Differential impacts of hemin and free iron on amoxicillin susceptibility in ex vivo gut microbial communities.

Introduction: The rise of antibiotic-resistant infections worldwide has created a need to enhance the efficacy of existing antibiotics. Modification of metabolism has been shown to potentiate antibiotic lethality. In this study, we employed a novel ex vivo microbiome culture approach to study the effects of different forms of iron on amoxicillin susceptibility. Methods: Synthetic and human stool-derived microbiota were cultured and treated with amoxicillin, with growth monitored by optical density. These samples were sequenced using an Oxford nanopore long-read 16S rRNA V4-V9 approach and computationally defined using the Emu algorithm. The validity of this pipeline was confirmed with consortia, murine cecal content, and a human stool sample. The stool-derived community was then cultured for 24 h with ranging concentrations of either hemin, FeSO4, or FeCl3 and concurrent amoxicillin dosage, then profiled to identify the effects of different forms of iron on amoxicillin susceptibility. Results: Alpha diversity, beta diversity, and normalized relative abundances confirmed the efficacy of the selected ex vivo pipeline, allowing for ~77% species retention over 24 h. Treatment of communities with hemin protected Bacteroides, Escherichia-Shigella, Parabacteroides, and Parasutterella against amoxicillin, while two forms of free iron did not. Discussion: This ex vivo pipeline enables reproducible assessment of how metabolic modulators like hemin alter amoxicillin susceptibility, highlighting a link between iron-sequestering genera and antibiotic-protection. Future mechanistic insights may support hemin-based strategies to boost antibiotic efficacy.