Background: Bacterial infections have become more challenging to treat due to the emergence of multidrug-resistant pathogenic bacteria. Combined antibiotics prove to be a relatively effective method to control such resistant strains. This study aim to investigate synergistic activity of eugenol combined with colistin against a collection of clinical isolatedEscherichia coli(E.coli) strains, and to evaluate potential interaction.Methods: Antimicrobial susceptibility, minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) of the bacteria were determined by disk diffusion assay, broth microdilution method and checkerboard assay, respectively. The mcr-1 mRNA expression was measured by Real-time PCR. To predict possible interactions between eugenol and MCR-1, molecular docking assay was taken.Results: For total fourteen strains including eight colistin-resistant strains, eugenol was determined with MIC values of 4 to 8 μg/mL. Checkerboard dilution test suggested that eugenol exhibited synergistic activity when combined with colistin (FICI ranging from 0.375 to 0.625). Comparison analysis of Real-time PCR showed that synergy could significantly down-regulate expression of mcr-1 gene. A metal ion coordination bond with catalytic zinc atom and a hydrogen bond with crucial amino acid residue Ser284 of MCR-1 were observed after molecular docking, indicating antibacterial activity and direct molecular interactions of eugenol with MCR-1 protein.Conclusions: Our results demonstrated that eugenol exhibited synergistic effect with colistin and enhanced its antimicrobial activity. This might further contribute to the antibacterial actions against colistin-resistantE.colistrains.Graphical Synergistic effect of eugenol with colistin against colistin-resistant Escherichia coli isolates.

The Rcs phosphorelay system is present in many members of the Enterobacteriaceae. The aim of this study was to illustrate the possible mechanisms of eugenol on ultimate targets of Klebsiella pneumoniae (K. pneumoniae) Rcs phosphorelay, rcsB, and impact on biofilm formation. The minimum inhibitory concentration (MIC) of eugenol against K. pneumoniae KP1 and KP1 ΔrcsB strain was determined using the 2-fold micro-dilution method. Biofilm was measured by crystal violet staining. Transcriptome sequencing was performed to investigate sub-MIC eugenol on K. pneumoniae, and gene expression at mRNA level was analyzed by RT-qPCR. In vitro biofilm formation test and molecular docking were used to evaluate the effect of eugenol and to predict potential interactions with RcsB. MicroScale Thermophoresis (MST) was conducted for further validation. MIC of eugenol against K. pneumoniae KP1 and KP1 ΔrcsB strain was both 200 μg/ml. Transcriptome sequencing and RT-qPCR results indicated that rpmg, degP, rnpA, and dapD were downregulated, while rcsB, rcsD, rcsA, yiaG, and yiaD were upregulated in the eugenol-treated group. ΔrcsB exhibited a weakened biofilm formation capacity. Additional isopropyl-β-d-thiogalactoside (IPTG) hinders biofilm formation, while sub-MIC eugenol could promote biofilm formation greatly. Docking analysis revealed that eugenol forms more hydrophobic bonds than hydrogen bonds. MST assay also showed a weak binding affinity between eugenol and RcsB. These results provide significant evidence that rcsB plays a key role in K. pneumoniae biofilm formation. Sub-MIC eugenol facilitates biofilm formation to a large extent instead of inhibiting it. Our findings reveal the potential risk of natural anti-biofilm ingredients at sub-MIC to treat drug-resistance bacteria.

Avian pathogenic Escherichia coli (APEC) infection poses a significant challenge to the poultry industry, severely threatening poultry health and industrial development. The emergence of antibiotic resistance in conventional treatments underscores the urgent need for novel alternative therapies. Fermented Chinese herbal residue solution, as a potential substitute, is rich in bioactive components and exhibits multifaceted effects, including anti-inflammatory, antioxidant, and immunomodulatory properties. This study investigates the anti-inflammatory mechanisms and key active compounds of the fermented herbal residue solution (BY3) in an APEC infection model. An in vitro APEC-infected HD11 cell model was established, with optimal infection conditions determined as a multiplicity of infection (MOI) of 0.1 and an infection duration of 6 hours, based on cytotoxicity assays and qPCR analysis. Results demonstrated that BY3 intervention significantly downregulated the expression of inflammatory cytokines. APEC infection markedly upregulated the expression of key target genes in the PI3K/AKT and NF-κB pathways, whereas BY3 treatment significantly reduced their expression. Western blot analysis further confirmed that BY3 significantly decreased the phosphorylation levels of AKT, P65, and IκB proteins, as well as the total PI3K protein content. These findings suggest that BY3 mitigates APEC-induced inflammation by modulating the PI3K/AKT and NF-κB pathways. To elucidate the active components of BY3, non-targeted metabolomics sequencing, database comparison, and molecular docking were employed, identifying four key bioactive compounds: Tangeretin, Arctigenin, Rhein, and Phloretin. All tested compounds significantly reduced APEC-induced inflammatory cytokine expression. qPCR and Western blot analyses revealed differential regulatory effects on the PI3K/AKT and NF-κB pathways. In the PI3K/AKT pathway, Tangeretin exhibited the most comprehensive inhibitory effect, significantly reducing both AKT phosphorylation and total PI3K levels. In the NF-κB pathway, all compounds except Rhein markedly decreased the phosphorylation of P65 and IκB. Collectively, BY3 and its identified compounds exert protective effects against APEC-induced HD11 cell damage by regulating the PI3K/AKT and NF-κB pathways, suggesting that the fermented Chinese herbal residue solution may represent a promising therapeutic approach for APEC-related diseases in poultry.