Study On The Mechanism Of Cetobacterium Somerae In Protecting Zebrafish Against Aeromonas Hydrophila Infection

Aquaculture is the fastest growing food production industry in the world.However,with the development of aquaculture,the problem of disease became more and more serious.The use of probiotics is one of the common methods to control the disease in aquaculture.The gut is one of the most important immune organs of fish,moreover,the gut microbes are important mediators for maintaining intestinal homeostasis and intestinal health.In addition,metabolites of gut microbiota also play an important role in maintaining fish health.Probiotics play an important role in regulating fish intestinal microbes and their metabolites.However,the mechanism by which probiotics regulate gut microbes to improve host disease resistance remains unclear.This study used zebrafish as an animal model to systematically investigate the mechanism of Bacillus velezensis 1704-Y regulating the Cetobacterium to affect the metabolism of vitamin B₁₂,thereby affecting the intestinal ecological network to improve the host’s resistance to pathogen infection.This study can provide scientific basis for promoting the green and healthy development of aquaculture.Our main results are shown as follows:1.Study on the effect of Bacillus velezensis 1704-Y in enhancing resistance to Aeromonas hydrophila infection in zebrafishIn this study,zebrafish were randomly divided into two groups.one was fed with common diet（group CK）,and another was fed with B.Velezensis 1704-Y supplemented diet（group Y）.Zebrafish were fed twice daily（3%total weight daily）for 28 days.At the end of feeding trial,three fish from each tank were euthanized.The gut,kidney,and liver were sampled,and stored at-80^oC until DNA extraction.Then the rest of zebrafish（25 fish in each replicate）were immersed in water containing Aeromonas hydrophila with a final concentration of 2×10⁸CFU/m L for 10 days.We found that dietary supplemented with B.velezensis could significantly inhibit the propagation of Aeromonas in the intestine,and significantly prevent the translocation of Aeromonas to other tissues.More importantly,dietary supplemented with B.velezensis significantly improved the survival rate of zebrafish after bath infection with A.hydrophila.The results of 16S r RNA gene sequencing analysis showed that B.velezensis significantly influenced the composition of gut microbiome in zebrafish（ANOSIM,R=0.647,P=0.001）.However,no protective effect was observed when the fish were fed a diet containing an antibiotic mix before oral administration of BV1704-Y,suggesting that gut microbiota played a key role in the protection of fish from A.hydrophila infection.Cetobacterium was strongly associated with the administration of B.velezensis,suggesting that Cetobacterium could be identified as the potential biomarker in group Y.These results raised the possibility that Cetobacterium have positive interaction in the protection of the host against pathogenic bacteria by B.velezensis.2.Whole genome sequencing and functional analysis of Cetobacterium somerae CS2105-BJA total of 126 strains of bacteria,including 34 strains of Cetobacterium,were isolated from the gut of zebrafish.Dietary supplemented with Cetobacterium could significantly increase the content of Cetobacterium in the gut of zebrafish,and protect zebrafish against the A.hydrophila infection.The results of complete genome of Cetobacterium showed that the genome size of Cetobacterium was 2.97 Mb,containing 1 ring chromosome,6 ring plasmids,and the GC content was 30.91%.After annotation by the KEGG database,the genes of carbohydrate metabolism had the highest abundance,followed by amino acid metabolism,energy metabolism,and metabolism of cofactors and vitamins,indicating that Cetobacterium had a strong ability to metabolize and produce vitamins.Subsequently,we determined the content of vitamin B₁₂ in the gut by HPLC,and the results showed that there was a significant positive correlation between vitamin B₁₂ and the content of Cetobacterium in the gut.At the same time,the in vitro culture test showed that the highest concentration of vitamin B₁₂produced by Cetobacterium was 140 ng m L^-1 OD600^-1.The result of correlation analysis showed that the content of vitamin B₁₂ was negatively correlated with Aeromonas in the gut.These results indicated that the protection of Cetobacterium against Aeromonas infection in zebrafish may depend on vitamin B₁₂.3.Response of gut microbiome to vitamin B₁₂and analysis of molecular ecological networkZebrafish were randomly divided into two groups.one was fed with common diet（group CK）,and another was fed with B.Velezensis 1704-Y supplemented diet（group Y）.Zebrafish were fed twice daily（3%total weight daily）for 28 days.At the end of feeding trial,three fish from each tank were euthanized.The gut,kidney,and liver were sampled,and stored at-80^oC until DNA extraction.Then the rest of zebrafish（25 fish in each replicate）were immersed in water containing Aeromonas hydrophila with a final concentration of 2×10⁸ CFU/m L for 10days.The results showed that dietary supplemented with vitamin B₁₂ significantly improved the survival rate of zebrafish after bath infection with A.hydrophila,and this supported by the gut microbiota.Dietary supplemented with vitamin B₁₂ significantly influenced the composition of gut microbiome in zebrafish（ANOSIM,R=0.782,P=0.003）.It could reduce the ratio of Firmicutes to Bacteroidetes and significantly increase the content of obligate anaerobes in the gut.The results of RDA analysis also showed that dietary supplemented with vitamin B₁₂ significantly affected the gut microbial composition（R²=0.9233,P=0.0005）.Then,we developed a gut ecological network based on the random matrix theory.The results showed that dietary supplemented with vitamin B₁₂ could increase the number of network nodes,connection edges,and modules,and improve the stability and complexity of the network,which could maintain the health of gut.By constructing a partial least squares pathway model,it was demonstrated that vitamin B₁₂acts mainly through an indirect mode of action,i.e.,influencing the gut microbial key flora and increasing the complexity of the gut microbial ecological network,thereby improving the host’s resistance to pathogen infections.4.Study on the pathway of vitamin B₁₂ to protect zebrafish against pathogen infectionWe measured the gut environment of zebrafish in different treatment groups.The results showed that dietary supplemented with vitamin B₁₂ could significantly improve the expression level of gut barrier related gene Occludin,Zo-1,and Claudin15（P<0.05）.And it also could significantly increase the expression levels of intestinal barrier related proteins ZO-1 and Claudin（P<0.05）,which could prevent Aeromonas from destroying the gut barrier.Meanwhile,dietary supplemented with vitamin B₁₂ could significantly increase the activities of antioxidant enzymes SOD and GSH-Px in the gut of zebrafish,and protect the host from the attack of reactive oxygen species.Dietary supplemented with vitamin B₁₂ could reduce the redox potential in gut and maintain the anaerobic state.However,no stimulating effect on gut barrier and antioxidant system was observed when the fish were fed a diet containing an antibiotic mix before oral administration of vitamin B₁₂,suggesting that the effect of vitamin B₁₂ on gut environment depended on gut microbiota.Subsequently,correlation network analysis demonstrated that the gut microbiota affected by vitamin B12 significantly impressed the integrity of the gut barrier,the activity of the gut antioxidant enzyme,and the redox potential of the gut.Finally,the results of the partial least squares path model proved that the protective effect of vitamin B12 on zebrafish was mainly achieved by improving the stability of the gut microbial ecological network,which could enhance the gut barrier and prevent the migration of the pathogens.In conclusion,dietary supplemented with B.velezensis could effectively increase the abundance of Cetobacterium,which could produce the vitamin B₁₂.And these further promoted the stability of gut ecological network and enhanced the interaction between microbes,and mediated the stability of the gut environment,including the decrease of the gut redox potential,the enhancement of the gut barrier,and the activation of the gut antioxidant system,which could prevent the colonization and translocation of pathogens in the gut and maintain the health of the host.Our study could provide scientific basis for promoting the green and healthy development of aquaculture.