Colonization And Development Of The Fecal Microflora In Infants With High-throughput Sequencing

Objective: Intestinal flora play an important role in nutrition, metabolism and the immune system maturity, and promote the improvement of the intestinal mucosa barrier,The changes of diversity and institutions of intestinal flora is associated with disease,Infants and young children period is the important stage of intestinal flora engraftment and succession,Early childhood intestinal flora planting pattern will last into adulthood,In the past for intestinal micro ecology research adopt cultivation or molecular biology techniques, has obvious limitations on the research of the intestinal flora,With the emergence of the second generation of high-throughput sequencing technologies,With its sequencing is fast, high accuracy, is gradually applied in the study of gut microbes,But a lot of research for the European and American countries, the domestic reported less, and most of the sample size is less,Not enough to explain our country infant colonization and evolution process of intestinal microflora,This study aims to Illumina Miseq high-throughput sequencing platform for infant gut bacteria multiple time sequencing, discusses the evolution process of fecal microflora after baby is born, and for the later provides the reference for the study of gut bacteria and disease.Methods: 30 term infants with natural delivery born between 1 Nov 2013 and 31 Mar 2015 at the The Third Hospital of Hebei MedicalUniversity or The Fourth Hospital of Shijiazhuang City were enrolled, All of them are vaginal delivery, breastfeeding, family members have noallergy, perinatal mother did not use of antibiotics, babies and their parents are living in shijiazhuang and its surrounding counties and cities, climate environment, living environment, eating environment are fairly。Collect the stool specimens of first day, third day, seventh day, firstmonth,third month, sixth months,Use the OMEGA E.Z.N.A kit to extract DNA from stool specimens,Use Illmina Miseq PE250 high-throughput sequencing platform to sequence the V3, V4 variable area of bacterial 16 s r RNA,and use Mothur application software to measure sequence OTU cluster, analyze the diversity and abundance of the intestinal flora in phylum and genus level,intestinal microflora are described in details.Results: 1 158 specimen samples was collected, of which 22 weren’t availible, at a rate of 12%, DNA couldn’t be extracted from 15 samples, and amplification failure occurred in 11 samples. As a result, 132 specimen samples were sequenced. The sequence was 5143376 long.containing 3096312352 bp, the optimized sequence was 2717040, containing 1206679948 bp, and the average length was 444.12 bp. 2 The 132 samples were sequenced and were found to belong to 17 phyla and 181 genera. At the phyla level, 5 phylum covered 99% microflora, named Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria. The main genus included Bifidobacterium, Lactococcus, Pseudomonas, Enterococcus, Escherichia-Shigella, Klebsiella,Psychrobacteretc. 3 Adopt the method of random sampling on the sequence, in order to draw the sequence number and the number of OTU build Rarefaction curve, the curve tends to smooth, explain the sequencing data amount is reasonable, the sequencing depth, enough to cover 97% of gut bacteria strains.Through Chao1 index, the index of ACE is gradually increasing with the passage of time, showing fecal microbes abundance increase gradually, Shannon index, Simpson index gradually decreases gradually over time after the birth, the stool samples of flora diversity gradually increase. 4.1 The succession of fecal microflora in the level of phylum:Proteobacteria and Firmicutes are advantaged when birth, respectively accounting for 46.5% and 45.3%, with the passage of time, the Proteobacteria and Firmicutes gradually declined, until sixth month Proteobacteria was24%, Firmicutes was 23.3%, Actinobacteria rapid growth from 3.4% to 45.7% from birth. 4.2 The succession of fecal microflora in the level of genus, in first dayafter born the intestinal flora is mainly composed of Lactococcus andPseudomonas, in third day the intestinal flora have individual differencesthere are many aerobic bacteria and facultative anaerobic bacteria, such as Enterococcus, Escherichia-Shigella. Escherichia-Shigellaand, and Lactococcus and Pseudomonas decreased dramatically, in seventh day Bifidobacterium colonization increasedly, In first month all specimens haveBifidobacterium colonization, and growth rapidly to become absolutelydominant bacteria, average of 30%- 40%,In third month, more Bifidobacterium colonization, and reach 40%- 60%, mainly constituent part are Bifidobacterium, Escherichia-Shigella.In sixth month who individuals add complementary food,Bifidobacteria decrease, others are same with third month. 5 PCA analysis shows that(1) Similarity analysis between groups: group A and group exists obvious difference.No obvious gathered groups, each group microbial composition differences.(2) All individual similarity analysis:visible individuals within the group A flora is similar, but different with other time points, with the extension of feeding time, the change of intestinal flora.But from the perspective of the individual samples of group A high similarity, B and C group of individuals is relatively concentrated, group D and group E differences between individuals individual and group F flora similar trends. 6 Mutation analysis: each group have variant individuals, in combinationwith follow-up information, insufficient breast milk baby is given priority to with Escherichia-Shigella engraftment, diarrhea babies such as Escherichia-Shigella, fusobacterium engraftment is given priority to, use of antibiotics, home a baby out of the neonatal period for staphylococcus aureus, Escherichia-Shigella after continuous engraftment.This study foundin the follow-up process, diarrhea, feeding antibiotics application to infants gut microbes colonize the diversity and structure, provide a basis for further analysis of multiple factors.Conclusion: 1 High-throughput sequencing technology could detect almost all the flora in the feces, and enables us to have a more comprehensive understanding of the composition of infant intestinal flora. 2 In this study, 17 phyla and 181 genera were sequenced. The 17 phyla were Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria. The 181 genera included Bifidobacterium, Lactococcus, Pseudomonas, Enterococcus, Escherichia, Shigella, Klebsiella,Psychrobacter etc. 3 The succession of fecal microflora : 3.1 In the level of phylum,Proteobacteria and Firmicutes are dominate bacteria at birth, with the passage of time, the Proteobacteria and Firmicutes gradually declined while the Actinobacteria rising up. 3.2 In the level of genus, the intestinal flora is mainly composed of Lactococcus and Pseudomonas after birth, with the passage of time, in seventh day Bifidobacterium colonization increased. In first month, Bifidobacterium grew rapidly to become absolute dominant bacteria, until the addition of side dish.Bifidobacteria decreased with the increase of side dish. On the contrary, Escherichia-Shigella increased. 4 Term health, breastfeeding, vaginal child birth intestinal microbial flora of variation analysis: analysis of variation of specimens from the intestinal microflora showed that, feeding, antibiotic use, and those with infectious diseases can affect the diversity and structure of infant’s gut microbial colonization.