Transmission Study Of Bifidobacterium Animalis Subsp.Lactis Probio-M8 On Mother And Infant Pairs During Lactation

Most previous studies have shown that the vertical transmission of maternal microbes plays an important role in the development of infant gut microbiota.However,the route by which maternal microbes are transmitted to infant remains unclear.Also,it remains unclear whether oral administration of probiotics by mother results in transmission to their infants.In the present study,Bifidobacterium animalis subsp.lactis Probio-M8（Probio-M8）was used as a biomarker strain,a strain isolated from the breast milk of a healthy woman,to directly demonstrate vertical transmission at the strain level and genetic variation of maternally ingested bacteria between mother-infant pairs.In addition,metagenomic deep sequencing was used to investigate the relationship between mother and infant gut microbiota.A total of 11 healthy lactating mothers and their infants were recruited for present study.During the experiment,mothers took Probio-M8 powder(6×10¹⁰CFU/d)orally every day.After a week of administration,samples of breast milk,maternal feces and infant’s feces were collected weekly for 8-15 weeks.The vertical bacterial transmission was studied by traditional culture-dependent techniques and metagenomic sequencing techniques.The results demonstrate as follow:（1）A total of 222 strains of Bifidobacterium animalis subsp.lactis were isolated from440 maternal feces,infant feces and breast milk samples by pure culture technology.195 of them were identified as Probio-M8 homologous strains by comparative genome analysis,of which 145,21 and 29 strains were isolated from maternal feces,breast milk,and infant feces,respectively.Homologous strains of Probio-M8 were identified in the three kinds of samples from five pairs of mother-infant,indicating that maternally ingested Probio-M8 could translocate to infant during lactation.（2）Metagenomic deep sequencing was performed on 44 fecal samples from mothers and infants at the 4th and 8th weeks of the experiment,and 16 high-quality Bifidobacterium animalis subsp.lactis metagenomic assembled genomes were assembled by Meta SPAdes software.Comparative genomic analysis identified 13 of these strains as Probio-M8homologous strains from feces of 3 infants and 5 mothers.Metagenomic analysis once again demonstrated that the Probio-M8 strain could vertically transmit between mother and infant.（3）A total of 499 SNPs were identified in 195 homologous isolates of Probio-M8recovered from mother-infant pairs,of which 91%were only detected once,suggesting that most of the mutations were random.However,non-synonymous mutations mainly occurred in genes related to carbohydrate and amino acid transport and metabolism.In addition,a high frequency mutant gene,glc U（encodes a glucose transporter）,was detected.Mutations in this gene were found in 96 Probio-M8 homologous strains isolated from 8 maternal and infant pairs.The use of 95 carbon sources by glc U mutants was detected by Phenotype Micro Arrays technology,and the metabolic ability of glc U mutants to D-glucose and a-methyl-D-glucoside and organic acids was significantly improved.These results indicated that Probio-M8 underwent adaptive evolution for better survival in human host environments.（4）Based on the above metagenomic deep sequencing data,the HUMAn N2 process was used to analyze maternal and infant gut microbiota,and the results showedα-diversity of mother gut microbiota was higher than infants and the composition of gut microbiota was more similar in paired mothers and infants,and the functional gene richness of gut microbiota in mothers was greater than that in infants.（5）The shared species in mother and infant gut were analyzed by metagenomic sequencing based on reads at the species level.It was found that Bifidobacterium longum,Escherichia coli,Streptococcus salivarius and Veillonella parvula were shared in all the of mother and infant pairs.（6）Metagenomic assembly method was used to analyze the genomic similarity and phylogenetic relationship of shared strains in mother and infant gut.The results showed that bacteria belonging to Bacteroides,Parabacteroides,Ruminococcus,Eubacterium,Eggerthella,Bilophila and Akkermansia were transmitted vertically from mother to infant.Among them,strains belonging to the genera Bacteroides,Parabacteroides,Ruminococcus and Akkermansia that were transmitted from mother to infant had different degrees of potential to utilize the breast milk oligosaccharides.In this study,a combination of culture-dependent and culture-independent method was used to confirm that maternally ingested Probio-M8 derived from breast milk could be transferred to the infant gut through lactation,which not only confirmed the vertical transmission of bacteria between mother and child at the strain level,but also provided a new idea for the supplementation of probiotics in early life.