Decoding The Human Gut Resistome: Screening For Antimicrobial Resistance In Gut Microbiota

Abstract

Background: Antimicrobial resistance (AMR) is a critical global health threat, exacerbated by the overuse and misuse of antibiotics, leading to treatment failures and complications. The human gut microbiota, a complex and diverse microbial ecosystem essential for health, serves as a major reservoir of antimicrobial resistance genes (ARGs), collectively referred to as the gut resistome. These genes, which are often embedded within microbial communities, can spread via horizontal gene transfer (HGT), exacerbating AMR and complicating efforts to control resistant infections. This study characterizes the gut resistome in healthy Lebanese individuals using advanced shotgun metagenomics, culturomics, and antimicrobial susceptibility testing.

Material and Methods: Fecal samples were collected from healthy Lebanese individuals with ethical approval from the Institutional Review Board (IRB). Samples were preserved in transport media at -80°C before microbial DNA extraction using the QIAmp PowerFecal Pro DNA kit. Shotgun metagenomics was performed on the Illumina MiSeq platform to profile the microbial communities and their associated resistomes. Culturomics was employed to isolate bacteria on various agars, followed by antimicrobial susceptibility testing (AST) against seven antibiotics: meropenem, ertapenem, cefoxitin, vancomycin, tigecycline, piperacillin-tazobactam, and clindamycin. Additionally, 16S rRNA gene sequencing was conducted to confirm bacterial diversity and identify specific genera involved in AMR.

Results: The shotgun metagenomics data revealed a highly diverse gut microbiome, with the phyla Firmicutes, Actinobacteria, and Bacteroidetes being the most abundant. The taxonomic composition included known SCFA-producing genera, such as Faecalibacterium, Ruminococcus, and Bifidobacterium. Analysis of the gut resistome identified a wide range of antimicrobial resistance genes, with efflux pumps (1089.43 CPM), unclassified resistance mechanisms (469.82 CPM), and beta-lactam (338.67 CPM) and macrolide resistance (303.65 CPM) being the most. Notably, multidrug-resistant (MDR) strains were detected, resistant to all seven tested antibiotics. Additionally, a proportion of unclassified species was detected, reflecting potentially novel, underexplored taxa within the gut microbiome that may contribute to AMR.

Conclusion & Impact: This study enhances our understanding of the gut resistome, highlighting its role as a critical source of antimicrobial resistance in the human microbiome. By integrating shotgun metagenomics, culturomics, and antimicrobial susceptibility testing, the findings underscore the importance of monitoring AMR profiles in the gut microbiota, especially in relation to multidrug-resistant organisms. These insights provide a foundation for future research on Minimum Inhibitory Concentration (MIC) testing and the development of targeted strategies to mitigate AMR in both clinical and community settings. The results stress the necessity for ongoing surveillance of the gut resistome, offering potential pathways for improving public health interventions and antimicrobial stewardship in Lebanon and beyond. Additionally, this study contributes to the development of new guidelines for monitoring and managing AMR in the human microbiome.