益生菌剂调整肠道疾病人群菌群结构丰度水平的研究

Probiotics Modulate the Structure and Abundance of Gut Mircrobiota in Populations with Intestinal Diseases

为探究益生菌剂对肠道疾病人群菌群多样性及丰度水平的调节能力。本研究以健康、便秘、腹泻及腹胀、肠易激综合征患者、排便不规律等人群为对象,使其定时、定量摄入益生菌剂(probotic preparations,PPrs)共6周,采集新鲜粪便样品并提取DNA,利用Ion Torrent PGM测序技术进行16S r RNA V3区扩增子测序,并联合生物信息学和多变量统计学分析方法对测序数据进行多样性分析。在测序深度满足要求的情况下,从门、科、属乃至种4个水平由浅至深挖掘益生菌剂对各受试人群肠道菌群失调的调节作用。所有测序序列在97%相似水平划分得到2 320个分类操作单元,拟杆菌门(Bacteroidetes)、硬壁菌门(Firmicutes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)为各组的优势菌门,占总序列数的99.81%。益生菌剂对各组受试人群的共性特征是:毛螺菌科(Lachnospiraceae)的调整均有长期的积极作用,而产碱杆菌科(Alcaligenaceae)、理研菌科(Rikenellaceae)、双歧杆菌科(Bifidobacteriaceae)等优势菌科在干预停止后又恢复至干预前的状态。益生菌剂对各种疾病状态人群的作用存在差异,便秘组的蓝细菌门(Cyanobacteria)和梭杆菌门(Fusobacteria)均从无到有。经主成分分析(principal component analysis,PCA)鉴定显示,在干预第1~4周期间,益生菌剂对腹泻组属水平的调整优于其他两组,柔嫩梭菌属(Faecalibacterium)、不动杆菌属(Acinetobacter)、布劳特氏菌属(Blautia)、梭菌属(Clostridium)、小类杆菌属(Dialister)、埃格特菌属(Eggerthella)、颗粒链菌属(Granulicatella)、乳杆菌属(Lactobacillus)、草酸杆菌属(Oxalobacter)、Pyramidobacter等10个属多样性及相对丰度均有明显的提升;便秘组仅有普氏菌属(Prevotella)、巨单胞菌属(Megamonas)、柯林斯菌属(Collinsella)3个属相对丰度变化;其他组的Adlercreutzia、Collinsella、克雷白氏杆菌属(Klebsiella)、Parabacteroides、萨特氏菌属(Sutterella)5个属有相对丰度的变化。PCA分析还揭示了益生菌剂在不同肠道疾病个体菌群结构的调整方面存在明显的差异,对不同肠道疾病的改善及治疗作用也不一样。另外,便秘组的Bacteroides和Odoribacter有降低的趋势,且回复到与对照组接近的状态。经热图分析鉴定发现,Akkermansia muciniphila、Bacteroides fragilis、Faecalibacterium prausnitzii等几种已被认定功效的关键菌阶段性地出现,但是停止干预后即消失,原有的Bacteroides ovatus开始降低,这些菌的出现对维护宿主健康具有价值。综上所述,该益生菌剂干预能起到改变肠道微生物多样性的功效,并具有调整菌群丰度水平的作用,表现出了抑制肠道有害微生物生长的作用,特别是对肠道健康有益菌的出现更体现出该益生菌剂的生理功效;因此,该益生菌剂应用于维持肠道菌群结构的稳态具有广阔的市场前景。

The aim of the present study was to explore the regulatory effect of probiotics on the intestinal microbial diversity and abundance in patient populations with intestinal diseases. A probiotic preparation（PPr） was regularly consumed at a constant amount for 6 weeks by healthy, constipated, diarrheal, abdominal bloating, irritable bowel syndrome and irregular defecation populations. Before, during and after the experiment, fecal samples were collected for bacterial genomic DNA extraction. The sequencing of 16 S r RNA V3 region was performed with the Personal Genome Machine（PGM, Ion Torrent）. The sequencing data were used for diversity analysis by bioinformatics and multivariate statistical analysis. When the sequencing depth was satisfactory, the effect of the probiotics in correcting gut microbiota imbalance in the subjects was evaluated at the four levels of phylum, family, genus and even species. All sequencing reads were divided into 2 320 operational taxonomic units at a 97% similarity level. Bacteroidestes, Firmicutes, Proteobacteria and Actinobacteria were the dominant phyla, accounting for 99.81% of the total number of sequences. For all the subjects, the probiotics had a longlasting positive impact on Lachnospiraceae whereas Alcaligenaceae, Rikenellaceae, Bifidobacteria（Bifidobacteriaceae） and other dominant bacteria returned to the pre-intervention level when the intervention was withdrawn. The effects of probiotics on gut microbiota were different among all patient populations, and both Cyanobacteria and Fusobacteria were detected in the constipation group after consumption of the priobiotics. Principal component analysis（PCA） analysis showed that the regulatory effect of the probiotics in the diarrheal group was superior to that in two other groups during the first to the fourth week of intervention. The diversity and abundance of 10 genera including Faecalibacterium, Acinetobacter, Blautia, Clostridium, Dialister, Eggerthella, Granulicatella, Lactobacillus, Oxalobacter, Pyramidobacter were significantly increased in the diarrheal group, while only three genera： Prevotella, Megamonas and Collinsella in the constipation group changed. Moreover, the other groups exhibited a change in the abundance of Adlercreutzia, Collinsella, Klebsiella, Parabacteroides, and Sutterella. PCA analysis also revealed obvious differences in the regulatory effect of the probiotics among different gut microbial populations and that the probiotics had different effects in improving and treating different intestinal diseases. In addition, Bacteroides and Odoribacter in the constipation group tended to decrease to normal levels. Heatmap analysis showed that several key functional fungi, such as Akkermansia muciniphila, Bacteroides fragilis and Faecalibacterium prausnitzii, were found to appear only at some stages, but disappear after the intervention was withdrawn. At this time, the original Bacteroides ovatus began to decrease. The appearance of these bacteria was beneficial to host health. In summary, probiotic intervention can play a role in altering the diversity and abundance of gut microbes and inhibit the growth of harmful microorganisms in the gut. More importantly, the appearance of beneficial bacteria manifests the physiological effectiveness of the probiotics. Therefore, the probiotics have promising applications in maintaining a healthy state of intestinal microflora.