利用单细胞扩增技术解析母婴间肠道微生物构成及功能

Analysis of intestinal microbial composition and function of mother-infant pairs by single cell amplification technique

微生物在人体中扮演重要角色,而婴儿早期微生物对日后健康发育至关重要.微生物可通过产道和母乳这2种主要途径从母亲传递给婴儿.本研究利用单细胞扩增技术对3对母婴粪便样品(共48份细菌悬液)进行宏基因组测序分析,旨在探究母婴间肠道微生物构成并对相关功能基因作出分析.结果显示,每对母婴间微生物尽管含量不同,但种类都高度相似(>99%).三婴儿中,一个顺产婴儿显示与其母亲有相似的高丰度物种,如Roseburia hominis,Roseburia intestinalis,Eubacterium rectale,Eubacterium eligens等;另2个剖腹产婴儿显示与皮肤、口腔相关的高丰度物种,如Propionibacterium acnes,Staphylococcus saprophyticus/S.haemolyticus,Delftia acidovorans等.剖腹产婴儿中拟杆菌少于顺产婴儿,如Bacteroides(B.)fragilis,B.helcogenes,B.salanitronis等.本研究共检测到1283种细菌,除常见的人体肠道微生物外,还检测到4个低丰度物种,分别是Lactobacillus(L.)amylovorus,L.kefiranofaciens,L.sanfranciscensis和Bifidobacterium asteroids.粪便样品功能宏基因组显示,婴儿粪便中富有与碳水化合物相关的磷酸转移酶系统(PTS)和β-葡萄糖苷酶基因.本研究初次使用单细胞扩增技术对母婴间肠道微生物组成及功能做出探索,获得了较高测序深度及物种多样性,发现了之前鲜有报道的低丰度物种,证实单细胞扩增技术可用于后续不同生态环境微生物领域的研究.

Microorganism plays an important role in human health, such as promoting digestion and absorption, regulating the host immune system, maintaining dental health, and resistance to pathogens invasion. The microbes of newborn babies are crucial to their health;it may be involved in infant brain development and neurodevelopment. Maternal genotype and diet, antibiotic use, maternal viral infections during pregnancy and mode of delivery are possible factors that can modulate the intestinal microbiome. Our previous studies have found that microbes can be vertically transferred from mother to infant through two main ways, birth canal and the breast milk. In the natural environment, only 1% of the microbes can be cultured. Thus, many low and non-cultivatable taxa cannot be accurately identified by traditional methods. Comparing with PCR-based whole genome amplification methods, the single cell multiple replacement amplification(MDA) technique can reduce amplification bias by 3~4 orders of magnitude, increase the minimal DNA of a single bacterial cell by about 5 billion times, and produce an average length of >12 kb amplification, which allows the gene fragment to be sequenced accurately. In this experiment, we applied the single-cell amplification technique to analyze 3 pairs of maternal and infant feces(a total of 48 bacterial suspensions) in order to explore the composition of intestinal microorganisms and analyze the related functional genes. The results showed that the fecal microorganisms of each pair of mother and infant were highly similar(>99%) despite in different abundance. Among three infants, one vaginally born infant showed similar high abundance species to its mother, such as Roseburia hominis, Roseburia intestinalis, Eubacterium Rectale, and Eubacterium eligens, while the high abundant species of other two C-sections infants were associated with the skin and mouth, such as Propionibacterium acnes, Staphylococcus saprophyticus, Haemolyticus, and Delftia Acidovorans. Bacteroides(B.), such as B. fragilis, B. helcogenes, and B. salanitronis, were less prevalent in C-section delivered infants compared to the vaginally born infant. A total of 1283 bacterial species were identified in this study. In addition to the previously reported human gut-related species, we detected three low-abundant taxa, namely Lactobacillus(L.) amylovorus, L. kefiranofaciens, L. sanfranciscensis, and Bifidobacterium asteroids. The functional metagenomes of fecal samples showed that the carbohydrate-related phosphotransferase system(PTS) genes and beta-glucoside genes were enriched in the infant microbiome. In this study, we applied the single-cell amplification technique to explore the composition and function of intestinal microorganisms of the mother and infant, with high sequencing depth and species diversity coverage. We also detected some low abundance species, which were rarely reported before. We believe the present approach is suitable for future analysis of microbiota of different ecological environments.