Robogut-based ecosystem screening of live biotherapeutic candidates for intestinal CRE suppression

Carbapenem-resistant Enterobacterales (CRE) can colonize the gastrointestinal tract and serve as a reservoir for infection and transmission. Live biotherapeutic products (LBPs) offer a potential strategy to suppress intestinal CRE colonization, but conventional plate-based screening may not reliably identify candidates that retain activity in a gut-like microbial ecosystem. This limitation hampers prioritization of therapeutically relevant LBP candidates and can lead to the advancement of false-positive candidates to animal testing. Here, we established a Robogut-based screening framework to evaluate anti-CRE candidate strains between primary plate-based screening and in vivo validation. Human fecal isolates were first screened against the CRE challenge strain Klebsiella pneumoniae PNUH40, and selected candidates were evaluated under Robogut-based in vitro CRE colonization conditions. Robogut-based screening distinguished candidates with activity retained under gut-like conditions from a plate-positive candidate that failed to retain activity, and the screening outcome was concordant with efficacy in a murine CRE colonization model. Microbial community analysis and targeted short-chain fatty acid quantification showed that Robogut culture established a fecal-derived microbial community and a metabolic environment characterized by acetate, propionate, and butyrate accumulation. These preliminary findings support the potential of Robogut-based screening as an intermediate step for prioritizing anti-CRE LBP candidates before animal validation.