Host-Microbe Metabolite Interactions in Asymptomatic C. Difficile Carriers: A Combined qPCR and SCFA Profiling Study.

Abstract

Background:Asymptomatic colonization with Clostridioides difficile (C. difficile) is common, yet the microbial and metabolic dynamics that distinguish silent carriage from overt infection remain poorly understood. Short-chain fatty acids (SCFAs) and calprotectin may serve as functional biomarkers reflective of gut homeostasis and inflammation.

Objective:This study aimed to evaluate host-microbe metabolic interactions in asymptomatic C. difficile carriers through combined quantitative PCR (qPCR), gas chromatography, and enzyme immunoassay analysis, with a focus on toxin gene loads, SCFA concentrations, and fecal calprotectin.

Methods:In this cross-sectional study, 50 adult participants were recruited—25 asymptomatic carriers and 25 healthy controls. Stool samples underwent qPCR to quantify total bacterial load and the toxin genes tcdA and tcdB. SCFA levels (acetate, propionate, butyrate) were measured via gas chromatography, while fecal calprotectin was assessed using ELISA. Statistical analysis included t-tests, chi-square tests, logistic regression, and multivariate linear regression.

Results:Toxin gene loads (tcdA, tcdB) were significantly higher in carriers compared to controls (p < 0.0001), whereas total bacterial load showed no significant difference (p = 0.38). SCFA concentrations were significantly reduced in carriers: acetate (50.6 vs. 60.6 mmol/kg), propionate (17.8 vs. 22.2 mmol/kg), and butyrate (11.1 vs. 16.1 mmol/kg); all p < 0.0001. Fecal calprotectin levels were significantly elevated in carriers (102.8 vs. 44.3 mg/kg, p < 0.0001). Logistic regression identified BMI, calprotectin, and lower butyrate and acetate as independent predictors of carriage. Multivariate analysis revealed tcdA load as the primary predictor of calprotectin elevation.

Conclusion:Asymptomatic C. difficile carriage is characterized by elevated toxin gene loads, reduced SCFA levels, and subclinical inflammation. These findings support a metabolic-inflammatory axis in asymptomatic colonization and suggest that integrating microbial, metabolic, and immune markers can improve risk stratification in C. difficile surveillance..