The Screening And Restoration Mechanism Of Lactobacillus Strains In Antibiotic-induced Dysbiosis

Acting as the huge “digestion organ”,the gut microbiome plays crucial roles in interaction and co-evolution relationships with human intestinal tract.Nowadays,the use of antibiotics is widespread and continues to accelerate,which as well accompanied by extensive public health benefits in recent decades.However,the overuse and misuse of antibiotics is a worldwide problem,and there are growing concerns that antibiotic exposure may lead to short-term and/or long-term negative health consequences.Antibiotic exposure could induce the gut disorder,such as loss of keystone taxa,blooms of pathogens,loss of diversity and shifts in metabolic capacity.Antibiotic-induced alteration of the gut microbiota can result in antibiotic-associated diarrhea,inflammatory bowel disease,irritable bowel syndrome and pseudomembranous colitis.In addition,antibiotic-induced dysbiosis could also lead to metabolic disorders or inflammation,such as obesity,allergy,asthma,diabetes and rheumatic arthritis.It is increasingly evident that Lactobacillus strains isolated from fermented foods can provide health benefits,some of which are genetically similar to strains used as probiotics,and there is evidence that Lactobacillus strains have been used to rebuild the antibiotic-induced dysfunction in gut microbiota or immune system,but whether the different strains of probiotics result in similar or reverse effects remains unclear.In this study,the ampicillin,cefixime and clindamycin were applied to establish a mice model of gut dysbiosis,and the combined or single effects of different Lactobacillus species on restoration of microbial ecology,recovery of metabolites,improvement of gut barrier function and regulation of immunity were evaluated,as well as the evidences of beneficial food supplement strategy.Firstly,the murine models of single antibiotic exposure(ampicillin,cefixime,high-dose of cefixime and clindamycin respectively)were created to explore the consequences of short-term therapeutic-doses of antibiotic use on gut microbial ecology and function,and any associated metabolic consequences.Based on 16 S rRNA gene sequencing data,all 4 antibiotic groups induced the gut dysbiosis including the alteration of diversity,composition and abundance of microbial taxa.The heatmap and LDA effect size(LEfSe)analysis were applied specifically to identify the key taxa that were differentially enriched at genus and family level after ampicillin exposure,particular taxa including Klebsiella,Anaeroplasma,Enterococcus and Enterobacter were over-represented in modelling faeces compared with the control while Lachnospiraceae and Akkermansia were loss in the ampicillin group.Based on the relationship between the biomarkers and gut dysbiosis,we applied a non-metric multidimensional scaling(NMDS)plot of the samples to establish an ampicillin dysbiosis model.In this model,a host-microbiome correlation exists and the tight-junction protein ZO-1 is negatively associated with specific gut microbiota of antibiotic-induced dysbiosis,while endotoxin and diamine oxidase are positively associated with such dysbiosis status.Secondly,the 20 Lactobacillus strains were isolated from fermented food origin including L.plantarum,L.casei,L.rhamnosus and L.helveticus.They were isolated and compared to characterise functional properties,including generation time,survival in the gastrointestinal environmental conditions,carbohydrate utilisation,drug resistance,adhesion to cells and colonisation in mice.The survival rates of L.rhamnosus GG in both gastric and duodenal juices were relatively high,and L.plantarum CCFM605,CGMCC12436 and L.casei CCFM5,CGMCC12435 maintained high cell numbers in simulated gastrointestinal conditions.The evaluation of their colonisation in the mouse gut indicated that the L.plantarum CCFM4,CCFM595,CGMCC12436,L.rhamnosus CCFM492 and L.casei CCFM30 could colonise relatively longer than other tested strains.Furthermore,based on the functional properties of Lactobacillus strains in vitro assays and colonisation in the mouse gut,the principal component analysis(PCA)was performed,and the strains which clustered closer to each component,were selected respectively in two groups for further analysis.The Lactobacillus cocktails A mainly advantage in generation time,colonisation and survival in gastric juice,while the cocktails B advantage in properties of adhesion to cells and utilisation of FOS.The different recovery effects of two cocktails(each contains four strains)of Lactobacillus against antibiotic-induced change of gut microbiota were evaluated in C57BL/6J mice.The results showed that Lactobacillus cocktails A(contains L.plantarum CCFM4,L.casei CCFM5,L.rhamnosus LGG and L.helveticus CCFM673)and B(contains L.plantarum CGMCC12436,L.casei CGMCC12435,L.rhamnosus CCFM492 and L.helveticus CCFM6)could effectively restore the structure of microbial communities destroyed by ampicillin or high-dose of cefixime on phylum level,which was difficult to get restored naturally in the short term.However,the clindamycin-induced dysbiosis was hardly to recover by these two cocktails.Both groups of Lactobacillus cocktails significantly increased the levels of short-chain fatty acids(SCFAs)reduced by ampicillin or cefixime in caecal content,but the decreased SCFAs in clindamycin group cannot be enhanced by Lactobacillus treatment.Moreover,the intestinal injury and inflammation in ileum and colon of mice were alleviated by administration of the Lactobacillus cocktails B,and the abundance of Mucispirillum?Enterococcus?Alistipes and Turicibacter was modified by Lactobacillus cocktails B in high-dose cefixime-induced dysbiosis.To further evaluate functions of single Lactobacillus strains,isolated from fermented food,in restoration of ampicillin-induced disruption based on mucosal barrier,gut microbial community and metabolome analyses,8 Lactobacillus strains belong to previous two cocktails groups were tested.The gut permeability and concentrations of endotoxin were remarkably recovered by almost all 8 strains,and the expression of tight junction protein Claudin-1 was significantly enhanced by L.casei CGMCC 12435(LacC)in colon while the levels of ZO-1,Occludin and Claudin-1 were remarkably increased by L.plantarum CGMCC12436(LacP).The LEfSe analysis revealed that the number of significantly altered taxa was lower in LacC,LacG(L.rhamnosus strain GG)and LacP than NaR,indicating they are effective in restoration of gut dysbiosis.Furthermore,the composition of faecal metabolites and levels of SCFAs could be recovered only by LacC strain,demonstrating the strain-specific restoration of metabolites.Correlation analysis between microbiota and metabolites indicated that the higher level of acetate in LacC group was positively correlated with increased relative abundance of Citrobacter,Bifidobacterium Eubacterium S24-7,Rikenellaceae and Clostridiaceae.The LacC strain was more effective in modulating the levels of sIgA,Reg?? and IFN-? whilst LacG administration reduced the level of sIgA and Reg??,there was no obvious observation in regulation of cytokines by LacP.Furthermore,the mechanism of Lactobacillus strains in alleviation of antibiotic-induced dysbiosis was further discussed through pathway,gut barrier and immune modulation.The LacC and LacP decreased the level of NF-?B in colon and down-regulated the expression of NF-?B p65 in immunofluorescence,indicating the ampicillin-induced inflammation may be modulated through NF-?B signal pathway.The LPS-induced gut barrier disruption was constructed in Transwell in Caco-2 cells,and no significant protective effects of LacC and LacP were observed in decrease of TEER,however,the expression of GPR109 A,which is a butyrate receptor in activation of Toll-like receptors 4,was up-regulated by the two strains.In addition,the presence of the cpsD gene cluster in L.plantarum CGMCC12436 which performed by whole genomic sequencing may be involved in its colonisation and beneficial adaptation in the gut.