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Objective To investigate the characteristics of intestinal microbiota in neonates on the first and third day after birth.Methods A total of 50 healthy singleton neonates who were born between June 15,2016 and August 3,2016 in Shanghai First Maternity and Infant Hospital were enrolled.Their stool samples were collected on the first and third day after birth and the samples were labeled according to the time of collection (D1 and D3 groups,n=50 each).Illumina NexSeq high-throughput sequencing platform was used to sequence the variable region 4 and 5 of all bacterial 16S rRNA genes in the samples.The composition of intestinal microbial communities was determined and the differences between the two groups were compared by Metastats analysis.Results (1) A total of 100 stool samples were sequenced and the retrieved sequences were from 25 bacterial phyla,119 families,227 genera and 159 species.(2) Major phyla in the two groups were the same,namely,Proteobacteria,Frimicutes,Bacteroidetes and Actinobacteria.The relative abundances of Frimicutes (0.27 ± 0.03 vs 0.41 ± 0.05) and Bacteroidetes (0.07 ± 0.01 vs 0.09 ± 0.03) increased over time,while that of Actinobacteria (0.10±0.01 vs 0.01 ±0.00) decreased on day 3.No significant difference in the relative abundance of Proteobacteria (0.51 ±0.03 vs 0.49± 0.05) was observed between D1 and D3 groups.There were significant difference in relative abundances of Frimicutes and Actinobacteria between the two groups (both q=-0.01,both P＜0.05).(3) Among the top ten most abundant families,Enterobacteriaceae,Staphylococcaceae,Enterococcaceae,Streptococcaceae and Lachnospiraceae were detected in both of the two groups.The relative abundances of Enterobacteriaceae (0.25 ± 0.02 vs 0.46 ± 0.06),Staphylococcaceae (0.07 ± 0.02 vs 0.12 ± 0.03),Enterococcaceae (0.04±0.02 vs 0.10±0.04),Streptococcaceae (0.03 ±0.02 vs 0.06±0.01) increased over time,while that of Lachnospiraceae (0.03 ± 0.01 vs 0.02 ± 0.02) decreased on day 3.Only the relative abundance of Enterobacteriaceae had statistical difference between the two groups (q=0.00,P＜0.05).(4) Among the top ten most abundant genera,Staphylococcus,Enterococcus,Streptococcus,Bacteroides and Pseudomonas were detected in both groups.The relative abundances of aerobic and facultative anaerobic bacteria which belonged to genera of Stenotrophomonas,Propionibacterium,Acinetobacter,Bacillus,Sphingomonas and so on decreased on day 3 as compared with those on day 1 (0.00±0.00 vs 0.07±0.02,0.00±0.00 vs 0.06±0.01,0.00±0.00 vs 0.03±0.01,0.00±0.00 vs 0.02±0.01,0.00±0.00 vs 0.02±0.00,all q=0.00,all P＜0.05).However,the relative abundances of anaerobic bacteria which belonged to Bacteroides,Veillonella,Parabacteroides and so on increased on day 3 (0.01 ±0.00 vs 0.08±0.03,0.00±0.00 vs 0.03±0.02,0.00±0.00 vs 0.01 ±0.00,q=0.01,0.01 and 0.00,all P＜0.05).(5) The most abundant species in intestinal microbiota was escherichia coli in both groups.Three less abundant species including lactobacillus gasseri,lactobacillus animalis and bifidobacterium bifidum were detected in both groups.(6) Regardless of the mode of delivery,Staphylococcus,was the highest predominant genera in meconium samples,followed by stenotrophomonas.Stool samples collected on the third day after birth were divided into four groups based on deliver modes and feeding patterns.Neonates who were born abdominally with exclusive breastfed thereafter were different from those of the other three groups in predominant intestinal bacteria,but the difference was not statistically significant.Bifidobacterium and Subdoligranulum were only detected in the vaginally born neonates.Conclusions Meconium is not sterile.Although the intestinal microbiota on the first day of life is different from that on the third day of life,the dominant bacteria are common.During the first three days of life,the relative abundances of aerobic and facultative anaerobic bacteria decreased significantly over time,while the relative abundance of anaerobic bacteria increased.