Bacterial DNAemia is associated with serum zonulin levels in older subjects

Abstract

The increased presence of bacteria in blood is a plausible contributing factor in the development and progression of aging-associated diseases. In this context, we performed the quantification and the taxonomic profiling of the bacterial DNA in blood samples collected from forty-three older subjects enrolled in a nursing home. Quantitative PCR targeting the 16S rRNA gene revealed that all samples contained detectable amounts of bacterial DNA with a concentration that varied considerably between subjects. Correlation analyses revealed that the bacterial DNAemia (expressed as concentration of 16S rRNA gene copies in blood) significantly associated with the serum levels of zonulin, a marker of intestinal permeability. This result was confirmed by the analysis of a second set of blood samples collected from the same subjects. 16S rRNA gene profiling revealed that most of the bacterial DNA detected in blood was ascribable to the phylum Proteobacteria with a predominance of the genus Pseudomonas. Several control samples were also analyzed to assess the influence of contaminant bacterial DNA potentially originating from reagents and materials. The data reported here suggest that para-cellular permeability of epithelial (and, potentially, endothelial) cell layers may play an important role in bacterial migration into the bloodstream. Bacterial DNAemia is likely to impact on several aspects of host physiology and could underpin the development and prognosis of various diseases in older subjects.

Figure 1

Bacterial load in blood samples of older volunteers (n = 43) and in technical controls as determined by qPCR with panbacterial primers targeting the 16S rRNA gene. Data are reported as copies of the gene per volume of blood in linear (A) and logarithmic (B) scale. C_extr, DNA extracted from ultrapure deionized water with the same protocol used for blood samples; C_EDTA, DNA extracted with the same protocol used for blood samples from phosphate buffered saline (PBS) into an EDTA tube, added directly (C_dEDTA) or after passage through a vacutainer blood collection system (C_vEDTA); C_qPCR, ultrapure deionized water directly used as template in qPCR reactions. Horizontal black lines indicate the median.

Figure 2

Correlations between bacterial DNAemia, age, BMI, and metabolic and functional markers determined in blood of the older subjects under study (n = 43). The heatmaps on the left (white-red color gradient) refers to the distribution among older subject of the item reported in the central column. The heatmap on the right (blue-white-red color gradient) represents the Spearman’s correlation coefficient, ρ. Asterisks indicate the Kendall rank correlation P value: ***P < 0.001; **P < 0.01; *P < 0.05; + , 0.05 < P ≤ 0.10.

Figure 3

Linear regression and correlation analysis carried out between blood bacterial load and serum zonulin levels. Correlation has been assessed through Pearson’s (r coefficient), Spearman’s (ρ), and Kendall’s (τ) analyses. Analyses were performed on the first (n = 43, panels A,B,C) and the second (n = 42, panel D,E,F) set of blood samples collected from older people. The second set of blood samples was collected approximately four months after the first draw from the same older volunteers. Panels A and D, tables showing correlation and regression indexes and p values. Panels B and E, linear regression plot; vertical dotted lines indicate the quartiles. Panels C and F, Tukey box plots showing levels of bacterial DNAema in blood samples grouped in quartiles based on zonulin level.

Figure 4

Taxonomic composition of the bacterial DNA detected in the blood of older subjects expressed as 16S rRNA gene copies per volume of blood. (A) distribution of phyla in each analyzed blood sample. (B) most abundant families and genera detected in the blood samples under study. Horizontal black lines indicate the median.

Figure 5

Correlations of the taxonomic units detected in blood (expressed as abundances normalized to 16S rRNA gene copies) toward age, BMI, and metabolic and functional markers determined in blood of the older subjects under study (n = 43). This figure only includes taxa whose abundance significantly correlated with at least one parameter. The heatmap on the left (black-yellow color gradient) shows the distribution among samples of the abundances of taxonomic units. White boxes in the black-yellow heatmap indicate that taxa that have not been detected in a specific sample. The heatmap on the right (blue-white-red color gradient) represents the Spearman’s correlation coefficient, ρ. Asterisks indicate the P value of the Kendall’s rank correlation: *P < 0.05; **P < 0.01; ***P < 0.001.