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, FeSO 4 , or FeCl 3 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.