Abstract
BACKGROUND: Diarrhea remains a major cause of morbidity worldwide, particularly in low- and middle-income countries. Hospital environments impose strong selective pressures on the gut microbiome through antimicrobial exposure, invasive procedures, and pathogen transmission, yet differences between hospital-onset and community-onset diarrhea remain poorly characterized at the microbiome level. This study aimed to compare the taxonomic and functional profiles of the gut microbiome in hospitalized (Hosp) and emergency room (ER) patients with diarrhea using shotgun metagenomics. RESULTS: Fecal samples from 41 patients (Hosp = 24; ER = 17) attending the Hospital Universitario Mayor-Méderi (Bogotá, Colombia) were analyzed. The gut microbiomes were dominated by Enterobacteriaceae, particularly Klebsiella pneumoniae and Escherichia coli, together with abundant bacteriophages from the families Myoviridae, Siphoviridae, Podoviridae, and crAss-like phages. Phages predicted to infect Escherichia and Klebsiella were significantly depleted in Hosp patients (p < 0.05). Read-based functional profiling revealed the presence of virulence factors associated with K. pneumoniae capsule biosynthesis, secretion systems, and toxins from Clostridioides difficile and Clostridium perfringens. In parallel, Hosp patients showed a higher diversity of antimicrobial resistance markers, with a marked increase in glycopeptide resistance determinants. A total of 492 high-quality metagenome-assembled genomes were reconstructed, including multiple diarrhea-associated taxa. Hosp patients exclusively harbored genomes of K. pneumoniae, Enterococcus faecium, and most reconstructed Clostridium species (C. symbiosum, C. saccharolyticum_A, C. innocuum, C. scindens, C. leptum, and Clostridium sp000435835). In contrast, ER patients harbored genomes classified as Escherichia coli, Escherichia flexneri, and Enterococcus faecalis. Genomes associated with hospitalization carried higher loads of antimicrobial resistance markers (e.g., oqxA and aac(6')-Ii) and virulence factors (e.g., iutA and traT), whereas ER genomes, particularly E. coli and E. flexneri, encoded diverse aminoglycoside resistance and adhesion traits. CONCLUSIONS: Hospital-onset diarrhea was associated with distinct microbiome features, including differences in phage-bacteria dynamics involving key diarrhea-associated taxa, as well as a higher abundance of virulence factors and antimicrobial resistance markers. These findings underscore the potential value of shotgun metagenomics as a complementary approach for infection surveillance and the development of precision diagnostic strategies in hospital settings.