Preclinical Development of Enterobacter ludwigii LS57, a Human-Derived Swarming Strain, as a Live Biotherapeutic Product for Inflammatory Bowel Disease

Abstract

Inflammatory Bowel Disease (IBD) remains an area of major unmet need, and the undefined composition and manufacturing burden of fecal microbiota transplantation have motivated a shift toward single-strain Live Biotherapeutic Products (LBPs). Swarming bacteria are enriched in the stressed gut and limit mucosal damage, but no human-derived swarming strain has been developed as an LBP. We therefore report the complete preclinical development of Enterobacter ludwigii LS57, a swarming strain isolated from human feces. Chemistry, Manufacturing and Controls (CMC) covered a three-tier cell bank, 30 L fed-batch fermentation and a colon-targeted enteric-coated capsule, enabling consistent pilot-scale production. No treatment-related findings emerged in safety pharmacology (Sprague-Dawley rats) or in single-dose and Good Laboratory Practice (GLP)-compliant 1-week repeat-dose toxicity studies (Sprague-Dawley rats and Beagle dogs). LS57 was identified as E. ludwigii by 16S rRNA and whole-genome sequencing and produced metabolites with reported anti-inflammatory activity in culture. In Dextran Sulfate Sodium (DSS)-induced colitis, LS57 reduced weight loss, preserved colon length, lowered colonic pro-inflammatory cytokines and remodeled the gut microbiota. It also attenuated Trinitrobenzene Sulfonic Acid (TNBS)-induced colitis and enhanced the effect of ciclosporin A. In vitro, LS57 was non-hemolytic, non-cytotoxic, and acid-labile, formed no biofilm, and its resistance to aminopenicillins, cephamycins and imipenem was not transferable by conjugation. In vivo, LS57 colonized the inflamed colon without translocation in wild-type and interleukin-10-deficient mice, and experimental bacteremia was treatable with gentamicin or levofloxacin. LS57 combines efficacy across two mechanistically distinct colitis models with a safety package that addresses the principal regulatory concerns for LBPs, and a scalable manufacturing process, compatible with current good manufacturing practice, in which enteric coating overcomes the strain's poor gastric survival. These data support clinical development of LS57 in IBD.