Evaluation of Microbiome Therapeutic Candidates for Carbapenem-Resistant Enterobacteriaceae Associated with Capsule Biosynthesis.

Carbapenem-resistant Enterobacteriaceae (CRE) are life-threatening multidrug-resistant superbugs with severely limited treatment options, highlighting the urgent need for next-generation therapeutics that employ novel mechanisms of action. Microbiome-based therapies have emerged as a promising alternative, with several development pipelines actively progressing. In this study, a comprehensive evaluation of efficacy and safety based on mechanistic evidence is critical for the optimal selection of therapeutic candidate strains. So, we evaluated Lactobacillus and Bacillus candidate strains for activity against CRE, assessing their efficacy and safety in the context of mechanistic insights. Cell-free supernatants derived from these strains exhibited dose-dependent growth inhibition and time-dependent bactericidal activity against carbapenem-resistant Klebsiella pneumoniae. All candidate strains exhibited acceptable safety profiles with no hemolytic activity, although bile salt deconjugation and antibiotic susceptibility patterns varied by strain. Mechanistic analyses revealed that treatment with cell-free supernatants significantly reduced the production of capsular polysaccharide (CPS) and downregulated the CPS biosynthesis-related gene galF, thereby enhancing susceptibility to human serum killing. Additionally, a strong correlation was observed between antibacterial potency and CPS suppression, and two relatively superior candidate strains were prioritized based on their dual functional advantages. Collectively, these findings provide a robust mechanistic rationale for strain prioritization and support the strategic advancement of capsule suppression-based microbiome therapeutics for controlling CRE infections into preclinical and clinical development.