基于16S核糖体RNA高通量测序前瞻性分析老年重度烧伤患者早期肠道菌群变化
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1.武汉科技大学医学部医学院;2.汉大学同仁医院暨武汉市第三医院消化科

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国家自然科学(82100559);湖北省自然科学基金面上项目(2019CFB749)


Prospective Analysis of Early Gut Microbiota Changes in Elderly Patients with Severe Burns based on high-throughput sequencing of 16S ribosomal RNA
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    摘要:

    目的:借助16S核糖体RNA(16S rRNA)高通量测序分析老年重度烧伤患者早期肠道菌群变化。方法:按照入排标准纳入13例重度烧伤患者(Burn组)和同期的12例健康志愿者(Control组)。收集两组临床资料及粪便样本,并对样本进行16S rRNA V4区基因测序,分析各类菌属的相对丰度;用Rarefy方法进行操作分类单元(Operational Taxonomic Unit,OTU)划分,使用Z- score方法对差异菌群进行标准化,构建差异菌群热图,通过QIIME(v.1.9.1)软件分析粪便菌群 OTU 数目及多样性指数;利用线性判别分析(LDA Effect Size,LEfSe)解析优势菌群;通过PICRUSt2软件预测菌群功能丰度;使用R语言中的igraph包对试验样本属水平差异菌行相关性可视化分析。数据通过独立样本t检验、Wilcoxon秩和检验、卡方检验和Kruskal-Wallis检验分析,P<0. 05 为差异有统计学意义。结果:重度烧伤患者早期肠道菌群以厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)为主。 23种菌属在两组中具有一定差异性(P<0.05)。差异菌菌群热图可见Control组的菌群丰度略优于Burn组。LEfSe分析表明Control组的梭菌纲(clostridia)与Burn组的芽孢杆菌纲(Bacilli)的LDA评分大于4分。与Control组相比,Burn组的肠道菌群物种数目在 1 个门、2个纲、6个目、6个科和10个属中显著增加,两组菌群微生物分布分支图提示Burn组以芽孢杆菌纲为主要区别标志菌群,Control组以梭菌纲为主要标志菌群。京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)功能预测分析提示两组间无显著差异(P>0.05)。联合网络可视化分析确定发现厚壁菌门影响最大,在可检测到的菌属中共有136例菌属彼此间具有一定的正相关或负相关性(|cor|>0.3;P<0.05),以协同作用为主。结论:老年重度烧伤患者早期肠道菌群与健康同龄人存在显著差异,其益菌种类及丰度减少,厌氧菌比例显著升高,菌群多样性降低,不同菌群间存在一定的协同及拮抗作用。

    Abstract:

    Objective: To analyze the early changes in the gut microbiota of elderly patients with severe burns using high-throughput sequencing of 16S ribosomal RNA (16S rRNA). Methods: Thirteen patients with severe burns (Burn group) and twelve healthy volunteers (Control group) were enrolled according to the inclusion and exclusion criteria. Clinical data and fecal samples were collected from both groups, and 16S rRNA V4 region gene sequencing was performed to assess the relative abundance of various bacterial taxa. The Rarefy method was employed to generate operational taxonomic units (OTUs), and Z-score normalization was applied to identify differentially abundant bacteria. A heatmap for differential bacterial communities was constructed. The number of fecal microbiota OTUs and diversity indices were analyzed using QIIME (version 1.9.1). Linear discriminant analysis effect size (LEfSe) was used to identify dominant bacterial groups. The functional abundance of the microbiota was predicted using PICRUSt2 software. The correlations among differential bacterial taxa at the genus level were visualized using the igraph package in R language. Data were analyzed using independent sample t-tests, Wilcoxon rank-sum tests, chi-square tests, and Kruskal-Wallis tests, with a significance threshold of P < 0.05. Results: The early gut microbiota of severe burn patients was predominantly composed of Firmicutes and Bacteroidota. Twenty-three bacterial genera exhibited significant differences between the two groups (P < 0.05). The heatmap of differential bacterial communities indicated that the Burn group had a slightly lower abundance of microbial taxa than the Control group. LEfSe analysis indicated that the LDA scores for Clostridia in the Control group and Bacilli in the Burn group were both greater than 4. Compared to the Control group, the Burn group exhibited a significant increase in bacterial species in one phylum, two classes, six orders, six families, and ten genera. The microbial distribution dendrogram suggested that Bacilli were the primary distinguishing marker for the Burn group, while Clostridia were the main marker for the Control group. KEGG functional prediction analysis indicated no significant differences between the two groups (P > 0.05). Combined network visualization analysis revealed that Firmicutes exerted the greatest influence, with 136 bacterial genera showing significant positive or negative correlations (| cor | > 0.3; P < 0.05), primarily through synergistic interactions. Conclusion: There are significant differences in the early gut microbiota of elderly patients with severe burns compared to their healthy peers, characterized by a reduction in beneficial bacterial species and abundance, an increase in the proportion of anaerobic bacteria, and decreased microbial diversity, along with certain synergistic and antagonistic interactions among different microbiota.

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  • 收稿日期:2024-10-22
  • 最后修改日期:2025-01-17
  • 录用日期:2025-03-27
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