| Part I DGGE analysis of neonatal intestinal flora diversity and influencing factorsObjective: To research dynamic characteristics of neonatal intestinal flora diversity and to analyze the related factors influencing the intestinal flora diversity by using PCR- DGGE technology.Method: Fecal samples were collected from 157 full term and 72 preterm neonates at 3rd, 5th, 7th days and 3rd week after birth. The DNA of stool samples were extracted and PCR amplification were performed targeting the variable regions 3 of the 16 Sr RNA gene. After examing by denaturing gradient gel eleclrophoresis(DGGE), richness and Shannon index were calculated and analyzed statistically accoriding to the band present on the DGGE gel.Results:(1) The nenoatal intestinal flora changed dynamically with time. Richness increased with time during the observation period(6.17±2.23, 10.34±2.60, 13.04±3.82, 13.86±3.81). Shannon index rised with time within 1 week after birth(1.33±0.34,1.74±0.25,1.80±0.31). There was no significant change of diversity between the 7th day and the 3rd week(P>0.05). Richness and Shannon index of the 5th day was significantly higher than those of the 3rd day after birth(P<0.001).(2) The effect of delivery mode: Comparing to richness and Shannon index of vaginal delivery group, those of cesarean section group were significantly low at 3rd day after birth(5.63±2.05, 1.24±0.35 vs 6.82±2.28, 1.44±0.30,P<0.001)as we can see the same result in Shannon index at the 5th day after birth(P<0.05). No diversity of two groups was found between the 1st week and the 3rd week.(3) The effect of feeding way: Shannon index of breast feeding group was lower than that of formula feeding group at the both time of the 1st week and the 3rd week after birth(1.75±0.32,1.79±0.32 vs 1.88±0.29, 1.92±0.35, P<0.05). The was no difference between these two groups in richness(P>0.05).(4) The effect of gestational age: Full term neonates' richness at the three time point during 1 week after birth and Shannon index of the 3rd day and the 5th day were significantly higher than those of preterm infants. There was no difference between these two groups at the 3rd week after birth.Conclusion: The intestional flora diversity of neonates changed dynamically with time and the most dramatic development period was between day 3 to day 5 after birth. The intestinal flora diversity of cesarean section newborn was lower than vaginal delivery baby in the early period after birth. Premature infants had low intestinal flora diversity and delayed colonization.Part II lllumina high-throughput sequencing technology analysis of the effect of gestational age and feeding mode on preterm neonatal intestinal microbiota profileObjective: To study the effect of gestational age and feeding type on preterm neonatal intestional microbiota distribution characteristics with Illumina high-throughput sequencing technology.Methods:(1) 35 premature infants were recruited into the feeding group who were deliveried by cesarean section and fed by breast milk and formula; the gestational age group including 22 preterm infants excluded the breast feeding infants based the feeding group.(2) After mixing up the DNA of fecal bacteria proportionally and with equal concentration, the amplification of the variable regions 3 of the 16 S r RNA gene was sequenced through Illumina Miseq sequencing platform and then analyzed the profile of the intestinal flora composition.Results:(1) The intestinal microbiota of premature infants was dominated by two phylums--Firmicutes, Proteobacteria and by three classes--Bacilli, Clostridia, Gammaproteobacteria. In the family and genus level, it was dominated by Streptococcaceae, Veillonellaceae and Enterobacteriaceae. We did not find Bacteroidetes and Bifidobacterium in the whole observation period.(2) The effect of feeding type: There was no significant difference in relative abundance of the main phylum and genus between the two feeding types. In the low abundance species level, breast feeding group owned more species than formula feeding group. The study found that the relative abundance of Lactobacillus of breast feeding group was significantly higher than that of formula feeding group(P<0.05). Escherichia was failed to find in formula feeding group. In addition, the composition of bacteria in these two groups at four time points is also different and the difference in the phylum level was found during 3 to 5 days after birth.(3) The influence of gestational age: Old gestational age group had more abundant species than young gestational age group. Veillonella which was one of the dominant genus was significantly higher in old gestational age group than young gestational age group(P<0.05).Conclusion: High-throughput sequencing revealed the intestinal flora profile of preterm infants: more facultative anaerobe colonized, strictly anaerobic bacteria colonized delay. Gestational age is the important factor influencing the intestinal microbiota distribution characteristics in preterm infants. Feeding type had less effect on the structure of main species. Formula feeding existed disadvange effect on the establishment of low abundance microbiota. |
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