Bacterial Analysis of the Whole Blood in Chinese Healthy Donors Using 16S rDNA-Targeted Metagenomic Sequencing

Abstract

Background: The presence of bacteria in the blood of healthy individuals remains controversial. This study explored the comprehensive bacterial profiles and specific biomarkers in different components of healthy Chinese blood donors. Methods: A total of 5230 whole blood (WB) specimens were collected. Among them, 5200 random samples were pooled into 26 mixed samples for bacterial profile analysis. The remaining 30 random samples were divided into 4 groups based on components: WB, plasma, red blood cells (RBCs), and buffy coat (BC). Subsequently, the amplicons of the bacterial 16S rDNA V3-V4 fragments were sequenced to measure the diversity and composition of the bacteria using next-generation sequencing. Results: The bacterial DNAs in the blood primarily originated from the Proteobacteria phylum. A total of 301 species of bacterial DNA were found in blood specimens, with 46 species being present among all groups. A significantly higher abundance of bacterial DNA was found in the plasma and RBCs compared to those in BC and WB. However, the plasma and RBC groups showed significantly higher species diversity and richness compared to the BC and WB groups. In addition, the WB group had a significantly different community structure and composition compared to the plasma and RBC groups but was similar to the BC group. Conclusion: The presence of bacterial DNA fragments was confirmed in blood from healthy Chinese donors. The bacterial DNA fragments enriched in plasma showed the highest diversity, followed by RBC, WB, and BC. These results provide a foundation for further research on the microbiome in the blood of healthy individuals.

Keywords: 16S rDNA; Chinese blood donors; blood components; blood microbiome; whole blood.

Figure 1

Schematic representation of the workflow for blood sample preparation, genomic extraction, library preparation, sequencing using Illumina MiSeq, and metagenomics analysis using the CLC Microbial Genomics Module.

Figure 2

The microbiota species and abundance analysis of mixed whole blood from 5200 whole blood donors. (a) Stacked bar of the microbial community at the phylum level. (b) Stacked bar of the microbial community at the species level. (c) Sunburst view of the microbial community showing all taxa belonging to the kingdom bacteria. (d) Heat map analysis of microbiota in 5200 whole blood donors.

Figure 3

The microbiota species and abundance analysis of WB, BC, plasma, and RBC groups from whole blood donors. (a) Venn diagram. (b) Statistical analysis of bacterial abundance among different blood components. (c) and (d) Stacked bar of the microbial community among WB, BC, plasma, and RBC at the phylum level and species level, respectively. (e) Sunburst view of the microbial community showing all taxa belonging to the kingdom bacteria. (f) Observed abundance of dominant bacteria among WB, BC, plasma, and RBC. Abbreviations: BC, buffy coat; RBC, red blood cell; WB, whole blood. ⁣^∗∗∗p < 0.001.

Figure 4

Heat map analysis of top 30 microbiota in WB, plasma, RBC, and BC groups. Abbreviations: J, plasma; Q, whole blood; R, red blood cells; W, buffy coat.

Figure 5

Alpha diversities shown in a box plot. (a) Total number. (b) Chao 1 bias-corrected. (c) Simpson's index. (d) Shannon entropy.

Figure 6

Beta diversity results seen as 2D PCoA, with coloring performed according to taxonomic abundance values. (a) Jaccard. (b) Bray–Curtis. Abbreviations: J, plasma; Q, whole blood; R, red blood cells; W, buffy coat.

Figure 7

Linear discriminant analysis (LDA) effect size (LEfSe) analysis revealed significant bacterial differences in the WB, plasma, RBC, and BC groups. The LDA scores (log 10) > 2 and p < 0.05 are listed. (a) WB vs plasma. (b) Plasma vs RBC. (c) WB vs BC. (d) WB vs RBC. (e) RBC vs BC. (f) Plasma vs BC. Abbreviations: BC, buffy coat; RBC, red blood cells; WB, whole blood.