Qualitative and semiquantitative analysis of the composition of the upper respiratory tract microbiota of patients with COVID-19 in relation to the intensity of SARS-CoV-2 virus replication

Katarzyna Talaga-Ćwiertnia^1*, Agnieszka Sroka-Oleksiak¹, Dominika Salamon¹, Agnieszka Krawczyk¹,
Barbara Zapala², Monika Brzychczy-Włoch¹, Tomasz Gosiewski¹

¹ Department of Molecular Medical Microbiology, Department of Microbiology, Faculty of Medicine Jagiellonian University.
Jagiellonian University Collegium Medicum, Poland
² Department of Diagnostics, Department of Clinical Biochemistry, Faculty of Medicine Jagiellonian University Collegium Medicum, Poland
^* E-mail: katarzyna.talaga@uj.edu.pl

 

The composition and role of the respiratory microbiome in the course of viral infections is still poorly understood. The aim of this study was to evaluate changes in the upper respiratory tract microbiota in relation to the intensity of SARS-CoV-2 virus replication, as expressed by the Cq parameter in quantitative PCR (qPCR). The study group consisted of 60 patients of the University Hospital in Krakow with COVID-19.

 

The material analyzed was nasopharyngeal swabs taken immediately after the patients' admission to the hospital, before antibiotic therapy. Viral RNA and bacterial DNA were isolated from clinical samples in parallel. In the first step, RNA isolates were subjected to qPCR amplification to confirm the presence of SARS-CoV-2 virus and determine the cycle threshold (Cq) value.

Depending on the Cq value obtained, the samples tested were assigned to one of three groups: I- Cq ≤31.0 (n=20; high level of replication), II- Cq > 31.0 (n=20; low level of replication), III- Cq = 0 (n=20; no viral RNA). In the second stage, isolates containing bacterial DNA were amplified by PCR (the V3-V4 region of the 16S RNA subunit was amplified). The obtained amplicons were used to prepare a genomic library for NGS sequencing in a MiSeq sequencer (Illumina).

 

It was observed that the diversity of α (expressed by Shannon index) and β (expressed by BrayCurtis and Jansen-Shannon indices) differed significantly between the study groups (p for α <0.044 and β <0.021 and <0.013, respectively). At the species level, using linear discriminant analysis (LDA score), bacteria unique to the groups were selected: Bacteroides uniformis (III), Prevotella maculosa, Corynebacterium pilbarense (II). On the other hand, in group I they included Pseudomonas beteli, Faecalibacterium prausnitzii, Bacteroides dorei, Akkermansia muciniphila, Enterococcus faecium and Bacteroides vulgatus. In the course of infection in the first group of patients, the presence of a significant percentage of pathogenic bacteria was characteristic, among which Bacteroides vulgatus and Akkermansia muciniphila were particularly noteworthy. These bacteria are responsible for the degradation of mucin and may contribute to the destruction of the mucosa of the upper respiratory tract, and consequently initiate transient colonization by other bacteria with pathogenic potential, accompanying SARS-CoV-2 infection.

 

Funding source: NCBiR funds within the framework of the project HOSPITALS-ONE/18/2020 implemented by a consortium of the University Hospital in Krakow and CM UJ Project description