Mechanisms Of Edwardsiella Piscicida Induced Enteritis In Big Belly Seahorses By Conjoint Analysis Of Metagenomics And Metabolomics

Seahorses(Hippocampus spp.)have extremely high medicinal and ornamental value.Actually,China takes the first attempt to industrial aquaculture of seahorse and now has the highest culture size throughout the world.While research on diseases,especially bacteria induced ones,is lagging behind the problem.Previous research mainly focused on pathogen isolation and pathological characterization,while mechanisms underlying these diseases especially the most common and harmful bacterial induced enteritis are still obscure.In this study,big-belly seahorses(H.abdominalis)and E.piscicida were used as the research object and pathogen,respectively.The research model was reconstructed by selecting the optimal combination of seahorse weight and injection concentration of the pathogen.Then the feasibility of the new model was evaluated and new parameters were supplemented in its evaluation system.Based on the upgraded model,metabolomics analysis was used to identify key virulence factors(KVFs)and core VFs(KVFs)to illustrate the effects of E.piscicida infection on intestinal microbiota and it functions,as well as antibiotic resistance organisms(AROs).Then metagenomic analysis was employed to determine the effects of pathogen infection on host metabolism,identify key metabolic biomarkers(KMBs)and functional pathways characterizing metabolic changes of the host.Finally,the pathogenesis of E.piscicida-induced enteritis driven by combined VFs was illustrated via conjoint analysis.And the results showed that:1.The optimal combination of seahorse weight(g)and challenge dose of E.piscicida(cfu/m L)suitable for modeling is 4.1–4.5 g,1×10~5 cfu/m L.The respiratory rate and expression of tight junction genes claudin 5 and occludin significantly decreased(P<0.05)on day 9,and their variation trend coincided with the pathogenesis process.After supplemented these parameters into the evaluation system,we found that the updated evaluation system(DAI)can accurately and effectively reflect the pattern and intensity of disease progression.2.Intestinal metagenomic analysis showed that(1)E.piscicida infection significantly increased the relative abundance of the opportunistic pathogens represented by Edwardsiella(70.10%)and E.piscicida(32.74%)(P<0.05),and the activities of their positively correlated 28 functional pathways represented by Flagella assembly,Bacterial chemotaxis,Bacterial secretion system,Lipopolysaccharide biosynthesis,ABC transporter and Two component system(TCS);while decreased the relative abundance of the autochonous microbiota represented by Enterococcus,Microbacterium and Lactobcillus and their positively correlated 11 functional activities,leading to dysbiosis and dysfunctions of intestinal microbiota;(2)Edwarsiella could express 123 VFs,of which 34 KVFs were highly significantly and positively correlated with the increased activities of the core functional pathways of the intestinal microbiota(P<0.01,|r|>0.7);(3)Fifteen CVFs(Flagella,Type IV pili,Lap,Bsa T3SS,Dot/Icm,Far AB,Capsule1,Hp-NAP,Legiobactin,Ira AB,Hpt,LPS3,Pho P,Bfm R,and Bop D)with significant positive correlation(P<0.01,|r|>0.7)were identified,and participated in all six pathogenic processes for the first time.(4)E.piscicida infection significantly increased the abundance of five AROs expressed by Edwarsiella,such as msb A,ade F,Hinf＿PBP3＿BCA,Ecol＿EFTu＿PLV and Ecol＿Glp T＿FOF.3.Metabolome data suggested that(1)E.piscicida infection disrupted host metabolism by mainly affecting Metabolism and Environmental Information Processing;(2)eleven KMBs were identified and enriched in ten significantly different functional pathways(P<0.05);(3)the activities of seven pathways related to nutrient transport and signal transduction,defense and translation mechanisms significantly decreased(P<0.05);(4)seven KMBs of host were identified for the first time for characterizing the infection of E.piscicida in H.abdominalis.4.Through the conjoint multi-omics analysis,we finally illustrated the mechanisms that 34 KVFs,including 15 CVFs,played a central role in coordinating other VFs to reshape the intestinal microbiota and host metabolites during motility,adherence,invasion,replication,avoidance of host defenses,and toxin release of E.piscicida.Therefore,this study successfully established a multi-omics analysis mode for analyzing pathogenesis of bacterial enteritis in seahorse,and systematically illustrated the mechanisms of combined VFs in E.piscicida induced enteritis by in H.abdominalis,which provides new research mode and theoretical references for exploring pathogenesis and preventing diseases of seahorses and even other fish-related diseases.