The increasing incidence of multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Tm), known for causing invasive enteric infections, presents a significant public health challenge. Given the diminishing efficacy of existing antibiotics, it is imperative to explore novel alternatives for the treatment of MDR S. Tm infections. Here, we identified esculetin (EST), a natural coumarin abundant in dietary foods and herbs, as a compound exhibiting broad-spectrum antibacterial properties against a range of MDR bacteria. Our findings demonstrate that EST effectively inhibited the proliferation and expansion of MDR S. Tm in both in vitro experiments and animal models. Specifically, EST significantly downregulated the type 3 secretion system-1 (T3SS-1) virulence expression of MDR S. Tm, thereby preventing its invasion into intestinal epithelial cells. In S. Tm-infected mice, we observed cecal injury characterized by the upregulation of inflammatory cytokines, a reduction in goblet cell numbers, a decreased expression of tight junction proteins, and microbial dysbiosis. Conversely, EST treatment ameliorated these pathological changes induced by S. Tm infection and reduced oxidative stress by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, thereby improving intestinal barrier function. These results suggest that dietary coumarins or a targeted plant-based diet may offer a promising strategy to counteract MDR bacteria-induced enteric diseases.

Introduction: The rapid progression of bacterial resistance and the dearth of novel antimicrobial drug development impose a significant public health burden on the treatment of bacterial infections. Drug combination therapy has become an attractive strategy for combating multidrug-resistant bacterial infections. More importantly, matching the physicochemical properties of multiple components in formulations is essential for clinical application. Methods: First, an enrofloxacin–colistin combination injection was developed using a tripartite strategy, defined as a three-step process involving the conversion of enrofloxacin to its salt form, the addition of 1,2-propanediol, and pH adjustment. Second, independent gradient model based on Hirshfeld surface (IGMH), nuclear magnetic resonance (NMR), and ultraviolet-visible spectroscopy (UV-vis) analysis were used to investigate the molecular mechanism of this process. Finally, the irritancy, toxicity, and efficacy of the combination injection were evaluated in vivo and in vitro. Results: The tripartite strategy increased the solubility of enrofloxacin by 1500-fold from 0.18 mg/mL to 272.76 mg/mL, thereby preventing enrofloxacin precipitation during 6 months at both 30°C and 4°C, maintaining colistin stability, and reducing injection-site irritation. 1,2-Propanediol enhanced hydrogen bonding with enrofloxacin and inhibited its self-aggregation. Importantly, the combination injection exhibited no significant liver and kidney toxicity while demonstrating outstanding therapeutic efficacy against Pasteurella multocida pneumonia with 62.5% survival rate. Discussion: The limited solubility of enrofloxacin has long hindered its co-formulation with pH-sensitive drugs. The tripartite strategy establishes a paradigm for overcoming challenges related to the crystal precipitation of insoluble drugs and the pH limitations in complex formulations. Our findings demonstrate that the tripartite strategy effectively enhances the solubility, stability, and therapeutic efficacy of enrofloxacin-colistin combinations, offering a novel solution to overcome challenges in developing complex antibacterial formulations for veterinary use. Plain Language Summary: A highly stable and weakly acidic enrofloxacin–colistin combination injection was produced.1,2-Propanediol enhanced enrofloxacin solubility and inhibited recrystallization.1,2-Propanediol increased hydrogen bonding with enrofloxacin and inhibited molecular self-aggregation.The tripartite strategy resolved the insoluble drug-pH conflict, enabling multi-drug formulations.The enrofloxacin–colistin combination injection demonstrated excellent therapeutic efficacy with negligible toxicity or irritation.