To the best of our knowledge, this is the first study to classify and investigate the correlation between the core microbiome of tonsils in Korean pediatric patients and tonsil hyperplasia based on the BMI percentile and the grade of tonsil hyperplasia. The first category of BMI percentile was divided into lean and obese groups based on the most up-to-date Korean National Growth Chart. The second category of tonsil hyperplasia was divided into three groups (grade 2, 3, and 4) using the Brodsky grading scale, classifying the palatine tonsil into five grades with high reliability21. All patients enrolled in our study had tonsil hyperplasia as we conducted microbiome analysis on the surgically resected palatine tonsil. Thus, we had no control data for healthy microbiomes as grade 0 and 1 children were excluded from this study. In addition, to exclude the effects of inflammation and antibiotic use22, we only enrolled children who had no recent clinical events of inflammation. Thus, our data is clinically significant because we analyzed a relatively homogenous whole palatine tonsillar microbiome cohort, and our study participants maintained a fasting period of at least 12 h before the surgery.
Our data revealed similar microbiome profiles in obese patients and in grade 4 patients with tonsillar hyperplasia (73% and 65% shared unique ASVs in obese and grade 4 hyperplasia, respectively). Moreover, the grade 4 group ASVs were positively correlated with body weight, BMI, percentile of body weight, and body surface area (Figs. 4a and 5), suggesting that the microbiome in enlarged tonsils may also influence the development of obesity. These findings are consistent with results from previous clinical studies and confirm the possible relationship between tonsillar hypertrophy and obesity from a metagenomic point of view23.
In a previous study, we examined the microbial communities in the saliva and tonsils of Korean children who underwent tonsillectomy due to tonsil hypertrophy7. The study found that the tonsil microbiota was dominated by Proteobacteria, Firmicutes, and Bacteroidetes, with the genera Haemophilus, Streptococcus, and Fusobacterium, Veillonella and Prevotella genera being the most abundant in both the tonsils and saliva24. Thus, the community structures (Haemophilus, Streptococcus, and Fusobacterium) were preserved regardless of BMI percentile and the grade of tonsil hyperplasia.
Several studies have reported that gut microbiota diversity and abundance differ between obese and non-obese individuals, but the results are inconsistent25,26. A microbial community study in Korean children found no significant difference between the two groups, while a study in Korean adults reported lower phylogenetic diversity in the obese group based on BMI27,28. Thus, the microbiome diversity in subjects with or without obesity varied depending on the participant age, growth stage, and living environment.
The relative abundances of Firmicutes (F) and Bacteroidetes (B) in the gut microbiome vary significantly across individuals within the same population due to various lifestyle factors including diet, physical activity, and antibiotic consumption29. The F/B ratio is an important factor for normal intestinal homeostasis. Several studies indicate that obese people have a higher F/B ratio than those with average weight, suggesting that the gut F/B ratio could serve as an obesity biomarker30,31,32. In particular, the F/B ratio is increased in people with obesity compared to that in people with average weight, and high rates of Firmicutes could serve a role in the development of obesity33. While previous studies have indicated that the gut microbiota could contribute to the development of obesity, the evidence linking obesity to alterations in the F/B ratio is unconvincing28,34,35. However, the F/B ratio in the oral microbiome remained relatively unexplored. Here, the observed F/B ratios were 2.51 and 2.78 in the lean and obese groups, respectively (Fig. 6a). Previous studies reported an increased F/B ratio in children with snoring or OSA36. We observed the highest F/B ratio in the grade 2 group in category 2, which was significantly decreased in grade 3 and 4 groups (4.96 vs. 1.91 and 2.62, respectively) (Fig. 6a). A decreased F/B ratio is associated with inflammatory bowel disease32, and our patient group was selected by excluding clinical features of inflammation, thus chronic inflammatory conditions might be partly involved in tonsillar hypertrophy. This result suggests that controlling chronic inflammation in grades 3 and 4 could alleviate symptoms by reducing the tonsil size, highlighting the need for early treatment.
Moreover, to simplify the complexity of microbiota composition, we focused on the two major bacterial phyla in the tonsil microbiome: Proteobacteria and Firmicutes. We observed a consistently decreased abundance of Proteobacteria in the obese and enlarged tonsil groups, with a consistent reduction in the Proteobacteria to Firmicutes (P/F) ratio (Fig. 6b). Although this study was limited to patients with snoring and did not measure OSA objectively, we were able to calculate the P/F ratio using data from previous studies, that confirmed reduced P/F ratio in OSA37,38. Proteobacteria is a more abundant phylum than Bacteroidetes in the tonsillar microbiome; however, there is no study on the P/F ratio in the tonsillar microbiome. Thus, further studies are needed to evaluate the significance of the P/F ratio in future oropharyngeal microbiome research, as Firmicutes and Bacteroidetes are the main predominant phyla in the gut microbiome39.
A recent study has suggested that OSA and microbiome dysbiosis may have bidirectional associations40. Reports have indicated that sleep-related factors are linked to microbiome dysbiosis, and pediatric OSA patients have severely dysregulated microbiomes. A study found a significant association between Streptococcus pyogenes and OSA in children41. We observed decreased Proteobacteria and increased Fusobacteria and Spirochetes in obese and grade 3 and 4 groups, similar to a previous study that identified Proteobacteria in patients with chronic tonsillitis, and Fusobacteria and Spirochetes in patients with OSA42. This study is distinct in that it examined the microbiome of tonsils in the absence of tonsillitis.
We investigated the correlation between 79 shared ASVs in the obesity and grade 4 groups and 25 variables (20 biochemical indices and five anthropometric variables). While there was no clinical difference between hematologic variables such as HCT and RBC for each group of categories 1 and 2, we observed a significant negative correlation between HCT and RBC with ASVs in both categories in the metagenomic analysis. A study using obese mice reported an association between the microbiota and significantly increased blood glucose levels and decreased RBC, HCT, and hemoglobin levels43. Moreover, an investigation of the gut microbiome and hematological parameters in Tibetans reported that age, BMI, RBC, hemoglobin, HCT, and platelet count were associated with the structure of the gut microbiome, whereas BMI and platelet count were significant explanatory variables44.
Among the various biochemical indices, ALT levels increased with obesity and tonsillar hypertrophy without AST elevation. As reported by Verrijken et al., elevated ALT levels without AST level increase in children with obesity can indicate non-alcoholic fatty liver disease45. Although pathologically elevated liver enzyme levels were not found in our pediatric cohort, we cannot give clinical significance to the AST/ALT ratio. The solitary elevation of ALT consistently observed in all categories has clinical relevance in that absolute ALT elevation (with an upper limit of normal) could be an early sign of fatty liver disease, even in overweight or obese children with normal liver enzymes46. This association is explainable in that ALT levels in overweight and obese patients were positively correlated with visceral adipose tissues, which has been associated with the pathogenesis of nonalcoholic fatty liver disease, a condition regarded as the hepatic component of the metabolic syndrome47. Interestingly, the increase in ALT according to tonsil size grade is a novel finding that requires further investigation. However, it is still understandable that these children underwent surgery mainly because of snoring and mouth breathing, which are related to OSA, a well-known risk factor for various metabolic syndromes, including non-alcoholic fatty liver disease. Recently, Kang et al. reported that higher severity OSA is strongly associated with ALT elevation in Korean children with obesity. Many such children undergo surgery due to snoring and mouth breathing, and it seems that OSA and obesity may have synergistic effects on ALT level elevation48. We believe that, based on the results of this study, further analysis of the changes in the oral microbiome and serum ALT after tonsillectomy in children in the future will be very interesting.
Furthermore, in our study, among 79 ASVs shared by obesity and tonsillar hypertrophy grade 4 groups, 10 ASVs, and 19 ASVs showed significant positive correlations with serum ALT levels in obesity and tonsillar hypertrophy grade 4 groups, respectively. Our result suggests a possible association of tonsillar microbiome and liver metabolism. There is some evidence connecting elevated serum ALT levels to altered gut microbiome composition. However, no report shows a significant association between serum ALT levels and the tonsillar microbiota. Despite the limitations, our findings demonstrate the association between systemic metabolism and the tonsillar microbiome, highlighting the possible influence of the tonsillar microbiome on the metabolic health of the host.
There are some limitations to this study. First, the patient number in subgroups based on body weight status in grade 2 is biased due to small sample size, which should be considered when interpreting the data (Supplementary Table 1). This imbalance in the ratio of obese to lean children in grade 2 is due to a sharp decrease in the number of pediatric tonsillectomies due to the COVID-19 pandemic. However, we were able to find clinical relevance by focusing on obesity and tonsil grade 4 data, which had a relatively balanced distribution in the enrolled patient numbers. The second limitation is that we did not perform polysomnography to objectively diagnose OSA, because when planning tonsillectomy in pediatric patients, the Korean insurance system does not provide routine PSG testing. However, since there is evidence that tonsil size and OSA severity are well correlated, it is possible that tonsil hypertrophy can be interpreted indirectly as OSA severity49. Third, other metabolic markers, including lipid profiles such as triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and central adiposity using waist circumference were not evaluated. Considering the increasing prevalence of obesity and OSA among children, known synergistic effect of OSA, and obesity on ALT elevation48, future studies on the effects of the tonsillar microbiome on dyslipidemia and systemic metabolic syndrome will be clinically relevant. Lastly, this study was cross-sectional and thus unable to conclude the direction of associations or causal effects in the association of obesity and tonsil hyperplasia. The relationships between the tonsillar microbiota and obesity or tonsillar hyperplasia need to be further explored. In addition, future prospective trials on the effects of tonsillectomy or tonsillar microbiome modulation using specific probiotics with a larger sample size will be of interest and provide further understanding of the tonsillar microbiome.
In conclusion, we showed that children with obesity and tonsil hypertrophy have comparable microbiome compositions and induce similar changes in the microbiome abundance and composition, confirming the association between obesity and tonsil hypertrophy from a metagenomic perspective. Moreover, these changes were well correlated with the P/F ratio rather than the F/B ratio, which is a well-accepted dysbiosis marker in the gut. Thus, we identify the P/F ratio as a novel and promising biomarker reflecting oral or tonsil dysbiosis, which should be verified in future studies. Finally, we identify a possible effect of the tonsillar microbiome on metabolic disorders of the host. These results shed light on the importance of dysbiosis of the tonsil and suggest the need for its regulation, especially during childhood.