Effects Of Stachyose/consortium Of Probiotics On Colonization Of Vancomycin Resistant Enterococcus And Gut Microbiota In Mice

Objective:Optimize the intestinal colonization model of Vancomycin Resistant Enterococcus(VRE)in mice by oral infection.On the basis of optimized VRE colonization mouse model,seek an alternative method to effectively treat or reduce VRE colonization.Methods:1.Optimization of VRE intestinal colonization model for oral infection in mice:C57BL/6J female mice were randomly divided into three groups:mixed antibiotic pretreatment group,ampicillin pretreatment group and vancomycin pretreatment group.On the 7th day before infection,the mice of three groups began to drink solution containing mixed antibiotics,ampicillin and vancomycin,respectively.The mixed antibiotic solution was changed to vancomycin-containing drinking water on the 2nd day before infection.Mice in mixed antibiotics and ampicillin pretreatment groups were given intraperitoneal injection of clindamycin on the day before infection.On day 0,three groups of mice received VRE gavage.On the third day after infection,the drinking water containing vancomycin and ampicillin were changed to normal drinking water until the end of the experiment.After screening the model,two different doses of 2.0×108 and 1.0×106 CFU/mL were administered on the basis of the vancomycin pretreatment model.Mouse feces were collected for viable counts within two weeks after infection,and changes in VRE colonization were observed.2.Effects of stachyose on the intestinal flora of healthy mice and VRE-infected mice.We conducted two independent experiments:non-infectious and infection experiment.In the non-infectious experiments:C57BL/6J mice were randomized into two groups,which were orally treated with stachyose or PBS for 7 consecutive days,respectively.In the infection experiment:all mice were infected with VRE according to vancomycin pretreatment procedure.Then,the two groups of VRE-infected mice were orally treated with stachyose or PBS for 7 consecutive days,respectively.During the period,mouse feces were collected and changes in VRE colonization were observed.The mice were euthanized,and the feces and colon of the mice in the non-infective and infection experiments were collected for 16s rRNA sequencing and colon RNA sequencing,respectively.And then evaluate the gut microbiota and gene expression of mice.3.Effects of probiotics consortium on VRE colonization and intestinal flora of mice infected with VRE.In vitro study:Seven probiotics were cultured in MRS broth and obtained sterile supernatants.PBS,sterile supernatant(adjusted/unadjusted-PH),mixed bacteria were added to VRE bacterial solution respectively,anaerobic culture for 24 hours,and viable counts were recorded to monitor the growth of VRE.In vivo experiments:C57BL/6J mice were randomly divided into three groups:blank group,PBS group,and probiotics group.The mice in PBS group and probiotics group were infected with VRE according to vancomycin pretreatment procedure,and no treatment was given to the blank group mice.On day 3 post-infection(p.i.),the mice in the probiotics group and PBS group were administered with mixed bacterial solution or PBS for 7 consecutive days,respectively.At different time points,fresh stool samples from mice were collected for viable counts.On day 12 p.i.,fresh mouse feces and colon tissue were collected for 16s rRNA sequencing and RNA-sequencing,respectively.Results:1.On the first day after infection,VRE was successfully colonized in the intestinal tract of three types of antibiotic-pretreated mice.The mouse fecal VRE counts in the three models was 105-108 CFU/mg in day 1 to 9 post-infection.On day 15 p.i.(end of the experiment),the VRE content in the feces decreased to 103-105 CFU/mg.Two mice from the ampicillin-treated group died on day 10 post-infection.There were no significant differences in VRE colonization between the two groups infected with different doses given by the vancomycin pretreatment model.2.On day 9 and 10 p.i.(intervention day 6 and day 7),the VRE colonization was significantly decreased in the VRE-ST group.16s RNA sequencing results showed that stachyose treatment did not alter the structure and diversity of the gut microbiota,but it selectively altered the relative abundance of special microorganisms in the host gut.In the uninfected experiment,the Lactobacillus family in VRE-ST group was significantly higher than the VRE-PBS group(P<0.05).In the infection experiment,the relative abundance of the Parabacteroides distasonis have increased significantly in VRE-ST group compared with VRE-PBS group(P<0.01).The abundance of Enterococcous in the VRE-ST group was significantly decreased compared with VRE-PBS group(P<0.05).RNA sequencing results showed that a total of 316 genes were differentially expressed between UN-ST and UN-PBS groups,and the most differentially expressed genes(DEGs)were enriched in metabolic pathways.Among them,up-regulated genes include Hsd17b14,Cyp3a44 and Argl,and down-regulated genes include Pnliprp2,Ces1c and Pla2g4c.In the VRE infection experiment,133 up-regulated genes and 70 down-regulated genes were differentially expressed in VRE-ST and VRE-PBS group.In the KEGG pathway analysis,differential genes were significantly enriched in 15 pathways.Among these pathways,the number of differential genes in the pancreatic secretory pathway is the highest.Among the TNF signaling pathway and the IL-17 signaling pathway,differential genes include Ccl2,Cxcl1,Cxcl2,Creb3,IL-1β,Sele,Ptgs2.In both infected and uninfected mice,stachyose treatment resulted in co-expression of 36 differential genes in the two different hosts.These 36 genes were significantly enriched in four pathways,namely fat digestion and absorption,linoleic acid metabolism,steroid hormone biosynthesis,and chemical carcinogenesis.Pnliprp1,Cyp2e1,and Cyp3a44 etc are shown in the protein network diagram to play an important role.3.Effects of probiotics consortium on VRE colonization and intestinal flora of mice infected with VRE.In vitro study,the probiotic combination can inhibit the growth of VRE,and the supernatant does not inhibit the growth of VRE whether or not the pH is adjusted.In vivo experiments,on day 9 after infection,the number of VRE in the probiotics group significantly reduced in the feces.The 16s rRNA results showed that the Shannon index of the PBS group and the probiotic group did not return to the level of normal mice.However,compared with PBS group,the Shannon index of the probiotic group was significantly higher than that of the PBS group(P<0.05).At the phylum level,the mouse gut flora is mainly composed of Bacteroidetes,Firmicutes,Proteobacteria,and Verrucomicrobia.Among them,Verrucomicrobia in the probiotic group were significantly higher than the PBS group(P<0.05).At the species level,the content of Clostridium difficile and Akkermansia muciniphila was significantly higher in PBS group blank group.However,compared with the PBS group,the relative content of Clostridium difficile decreased slightly,and Parabacteroides distasonis and Akkermansia muciniphila increased significantly.The results of RNA-sequencing showed that there were 432 DEGs between the probiotics consortium and the PBS group,including 324 up-regulated genes and 108 down-regulated genes.These differential genes were significantly enriched in six KEGG pathways,including:digestion and absorption of fat,absorption of linoleic acid,etc.Conclusion:Our study established an optimized a vancomycin pretreatment VRE colonization model that is stable and feasible.Supplementation of probiotics consortium and stachyose can effectively reduce the colonization of VRE.Consortium of probiotics can improve the diversity of the flora,while stachyose treatment did not alter the structure of the gut microbiota.Both stachyose and probiotics consortium can selectively alter the abundance of several key species and several gene expression related to metabolic process.