The Establishment And Influence Of The Intestinal Microflora Of Infants Within 6 Months With Different Feeding Mode

Objective:Infancy is the key period for intestinal flora implantation. The mode of implantation will affect the physiological development and immunological function of the neonatal gut after birth and reduce the risk of disease in the future. The way of feeding affects the implantation of intestinal flora. Breastfeeding is considered the best and the main source of intestinal flora in the infant is from the mother. We use a16 Sr RNA sequencing method to reveal the process of establishment of intestinal flora in infants within the first six months of age, evaluate the effect of different feeding methods on microflora implantation and its progression and provide evidence with which the relationship between intestinal flora and occurrence of disease could be inferred.Methods:wenty babies born at the Jilin University First Hospital during the period of April 2015 to October 2016 meeting the inclusion criteria were admitted into the study. These were fullterm babies delivered via vaginal deliveries of mothers of good physical health, either exclusively breast fed or formula fed, with no history of receiving antibiotics in the perinatal period, and have the families living in Changchun with similar climatic, dietary and living conditions. Divided into breast-fed(10cases)and formula-fed group(10cases).Stool samples were collected on day 14,2 months, 4 months and 6 months after birth, and were allocated to groups A to D and stored in a-80℃ refrigerator. stools samples from the exclusively breast feeding group were allocated to groups E and H respectively. The health and developmental conditions of the babies were noted in the follow-up visits. The MOBIO kit was used to extract DNA from stool specimens. A high-throughput sequencing platform was used to sequence the V3, V4 variable area of bacterial 16 srRNA. The diversity and abundance of intestinal flora were analyzed and identified in their respective phylum, genus, family, class and order.Results:1. 80 specimens were collected. DNA could not be extracted from one of them, and amplification failure occurred in one. As a result, 62 specimens were sequenced. The optimized sequence was 3641096,containing1524372983 bp, and the average length was 418.23 bp.2. The 62 samples were sequenced and were found to belong to 459 phyla, 318 genera, 378 families, 410 orders, and 430 classes. At the phyla level, 5 phyla covered 100% of the microflora, namely:Acinetobacter,Enterobacteriaceae,Veillonella,Escherichia/Shigella,Klebs iella.3. Breast-fed group main advantage Intestinal flora : The main phyla were Firmicutes, Protebacteria, and Actinobacteria. At the class level,the Enterobacter classes are the main advantageous bacteria, followed byNegativicutes, Clostridium classes and Actinomycetes. At the order level,the Enterobacteriaceae are the main advantageous bacteria, follwed by Selenomonadales, Clostridiuman, and Bifidobacteriales. At the family level, the Enterobacteriaceae are the main advantegeous bacteria,followed by Veillonellaceae, and Bifidobacterium; and at the genus level,Veillonella, Escherichia-Shigella and Klebsiella are the main advantageous bacteria.4. formula-fed groupmain advantage Intestinal flora:At the phylum level, Proteobacteria, Firmicutes and Actinobacteria are the predominant bacteria. At the class level, Proteobacteria are the dominant bacteria,followed by Clostridia,and Actinobacteria. At the order level,Enterobacteriales and Bifidobacteriales,and at the family level,Enterobacteriaceae are the dominant bacteria,followed by Veillonellaceae and Bifidobacteriaceae. Bifidobacterium. At the level of the genus,Veillonella and Escherichia-Shigella are predominant bacterial.5. 0-6 montha of breastfeeding group changes of intestinal flora;Firmicutes、Clostridia、Veillonellaceae、Clostridiales、Veillonella,these bacteria gradualy increased from birth to four months,6 months have a downward trend. Proteobacteria 、 Gammaproteobacteria 、Enterobacteriaceae 、 Enterobacteriales 、 Escherichia-Shigella, these bacteria gradualy decreased from birth to four months,6 months have arising trend.Actinobacteria、the bacteria gradualy increased from birth to four months,6 months have a rising trend.Actinobacteriia,the bacteria gradualy increased from birth to six months. Bifidobacteriaceae 、Bifidobacterium、Bifidobacteriales,these bacteria significantly increased from birth to two months,4 moths ofdecline,6 moths to rise again.6.0-6 montha of formula-feeding group changes of intestinal flora;Firmicutes、Clostridia、Veillonellaceae、Clostridiales、Veillonella,these bacteria gradualy decreased from birth to four months,6 months have a rising trend.Proteobacteria,Gammaproteobacteria、Enterobacteriaceae、Enterobacteriales、Escherichia-Shigella,these bacteria gradualy increased from birth to four months,6 months have a downward trend.Bifidobacteriaceae、Bifidobacterium、Bifidobacteriales,these bacteria significantly increased from birth to four months,6 moths to down.In conclusion:1. Using the Illumina high-throughput 16 SrRNA sequencing technology to identify the class, order, family, genus, and species of the intestinal flora is reliable, efficient and of high accuracy.2.Baby feces micronial diversity is very high,detect 491 species.Formula-fed infants species abundance than breast-feding group. the difference was statistically significant between them.3. In the intestinal flora of both breastfed and formula-fed groups of infants, at the level of the phylum, are predominantly protebacteria,formicates, and actinobacteria. At the class, order, family and genus levels, predominant bacterial compositions were similar. With increase in age, in both groups of infants, bifidobacteriaceae and bifidobacteriales gradually increased in number, though in the breastfeeding group, the increase appeared to be more pronounced.