When adjusted for age and sex, subjects with appendectomy were less likely to produce CH 4 . However, among those for whom CH 4 was present, the magnitude of CH 4 did not differ between the two groups, despite similar H 2 levels. To our knowledge, this is the first study to show an association between appendectomy and a decreased rate of excess exhaled CH 4 .

It has been hypothesized that the appendix may serve as a reservoir for the gut microbiome due to its location and shape, making it relatively sheltered from microbial changes that occur in the rest of the colon19,20. During diarrhoeal illnesses, the appendix may function to repopulate the gut with its own luminal and mucosal microbiome19. Although causality cannot be establish, the theory that the appendix may act as a microbial reservoir is supported by our finding that the number of patients with detectable or excess CH 4 was decreased in the appendectomy group, but the increase in magnitude in those for whom CH 4 was present did not differ between appendectomy groups. Previous studies have shown that methanogens exist in the colonic walls and stool21, and given the notable difference in the surface area and volume of the colon vs the appendix, the bulk of CH 4 production likely occurs in the rest of the gut as opposed to the appendix, which may act only as a reservoir. Interestingly, diverticulosis (a form of diverticula) has been linked to an increase in methanogens, and the appendix may have a similar function22. Although of note, the appendix, unlike the diverticulum, has a muscle layer and can perform antegrade peristalsis23; hence, it can potentially act as an active reservoir for the gut microbiome. Another potential explanation for the high rates of CH 4 is that a particular composition of the microbiome is associated with appendectomies, and those who do not require surgery have increased amounts of CH 4 .

This association between CH 4 and the appendix may have clinical implications. CH 4 is associated with constipation, and studies have attempted to treat this based on eliminating methanogens. Non-systemic (i.e., poorly absorbed) oral antibiotic for the treatment of methanogenic archaea in the gut appears to have a high rate of recurrence in human subjects24. This phenomenon can be potentially be explained by the theory that the appendix serves as an active reservoir of methanogenic archaea with a thick biofilm resistant to antibiotic penetrance. Future studies should consider measuring the response and recurrence rates after antibiotic treatments between those with and without an appendix to determine whether the appendix is indeed acting as a reservoir.

Additionally, a history of appendectomy has been associated with a decreased risk for developing ulcerative colitis25 and an increased risk for Crohn’s disease26. In fact, appendectomy has been proposed as a potential treatment for ulcerative colitis27. In line with our hypothesis, the importance of the role of the appendix in inflammatory bowel diseases may in part be explained by the appendix acting as a reservoir to maintain the host gut microbiome.

Another interesting observation in this study was the association between methanogens (detected by the presence of CH 4 in the breath) and age. Older patients are known to have increased CH 4 28 and since the prevalence of appendectomy increases with an individual’s age, this confounder warranted adjustment in this analysis. In addition, we found that age may be a significant contributor to increased CH 4 levels. For every 5-year increase in age, there was approximately a 1 ppm increase in the CH 4 Max, and there was a 4-fold increase in the percentage of CH 4 producers from the patient’s first decade of life to the ninth decade of life (Fig. 1). This has been reported previously18. One possible explanation is that pockets in the intestine, such as the appendix and diverticula, may contribute to housing methanogens. This is evidenced by the fact that the prevalence of diverticulosis increases with age29. Alternatively, subjects with higher levels of methanogens may have increased archaeal compositions in the gut or have longer life expectancies than those with low levels of methanogens.

This study has several strengths and weaknesses. Given the referral status of the subjects, the results of the study may not be generalizable to the general population. Due to the retrospective design, we did not have detailed information regarding patients’ symptoms at the time of BT. Therefore, we were not able to correlate symptoms with the presence or absence of CH 4 and appendectomy. Given that CH 4 gas has been shown to decrease gut motility in humans and animal models30, it would be worthwhile to design a prospective study with symptom correlations. This new finding may hold clinical significance, as a reduction of CH 4 levels has been shown to reduce constipation in humans17. There are several strengths to our study, including the large sample size and the use of the same fermentable sugar substrate (lactulose) with the same device.