Isolation And Identification Of Bacillus Equi-derived Bacteria And Their Modulating Effects On Colitis And Depression-like Behavior In Mice

Ulcerative colitis(UC)and Crohn’s disease(CD)together form inflammatory bowel disease(IBD).UC is an intestinal disease caused by a variety of causes,and the disease mainly contains inflammation,impaired mucosal barrier function and dysbiosis,which can cause colon cancer in severe cases,endangering human and animal health.Although some medications help control its symptoms,long-term application can produce various side effects.Also,ulcerative colitis involves activation of the immune response during its onset,inducing psychiatric symptoms,such as depression.Therefore,alternative therapies for ulcerative colitis need to be investigated,and the effectiveness and safety of probiotic interventions for colitis and the resulting depression-like behaviors have stirred the interest of the scientific community.In this study,we firstly screened Bacillus equi with good probiotic properties,then evaluated the intervention effect of Bacillus on colitis and depression-like behavior in mice by clinical indexes,biochemical indexes and inflammatory factor indexes,and finally analyzed the flora changes of cecum contents by16S amplicon sequencing and combined analysis of colonic transcriptomics to obtain biomarkers and potential therapeutic targets.The study methods and results are as follows:1.Isolation and identification of Bacillus equi originSix Bacillus strains were isolated from fresh feces of healthy horses with negative peroxidase test and positive Gram stain,strain numbers W1.1,W2.4,Y4.2,Y4.3,L6.2and L7.1.The 16S rDNA sequencing results of the six strains were analyzed by BLAST comparison on NCBI,and the results were W1.1 for Bacillus licheniformis,W2.4 and Y4.2 were Bacillus subtilis,Y4.3 were Bacillus subtilis,L6.2 were Bacillus secureus and L7.1 were Bacillus megaterium.Their probiotic potential was also evaluated and the survival of all strains in pH=3.0 broth and in 0.3%bile salt broth was more than 20%in tolerance tests.Among them,Y4.3 and L7.1 showed high hydrophobicity and self-agglutination ability in the adhesion test assessment,and also reached more than 40%and 60%adhesion to Caco-2 cells.In terms of antioxidant capacity,Y4.3 and L7.1achieved more than 80%DPPH scavenging.In addition,six strains of Bacillus are sensitive to some common antibiotics and are not hemolytic,which can prove the safety of this Bacillus.After a comprehensive evaluation,Y4.3 Bacillus subtilis and L7.1Bacillus megaterium were selected to study their modulatory effects on colitis in mice.2.Modulatory effects of Bacillus subtilis and Bacillus megaterium of equine origin on colitis and depression-like behavior in miceFive-week-old male C57BL/6 mice were randomly divided into control group(Normal Treatment group,NT group),DSS group,5-aminosalicylic acid group(PC group),Bacillus subtilis Y4.3-DSS group(DSS＿BS group),Bacillus megaterium L7.1-DSS group(DSS＿BM group),Bacillus subtilis Y4.3+Bacillus megaterium Bacillus subtilis L7.1 group(DSS＿S＿M group).On days 1-7,except for the NT group,which maintained normal drinking water,the remaining five groups of mice were given drinking water containing 3%(w/v)DSS for 7 days,of which the freshly prepared DSS solution was changed every 2 days;on days 8-14,the DSS＿BS group was gavaged with Bacillus subtilis Y4.3(5×10~8CFU/10g body weight),the DSS＿BM group was gavaged with Bacillus megaterium L7.1(5×10~8CFU/10g body weight),the DSS＿S＿M group was gavaged with Bacillus subtilis and Bacillus megaterium(5×10~8CFU/10g body weight),the PC group was gavaged with an equal volume of 5-aminosalicylic acid(3mg/10g body weight)as a positive control,and the NT group was given an equal volume of saline as a blank control.given an equal volume of saline.The results showed that the body weight of the mice gradually decreased and reached the lowest at the 7th d when the DSS intervention started and was significantly lower than that of the control mice(p<0.001),but there was no significant difference at the 14th day;the DAI of the mice was significantly higher than that of the control mice(p<0.05).In addition,compared to the control group,mice in the DSS group had significantly higher spleen index(p<0.05)and shorter colon length(p<0.05),and the color of colon contents was red-black;the results of H&E staining of colon tissues showed more colonic epithelial cells were shed,and there was a large number of inflammatory cells infiltrating the mucosal and submucosal layers;the pathological histological score was significantly lower(p<0.01);serum biochemical parameters showed significantly elevated myeloperoxidase(MPO)and malondialdehyde(MDA)(p<0.0001)and significantly decreased superoxide dismutase(SOD)(p<0.05);significantly elevated expression of cytokines IL-1β,IL-6,IL-8,TNF-α,TLR4,Myd88and NF-κB(p<0.05),the expression of intestinal tight junction proteins ZO-1,Occludin and Claudin-1 were significantly decreased(p<0.05).The PC group,DSS＿BS group,DSS＿BM group,and DSS＿S＿M group,although not significantly different in body weight,significantly delayed the increase of DAI index,especially at the 14th d.A significant difference(p<0.05)was observed in the recovery of spleen index and colon length;the results of H&E staining of colon tissue also showed a decrease in the number of inflammatory cell infiltration;in addition,both serum biochemical indices and the expression of inflammatory factors were closer to the Control group.The results showed that the DSS group significantly increased the immobility time of mice in the tail suspension test(TST)and forced swimming test(FST)and significantly decreased the preference for sucrose in the sucrose preference test compared to the NT group.In contrast,compared to the DSS group,the DSS＿BS,DSS＿BM and DSS＿S＿M groups showed significantly lower immobility time in the FST and TST tests(p<0.05)and significantly higher preference for sucrose in the sucrose preference test.Immunohistochemical staining of neuroglia and microglia in the hippocampal CA3horn region of the brain revealed significantly higher cell expression in the DSS group compared to the NT group(p<0.05),while the expression in the DSS＿BS,DSS＿BM and DSS＿S＿M groups was significantly lower(p<0.05)basically returning to the NT group level.Compared with the NT group,the expression of inflammatory cytokines IL-1βand TNF-αwas significantly higher in the DSS group(p<0.05)and the expression of BDNF was significantly lower in the DSS group(p<0.05),while the expression in the remaining three groups significantly returned to the level of the NT group3.Modulatory effects of Bacillus subtilis and Bacillus megaterium of equine origin on intestinal flora and colonic tissues in mice with colitisTo further investigate the regulatory effects of Bacillus subtilis Y4.3 and Bacillus megaterium L7.1 on intestinal microorganisms and colonic tissues,16S amplicon sequencing analysis was performed on the cecum contents of NT,DSS,DSS＿BS,DSS＿BM and DSS＿S＿M groups.The results of 16S amplicon sequencing analysis showed no significant differences inα-diversity and significant differences inβ-diversity in the DSS＿BS,DSS＿BM and DSS＿S＿M groups compared to the DSS group(p<0.05);in addition,the abundance of harmful bacteria Noviherbaspirillum in mouse intestinal microorganisms at the genus level was elevated after DSS induction(p<0.05),while the abundance of beneficial bacteria Turicibacter,prerotellaceae＿UCG＿001 and Lactobacillus increased at the genus level after Bacillus intervention(p<0.05).Transcriptome analysis of colon tissue revealed regulatory pathways focused on immune response,inflammatory processes and neurological development.In summary,in this study,six strains of Bacillus sp.were isolated from healthy horse feces,and two strains with good probiotic potential were selected to observe their regulating effects on mouse colitis,and it was found that Bacillus subtilis and Bacillus megaterium of equine origin had regulating effects on mouse colitis,mainly in clinical indexes,biochemical indexes and inflammatory cytokine indexes,and could alleviate colitis by regulating intestinal flora and their own related pathways.