Profile of the Nasopharyngeal Microbiota Affecting the Clinical Course in COVID-19 Patients

Abstract

While populations at risk for severe SARS-CoV-2 infections have been clearly identified, susceptibility to the infection and its clinical course remain unpredictable. As the nasopharyngeal microbiota may promote the acquisition of several respiratory infections and have an impact on the evolution of their outcome, we studied the nasopharyngeal microbiota of COVID-19 patients in association with baseline disease-related clinical features compared to that of patients tested negative. We retrospectively analyzed 120 nasopharyngeal pseudonymized samples, obtained for diagnosis, divided into groups (infected patients with a favorable outcome, asymptomatic, and deceased patients) and patients tested negative for SARS-CoV-2, by using Illumina-16S ribosomal ribonucleic acid (rRNA) sequencing and specific polymerase chain reaction (PCR) targeting pathogens. We first found a depletion of anaerobes among COVID-19 patients, irrespective of the clinical presentation of the infection (p < 0.029). We detected 9 taxa discriminating patients tested positive for SARS-CoV-2 from those that were negative including Corynebacterium propinquum/pseudodiphtericum (p ≤ 0.05), Moraxella catarrhalis (p ≤ 0.05), Bacillus massiliamazoniensis (p ≤ 0.01), Anaerobacillus alkalidiazotrophicus (p ≤ 0.05), Staphylococcus capitis subsp. capitis (p ≤ 0.001), and Afipia birgiae (p ≤ 0.001) with 16S rRNA sequencing, and Streptococcus pneumoniae (p ≤ 0.01), Klebsiella pneumoniae (p ≤ 0.01), and Enterococcus faecalis (p ≤ 0.05) using real-time PCR. By designing a specific real-time PCR, we also demonstrated that C. propinquum is decreased in asymptomatic individuals compared to other SARS-CoV 2 positive patients. These findings indicate that the nasopharyngeal microbiota as in any respiratory infection plays a role in the clinical course of the disease. Further studies are needed to elucidate the potential role in the clinical course of the disease of M. catarrhalis, Corynebacterium accolens, and more specifically Corynebacterium propinquum/diphteriticum in order to include them as predictors of the severity of COVID-19.

Keywords: 16S rRNA sequencing; COVID-19; SARS-CoV-2; metagenomics; nasopharyngeal microbiota.

FIGURE 1

Principal coordinate analysis showing the change in the nasopharyngeal microbiota profile during COVID-19 airway infection in clinical patient groups versus COVID-19 negative group. Principal component analysis (PCoA) was performed on species from raw data (number of reads) using the Jensen Shannon divergence distance matrix and the Multivariate Analysis of Variance (MANOVA) statistical test. Each sample is represented by a point: (A) the microbiota profile of COVID-19 negative patients and cases (p ≤ 0.006); (B) the microbiota profile in asymptomatic patients and deceased patients (p ≤ 0.001); (C) the microbiota profile in patients with a favorable outcome and deceased patients (p ≤ 0.001); (D) the microbiota profile in patients with a favorable outcome and COVID-19 negative individuals (p ≤ 0.001).

FIGURE 2

Association of specific taxa in the airway microbiota of COVID-19 patients. The relative abundance of differentially enriched taxa in the four clinical groups of COVID-19 patients (negative, asymptomatic, deceased patients, and patients with a favorable outcome). Five pathogens enrichment in groups of SARS-CoV-2 positive subjects: Corynebacterium propinquum/pseudodiphtericum (p ≤ 0.05), Moraxella catarrhalis (p ≤ 0.05), Bacillus massiliamazoniensis (p ≤ 0.01), Anaerobacillus alkalidiazotrophicus (p ≤ 0.05), and Staphylococcus capitis subsp. capitis (p ≤ 0.001). One-way ANOVA (Kruskal–Wallis statistic). *p ≤ 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001.

FIGURE 3

Association of specific taxa in the airway microbiota of COVID-19 patients. Enrichments of specific taxa in the respiratory microbiota of COVID-19 patients in asymptomatic and deceased patients (A) and in deceased patients and patients with a favorable outcome (B) by linear discriminant analysis (LDA) effect size (LEfSe).

FIGURE 4

All respiratory pathogens routinely detected in our COVID-19 patients and by specific PCR. The pathogenic bacteria PCR shows the significant enrichment of Streptococcus pneumoniae (p ≤ 0.01) and Corynebacterium propinquum (p ≤ 0.000006) in the group of COVID-19 positive patients, and Klebsiella pneumoniae (p ≤ 0.01) in deceased patients and patients with a favorable outcome group compared to COVID-19 negative subjects, and Enterococcus faecalis (p ≤ 0.05) in deceased patients compared to negative individuals.

FIGURE 5

The enriched nasopharyngeal microbiota bacteria in the COVID-19 positive group and COVID-19 negative group. Generated using Biorender (https://biorender.com).