province was a new strain by classifying and identifying with genome sequencing,which promoting grass carp disease-resistance,growth and immunity.It is found that it may contain coagulin biosynthesis gene clusters from the genomic function annotation,indicating that exploit this strain as a probiotic for food additives has potential application value in fish aquaculture.