Antimicrobial Activity of Eugenol Against NDM-Producing <i>Escherichia coli</i> : An <i>In Vitro</i> and <i>In Vivo</i> Study

Background: The global dissemination of New Delhi metallo- β -lactamase-1 (NDM-1)-producing Escherichia coli (carbapenem-resistant Escherichia coli [CREC]) has escalated into a pressing public health crisis, driven by the rapid evolution of multidrug resistance and the scarcity of effective therapies. To address this challenge, this study focused on evaluating the therapeutic potential of eugenol, a natural phenolic compound, against NDM-1-CREC through integrated in vitro and in vivo approaches. Methods: The in vitro antibacterial activity of eugenol was evaluated using the microbroth dilution method, growth curve, and time-antibacterial curve. The in vivo antibacterial activity of eugenol was studied by drawing survival curves, counting the live bacterial load in each tissue, measuring the expression of inflammatory factors, and histopathological observation. Morphology and ultrastructure were analyzed by SEM/TEM, cell wall integrity by alkaline phosphatase, biofilm by crystal violet staining, and gene expression by RNA-seq with RT-qPCR validation. Results: In vitro experiments demonstrated that eugenol exerted rapid bactericidal effects, achieving complete eradication of CREC isolates within 2 hours, and enhanced the sensitivity of imipenem and meropenem. In vivo , eugenol significantly improved survival rates in a murine intraperitoneal infection model, reducing bacterial loads in peritoneal fluid and visceral organs (liver, lung, kidney, spleen; 2–3log10 colony-forming units [CFU]/mL reduction) while attenuating systemic inflammation (IL-6, TNF-α, IL-1β). Histopathological analysis further corroborated these results, showing marked alleviation of tissue damage in eugenol-treated cohorts. Mechanistic studies revealed that eugenol disrupted membrane integrity via structural damage (Scanning electron microscopy/transmission electron microscopy), suppressed biofilm formation (crystal violet assay), and downregulated resistance/virulence genes ( mdtJ, ompA, csgD, flhD, fimB ; RNA-seq/qRT-PCR validation). Conclusions: These findings not only highlight eugenol’s potential as a candidate drug but also provide a framework for repurposing natural compounds to combat carbapenemase-producing pathogens, offering a pragmatic strategy to mitigate the global antimicrobial resistance crisis.