Illumina高通量测序技术分析早产儿出生后肠道菌群变化的初步研究

目的 初步探讨NICU早产儿出生后肠道菌群变化特征及其与败血症的关系。方法 以复旦大学附属儿科医院NICU住院的早产儿为研究对象，于出生后第1天采集胎粪，之后于每周龄时或评估败血症时采集粪便样本，直至出院或生后8周。采用Illumina高通量测序技术对粪便样本中所有细菌的16S rRNA-V3区进行DNA测序，应用MG-RAST V3.3.6分析和统计样品序列数目、操作分类单元（OTU）数量，分析肠道菌群物种丰度和分布，并进行聚类分析。结果 3例早产儿共采集生后1、7、14和21 d的12份粪便样本，其中5份样本PCR扩增失败，7份样本(例1生后14、21 d；例2生后7、21 d；例3生后7、14、21 d)DNA测序成功进入分析。3例早产儿平均胎龄为（31.3±0.8）周，平均出生体重为（1 540±144） g。3例均生后应用抗生素，其中例1母亲有产前抗生素暴露史；例2在住院过程中发生全身炎症反应综合征。①7份样本稀疏曲线表明测序深度充分。物种多样性分析显示OTU值为381~608，微生物丰度较高，且与日龄呈正相关，其中例2生后7 d样本肠道菌群多样性最低；②7份样本共检测到18个菌门，均以放线菌门、拟杆菌门、厚壁菌门和变形菌门为优势菌门；变形菌门在例1生后14和21 d样本分别占97.52％和49.11％，放线菌门在例2生后7 d样本占99.46％；③共检测到172个科，其中63科为7份样本共有；相对丰度≥1％的科共检测到10个，例2生后7 d样本棒状杆菌科占97.90％，21 d样本中葡萄球菌科占27.16％；④聚类分析显示，同一研究对象不同时点肠道微生物相似性较高。结论 早产儿产前抗生素暴露及出生后早期抗生素暴露可能会显著降低肠道微生物的多样性，影响正常菌群的定植。发展为败血症的早产儿生后肠道微生物多样性较低，以致病菌占优势的肠道微生物区可能与败血症的发生相关。

Changes of postnatal gut microbiota in preterm infants using Illumina high-throughput sequencing technology

Objective To explore changes of postnatal gut microbiota in preterm infants in NICU and its association with neonatal sepsis.Methods Preterm infants hospitalized in NICU of Children's Hospital of Fudan University were enrolled in the study. Fecal samples were collected at day 1, 7, 14 and 21 after birth, respectively. Illumina high-throughput sequencing techno-logy was used to sequence 16S rRNA-V3 hypervariable region of all microbes in 7 fecal samples of 3 recruited subjects. MG-RAST V3.3.6 was used to analyze and calculate the numbers of sequences and operational taxonomic units （OTUs） for each sample, then the species abundance and distribution were analyzed and followed by cluster analysis.Results Seven samples had been analyzed. Three subjects had an average gestational age of (31.3±0.8) weeks and an average birth weight of (1 540±144) g. They all received antibiotic administration after birth. Maternal antibiotic exposure occured in 1 case. Another one case developed SIRS during hospitalization. ①The rarefaction curves showed that adequate sequencing depth was achieved. Analysis of species abundance showed that the OTUs number ranged from 381 to 608 indicating high microbial diversity. Meanwhile, the microbial diversity had a positive correlation with the postnatal age. ②Eighteen phyla were detected from all samples. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were predominant in all samples. However, the dominant phylum of case 1 belonged to Proteobacteria, with a percentage of 97.5％(day 14) and 49.1％(day 21) , while Actinobacteria predominated in day-7 sample of case 2, with a percentage of 99.5％. ③172 families were detected altogether, 63 of them were detected in all samples. Ten families were in higher relative abundance. In case 2, Corynebacteria occupied 97.9％ in day-7 sample, and Staphylococcaceae occupied a higher proportion in day-21 sample(27.2％) than the other two samples. ④The cluster analysis showed high similarity of intestinal microflora in different time points of one subject.Conclusion Gut microbial colonization and development in preterm infants in NICU were altered by various factors. Prolonged broad-spectrum antibiotics at prenatal and early postnatal age might profoundly decrease microbial diversity and affect microbial colonisation. Microbiota was less diverse from birth in infants who developed sepsis. There may be a microbiome predominanted of pathogens that may be associated with sepsis in preterm infants.