Oropharyngeal microbial ecosystem perturbations influence the risk for acute respiratory infections in common variable immunodeficiency

Abstract

Background: The respiratory tract microbiome is essential for human health and well-being and is determined by genetic, lifestyle, and environmental factors. Patients with Common Variable Immunodeficiency (CVID) suffer from respiratory and intestinal tract infections, leading to chronic diseases and increased mortality rates. While CVID patients' gut microbiota have been analyzed, data on the respiratory microbiome ecosystem are limited.

Objective: This study aims to analyze the bacterial composition of the oropharynx of adults with CVID and its link with clinical and immunological features and risk for respiratory acute infections.

Methods: Oropharyngeal samples from 72 CVID adults and 26 controls were collected in a 12-month prospective study. The samples were analyzed by metagenomic bacterial 16S ribosomal RNA sequencing and processed using the Quantitative Insights Into Microbial Ecology (QIME) pipeline. Differentially abundant species were identified and used to build a dysbiosis index. A machine learning model trained on microbial abundance data was used to test the power of microbiome alterations to distinguish between healthy individuals and CVID patients.

Results: Compared to controls, the oropharyngeal microbiome of CVID patients showed lower alpha- and beta-diversity, with a relatively increased abundance of the order Lactobacillales, including the family Streptococcaceae. Intra-CVID analysis identified age >45 years, COPD, lack of IgA, and low residual IgM as associated with a reduced alpha diversity. Expansion of Haemophilus and Streptococcus genera was observed in patients with undetectable IgA and COPD, independent from recent antibiotic use. Patients receiving azithromycin as antibiotic prophylaxis had a higher dysbiosis score. Expansion of Haemophilus and Anoxybacillus was associated with acute respiratory infections within six months.

Conclusions: CVID patients showed a perturbed oropharynx microbiota enriched with potentially pathogenic bacteria and decreased protective species. Low residual levels of IgA/IgM, chronic lung damage, anti antibiotic prophylaxis contributed to respiratory dysbiosis.

Keywords: Haemophilus; IgA; IgM; Pneumococcus; chronic obstructive pulmonary disease; common variable immunodeficiency; microbiome and dysbiosis; oropharyngeal microbiome.

Figure 1

Study design.

Figure 2

Alpha and beta diversity (A, B) and bacterial community composition of the oropharynx in CVID and controls (C). Species richness and diversity index were estimated by Chao1 (alpha diversity) and represented in CVID vs. controls (A). Bars indicate the median. Non-parametric Mann–Whitney t-test was used to evaluate statistical significance. Beta diversity by Principal Component Analysis (PCA, B) was calculated to capture inter-sample variation in microbial composition. Two-tailed P value significances are shown as **p< 0.01. A cladogram (C) illustrating the phylogenetic relationship between taxa, the central dot representing the kingdom Bacteria, the first circle representing Phylum, then the Class, Order, Family, and Genus levels. Taxa that are increased in CVID compared with controls are in red, and taxa that are reduced in CVID compared with controls are in green. Named taxa are significant according to both univariate and multivariate statistics and are marked as small letters in the cladogram referring to corresponding taxa names in the legend at the right side of the figure. The vertical lines on the left side of the legend define taxa representing different levels of the same branch. The phylogenetic tree and coloring were made using LEfSe.

Figure 3

Global microbiome classifier by CVID status. Relative abundances of oropharyngeal microbial taxa associated with CVID status are displayed as a heatmap of log-abundance z-scores with the direction of association indicated to the left (A). The mean contribution of each marker species to the classification is shown to the left (bars correspond to the log-odds ratio in logistic regression). Below the heatmap, the classification score of the microbial signature from cross-validation is shown as a gray scale. (B) The cross-validation accuracy of the microbiota classifier is depicted as a receiver–operator-characteristic (ROC) curve summarizing mean test predictions made in ten times resampled tenfold cross-validation with the area under the curve (AUC) indicated inside each plot.

Figure 4

Intra-CVID differences in alpha diversity in oropharyngeal microbiome. Chao1 (alpha diversity) was compared in CVID patients grouped according to their age (A), IgM (B) and IgA serum levels (C), EUROCLASS groupin0g (D), COPD status (E), and whether to take or not antibiotic prophylaxis (F). Bars indicate the median. Non-parametric Mann–Whitney t-test was used to evaluate statistical significance. Two-tailed P value significances are shown as * p<0.05, **p< 0.01, ***p< 0.001. ****p<0.0001. HD, healthy donors; yrs, years; B-, B cells<1% of lymphocytes; B+Sm-, B cells>1% and Switched Memory B cells >=2% of lymphocytes; B+Sm+, B cells>1% and Switched Memory B cells >2% of lymphocytes; COPD, chronic obstructive pulmonary diseases; mod/sev, moderate-severe; AB, prophylaxis antibiotic prophylaxis.

Figure 5

Bacterial community composition of the oropharynx in CVID patients and controls. Patients were grouped by IgM and IgA serum levels (A, B) and COPD status (C). The first six most frequently identified genera in CVID patients are shown. The horizontal line inside the box represents the median. The whiskers represent the 10 and 90 percentiles. The non-parametric Mann–Whitney test was used to evaluate statistical significance. Two-tailed P value significances are shown as *p<0.05, **p< 0.01, ***p< 0.001, ****p<0.0001.

Figure 6

Summary of the main results.