In recent years, with the improvement of China’s economic level, culture and material life have undergone great changes, coupled with the change of dietary structure and the influence of bad habits, digestive system diseases are on the rise year by year, and the incidence rate of digestive system tumors is also increasing year by year. Colorectal cancer is a common malignant tumor that ranks fourth among malignant tumors of the digestive system, and its mortality rate ranks among the top three in the world, with more than 900,000 people dying of colorectal cancer every year (1, 2). It is expected that by 2023, the number of colorectal cancer cases worldwide could increase by about 60% compared to the current level, with 2.2 million new cases and 1.1 million deaths due to colorectal cancer (3). There is evidence of a strong association between colorectal cancer and colorectal polyps. Colorectal polyps are prominent growths on the mucosal surface of the intestinal tract, the incidence of which increases with age and is particularly prevalent in people over the age of 40 Although most polyps are benign (4), adenomatous polyps have been shown to be an early stage of colon cancer, with the cancer rate of these polyps ranging from 0.2 to 5.0% (5). Most colorectal cancers are the gradual evolution of benign polyps, a process that involves genetic, histologic, morphologic, and intestinal microbiome changes and usually takes more than 10 years (6, 7). Emphasizing the early diagnosis of colorectal polyps, especially the prevention, early diagnosis and treatment of adenomatous polyps, is important for improving the overall prognosis of colorectal cancer patients. Currently including fecal occult blood test (FOBT), flexible sigmoidoscopy, and colonoscopy are being used for colorectal cancer screening with different advantages and disadvantages. FOBT and sigmoidoscopy screening reduced the mortality rate of colorectal cancer by 16 and 30%, respectively (6); observation of the lesion, biopsy and diagnosis is preferred to colonoscopy (7), but the modality is invasive diagnosis, expensive, limited frequency of examination, and low patient compliance. There is an urgent need to explore an accurate, easy to perform, non-invasive and highly reproducible screening tool for colorectal cancer and colorectal polyps. Gut microbiota, as an important player in intestinal health, has gradually gained attention for its association with gastrointestinal diseases, especially colorectal cancer and its precancerous lesions. In recent years, it has been found that dysregulation of the composition of the intestinal flora is closely associated with the development of adenomatous polyps. MHBT is a reliable way to measure GI bacteria (8), and although jejunal aspirate culture is still regarded as the gold standard for the diagnosis of small intestinal bacterial overgrowth (SIBO), the test is complicated and invasive and MHBT has been, by virtue of its safety, cost-effectiveness, and simplicity of operation, been widely used for clinical screening of SIBO. Although some studies have questioned its accuracy and specificity, especially its consistency across different substrates and populations, it still has some clinical reference value as a non-invasive tool. Recent studies have shown (9–11) an association between intestinal microorganisms and the development of adenomatous polyps and direct bowel cancer. In this study, we combined the molecular detection techniques of intestinal flora to analyze the relative abundance of Bifidobacterium spp. (Bb), Lactobacillus spp. (Lb), Enterotoxin-producing Bacteroidetes fragilis (ETBF), Clostridium nucleatum (Fn), and anaerobic Streptococcus alginolyticus (Panaerobius) by real-time fluorescence quantitative PCR (qPCR), and then to explore the correlation between the two species and the development of colorectal polyps, and then evaluate the clinical value of MHBT combined with intestinal flora testing for colorectal polyp screening was evaluated. The results of the study aim to provide a more comprehensive and precise non-invasive strategy for colorectal polyp screening, optimize the existing pathway for colorectal polyp screening, and provide a new evidence-based basis for early detection and prevention of colorectal cancer.

Although there was no significant difference (P > 0.05) between the observation and control groups in systemic barrier function indicators such as DAO, D-lac, and LPS, the significantly higher CH₄+H₂ positivity may involve the following mechanisms: local barrier damage is prioritized over systemic alterations: colorectal polyps may mainly cause down-regulation of localized tight junction proteins in the mucosa (e.g., ZO-1) rather than widespread leaky gut. Animal experiments showed that claudin-3 expression was reduced by 50% within 5 mm of polyp margins, but serum DAO remained normal (14), consistent with the present findings. Selective diffusion of flora metabolites: CH₄ and H₂, as small molecule gases, can diffuse directly into the bloodstream through the locally damaged mucosa, whereas large molecule barrier markers (e.g., LPS) need to be extensively leaked to be significantly elevated. This may explain why there was a significant difference in breath positivity (CH₄+H₂ positivity 67/87 in the observation group vs. 64/109 in the control group, P = 0.007), but only a trend of elevation in LPS (10.48 vs. 8.53, P = 0.154). AFP, CA19-9, CA-125, and CEA did not differ between the two groups (P > 0.05). Combined with the pathology, it was confirmed that all of the observation groups were benign polyps, and the following mechanisms could explain the above results: MHBT positivity precedes tumor marker changes: dysbiosis may be an early event in colorectal carcinogenesis, while traditional tumor markers tend to be elevated after malignant transformation. Polimeno et al. (15) showed that p-STAT3 and ALR were found to be significantly up-regulated in the tissues of G1-stage CRC, while PIAS3 suppressed the expression of p-STAT3 and ALR. While PIAS3 suppressed expression, supporting the idea that colony-initiated signaling pathways are activated early in carcinogenesis, suggesting that colony dysregulation and its signaling pathways precede changes in conventional tumor markers. This study suggests that MHBT may serve as an early warning indicator of the malignant tendency of polyps, earlier than the abnormalities of markers such as CEA. The independent pathways of flora metabolism and tumor markers: MHBT reflects the functional status of the flora, while CEA and other markers are mainly associated with epithelial cell malignancy, and their synergistic effects have not yet formed in the polyp stage, so long-term follow-up is needed to verify their dynamic association. Although systemic barrier markers did not show any difference, the following mechanisms may link the local changes with the breath test results: regional immune microenvironment regulation: localized Th17/Treg imbalance in polyps may promote the proliferation of methanogenic bacteria, while IL-22 restricts the systemic diffusion of bacterial products through the up-regulation of MUC2, resulting in the phenotype of “localized bacterial flora activity—normal systemic barrier” (Figure 2) “Phenotype (16). Self-sustaining effects of bacterial metabolism (17): methane slows peristalsis by inhibiting smooth muscle contraction, prolonging the contact time between hydrogen-producing bacteria and substrate, resulting in a “low peristalsis-high gas production” cycle, a process that may be independent of classical barrier markers.

Although univariate analysis showed that the observation group was older (61.85 vs. 52.47 years) and had a higher proportion of males (64.94% vs. 49.54%), there was no significant effect of age (CH₄: P = 0.481; H₂: P = 0.593) and gender (CH₄: P = 0.428; H₂: P = 0.554) in the multivariate model. This may be explained by the fact that age is not a driver of breath positivity: despite the fact that aging is accompanied by a decline in the diversity of the intestinal flora, the present study confirms, by statistical correction, that the increased rate of breath positivity in patients with polyps is mainly attributable to the disease state and not to age-related changes in the physiological flora. The clinical significance of gender differences is limited: the high prevalence of males in colorectal polyps may stem from differences in hormone levels (e.g., androgens promote cell proliferation) or lifestyle habits (e.g., smoking/alcohol consumption) (18), but these factors did not directly interfere with flora gas-producing functions, and thus gender was not statistically significant in the multifactorial model. Multifactorial logistic regression analysis showed that after controlling for age and gender confounders, group (observation vs. control) remained an independent risk for positive CH₄ (OR = 2.32, 95%CI = 1.15-4.67, P = 0.019) and H₂ (OR = 2.14, 95%CI = 1.09-4.22, P = 0.027) Factors. This result suggests that the elevated breath test positivity in patients with colorectal polyps is not due to age or gender differences, but is closely related to pathophysiologic changes induced by the polyps themselves. Possible mechanisms include: colony-specific enrichment: the polyp microenvironment (e.g., local hypoxia, altered mucus secretion) may selectively promote the colonization of methanogenic bacteria (e.g., Methanobrevibacter) and hydrogen-producing bacteria (e.g., Bacteroides) (19). Girardi et al. (20) demonstrated in an animal model that adenomatous polyps The abundance of Pseudomonas aeruginosa (Methanobrevibacter) in tissues was 5∼ 8-fold higher than in normal mucosa. Metabolite feedback regulation: H₂ produced by polyp-associated flora may promote the proliferation of pathogenic bacteria (e.g., Fusobacterium nucleatum) via the hydrogenase pathway, whereas CH₄ may inhibit the contraction of intestinal smooth muscle and delay the transport of contents, forming a positive feedback loop (e.g., “dysbiosis-increased gas production”). Positive feedback loop (21).

The significant enrichment of Clostridium perfringens and Streptococcus anaerobius digestans in the observation group (P < 0.001) may have driven the increase in CH₄/H₂ positivity through the following mechanism: metabolic substrate competition: as a hydrogenotroph, Fn can consume H₂ to produce H2S via hydrogenase, while methanogenic bacteria such as Panaerobius utilize H₂/CO₂ to synthesize CH₄ (21), which together resulted in a simultaneous increase in H2 positivity (59.77% vs. 40.37%, P = 0.007) and CH₄ positivity (72.41 vs. 58.72%, P = 0.047) in the observed group. Local barrier damage: although there was no difference in systemic barrier indicators (DAO, LPS), Fn-secreted FadA protein disrupted colonic epithelial tight junctions (22) and facilitated diffusion of small-molecule gases (CH₄/H₂₎ into the bloodstream, whereas large-molecule toxins (e.g., LPS), which were blocked by intact mucosa, were not significantly elevated (10.48 vs. 8.53, P = 0.154). Notably, conventional probiotics such as Bifidobacterium spp. and Lactobacillus spp. proliferated abnormally in the observed group, possibly related to the altered pH of the polyp microenvironment or disruption of the mucus layer (23). Whether the overcolonization of such “probiotics” has pro-inflammatory or carcinogenic potential needs to be further verified by functional experiments.

Although the AUC of single CH₄ assay was only 0.537 (P = 0.300), its combination with H₂ elevated the AUC to 0.663 (P < 0.001), with a sensitivity of 79.31%, suggesting the following optimization logic: functional complementarity: CH₄ labels the chronic dysbiosis of methanogens (specificity 52.29%), while H₂ reflects the parthenogenicity of the acute metabolic activation of anaerobic bacteria (sensitivity 48.28%), and the combination of the two can cover a broader range of flora functional states. Enhanced specificity by flora markers: the introduction of genera strongly associated with polyps, such as Fn (AUC = 0.717) and Panaerobius (AUC = 0.783), significantly increased the AUC of the combined model to 0.831 (sensitivity 79.27%, specificity 82.90%). For example, Panaerobius ≥ –15.94 identified patients with high-risk polyps (sensitivity 92.68%), compensating for the high false-positive rate of a single breath test.

This study suggests that MHBT combined with flora analysis can be used as a stratified screening tool before colonoscopy: initial screening of high-risk groups: those with positive CH₄+H₂ (AUC = 0.663) or abnormal Panaerobius abundance (AUC = 0.783) are prioritized to undergo colonoscopy, reducing overuse of invasive tests. Value of dynamic monitoring: longitudinal monitoring of CH₄/H₂ changes may warn of malignant transformation of polyps, but needs to be validated in follow-up studies. Limitations include: threshold controversy: current CH₄ diagnostic cutoff ( ≥ 10 ppm) has low specificity (52.29%) and needs to be recalibrated based on colony characteristics (e.g., specificity increased to 75% for CH₄ ≥ 15 ppm). Undifferentiated polyp subtypes: possible differences in colony-breath characteristics between adenomatous and hyperplastic polyps, requiring subsequent stratified analysis.

In conclusion, MHBT can reflect the possible dysfunction of flora in patients with colorectal polyps by detecting the status of intestinal gas production and has certain screening value. The combined test of CH₄ and H₂ is superior to the single index in diagnostic efficacy, and has certain screening potential. Although its diagnostic specificity is still limited and it cannot yet replace colonoscopy, it can be used as a non-invasive aid for early detection of colorectal polyps in specific populations. Follow-up studies may further explore individualized threshold optimization, dynamic detection modes and their combined application with other biomarkers to improve screening accuracy and clinical utility.