Mechanistic Evaluation of LolA and β-Sliding Clamp Inhibition by Silybin and Diclofenac as Gentamicin Adjuvants Against Enteropathogenic Escherichia coli

To address the challenges of antimicrobial resistance, one potential approach is to repurpose existing drugs. In the current study, structurally distinct antibiotics were combined with the repurposed drugs silybin and diclofenac to evaluate the potential of these combinations against resistant bacterial strains. The investigation encompassed in vitro determination of the minimum inhibitory concentration (MIC) and time-kill kinetics, as well as in vivo assessments of percentage change in body weight, total white blood cell (WBC) count, colitis and edema, histopathological changes, stool scores, and blood culture in an enteropathogenic Escherichia coli (EPEC)-infected mouse model. Both silybin and diclofenac potentiated the activity of several antibiotics, with gentamicin showing the most promising results. The gentamicin-silybin (Gen + Sily) and gentamicin-diclofenac (Gen + Dic) combinations not only demonstrated sustained antibacterial activity in the time-kill assay compared with gentamicin monotherapy but also provided greater protection against crypt cell damage and more effectively alleviated colitis and edema in the EPEC-infected mouse model. In silico molecular docking and 250-ns molecular dynamics simulations hypothesized two potential targets implicated in antibiotic adjuvant activity: LolA and the β-sliding clamp protein. Molecular docking and MM/PBSA analyses indicated that silybin and diclofenac exhibited stronger binding affinities and more favorable total binding free energies toward LolA and the β-sliding clamp protein, respectively, than the corresponding reference compounds.