Characterizing the gut microbiota composition in Taiwanese hypertensive patients using 16S rRNA sequencing analysis

Abstract

Hypertension (HTN) is a significant risk factor for cardiovascular and cerebrovascular diseases. Accumulating evidence suggests a close relationship between HTN and alterations in the gut microbiota composition and abundance. We recruited 23 HTN patients and 17 controls matched for demographic characteristics. DNA extracted from fecal samples of patients was subjected to Illumina MiSeq sequencing, targeting the V3-V4 region of the bacterial 16S rRNA gene for analysis. We compared the diversity and composition of gut microbiota between the two groups. The α-diversity of gut microbiota in HTN patients was similar to that in the control group. β-diversity analysis showed slight differences in microbial composition between the HTN and control groups. We used Welch's t-test to evaluate the significant difference in the bacterial composition of the top 20 ASVs between the HTN group and the control group, and the results showed that Tyzzerella was significantly increased, while Faecalibacterium was significantly decreased in the HTN group. We performed PCR using Faecalibacterium-specific primers and analyzed their levels through agarose gel electrophoresis, confirming the reduced abundance of Faecalibacterium in the HTN group. In addition, Tax4Fun2 analysis was employed to examine differences in microbial functionality between the HTN group and the control group. In conclusion, we studied the fecal microbiota of HTN population in Taiwan through 16S rRNA gene sequencing, and found that Faecalibacterium has a lower abundance in HTN patients. This unique alteration in gut microbiota may provide insights into the pathogenesis of HTN and aid in the development of novel biomarkers and therapeutic targets.

Keywords: 16S-rRNA sequencing; cardiocerebrovascular diseases; gut microbiota; hypertension; taxonomic biomarkers.

Figure 1

The relative abundance of the most abundant taxa at various taxonomic levels (phylum, class, order, family, genus, and species) between the HTN and control groups. (A) Top 10 taxa with the highest relative abundance of ASV at different taxonomic levels were compared between the two groups. At each taxonomic level, different colors were used to represent the distribution and proportion of distinct microbial taxa. (B-E) The box plot illustrates the Mann-Whitney U test comparing the relative abundances of each taxonomic unit with significant differences between the two groups. The bacteria with significant differences between the two groups included: (B) g_Faecalibacterium (C) g_Lachnospiraceae_UCG-004, (D) g_Coprobacter, and (E) c_Alphaproteobacteria. The y-axis represents the relative abundance, while the x-axis represents the different taxa at specific taxonomic levels. Boxplots display the median (center line), interquartile range (box), and whiskers representing the minimum and maximum values within 1.5 times the interquartile range. Outliers are represented as individual data points.

Figure 2

Comparison of gut microbiota abundance between the HTN and control groups by Welch's t-test and LEfSe (A) Differential abundances of the top 20 ASVs between the HTN and control groups. Pair-wise Welch's t-tests were performed to identify statistically significant differences in ASV abundances. The X-axis represents the absolute differential means value, while the y-axis displays the taxa names, ranging from species to higher taxonomic ranks. The asterisk (*) indicates statistically significant differences based on the p-value. (B) LEfSe analysis for identification of fecal microbiome members enriched in HTN or control groups. The bar plot represents the effect size (LDA score) of significant differential taxa identified using LEfSe analysis. The length of the bar corresponds to a log10 transformed LDA score, indicating the magnitude of the effect. The colors of the bars indicate the group: dark green represents HTN, and light green represents the control group. The histogram of LDA scores highlights the biomarkers with statistical differences between the HTN and control groups, with the length of the bars reflecting their respective influence levels.

Figure 3

The relative abundance of the genus Faecalibacterium in the HTN and control groups was quantified using species-specific PCR and agarose gel electrophoresis. (A) PCR and agarose gel electrophoresis of fecal DNA from two groups of participants using 16S primers (Fprau223F/Fprau420R). M, DNA marker; panel left: lanes 1-17, DNA of control group; panel right: lanes 1-23, DNA of HTN group; N, negative control. (B) The PCR bands were quantified and presented using a box plot.

Figure 4

Predicting functional composition and KEGG pathway differences in prokaryotic microbiome between HTN and control groups using Tax4Fun2. (A) Analysis of potential functional differences between the two groups. (B) The correlation in KEGG pathway abundances was investigated.

Figure 5

Graphical summary of gut microbiota analysis in HTN and control groups. Alpha diversity showed no significant differences, while beta diversity analysis revealed microbial shifts. In HTN, Tyzzerella increased, whereas Faecalibacterium decreased among the top 20 ASVs. Taxonomic analysis (Mann-Whitney U test) identified reductions in Faecalibacterium, Lachnospiraceae_UCG-004, Coprobacter, and Alphaproteobacteria. LEfSe highlighted increased Enterobacteriaceae and decreased Alphaproteobacteria, Lachnospiraceae_UCG-004, and Faecalibacterium. Functional predictions suggested metabolic alterations.