Purpose: Previous culture-based studies suggest three significant pathogens in acute tonsillitis (AT): Streptococcus pyogenes, Fusobacterium necrophorum, and Streptococcus dysgalactiae. Next-generation sequencing (NGS) provides further insights into the human microbiome and may pinpoint additional pathogens in bacterial infections. We aimed to investigate the tonsillar microbiome and identify pathogens associated with AT by applying NGS to tonsillar swabs from patients with severe AT, comparing the findings with both healthy controls and culture-based results.
Methods: Full-length sequencing of the 16S rRNA gene (16S tNGS) was performed on tonsillar swabs from 64 AT patients and 55 controls, who were prospectively enrolled at two Danish Ear-Nose-Throat Departments between June 2016 and December 2019.
Results: The mean number of detected bacteria was significantly higher in patients analysed with 16S tNGS (36) than with culture methods (6.5, p < 0.001). The alpha diversity was lower in patients compared to controls (p < 0.001) and beta diversity showed separation of the two groups (p = 0.001). S. pyogenes (p = 0.001) and Bifidobacteriaceae (p = 0.002) were significantly more abundant in patients compared to controls. The three suggested pathogens were detected more frequently using 16S tNGS compared to culture: S. pyogenes (38% vs. 27%, p = 0.26), F. necrophorum (19% vs. 11%, p = 0.32), and S. dysgalactiae (14% vs. 11%, p = 0.79).
Conclusion: The tonsillar microbiome differed significantly between AT patients and healthy controls. Our findings confirm the role of S. pyogenes in AT, but did not identify additional likely pathogens. The addition of 16S tNGS to cultures increased the collective detection rate of three previously suggested pathogens from 48 to 70%.
Keywords: 16S tNGS; Acute tonsillitis; Nanopore; Next-generation sequencing; Pathogens.
© 2025. The Author(s).