Oropharyngeal Microbiome Analysis in Patients with Varying SARS-CoV-2 Infection Severity: A Prospective Cohort Study

Abstract

Patients with COVID-19 infection have distinct oropharyngeal microbiota composition and diversity metrics according to disease severity. However, these findings are not consistent across the literature. We conducted a multicenter, prospective study in patients with COVID-19 requiring outpatient versus inpatient management to explore the microbial abundance of taxa at the phylum, family, genus, and species level, and we utilized alpha and beta diversity indices to further describe our findings. We collected oropharyngeal washing specimens at the time of study entry, which coincided with the COVID-19 diagnosis, to conduct all analyses. We included 43 patients in the study, of whom 16 were managed as outpatients and 27 required hospitalization. Proteobacteria, Actinobacteria, Bacteroidetes, Saccharibacteria TM7, Fusobacteria, and Spirochaetes were the most abundant phyla among patients, while 61 different families were detected, of which the Streptococcaceae and Staphylococcaceae families were the most predominant. A total of 132 microbial genera were detected, with Streptococcus being the predominant genus in outpatients, in contrast to hospitalized patients, in whom the Staphylococcus genus was predominant. LeFSe analysis identified 57 microbial species in the oropharyngeal washings of study participants that could discriminate the severity of symptoms of COVID-19 infections. Alpha diversity analysis did not reveal a difference in the abundance of bacterial species between the groups, but beta diversity analysis established distinct microbial communities between inpatients and outpatients. Our study provides information on the complex association between the oropharyngeal microbiota and SARS-CoV-2 infection. Although our study cannot establish causation, knowledge of specific taxonomic changes with increasing SARS-CoV-2 infection severity can provide us with novel clues for the prognostic classification of COVID-19 patients.

Keywords: SARS-CoV-2 infection; bacterial abundance; bacterial diversity; oropharyngeal microbiome.

Figure 1

Relative abundance of dominant phyla (A), families (B), and genera (C) in patients with COVID-19 requiring hospitalization vs. patients treated on an outpatient basis.

Figure 2

Relative abundance of dominant species in patients with COVID-19 requiring hospitalization vs. patients treated on an outpatient basis. Only statistically significant differences in the relative abundance of species are depicted. Note: Streptococcus AFUC_s and Streptococcus ALIF_s correspond to the Streptococcus sinensis group and Streptococcus salivarius group, respectively.

Figure 3

Alpha diversity analysis using the Chao1, Shannon, Simpson, and Fisher indices to evaluate comparative bacterial richness between patients with COVID-19 requiring hospitalization and patients treated on an outpatient basis.

Figure 4

Visualization of β-diversity analysis using the Multi-Dimensional Scaling (MDS) (A) and the respective non-metric version (metaNMDS) (B).

Figure 5

(A) Cladogram and (B) LDA score of the microbial species in the pharyngeal microbiomes of study patients, based on which the patients could be classified as those requiring inpatient management and those with mild symptoms treated on an outpatient basis. (C) Visualization of LEfSe analysis in the form of a bar chart of the main microbial species that could help to distinguish hospitalized patients with COVID-19 from patients with mild symptoms requiring treatment on an outpatient basis. The dotted and continuous horizontal lines indicate the median and mean values of each group, respectively.