CHIR-090

Fermented foods (FFs) represent complex living ecosystems that deliver viable microbes and bioactive metabolites linked to human health benefits. However, many probiotic strains isolated from FFs fail to reproduce these effects in vivo, likely due to the disruption of their natural ecological synergy during isolation. Here, we employed a systematic, ecology-aware culturomics framework to transform the Kimchi microbiome into genome-vetted, multi-species probiotic candidates while preserving ecological fidelity. Specifically, 56 distinct enrichment culture conditions were established using six liquid media (In-situ, MRS, NB, TSB, BHI, BB) across varied redox states (aerobic, anaerobic, microaerophilic), incubation periods (12 h, 66 h), and selective suppressants (CHIR-090, nalidixic acid). Results indicated that In-situ and MRS media under microaerophilic conditions effectively preserved the lactobacilli core, whereas generalist media and aeration expanded taxonomic breadth to include rare taxa. Furthermore, extended incubation (66h) successfully unlocked 107 unique taxa compared to the limited diversity of short incubation (12h). Shotgun metagenomic mining further revealed promising functional properties, including acid tolerance, adhesion modules, and diverse bacteriocin-skewed biosynthetic gene clusters. Crucially, the collection exhibited a strong safety profile: only 1 % of identified risk factors were antibiotic resistance genes (ARGs) on mobile genetic elements (MGEs), and only 4 % represented colocalized ARGs, virulence factors, and MGEs. Systematic-culturomic isolation later yielded over 90 strains, including Weissella, Bacillus, and Lactococcus, significantly expanding beyond standard lactobacilli-centric portfolios. Overall, this study confirms that ecology-aware culturomics captures the functional diversity of the Kimchi microbiome, providing a scalable model for realizing the full therapeutic potential of FFs.

The postantibiotic effect (PAE) is the delay in bacterial regrowth following antibiotic removal. It has important implications for dosing regimens since drugs that have extended activity following their elimination can be dosed less frequently, widening the therapeutic window. While the PAE has been associated with target vulnerability and the rate of target turnover, little is known about the genetic components that modulate the PAE. Here, we developed a high-throughput assay to screen the Escherichia coli Keio collection of ∼4000 deletion strains, identifying genes that enhance the PAE for CHIR-090, an inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). This screen revealed approximately 400 gene knockouts that enhanced the PAE of CHIR-090. The list of PAE enhancers was enriched for genes involved in transmembrane transport and outer membrane synthesis. Notably, deletion of the rfaE gene, which is involved in lipopolysaccharide (LPS) biosynthesis, increased the PAE of the LpxC inhibitors CHIR-090 and LPC-058 by 2 and 3 h, respectively. Consistent with this phenotype, cotreatment of wild-type E. coli with an RfaE inhibitor increased the PAE of CHIR-090 or LPC-058 by 1 h. To probe the mechanism of this interaction, we measured the rate of LpxC turnover and found that knocking out rfaE extended the half-life of LpxC by 2-fold, suggesting that disrupting RfaE increases the stability of LpxC, increasing target vulnerability and enhancing the PAE of LpxC inhibitors.