Case-Control Microbiome Study of Chronic Otitis Media with Effusion in Children Points at Streptococcus salivarius as a Pathobiont-Inhibiting Species

Jennifer Jörissen, Marianne F L van den Broek, Ilke De Boeck, Wannes Van Beeck, Stijn Wittouck, An Boudewyns, Paul Van de Heyning, Vedat Topsakal, Vincent Van Rompaey, Ine Wouters, Liesbet Van Heirstraeten, Pierre Van Damme, Surbi Malhotra-Kumar, Heidi Theeten, Olivier M Vanderveken, Sarah Lebeer, Jennifer Jörissen, Marianne F L van den Broek, Ilke De Boeck, Wannes Van Beeck, Stijn Wittouck, An Boudewyns, Paul Van de Heyning, Vedat Topsakal, Vincent Van Rompaey, Ine Wouters, Liesbet Van Heirstraeten, Pierre Van Damme, Surbi Malhotra-Kumar, Heidi Theeten, Olivier M Vanderveken, Sarah Lebeer

Abstract

Chronic otitis media with effusion (OME) has been associated with a shift in microbiome composition and microbial interaction in the upper respiratory tract (URT). While most studies have focused on potential pathogens, this study aimed to find bacteria that could be protective against OME through a case-control microbiome study and characterization of isolates from healthy subjects. The URT and ear microbiome profiles of 70 chronic OME patients and 53 controls were compared by 16S rRNA amplicon sequencing. Haemophilus influenzae was the most frequent classic middle ear pathobiont. However, other taxa, especially Alloiococcus otitis, were also frequently detected in the ear canal of OME patients. Streptococci of the salivarius group and Acinetobacter lwoffii were more abundant in the nasopharynx of healthy controls than in OME patients. In addition to the microbiome analysis, 142 taxa were isolated from healthy individuals, and 79 isolates of 13 different Streptococcus species were tested for their pathobiont-inhibiting potential. Of these, Streptococcus salivarius isolates showed a superior capacity to inhibit the growth of H. influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, A. otitis, and Corynebacterium otitidis S. salivarius strains thus show potential as a probiotic for prevention or treatment of OME based on their overrepresentation in the healthy nasopharynx and their ability to inhibit the growth of respiratory pathobionts. (This study has been registered at ClinicalTrials.gov under registration no. NCT03109496.)IMPORTANCE The majority of probiotics marketed today target gastrointestinal health. This study searched for bacteria native to the human upper respiratory tract, with a beneficial potential for respiratory and middle ear health. Comparison of the microbiomes of children with chronic otitis media with effusion (OME) and of healthy controls identified Streptococcus salivarius as a health-associated and prevalent inhabitant of the human nasopharynx. However, beneficial potential should be assessed at strain level. Here, we also isolated specific S. salivarius strains from the healthy individuals in our study. These isolates showed a beneficial safety profile and efficacy potential to inhibit OME pathogens in vitro These properties will now have to be evaluated and confirmed in human clinical studies.

Keywords: 16S rRNA; Streptococcus salivarius; ear canal; microbiome; middle ear; otitis media; otitis media with effusion; pediatric; probiotics; upper respiratory tract.

Copyright © 2021 Jörissen et al.

Figures

FIG 1
FIG 1
The bacterial microbiome of the human upper respiratory tract and ears. (A) Genus-level microbiome composition of multiple upper respiratory tract and ear niches in health (“H”) and during chronic otitis media with effusion (“D”). Each bar represents one sample, and the full height of a bar represents 100% of reads. The 11 most abundant genera across all samples are shown, with all other genera summarized under “Residual.” Health status is indicated by header bar color and letter, and the number of successfully sequenced samples per location and group is provided where possible. For the healthy adenoid swabs and healthy middle ear rinses, 1 and 4 samples were available for analysis, respectively. Adenoid swabs and tissue samples were grouped into one graph, as they did not differ significantly. Note that by “healthy,” we refer to the absence of inflammation and infection at the studied anatomical site. For the nasopharynx, children attending day care were included, in addition to cochlear implant recipients who were sampled as reference for the other anatomical sites. (B) Similarity (1 − Bray-Curtis dissimilarity) of middle ear effusion samples to matched nasopharynx versus side-matched ear canal samples. A high score indicates high similarity between two locations. Symbols are sized based on the number of reads remaining in the middle ear sample after contaminant filtering. Colors indicate which ASV is dominant (>50% relative abundance) in the middle ear. (C) Stacked bar charts of healthy middle ear samples. Only ASVs detected in more than one sample are shown.
FIG 2
FIG 2
ASVs significantly differentially abundant in the nasopharynx of chronic OME (“D”) versus controls (“H”) by Analysis of Composition of Microbiomes (ANCOM) analysis with stringent correction for multiple testing (see Fig. S1 in the supplemental material for an alternative analysis approach).
FIG 3
FIG 3
Mean inhibition of classic otopathogens by Streptococcus species isolated from the URT of healthy children and adults, as measured through the spot assay method. Each point represents a different isolate.
FIG 4
FIG 4
Inhibition of upper respiratory tract and classic and suspected otopathogens by 7 S. salivarius isolates, as measured through the spot assay method. Isolates were compared to S. salivarius 24SMB and S. oralis 89a, isolated from the Rinogermina probiotic nasal spray, and Hextril mouthwash (0.1% hexetidine) served as a positive control. For each pathobiont, the tested isolates are sorted from smallest (left) to largest (right) mean inhibition zone diameter. Statistical comparison of inhibition zones was performed with 0.1% hexetidine as a reference. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 5
FIG 5
Adhesion of bacterial isolates to respiratory epithelial cells (Calu-3). The adhesion of Streptococcus salivarius (Ssal) isolates obtained in the present study was statistically compared to that of S. salivarius 24SMB (38–40) of the Rinogermina probiotic nasal spray using an unpaired Wilcoxon rank sum test. S. oralis (Soral) 89a was also isolated from Rinogermina, but it showed a lower adhesion capability than all S. salivarius strains. Lacticaseibacillus casei (Lcas) AMBR2 and Lacticaseibacillus rhamnosus (Lrham) GG were included as examples of lactobacilli with a higher and a lower ability to adhere to respiratory epithelium, respectively (64). Isolates are sorted from lowest to highest median adhesion percentage. ns, P > 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. P values are adjusted by the Holm method.

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