PCR-变性梯度凝胶电泳法初探婴儿期肠道菌群菌种演替

A preliminary analysis of changes in composition of intestinal microbiota during infancy using polymerase chain reaction-denaturing gradient gel electrophoresis

目的通过研究婴儿粪便菌种组成,探索婴儿期肠道菌群定植规律。方法收集15例健康婴儿出生后第0、2、4、7、10、14、28天,以及3、6、12月时的新鲜粪便,采用PCR-变性梯度凝胶电泳(DGGE)检测肠道菌群结构,对其优势菌种进行测序,分析婴儿期肠道菌群菌种演替过程。结果 DGGE指纹图谱可见,婴儿期的肠道菌群结构随出生后时间变化显著;克隆测序结果显示,变形菌门的细菌定植最早,以专性需氧菌肠杆菌、假单胞菌为主,其次是厚壁菌门的专性厌氧菌(梭状芽孢杆菌、小韦荣球菌)和兼性厌氧菌(多枝梭菌)定植,随后疣微菌门定植,放线菌门定植最晚,以双歧杆菌为主,并逐渐成为优势菌群。结论婴儿期肠道中专性需氧菌首先定植,随后是专性厌氧菌和兼性厌氧菌。变形菌门细菌定植最早,厚壁菌门细菌次之,随后是疣微菌门细菌,以双歧杆菌为主的放线菌门细菌定植最晚。

Objective To investigate the composition of bacteria in the stools of infants and the colonization of intestinal microbiota during infancy. Methods Fresh stools were collected from 15 healthy infants at 0, 2, 4, 7, 10, 14, and 28 days and 3, 6, and 12 months after birth. Polymerase chain reaction-denaturing gradient gel electrophoresis（PCRDGGE） was used to analyze the composition of intestinal microbiota, perform sequencing of dominant bacteria, and to analyze the changes in the composition of intestinal microbiota during infancy. Results DGGE fingerprint showed that the composition of intestinal microbiota during infancy changed significantly over time after birth. The cloning and sequencing results indicated that Proteobacteria colonized the earliest, mainly the obligate aerobes Enterobacter and Pseudomonas, followed by the obligate anaerobes（Clostridium hathewayi and Veillonella parvula） and the facultative anaerobe Clostridium ramosum in Firmicutes, and Verrucomicrobia. Actinobacteria colonized the latest, mainly Bifidobacterium, and gradually became dominant bacteria. Conclusions During infancy, obligate aerobes colonize the intestinal tract the earliest, followed by obligate anaerobes and facultative anaerobes. Proteobacteria colonizes the earliest, followed by Firmicutes and Verrucomicrobia, and Actinobacteria, mainly Bifidobacterium, colonizes the latest.