Cohort Analysis Of Maternal-infant Flora In Infants Complementary Food Induced Diarrhea And Study On Mechanism Of Anti-diarrhea Strains

At complementary feeding period,infants are prone to suffer from complementary food-induced diarrhea(CFID),which causes electrolyte imbalance and malnutrition in infants,and affects infant growth and development.Infant diarrhea is closely related to gut microbiota.In order to explore the relationship between CFID and beneficial bacteria in the gut,and to discover the probiotics that could alleviate CFID,the correlation between CFID,breast milk flora and infant intestinal flora were analyzed,and the characteristic bacteria of CFID in infant gut and breast milk were explored.Based on the analysis of characteristic bacteria,the anti-CFID probiotics were screened from healthy infants and breast milk,and the mechanism was explored.The correlation between CFID and microbiota of breast milk and infant intestine was discussed firstly.12 pairs of mothers and infants in CFID group and 13 pairs of mothers and infants in healthy group were enrolled in this study.High-throughput sequencing of16 S r RNA V4 region was used to analyze the composition of infant feces and breast milk at different time.The species diversity and richness of the samples were investigated by OTU clustering and diversity analysis.The OTUs of breast milk and fecal in CFID group were lower than that in healthy group.The alpha diversity of fecal microbiota between two groups was not different,but the alpha diversity of breast milk in healthy group was higher than that in the CFID group.Species profiling and LEf Se were used to analyze the microbiota composition and succession.Along with the occurrence of diarrhea,Bacteroides and Clostridium become dominant bacteria in infant feces of CFID group.The abundances of Bacillus,Escherichia and Bifidobacterium decreased,while Prevotella and Ruminococcus increased.The abundances of Bifidobacterium and Escherichia in breast milk of CFID group decreased,and Veillonella increased.The microbiota structure of infant feces and breast milk of healthygroup was relatively stable.Enterococcus,Escherichia,Veillonella,and Streptococcus in infant feces were negatively correlated with the occurrence of CFID,and Rothia and Prevotella were positively correlated with the occurrence of CFID.Lactobacillus,Enterococcus and Veillonella in breast milk were negatively correlated with CFID.The occurrence of CFID is associated with decreased transmission of breast milk microbes to infants’ gut.The comparison results of OTU,Sourcetracker and strains identification showed that the number of shared OTUs in feces and breast milk of CFID group was lower than that of healthy group,and the breast milk-derived microbiota in feces of CFID infants was significantly reduced,and Lactobacillus and Enterococcus isolated from infant feces and breast milk had high homology on the phylogenetic tree.Therefore,it is believed that CFID is related to the reduction of breast milk derived strains.Potentially anti-CFID strains Enterococcus and Lactobacillus can be transferred from breast milk to infant intestine.Gut microbes can metabolize tryptophan to produce indole derivatives which could activate promote pregnane X receptor to strengthen the gut barrier.320 strains of Lactobacillus and Enterococcus isolated from breast milk and feces were tested for the ability to produce indole derivatives and promote PXR expression.Four potentially functional strains were obtained.Gastrointestinal colonization analysis showed that four strains had a certain tolerance and colonization in the gut.The anti-CFID analysis showed that only the MN-431 tryptophan broth culture(MN-431TBC)could significantly reduce the occurrence of CFID and intestinal permeability.The anti-CFID effect of different components in MN-431 TBC were analysed.It was found that MN-431 cells and unfermented tryptophan broth had no anti-CFID effect,and only the supernatant obtained from MN-431 fermented tryptophan(MN-431TBS)played an anti-CFID role.The composition of MN-431 TBS was analyzed by HPLC,and the indole derivatives in MN-431 TBS were IAld and Skatole,they could activating PXR and aryl hydrocarbon receptors(AHR).The anti-CFID mechanism of MN-431 TBS was explored by AHRTh17 response pathway and PXR/NF-κB signaling pathway.MN-431 TBS can significantly activate the AHR-Th17 response pathway in rats,promote the expression of AHR,reduce the expressions of intestinal IL-17 A,IL-17 F,IL-21 and serum IL-17 F,IL-21,and IL-22,and reduce the intestinal inflammatory response of infants during the complementary feeding period.MN-431 TBS can also activate the PXR/NF-κB signaling pathway,reduce the expression of inflammatory cytokines TNF-α and IL-6,especially TNF-α,and enhance colon tight junctions Protein ZO-1 to reduce intestinal mucosal injury in rats.Colony stimulating factor 1 receptor(CSF1R)can regulate the homeostasis of intestinal cell function and epithelial structure.Probiotic exopolysaccharide(EPS)can affect the expression of CSF1 R.This study screened EPS producing probiotics that could regulate intestinal epithelial cell structure and function.The EPS-producing ability and CSF1R-promoting ability in RAW264.7 cells of 320 strains were analyzed.Four EPSproducing strains that could promote CSF1 R expression in macrophages were obtained.Among them,FN-345 and MN-169 can colonize the intestine for 1 day,increase the polysaccharide content in the colon content,promote the expression of intestinal CSF1 R,and significantly reduce the occurrence of CFID.HE staining showed FN-345 and MN-169 could increase the height of small intestinal villi and decrease the shedding of small intestinal mucosa and the secretion of intestinal fluid.The crude exopolysaccharide of FN-345 and MN-169 were extracted,and the anti-CFID function was verified.The results showed that only the crude exopolysaccharide of FN-345(c-eps345)had significant antiCFID effect.The target of c-eps345 is intestinal CSF1R-dependent macrophages.In order to explore the anti-CFID mechanism of c-eps345,four key proteins in the CSF1R-MAPK pathway that regulate cell proliferation and differentiation were analyzed.c-eps345 significantly promoted the transcription and expression of CSF1 R,ERK,SRF,and CREB factors in intestine of rats,thereby regulating cell proliferation in crypts and the differentiation of paneth cells and goblet cells,and ultimately promoting the homeostasis of intestinal structure and function.