Since the appearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in 2019, cases of coronavirus disease 2019 (COVID-19) have rapidly spread around the world infecting over 113 million people and claimed over 2.5 million lives, as of March 1, 2021 (https://covid19.who.int/). COVID-19 is transmitted through the respiratory tract via aerosolized droplets containing viral particles1. Severity of COVID-19 is dictated by immune-inflammatory response of the host to the virus (i.e., cytokine storm). Host-associated microorganisms can influence viral infectivity2 and is the major player in mediating immune-inflammation3. Conversely, studies have shown that viruses can modulate microbiota community in the oropharyngeal and respiratory tract4. However, few studies have examined the nasopharyngeal microbiota in COVID-19 patients. One study found no significant microbial diversity outcomes5, while another study found a significant decrease in microbial diversity, differences between microbial communities, and a higher abundance of species Propionibacteriaceae and a reduction in Corynebacterium accolens in COVID-19-positive relative to -negative patients6. This proof-of-concept study aimed to identify nasopharyngeal microbiota alterations in COVID-19-positive and –negative patients to determine if: (1) nasopharyngeal swab samples collected for PCR testing of SARS-CoV-2 and the associated viral transport media (VTM) are suitable for microbiome analysis and (2) COVID-19-positive and -negative patients have dissimilar nasopharyngeal dysbiotic microbial communities.
We examined COVID-19-positive (n = 9) and -negative (n = 10) subjects recruited from Rush University Medical Center (RUMC) located in Chicago, IL in the early stage of the pandemic (April 2020). The nine COVID-19-positive subjects had mild COVID-19 with no hospitalizations or deaths reported (Table S1). The study was approved by RUMC Institutional Review Board [COVID-19 Biorepository (ORA #20032309)], to use random de-identified remnant specimens, from standard care testing of COVID-19, with no a priori exclusion criteria.
Analysis of communities using 16S ribosomal RNA gene amplicon sequencing revealed that microbial alpha-diversity indices were not significantly different between patient groups, but taxonomic feature richness was lower in COVID-19-positive compared to -negative patients (Table S2). Significant differences (PERMANOVA: q = 0.016) in nasopharyngeal microbial community structure were observed between positive and negative patients in beta diversity analyses conducted on microbial features (i.e., amplicon sequence variant, ASV) (Figure 1A; Table S3). Microbial communities in both groups were dominated by the phyla Proteobacteria, Actinobacteria and Firmicutes, and the genera Corynebacterium, Morganella, Moraxella, Escherichia-Shigella, Proteus, and Staphylococcus (˃ 50% of all sequences; Figure 1B-C; Table S4). The microbial community in COVID-19-positive patients can be characterized as pro-inflammatory as exemplified by significantly higher (Mann-Whitney test: p = 0.0002) Proteobacteria-to-Actinobacteria ratio relative to COVID-19-negative patients (Figure 1D). Between-group differences in taxon abundances were identified using ANCOM (Table 1) and DeSeq2 (Tables S5, S6). ANCOM identified the genus Streptococcus as significantly less abundant in COVID-19-positive relative to -negative patients, with nine genera trending towards significance (Table 1; Figure 1E). DESeq2 analysis identified 25 significantly differentially abundant genera, including lower abundances of Rothia and Prevotella in COVID-19-positive patients (Table S6). A machine learning approach (Boruta) was employed for feature selection to identify taxa driving differences between COVID-19-positive and -negative patients. This analysis identified nine genera, many of which were also identified in DESeq2 and ANCOM analyses (Figure 2).
This study provides evidence that current biorepositories with nasopharyngeal VTM can be utilized to assess microbiome communities. Importantly, this proof-of-concept study is the first to show that COVID-19-positive patients have a dysbiotic “pro-inflammatory” nasopharyngeal microbial community characterized by loss of putative nasal commensal bacteria and an increase in putative pro-inflammatory bacteria. Specifically, this study revealed: (1) a characteristic nasal microbiome in COVID-19-positive individuals with reduced microbial diversity, (2) an increased relative abundance of multiple putative pathogenic Proteobacteria taxa, and (3) decreased relative abundance of nasal commensal organisms such as Corynebacterium (Actinobacteria)7 and Streptococcus (Firmicutes), previously shown to be affected by influenza virus infection8. These differences need to be further evaluated in future studies, but could reflect differences prior to infection leading to susceptibility to SARS-CoV-2 infection or contributing to COVID-19 severity or may be a result of post-infection microbiome alteration.
In conclusion, this proof-of-concept study provides preliminary data suggesting the presence of a dysbiotic and pro-inflammatory nasopharyngeal microbiota in COVID-19-positive patients. The relatively small sample size may have limited our ability to identify additional significant differences between groups. Nonetheless, our study provides a strong scientific rationale for future studies to investigate the relationship between nasal microbiome and SARS-CoV-2 infection and COVID-19 severity, and also the relationship to the long-lasting effects of COVID-19.