The rise of antibiotic resistance, particularly in multidrug-resistant (MDR) gram-negative bacteria, poses a significant threat to global health. Colistin, a polymyxin antibiotic, is a last-resort treatment option for infections caused by these pathogens. However, the emergence of colistin resistance, driven by chromosomal mutations and the spread of mobile colistin resistance genes like *mcr-1*, severely limits its clinical utility. The development of colistin adjuvants, compounds that enhance the activity of colistin, offers a promising strategy to address this challenge. These adjuvants could potentially reduce the necessary colistin dosage, mitigating toxicity and slowing the development of resistance. Previous research has identified several potential colistin adjuvants, including various phytochemicals and the FDA-approved drug otilonium bromide. This study explores the potential of another FDA-approved drug, valnemulin (Val), as a colistin adjuvant. Val, a pleuromutilin antibiotic, exhibits activity against mycoplasmas and *Pasteurella* species, and has shown synergistic effects with doxycycline against *Acinetobacter baumannii*. Its established safety profile and lack of activity against Enterobacteriaceae make it a promising candidate for investigation as a colistin enhancer. This research investigates the synergistic effects of Val and colistin against various gram-negative pathogens, both *in vitro* and *in vivo*, and explores the underlying mechanisms of this synergistic activity.

The escalating problem of antibiotic resistance necessitates the exploration of novel therapeutic strategies. Colistin, a cationic lipopeptide antibiotic, has emerged as a last-line defense against multidrug-resistant Gram-negative bacteria. Its mechanism of action involves binding to the lipopolysaccharide (LPS) in the bacterial outer membrane, leading to membrane disruption and cell death. However, colistin resistance mechanisms, including modifications to lipid A and the emergence of mobile resistance genes (e.g., *mcr-1*), have significantly hampered its effectiveness. The search for colistin adjuvants has intensified, focusing on compounds that can either restore or enhance colistin's activity against resistant bacteria. Studies have explored the potential of natural compounds, such as flavonoids and melatonin, which have demonstrated synergistic effects with colistin by disrupting bacterial iron homeostasis or enhancing oxidative damage. The repurposing of FDA-approved drugs as colistin adjuvants also holds promise, offering a more streamlined and safer approach to developing new therapeutics. Otilonium bromide, an antispasmodic drug, has shown potential as a colistin adjuvant by dissipating the bacterial proton motive force (PMF). This study builds upon these previous findings by investigating the synergistic potential of valnemulin, another FDA-approved drug, as a novel colistin adjuvant.

This study employed a comprehensive approach to evaluate the synergistic effects of valnemulin (Val) and colistin against colistin-resistant and -susceptible gram-negative bacteria. The methodology encompassed several key techniques: 1. **Antimicrobial Susceptibility Tests:** Minimum inhibitory concentrations (MICs) of Val and colistin were determined individually and in combination using broth dilution and checkerboard assays. The fractional inhibitory concentration index (FICI) was calculated to assess synergy. 2. **Time-Kill Kinetic Studies:** Time-dependent killing curves were generated to monitor the bactericidal activity of Val, colistin, and their combination over 24 hours. 3. **Resistance Development Assay:** The emergence and development of colistin resistance in the presence and absence of Val were assessed using sequential passaging of susceptible strains. 4. **Scanning Electron Microscopy (SEM):** SEM imaging was used to visualize the morphological changes in bacterial cells following treatment with Val, colistin, and their combination. 5. **Cytotoxicity Evaluation:** MTT assays were performed to assess the cytotoxicity of Val and colistin on mammalian HEK293T cells. 6. **Intracellular Bacterial Determination:** A cell infection model (Vero cells infected with *E. coli* P47) was used to evaluate the synergistic effects of Val and colistin on intracellular bacteria. 7. **Mouse Infection Model:** A mouse sepsis model was employed to investigate the *in vivo* synergistic effects of Val and colistin against colistin-resistant *E. coli*. 8. **Membrane Permeability Test:** SYTOX Green staining was used to assess the effects of Val and colistin on bacterial membrane permeability. 9. **Membrane Depolarization:** DiSC3(5) staining was used to evaluate the impact of Val and colistin on bacterial membrane potential (PMF). 10. **Intracellular ATP Measurement:** An ATP assay was performed to determine the effect of Val and colistin on intracellular ATP levels. 11. **Swimming Assay:** A swimming motility assay was used to assess the effect of Val and colistin on bacterial motility. 12. **Transcriptomic Analysis:** RNA sequencing was conducted to analyze gene expression changes in *E. coli* P47 treated with colistin alone and in combination with Val.

The study demonstrated several key findings: 1. **Synergistic Activity *in vitro* and *in vivo*:** Valnemulin exhibited a strong synergistic effect with colistin against both colistin-resistant and colistin-susceptible gram-negative pathogens (*E. coli*, *K. pneumoniae*, *A. pittii*) in both *in vitro* and *in vivo* models. FICIs consistently indicated synergy. Time-kill curves showed a significantly greater reduction in bacterial load with the combination therapy compared to monotherapy. The mouse sepsis model demonstrated a significant increase in survival rates with the combination treatment. 2. **Prevention of Colistin Resistance Development:** The combination of Val and colistin significantly slowed or prevented the development of colistin resistance *in vitro*, as evidenced by reduced increases in colistin MICs compared to colistin alone. 3. **Lack of Cytotoxicity:** Valnemulin displayed no significant cytotoxicity against mammalian HEK293T cells at concentrations tested, suggesting a favorable safety profile. 4. **Enhanced Membrane Permeabilization:** SEM imaging revealed membrane disruption in *E. coli* P47 treated with the Val-colistin combination. SYTOX Green staining showed significantly increased bacterial membrane permeability with the combination treatment compared to colistin alone. 5. **Dissipation of Proton Motive Force (PMF):** The combination of Val and colistin caused a significant increase in fluorescence intensity with DiSC3(5) staining, indicating dissipation of bacterial membrane potential (PMF). This was further supported by a decrease in intracellular ATP levels. The swimming motility assay also demonstrated a significant reduction in bacterial motility with the combination treatment. 6. **Transcriptomic Changes:** Transcriptomic analysis of *E. coli* P47 revealed significant changes in gene expression upon treatment with the Val-colistin combination. Upregulation of genes related to membrane components and efflux pumps was observed, potentially as a bacterial response to the combined stress. Conversely, downregulation of flagellum-related genes was consistent with the observed reduction in bacterial motility.

This study provides compelling evidence for the potential of valnemulin as a colistin adjuvant. The observed synergistic bactericidal activity against both colistin-resistant and -susceptible strains, both *in vitro* and *in vivo*, addresses the urgent clinical need for novel strategies to combat MDR gram-negative infections. The combination's mechanism of action appears multifaceted, involving enhanced membrane permeabilization, PMF dissipation, and suppression of bacterial motility. The lack of cytotoxicity observed with Val is a crucial advantage, potentially allowing for reduced colistin dosage and decreased toxicity. The transcriptomic data provide further insights into the underlying mechanisms, showing both upregulation of membrane-related genes and downregulation of motility-related genes. Future research could focus on exploring the clinical potential of this combination therapy through well-designed clinical trials to assess efficacy and safety in human patients. Further investigation into the specific interactions between Val and colistin at a molecular level is also warranted. The findings could also have implications for the treatment of intracellular bacterial infections and potentially even cancer, given the demonstrated efficacy against intracellular bacteria.

This study successfully demonstrated the synergistic antibacterial effects of valnemulin (Val) and colistin against multidrug-resistant gram-negative pathogens. The combination effectively combats colistin resistance, enhances membrane permeabilization and PMF dissipation, reduces ATP levels, and suppresses bacterial motility. Val’s lack of cytotoxicity presents a significant advantage. These findings strongly suggest the clinical potential of Val as a colistin adjuvant and warrant further investigation through pre-clinical and clinical studies. Future work could explore the optimization of Val and colistin dosing regimens and evaluate the efficacy of this combination against a broader range of bacterial species and infection models.

While this study provides strong evidence for the synergistic efficacy of valnemulin and colistin, certain limitations should be acknowledged. The *in vivo* study used a relatively small sample size, and further research with a larger cohort is necessary to confirm the findings. The study mainly focused on a limited number of bacterial strains, and additional testing with a broader range of clinical isolates is needed to determine the generalizability of the results. Moreover, the specific molecular interactions between Val and colistin remain to be fully elucidated.