Saliva microbiome in relation to SARS-CoV-2 infection in a prospective cohort of healthy US adults

Abstract

Background: The clinical outcomes of SARS-CoV-2 infection vary in severity, potentially influenced by the resident human microbiota. There is limited consensus on conserved microbiome changes in response to SARS-CoV-2 infection, with many studies focusing on severely ill individuals. This study aimed to assess the variation in the upper respiratory tract microbiome using saliva specimens in a cohort of individuals with primarily mild to moderate disease.

Methods: In early 2020, a cohort of 831 adults without known SARS-CoV-2 infection was followed over a six-month period to assess the occurrence and natural history of SARS-CoV-2 infection. From this cohort, 81 participants with a SARS-CoV-2 infection, along with 57 unexposed counterparts were selected with a total of 748 serial saliva samples were collected for analysis. Total bacterial abundance, composition, population structure, and gene function of the salivary microbiome were measured using 16S rRNA gene and shotgun metagenomic sequencing.

Findings: The salivary microbiome remained stable in unexposed individuals over the six-month study period, as evidenced by all measured metrics. Similarly, participants with mild to moderate SARS-CoV-2 infection showed microbiome stability throughout and after their infection. However, there were significant reductions in microbiome diversity among SARS-CoV-2-positive participants with severe symptoms early after infection. Over time, the microbiome diversity in these participants showed signs of recovery.

Interpretation: These findings demonstrate the resilience of the salivary microbiome in relation to SARS-CoV-2 infection. Mild to moderate infections did not significantly disrupt the stability of the salivary microbiome, suggesting its ability to maintain its composition and function. However, severe SARS-CoV-2 infection was associated with temporary reductions in microbiome diversity, indicating the limits of microbiome resilience in the face of severe infection.

Funding: This project was supported in part by Danone North America and grants from the National Institutes of Health, United States.

Keywords: Microbiome; SARS-CoV-2; Saliva; Upper respiratory track.

Fig. 1

Study design schematic. Study participants are a subset from the larger population (n = 829) of the Rutgers Corona Cohort (RCC). COVID-negative subjects were selected from a larger pool of negative participants and matched 2:1 with the cases based on age, sex, BMI, and presence of co-morbidities.

Fig. 2

Total bacterial populations in the saliva samples in relation to the baseline sample, based on enumeration of 16S rRNA gene copies.A. SARS-CoV-2-infected subjects before, during, and after viral positivity (n = 34) and their 31 matched unexposed counterparts, with the study week shown relative to COVID infection with the week of the first positive test defined as week 0. B. SARs-CoV-2-infected subjects who were virus-positive at the first study visit (n = 47) and their 41 matched unexposed counterparts. There were no significant differences in either analysis between the exposed or unexposed subjects (Student's T test, FDR-corrected p > 0.05).

Fig. 3

Normalized alpha diversity of the salivary microbiome over time. Alpha diversity as measured by Faith's phylogenetic diversity (PD) normalized based on proportion of the first timepoint for each individual. A. SARS-CoV-2 infected subjects (n = 34) before, during, and after viral positivity and their 31 matched unexposed counterparts, with the study week shown relative to COVID infection with the week of the first positive test defined as week 0. B. SARS-CoV-2-infected subjects (n = 47) who were virus-positive at the first study visit and their 41 matched unexposed counterparts. There were no significant differences in either analysis between exposed and unexposed subjects (p > 0.05) when controlling for age and week of sampling, using linear mixed effects modeling.

Fig. 4

Beta diversity of the salivary microbiome over time. Panels: A. PCoA plots of unweighted and B. weighted UniFrac analyses of all timepoints for the 34 exposed subjects from before, during, and after viral positivity [colored by status relative to SARS-CoV2 infection] and their 31 matched unexposed subjects. C. Resilience of microbiome composition assessed by within-subject pairwise unweighted and D. weighted UniFrac distances between the first sample and later time points. Left panels: SARS-CoV-2-infected subjects (n = 34) before, during, and after viral positivity and their 31 matched unexposed subjects, with the study week shown relative to COVID infection with the week of the first positive test defined as week 0. Right panels: SARs-CoV-2-infected subjects (n = 47) who were virus-positive at the first study visit and their 41 matched unexposed subjects. No significant differences were found between exposed and unexposed subjects (p > 0.05) when controlling for age and week of sampling, using linear mixed effects modeling.

Fig. 5

Bar charts of taxon abundances in the salivary microbiome of subjects before, during, and after SARS-CoV-2 infection. Mean relative abundance of taxa at the phylum, genus, and species level of 34 exposed subjects before, during, and after viral positivity and their 31 matched unexposed subjects using one sample per subject for each period in relation to infection. Taxa with mean relative abundance across all samples <1.5% are binned into ‘Other’. No significant differences were found between exposed and unexposed subjects (p > 0.05).

Fig. 6

Alpha diversity analysis in symptom severity and illness duration. A. Change in alpha diversity as measured by Faith's phylogenetic diversity of the salivary microbiome comparing the values before SARS-CoV-2 infection in 81 subjects with those obtained during and after infection, according to the severity of their symptoms. Kruskal–Wallis test with Dunn's post hoc test FDR-correct p-values: ∗p < 0.05 B. Alpha diversity as measured by Faith's PD of the salivary microbiome of 79 SARS-CoV-2 infected subjects in samples obtained before, during, and after infection, according to the duration of the clinical illness. Mann–Whitney U test p-values: ∗p < 0.05. Before samples were obtained 14 ± 14 days before infection, early after were 14 ± 14 days after infection, and late after 154 ± 7 days after infection.

Fig. 7

Diversity analyses of functional gene pathways from metagenomics. A) Alpha diversity metrics B) Beta diversity metrics.