Characterize The Succession Of Gut Microbiota Of Caesarean Infants With High Throughput Sequencing Technology

Objective: The human gut harbors diverse microbes that play afundamental role in the well-being of their host. Humans and microbes coexistin a multifaceted symbiotic relationship that confers benefits to both the hostand the microbe. The gut microbiota aids in the digestion of foods andacquisition and absorption of nutrients from otherwise indigestible foods. Inaddition, the microbiota provides resistance to colonization and prevents theovergrowth of harmful pathogenic organisms. The gut microbiota is alsoinstrumental in promoting the development of the gut immune system. Infantperiod was a critical stage of the establishment of intestinal flora system, andmode of delivery was a key factor to influence the initial colonization of gutmicrobiota, the patterns of which could effect the risk of subsequent allergicdiseases and obesity by disturbing the development of gastrointestinalphysiology and the maturity of the immune system. It is obviously defective toinvestigate complex intestinal microflora with traditional molecular biologicaltechnology, such as real time-PCR and denaturing gradient gel electrophoresis.High-throughput sequencing technology could detect almost all the flora in thefeces. Therefore we apply Illumina high-throughput sequencing technology toclearly identify caesarean infant intestinal flora to more comprehensivelyunderstand the intestinal flora and to illustrate the relationship between modeof delivery and infant health and disease.Methods: 30 full-term infants born by caesarean delivery satisfying theinclusion criteria were selected in the obstetrics department of the ThirdHospital of Hebei Medical University and the Fourth Hospital of ShijiazhuangCity from Jan 2013 to Dec 2014. The infants and their parents were living inShijiazhuang city or its surrounding counties. The climate environment, livingenvironment, living habits were similar. All subjects signed informed consentprior to the start of this study and were accepted by the Ethics Committee ofthe Third Hospital of Hebei Medical University. Fecal samples were collectedat 1(meconium), 3,7 days and 1,3,6 months after birth(respectively set asgroup T1,T2,T3,T4,T5 and T6) and were stored at-80 ℃.The clinicalfollow-ups to evaluate feeding situation, growth and development, andprevalence of illness and subsequent medicine intake(antibiotics andprobiotics) were performed from birth to 6 months of age. The total DNA offecal samples were extracted by using E.Z.N.A.® Stool DNA Kit andamplification were performed targeting the variable regions 3 and 4 of the 16 Sr RNA gene. The abundance and alpha diversity and the following clusteranalysis of fecal microbiota were analyzed by pyrosequencing of 16 S r RNAgenes on the Illumina Mi Seq platform.Results:153 samples were analyzed, the rarefaction curves showed thatadequate sequencing depth was achieved. Analysis of alpha diversity indicatedhigh microbial diversity. Meanwhile,the microbial diversity had a positivecorrelation with the postnatal age. 14 phyla and 183 genera were detectedfrom all samples. About 97%-99% of the sequences belong to five mostpopulated bacterial phyla: Firmicutes, Proteobacteria, Bacteroidetes,Actinobacteria and Candidate division TM7. At the level of phylum,Firmicutes and Proteobacteria were dominant bacterial phyla within 6 monthsafter birth. At the level of genus, at the 1st day, Lactococcus, Bacillus,Pseudomonas and Solibacillus which were aerobe and facultative anaerobeswere the main bacterial groups, a small amount of Bifidobacterium andStreptococcus started to colonized. At 3rd days after birth, Klebsiella, andEscherichia-Shigella which were facultative anaerobes became thepredominant bacterial groups. At 7th days after birth, the abundance ofanaerobe and facultative anaerobes, Clostridium and Enterococcus, appear torise. From 1month to 6months of age, the abundance of Bifidobacteriumincreased rapidly and truned into the advantage bacterium group.Conclusions:1 High-throughput sequencing technology could detect almost all the flora inthe feces, and enables us to have a more comprehensive understanding of thecomposition of infant intestinal flora.2 At the level of phylum, fourteen bacterial phyla were detected in all samplesby using Illumina sequencing of the 16 S r RNA-V6 region, and more than 97%of the sequences in all samples were found to belong to the five mostpopulated bacterial phyla; namely, Firmicutes, Proteobacteria, Bacteroidetes,Actinobacteria and Candidate division TM7. At the level of genus, 183 bacterial genera were detected in all samples, including Lactococcus, Bacillus,Bifidobacterium, Escherichia-Shigella and Klebsiella etc.3 The succession of gut microbiota of Caesarean infants:3.1 At the level of phylum, Firmicutes and Proteobacteria were dominantbacterial phyla within 6 months after birth.3.2 At the first day after birth, large amounts of bacteria were detected in thefecal samples, and Lactococcus, Bacillus, Pseudomonas and Solibacilluswhich were aerobe and facultative anaerobes were main bacterial groups.They cost the oxygen in the intestinal tract, the facultative anaerobes andanaerobe, Klebsiella, Escherichia-Shigella, Clostridium and Enterococcus,started to colonized increasingly. At the first month after birth, the abundanceof Bifidobacterium increased rapidly and turned into the advantage bacteriumgroup at 6month of age.