Application of Incyclinide and colistin in preparation of bactericidal drugs

Abstract

The invention discloses an application of Incyclinide and colistin in preparation of a bactericidal drug for enterobacteriaceae bacteria carrying a mcr (mcr) gene. Incyclinide (Ini) is a chemically modified tetracycline derivative, the invention proves that Ini can recover the bactericidal effect of colistin on enterobacteriaceae bacteria carrying mcr genes in vitro and in animal models, and mechanism analysis shows that Ini recovers the drug effect of colistin by inhibiting lipid A modification, inducing oxidative damage and dissipating proton power (PMF). As a colistin adjuvant, Ini has huge potential in the aspect of resisting enterobacteriaceae bacteria carrying mcr genes.

Description

Technical Field

[0001] The present invention belongs to the technical field of bactericidal drug development, and particularly relates to the application of Incyclinide in combination with colistin in the preparation of a bactericidal drug for Enterobacteriaceae bacteria carrying the mcr gene. Background Art

[0002] The widespread use of antibiotics in the medical field has greatly reduced the mortality and morbidity associated with infections, bringing about a revolutionary change in healthcare. However, the overuse and abuse of antibiotics in human medicine and agriculture have created a selection pressure that has promoted the widespread dissemination of drug-resistant bacteria, which has significantly exacerbated the escalating crisis of antibiotic resistance (AMR). Antibiotic resistance not only threatens the health of individual patients but also constitutes a global health crisis that could reverse decades of medical progress, highlighting the urgent need for global coordinated action led by the World Health Organization. In recent years, antibiotic resistance has spread rapidly, especially resistance to last-line antibiotics such as colistin, tigecycline, carbapenems, and cefepime.

[0003] Colistin (also known as polymyxin E) is a polypeptide cationic antibiotic that exhibits significant antibacterial activity against most Gram-negative pathogens, particularly Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. For these bacterial species, once multidrug resistance is exhibited, colistin often becomes the only effective antibiotic option. Colistin exerts its antibacterial effect by binding electrostatically to the anionic lipid A residues of lipopolysaccharide (LPS) in the outer membrane of Gram-negative pathogens. This binding leads to increased cell membrane permeability, leakage of cell contents, and ultimately lysis of the cells. However, the clinical use of colistin is significantly limited by its side effects, particularly nephrotoxicity and neurotoxicity, and also by the rapidly emerging and spreading resistance mechanisms in pathogens. The acquisition of colistin resistance is mainly due to a reduction in the electrostatic interaction between colistin and the negatively charged residues of lipid A. This process is usually mediated by the binding of cationic substituents, such as phosphoethanolamine (pEtN) or 4-amino-4-deoxy-L-arabinose groups, to the phosphate groups of the LPS lipid A moiety. This modification is catalyzed by the corresponding transferases EptA and ArnT, thus reducing the overall negative charge on the bacterial cell surface. Previously, these LPS modifications were thought to be caused by chromosomal mutations that activate the two-component regulatory systems PhoP-PhoQ and PmrA-PmrB, which regulate the expression of eptA or arnT. Since the spread of chromosomal colistin resistance is limited, its clinical impact has been mainly confined to outbreak events. However, the mobilizable colistin resistance (mcr) genes, which can be transferred horizontally by plasmids, have been identified as a key mechanism that facilitates the rapid spread of resistance within and between different species. Multidrug-resistant strains, especially those carrying resistance genes to other last-line antibiotics, may coexist with the mcr genes, and this trend of gene coexistence poses a severe challenge to clinical treatment as it may render existing antibiotic treatment regimens ineffective and accelerate the depletion of effective antibiotic resources.

[0004] Developing new therapeutic strategies using adjuvants to restore the sensitivity of drug-resistant bacteria to existing antibiotics is a method that holds promise for extending the lifespan of well-studied and clinically validated drugs. In currently clinically used combination therapies, a prominent example is the combination of β-lactam antibiotics with β-lactamase inhibitors. In addition to using inhibitors targeting specific proteins encoded by resistance genes, inhibiting key pathways using adjuvants can also restore the potency of antibiotics. For example, it has been found that natural flavonoids can restore the potency of colistin by interfering with the iron balance in bacteria. However, there are currently no commercially available colistin adjuvants for clinical use, which highlights the urgent need to develop potential colistin adjuvants. Summary of the Invention

[0005] To overcome the drawbacks and deficiencies of the prior art, the purpose of the present invention is to provide the use of Incyclinide in combination with colistin in the preparation of a bactericidal drug for Enterobacteriaceae bacteria carrying the mcr gene.

[0006] The present invention is realized as follows: the use of Incyclinide in combination with colistin in the preparation of a bactericidal drug for Enterobacteriaceae bacteria carrying the mcr gene.

[0007] The present invention overcomes the deficiencies of the prior art and provides the use of Incyclinide in combination with colistin in the preparation of a bactericidal drug for Enterobacteriaceae bacteria carrying the mcr gene. Incyclinide (Ini) is a chemically modified tetracycline derivative. The present invention demonstrates that Ini can restore the bactericidal effect of colistin on Enterobacteriaceae bacteria carrying the mcr gene in vitro and in animal models. Mechanistic analysis shows that Ini restores the efficacy of colistin by inhibiting lipid A modification, inducing oxidative damage, and dissipating the proton motive force (PMF). As a colistin adjuvant, Ini has great potential in combating Enterobacteriaceae bacteria carrying the mcr gene.

[0008] Preferably, the Enterobacteriaceae bacteria include Escherichia coli P47 and Escherichia coli P80.

[0009] Compared with the disadvantages and deficiencies of the prior art, the present invention has the following beneficial effects: Through in vitro experiments, zebrafish infection models, and mouse infection models, the present invention demonstrates that Incyclinide (Ini) synergistically acts with colistin to eliminate Enterobacteriaceae bacteria carrying mcr. Mechanistic studies show that Ini inhibits the modification of lipid A by inhibiting the expression of mcr, thereby promoting the entry of colistin into the bacterial cell membrane. In addition, Ini also dissipates the proton motive force (PMF), thereby impairing the function of PMF-dependent efflux pumps, leading to the accumulation of colistin within the membrane. The accumulated colistin induces oxidative stress, exacerbates membrane damage, and ultimately results in cell death. Therefore, Ini has great potential to become an efficient colistin adjuvant, capable of effectively addressing the growing threat posed by infections caused by pathogens carrying the mcr gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is an in vitro experiment on the synergistic effect of Ini and colistin in eliminating Enterobacteriaceae bacteria carrying the mcr gene and inhibiting biofilm formation; among them, Figures A - B are isobolograms showing the synergistic effect of Ini and colistin on Enterobacteriaceae bacteria carrying the mcr gene, with the X-axis and Y-axis being FIC Ini and FIC 黏菌素 ; Figure C is a heat map of the FIC index of Ini and colistin on Enterobacteriaceae bacteria carrying the mcr gene; Figures D - K are time-kill curves of Enterobacteriaceae bacteria carrying the mcr gene under treatment with Ini, colistin, and their combination; Figures L - N are the biofilm formation of Escherichia coli P47 after treatment with Ini, colistin, and their combination for 24 hours, 48 hours, and 72 hours; Figure O is a CLSM imaging of the biofilm structure of Escherichia coli P47 after treatment with Ini, colistin, and their combination for 5 days; all experiments were independently repeated three times, and the data are expressed as mean ± standard deviation (SD). In the figures, Ini is incyclinide; CT is colistin; statistical analysis was performed using GraphPad Prism 9.0 software, and unpaired t-tests between two groups were used to determine P (ns, not statistically significant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

[0011] Figure 2 is a checkerboard broth microdilution test of Ini and colistin on Enterobacteriaceae bacteria carrying mcr; among them, the dark green area represents a higher bacterial cell density, and the figure shows the average OD value of biological replicates at a wavelength of 600 nm.

[0012] Figure 3is the synergistic bactericidal effect of the combination of Ini and colistin in mammalian cells and animal infection models. Among them, Panel A shows the results of MTT assay for the cytotoxicity of Ini on Vero cells, and Vero cells were treated with Ini, colistin, and their combination respectively. Panels B - C show the intracellular bacterial load of Escherichia coli P47 in Vero cells after treatment with Ini, colistin, and their combination. Panels D - F show the inflammatory response of RAW264.7 cells induced by bacterial LPS (1 μg / ml) in the presence of different concentrations of Ini. After treatment with Ini, the levels of TNF-α, IL-1β, and IL-10 cytokines in the culture samples were quantitatively determined by ELISA analysis. Panel G shows the schematic diagram of the experimental protocol for two animal models. Panel H shows that 16 zebrafish in each group were infected with clinically isolated Escherichia coli P80 carrying the mcr gene and then received treatment with Ini, colistin, and their combination respectively, and their survival rates were recorded. The P value was determined by Log-rank (Mantel-Cox) test. Panel I shows that 8 mice in each group were infected with clinically isolated Escherichia coli P80 carrying the mcr gene and then received treatment with Ini, colistin, and their combination respectively, and their survival rates were recorded. The P value was determined by Log-rank (Mantel-Cox) test. Panels J - L show the amount of bacteria in the liver, spleen, and kidney of the mouse peritonitis-sepsis infection model. In the figure, Ini is incyclinide; CT is colistin. All experiments were performed in triplicate independently, and the data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9.0 software, and unpaired t-test between two groups was used to determine P (ns, no statistical significance, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

[0013] Figure 4 is the histological analysis of the main organs of mice. In the figure, on the 7th day after injecting mice with Ini (64 mg / kg), colistin (1 mg / kg), and their combined preparation, the main organs (liver, spleen, and kidney) excised from each group of mice were stained with hematoxylin and eosin (H&E).

[0014] Figure 5Ini enhanced colistin-induced damage to the cell membrane of Escherichia coli P47 bacteria; among them, Figure A shows the morphological changes of bacteria after treatment with Ini, colistin, and their combination observed by scanning electron microscopy; Figures B-C show the determination of bacterial membrane permeability treated with Ini, colistin, and their combination using SYTOX Green staining; Figures D-E show the determination of bacterial membrane permeability treated with Ini, colistin, and their combination by evaluating the activity of β-galactosidase; Figures F-G show the determination of membrane fluidity in the presence of Ini, colistin, and their combination using Laurdan staining; Figures H-I show the evaluation of the bacterial ΔΨ after treatment with Ini or colistin alone and their combination therapy; Figures J-K show that Ini and / or colistin have no effect on the bacterial ΔpH by BCECF-AM staining; Figures L-M show the intracellular ATP levels after treatment with Ini or colistin alone and their combination; Figures N-O show the detection of bacterial efflux pump activity using Nile red staining; Figure P shows the increased accumulation of colistin in bacteria caused by Ini; Figure Q shows the synergistic mechanism of Ini in restoring membrane damage caused by colistin; in the figures, Ini is incyclinide; CT is colistin. All experiments were performed in three independent replicates, and the data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9.0 software, and unpaired t-tests between two groups were used to determine P (ns, not statistically significant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

[0015] Figure 6 Ini and colistin can inhibit the motility of Escherichia coli P47; among them, Figure A shows the motility analysis using semi-solid agar plates containing Ini, colistin, and their combination; Figure B shows the measurement of the migration distance (centimeters) after the inoculated plates were cultured at 37°C; in the figures, Ini: incyclinide; CT: colistin. Statistical analysis was performed using GraphPad Prism 9.0, and the P value was calculated using unpaired t-tests between two groups (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

[0016] Figure 7 Ini enhanced the antibacterial effect of colistin by targeting the flagellar formation and motility of bacteria; among them, Figure 7Figure A is a volcano plot of the distribution of gene expression differences. The horizontal axis represents the fold change in expression level (fold change value), and the vertical axis represents the relevant statistical value. Genes with significant expression differences (DEGs) are determined using an adjusted P-value less than 0.05 as the threshold (Student’s t-test). Figure 7 C, Figure 7 Figure E shows the GO annotation analysis of DEGs and genes involved in the most significantly enriched pathways such as bacterial flagella and cell motility. Figure 7 B, Figure 7 Figure D shows the KEGG enrichment analysis of DEGs and genes involved in the most important enriched pathways such as flagellar assembly and bacterial chemotaxis. Figure 7 Figure F shows the quantitative determination of the number of Escherichia coli P47 bound to macrophage RAW 264.7 cells after 1 hour of in vitro infection. In the figure, Ini refers to incyclinide; CT refers to colistin; all experiments were performed in triplicate, and the data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9.0 software, and an unpaired t-test between two groups was used to determine P (ns, not statistically significant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

[0017] Figure 8 It is the synergistic effect of Ini and colistin that exacerbates the oxidative damage of the cell membrane of Escherichia coli P47. Among them, Figures A - B show that Ini or its combination with colistin accelerates the TCA cycle. Figure C shows the fold change in the mRNA expression level of DEGs in the TCA cycle through transcriptome analysis. Figures D and E show the ROS levels of bacteria after treatment with Ini, colistin, and their combination. Figures F - G show the determination of SOD activity after adding colistin, Ini, and their combination. Figures H - I show the hydrogen peroxide content of bacteria under the action of Ini, colistin, and their compound preparation. Figure J shows the determination of the intracellular iron content of bacteria after treatment with Ini, colistin, or their combination. Figure K shows that Ini converts intracellular iron from the ferric form to the ferrous form. Figure L shows the synergistic mechanism of Ini and colistin in inducing membrane oxidative damage. In the figure, Ini refers to incyclinide; CT refers to colistin; all experiments were performed in triplicate, and the results are expressed as the mean ± SD. Statistical analysis was performed using GraphPad Prism 9.0, and an unpaired t-test between two groups was used to determine the P-value (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

[0018] Figure 9 It was Ini that inhibited the modification of lipid A; among them, A and B were the quantitative analysis of the expression level of MCR-1 using Western blotting; Figure C was the determination of the LPS content in bacteria treated with different concentrations of Ini; Figure D was the negative-ion MALDI-TOF mass spectra of purified lipid A and lipid A-pEtN in Escherichia coli P47 in the presence of different concentrations of Ini; Figure E was the quantitative analysis of the contents of lipid A and lipid A-pEtN; Figure F was the determination of the binding of Escherichia coli P47 to the colistin membrane in the presence of Ini; Figure G was the change in the positive charge on the surface of the membrane of Escherichia coli P47 after treatment with Ini; Figure H was the synergistic mechanism of Ini in inhibiting the modification of lipid A and promoting the absorption of colistin by bacteria into the cell membrane; in the figure, Ini: incyclinide; CT: colistin; all experiments were performed in triplicate, and the results were expressed as mean ± SD; statistical analysis was performed using GraphPad Prism 9.0, and the P value was determined using unpaired t-test between two groups (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

[0019] Figure 10 It was the checkerboard broth microdilution test of Ini and colistin against Enterobacteriaceae bacteria; among them, the dark green area represented a higher bacterial cell density, and the figure showed the average OD value of biological replicates at a wavelength of 600 nm.

[0020] Figure 11 It was the synergistic antibacterial mechanism of Ini and colistin against Enterobacteriaceae bacteria carrying mcr; among them, Ini inhibited the modification of lipid A by inhibiting the expression of mcr, thereby promoting the absorption of colistin by bacteria into the cell membrane ①; Ini could also dissipate the proton motive force and damage the proton motive force-dependent efflux pump, resulting in the accumulation of colistin inside the cell membrane ②; the increase in the intracellular colistin content led to bacterial membrane damage, and the accumulated colistin induced oxidative stress, exacerbating membrane damage, and ultimately leading to cell death ③. Detailed implementation manners

[0021] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] I. Materials and methods

[0023] 1. Strains and reagents

[0024] All strains used in the present invention are shown in Table 1 below.

[0025] Table 1. Bacterial strains

[0026]

[0027] All strains were grown overnight at 37 °C in Luria - Bertani (LB) broth or on LB agar plates. Escherichia coli P47, which is derived from Escherichia coli J53 and carries an IncI2 - type plasmid containing the mcr - 1 gene, was selected as the model strain for mechanism studies. Clinically isolated Escherichia coli P80 was used for in - vivo evaluation. Ini (MedChem Express, Shanghai, China) was first dissolved in dimethyl sulfoxide (DMSO) (Meilunbio, Dalian, China) and then diluted to the required working concentration in the medium.

[0028] 2. Antibiotic susceptibility testing

[0029] The minimum inhibitory concentration (MIC) of Ini and colistin against mcr - 1 - carrying Enterobacteriaceae was determined using the broth dilution method and referring to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (Reference: CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 34th ed. Clinical and Laboratory Standards Institute (2024)). Subsequently, the present invention evaluated the synergistic effect of Ini and colistin in eliminating mcr - gene - carrying Enterobacteriaceae using checkerboard analysis (Reference: Xu, C. et al. Valnemulin restores colistin sensitivity against multidrug - resistant gram - negative pathogens. Communications Biology 7, 1122 (2024)). By calculating the fractional inhibitory concentration (FICI) index, the present invention plotted contour maps and heat maps to visually display the interaction of the combination of Ini and colistin. The FIC index indicates synergistic effects.

[0030] 3. Time - dependent killing assay

[0031] The synergistic antibacterial effect of Ini and colistin was determined by time - dependent killing assay. Enterobacteriaceae carrying the mcr gene in the exponential growth phase were diluted to 10 6CFU / ml. Then, bacterial cells were treated with sub-inhibitory concentrations of colistin, Ini, or a combination of both for 24 hours. Viable cells were counted at time points of 0, 3, 6, 18, and 24 hours, and each experiment was repeated three times. Log 10 CFU / mL vs. time curves were plotted using GraphPad Prism 9.0 (San Diego, CA, USA) to generate time-kill curves.

[0032] 4. Determination and visualization of biofilm formation

[0033] The synergistic effect of Ini and colistin in preventing biofilm formation was detected by crystal violet staining for colistin. A bacterial suspension of Escherichia coli P47 (OD 600nm = 0.1) was cultured with TSB medium containing sub-inhibitory concentrations of colistin, Ini, and their combination at 37 °C for 24, 48, and 72 hours. After incubation, the bacterial suspension in the wells was discarded and washed with PBS. The biofilm was fixed with methanol, and after discarding the methanol, the residual methanol was washed again with PBS. Subsequently, 0.1% crystal violet solution was added for staining. After discarding the staining solution, the well plate was slowly rinsed to avoid washing away the biofilm. After the 96-well plate was naturally dried, 95% ethanol was added and slowly shaken to dissolve the crystal violet. After the incubation, a Tecan Spark multimode microplate reader (Tecan Australia Pty Ltd) was used to quantify the biofilm quality. The effects of Ini and colistin were observed using a confocal laser scanning microscope (CLSM). An overnight culture was inoculated onto glass slides placed in 24-well cell culture plates (BeyoGold TM , China), and then treated with Ini, colistin, and their combination at 37 °C for 5 days. After the treatment, the cells adhered to the glass slides were washed and fixed, and then stained with PI for bacterial nucleic acids. The biofilm was imaged using CLSM (Leica, TCS SP8, Germany) with an excitation wavelength of 535 nm.

[0034] 5. Cell lines

[0035] RAW264.7 cells, Hela cells, and Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone) supplemented with 10% (v / v) fetal bovine serum (HyClone).

[0036] 6. Safety assessment

[0037] The cytotoxicity of Ini and colistin against mammalian cells was evaluated by MTT assay (Reference: Zeng, P. et al. Investigation of antibiofilm activity, antibacterial activity, and mechanistic studies of an amphiphilic peptide against Acinetobacter baumannii. Biochimica et Biophysica Acta (BBA)-Biomembranes 1863, 183600 (2021)). Briefly, Vero cells were cultured to a cell density of 8000 cells per well. Then, the cells were co-cultured with Ini, colistin, and their combination for 24 hours. According to the manufacturer's instructions, the MTT Cell Proliferation and Cytotoxicity Detection Kit (Beyotime, Shanghai, China) was used to evaluate cell viability. Finally, the absorbance was measured at a wavelength of 570 nm using a microplate reader to determine cell viability.

[0038] The toxicity of Ini was further evaluated using 5 - 8-week-old BALB / c mice. The mice were divided into four groups and were intravenously injected with Ini, colistin, and their combination, respectively. The control group was given the same volume of solvent. After seven days, the mice were euthanized and dissected, and the spleen, kidney, and liver were removed and immediately fixed with 4% paraformaldehyde phosphate buffer. Subsequently, the organs were embedded in paraffin, stained with hematoxylin and eosin (H&E), and finally, the organ sections were observed using an optical microscope to evaluate the histopathological changes of each organ.

[0039] 7. Intracellular bacterial load determination

[0040] A cell infection model was used to evaluate the efficacy of Ini and colistin in eliminating intracellular bacteria. Vero cells were seeded into 24-well plates at a density of 1×10 5 cells per well and cultured overnight at 37°C and 5% CO2. The cells were co-cultured with Escherichia coli P47 during the exponential growth phase at a multiplicity of infection (MOI) of 100:1. After 1 hour of culture, the supernatant was removed, and the cells were transferred to fresh medium containing Ini, colistin, and their combination and continued to be cultured. After 6 hours of culture, the cells were rinsed twice with sterile PBS, and then cultured with fresh medium supplemented with meropenem (8 μg / mL) to eliminate extracellular bacteria. After 15 minutes of incubation, the cells were rinsed twice again with sterile PBS, and the cells were lysed with 0.1% (v / v) Triton X-100 (Beyotime, China) to quantify the number of viable bacteria in the cells.

[0041] 8. In vitro cell adhesion assay

[0042] HeLa cells were seeded into 24-well plates at a density of 0.8 - 1×10 5 cells / well and cultured overnight at 37 °C in a 5% CO2 incubator. Escherichia coli P47 was cultured in fresh LB broth at a ratio of 1:100 for approximately 2 hours until the OD 600nm reached 0.5. Different concentrations of Ini and colistin were added to the cell medium. Then, the cells were co-cultured with Escherichia coli P47 at a multiplicity of infection (MOI) of 100:1 for 1 hour. After 1 hour of culture, the cells were washed three times with sterile PBS and lysed in 0.2% (v / v) Triton X-100 for 20 minutes. The viable bacteria were quantitatively analyzed using GraphPad Prism 9.0.

[0043] 9. In vitro cytokine measurement

[0044] The effect of Ini on cytokine production in RAW264.7 cells was evaluated using a cytokine assay (Reference: Harimoto, T. et al. A programmable encapsulation system improves delivery of therapeutic bacteria in mice. Nature Biotechnology 40, 1259 - 1269 (2022)). RAW264.7 cells were cultured to a density of 2×10 4 cells / well. The levels of cytokines such as TNF-α, IL-1β, and IL-10 in the cell culture supernatant were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Boster Biological Technology, China), and the operation steps were performed according to the manufacturer's instructions.

[0045] 10. Zebrafish infection model

[0046] The zebrafish infection model was used to evaluate the synergistic effect of Ini and colistin. Bacterial cells of Escherichia coli P80 in the exponential phase were washed and resuspended with sterile PBS. Adult zebrafish were divided into 6 groups (16 fish per group) and intraperitoneally injected with 0.1 mL of Escherichia coli P80 at a concentration of 3.0×10 9 CFU / ml. One hour after infection, different concentrations of Ini, colistin, and their combination were intraperitoneally injected into the zebrafish. Untreated zebrafish were only injected with the solvent as a control group.

[0047] 11. Mouse infection model

[0048] The synergistic effect of treating infections caused by Enterobacteriaceae bacteria carrying the mcr gene was further evaluated using a murine infection model (References: Cai, J. et al. Structural-Activity Relationship-Inspired the Discovery of Saturated Fatty Acids as Novel Colistin Enhancers. Advanced Science 10, 2302182 (2023). and Xu, C. et al. Imidazole Type Antifungal Drugs Are Effective Colistin Adjuvants That Resensitize Colistin-Resistant Enterobacteriaceae. Advanced Therapeutics 3, 2000084 (2020).). Briefly, BALB / c mice aged 5 - 8 weeks were randomly divided into 6 groups (8 mice per group). Subsequently, all mice were infected with the clinically isolated Escherichia coli P80 carrying the mcr gene at an infection dose of 3×10 8 CFU / ml to establish a septicemia infection model for further study. One hour after infection, the mice were treated with Ini, colistin, and their combination formulations respectively. All animal experiments were approved by the Animal Care and Use Committee of Zhejiang University, and the present invention strictly adhered to all relevant animal use ethical regulations.

[0049] 12. Scanning Electron Microscope (SEM) Detection

[0050] The cell morphology of Enterobacteriaceae bacteria carrying mcr during the treatment with Ini, colistin, and their combination formulations was observed using scanning electron microscopy imaging technology (References: Xu, C. et al. Bactericidal, anti-biofilm and anti-virulence activity of vitamin C against carbapenem-resistant hypervirulent Klebsiella pneumoniae. iScience 25, 103894 (2022)). After rinsing the bacterial cells twice with sterile PBS, they were fixed overnight with 2.5% glutaraldehyde. After fixation, the bacterial cells were centrifuged at 10000 rpm for 1 minute, and then dehydrated with 100% ethanol. Finally, the cell morphology was observed using a Hitachi SU8010 scanning electron microscope (Tokyo, Japan).

[0051] 13. Determination of Cell Membrane Permeability

[0052] The synergistic effect of Ini and colistin on bacterial membrane permeability was determined using SYTOX Green (Thermo Fisher Scientific, Waltham, USA) (Reference: Sochacki, K. A., Barns, K. J., Bucki, R. & Weisshaar, J. C. Real-Time Attack On Single Escherichia Coli Cells By The Human Antimicrobial Peptide Ll-37. Proceedings of the National Academy of Sciences of the United States of America 108, E77-81, doi:10.1073 / pnas.1101130108 (2011)). Briefly, Escherichia coli P47 in the exponential phase was treated with different concentrations of Ini, colistin, and their combination for 2 h. After treatment, the bacterial cells were collected by centrifugation and resuspended in sterile saline to an OD 600nm of 0.2. The bacterial suspension was stained with SYTOX Green at a final concentration of 1 μM for 10 min. The fluorescence intensity of each test group was detected using a Tecan Spark multimode microplate reader.

[0053] 14. Determination of β-galactosidase activity

[0054] In addition, the β-galactosidase activity assay was also used to evaluate the bacterial membrane permeability of Ini, colistin, and their combination during the treatment process (Reference: Cai, J. et al. Structural-Activity Relationship-Inspired the Discovery of Saturated Fatty Acids as Novel Colistin Enhancers. Adv Sci (Weinh) 10, e2302182, doi:10.1002 / advs.202302182 (2023)). Escherichia coli P47 cultured overnight was diluted 100-fold with fresh medium and then cultured for another 4 h. The bacterial cells were collected by centrifugation and resuspended in sterile PBS to an OD 600nm of 0.2. O-Nitrophenyl-β-D-galactopyranoside (ONPG) at a final concentration of 3 mM was added to the bacterial suspension, and then incubated at 37 °C for 1 h. Finally, the absorbance was measured at a wavelength of 420 nm to evaluate the β-galactosidase activity.

[0055] 15. Membrane fluidity measurement

[0056] The fluidity of the membrane was evaluated using the Laurdan staining method (Reference: Müller, A. et al. Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains. Proceedings of the National Academy of Sciences 113, E7077 - E7086 (2016)). The Escherichia coli P47 bacterial suspension in the exponential phase was centrifuged and resuspended with sterile PBS. Then it was stained with laurdan at a final concentration of 10 μM for 30 minutes at 37 °C in the dark. The stained cell culture was washed twice with PBS and then incubated with different concentrations of Ini, colistin, and their combination preparations at 37 °C for 2 hours. Laurdan GP was used to evaluate membrane fluidity, and its calculation formula is: GP = (I435 - I490) / (I435 + I490).

[0057] 16. Membrane depolarization

[0058] The bacterial membrane potential after treatment with Ini and colistin was measured using DiSC3(5) fluorescence staining Escherichia coli P47 cells in the exponential phase were treated with different concentrations of Ini, colistin, and their combinations for 2 hours. After washing with PBS buffer, they were resuspended with PBS containing 100 mM KCl at a final concentration to OD 600nm = 0.2, stained with DiSC3(5) at a final concentration of 1 μM, incubated at 37 °C in the dark, and the fluorescence level was measured using a Tecan Spark multimode microplate reader with an excitation wavelength of 610 nm and an emission wavelength of 660 nm

[0059] 17. Proton gradient (ΔpH)

[0060] The BCECF-AM staining method was used to evaluate the ΔpH of bacteria (Reference: Worthan, S. B. et al. Evolution of pH-sensitive transcription termination in Escherichia coli during adaptation to repeated long-term starvation. Proceedings of the National Academy of Sciences 121, e2405546121 (2024)). Escherichia coli P47 bacterial cells in the exponential phase were rinsed with PBS and then stained with BCECF-AM (Beyotime, Shanghai, China) at a final concentration of 10 μM for 30 minutes in the dark. After staining, the bacteria were treated with different concentrations of Ini, colistin, and their combination for 10 minutes. The fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 535 nm was monitored using a Tecan Spark multimode microplate reader.

[0061] 18. Intracellular adenosine triphosphate (ATP) level

[0062] The bacterial cells of Escherichia coli P47 were centrifuged and resuspended in PBS (OD 600nm = 0.5). After treatment with Ini, colistin, and their combination for 2 hours, the bacterial cells were centrifuged and collected for lysis. The ATP content in the supernatant was determined using an enhanced ATP detection kit (Beyotime, Shanghai, China) and a Tecan Spark multimode microplate reader (Reference: Jia, Y. et al. Melatonin prevents conjugative transfer of plasmid-mediated antibiotic resistance genes by disrupting proton motive force. Pharmacological Research 175, 105978 (2022)).

[0063] 19. Swimming assay

[0064] Escherichia coli P47 in the exponential phase was inoculated into semi-solid LB agar medium (0.3% agar) containing different concentrations of Ini, colistin, and their combination. After culturing at 37 °C for 24 hours, the formed swimming halos were imaged and their migration distances were measured. This method was used to evaluate the effects of Ini and colistin on the swimming ability of bacteria.

[0065] 20. Nile red efflux assay

[0066] Escherichia coli P47 bacterial cells in the stationary phase were centrifuged and resuspended in sterile PBS buffer containing 1 mM MgCl2 (OD 600nm = 1.0). The bacterial suspension was pre-cultured with CCCP and then treated with different concentrations of Ini, colistin, and their combinations. After treatment, the bacterial cells were stained with 5 μM Nile red for 1 hour. The fluorescence in the bacterial suspension was monitored for 7 minutes using a Tecan Spark multimode microplate reader (Reference: Bohnert, J.A., Karamian, B. & Nikaido, H. Optimized Nile Red efflux assay of AcrAB-TolC multidrug efflux system shows competition between substrates. Antimicrob Agents Chemother 54, 3770 - 3775, doi:10.1128 / AAC.00620-10 (2010).).

[0067] 21. Colistin quantification

[0068] Escherichia coli P47 bacterial cells were resuspended in sterile PBS containing 16 μg / ml colistin with or without Ini (OD 600nm = 0.6) and incubated at 37 °C for 30 minutes. The cells were harvested by centrifugation, washed three times with sterile PBS, and then resuspended in sterile physiological saline. 200 μl of glycine-hydrochloric acid buffer with a pH of 2.4 was added and incubated for 30 minutes to dissociate the colistin bound to the bacterial membrane. Subsequently, the bacterial cells were collected and lysed by sonication to release the intracellular colistin accumulated in the bacteria. The content of colistin was quantified using a colistin enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions.

[0069] 22. Reactive oxygen species (ROS) level

[0070] The intracellular ROS level was measured using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Reference: Xu, C. et al. Bactericidal, anti-biofilm, and anti-virulence activity of vitamin C against carbapenem-resistant hypervirulent Klebsiella pneumoniae. Iscience 25, 103894 (2022).). After staining the logarithmic-phase Escherichia coli P47 bacterial cells with 10 mM DCFH-DA at 37 °C for 30 minutes, they were washed three times and resuspended in sterile PBS containing Ini, colistin, and their complexes. The fluorescence intensity was detected using a Tecan Spark multimode microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0071] 23. Superoxide dismutase (SOD) activity

[0072] The intracellular SOD activity was measured using a WST-8 total superoxide dismutase detection kit (S0101, Beyotime, China). The overnight-cultured Escherichia coli P47 was diluted 100-fold and then cultured for another 4 hours. The bacterial cells were harvested and resuspended in sterile PBS (OD 600nm = 0.5). Then, they were treated with Ini, colistin, and their combinations for 2 hours. The intracellular SOD activity was measured according to the manufacturer's instructions.

[0073] 24. NAD + / NADH ratio quantification

[0074] The exponential-phase Escherichia coli P47 bacterial cells were cultured with Ini, colistin, and their complexes for 2 hours. After the culture, the bacterial cells were collected and resuspended in sterile PBS to OD 600nm = 0.2, and NAD + / NADH detection was performed using a NAD+ / NADH detection kit (Beyotime, China).

[0075] 25. Hydrogen peroxide content test

[0076] The Escherichia coli P47 bacterial culture was co-cultured with Ini, colistin, and their combinations until the OD 600nm reached 0.5. Subsequently, the culture was harvested and homogenized by ultrasound. The cell lysate was collected, and the hydrogen peroxide content was determined using a hydrogen peroxide detection kit (S0038, Beyotime, China) according to the manufacturer's instructions.

[0077] 26. Intracellular iron content determination

[0078] With and without Ini, Escherichia coli P47 was treated with colistin for 47 h, and the bacterial cultures were harvested and homogenized by sonication for 10 7 cells. Cell lysates were collected to determine intracellular iron content. Total iron and ferrous iron were quantified using a total iron colorimetric kit (E-BC-K139-M, Elabscience, USA) and a cellular ferrous iron colorimetric kit (E-BC-K881-M, Elabscience, USA), respectively.

[0079] 27. Transcriptome analysis

[0080] Transcriptome analysis was performed on Escherichia coli P47 exposed to 0.5 μg / mL colistin without Ini or with 2 μg / mL Ini (Reference: Chen, C., Cai, J., Shi, J., Wang, Z. & Liu, Y. Resensitizing multidrug-resistant Gram-negative bacteria to carbapenems and colistin using disulfiram. Communications Biology 6, 810 (2023)). Total RNA was extracted using a bacterial RNA extraction kit (Vazyme, Nanjing, China) and sent to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for sequencing on the Illumina Hiseq system. Bioinformatics analysis methods refer to: Xu, C. et al. Valnemulin restores colistin sensitivity against multidrug-resistant gram-negative pathogens. Communications Biology 7, 1122 (2024).

[0081] 28. Western Blot analysis

[0082] The expression of mcr-1 was evaluated in Escherichia coli BL21 cells transformed with the recombinant plasmid pET28a-mcr-1 (GenBank accession number: KY283125.1) (hereinafter referred to as BL21 / pMCR-1) (Reference: Ye, X. et al. Peptide MSI-1 inhibited MCR-1 and regulated outer membrane vesicles to combat immune evasion of Escherichia coli. Microbial Biotechnology 16, 1755-1773 (2023)). BL21 / pMCR-1 bacterial cells were cultured with different concentrations of Ini. After the culture was completed, the cells were lysed by sonication and centrifuged at 12,000 g for 2 minutes, and the supernatant rich in soluble MCR-1 protein was collected. The protein samples were loaded onto 10% or 12% SDS-PAGE gels for Western blotting. Enhanced chemiluminescence substrates were used for imaging, and quantification was performed using ImageJ software.

[0083] 29. Determination of LPS content

[0084] The LPS content was determined by the Limulus amebocyte lysate (LAL) test (Reference: Ma, Y. et al. Deletion of CD38 mitigates the severity of NEC in experimental settings by modulating macrophage-mediated inflammation. Redox Biology 77, 103336 (2024)). Escherichia coli P47 cultured overnight was diluted 100-fold and then cultured for another 4 hours. Bacterial cells were collected by centrifugation (5000 rpm, 5 minutes) and resuspended in PBS to an OD 600nm = 0.5. After treatment with different concentrations of Ini for 2 hours, the LPS content in the bacterial cells was determined using a chromogenic LAL endotoxin detection kit (C0276S, Beyotime, Shanghai, China) according to the manufacturer's instructions.

[0085] 30. Membrane charge

[0086] Membrane charge was measured by FITC-labeled poly-L-lysine (PLL) (Reference: Cai, J. et al. Structural-Activity Relationship-Inspired the Discovery of Saturated Fatty Acids as Novel Colistin Enhancers. Advanced Science 10, 2302182 (2023).). After incubation with different concentrations of Ini until the OD 600nm reached 0.5, bacterial cells were collected by centrifugation and washed twice with sterile saline. Subsequently, the cells were resuspended in an equal volume of Tris buffer (0.03 M, pH 8.0 Tris, 20% sucrose, 20 μg / ml FITC) and incubated in the dark for 10 minutes. Then, the cells were washed three times with Tris buffer (0.03 M, pH 8.0 Tris, 20% sucrose, without FITC). The fluorescence level of the samples was measured using a Tecan Spark multimode microplate reader at an excitation wavelength of 490 nm and an emission wavelength of 525 nm.

[0087] 31. Extraction and quantification of lipid A

[0088] Escherichia coli P47 was cultured in 200 ml of MH broth containing Ini (concentration range 0 - 8 μg / mL) until the OD 600nm reached 0.8 - 1.0. Cells were collected by centrifugation (5000 rpm, 5 minutes) and washed twice with sterile PBS. Then the cells were incubated in 100 ml of single-phase Bligh-Dyer mixture at room temperature for 20 minutes. The mixture was centrifuged (2000g, 20 minutes), and the pellet was washed twice with single-phase Bligh-Dyer mixture. The insoluble residue was then resuspended in 27 ml of sodium acetate buffer (pH 4.5) containing 1% SDS and boiled at 100 °C for 30 minutes. After adding 30 ml of chloroform and 30 ml of methanol to the mixture, it was centrifuged again (2000g, 20 minutes), and the lower phase was collected. The lower phase was washed twice with pre-equilibrated two-phase Bligh-Dyer upper phase and dried using a nitrogen dryer. The sample was finally dissolved in 100 μL of chloroform:methanol (9:1, v / v) for quantitative analysis. An equal volume of the lipid sample was mixed with 2,5-dihydroxybenzoic acid (Sigma-Aldrich, USA). The contents of lipid A and lipid A pEtN were determined using a Bruker Autoflex Speed MALDI system (Bruker, Germany) in negative ion mode. The modification of lipid A was quantified by calculating the peak intensity ratio of lipid A (m / z 1796.2) to lipid A pEtN (m / z 1919.2).

[0089] II. Statistical analysis

[0090] All data were from at least three independent repeated experiments and were expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9.0 software. P values were used to determine statistical significance, and the specific thresholds were: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For comparisons between two groups, an unpaired t-test was used; for multiple group comparisons, one-way analysis of variance (one-way ANOVA) was adopted. In the animal infection model, the Log-rank (Mantel-Cox) test was used to evaluate the significance of differences in overall survival rates.

[0091] III. Results

[0092] 1. Synergistic bactericidal effect of Ini and colistin in eliminating mcr-carrying Enterobacteriaceae bacteria in vitro and inhibiting biofilm formation

[0093] In the primary screening, checkerboard analysis was used to evaluate the synergistic effect of Ini and colistin in eradicating mcr-carrying Enterobacteriaceae bacteria (including Escherichia coli (n = 6), Enterobacter cloacae (n = 2), and Klebsiella pneumoniae (n = 2)). As shown in the contour plot and heat map, when Ini and colistin were used in combination, a synergistic bactericidal effect was observed, which was reflected in the fractional inhibitory concentration (FIC) index being lower than 0.5 ( Figure 1 A, 1B, 1C, and Figure 2 . The FIC indices of all strains are shown in Table 1 above.

[0094] Time-dependent bactericidal experiments were used to further evaluate the effect of the combination of Ini and colistin in treating mcr-carrying Enterobacteriaceae bacteria ( Figure 1 D - 1K). Within 24 hours of incubation, Ini alone could not inhibit the growth of most of the tested strains. For Escherichia coli 57TC, Klebsiella pneumoniae TER244, Enterobacter cloacae ECC-A6, and Enterobacter cloacae SXJ55, colistin alone inhibited the growth of bacteria within the first 6 hours, but the inhibitory effect weakened after 18 hours of extended treatment. In contrast, the combination of Ini and colistin significantly reduced the bacterial load by approximately 10 7 -fold within 24 hours of treatment, effectively eliminating almost all bacterial cells.

[0095] Since infections caused by Enterobacteriaceae bacteria such as Escherichia coli are usually associated with the formation of biofilms, inhibiting this process can improve the efficacy of antibiotics (References: U. & Balsalobre, C. Biofilm infections, their resilience to therapy and innovative treatment strategies. Journal of internal medicine 272, 541 - 561 (2012).

[0096] In this invention, the crystal violet staining method was used to study whether Ini could restore and enhance the inhibitory effect of colistin on biofilm formation ( Figure 1 L, 1M, and 1N). Using Ini alone at a sub - inhibitory concentration (4 μg / mL) significantly inhibited the biofilm formation of Escherichia coli P47. Compared with using colistin alone, adding 4 μg / mL of Ini significantly enhanced the inhibitory effect of colistin, and it was time - dependent. Similarly, using confocal laser scanning microscopy (CLSM) to image the bacterial biofilms after treatment with Ini, colistin, and their combination further verified the synergistic inhibitory effect. CLSM images showed that the combination could significantly eliminate the biofilm structure compared with using Ini or colistin alone ( Figure 1 O).

[0097] In summary, the results showed that Ini is a promising adjuvant that can restore and enhance the activity of colistin in vitro.

[0098] 2. Ini restored the bactericidal effect of colistin in mammalian cells and a mouse infection model

[0099] Considering that the role of Escherichia coli as a model organism in microbial research has been widely recognized, this invention selected Escherichia coli P47 as the model strain for further research. Given the synergistic bactericidal effect of Ini and colistin against mcr - carrying Enterobacteriaceae bacteria, this invention evaluated the efficacy of this combination against intracellular bacteria. First, this invention used Vero cells as a model to evaluate the cytotoxicity of Ini and colistin on mammalian cells. The results showed that more than 93% of Vero cells remained viable after treatment with Ini, colistin, and their combination, indicating that the cytotoxic effects of using Ini and colistin alone or their combination on mammalian cells were very small ( Figure 3 A). In addition, histopathological analysis showed that there were no obvious pathological changes in the liver, spleen, and kidney tissues of mice treated with Ini and colistin alone or their combination. These findings further confirmed that at the working concentration, monotherapy with Ini or colistin or their combination therapy would not be toxic to mammalian organs ( Figure 4 ).

[0100] In the cell infection model, the ability of Ini to clear intracellular bacteria was dose - dependent (Figure 3 B), while colistin at a concentration of 0.5 - 2 μg / mL has no obvious bactericidal effect. It is worth noting that, compared with using colistin alone, adding 4 μg / mL of Ini to colistin can significantly reduce the bacterial load by 100 - fold, indicating that combination therapy has better antibacterial effects against intracellular pathogens ( Figure 3 C).

[0101] Subsequently, the present invention explored the immunomodulatory properties of Ini because it has been previously reported that tetracycline drugs can down - regulate the production of LPS - induced cytokines. This property can reduce excessive inflammatory responses and help prevent tissue damage. After inducing an inflammatory response in RAW264.7 cells with LPS, the present invention treated the cells with different concentrations of Ini. The results showed that the production of pro - inflammatory cytokines tumor necrosis factor - α (TNF - α) and interleukin - 1β (IL - 1β) was inhibited, while the production of the anti - inflammatory cytokine interleukin - 10 (IL - 10) was stimulated ( Figure 3 D - 3F). This dual effect indicates that Ini can not only inhibit the excessive production of harmful pro - inflammatory mediators but also promote the release of protective anti - inflammatory factors. These immunomodulatory properties of Ini are crucial for reducing tissue damage associated with excessive inflammation and may bring benefits to the treatment of inflammatory diseases and bacterial infections.

[0102] The present invention further evaluated how Ini affects the efficacy of treating in - vivo infections caused by Escherichia coli carrying mcr. In a zebrafish infection model ( Figure 3 G, 3H), zebrafish were initially infected with 3×10 8 CFU / ml of Escherichia coli P80. Zebrafish that were not treated and those treated with colistin (1 mg / kg) or Ini (16 mg / kg) alone all died from the infection within 36 hours. In contrast, the combination therapy of Ini (16 mg / kg) and colistin (1 mg / kg) increased the survival rate and saved 25% of the animals 7 days after infection. In addition, compared with the survival rate of only 6.25% for colistin (4 mg / kg) monotherapy, the survival rate of the combination therapy of Ini and colistin was significantly increased to 56.25%. This obvious improvement indicates that in the zebrafish infection model, Ini can significantly restore the bactericidal effect of colistin (p = 0.0042).

[0103] To further evaluate the efficacy, the present invention constructed a mouse peritoneal infection model ( Figure 3 G, 3I). Mice were infected with 3×10 8After Escherichia coli P80 at CFU / ml, mice treated with Ini monotherapy (4 mg / kg) died within 12 hours after infection, while mice treated with colistin monotherapy (0.25 mg / kg) died within 24 hours after infection. In addition, the survival rate of colistin monotherapy (1 mg / kg) was only 12.5%. In contrast, the combination therapy of 4 mg / kg Ini and 1 mg / kg colistin significantly increased the survival rate to 62.5% (p = 0.0303), highlighting the higher efficacy of the combination therapy.

[0104] 3. Ini and colistin have a synergistic effect in disrupting the bacterial membrane

[0105] After demonstrating the synergistic effect of Ini and colistin in eliminating mcr-carrying Enterobacteriaceae in vitro and in vivo, the present invention then used scanning electron microscopy (SEM) to observe the morphological changes of Escherichia coli P47 after treatment with Ini and colistin ( Figure 5 A). When Escherichia coli P47 was treated with sub-inhibitory concentration of Ini (8 μg / mL) or colistin (1 μg / mL) alone, no obvious changes in bacterial morphology were observed. However, the simultaneous use of 8 μg / mL Ini and 1 μg / mL colistin led to complete disruption of the bacterial cell membrane, manifested as obvious visible shrinkage and rupture of the cell membrane. Most cells showed signs of complete rupture, resulting in leakage of cell fluid and ultimately cell death.

[0106] Previous studies have shown that the MCR enzyme present in Enterobacteriaceae carrying the mcr gene can promote specific modifications of lipid A in the cytoplasmic membrane. These alterations effectively reduce the ability of colistin to induce bacterial membrane permeation and disruption. In the present invention, the SYTOX Green staining method was used to preliminarily evaluate the permeability of the bacterial membrane after treatment with Ini and colistin. This fluorescent nucleic acid stain fluoresces more strongly when penetrating bacteria with permeable membranes and can serve as an effective indicator of membrane integrity. Compared with colistin alone, the addition of Ini significantly increased the intensity of SYTOX Green, indicating that Ini can significantly restore the membrane permeation effect of colistin ( Figure 5 B, 5C). Inner membrane permeability was also evaluated by measuring β-galactosidase activity because this enzyme can only cleave ONPG after the bacterial membrane is permeable. Compared with Ini or colistin alone, the combination of Ini and colistin increased the activity of β-galactosidase. In addition, the present invention also evaluated the changes in membrane fluidity after treatment with Ini and colistin ( Figure 5 D and 5E). After the combined use of Ini and colistin, the Laurdan generalized polarization (GP) value of the bacterial cell membrane decreased significantly, indicating that the presence of Ini can increase membrane fluidity. Figure 5F, 5G). Given that the overall increase in membrane fluidity may result from the dissipation of the bacterial membrane potential, and considering that several proton motive force dissipating agents (such as CCCP) have been found to synergistically eliminate bacteria with colistin, the present invention proposes the hypothesis that Ini may also have the function of a proton motive force dissipating agent.

[0107] 4. Ini dissipates the proton motive force of bacteria

[0108] The proton motive force (PMF) consists of two basic components: the transmembrane electrical potential difference (ΔΨ) and the transmembrane proton concentration gradient (ΔpH). Since this probe accumulates in polarized bacterial cells but is rapidly released into the culture medium, it fluoresces when ΔΨ is disrupted. As Figure 5 shown in Figures 5H and 5I, Ini has the ability to disrupt the bacterial ΔΨ and synergistically disrupts ΔΨ with colistin. In addition, the effect of the combined treatment of Ini and colistin on the bacterial ΔpH was evaluated using the pH-sensitive fluorescent probe BCECF-AM ( Figure 5 Figures 5J and 5K). After adding Ini, there was no significant change in the bacterial ΔpH whether colistin was present or not. These findings indicate that Ini neither affects ΔpH alone nor regulates ΔpH together with colistin. In summary, the research results of the present invention show that Ini acts as a proton motive force dissipating agent, enhancing the proton motive force dissipating effect of colistin.

[0109] Given that the proton motive force drives various cellular processes, including adenosine triphosphate (ATP) synthesis, active transport of molecules, bacterial motility, and rotation of bacterial flagella, four functional assays were used to indirectly verify the proton motive force dissipation caused by Ini and colistin. First, the present invention quantified the intracellular ATP levels of Escherichia coli P47 carrying mcr when treated with Ini and different concentrations of colistin (0 - 4 μg / mL) simultaneously. The data showed that after adding Ini, the intracellular ATP levels responded significantly less to colistin, indicating that Ini has a synergistic effect on the activity of colistin in inhibiting ATP synthesis ( Figure 5 Figures 5L and 5M). In the bacterial motility assay, Ini and colistin synergistically inhibited bacterial motility. The migration distance of bacteria was significantly reduced when treated with a combination of Ini (1 - 2 μg / mL) and colistin (1 μg / mL) compared to treatment with Ini alone or colistin alone ( Figure 6 ). Nile red is a substrate for various efflux pumps in bacteria. After inhibiting these efflux pumps, Nile red accumulates in bacterial cells, resulting in increased intracellular fluorescence. As Figure 5 shown in Figure 5O and Figure 5As shown in , Ini inhibited the activity of the efflux pump, indicating that it could also inhibit the efflux of colistin. This inhibitory effect led to an increase in the intracellular concentration of colistin, thereby enhancing its antibacterial activity. To further verify these findings, the present invention quantified the intracellular concentration of colistin in bacterial cells treated with different concentrations of Ini (0 to 8 μg / mL). The results showed that the accumulation of colistin in bacterial cells increased significantly.

[0110] In summary, the research results of the present invention show that Ini acts synergistically with colistin to effectively disrupt the proton motive force of bacteria. This disruption leads to severe membrane damage and ultimately results in the death of bacterial cells ( Figure 3 Q).

[0111] 5. Ini can inhibit the adhesion and invasion of macrophages

[0112] To elucidate the molecular mechanism of the synergistic bactericidal effect of Ini and colistin, the present invention performed transcriptomic analysis on Escherichia coli P47 after treatment with colistin alone or in combination with Ini and colistin. The results showed that when treated with Ini and colistin in combination, 507 differentially expressed genes (DEGs) were up-regulated and 457 differentially expressed genes were down-regulated respectively ( Figure 7 A). Gene Ontology (Go) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that the differentially expressed genes were mainly involved in bacterial flagellar assembly, cell motility, and chemotaxis ( Figure 7 B, Figure 7 7C). Specifically, as shown in Figure 7 E, the differentially expressed genes involved in these biological processes showed a high degree of similarity. Notably, almost all differentially expressed genes were down-regulated except for fdrA, yfiR, and dppA. This indicates that the combined treatment of colistin and Ini can inhibit flagellar formation and bacterial motility, which is consistent with the previously discovered synergistic inhibitory effect of Ini and colistin on bacterial swimming motility ( Figure 6 ). Flagella play a key role in multiple infection processes such as biofilm formation, adhesion, and invasion. As shown in Figure 7 F, the addition of 4 μg / mL Ini significantly reduced the number of intracellular bacterial cells, indicating that Ini can inhibit the adhesion and invasion of bacteria to mammalian cells.

[0113] 6. Ini interfered with the redox process and metabolism of bacteria

[0114] ​​The synergistic bactericidal effect of Ini and colistin prompted this invention to study whether adding Ini to colistin treatment would disrupt the redox process, as colistin generates ROS in Gram-negative bacteria. First, this invention measured the NAD + / NADH ratio regulated by the TCA cycle, as the acceleration of the TCA cycle promotes ROS production. Although 4 μg / mL Ini alone did not affect the NAD + / NADH ratio, its combination with colistin significantly decreased the NAD + / NADH ratio, indicating a synergistic effect between Ini and colistin, accelerating the TCA cycle and inducing ROS generation ([[]] Figure 8 A, 8B). Transcriptome analysis of Escherichia coli P47 treated with the combination of Ini and colistin compared to that treated with colistin alone also showed that among six differentially expressed genes related to the TCA cycle, five genes had upregulated expression levels, including fumB, fumC, sdhA, sdhB, sdhC, and sdhD. The downregulation of acnA might inhibit the translation of AcnA, and AcnA seems to be an enzyme that plays a role in maintaining or survival during the oxidative stress process. These results are consistent with the accelerated TCA cycle observed after adding Ini ( Figure 8 C). In this regard, adding Ini increased the ROS levels in untreated bacterial samples and those treated with colistin alone ( Figure 8 D, 8E). In addition, superoxide dismutase (SOD) is a metalloenzyme that catalyzes the dismutation of superoxide radicals into oxygen molecules and hydrogen peroxide, which can be removed by catalase 68 . Treatment with Ini alone and the combination of Ini and colistin both significantly inhibited the activity of SOD in bacteria and increased the level of hydrogen peroxide, indicating that treatment with Ini alone or in combination with colistin can promote the accumulation of ROS and induce oxidative stress damage in bacteria ( Figure 8 F, 8J, 8K, 8I).

[0115] In addition, it was observed that treatment with Ini alone or the combination of Ini and colistin decreased the intracellular Fe 2+ / Fe 3+ ratio ( Figure 8 J, 8K). Consistent with the redox imbalance caused by the Fenton reaction, the elevated hydrogen peroxide was converted into highly reactive hydroxyl radicals (-OH), during which ferrous iron was oxidized to iron.

[0116] In summary, adding Ini to colistin treatment can promote the production and accumulation of ROS, leading to oxidative damage to DNA, proteins, and bacterial cell membranes ( Figure 8 L).

[0117] 7. Ini inhibits the expression of mcr and the modification of lipid A

[0118] Since the expression of mcr can alter lipid A by adding phosphoethanolamine (pEtN) groups, resulting in reduced sensitivity to colicins, the present invention determined the expression level of mcr-1 after treatment with different concentrations of Ini. The results showed that Ini could significantly inhibit the expression level of mcr-1 in Escherichia coli BL21-MCR-1 in a dose-dependent manner. Therefore, Ini restored the bactericidal effect of colistin on this strain, with an FIC index value of 0.1875( Figure 9 A, 9B, Figure 10 ). Similarly, when Escherichia coli J53 was treated with Ini and colistin, an FIC index of 0.75 was observed, and no synergistic effect was detected( Figure 10 ). After demonstrating that the addition of Ini did not affect LPS synthesis( Figure 9 C), the present invention quantified the proportion of modified lipid A (lipid A-phosphoethanolamine). First, the present invention obtained the mass spectra of lipid A and lipid A-phosphoethanolamine using MALDI-TOF mass spectrometry and found that their masses were 1796.2 Da and 1919.2 Da, respectively( Figure 9 D). Then, the change in lipid A after the addition of Ini was quantified by calculating the peak intensity ratio of lipid A and lipid A-phosphoethanolamine. As expected, in the presence of Ini at a concentration of 4 to 8 μg / mL, the proportion of lipid A-phosphoethanolamine decreased significantly( Figure 9 E). Subsequently, after the addition of Ini, the colistin bound to the bacterial cell membrane increased significantly, indicating that the inhibitory effect of Ini on lipid A modification could restore the affinity between colistin and lipid A( Figure 9 F). In addition, the present invention also measured the membrane charge using FITC-labeled poly-L-lysine (PLL). After the addition of Ini, the fluorescence intensity increased, indicating that Ini increased the positive charge on the bacterial cell membrane( Figure 9 G). In summary, Ini inhibits the modification of lipid A by suppressing the expression of mcr, thereby restoring the binding of colistin to the outer membrane( Figure 9 H).

[0119] In summary, Ini restricts mcr expression, thereby inhibiting the modification of lipid A and promoting the entry of colistin into the bacterial cell membrane. In addition, Ini dissipates the PMF, which impairs the function of the PMF-driven efflux pump, leading to the accumulation of colistin within the membrane. The accumulated colistin induces oxidative stress and exacerbates membrane damage, ultimately resulting in cell death( Figure 11 ).

[0120] IV. Conclusion

[0121] In summary, the research of the present invention shows that in in vitro experiments and in vivo infection models (including zebrafish and mouse models), Ini can enhance the efficacy of colistin against mcr-carrying Enterobacteriaceae bacteria. Mechanistic studies have shown that Ini inhibits the modification of lipid A by inhibiting the expression of mcr, thereby promoting the uptake of colistin by the bacterial cell membrane. In addition, Ini can also dissipate the proton motive force and damage the proton motive force-dependent efflux pump, resulting in the accumulation of colistin in the membrane. Therefore, the accumulation of colistin induces oxidative stress and membrane damage, ultimately leading to cell death( Figure 11 ). The discovery of Ini as a new colistin adjuvant provides a promising treatment method for combating the increasingly serious infections caused by mcr-carrying Enterobacteriaceae bacteria.

[0122] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of incyclinide in combination with colistin in the preparation of bactericidal drugs for Enterobacteriaceae carrying mcr gene.

2. The use according to claim 1, characterized in that The Enterobacteriaceae bacteria include Escherichia coli P47 and Escherichia coli P80.

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