Application of combination of valnemulin and polymyxin in preparation of bactericide

Abstract

The invention discloses an application of combination of valnemulin and polymyxin in preparation of a bactericide. The valnemulin and the polymyxin cooperate to kill bacteria, the antibacterial activity of the polymyxin on gram-negative bacteria which are drug-resistant and sensitive to the polymyxin such as escherichia coli, klebsiella pneumoniae and acinetobacter pideri can be effectively recovered in vitro and in vivo, in addition, the valnemulin has no cytotoxicity to mammalian cells, and the antibacterial activity of the polymyxin on the gram-negative bacteria can be effectively recovered. The application of valnemulin effectively reduces the therapeutic dose of polymyxin, and reduces the side effect risk of polymyxin on mammals.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial drug development, and in particular relates to an application of valnemulin combined with polymyxin in the preparation of a bactericide. Background Art

[0002] As the foundation of modern medicine, the clinical use of antibiotics has increased human life expectancy and is essential for the prevention and treatment of postoperative infections caused by invasive surgery. However, the cumulative consumption of antibiotics has promoted the emergence and development of antibiotic resistance. To date, many mobile element-mediated antibiotic resistance (AMR) genes have been discovered and reported, even including resistance genes to "last line of defense" antibiotics such as carbapenems, polymyxins, tigecycline and other antibiotics. Bacteria have a significant tendency to transfer genetic elements within and between genera, leading to the emergence of multidrug resistance (MDR), extensive drug resistance (XDR) and even pan-drug resistance (PDR), which further limits the choice of clinical treatment. Colistin is a cationic polypeptide antibiotic, also known as polymyxin E. It is currently considered to be one of the last line of defense antibiotics because of its high efficiency and low resistance rate against severe infections caused by multidrug-resistant or even pan-resistant Gram-negative strains. The positively charged polymyxin residues bind to the negatively charged lipid A phosphate groups on the bacterial cell membrane lipopolysaccharide, resulting in cell membrane destruction and leakage of cell contents, ultimately leading to cell death. However, the clinical application of polymyxins is limited due to the adverse reactions of nephrotoxicity and neurotoxicity. In addition, the polymyxin resistance of Gram-negative bacteria is mainly related to chromosomal mutations in genes encoding the two-component system, such as phoPQ, pmrAB, and ccrAB, and mutations in the mgrB gene encoding lipid A phosphoethanolamine (pEtN) transferase, which catalyzes the transfer of phosphoethanolamine to the head gene of lipid A, resulting in a decrease in the affinity of polymyxins for lipid A.

[0003] In recent years, a new type of mobile gene mcr encoding pEtN transferase mediating polymyxin resistance has been reported at home and abroad. Moreover, there are more and more reports that the mcr gene coexists with multidrug-resistant strains, such as those carrying bla KPC ,bla NDM and bla VIM Gene-specific carbapenem-resistant Enterobacteriaceae have led to the emergence of true superbugs that are resistant to almost all known antibiotics.

[0004] Antibiotic adjuvants are compounds that have no or weak antimicrobial activity but can restore or enhance the activity of antibiotics against bacteria that exhibit an antibiotic-resistant phenotype. The development of adjuvants for polymyxins offers several advantages, including minimizing the dose of antibiotics used in treatment. This reduction is critical to curbing the emergence of antibiotic resistance, a key issue in contemporary medicine. In addition, it addresses the issue of drug toxicity, thereby improving the overall safety of antibiotic regimens. For example, several natural phytochemicals have been identified to have synergistic effects with polymyxins. Catechol flavonoids and kaempferol have been reported to enhance the activity of polymyxins against Gram-negative bacteria by disrupting iron homeostasis. In addition, melatonin can enhance the activity of polymyxins against MCR-producing polymyxin-resistant pathogens by promoting oxidative damage and inhibiting efflux pumps. Otilonium bromide, an anticonvulsant approved by the U.S. Food and Drug Administration (FDA), has been reported to enhance the activity of polymyxins against Gram-negative pathogens and their persisters by dissipating bacterial membrane proton motive force (PMF) and inhibiting efflux pumps. These findings suggest that the strategy of identifying polymyxin adjuvants from the U.S. Food and Drug Administration-approved library is safer and more cost-effective than developing new antibiotics. So far, there are no commercially available polymyxin adjuvants, so the development of potential polymyxin adjuvants remains urgent. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide an application of valnemulin in combination with polymyxin in the preparation of a fungicide.

[0006] The present invention is achieved by using valnemulin or a pharmaceutically acceptable salt thereof in combination with polymyxin in the preparation of a bactericide for polymyxin-resistant and -sensitive Gram-negative pathogens.

[0007] Preferably, the Gram-negative pathogens include Escherichia coli, Klebsiella pneumoniae and Acinetobacter pittii.

[0008] Preferably, when the concentration range of valnemulin is 2 μg / mL, the concentration range of polymyxin is 1-2 μg / mL.

[0009] The present invention overcomes the deficiencies of the prior art and provides an application of valnemulin in combination with polymyxin in the preparation of a bactericide. Valnemulin is a pleuromutilin antibiotic that has high antibacterial activity against mycoplasma and Pasteurella, but lacks antibacterial activity against bacteria of the Enterobacteriaceae family. Valnemulin has been widely used in the prevention and treatment of swine dysentery, colitis and ileitis. Studies have shown that valnemulin can bind to the 50S ribosomal subunit, thereby inhibiting protein synthesis. In addition, valnemulin has no cytotoxicity to mammalian cells within the range of 0 to 25 μg / mL, indicating that the clinical use of valnemulin within this dosage range is safe. The present invention found in vitro and in mouse infection models that valnemulin can synergistically kill polymyxin-resistant and sensitive Gram-negative pathogens, including Escherichia coli, Klebsiella pneumoniae and Acinetobacter pittii. In addition, the present invention further discloses the mechanism of the synergistic bactericidal effect.

[0010] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: the present invention uses valnemulin and polymyxin to synergistically kill bacteria, and can effectively restore the antibacterial activity of polymyxin against polymyxin-resistant and sensitive Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae and Acinetobacter pituitus both in vitro and in vivo; in addition, valnemulin has no cytotoxicity to mammalian cells, and the use of valnemulin effectively reduces the therapeutic dose of polymyxin and reduces the risk of side effects of polymyxin on mammals. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The results of valnemulin and polymyxin synergistically killing polymyxin-resistant strains and polymyxin-sensitive strains; (A) is a contour plot showing the synergistic effect curve of valnemulin and polymyxin in polymyxin-resistant strains and polymyxin-sensitive strains; (B) is a time-killing curve of polymyxin-resistant strains when valnemulin, polymyxin and the combination of the two are used; (C) is a curve showing the change of in vitro polymyxin resistance before and after adding valnemulin (Val is valnemulin hydrochloride; CT is polymyxin);

[0012] Figure 2 Checkerboard broth microdilution assay of valnemulin and colistin against colistin-resistant and -susceptible strains. The figure shows the average OD at 600 nm of biological replicates, where dark blue areas represent higher bacterial density.

[0013] Figure 3The results are as follows: (A) is the MTT assay to determine the cytotoxicity of valnemulin to HEK293T cells, and HEK293T cells were incubated in the presence of valnemulin, polymyxin or a combination of the two. (B) is the result of the determination of the amount of intracellular Escherichia coli P47 in Vero cells after treatment with valnemulin, polymyxin and their combination; (C) is the survival rate of mice infected with polymyxin-resistant clinical Escherichia coli P80 treated with valnemulin, polymyxin and their combination (n = 5 per group); the experiment was performed in triplicate, and the data are expressed as mean ± standard deviation. GraphPadPrism 9.0 was used for statistical analysis, and p values ​​(ns, not statistically significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001), unpaired t test between two groups;

[0014] Figure 4 The effect of valnemulin on the membrane permeability of polymyxin; (A) is the scanning electron microscope image of Escherichia coli P47 treated with valnemulin, polymyxin and the combination of the two; (B) is the bacterial membrane permeability of Escherichia coli P47 treated with valnemulin, polymyxin and the combination of the two using SYTOX Green staining method; the experiment was performed in triplicate, and the data are expressed as mean ± standard deviation. The statistical significance of polymyxin + valnemulin and polymyxin was analyzed by unpaired t test (* p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001); the statistical significance of polymyxin + valnemulin and valnemulin was determined by one-way ANOVA and is shown as #### p<0.0001;

[0015] Figure 5 The results of the study on the dissipation of bacterial proton motive force (PMF) by valnemulin; (A) is the DiSC3(5) staining method to measure the membrane potential of Escherichia coli P47 after the addition of valnemulin, polymyxin and their combination; (B) is the result of the determination of the intracellular ATP level of Escherichia coli P47 after the addition of valnemulin, polymyxin and their combination; (C) is the result of the bacterial flagellar motility test of Escherichia coli P47 inoculated on a semi-solid medium containing valnemulin, polymyxin and their combination; the experiment was repeated three times and the data are expressed as mean ± standard deviation. The unpaired t-test was used to analyze the statistical significance of polymyxin + valnemulin and polymyxin ( * p<0.05, ** p<0.01, ***p<0.001, **** p < 0.0001). The statistical significance of polymyxin + valnemulin versus valnemulin was determined by one-way ANOVA and is shown as #### p<0.0001;

[0016] Figure 6 is the transcriptomic analysis of Escherichia coli P47 under the combined action of polymyxin (1μg / mL) and valnemulin (2μg / mL) and polymyxin (1μg / mL); (A) is the volcano plot gene expression difference distribution diagram, the x-axis represents the difference in expression level (FC value), the y-axis represents the corresponding statistical value, and the adjusted p value <0.05 (Student's t test) is used as the cutoff value for significantly differentially expressed genes (DEGs); (B) is the GO (gene ontology) annotation analysis result of DEGs; (C) is the GO enrichment analysis result of up-regulated DEGs; (D) is the GO enrichment analysis result of down-regulated DEGs; (E) is some genes involved in membrane composition and cell motility.

[0017] Figure 7 This is a demonstration of the synergistic antibacterial mechanism of valnemulin and polymyxin against polymyxin-resistant bacteria; among them, the combined use of valnemulin and polymyxin causes enhanced bacterial membrane permeability① and cell membrane destruction②, leading to cytoplasmic leakage and metabolic disorders; valnemulin can also synergize with polymyxin to dissipate bacterial PMF③; the dissipation of PMF leads to a decrease in intracellular ATP levels④ and inhibition of flagella-dependent motility⑤; the decrease in intracellular ATP levels causes disturbances in bacterial metabolic homeostasis and even death⑥; the combined action of valnemulin and polymyxin can downregulate the expression of cell motility-related genes; inhibition of bacterial motility can inhibit the colonization and immune escape of pathogens, ultimately leading to cell death; Val and polymyxin jointly downregulate the expression of cell motility-related genes⑦; inhibition of bacterial motility can inhibit the colonization⑧ and immune escape of pathogens⑨, ultimately leading to cell death⑩. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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 intended to limit the present invention.

[0019] 1. Materials and Methods

[0020] 1. Bacterial strains and reagents

[0021] All strains used in the present invention are shown in Table 1 below.

[0022] Table 1

[0023]

[0024]

[0025] The strains were incubated in LB (Luria-Bertani) broth or on LB agar plates overnight at 37° C. Walnemulin (Meilunbio, Dalian, China) was dissolved in double distilled water and then diluted to different working concentrations in culture medium.

[0026] 2. Antimicrobial sensitivity test

[0027] The minimum inhibitory concentration (MIC) of valnemulin and colistin was determined by broth dilution method, and the results were analyzed according to the Clinical and Laboratory Standards Institute guidelines (reference: CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 33th ed. Clinical and Laboratory Standards Institute. 2023.). The checkerboard assay was used to further analyze the synergistic antibacterial effect of valnemulin and colistin (reference: Xu C, Chen K, Chan KF, Chan EWC, Guo X, Chow HY, et al. Imidazole Type Antifungal Drugs Are Effective Colistin Adjuvants That Resensitize Colistin-Resistant Enterobacteriaceae. Advanced Therapeutics. 2020; 3(9): 2000084.). Briefly, valnemulin and colistin were serially diluted in 100 μL of cation-adjusted MH broth (CAMHB) to prepare an 8 × 6 matrix. 6 CFU / mL of bacterial suspension was inoculated into broth containing different concentrations of valnemulin and colistin and incubated at 37°C for 16 h. TM The OD of the bacterial suspension was measured by an ELISA reader (PerkinElmer, Waltham, MA, USA). 600nm The fractional inhibition concentration index (FICI) was calculated as follows to plot the isotherm: FICI = FIC 沃尼妙林 +FIC 多黏菌素=(MIC of polymyxin combined with valnemulin) / (MIC of valnemulin alone)+(MIC of polymyxin combined with valnemulin) / (MIC of polymyxin alone). FICI ≤ 0.5 indicates synergy. All experiments were repeated three times. FIC valnemulin and FIC polymyxin were used as x- and y-coordinates to draw isotherms. FICI values ​​≤ 0.5 indicate synergy.

[0028] 3. Study on time-killing kinetics

[0029] After some modifications according to the previously reported method, the synergistic bactericidal effect of valnemulin and polymyxin was evaluated by contraction and time-dependent killing curves (reference: Liu, Y. et al. Metformin restorestetracyclines susceptibility against multidrug resistant bacteria. Advanced Science, 1902227 (2020).). Specifically, the overnight culture of polymyxin-resistant strains was diluted 100 times with 3 mL LB broth and then incubated at 37 ° C and 200 rpm for 2 h. The bacterial suspension was treated with different concentrations of valnemulin, polymyxin and their combination for 24 h. The viable counts at each time point were plotted using GraphPad Prism 9.0 (San Diego, CA, USA) Log 10 Time-kill curve of CFU / mL versus time. This experiment was performed with three biological replicates.

[0030] 4. Drug resistance induction experiment

[0031] The development of polymyxin resistance was evaluated according to a previously reported method (reference: Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, et al. A new antibiotic kills pathogens without detectable resistance. Nature. 2015; 517(7535): 455.). Briefly, polymyxin-sensitive Escherichia coli Bw25113, Klebsiella pneumoniae 80, and Acinetobacter pituitarius P87 were cultured for 10 days with subinhibitory levels of polymyxin treatment in the absence and presence of 2 μg / mL valnemulin to obtain resistant mutants.

[0032] 5. Scanning electron microscopy (SEM) analysis

[0033] Scanning electron microscopy images show the cell morphology of polymyxin-resistant Escherichia coli treated with valnemulin, polymyxin, and the combination of the two (reference: Xu C, Dong N, Chen K, Yang X, Zeng P, Hou C, et al. Bactericidal, anti-biofilm and anti-virulence activity of vitamin C against carbapenem-resistant hypervirulent Klebsiella pneumoniae. iScience. 2022; 25(3): 103894.). Briefly, E. coli P47 carrying mcr-1 was treated with valnemulin, polymyxin, or the combination of the two for 2 h in the late exponential phase. The cells were washed twice with sterile PBS and then fixed with 2.5% glutaraldehyde overnight. The fixed cells were centrifuged (10,000 rpm, 1 min) and dehydrated with 100% ethanol. The cell morphology was then observed using a Hitachi SU8010 scanning electron microscope (Tokyo, Japan).

[0034] 6. Cytotoxicity Assessment

[0035] The cytotoxicity of valnemulin and polymyxin on mammalian cells was studied using HEK293T cells (reference: Zeng P, Yi L, Xu J, Gao W, Xu C, Chen S, et al. Investigation of antibiofilm activity, antibacterial activity, and mechanistic studies of anamphiphilic peptide against Acinetobacterbaumannii. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2021; 1863 (6): 183600.). Briefly, HEK293T cells were cultured in a 37°C, 5% CO2 incubator until the density reached 6000-8000 cells per well in a 96-well microplate. After that, different concentrations of valnemulin, polymyxin, and a combination of the two were added to the culture medium and incubated for 24 hours. The culture medium without cells and drugs was used as a blank control. Cell viability was determined using the MTT cell proliferation and cytotoxicity assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions, and EnSight TM The absorbance at 570 nm was measured by a microplate reader.

[0036] 7. Intracellular Bacteria Assay

[0037] The synergistic bactericidal effect of valnemulin and polymyxin against intracellular bacteria was determined using a cell infection model and followed a previously described method with minor modifications (reference: Liu, Y. et al. Metformin restores stetracyclines susceptibility against multidrug resistant bacteria. Advanced Science. 2020; 1902227. Ernstsen CL, Login FH, Jensen HH, R, J, Nejsum LN. Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy. Journal of microbiological methods. 2017; 139: 37-44.). Briefly, Vero cells were infected with Escherichia coli P47 at an MOI of 100. The plates were centrifuged at 1000 rpm for 5 minutes and incubated at 37°C and 5% CO2 for 1 hour. Vero cells were cultured with valnemulin, polymyxin, and a combination of the two for 6 hours at 37°C and 5% CO2. Extracellular bacteria were removed with 2 μg / mL meropenem and washed twice with PBS. Subsequently, the cells were lysed with DMEM supplemented with 0.1% BSA and 0.1% Triton X-100, and the serially diluted lysates were plated on MH agar plates for CFU counting.

[0038] 8. Mouse infection model

[0039] The synergistic antibacterial effect of valnemulin and colistin in vivo was evaluated in a mouse infection model (reference: Xu C, Chen K, Chan KF, Chan EWC, Guo X, Chow HY, et al. Imidazole Type Antifungal Drugs Are Effective Colistin Adjuvants That Resensitize Colistin-Resistant Enterobacteriaceae. Advanced Therapeutics. 2020; 3(9): 2000084.). Male BALB / c mice aged 5 to 8 weeks, weighing 18 g, were purchased from the Experimental Animal Center of Soochow University. Briefly, the mice were divided into 4 groups (5 mice in each group), and all mice were immunosuppressed by cyclophosphamide treatment with 4.0×10 8 CFU / mL dose infection of clinical polymyxin-resistant and carbapenem-resistant Escherichia coli P80 was used to establish a sepsis infection model for subsequent studies. 1 hour after infection, 2 mg / kg of valnemulin, 4 mg / kg of polymyxin, and the combination of the two were given once every 12 hours for 48 hours. All animal experiments were approved by the Experimental Animal Ethics Committee of Jiangsu University.

[0040] 9. Cell membrane permeability detection

[0041] The synergistic effect of valnemulin and polymyxin on bacterial membrane permeability was determined using SYTOX Green (Thermo Fisher Scientific, Waltham, USA) (Reference: Sochacki KA, Barns KJ, Bucki R, Weisshaar JC. Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37. Proceedings of the National Academy of Sciences. 2011; 108(16): E77-E81.). Briefly, logarithmic phase Escherichia coli P47 was treated with different concentrations of valnemulin, polymyxin, and the combination of the two for 2 hours. After treatment, bacterial cells were collected by centrifugation, washed twice with sterile PBS, resuspended in PBS, and the OD was adjusted. 600 to 0.2. The bacterial suspension was stained with SYTOX Green at a final concentration of 1 μM and incubated in the dark for 10 min. TMThe fluorescence intensity of each group was measured by microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 523 nm.

[0042] 10. Membrane depolarization assay

[0043] The bacterial membrane potential was measured using the fluorescent probe DiSC3(5) (Thermo Fisher Scientific, Waltham, USA) (Reference: Te Winkel JD, Gray DA, Seistrup KH, Hamoen LW, Strahl H. Analysis of antimicrobial-triggered membrane depolarization using voltage sensitive dyes. Frontiers in cell and developmental biology. 2016: 29.). Overnight cultured Escherichia coli P47 was diluted 100-fold in 3 mL LB broth and incubated until the logarithmic phase. The bacterial cells were collected by centrifugation and resuspended in the same volume of sterile PBS. The bacterial suspension was labeled with DiSC3(5) at a final concentration of 1 μM and continuously shaken at 100 rpm for 5 min at 37°C in the dark. Different concentrations of valnemulin, polymyxin, and a combination of the two were added to the stained bacterial cells, and then EnSight TM The fluorescence intensity of each group was measured by a microplate reader.

[0044] 11. Determination of intracellular adenosine triphosphate (ATP)

[0045] The effects of valnemulin and polymyxin on intracellular ATP were determined using an enhanced ATP assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions with some modifications (reference: Jia Y, Yang B, Shi J, Fang D, Wang Z, Liu Y. Melatonin prevents conjugative transfer of pla smid-mediated antibiotic resistance genes by disrupting proton motive force. Pharmacological Research. 2022; 175: 105978.). Briefly, after 2 h of treatment with valnemulin, polymyxin, and the combination of the two, bacterial cells were lysed and the supernatant was taken to measure ATP levels using an EnSight microplate reader.

[0046] 12. Flagellar movement experiment

[0047] The effects of valnemulin and colistin on the swimming activity of Escherichia coli P47 were studied according to a previously reported method with slight modifications (reference: Lee JH, Kim YG, Ryu SY, Cho MH, Lee J. Ginkgolic acids and Ginkgo biloba extract inhibit Escherichia coli O157:H7 and Staphylococcus aureus biofilm formation. International Journal of Food Microbiology. 2014; 174: 47-55.). Briefly, Escherichia coli P47 was inoculated into LB medium supplemented with 0.3% agar and different concentrations of valnemulin, colistin or a combination of the two, and after incubation at 37°C for 48 h, the bacterial swimming circles were photographed.

[0048] 13. Transcriptome analysis

[0049] Transcriptomic analysis was performed on Escherichia coli P47 treated with 1 μg / mL polymyxin without or with 2 μg / mL valnemulin (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. 2023; 6(1): 810.). Briefly, an overnight culture of Escherichia coli P47 was diluted 100-fold with 3 mL of CAMHB, and then the bacteria were treated with valnemulin or valnemulin combined with polymyxin and incubated at 37°C until OD 600nmThe total RNA of the samples was extracted using a bacterial RNA extraction kit (Vazyme, Nanjing, China) and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. (Shanghai, China) for sequencing using the Illumina HiSeq system. Bioinformatics analysis was performed using the Majorbio cloud platform (www.majorbio.com). Differential expression analysis was performed using the DESeq2 package (reference: Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genomebiology. 2014; 15(12): 1-21.). Functional annotation was performed using the GO and KEGG databases (references: Consortium GO. The Gene Ontology (GO) database and informatics resource. Nucleic acids research. 2004; 32 (suppl_1): D258-D61. and references: Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic acids research. 2017; 45 (D1): D353-D61.).

[0050] 14. Statistical analysis

[0051] All data collected from at least three replicates are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 9.0, and p values ​​were calculated by unpaired t-test between two groups or one-way ANOVA ( * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

[0052] 2. Results

[0053] 1. Vulnemulin works synergistically with polymyxin to eliminate polymyxin-resistant and -susceptible isolates in vitro and slow the development of polymyxin resistance

[0054] In the present invention, a chessboard method was constructed to determine the synergistic bactericidal effect of valnemulin and polymyxin, and in vitro experiments were performed using polymyxin-resistant strains and polymyxin-sensitive strains ( Figure 1 A and Figure 2 ). Four clinical polymyxin-resistant strains were screened, including Escherichia coli P47 carrying mcr-1, Klebsiella pneumoniae P30 and 80 with mgrB mutations, and Acinetobacter pituitii CS17 with pmrC mutations. From the isobologram, it can be seen that valnemulin and colistin have a synergistic bactericidal effect on the four polymyxin-resistant strains, with FICIs of 0.312, ≤0.0625, ≤0.0938, and ≤0.0156, respectively. The effects of valnemulin and colistin on polymyxin-susceptible strains were tested using Escherichia coli J53, Klebsiella pneumoniae MGH78578, and Acinetobacter pituitii P873 strains. From the isobologram, it can be seen that valnemulin and colistin also have a synergistic effect in killing polymyxin-susceptible strains, with FICIs of 0.3125, ≤0.28125, and ≤0.375, respectively, but the bactericidal effect is lower than that on polymyxin-resistant strains.

[0055] In order to further evaluate the efficacy of the combined use of valnemulin and polymyxin against polymyxin-resistant strains, the present invention conducted a bactericidal kinetic test on the above three polymyxin-resistant strains, including Escherichia coli P47, Klebsiella pneumoniae P30 and Acinetobacter pituitus CS17, with 8 μg / mL, 64 μg / mL and 16 μg / mL polymyxin MICs, respectively ( Figure 1 B). Neither valnemulin nor polymyxin alone could inhibit the growth of the test strains, indicating that valnemulin at a concentration of 2 μg / mL or polymyxin at a concentration of 2 μg / mL (polymyxin concentration of 32 μg / mL for Klebsiella pneumoniae P30 and 16 μg / mL for Acinetobacter pituitus CS17) could effectively kill the strains within 24 hours, while the combined use of valnemulin and polymyxin could effectively kill almost all strains within 24 hours, reducing the bacterial load by about 10 9 times.

[0056] In order to explore the possibility of valnemulin in preventing the emergence and development of polymyxin resistance, the present invention used polymyxin-sensitive strains to conduct resistance induction experiments ( Figure 1 C). Although E. coli J53 did not develop polymyxin resistance either alone or in combination with valnemulin, serial passages of K. pneumoniae MGH78578 and A. pituitien P87 treated with subinhibitory concentrations of polymyxin resulted in rapid increases in polymyxin MICs by 64-fold and 16-fold, respectively. In contrast, addition of 2 μg / mL valnemulin resulted in 16-fold and 1-fold decreases in polymyxin MICs, respectively, suggesting that the combination of valnemulin and polymyxin may be a promising strategy to prevent the development of polymyxin resistance.

[0057] 2. Bactericidal effect of valnemulin combined with polymyxin in mammalian cells and mouse infection models

[0058] In view of the good utility of Escherichia coli as a model organism in microbiological research, we selected the strain Escherichia coli P47 for subsequent research. After observing the synergistic effect of valnemulin and polymyxin on polymyxin-resistant strains, the present invention further evaluated the bactericidal effect of combined treatment on intracellular bacteria in a cell infection model. First, the cytotoxicity of valnemulin and polymyxin on mammalian cells was evaluated by the MTT method. The results showed that after adding valnemulin and polymyxin and the combination of the two, the survival rate of HEK293T cells exceeded 94%, indicating that valnemulin and polymyxin had no significant cytotoxic effect on mammalian cells ( Figure 3 A). In the cell infection model, the number of bacteria in Vero cells treated with valnemulin, polymyxin, and their combination was compared by counting the number of viable bacteria ( Figure 3 B). Compared with the untreated group, 2 μg / mL valnemulin could not kill intracellular bacteria, and 2 μg / mL polymyxin slightly reduced the number of viable intracellular bacteria. However, the combination of 2 μg / mL valnemulin and 2 μg / mL polymyxin dramatically reduced the number of viable bacteria by 1000-fold, indicating that the combination had a significant antibacterial effect against intracellular pathogens.

[0059] The present invention uses a mouse sepsis infection model to further evaluate the in vivo bactericidal effect of the combined treatment of valnemulin and polymyxin ( Figure 3 C) 4×10 8 After mice were infected with 100 CFU / mL polymyxin-resistant Escherichia coli P80, all died within 36 h after treatment with saline, polymyxin (2 mg / kg), and valnemulin (2 mg / kg). The combined treatment of valnemulin and polymyxin significantly improved the survival rate of mice, rescuing 60% of the mice at 48 h after infection, indicating that valnemulin can effectively restore the bactericidal effect of polymyxin in vivo. These data emphasize that the combination of valnemulin and polymyxin may be a promising potential strategy to combat polymyxin-resistant pathogens.

[0060] 3. Vonilamide enhances the transmembrane activity of polymyxin, leading to membrane damage

[0061] After demonstrating that valnemulin can restore the bactericidal effect of polymyxin in vitro and in vivo, the present invention used scanning electron microscopy to visualize the morphology of Escherichia coli P47 during valnemulin and polymyxin treatment ( Figure 4A). No morphological changes were observed after treatment of E. coli P47 with subinhibitory concentrations of valnemulin (2 μg / mL) and colistin (2 μg / mL). However, when valnemulin and colistin were used in combination, wrinkling of the cell membrane was seen, suggesting that their combination led to leakage of cellular contents and ultimately cell death.

[0062] Previous studies have reported that the bactericidal effect of polymyxins depends on the specific binding to lipid A in lipopolysaccharide in the bacterial cell membrane, resulting in enhanced cell membrane permeability, leakage of cell contents, and ultimately cell death. At the same time, resistance to polymyxins mainly occurs on the basis of lipid A modification, thereby preventing the binding of polymyxins. Therefore, the present invention speculates that valnemulin may promote the damage of polymyxins to bacterial cell membranes by enhancing the membrane permeability activity of polymyxins. The SYTOX Green staining method was used to detect polymyxin-resistant strains Escherichia coli P47, Klebsiella pneumoniae P30 and Acinetobacter pituitarius CS17 to evaluate the synergistic effect of valnemulin and polymyxin on bacterial membrane permeability. Although valnemulin (8 μg / mL) alone cannot enhance bacterial cell membrane permeability, the combination of valnemulin (8 μg / mL) and polymyxin can significantly improve bacterial membrane permeability compared with polymyxin treatment alone ( Figure 4 B) These data suggest that valnemulin can restore the membrane permeabilization effect of colistin against colistin-resistant strains.

[0063] 4. Walnemulin dissipates bacterial proton motive force (PMF)

[0064] Previous studies have reported that PMF is a fundamental driving force for a variety of physiological functions and is an unprecedented antibacterial target on the bacterial cell membrane. Considering that polymyxin is a membrane-targeted antibacterial cyclic peptide, valnemulin may enhance the bactericidal effect of polymyxin by dissipating PMF. To verify this hypothesis, the present invention measured bacterial PMF using three different methods.

[0065] First, since PMF is maintained by pH homeostasis controlled by proton pumps and depends on membrane potential, the membrane potential of bacteria was determined using the membrane-permeable fluorescent probe DiSC3(5). DiSC3(5) can accumulate in untreated polarized E. coli P47 cells. After dissipation of the bacterial membrane potential, DiSC3(5) will be rapidly released into the culture medium, and a strong signal can be monitored by fluorescence. Figure 5As shown in A, compared with the treatment with 1-16 μg / mL polymyxin alone, the fluorescence intensity increased significantly after adding valnemulin (8 μg / mL) and polymyxin, indicating that valnemulin can enhance the activity of polymyxin in dissipating bacterial membrane potential. At the same time, the combination of polymyxin and valnemulin has a stronger depolarizing effect than valnemulin alone, further confirming that valnemulin and polymyxin have a synergistic effect in dissipating bacterial membrane potential.

[0066] Since PMF is the driving force for ATP synthesis, the present invention then evaluated the ATP level in E. coli P47 treated with valnemulin, polymyxin and their combination ( Figure 5 B). Although valnemulin (8 μg / mL) could not significantly reduce the ATP content of bacterial cells, the combination of valnemulin and polymyxin significantly reduced the intracellular ATP level compared with valnemulin or polymyxin alone, indicating that valnemulin and polymyxin have a synergistic inhibitory effect on ATP synthesis.

[0067] Subsequently, since PMF can also drive bacterial motility, the present invention performed a bacterial swimming motility assay. Figure 5 As shown in C, the migration distance of E. coli P47 did not change after being inoculated on a semi-solid medium containing valnemulin (2 μg / mL) or polymyxin (1 μg / mL). However, the combined use of valnemulin (2 μg / mL) and polymyxin (1 μg / mL) can effectively shorten the bacterial migration distance, indicating that valnemulin and polymyxin synergistically inhibit bacterial movement.

[0068] In order to confirm the synergistic bactericidal effect of polymyxin and PMF dissipant, the present invention verifies the synergistic bactericidal effect of PMF dissipant and polymyxin by measuring the MIC of the classic PMF interferor - carbonyl cyanide m-chlorophenylhydrazone (CCCP) and polymyxin against Escherichia coli P47 and Escherichia coli J53, as shown in Table 2 below.

[0069] Table 2

[0070]

[0071]

[0072] In the detection concentration range (MIC>16ug / mL), CCCP had no inhibitory effect on E. coli P47 and E. coli J53. The MIC of polymyxin decreased in a dose-dependent manner with the addition of CCCP. CCCP and polymyxin had a synergistic inhibitory effect on E. coli P47 and E. coli J53, with FICI <0.25 and ≤0.375, respectively.

[0073] Taken together, these findings suggest that valnemulin and colistin have a synergistic effect in dissipating PMF, thereby inhibiting multiple physiological functions such as ATP synthesis and bacterial motility, ultimately enhancing bactericidal and antibacterial activities.

[0074] 5. Vulnemulin-induced changes in gene expression

[0075] In order to reveal the molecular mechanism of the combined bactericidal effect of valnemulin and polymyxin, the present invention conducted transcriptomic analysis on Escherichia coli P47 treated with polymyxin alone and polymyxin combined with valnemulin to evaluate the changes in gene expression levels. Compared with polymyxin alone, 1545 genes were upregulated and 318 genes were downregulated in Escherichia coli P47 treated with valnemulin combined with polymyxin ( Figure 6 A). GO annotation analysis showed that these upregulated differentially expressed genes (DEGs) were related to biological processes (such as cell motility and metal ion homeostasis), cellular components (such as cell projections), and molecular functions (such as carbohydrate transmembrane transporter activity) ( Figure 6 B). GO enrichment analysis showed that these up-regulated DEGs were involved in cell membrane and transport, while the down-regulated DEGs were enriched in cell motility, especially flagella-dependent cell motility ( Figure 6 C, 6D). Specifically, genes related to membrane components were significantly upregulated after combined treatment. In particular, transcription of genes encoding efflux pumps was upregulated, including members of the ATP-binding cassette (ABC) superfamily, including yddA, malK, malF, malG, phnC, yciO, and malA. In addition, expression of genes from the major facilitator superfamily (MFS), such as ydiM, setC, ydjE, entS, ydiN, and genes belonging to the resistance-regulated division (RND) family (acrE, acrF) were also upregulated. The induction of the bacterial efflux machinery is a response to the synergistic stress imposed by the combination of polymyxin and valnemulin. The expression levels of flagellar biosynthesis proteins (fliT, fliS, flgN, fliQ, fliO, fliR, fliH, fliL, fliJ, and flgD) and flagellar structural proteins (flgB, flgA, yegR, fliN, fliM, fliD, flgK, flgL, fllie, flgG, flgH, flgC, fliF, flgF, flgE, and fliC) were significantly downregulated, which is consistent with the inhibitory effect of the combined use of valnemulin and polymyxin on bacterial cell motility ( Figure 6 E). Taken together, these data suggest that valnemulin restores the activity of colistin in membrane permeabilization and membrane potential dissipation and inhibits cell motility.

[0076] Discussion

[0077] Currently, the evolution of antibiotic resistance poses a certain threat to antibiotic defense, and the discovery of new antibiotic adjuvants provides a potential strategy to compensate for the ineffectiveness of antibiotics to meet clinical needs. Previous studies have reported that the combined use of polymyxin and adjuvants can effectively prolong the effect of polymyxin by restoring the bactericidal activity of polymyxin, thereby treating infections caused by polymyxin-resistant pathogens.

[0078] In the present invention, it is found that valnemulin and polymyxin act synergistically to dissipate the bacterial membrane potential, resulting in the inability to transport ions and carbohydrates. It is reported that polymyxin binds to lipopolysaccharide and replaces the magnesium and calcium ions that stabilize the membrane, resulting in increased membrane permeability and the uptake of polymyxin into the periplasm. Divalent cations (Mg 2+ and Ca 2+ ) with the head group of LPS depends on the electric field. Warnemulin is a PMF dissipator that disrupts the outer membrane potential of bacteria. The addition of warnemulin weakens the electrostatic interaction between divalent ions and LPS, resulting in increased disorder of chain stacking in lipids and thinning of the membrane. In addition, the weakening of divalent ion binding can enhance the binding of polymyxin to LPS. For this reason, warnemulin and polymyxin synergistically kill bacteria. Although warnemulin itself has a weak antibacterial effect, it can effectively restore the antibacterial activity of polymyxin against polymyxin-resistant and sensitive Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae and Acinetobacter pitei both in vitro and in vivo. It is worth noting that the strong nephrotoxicity and neurotoxicity mediated by polymyxin to mammalian cells are the main limiting factors for the clinical use of colistin. However, warnemulin is not cytotoxic to mammalian cells, so the application of warnemulin effectively reduces the therapeutic dose of polymyxin, thereby further reducing the risk of side effects of polymyxin to mammals.

[0079] Intracellular pathogens such as enteroinvasive Escherichia coli are able to invade host cells, which helps them escape the host immune defense or clearance of antimicrobial drugs and survive, leading to the establishment, persistence and spread of infection. In addition, intracellular bacteria contribute to the survival of tumor cells because tumor-resident bacteria play an important role in regulating and promoting tumor metastasis. Therefore, intracellular bacteria are considered to be a new potential therapeutic target for cancer treatment. In the present invention, the synergistic antibacterial effect of valnemulin and polymyxin against intracellular bacteria also shows the potential of combined use in cancer treatment.

[0080] In the mechanism study of the present invention, it was found that valnemulin can enhance the activity of polymyxin in penetrating bacterial cell membranes, ultimately destroying the bacterial cell membrane and leading to cell death. According to transcriptomic analysis, the upregulation of membrane-related genes may be to compensate for the attack of the combination of valnemulin and polymyxin. In addition to cell membrane damage, the PMF dissipation caused by the combination of valnemulin and polymyxin leads to a decrease in intracellular ATP levels. Considering that ATP is the main source of energy for almost all biological processes, ATP levels can be used to observe bacterial metabolic levels. The decrease in intracellular ATP levels caused by the combination of valnemulin and polymyxin can cause disturbances in bacterial metabolic homeostasis and even death.

[0081] In addition, the combination of valnemulin and polymyxin also inhibited bacterial cell motility. PMF and ATP are essential for the formation of flagella, which is crucial for bacterial motility. ATP is required for the initial step of flagellar protein transport, while PMF accelerates the transport process and drives flagellar rotation. At the same time, the expression levels of motility-related genes decreased, including genes related to flagellar assembly and genes related to flagellar-dependent clustering and flagellar motility. In summary, considering that flagella-mediated motility contributes to the virulence of strains by enabling them to colonize and evade host immune responses, the synergistic inhibition of bacterial motility by valnemulin and polymyxin may improve the therapeutic effect in clinical trials ( Figure 7 ).

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of valnemulin or a pharmaceutically acceptable salt thereof in combination with polymyxin in the preparation of a bactericide for polymyxin-resistant and -sensitive Gram-negative pathogens.

2. The use according to claim 1, characterized in that The Gram-negative pathogens include Escherichia coli, Klebsiella pneumoniae and Acinetobacter pittii.

3. The use according to claim 1, characterized in that When the concentration range of valnemulin is 2 μg / mL, the concentration range of polymyxin is 1-2 μg / mL.

Images