Comparative gut microbiome in diarrheal and non-diarrheal children: an individually matched case-control study

Diarrhea, a leading cause of under-five mortality in developing countries, drives therapeutic challenges amid global antibiotic resistance. This study aimed to compare gut microbiota, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and virulence factors (VFs) in diarrheal and non-diarrheal children, explore reasons for asymptomatic pathogen carriage, and develop a microbiome-based model to predict the potential etiology of diarrhea. This individually matched case-control study analyzed 42 paired fecal samples (selected from 716 diarrhea/non-diarrhea cases) to compare gut microbiome profiles, ARGs, MGEs, and VFs using metagenomic sequencing and pathogen-specific PCR. Diarrheal children showed reduced alpha diversity, increased Proteobacteria, and elevated VFs and MGEs. Surprisingly, ARGs were more abundant in non-diarrheal children, suggesting ARG colonization in healthy hosts. Actinomyces was enriched in non-diarrheal pathogen carriers and may be associated with asymptomatic bacterial pathogen carriage. A random forest (RF) model incorporating non-pathogenic bacteria achieved high accuracy in predicting diarrheal status and pathogen carriage. This study reveals distinct microbial ecologies between diarrheal and healthy children. The enrichment of specific bacterial taxa in asymptomatic carriers may be associated with pathogen tolerance. The unexpected ARG abundance in healthy children highlights a hidden antimicrobial resistance reservoir. These findings inform microbiome-based diagnostics and antibiotic stewardship in pediatric diarrhea.