Network analysis of nitrate-sensitive oral microbiome reveals interactions with cognitive function and cardiovascular health across dietary interventions

Anni Vanhatalo, Joanna E L'Heureux, James Kelly, Jamie R Blackwell, Lee J Wylie, Jonathan Fulford, Paul G Winyard, David W Williams, Mark van der Giezen, Andrew M Jones, Anni Vanhatalo, Joanna E L'Heureux, James Kelly, Jamie R Blackwell, Lee J Wylie, Jonathan Fulford, Paul G Winyard, David W Williams, Mark van der Giezen, Andrew M Jones

Abstract

Many oral bacteria reduce inorganic nitrate, a natural part of a vegetable-rich diet, into nitrite that acts as a precursor to nitric oxide, a regulator of vascular tone and neurotransmission. Aging is hallmarked by reduced nitric oxide production with associated detriments to cardiovascular and cognitive function. This study applied a systems-level bacterial co-occurrence network analysis across 10-day dietary nitrate and placebo interventions to test the stability of relationships between physiological and cognitive traits and clusters of co-occurring oral bacteria in older people. Relative abundances of Proteobacteria increased, while Bacteroidetes, Firmicutes and Fusobacteria decreased after nitrate supplementation. Two distinct microbiome modules of co-occurring bacteria, that were sensitive to nitrate supplementation, showed stable relationships with cardiovascular (Rothia-Streptococcus) and cognitive (Neisseria-Haemophilus) indices of health across both dietary conditions. A microbiome module (Prevotella-Veillonella) that has been associated with pro-inflammatory metabolism was diminished after nitrate supplementation, including a decrease in relative abundance of pathogenic Clostridium difficile. These nitrate-sensitive oral microbiome modules are proposed as potential pre- and probiotic targets to ameliorate age-induced impairments in cardiovascular and cognitive health.

Keywords: Aging; Nitric oxide; Oral microbiome.

Conflict of interest statement

None.

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1
Fig. 1
Flowchart illustrating the trial stages from participant enrolment to data analysis. PL, placebo; BR, nitrate.
Fig. 2
Fig. 2
The Shannon diversity index (A) and Chao 1 (B) indicated no differences in species diversity between placebo (PL) and nitrate (BR) conditions. Non-metric multidimensional scaling (NMDS) revealed that the overall salivary microbiome composition differed between PL and BR (C). Significant differences in physiological and cognitive traits between PL and BR included increases in plasma nitrate ([NO3−]) and nitrite ([NO2−]) concentration (D, E); decrease in plasma [NO2−]/[NO3−] ratio (F); and decreases in systolic blood pressure (SBP) (G), pulmonary O2 uptake relative to walking speed (H), and number of errors in the RVP test of sustained attention (I). In panels D-I blue symbols and lines indicate mean ± SD and red dashed lines show individual responses. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify operational taxonomic units (OTU) driving the differences between placebo (PL) and nitrate (BR) conditions within oral microbiome modules 1–6 (MM1-MM6). Within MM3, MM7 and MM8 no OTU exceeded the logarithmic LDA score threshold of 2.
Fig. 4
Fig. 4
A: Eigengene correlation network between the Addenbrooke's Cognitive Examination (ACE-III) cognitive traits at baseline and microbiome modules (MM1-MM8) within the PL condition. B: Consensus correlation network between oral microbiome modules and 32 cognitive and physiological traits. Only three significant consensus module-trait relationships persisted across placebo and nitrate conditions: MM5 correlated with reaction time in the ‘information processing’ cognitive function test, MM6 correlated with MAP, and MM7 correlated with pulmonary O2 uptake at rest. The colour scale on the right-hand side of each panel indicates the strength of positive (green) and inverse (red) correlations. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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