Oral Microbiome Dysbiosis Is Associated With Symptoms Severity and Local Immune/Inflammatory Response in COVID-19 Patients: A Cross-Sectional Study

Abstract

The human oral microbiome (HOM) is the second largest microbial community after the gut and can impact the onset and progression of several localized and systemic diseases, including those of viral origin, especially for viruses entering the body via the oropharynx. However, this important aspect has not been clarified for the new pandemic human coronavirus SARS-CoV-2, causing COVID-19 disease, despite it being one of the many respiratory viruses having the oropharynx as the primary site of replication. In particular, no data are available about the non-bacterial components of the HOM (fungi, viruses), which instead has been shown to be crucial for other diseases. Consistent with this, this study aimed to define the HOM in COVID-19 patients, to evidence any association between its profile and the clinical disease. Seventy-five oral rinse samples were analyzed by Whole Genome Sequencing (WGS) to simultaneously identify oral bacteria, fungi, and viruses. To correlate the HOM profile with local virus replication, the SARS-CoV-2 amount in the oral cavity was quantified by digital droplet PCR. Moreover, local inflammation and secretory immune response were also assessed, respectively by measuring the local release of pro-inflammatory cytokines (L-6, IL-17, TNFα, and GM-CSF) and the production of secretory immunoglobulins A (sIgA). The results showed the presence of oral dysbiosis in COVID-19 patients compared to matched controls, with significantly decreased alpha-diversity value and lower species richness in COVID-19 subjects. Notably, oral dysbiosis correlated with symptom severity (p = 0.006), and increased local inflammation (p < 0.01). In parallel, a decreased mucosal sIgA response was observed in more severely symptomatic patients (p = 0.02), suggesting that local immune response is important in the early control of virus infection and that its correct development is influenced by the HOM profile. In conclusion, the data presented here suggest that the HOM profile may be important in defining the individual susceptibility to SARS-CoV-2 infection, facilitating inflammation and virus replication, or rather, inducing a protective IgA response. Although it is not possible to determine whether the alteration in the microbial community is the cause or effect of the SARS-CoV-2 replication, these parameters may be considered as markers for personalized therapy and vaccine development.

Keywords: COVID-19; inflammatory cytokines; oral microbiome; secretory IgA; symptom severity.

FIGURE 1

SARS-CoV-2 load in control and COVID-19 subjects, as measured by ddPCR. (A) Graphical representation of the values detected by the use of three different molecular probes: negative samples, only the clouds corresponding to the housekeeping control genes are detectable (gray and purple clouds); low- and high-positive samples, the clouds corresponding to the virus genes are detectable and counted (positives to individual FAM probes: gray, red, and yellow; positives to individual HEX probes: purple, blue, and pink; double positives to FAM/HEX probes: beige and orange). (B) Virus load, expressed as genome copy number per analyzed sample (20 μl of extracted nucleic acid); left y axis refers to control, negative and low-positive values, whereas right y axis refers to high-positive COVID-19 subjects. Mean value ± SEM is also reported.

FIGURE 2

Alpha-diversity values in the oral microbiomes of control and COVID-19 subjects. (A) Comparison between control and COVID-19 patients, expressed as median and range values. (B) Comparison between controls and COVID-19 asymptomatic, pauci-symptomatic, and symptomatic subjects; median and range values are shown. (C) Comparison between genders (M, male; F, female) in the control and COVID-19 groups; median values with interquartile range are shown for each group.

FIGURE 3

Relative abundance and distribution of microorganisms in the oral cavity of control (CTR) and COVID-19 subjects. (A) Percentage distribution of most detected microbial genera. (B) Percentage distribution of most detected microbial species.

FIGURE 4

Significantly altered taxa in control (CTR) and COVID-19 subjects. The results are expressed as normalized counts ± SEM values. Significance p-values for each comparison are also displayed.

FIGURE 5

Mycome profile in the oral cavity of control and COVID-19 subjects. (A) Abundance of fungi expressed as total normalized counts for each individually detected mycetes. (B) Percentage distribution of the fungal genera in controls and COVID-19 patients.

FIGURE 6

Virome profile in the oral cavity of control (CTR) and COVID-19 subjects. The results are expressed as relative abundance (%). Left y axis refers to Human gammaherpesvirus 4 and Human alphaherpesvirus 1 (green bars), whereas right y axis refers to the amount of the four detected bacteriophage (orange-red bars).

FIGURE 7

Mucosal sIgA response in the oral cavity of COVID-19 and control (CTR) subjects. The positivity is expressed as the ratio (R) between the value detected in the sample and the threshold control value, following manufacturer’s instructions; mean value with range is also shown.

FIGURE 8

Presence of pro-inflammatory cytokines/chemokines in the oral cavity of COVID-19 patients and controls (CTR). The results are expressed as the mean values ± SEM of the concentration (pg/ml) for each indicated cytokine.