Use of valnemulin in combination with polymyxin in the preparation of a bactericide

Abstract

The present invention discloses the application of valnemulin in combination with polymyxin in the preparation of a bactericide. The present invention synergistically kills bacteria through valnemulin and polymyxin, 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 pittii in vitro and in vivo. In addition, valnemulin has no cytotoxicity to mammalian cells, and the application of valnemulin effectively reduces the treatment dose of polymyxin and reduces the risk of side effects of polymyxin on mammals.

Description

Technical Field

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

[0002] As the basis of modern medicine, the clinical use of antibiotics has increased human life expectancy and is crucial for preventing and treating postoperative infections caused by invasive surgeries. 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, including even resistance genes for "last resort" antibiotics such as carbapenems, polymyxins, tigecycline, etc. Bacteria have a significant tendency to transfer genetic elements within and between genera, leading to the emergence of multidrug resistance (MDR), extensively drug resistance (XDR), and even pandrug resistance (PDR), which further limits the options for clinical treatment. Colistin is a cationic polypeptide antibiotic, also known as polymyxin E, and is currently considered one of the last resort antibiotics due to its high efficacy and low resistance rate against severe infections caused by multidrug-resistant or even pandrug-resistant Gram-negative strains. The positively charged residues of polymyxin E bind to the negatively charged lipid A phosphate groups on the lipopolysaccharide of the bacterial cell membrane, resulting in cell membrane disruption and leakage of cell contents, ultimately leading to cell death. However, the clinical application of polymyxin E is limited due to adverse reactions such as nephrotoxicity and neurotoxicity. In addition, the emergence of polymyxin E resistance in Gram-negative bacteria is mainly related to chromosomal mutations in genes encoding two-component systems such as phoPQ , pmrAB and ccrAB as well as gene mutations in mgrB encoding lipid A phosphoethanolamine (pEtN) transferase, which catalyzes the transfer of phosphoethanolamine to the head group of lipid A, resulting in a reduced affinity of polymyxin E for lipid A.

[0003] In recent years, a novel mobile gene mcr encoding pEtN transferase-mediated polymyxin E resistance has been reported both at home and abroad. Moreover, more and more reports have shown that mcr genes coexist with multidrug-resistant strains, such as carbapenem-resistant Enterobacteriaceae carrying bla KPC , bla NDM and bla VIM genes, leading to the emergence of true superbugs that are resistant to almost all known antibiotics.

[0004] Antibiotic adjuvants are compounds with no or very weak antibacterial activity, but can restore or enhance the activity of antibiotics against bacteria exhibiting an antibiotic-resistant phenotype. The development of polymyxin E adjuvants offers several advantages, including minimizing the antibiotic dose used in treatment. This reduction is crucial for suppressing the emergence of antibiotic resistance, a key issue in contemporary medicine. In addition, it also addresses the problem of drug toxicity, thereby improving the overall safety of antibiotic treatment regimens. For example, several natural plant chemical constituents have been identified to have synergistic effects with polymyxin E. Catechol flavonoids and kaempferol have been reported to enhance the activity of polymyxin E against Gram-negative bacteria by disrupting iron homeostasis. In addition, melatonin can enhance the activity of polymyxin E against polymyxin E-producing mcr polymyxin E-resistant pathogens by promoting oxidative damage and inhibiting efflux pumps. Otilonium bromide has been reported to enhance the activity of polymyxin E against Gram-negative pathogens and their persister cells by dissipating the bacterial membrane proton motive force (PMF) and inhibiting efflux pumps. These findings suggest that the strategy of identifying polymyxin E adjuvants is safer and more cost-effective than developing new antibiotics. So far, there are no commercially available polymyxin E adjuvants, so there is still an urgent need to develop potential polymyxin E adjuvants. Summary of the Invention

[0005] To overcome the disadvantages and deficiencies of the prior art, the object of the present invention is to provide an application of valnemulin combined with polymyxin E in the preparation of a bactericide.

[0006] The present invention is realized as follows: an application of valnemulin or a pharmaceutically acceptable salt thereof combined with polymyxin E in the preparation of a bactericide against Gram-negative pathogenic bacteria resistant and sensitive to polymyxin E.

[0007] Preferably, the Gram-negative pathogenic bacteria include Escherichia coli ( Escherichia coli ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), and Acinetobacter pittii ( Acinetobacter pittii ).

[0008] Preferably, when the concentration range of valnemulin is 2 μg / mL, the concentration range of polymyxin E 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 E in the preparation of a bactericide. Valnemulin is a pleuromutilin antibiotic with high antibacterial activity against mycoplasma and Pasteurella, but lacks antibacterial activity against Enterobacteriaceae bacteria. 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 in the range of 0-25 μg / mL, indicating that valnemulin is safe for clinical use within this dose range. The present invention found in vitro and in a mouse infection model that valnemulin can synergistically kill polymyxin E-resistant and sensitive Gram-negative pathogenic bacteria with polymyxin E, including Escherichia coli ( Escherichia coli ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), and Acinetobacter pittii ( Acinetobacter pittii ). In addition, the present invention further discloses the mechanism of this synergistic bactericidal effect.

[0010] Compared with the disadvantages and deficiencies of the prior art, the present invention has the following beneficial effects: The present invention synergistically kills bacteria through valnemulin and polymyxin E, and can effectively restore the antibacterial activity of polymyxin E against polymyxin E-resistant and sensitive Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae and Acinetobacter pittii in vitro and in vivo. In addition, valnemulin has no cytotoxicity to mammalian cells, and the application of valnemulin effectively reduces the treatment dose of polymyxin E and reduces the risk of side effects of polymyxin E on mammals. Description of the Drawings

[0011] Figure 1 shows the results of the synergistic killing of polymyxin E-resistant strains and polymyxin E-sensitive strains by valnemulin and polymyxin E; among them, (A) is a contour map showing the synergistic action curves of valnemulin and polymyxin E in polymyxin E-resistant strains and polymyxin E-sensitive strains; (B) is the time-kill curve of polymyxin E-resistant strains when treated with valnemulin, polymyxin E and their combination; (C) is the change curve of in vitro polymyxin E resistance before and after adding valnemulin (Val is valnemulin hydrochloride; CT is polymyxin E);

[0012] Figure 2 is the checkerboard broth microdilution test of valnemulin and polymyxin E against polymyxin E-resistant and susceptible strains. The figure shows the average OD at 600 nm of biological replicates. Among them, the dark blue area represents a higher bacterial density;

[0013] Figure 3The bactericidal effects of valnemulin and polymyxin E in combination on mammalian cells and mouse infection models; among them, (A) is the cytotoxicity of valnemulin on HEK293T cells determined by the MTT method, and HEK293T cells were incubated in the presence of valnemulin, polymyxin E, or both in combination. (B) is the measurement result of the amount of intracellular Escherichia coli P47 after treatment of Vero cells with valnemulin, polymyxin E, and their combination; (C) is the survival rate of mice infected with polymyxin E-resistant clinical Escherichia coli P80 treated with valnemulin, polymyxin E, and their combination (n = 5 in each group); the experiment was performed in triplicate, and the data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9.0, and the p value was calculated (ns, not statistically significant, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001), and unpaired t tests were performed between the two groups;

[0014] Figure 4 The effect of valnemulin on the membrane permeability activity of polymyxin E; among them, (A) is the scanning electron microscopy imaging of Escherichia coli P47 treated with valnemulin, polymyxin E, and their combination; (B) is the measurement of the bacterial membrane permeability of Escherichia coli P47 treated with valnemulin, polymyxin E, and their combination using SYTOX Green staining; the experiment was performed in triplicate, and the data are expressed as mean ± standard deviation. Unpaired t tests were used to analyze the statistical significance between polymyxin E + valnemulin and polymyxin E （* p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001); the statistical significance between polymyxin E + valnemulin and valnemulin was determined by one-way ANOVA and shown as #### p <0.0001;

[0015] Figure 5Results of valnemulin dissipating the proton motive force (PMF) of bacteria; among them, (A) shows the membrane potential of Escherichia coli P47 after treatment with valnemulin, polymyxin E, and their combination determined by DiSC3(5) staining method; (B) shows the measurement results of intracellular ATP levels of Escherichia coli P47 after adding valnemulin, polymyxin E, and their combination; (C) shows the results of the bacterial flagellar motility test of Escherichia coli P47 inoculated on semi-solid medium containing valnemulin, polymyxin E, and their combination; The experiment was repeated three times biologically, and the data are expressed as mean ± standard deviation. Unpaired t t-tests were used to analyze the statistical significance between polymyxin E + valnemulin and polymyxin E ( * p p < 0.05, ** p p < 0.01, *** p p < 0.001, **** p p < 0.0001). The statistical significance between polymyxin E + valnemulin and valnemulin was determined by one-way ANOVA and shown as #### p p < 0.0001;

[0016] Figure 6 is the transcriptomic analysis of Escherichia coli P47 under the combined action of polymyxin E (1 μg / mL) and valnemulin (2 μg / mL) and polymyxin E (1 μg / mL); among them, (A) is the distribution map of gene expression differences in the volcano plot, the x-axis represents the difference in expression level (FC value), and the y-axis represents the corresponding statistical value. The adjusted p p-value < 0.05 (Student's t t-test) was used as the cut-off 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) are some genes involved in membrane components and cell motility.

[0017] Figure 7It is a demonstration of the synergistic antibacterial mechanism of valnemulin and colistin E against colistin E-resistant bacteria. Among them, the combined use of valnemulin and colistin E causes enhanced bacterial membrane permeability ① and cell membrane damage ②, resulting in cytoplasmic leakage and metabolic disorders; valnemulin can also synergistically dissipate the bacterial PMF ③ with colistin E; the dissipation of PMF leads to a decrease in intracellular ATP levels ④ and the inhibition of flagella-dependent motility ⑤; the decrease in intracellular ATP levels causes disorders and even death of bacterial metabolic homeostasis ⑥; the combined action of valnemulin and colistin E can down-regulate the expression of genes related to cell motility; the inhibition of bacterial motility can inhibit the colonization and immune escape of pathogenic bacteria, ultimately leading to cell death; the combined use of Val and colistin E down-regulates the expression of genes related to cell motility ⑦; the inhibition of bacterial motility can inhibit the colonization ⑧ and immune escape ⑨ of pathogens, ultimately leading to cell death ⑩. Detailed implementation manners

[0018] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, 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.

[0019] I. 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] The strains were incubated overnight at 37 °C in LB (Luria-Bertani) broth or on LB agar plates. Valnemulin (Meilunbio, Dalian, China) was dissolved in double-distilled water and then diluted to different working concentrations in the medium.

[0025] 2. Antibacterial drug susceptibility test

[0026] The broth dilution method was used to determine the minimum inhibitory concentration (MIC) of valnemulin and polymyxin E, and the results were analyzed with reference to the guidelines of the Clinical and Laboratory Standards Institute (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 polymyxin E (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 polymyxin E were serially diluted with 100 μL of cation-adjusted MH broth (CAMHB) to prepare an 8 × 6 matrix. Then, a bacterial suspension of 10 6 CFU / mL was inoculated into the broth containing different concentrations of valnemulin and polymyxin E and incubated at 37 °C for 16 h. The absorbance of the bacterial suspension at OD 600 nm was measured using an EnSight™ microplate reader (PerkinElmer, Waltham, MA, USA). The fractional inhibitory concentration index (FICI) was calculated as follows to plot the isobologram: FICI = FIC 沃尼妙林 + FIC 多黏菌素E = (MIC of polymyxin E combined with valnemulin) / (MIC of valnemulin alone) + (MIC of polymyxin E combined with valnemulin) / (MIC of polymyxin E alone). An FICI ≤ 0.5 indicates synergistic effect. All experiments were repeated three times. The FIC of valnemulin and the FIC of polymyxin E were plotted on the x-axis and y-axis, respectively, to draw the isobologram. An FICI value ≦ 0.5 indicates synergy.

[0027] 3. Time-kill kinetic study

[0028] After some modifications based on the previously reported method, the synergistic bactericidal effect of valnemulin and polymyxin E was evaluated by shrinkage and time-kill curves (Reference: Liu, Y. et al. Metformin restores tetracyclines susceptibility against multidrug resistant bacteria. Advanced Science, 1902227 (2020).). Specifically, the overnight culture of polymyxin E-resistant strains was diluted 100-fold with 3 mL of 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 E, and their combinations for 24 h. The viable cell count at each time point was used to plot the time-kill curve of log 10 CFU / mL over time using GraphPad Prism 9.0 (San Diego, CA, USA). This experiment was performed with three biological replicates.

[0029] 4. Resistance induction experiment

[0030] The development of polymyxin E resistance was evaluated according to the 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, sub-inhibitory levels of polymyxin E treatment were performed in the absence and presence of 2 μg / mL valnemulin, and Escherichia coli Bw25113, Klebsiella pneumoniae 80, and Acinetobacter pittii P87 sensitive to polymyxin E were passaged continuously for 10 days to obtain resistant mutants.

[0031] 5. Scanning electron microscopy (SEM) analysis

[0032] Scanning electron microscopy imaging showed the cell morphology of colistin E-resistant Escherichia coli under valnemulin, colistin E, and combined treatment of both (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, Escherichia coli P47 carrying mcr-1 was treated with valnemulin, colistin E, or a combination of both for 2 h at the late exponential phase. Washed twice with sterile PBS and then fixed overnight with 2.5% glutaraldehyde. The fixed cells were centrifuged (10,000 rpm, 1 min) and dehydrated with 100% ethanol. Then, the cell morphology was observed using a Hitachi SU8010 scanning electron microscope (Tokyo, Japan).

[0033] 6. Cytotoxicity assessment

[0034] Cytotoxicity studies of valnemulin and colistin E on mammalian cells were performed 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 an amphiphilic peptide against Acinetobacter baumannii. 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. Thereafter, different concentrations of valnemulin, colistin E, and their combination were added to the medium and incubated for 24 h. The medium without cells and drugs was used as a blank control. According to the manufacturer's instructions, the cell viability was measured using an MTT cell proliferation and cytotoxicity assay kit (Beyotime, Shanghai, China), and the absorbance at 570 nm was measured using an EnSight™ microplate reader.

[0035] 7. Intracellular Bacteria Assay

[0036] The synergistic bactericidal effect of valnemulin and colistin E against intracellular bacteria was determined using a cell infection model with minor modifications according to the method described previously (References: Liu, Y. et al. Metformin restores tetracyclines susceptibility against multidrug resistant bacteria. Advanced Science. 2020;1902227. Ernstsen CL, Login FH, Jensen HH, Nørregaard R, Møller-Jensen 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, colistin E, and their combination for 6 hours at 37 °C and 5% CO2. Extracellular bacteria were removed with 2 μg / mL meropenem and the cells were washed twice with PBS. Subsequently, the cells were lysed with DMEM supplemented with 0.1% BSA and 0.1% Triton X-100, and serial dilutions of the lysates were plated on MH agar plates for CFU counting.

[0037] 8. Mouse Infection Model

[0038] The in vivo synergistic antibacterial effect of valnemulin and polymyxin E was evaluated in a murine infection model (Reference: Xu C, Chen K, Chan KF, Chan EWC, Guo X, Chow HY, et al. Imidazole TypeAntifungal Drugs Are Effective Colistin Adjuvants That Resensitize Colistin‐Resistant Enterobacteriaceae. Advanced Therapeutics. 2020;3(9):2000084.). Male BALB / c mice, 5 - 8 weeks old and 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 and infected with clinically polymyxin E-resistant and carbapenem-resistant Escherichia coli P80 at a dose of 4.0×10 8 CFU / mL to establish a sepsis infection model for subsequent studies. One hour after infection, 2 mg / kg valnemulin, 4 mg / kg polymyxin E, and the combination of both were administered respectively, once every 12 h for 48 h. All animal experiments were approved by the Experimental Animal Ethics Committee of Jiangsu University.

[0039] 9. Detection of cell membrane permeability

[0040] The synergistic effect of valnemulin and polymyxin E 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 humanantimicrobial peptide LL-37. Proceedings of the National Academy of Sciences.2011;108(16):E77-E81.). Briefly, exponentially growing Escherichia coli P47 was treated with different concentrations of valnemulin, polymyxin E, and the combination of both for 2 h. After treatment, the bacterial cells were collected by centrifugation, washed twice with sterile PBS, resuspended in PBS, and the OD was adjusted 600To 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. The fluorescence intensity of each group was measured using an EnSight™ microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 523 nm.

[0041] 10. Membrane depolarization assay

[0042] 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). Escherichia coli P47 cultured overnight was diluted 100-fold in 3 mL of LB broth and incubated until the logarithmic phase. 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 under dark conditions at 37°C. Different concentrations of valnemulin, polymyxin E, and their combination were added to the stained bacterial cells, and then the fluorescence intensity of each group was measured using an EnSight™ microplate reader.

[0043] 11. Intracellular adenosine triphosphate (ATP) assay

[0044] According to the manufacturer's instructions with some modifications, an enhanced ATP assay kit (Beyotime, Shanghai, China) was used to determine the effects of valnemulin and polymyxin E on intracellular ATP (Reference: Jia Y, Yang B, Shi J, Fang D, Wang Z, Liu Y. Melatonin prevents conjugative transfer of plasmid-mediated antibiotic resistance genes by disrupting proton motive force. Pharmacological Research. 2022;175:105978.). Briefly, after treating with valnemulin, polymyxin E, and their combination for 2 h, bacterial cells were lysed, and the supernatant was used to measure the ATP level with an EnSight microplate reader.

[0045] 12. Flagellar motility assay

[0046] The effects of valnemulin and polymyxin E on the swimming of Escherichia coli P47 were investigated according to a previously reported method with minor modifications (Reference: Lee J-H, Kim Y-G, 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 their combination. After incubation at 37 °C for 48 h, the bacterial swimming circles were photographed.

[0047] 13. Transcriptome analysis

[0048] Transcriptomic analysis was performed on Escherichia coli P47 treated with 1 μg / mL polymyxin E 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 CAMHB, and then the bacteria were treated with valnemulin or a combination of valnemulin and polymyxin E and incubated at 37 °C until the OD 600nm reached 0.6. Total RNA of the samples was then extracted using a bacterial RNA extraction kit (Vazyme, Nanjing, China) and sent to Shanghai Majorbio Bio-pharm 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 carried out using the DESeq2 package (Reference: Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome biology. 2014;15(12):1-21.). Functional annotation was performed using the GO and KEGG databases (Reference: Consortium GO. The Gene Ontology (GO) database and informatics resource. Nucleic acids research. 2004;32(suppl_1):D258-D61. and Reference: 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.).

[0049] 14. Statistical analysis

[0050] All data collected from at least three replicates were expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 9.0, and calculated by unpaired t test or one-way ANOVA between two groups p values ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

[0051] II. Results

[0052] 1. Tiamulin and polymyxin E have a synergistic effect, eliminating polymyxin E-resistant and -sensitive isolates in vitro and slowing the development of polymyxin E resistance

[0053] In the examples of the present invention, a checkerboard method was constructed to determine the synergistic bactericidal effect of tiamulin and polymyxin E, and in vitro experiments were carried out using polymyxin E-resistant strains and polymyxin E-sensitive strains ( Figure 1 A and Figure 2 ). Four clinically polymyxin E-resistant strains were screened, including Escherichia coli P47 carrying mcr-1 , Klebsiella pneumoniae P30 and 80 with mgrB mutations, and Acinetobacter pittii CS17 with pmrC mutations. It can be seen from the isobologram that tiamulin and colistin have a synergistic bactericidal effect on 4 polymyxin E-resistant strains, and the FICIs are 0.312, ≤0.0625, ≤0.0938 and ≤0.0156 respectively. Three strains, Escherichia coli J53, Klebsiella pneumoniae MGH78578 and Acinetobacter pittii P87, were used to detect the effect of tiamulin and polymyxin E on polymyxin E-sensitive strains. It can be seen from the isobologram that tiamulin and polymyxin E also have a synergistic effect in killing polymyxin E-sensitive strains, and the FICIs are 0.3125, ≤0.28125 and ≤0.375 respectively, but the bactericidal effect is lower than that on polymyxin E-resistant strains.

[0054] To further evaluate the efficacy of the combination of tiamulin and polymyxin E against polymyxin E-resistant strains, the present invention performed bactericidal kinetic tests on the above three polymyxin E-resistant strains, including Escherichia coli P47, Klebsiella pneumoniae P30 and Acinetobacter pittii CS17, at 8 μg / mL, 64 μg / mL and 16 μg / mL polymyxin E MIC respectively ( Figure 1B). The growth of the test strains could not be inhibited by valnemulin or polymyxin E alone, indicating that neither a valnemulin concentration of 2 μg / mL nor a polymyxin E concentration of 2 μg / mL (32 μg / mL for Klebsiella pneumoniae P30 and 16 μg / mL for Acinetobacter pittii CS17) could effectively kill the strains within 24 h. However, the combination of valnemulin and polymyxin E could effectively kill almost all strains within 24 h, reducing the bacterial load by approximately 10 9 -fold.

[0055] To explore the possibility of valnemulin in preventing the emergence and development of polymyxin E resistance, the present invention conducted a resistance induction experiment using polymyxin E-sensitive strains ( Figure 1 C). Although Escherichia coli J53 did not develop polymyxin E resistance alone or in combination with valnemulin, serial passage of Klebsiella pneumoniae MGH78578 and Acinetobacter pittii P87 treated with sub-inhibitory concentrations of polymyxin E rapidly increased the polymyxin E MIC by 64-fold and 16-fold, respectively. In contrast, the addition of 2 μg / mL valnemulin reduced the polymyxin E MIC by 16-fold and 1-fold, respectively, indicating that the combination of valnemulin and polymyxin E may be a promising strategy for preventing the development of polymyxin E resistance.

[0056] 2. Bactericidal effects of the combination of valnemulin and polymyxin E on mammalian cells and mouse infection models

[0057] Given the good utility of Escherichia coli as a model organism in microbiological research, we selected the strain Escherichia coli P47 for subsequent studies. After observing the synergistic effect of valnemulin and polymyxin E on polymyxin E-resistant strains, the present invention further evaluated the bactericidal effect of combination therapy on intracellular bacteria in a cell infection model. First, the cytotoxicity of valnemulin and polymyxin E on mammalian cells was evaluated by the MTT method. The results showed that the viability of HEK293T cells exceeded 94% after the addition of valnemulin, polymyxin E, and their combination, indicating that valnemulin and polymyxin E had no significant cytotoxic effect on mammalian cells ( Figure 3 A). In the cell infection model, the number of bacteria in Vero cells was compared by counting the viable bacteria when treated with valnemulin, polymyxin E, and their combination ( Figure 3 B). Compared with the untreated group, 2 μg / mL valnemulin could not kill intracellular bacteria, and 2 μg / mL polymyxin E could slightly reduce the number of viable bacteria in cells. However, the combination of 2 μg / mL valnemulin and 2 μg / mL polymyxin E could reduce the number of viable bacteria by 1000-fold, indicating that the combination had a significant antibacterial effect on intracellular pathogens.

[0058] The present invention uses a murine sepsis infection model to further evaluate the in vivo bactericidal effect of the combination therapy of valnemulin and polymyxin E ( Figure 3 C). After mice were infected with 4 × 10 8 CFU / mL of polymyxin E-resistant Escherichia coli P80, all of them died within 36 h after treatment with normal saline, polymyxin E (2 mg / kg), and valnemulin (2 mg / kg). The combination therapy of valnemulin and polymyxin E can significantly improve the survival rate of mice, saving 60% of the mice 48 h after infection, indicating that valnemulin can effectively restore the bactericidal effect of polymyxin E in vivo. These data emphasize that the combined use of valnemulin and polymyxin E may be a promising potential strategy against polymyxin E-resistant pathogens.

[0059] 3. Valnemulin enhances the membrane-permeabilizing activity of polymyxin E, leading to membrane disruption

[0060] After demonstrating that valnemulin can restore the bactericidal effect of polymyxin E in vitro and in vivo, the present invention visualized the morphology of Escherichia coli P47 during the treatment with valnemulin and polymyxin E using a scanning electron microscope ( Figure 4 A). After treatment of Escherichia coli P47 with sub-inhibitory concentrations of valnemulin (2 μg / mL) and polymyxin E (2 μg / mL), no morphological changes were observed. However, when valnemulin and polymyxin E were used in combination, shrinkage of the cell membrane was seen, indicating that their combination led to leakage of cell contents and ultimately cell death.

[0061] Previous studies have reported that the bactericidal effect of polymyxin E depends on 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 polymyxin E mainly occurs on the basis of lipid A modification, which prevents the binding of polymyxin E. Therefore, the present invention speculates that valnemulin may promote the damage of polymyxin E to the bacterial cell membrane by enhancing the membrane-permeating activity of polymyxin E. The SYTOX Green staining method was used to detect polymyxin E-resistant strains Escherichia coli P47, Klebsiella pneumoniae P30, and Acinetobacter pittii CS17 to evaluate the synergistic effect of valnemulin and polymyxin E on bacterial membrane permeability. Although valnemulin alone (8 μg / mL) could not enhance bacterial cell membrane permeability, the combination of valnemulin (8 μg / mL) and polymyxin E significantly increased the permeability of the bacterial membrane compared with treatment with polymyxin E alone ( Figure 4 B). These data indicate that valnemulin can restore the membrane-permeabilizing effect of polymyxin E on polymyxin E-resistant strains.

[0062] 4. Valnemulin dissipates the bacterial proton motive force (PMF)

[0063] Previous studies have reported that PMF is a fundamental driving force for various physiological functions and is an unprecedented antibacterial target on the bacterial cell membrane. Considering that polymyxin E is a membrane-targeting antibacterial cyclic peptide, valnemulin may enhance the bactericidal effect of polymyxin E by dissipating PMF. To verify this hypothesis, the present invention measured bacterial PMF using three different methods.

[0064] First, since PMF is maintained by pH homeostasis controlled by proton pumps and depends on the membrane potential, the membrane-permeable fluorescent probe DiSC3(5) was used to measure the membrane potential of bacteria. DiSC3(5) can accumulate in untreated polarized Escherichia coli P47 cells. After dissipating the bacterial membrane potential, DiSC3(5) will be rapidly released into the culture medium, and the strong signal can be monitored by fluorescence. As Figure 5 shown in A, compared with treatment with polymyxin E alone at 1 - 16 μg / mL, the fluorescence intensity increased significantly after adding valnemulin (8 μg / mL) and polymyxin E, indicating that valnemulin can enhance the activity of polymyxin E in dissipating the bacterial membrane potential. At the same time, the combined use of polymyxin E and valnemulin had a stronger depolarizing effect than valnemulin alone, further confirming the synergistic effect of valnemulin and polymyxin E in dissipating the bacterial membrane potential.

[0065] Given that PMF is the driving force for ATP synthesis, the present invention next evaluated the ATP levels in Escherichia coli P47 treated with valnemulin, polymyxin E, and their combination ( Figure 5 B). Although valnemulin (8 μg / mL) could not significantly reduce the ATP content in bacterial cells, the combined use of valnemulin and polymyxin E significantly reduced the intracellular ATP level compared with treatment with valnemulin or polymyxin E alone, indicating that valnemulin and polymyxin E have a synergistic effect on inhibiting ATP synthesis.

[0066] Subsequently, since PMF can also drive bacterial motility, the present invention conducted a bacterial swimming motility assay. As Figure 5 shown in C, after inoculating Escherichia coli P47 on semi-solid medium containing valnemulin (2 μg / mL) or polymyxin E (1 μg / mL), the migration distance did not change. However, the combined use of valnemulin (2 μg / mL) and polymyxin E (1 μg / mL) effectively shortened the bacterial migration distance, indicating that valnemulin and polymyxin E synergistically inhibit bacterial motility.

[0067] To confirm the synergistic bactericidal effect of polymyxin E and the PMF dissipator, the present invention verified the synergistic bactericidal effect of the PMF dissipator and polymyxin E by measuring the MIC of the classical PMF interferent - carbonyl cyanide m-chlorophenylhydrazone (CCCP) and polymyxin E against Escherichia coli P47 and Escherichia coli J53, as shown in Table 2 below.

[0068] Table 2

[0069]

[0070] Within the tested concentration range (MIC > 16 ug / mL), CCCP had no antibacterial effect on Escherichia coli P47 and Escherichia coli J53. The MIC of polymyxin E decreased in a dose-dependent manner with the addition of CCCP. CCCP and colistin had a synergistic antibacterial effect on Escherichia coli P47 and Escherichia coli J53, with FICI values < 0.25 and ≤ 0.375, respectively.

[0071] In summary, these findings indicate that valnemulin and polymyxin E have a synergistic effect in dissipating PMF, thereby inhibiting various physiological functions such as ATP synthesis and bacterial motility, and ultimately enhancing the bactericidal and antibacterial activities.

[0072] 5. Gene expression changes induced by valnemulin

[0073] To reveal the molecular mechanism of the combined bactericidal effect of valnemulin and polymyxin E, the present invention performed transcriptomic analysis on Escherichia coli P47 treated with polymyxin E alone and in combination with valnemulin to evaluate changes in gene expression levels. Compared with treatment with polymyxin E alone, a total of 1545 genes were upregulated and 318 genes were downregulated in Escherichia coli P47 treated with the combination of valnemulin and polymyxin E ( 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 upregulated DEGs were involved in cell membrane and transport, while the downregulated 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 combination treatment. In particular, the 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 malAIn addition, the expression of genes from the major facilitator superfamily (MFS), such as ydiM , setC , ydjE , entS , ydiN , as well as genes belonging to the resistance-nodulation-cell division (RND) family ( acrE , acrF ), was also upregulated. The induction of the bacterial efflux mechanism was a response to the synergistic stress imposed by polymyxin E and valnemulin. The expression levels of flagellar biogenesis proteins ( fliT , fliS , flgN , fliQ , fliO , fliR , fliH , fliL , fliJ , flgD ) and flagellar structure proteins ( flgB , flgA , yegR , fliN , fliM , fliD , flgK , flgL , fllie , flgG , flgH , flgC , fliF , flgF , flgE , fliC ) were significantly downregulated, which was consistent with the inhibitory effect of the combination of valnemulin and polymyxin E on bacterial cell motility ( Figure 6 E). Collectively, these data indicate that valnemulin restores the activity of polymyxin E in membrane permeation and membrane potential dissipation and inhibits cell motility.

[0074] III. Discussion

[0075] Currently, the evolution of antibiotic resistance poses a 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 reported that the combination of polymyxin E and an adjuvant could effectively extend the effect of polymyxin E by restoring its bactericidal activity, thereby treating infections caused by polymyxin E-resistant pathogens.

[0076] In the present invention, it was found that valnemulin synergistically acts with polymyxin E to dissipate the bacterial membrane potential, resulting in the inability to transport ions and carbohydrates. It has been reported that polymyxin E binds to lipopolysaccharide and replaces the magnesium and calcium ions that stabilize the membrane, leading to increased membrane permeability and the uptake of polymyxin E into the periplasm. Divalent cations (Mg2+ and Ca 2+ (3) The strength of the electrostatic interaction between divalent cations and the LPS head group depends on the electric field. Valnemulin is a PMF dissipator that disrupts the bacterial outer membrane potential. The addition of valnemulin weakens the electrostatic interaction between divalent cations and lipopolysaccharide, leading to an increase in the disorder of the chain packing in the lipid and a thinning of the membrane. In addition, the weakened binding of divalent cations can enhance the binding of polymyxin E to LPS. Therefore, valnemulin and polymyxin E synergistically kill bacteria. Although valnemulin itself has weak antibacterial activity, it can effectively restore the antibacterial activity of polymyxin E against polymyxin E-resistant and -sensitive Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Acinetobacter pittii in vitro and in vivo. It is worth noting that the strong nephrotoxicity and neurotoxicity mediated by polymyxin E to mammalian cells are the main limiting factors for the clinical use of colistin. However, valnemulin has no cytotoxicity to mammalian cells. Therefore, the application of valnemulin effectively reduces the therapeutic dose of polymyxin E, thereby further reducing the risk of side effects of polymyxin E on mammals.

[0077] Intracellular pathogens such as enteropathogenic Escherichia coli can invade host cells, which helps them escape host immune defenses or clearance by antibacterial drugs and survive, leading to the establishment, persistence, and spread of infections. 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 a new potential therapeutic target for cancer treatment. In the present invention, the synergistic antibacterial effect of valnemulin and polymyxin E against intracellular bacteria also indicates the potential of the combination in cancer treatment.

[0078] In the mechanism study of the present invention, it was found that valnemulin can enhance the activity of polymyxin E in penetrating the bacterial cell membrane, ultimately disrupting 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 valnemulin and polymyxin E combination. In addition to cell membrane disruption, the PMF dissipation caused by the combination of valnemulin and polymyxin E leads to a decrease in intracellular ATP levels. Considering that ATP is the main energy source for almost all biological processes, ATP levels can be used to observe the bacterial metabolic level. The decrease in intracellular ATP levels caused by the combination of valnemulin and polymyxin E can cause disorder of bacterial metabolic homeostasis and even death.

[0079] In addition, the combined use of valnemulin and colistin E can also inhibit the cell motility of bacteria. PMF and ATP are essential for flagella formation, which is crucial for bacterial motility. ATP is required for the initial step of flagellin transport, while PMF accelerates the transport process and drives flagellar rotation. Meanwhile, the expression levels of motility-related genes decrease, including those related to flagella assembly and those related to flagella-dependent swarming and flagellar motility. In summary, considering that flagella-mediated motility contributes to virulence by enabling strains to colonize and evade host immune responses, the synergistic inhibitory effect of valnemulin and colistin E on bacterial motility may improve the therapeutic efficacy in clinical trials ( Figure 7 ).

[0080] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of valnemulin or a pharmaceutically acceptable salt thereof in combination with polymyxin E in the preparation of a bactericide for polymyxin E-resistant and -sensitive Gram-negative pathogens; the Gram-negative pathogens are Escherichia coli, Klebsiella pneumoniae and Acinetobacter pituitarius.

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