Nasopharyngeal microbial dysbiosis in COVID-19 patients revealed by 16S rRNA gene sequencing

Filippos S Kardaras^1,2Eirini Siatravani³Katerina Tsilipounidaki⁴Efthymia Petinaki⁴Artemis G Hatzigeorgiou^1,2Vivi Miriagou^3*

• ¹DIANA-Lab, Dept. of Computer Science and Biomedical Informatics, University of Thessaly, Lamia, Greece
• ²Pasteur Hellenic Institute, Athens, Greece
• ³Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens, Greece
• ⁴Department of Medical Biopathology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Larissa, Greece

The coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2, has prompted intensive investigation into the factors influencing disease onset and severity. Among these, the role of the nasopharyngeal microbiome-an ecosystem critical for respiratory health and immune modulation-remains incompletely understood. This study aimed to elucidate the relationship between nasopharyngeal microbiota composition and COVID-19 clinical presentation during the initial phase of infection.Materials: A total of 81 nasopharyngeal swab samples were collected from individuals in Central Greece between January and February 2021. Following quality control, 77 samples were selected for microbiome analysis. These included SARS-CoV-2-negative controls (NE, n=26) and SARS-CoV-2positive patients classified as asymptomatic (AS, n=19), mild (MI, n=16), or severe (SE, n=16) based on clinical criteria. All COVID-19-positive samples were obtained within two days of symptom onset, and participants with recent hospitalization or antibiotic use were excluded. Microbiome profiling was performed using 16S rRNA gene-targeted metagenomic sequencing, followed by comprehensive bioinformatics and statistical analyses.Results: Significant differences were observed in alpha and beta diversity measures, with alpha diversity decreasing as COVID-19 severity increased. Three of the four individual study groups-NE, MI, and SE-exhibited distinct microbial profiles, while the asymptomatic group displayed greater heterogeneity. Significant variations in the abundance of specific phyla, families, and genera were identified between the different study groups. Comparing NE and SE groups, we observed a significant overrepresentation of the Proteobacteria phylum in SE, while the abundance of Fusobacteria was significantly lower in SE. In symptomatic COVID-19 patients, we observed significantly reduced abundances of important family constituents of the nasopharyngeal microbiota, such as Fusobacteriaceae, Prevotellaceae, and Streptococcaceae, suggesting disruption of microbial homeostasis during infection. Conversely, an increased prevalence of families associated with pathogenic or opportunistic pathogenic bacteria, including Enterobacteriaceae and Bacillaceae, was detected in the SE group, suggesting a potential role of these taxa in the disease progression of COVID-19.Conclusions: These findings shed light on specific genera that undergo significant changes during COVID-19 infection and contribute to our understanding of the dynamic nature of the nasopharyngeal microbiome in relation to disease progression and severity.