Study On The Changing Characteristics Of Intestinal Flora And Its Co-metabolites In Children Aged 0 To 6 Years And Infants With Abnormal Defecatio

Objective:To analyze the composition of intestinal microflora and the content of fecal bile acids and short chain fatty acids,find correlations between them.To look for characteristic microbiota closely related to metabolites of intestinal microflora.These data will provide information on the change characteristics and correlation of children’s intestinal microflora and its co-metabolites from early life to preschool.These results will provide basic information and convenience for future research on various aspects of human intestinal microflora,can provide reference for the prevention and treatment of many diseases,and contribute to a better understanding of the relationship between child health and intestinal microbiota maturation,so as to better safeguard children’s health from the perspective of intestinal health in the future.Methods:120 healthy newborns（0 to 28 days）and 150 healthy children aged 6 months to 6 years from the First Affiliated Hospital of Kunming Medical University were selected as the participants of this study.The 270 participants were divided into 9 groups according to age（n=30）,which were as follows:3 days group,7 days group,14 days group,25 days group,6 months group,1 year group,2 years group,3 years group,6 years group.The collected feces of healthy neonates and children were taken as samples,and 16S rDNA was used to analyze the composition of intestinal microflora.The contents of short-chain fatty acids and bile acids in feces were determined by gas chromatography and liquid mass spectrometry using the same grouping method and samples.R and SPSS statistical software were used to analyze the composition and correlation of intestinal microflora and its co-metabolites in children of different ages.Results:1.The richness and diversity of intestinal microflora gradually increased with age.They were lowest in the neonatal period,increased significantly from 6 months of age until stable after 3 years of age.The phylum of intestinal microbes in children aged 0-6 years old mainly consisted of Proteobacteria,Bacteroidetes,Firmicutes and Actinobacteria.The abundance of Proteobacteria and Actinomycetes decreased gradually from birth to 6 years old,while the abundance of Bacteroidetes and Firmicutes increased.Proteobacteria is the main component of the intestinal microflora in the newborn period.At 6 months,Bacteroidetes,Firmicutes and proteobacteria were found in equal abundance.Bacteroidetes and Firmicutes were the main phylum in intestinal microflora of children aged 1-6 years.In the level of genus,Escherichia-Shigella is the dominant genus in the neonatal period.At 6 months,Bacteroides and Escherichia-Shigella were the dominant genus of intestinal microflora,the relative abundance of Bifidobacteria reached the highest in all age groups.Since then,the abundance of Escherichia-Shigella decreased,while the abundance of Bacteroides continued to increase,becoming the main genus of intestinal microflora in children aged 1-6 years.2.Enterotypes 1,2 and 3 respectively clustered with Escherichia-Shigella,Bifidobacteria and Bacteroides showed a gradually increased richness and diversity of intestinal microflora in this order.In the neonatal period,E1 is dominant.At 6 months of age,the proportion of E2 was the highest,and at 1 year of age,they switched from E2 supplemented by E1 to E2 supplemented by E3.From then on,E2 decreased with the increase of age,while E3 was on the contrary and Enterotype gradually stabilized after 3 years old.3.The emergence windows,abundance change and function analysis of 40 important intestinal microflora that can reflect different age stages of host suggest that the functional transformation and structural change of intestinal microflora are a common trend,and the function of intestinal microflora gradually becomes mature and stable with the increase of age.4.The contents of acetate,propanoate and butyrate in feces were significantly higher than those of isobutyrate and isovalerate,and acetate was the highest.The content of fecal acetate,propionate and butyrate was the lowest at 3 days and increased significantly at 7 days,but there was no significant difference from 7 to 25 days.At 6 months of age,they increased significantly again,then the changes tended to be gentle,and gradually stabilized after 3 years of age.Fecal isobutyrate and isovalerate remain at very low levels during the neonatal period,especially the concentration of isovalerate,they increases significantly at 6 months of birth and remains at this level thereafter.The variation of fecal short-chain fatty acids was consistent with abundance of Flavonifractor,Lachnoclostridium,Faecalibacterium,Lachnospiraceae_ge and Enterobacter,which were positively correlated with them.5.The mean percentage of fecal PBAs in neonatal period accounting for 98%,fecal SBAs is rarely detected during neonatal period,accounting for only 2%of the total BAs.Both fecal PBAs and SBAs were significantly increased at 6 months,fecal PBAs tended to be stable from then on,while fecal SBAs continued to increase until 3 years old.The variation trend of fecal SBAs was opposite to abundance of Escherichia-Shigella,Enterococcus,Enterobacter and Streptococcus,which were negatively correlated with them,the variation trend was consistent with abundance of Flavonifractor,Faecalibacterium,Lachnospiraceae_ge,Blautia,Lachnospiraceae_UCG₀04,Lachnospira,Roseburia,Anaerostipes and Intestinibacter,which were positively correlated with them.Conclusions:1.The development of intestinal microflora in children aged 0-6 can be roughly divided into three stages:developmental stage（0-6months old）,transitional stage（6 months-2 years old）and stable stage（3-6 years old）.The composition and function of intestinal microflora were unstable in the neonatal period,and improved significantly in 6 months after birth,which is the critical period for the development of intestinal microflora,at this time,the abundance and function of intestinal microflora began to increase and improve,significantly promoting the metabolism of short-chain fatty acids and bile acids.2.The Escherichia-Shigella Enterotype was dominant in neonates,and the Bifidobacteria Enterotype was the highest at the age of 6 months,the Bacteroides Enterotype did not appear until the age of 1 year,and the Enterotype gradually stabilizes after 3 years old.3.The content of fecal SCFAs remained low in the neonatal period,and increased significantly in 6 months of birth with the maturity of intestinal microflora,and tended to be stable after 3 years old.4.The liver can effectively synthesize PBAs science 7 days after birth,the proportion of fecal PBAs in total BAs was higher in neonatal period,and the content of fecal SBAs was close to 0.At 6 months,the levels of both fecal PBAs and SBAs increased,and fecal PBAs tended to be stable from 6 months later as the child’s liver function matures.With the maturation of intestinal microflora,the content of fecal SBAs begins to increase significantly at 6 months of age and stabilizes after 3 years old.Objective:To analyze the changes of intestinal microflora and fecal short-chain fatty acids and bile acids in infants with different defecation frequency who were exclusively breastfed from 1 to 6 months of age.To explore the mechanism of intestinal microflora and its metabolites in different defecation frequency,look for the differential intestinal microflora and its co-metabolites of infants with different defecation frequency and explore microbial biomarkers.To provide more biological targets for clinical treatment,and provide a strong theoretical basis for clinical use of the most effective probiotics for the treatment these babies.Methods:30 infants aged 1 to 6 months who were exclusively breastfed were selected from the First Affiliated Hospital of Kunming Medical University,they were divided into 3 groups according to their defecation frequency（n=10）.Group A:defecate once within 3 days or defecate 3 times a day,that is normal group;Group B:defecation more than 3 times a day,that is increased defecation frequency group;Group C:defecate once more than every 4 days,that is decreased defecation frequency group.The collected fecal samples were used to analyze the composition of intestinal microflora by 16S rDNA,and the contents of short-chain fatty acids and bile acids in feces were determined by gas chromatography and liquid phase mass spectrometry.R and SPSS statistical software were used to analyze the composition and correlation of intestinal microflora and its co-metabolites in infants with different defecation frequencies.Results:1.The richness of intestinal microflora in the group with reduced defecation frequency was significantly decreased（P<0.05）,the heterogeneity of microbial community among individuals in the group with increased defecation frequency was greater.Firmicutes decreased significantly in infants with abnormal defecation frequency（P<0.05）.At the genus level,the dominant genus in the feces of infants with the normal and abnormal defecation groups were different.Bifidobacterium was the dominant genus in the normal defecation group and the reduced defecation frequency group,while Escherichia_Shigella was the dominant genus in the increased defecation frequency group,and the B/E value was<1.In Top20 of genus level,the difference microorganisms in the normal group were Phocaeicola,Blautia and Streptococcus,the difference microorganisms in the group with increased defecation frequency were Clostridium_Sensu_Stricto,the difference microorganisms in the group with reduced defecation frequency were Ruthenibacterium and Anaeromassillibacillus.2.The highest content of SCFAs in feces was acetate,which was more than 80%in the normal group,higher than abnormal defecation groups.There was no significant difference in fecal SCFAs between the increased defecation frequency group and the normal control group,but both of them were lower than the normal defecation group.The content of isovalerate in the reduced defecation frequency group was significantly higher than that in the normal defecation frequency group and the increased defecation frequency group（P<0.05）.3.The content of CA,CDCA and GCDCA in the group with increased defecation frequency was higher than that in the normal group,and the content of CDCA in the group with increased defecation frequency was significantly higher than that in the normal group（P<0.05）,the content of CA in the reduced defecation frequency group was significantly higher than that in the normal defecation group（P<0.05）.4.Correlation analysis indicated that Blautia,Paraprevotella and Desulfovibrio in the normal group correlated with isovalerate,showing a negative correlation.There was a negative correlation between the differential intestinal metabolites CDCA,CA and Ruminococcus、Agathobacter、Anaerobutyricum、Anaerostipes、Paraprevotella.Conclusions:1.There were significant differences in intestinal microflora and its co-metabolism among the groups with abnormal defecation frequency and the group with normal defecation frequency.2.Anaerobutyricum,Ruminococcus,Blautia,Agathobacter,and a certain proportion of enterococcus and Phocaeicola play a crucial role in maintaining the balance of intestinal micro flora of infants.3.CDCA is a differential metabolite in the group with increased defecation frequency.Isovalerate and CA were differential metabolites in the reduced defecation frequency group.4.Blautia,Paraprevotella,Desulfovibrio,Ruminococcus,Agathobacter,Anaerobutyricum and Anaerostipeswere are negatively correlated with differential metabolites.These provide the basis for future studies to use the most effective probiotics in clinical practice and/or alter the content of differential metabolites distribution in the colon to restore normal bowel movement in these infants.