腹泻柴达木马亚成体粪便微生物多样性分析及其生物标记物的筛选

doi:10.16829/j.slxb.150637

摘要/Abstract

摘要：

为比较青藏高原柴达木马亚成体腹泻与健康个体粪便微生物群落多样性和结构组成的差异, 我们利用16S rRNA测序技术对采集的腹泻 (n = 3) 和健康 (n = 13) 个体粪便样本细菌的组成与分布进行分析比较，并利用实时荧光定量PCR测定相关菌属的含量。结果显示，无论健康还是腹泻，厚壁菌门、拟杆菌门、疣微菌门、变形杆菌门和螺旋体门是柴达木马亚成体粪便中的优势菌门。相比健康组，腹泻组粪便微生物的Alpha多样性显著下降 (P < 0.05)，厚壁菌门相对丰度下降而变形杆菌门的相对丰度显著增加 (P < 0.05)，推断这两个门中的梭菌属、普雷沃菌属、纤杆菌属等丰度的失衡可能是导致柴达木马腹泻的原因之一。此外，通过机器学习的随机森林算法筛选出12个对健康和腹泻柴达木马亚成体粪便微生物差异具有较大影响的特征菌属，包括甲烷短杆菌属、纤杆菌属、Paludibacter、肉食杆菌属和迷踪菌属等。研究揭示了健康和腹泻柴达木马亚成体粪便微生物组的变化，为进一步研究青藏高原地区家畜腹泻提供一定的数据支持。

关键词: 16S rRNA测序, 实时荧光定量PCR, 粪便菌群, 生物标记物, 腹泻

Abstract:

The study aims to compare the difference in diversity and composition of fecal microbiota between healthy and diarrheal sub-adult Qaidam horses. We analyzed feces of healthy (n = 13) and diarrheal (n = 3) sub-adult Qaidam horses based on 16S rRNA high-throughput sequencing, and determined the absolute abundance of related differential bacteria genera by real-time qPCR. The results indicated that Firmicutes, Bacteroidetes, Verrucomicrobia, Proteobacteria and Spirochaetes were the dominant phyla in feces from both healthy and diarrheal Qaidam horses. Compared to healthy Qaidam horses, the fecal microbial Alpha diversity was significantly lower (P < 0.05), the relative abundance of Firmicutes was relatively lower (P < 0.05), and the ralative abundance of Proteobacteria was significantly higher (P < 0.05) in diarrheal Qaidam horses. It was then deduced that the dysbiosis of these two phyla (Clostridium, Prevotella, Fibrobacter, etc.) in microecology might play an important role in Qaidam horses’ diarrhea. Additionally, 12 biomarkers (feature genera), such as Methanobrevibacter, Fibrobacter, Paludibacter, Carnobacterium and Elusimicrobium, which had significant influence on the difference of fecal bacteria composition between the two groups, were screened by a machine learning algorithm (random forest). The study compared the characteristics of fecal microbial changes in healthy and diarrheal Qaidam horses and provided a data basis for the diarrhea of livestock in the Qinghai-Tibet Plateau.

Key words: 16S rRNA sequencing, Real-time qPCR, Fecal microbiota, Biomarker, Diarrhea

中图分类号:

Q933

王小琪, 郝文静, 张志超, 韩晶, 王儒敬, 段子渊. 腹泻柴达木马亚成体粪便微生物多样性分析及其生物标记物的筛选[J]. 兽类学报, 2022, 42(4): 432-441.

Xiaoqi WANG, Wenjing HAO, Zhichao ZHANG, Jing HAN, Rujing WANG, Ziyuan DUAN. Fecal microbiota diversity analysis of the diarrheal sub-adult Qaidam horses and biomarkers screening[J]. ACTA THERIOLOGICA SINICA, 2022, 42(4): 432-441.

图/表 8

表1 柴达木马粪便细菌16S rRNA 基因PCR扩增引物

Table 1 PCR amplification primers of bacterial 16S rRNA gene in Qaidam horses’ feces

属Genera	引物Primers （5'-3'）	参考文献References
拟杆菌属 Bacteroides	F： CTGAACCAGCCAAGTAGCG	蒋曼，2014
拟杆菌属 Bacteroides	R： CCGCAAACTTTCACAACTGACTTA	蒋曼，2014
普雷沃菌属 Prevotella	F： GGTTCTGAGAGGAAGGTCCCC	Moreno-Indias et al.， 2016
普雷沃菌属 Prevotella	R： TCCTGCACGCTACTTGGCTG	Moreno-Indias et al.， 2016
甲烷短杆菌属 Methanobrevibacter	F： CCTCCGCAATGTGAGAAATCGC	丽丽，2020
甲烷短杆菌属 Methanobrevibacter	R： TCWCCAGCAATTCCCACCAGTT	丽丽，2020
双歧杆菌属 Bifidobacterium	F： CGCGTCYGGTGTGAAAG	Kameoka et al.， 2021
双歧杆菌属 Bifidobacterium	R： CCCCACATCCAGCATCCA	Kameoka et al.， 2021

表2 柴达木马粪便细菌16S rRNA测序数据

Table 2 16S rRNA sequencing data of feces from Qaidam horses

组别

Groups

有效序列片段数

No. of filtered reads

操作分类单元数

OTUs

分类单元（属）

Taxa （genus）

样本覆盖度

Coverage

图1 柴达木马粪便微生物物种多样性.A：粪便微生物群落Alpha多样性 (均值 ± 标准差)；B：基于非加权Unifrac矩阵的主坐标分析图.* P < 0.05

Fig. 1 The fecal microbial diversity of Qaidam horses. A: The Alpha diversity of fecal bacteria (mean ± SD); B: PCoA plot based on Unweighted Unifrac matrix.* P < 0.05

图2 腹泻组和健康组柴达木马粪便微生物门水平的相对丰度比较. A：粪便细菌相对丰度组成 (门水平)；B：两组间有显著性差异的菌门. * P < 0.05

Fig. 2 Comparison of relative abundance of fecal microbial in diarrhea (Dia) group and naive control (NC) group of Qaidam horses at phylum level. A: The histograms of microbiota abundances at phylum level; B: The phyla with significant difference between two groups. * P < 0.05

图3 腹泻组和健康组柴达木马粪便微生物属水平的相对丰度比较. A：粪便细菌相对丰度组成 (属水平)；B：热图展示的两组间有显著性差异的菌属

Fig. 3 Comparison of relative abundance of fecal microbial in diarrhea (Dia) group and naive control (NC) group of Qaidam horses at genus level. A: The histograms of microbiota abundance at genus level; B: Clustering heatmap of genera with significant difference between two groups

图4 腹泻组和健康组柴达木马粪便主要菌属16S rRNA基因拷贝数差异分析

Fig. 4 Compared average copy number of 16S rRNA gene from the main fecal bacteria between diarrhea group and naive control group of Qaidam horses

图5 基于随机森林的健康和腹泻柴达木马粪便微生物差异分析 (A) 及ROC曲线分析 (B)

Fig. 5 Random Forest bar chart of variable importance on genus level (biomarkers) (A) and ROC curve analysis (B)

图6 腹泻组和健康组柴达木马粪便微生物表型相对丰度

Fig. 6 The microbiota phenotype relative abundance in Qaidam horses of diarrhea (Dia) group and naive control (NC) group

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