Discovery of oleanolic acid-derived amides as synergistic adjuvants against multidrug-resistant Escherichia coli

The global surge in antimicrobial resistance (AMR) within bovine-origin Escherichia coli represents a critical One Health challenge, severely compromising the efficacy of frontline veterinary antibiotics such as cefquinome (CEQ). In this study, we report the rational design and synthesis of a series of Oleanolic Acid (OA)-based amides as potent synergistic adjuvants to combat multidrug-resistant (MDR) E. coli . Leveraging a structure-guided approach, we exploited the superior 2α, 3β-dihydroxy pharmacophore of the OA scaffold and implemented site-specific amination at the C-28 position to enhance both metabolic stability and synergistic anti-bacterial activation. Among the synthesized candidates, derivative #3 emerged as the lead adjuvant. It significantly restored CEQ susceptibility across a panel of twelve bovine clinical isolates, exhibiting a remarkable fractional inhibitory concentration index (FICI) of 0.187, and effectively eradicating robust biofilms. Mechanistically, compound #3 disrupts bacterial bioenergetic homeostasis. It induces profound oxidative stress via superoxide dismutase (SOD) depletion and reactive oxygen species (ROS) accumulation, which subsequently triggers NAD+ depletion and aberrant ATP accumulation. This metabolic paralysis disrupts membrane integrity and effectively dismantles the architectural framework of mature biofilms. In a murine infection model, the synergistic combination of #3 and CEQ markedly reduced bacterial burdens in the liver and colon, while simultaneously modulating the host inflammatory response by downregulating pro-inflammatory cytokines (IL-6, TNF-α) and preserving tissue integrity. Overall, our findings position derivative #3 as a promising translational candidate for mitigating the agricultural AMR crisis by simultaneously overcoming bacterial resistance and alleviating infection-induced inflammation.