A Study On Human Milk Microbiota And Their Dominant Lactic Acid Bacteria Influencing Gut Mucosal Immune Development In Neonatal Mice

Microorganisms in breast milk,mainly derived from maternal intestine,skin and neonatal mouth,shape the structure and dynamics of microbiota in neonatal intestine.The microbes in breast milk are transmitted vertically to the infant's intestines,affecting the development of the intestinal immune system of the offspring.This study aimed to determine the mother-infant vertical transmission of beneficial bacteria and to explore the mechanism of the effect of isolated strains on the intestinal immunity of the offspring.The main contents are as follows:We recruited thirteen lactation mothers,and their breastmilk was undergone collection in three stages(colostrum,transition and mature)and corresponding neonatal feces,as well as maternal feces(mature stage),and sIgA-coated bacteria were enriched firstly.According to the results of 16 s rDNA sequencing,mature milk was most similar to the composition and structure of neonatal fecal in the same period.Cluster analysis showed that Streptococcus,Staphylococcus,Lactobacillus(Lactobacillus-gasseri),Enterococcus,Bifidobacterium(Bifidobacterium_longum_subsp_longum),Klebsiella,Ambigous and Escherichia-Shigelle were mainly transferred in sIgA coated bacteria.In addition,intake of antibiotics during neonatal period affects breast milk,infant feces and their sIgA coated bacterial composition,mainly increased the relative abundance of Staphylococcus,Pseudomonas and Gemmiger in breast milk significantly,while decreased the relative abundance of Escherichia-Shigelle,Klebsiella and Chryseobacterium significantly.Antibiotics significantly increased the relative abundance of Klebsiella and Escherichia-Shigelle in neonatal feces,while decreased the relative abundance of Enterococcus and Rothia.Pediococcus and Carnobacterium increased significantly after antibiotic treatment,while Streptococcus and Klebsiella decreased significantly in breastmilk of sIgA coated bacteria.Klebsiella and Lactobacillus were decreased,while Carnobacterium increased in group neonatal feces with sIgA coated after antibiotic treatment.Secondly,42 nd days of breast milk of eleven cities were collected and were cultured with a combination medium(MRS+YCFA).Results of 16 s rDNA sequencing showed that the microbiota of these samples had geographical specificity,and the highest occurrence of S.salivarius(26%),L.reueri,L.gasseri,L.casei and L.fermantation had regional specificity and the remaining Lactobacillus had no regional specificity.Four potential vertical transmission strains S.salivarius,one strain L.reuteri were isolated in breastmilk samples,and four strains L.plantarum were isolated from neonatal feces.Strains with strong hydrophobicity,adhesion and auto-aggregation abilities were subjected to animal experiments.The animal experiment is a breeding experiment.After female and male mice are mated in a 3:1 cage,the lactating mother mice were divided into four groups,the control group(CON),the S.salivarius gavage group(SS),the L.plantarum gavage group(LP),and the L.reuteri gavage group(LR).The female mice(n = 5)and the corresponding neonatal male and female mice(n = 5)were sacrificed after three weeks weaning.The other neonatal mice were classified in groups as followed,only interfered with the mother(SS-C,LP-C,and LR-C),only intervening offspring(C-SS,C-LP,and C-LR),intervening both in mother and offspring(SSSS,LP-LP,and LR-LR)until five weeks.The results showed that bacterial treatment increased the levels of related immunoglobulinin the serum of lactating mothers,decreased the permeability of intestinal mucosa(FD4 reduced)and increased the content of secretory immunoglobulin A(sIgA)in the small intestine.Three strains increased the expression of mRNA of the intestinal mucosal barrier,the antibacterial peptide in the female mice,but only LR increased the mRNA expression of some genes in the intestinal sIgA production pathway.After intervention in lactation mother,the serum levels of IgA,IgE,and IgG2 a in the offspring of the 3-week-old children increased,the content of FD4 decreased,the content of sIgA in the small intestine increased,and the expression of mRNA in intestinal mucosal barrier,mucosal barrier,and IgA pathway were up-regulated.These effects were more pronounced in male offspring mice.Three strains in three intervention mode had a similar effect on 5-week-old offspring mice as the 3-week-old offspring mice.However,it is more effective to intervene in the mother and offspring mice simultaneously.In conclusion,the potential beneficial transmission bacteria coated by sIgA from mother to infant are Streptococcus,Lactobacillus(Lactobacillus-gasseri),Enterococcus and Bifidobacterium(Bifidobacterium_longum_subsp_longum).Antibiotics treated in mothers or infants can reduce diversity and increase the abundance of bacteria in breast milk and neonatal feces and change the relative abundance of vertical transmission bacteria.The composition and diversity of the cultured breastmilk microbiota collected from eleven cities in China are geographically different.The dominant occurrence of potential vertical transmission species was S.salivarius,and L.reuteri,L.gasseri,L.fermentation,and L.casei was regionally specific.Potential vertical transmission strains: S.salivarius,L.reuteri(isolated from breastmilk)and L.plantarum(isolated from neonatal feces)can indirectly promote progeny mucosal immunity and intestinal villus development.It provides a guideline for regulating the development of special nutrition for lactating mothers and infants.