To Örebro University

Background: Sore throat/pharyngotonsillitis is a very common condition. While most cases are viral, the primary bacterial pathogen is group A beta-hemolytic streptococcus (Streptococcus pyogenes). Further, Fusobacterium necrophorum has over the last decade attracted attention. rnrnSequence-based techniques continue to gain ground in medical microbiology. To describe the microbiota in a sample, either the whole genomes (metagenomics) or marker genes/genomic regions (metataxonomics), such as the 16S rRNA gene, can be sequenced. Some studies have investigated how findings from these methods correspond to conventional microbiological methods for infectious diseases, such as cultures. However, no previous study has approached the condition acute pharyngotonsillitis this way.

Methods: Throat samples from patients with acute sore throat (n=129) and controls (n=86), both groups aged 15-45, were collected. DNA was extracted and the V3-V4 regions of the 16S rRNA genes were amplified using PCR. After normalization based on fragment analysis, and size selection with Ampure beads and PCR against adapter sequences coupled to the V3-V4 fragments, clonal amplifiction was performed with isothermal PCR. Finally, sequencing was performed on the Ion Torrent S5 XL. The SILVA database was used for taxonomic classification and the results were compared to culture findings for S. pyogenes and F. necrophorum, using Mann Whitney U tests.

Results: Among the 215 samples, 46 patients and 1 of the controls were culture-positive for S. pyogenes. For F. necrophorum, 20 patients and 3 controls were culture-positive. Seven of the samples were culture-positive for both S. pyogenes and F. necrophorum. rnrnIn the metataxonomic analysis, S. pyogenes were significantly more abundant among patients than controls (p=0.0046), and in samples culture-positive for S. pyogenes, compared to culture-negative (p<0.0001).

The percent of reads representing F. necrophorum were significantly higher in patients compared to controls (p<0.001), as well as in culture-positive samples compared to culture-negative (p<0.0001). rnrnAlthough significant differences between culture-positive and culture-negative samples were seen, even among culture-positive samples the abundance of S. pyogenes or F. necrophorum were on average low (2,1% and 10,6%, respectively) and with large variation (0-49,8% and 0-76,1%, respectively).

Conclusions: Findings from a metataxonomic 16S rRNA gene analysis differed regarding species of interest between groups based on symptoms of a sore throat or culture findings. However, the results were heterogeneous and difficult to interpret for a single sample.