The rising incidence of colistin (COL) resistance poses a significant challenge, undermining the therapeutic efficacy of COL against life-threatening bacterial infections. Therefore, the urgent identification and development of new therapeutics are imperative. It has been proven that combinations of antibiotics and promising non-antibiotic agents could be a potential strategy to combat infections caused by MDR pathogens. Due to various antimicrobial properties, medicinal plants have attracted significant attention, which could be promising adjuvant. In this study, we investigated the synergistic effects of combining COL with resveratrol (RST) and baicalin (BAI) against mcr-1-positive Escherichia coli through antibiotic susceptibility testing, checkerboard method and time-killing assays. The mechanisms of combination treatment were analyzed using SEM, fluorometric assays and transcriptome analysis. The molecular docking assay was conducted to elucidate potential interactions between RST, BAI and the MCR-1 protein. Finally, we assessed the in vivo efficacy of combination against mcr-1-positive Escherichia coli. The results demonstrated that the combination of RST, BAI and COL showed significant synergistic activity both in vitro and in vivo. Further mechanistic study revealed that the combination could increase the membrane-damaging ability of COL, disrupt the homeostasis of proton motive force (PMF), inhibit the activity of efflux pumps and impair ATP supply. The molecular docking revealed that RST and BAI could bind to MCR-1 stably, indicating the combination of RST and BAI may be an effective MCR-1 inhibitor. Our findings demonstrated that the combination of RST and BAI might be potential COL adjuvant, providing an alternative approach to address mcr-1-positive Escherichia coli infections.

Bacterial infections pose a major threat to human health and economic stability. In particular, intracellular bacterial infections present significant clinical challenges due to antibiotic resistance and poor drug penetration. Therefore, there is an urgent need to develop novel therapeutic strategies to address the problem of intracellular bacterial infections. Extracellular bacterial vesicles become ideal delivery systems due to their natural targeting properties. Here, we developed a bacteria-derived extracellular vesicles (EVs)-based drug delivery platform to enhance the therapeutic efficacy of antibiotics against intracellular infections. EVs were successfully isolated and increased production from S. aureus by ultracentrifugation, then loaded with ceftiofur (CEF) via co-incubation. In vitro tests demonstrated the potent antibacterial activity of CEF, achieving complete growth inhibition within 24 h and a 4-log viability reduction in 4 h. Furthermore, confocal microscopy revealed efficient CEV internalization in IEC-6 cells, with 12-fold greater intracellular bacterial clearance than free CEF. In vivo, CEV-incorporated hydrogel (CEVH) significantly reduced both intra- and extracellular bacterial loads and accelerated wound healing. These findings demonstrate that bacterial EVs serve as a universal delivery platform to significantly enhance the efficacy of existing antibiotics against intracellular infections.

The emergence and widespread of tigecycline resistance undoubtedly poses a serious threat to public health globally. The exploration of combination therapies has become preferred antibacterial strategies to alleviate this global burden. In this study, tigecycline-resistant tet(X4)-positive Escherichia coli were selected for adjuvant screening. Interestingly, 9-aminominocycline (9-AMC), one of the tigecycline metabolites, exhibits synergistic antibacterial activity with tigecycline using checkerboard assay. The efficacy in vitro and in vivo was evaluated, and the synergistic mechanism was further explored. The results suggested that 9-AMC combined with tigecycline could inhibit the growth of antibiotic resistant bacteria, efficiently retard the evolution of tet(X4) gene and narrow the drug mutant selection window. In addition, the combination of tigecycline and 9-AMC could destroy the normal membrane structure of bacteria, inhibit the formation of biofilm, remarkably reduce the level of intracellular ATP level, and accelerate the oxidative damage of bacteria. Furthermore, 9-AMC is more stable in the bind of Tet(X4) inactivating enzyme. The transcriptomics analysis revealed that the genes related to the 9-AMC and tigecycline were mainly enriched in ABC transporters. Collectively, the results reveal the potentiation effects on tigecycline and the probability of 9-AMC as a novel tigecycline adjuvant against tet(X4)-positive Escherichia coli, which provides new insights for adjuvant screening.

Antimicrobial resistance (AMR) is a growing public health crisis that requires innovative solutions. Emerging multidrug resistant (MDR) Salmonella typhimurium has raised concern for its effect on pathogenic infection and mortality in humans caused by enteric diseases. To combat these MDR Salmonella typhimurium pathogens, highly effective and broad-spectrum antibiotics such as flufenicol (FFC) need to be evaluated for their potent antibacterial activity against Salmonella typhimurium. However, the low solubility and low oral bioavailability of flufenicol need to be addressed to better combat AMR. In this work, we develop a novel nano-formulation, flufenicol nano-micelles (FTPPM), which are based on d-α-tocopherol polyethylene glycol 1,000 succinate (TPGS)/poloxamer 188 (P188), for the targeted treatment of biofilms formed by drug-resistant Salmonella typhimurium in the intestine. Herein, FTPPM were prepared via a thin film hydration method. The preparation process for the mixed micelles is simple and convenient compared with other existing nanodrug delivery systems, which can further decrease production costs. The optimized FTPPM demonstrated outstanding stability and sustained release. An evaluation of the in vivo anti-drug-resistant Salmonella typhimurium efficacy demonstrated that FTPPM showed a stronger efficacy (68.17 %) than did florfenicol-loaded TPGS polymer micelles (FTPM), flufenicol active pharmaceutical ingredients (FFC-API), and flufenicol commercially available medicine (FFC-CAM), and also exhibited outstanding biocompatibility. Notably, FTPPM also inhibited drug-resistant Salmonella typhimurium from forming biofilms. More importantly, FTPPM effectively restored intestinal flora disorders induced by drug-resistant Salmonella typhimurium in mice. In summary, FTPPM significantly improved the solubility and oral bioavailability of florfenicol, enhancing its efficacy against drug-resistant Salmonella typhimurium both in vitro and in vivo. FTPPM represent a promising drug-resistant Salmonella typhimurium treatment for curbing bacterial resistance via oral administration.

The dissemination of antibiotic resistance genes (ARGs) via conjugative plasmids is a major driver of the antimicrobial resistance crisis. The optrA gene, which confers cross-resistance to linezolid and florfenicol, is frequently detected in Enterococcus isolates and disseminated via plasmids. This study investigated the inhibitory effect of carvacrol on horizontal transfer of an optrA-carrying plasmid in porcine Enterococcus faecalis. In vitro conjugation assays showed that subinhibitory carvacrol (1/32 MIC, 16 µg/mL) significantly reduced conjugation efficiency in a dose- and time-dependent manner. In vivo (mouse model) treatment with 32 μg/mL carvacrol decreased transconjugant recovery by 62.8%. Mechanistically, carvacrol disrupted membrane integrity, depleted intracellular ATP, sup-pressed ROS production, reduced biofilm formation, and downregulated conjugation-associated genes (type VI secretion protein, conjugal transfer protein, prgB, asa1) as shown by transcriptomics and RT-qPCR. Collectively, carvacrol inhibits optrA plasmid transfer via multi-target mechanisms involving membrane damage, energy metabolism interference, and suppression of conjugation gene expression. This study highlights carvacrol as a natural agent to control the transfer of ARGs.