Robogut-Based Evaluation of Living Biopharmaceuticals Active Against Carbapenem-Resistant Enterobacteriaceae

Abstract

Antibiotic resistance among gut-associated pathogens, especially carbapenem-resistant Enterobacteriaceae (CRE), poses a significant global health challenge. To address the urgent need for novel, microbiome-based therapeutics and reduce reliance on animal models, we established and validated a human-derived Robogut fermentative system for screening live biopharmaceutical products (LBPs). The Robogut, a single-stage anaerobic bioreactor, was inoculated with healthy donor feces and supported a stable and resilient microbial community over time. We isolated five candidate LBPs from healthy donors and evaluated their safety and antimicrobial activity against clinical CRE isolates in vitro, in the Robogut, and in vivo using a murine model. Three lactic acid bacteria (LAB) strains exhibited potent and selective activity against CRE Klebsiella pneumoniae in both Robogut and murine assays, while two Escherichia strains were effective only against CRE E. coli. The Robogut recapitulated colonization resistance dynamics, with PNUH40 (K. pneumoniae) rapidly suppressed in the presence of a stable microbiota and selectively cleared upon LBP treatment. Notably, results from Robogut screening reliably predicted murine outcomes, highlighting the system's translational utility. This study underscores the value of Robogut models as ethical, high-fidelity platforms for preclinical testing of LBPs and for advancing microbiome-based therapeutics against multidrug-resistant gut pathogens.