New use of chlorpromazine in enhancing the antibacterial effect of colistin

Abstract

The present application relates to the new use of chlorpromazine in preparing colistin bacterial antibacterial effect synergist. Colistin (COL) belongs to polymyxin antibiotics, and is a kind of unique non-ribosomal cyclic oligopeptide antibacterial drug. The chlorpromazine of the present application shows significant synergistic antibacterial effect when combined with colistin, and the FICI is 0.1533-0.3691, both less than 0.5, that is, the combination of the two shows significant synergistic antibacterial effect. The present application can restore the sensitivity of bacteria to colistin antibiotics, reduce the dosage of colistin, improve the antibacterial effect, reduce the toxic side effects, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel use of chlorpromazine in enhancing the antibacterial activity of colistin. Background Technology

[0002] Colistin (COL) belongs to the polymyxin class of antibiotics, a unique class of non-ribosomal cyclic oligopeptides. Due to its significant nephrotoxicity and neurotoxicity, the clinical application of COL was once severely limited and gradually decreased with the development of other antibacterial drugs. However, since the mid-to-late 1990s, with the widespread use of antibacterial drugs and the emergence of multidrug-resistant (MDR) Gram-negative bacteria, the number of effective antibacterial drugs available clinically has become increasingly limited. Because COL still possesses strong antibacterial activity against multiple MDR and extensively drug-resistant Gram-negative bacteria, its clinical value has been re-emphasized, and it has gradually become one of the important "last line of defense" drugs for treating severe drug-resistant Gram-negative bacterial infections. At the same time, the increased use of COL has also accelerated the emergence and spread of bacterial resistance, posing a serious threat to human and animal health. Therefore, how to improve the antibacterial efficacy of COL while reducing its dosage, toxicity, and resistance selection pressure has become an urgent problem to be solved. Combination therapy can improve the treatment effect of COL by enhancing antibacterial activity, reducing the effective dose and delaying the development of drug resistance. Therefore, it is considered one of the important strategies to restore or enhance the antibacterial activity of COL.

[0003] Chlorpromazine (CPZ) is a classic phenothiazine antipsychotic drug that exerts its pharmacological effects primarily by antagonizing central dopamine D2 receptors and reducing dopaminergic neurotransmission. Clinically, it is mainly used to treat mental illnesses such as schizophrenia. Its structural formula is shown below: Summary of the Invention

[0004] The purpose of this invention is to provide an application of chlorpromazine in the preparation of a colistin-based antibacterial synergist, wherein the ratio of chlorpromazine to colistin is 8:1, 16:1, 64:1, 256:1, or 1024:1. Preferably, the ratio of chlorpromazine to colistin is between 8:1 and 1024:1; more preferably, the ratio is between 8:1 and 256:1.

[0005] The present invention also provides an antimicrobial composition for enhancing the antimicrobial activity of colistin, the antimicrobial composition comprising chlorpromazine and colistin, wherein the ratio of chlorpromazine to colistin is between 8:1 and 1024:1. An antimicrobial composition for enhancing the antibacterial activity of colistin against Klebsiella pneumoniae, the antimicrobial composition comprising chlorpromazine and colistin, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1, 256:1, or 1024:1.

[0006] An antimicrobial composition for enhancing the activity of colistin against Salmonella and Klebsiella pneumoniae, the antimicrobial composition comprising chlorpromazine and colistin, wherein the ratio of chlorpromazine to colistin is between 8:1 and 256:1. An antimicrobial composition for enhancing the activity of colistin against Klebsiella pneumoniae, the antimicrobial composition comprising chlorpromazine and colistin, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1, 256:1, or 1024:1. Preferably, the ratio of chlorpromazine to colistin is between 64:1 and 1024:1.

[0007] An antimicrobial composition for enhancing the anti-Salmonella activity of colistin, the antimicrobial composition comprising chlorpromazine and colistin, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1 or 1024:1, preferably, the ratio of chlorpromazine to colistin is between 16:1 and 64:1.

[0008] The application of chlorpromazine in the preparation of a colistin synergist for the antibacterial activity of Salmonella and Klebsiella pneumoniae, wherein the ratio of chlorpromazine to colistin is 8:1, 16:1, 64:1, 256:1, 1024:1, 2048:1, 8195:1, 16368:1, or 533333:1. Preferably, the ratio of chlorpromazine to colistin is between 8:1 and 533333:1; preferably, the ratio is between 16:1 and 533333:1; preferably, the ratio is between 64:1 and 1024:1. Preferably, the ratio is between 1024:1 and 2048:1.

[0009] The application of chlorpromazine in the preparation of a colistin synergist against Salmonella and Klebsiella pneumoniae, wherein the Salmonella strains are F30, 11R, S12, and the Klebsiella pneumoniae strains are E4, 7G, CTR26, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1, 1024:1, 2048:1, 8195:1, 16368:1, or 533333:1. Preferably, the ratio of chlorpromazine to colistin is between 64:1 and 2048:1. More preferably, the ratio of chlorpromazine to colistin is between 1024:1 and 533333:1.

[0010] The application of chlorpromazine in the preparation of a colistin synergist against Salmonella and Klebsiella pneumoniae, wherein the Salmonella strains are F30, 11R, S12, and the Klebsiella pneumoniae strains are E4, 7G, CTR26, and the ratio of chlorpromazine to colistin is 8:1, 16:1, 64:1, 256:1, or 1024:1. Preferably, the ratio of chlorpromazine to colistin is between 16:1 and 1024:1. More preferably, the ratio of chlorpromazine to colistin is between 16:1 and 64:1.

[0011] The application of chlorpromazine in the preparation of a colistin-enhancing agent against Klebsiella pneumoniae, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1, 1024:1, 8195:1, 16368:1, or 533333:1. Preferably, the ratio of chlorpromazine to colistin is between 8195:1 and 533333:1.

[0012] The application of chlorpromazine in the preparation of a colistin-enhancing agent against Salmonella, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1, 1024:1, 2048:1, or 16368:1. Preferably, the ratio of chlorpromazine to colistin is between 64:1 and 2048:1. The application of chlorpromazine in the preparation of a colistin-enhancing agent against Salmonella, wherein the ratio of chlorpromazine to colistin is 8:1, 16:1, 64:1, 256:1, 1024:1, or 2048:1.

[0013] A novel application of chlorpromazine in enhancing the antibacterial effect of colistin against Klebsiella pneumoniae. Chlorpromazine is used to enhance the antibacterial effect of colistin, wherein the ratio of chlorpromazine to colistin is 256:1, 1024:1, 2048:1, 8195:1, 16368:1, or 533333:1. Preferably, the ratio of chlorpromazine to colistin is between 8195:1 and 533333:1. A novel use of chlorpromazine in enhancing the antibacterial effect of colistin, wherein the ratio of chlorpromazine to colistin is 8:1, 16:1, 64:1, 256:1, 1024:1, 2048:1, 8195:1, or 16368:1. Preferably, the ratio of chlorpromazine to colistin is between 64:1 and 256:1.

[0014] A novel application of chlorpromazine in enhancing the antibacterial activity of colistin against Salmonella and Klebsiella pneumoniae, wherein the ratio of chlorpromazine to colistin is between 16:1 and 256:1; preferably, the ratio is between 64:1 and 256:1; and more preferably, the ratio is between 256:1 and 8195:1. A novel application of chlorpromazine in enhancing the antibacterial activity of colistin, wherein chlorpromazine is used to enhance the antibacterial activity of colistin, and the ratio of chlorpromazine to colistin is 16:1, 64:1, 1024:1, or 2048:1. Preferably, the ratio of chlorpromazine to colistin is between 64:1 and 2048:1.

[0015] A novel use of chlorpromazine in enhancing the anti-Klebsiella pneumoniae activity of colistin, wherein the dosage of chlorpromazine is 32 μg / mL, and the dosage of colistin is 0.03125 μg / mL, 0.5 μg / mL, or 2 μg / mL; or the dosage of chlorpromazine is 64 μg / mL, and the dosage of colistin is 0.00781 μg / mL, 0.00012 μg / mL, or 0.00391 μg / mL.

[0016] The application of chlorpromazine in the preparation of a colistin-enhancing agent against Klebsiella pneumoniae, wherein the Klebsiella pneumoniae strain is E4, 7G, CTR26, wherein the dosage of chlorpromazine is 32 μg / mL, and the dosage of colistin is 0.03125 μg / mL, 0.5 μg / mL, or 2 μg / mL. Preferably, when the dosage of chlorpromazine is 32 μg / mL, the dosage of colistin is 0.03125-2 μg / mL.

[0017] The application of chlorpromazine in the preparation of a colistin-enhancing agent against Salmonella, wherein the Salmonella strains are F30, 11R, and S12, and the dosage of chlorpromazine is 32 μg / mL, and the dosage of colistin is 0.125 μg / mL, 2 μg / mL, or 4 μg / mL.

[0018] The application of chlorpromazine in the preparation of a colistin-based anti-Klebsiella pneumoniae synergist, wherein the Klebsiella pneumoniae strain is E4, 7G, CTR26, and the dosage of chlorpromazine is 64 μg / mL, and the dosage of colistin is 0.00012 μg / mL, 0.00391 μg / mL, or 0.00781 μg / mL. Preferably, when the dosage of chlorpromazine is 64 μg / mL, the dosage of colistin is 0.00012-0.00781 μg / mL.

[0019] The application of chlorpromazine in the preparation of colistin as an antibacterial synergist against Salmonella F30, 11R, and S12, wherein the dosage of chlorpromazine is 64 μg / mL, and the dosage of colistin is 0.03125 μg / mL, 0.0625 μg / mL, or 1 μg / mL.

[0020] The application of chlorpromazine in the preparation of a colistin-enhancing agent against Klebsiella pneumoniae E4, 7G, and CTR26, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1, 8195:1, or 533333:1. Preferably, the ratio of chlorpromazine to colistin is between 8195:1 and 533333:1. More preferably, the ratio of chlorpromazine to colistin is between 64:1 and 533333:1. The application of chlorpromazine in the preparation of a colistin-enhancing agent against Klebsiella pneumoniae E4, 7G, and CTR26, wherein the ratio of chlorpromazine to colistin is 16:1 or 64:1.

[0021] The use of chlorpromazine in the preparation of a compound formulation for enhancing the antibacterial activity of colistin against Klebsiella pneumoniae E4,7G,CTR26, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1, 256:1, 1024:1, 2048:1, 8195:1, 16368:1, or 533333:1. Preferably, the ratio of chlorpromazine to colistin is between 16368:1 and 533333:1.

[0022] The use of chlorpromazine in the preparation of a compound formulation for enhancing the anti-Salmonella F30, 11R, and S12 activity of colistin, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1, 256:1, 1024:1, or 2048:1. Preferably, the ratio of chlorpromazine to colistin is between 1024:1 and 2048:1. More preferably, the ratio of chlorpromazine to colistin is between 16:1 and 256:1.

[0023] The application of chlorpromazine in the preparation of a colistin synergist against Salmonella F30, 11R, and S12, wherein the ratio of chlorpromazine to colistin is 16:1, 64:1, 256:1, 1024:1, or 2048:1. Preferably, the ratio of chlorpromazine to colistin is between 16:1 and 2048:1. More preferably, the ratio is between 64:1 and 2048:1.

[0024] The application of chlorpromazine in the preparation of colistin as an antibacterial synergist against Salmonella F30, 11R, and S12: chlorpromazine was used at a dosage of 32 μg / mL, and colistin at dosages of 0.125 μg / mL, 2 μg / mL, or 4 μg / mL; chlorpromazine was used at a dosage of 64 μg / mL, and colistin at dosages of 0.03125 μg / mL, 0.0625 μg / mL, or 1 μg / mL.

[0025] The present invention also provides an antibacterial test method, or a target analyte test method, for the use of chlorpromazine in enhancing colistin against Salmonella, the method comprising the following steps: S1: Dissolve chlorpromazine and colistin in a solvent; S2: Chlorpromazine and colistin are mixed in a certain proportion to obtain a composition; S3: Use the composition from step S2 for MIC testing, FICI testing, time-sterilization curve testing, or target analyte testing.

[0026] The solvent mentioned in step S1 includes dimethyl sulfoxide or deionized water.

[0027] In step S2, the ratio of chlorpromazine to colistin is 64:1.

[0028] In this article, the term "synergistic effect" refers to the effect of two drugs used together being greater than the algebraic sum of the effects of the two drugs alone.

[0029] In this article, the term "additive effect" means that the combined effect of two drugs is equal to the algebraic sum of their individual effects.

[0030] In this article, the term "antagonistic effect" refers to the combined effect of two drugs being less than the sum of their individual effects.

[0031] In this article, the term "anti-Salmonella effect" refers to the inhibition of Salmonella proliferation or growth, or the killing of Salmonella.

[0032] In this article, the term "anti-Klebsiella pneumoniae effect" refers to inhibiting the proliferation or growth of Klebsiella pneumoniae, or killing Klebsiella pneumoniae.

[0033] The present invention has the following beneficial effects: 1. Chlorpromazine and colistin exhibit a significant synergistic effect when used in combination, with FICI values ​​ranging from 0.1533 to 0.3691, all less than 0.5, indicating a significant synergistic effect. 2. Chlorpromazine can significantly enhance the antibacterial activity of colistin against Klebsiella pneumoniae and Salmonella. 3. Combining chlorpromazine with colistin can effectively reduce the amount of colistin used. Compared with the MIC values ​​when colistin is used alone, when 32 μg / mL of chlorpromazine is added, the MIC of colistin against Salmonella and Klebsiella pneumoniae decreases by 8-2048 times; when 64 μg / mL of chlorpromazine is added, the MIC of colistin against Salmonella and Klebsiella pneumoniae decreases by 32-533333 times. Attached Figure Description

[0034] Figure 1 Growth curves of chlorpromazine and its combination with colistin. Detailed Implementation

[0035] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0036] Example 1: MIC Test Materials and methods 1. Drug testing Chlorpromazine (CPZ): Shanghai Beyotime Biotechnology Co., Ltd., Purity: >99% Colistin (COL): Dalian Meilun Biotechnology Co., Ltd., Purity: 88.7% Chlorpromazine was prepared to a concentration of 5000 μg / mL using deionized water. Colistin was prepared to a concentration of 2560 μg / mL using deionized water. The test drugs were stored at -20 ℃. Before the test, the drug stock solution was removed and allowed to thaw at room temperature, thoroughly mixed, and then pharmacodynamic tests were performed separately.

[0037] 2. Strains Clinically isolated Enterobacterial strains: Salmonella (F30, 11R, S12) and Klebsiella pneumoniae (E4, 7G, CTR26) were provided by the Pharmacology Laboratory of Henan Agricultural University. Salmonella was activated by streaking on SS medium, and Klebsiella pneumoniae was activated by streaking on MacConkey medium. Both were incubated at 37 ℃ for 16-18 h and stored at 4 ℃ for later use.

[0038] 3. Culture medium LB broth medium: Produced by Beijing Aoboxing Biotechnology Co., Ltd. Prepare 1000 mL according to the product instructions, autoclave at 121℃ for 30 minutes, and store at 4℃ for later use. SS and MacConkey broth media: Produced by Beijing Aoboxing Biotechnology Co., Ltd. Prepare 1000 mL according to the product instructions, heat to boiling to fully dissolve and sterilize, cool to 60℃, pour into sterile Petri dishes, and store at 4℃ for later use. MHB broth medium: Produced by Beijing Aoboxing Biotechnology Co., Ltd. Prepare 1000 mL according to the product instructions, autoclave at 121℃ for 30 minutes, and store at 4℃ for later use.

[0039] 4. Instruments High-pressure steam sterilizer (Shanghai Shenan Medical Instrument Factory); AB204-N electronic analytical balance (Mettler-Tolly Instruments Shanghai Co., Ltd.); HZQ-R air bath shaker (Beijing Liuyi Instrument Factory); DH-600 electric thermostatic incubator (Beijing Kewei Yongxing Instrument Co., Ltd.); DHG-9140A vacuum drying oven (Shanghai Yiheng Experimental Instrument Co., Ltd.); SW-CJ-JC clean bench (Suzhou Antai Air Technology Co., Ltd.).

[0040] 5. Preparation of bacterial culture Single colonies of Salmonella and Klebsiella pneumoniae were picked from MacConkey agar stored at 4 °C and inoculated into 5 mL of LB broth. The cultures were then activated by shaking at 180 rpm at 37 °C to induce the bacteria to enter the late exponential growth phase. The bacterial suspension was then diluted to 10⁵ CFU / mL in MHB broth. Chlorpromazine was added to the bacterial suspension at the appropriate concentrations for later use. The final concentrations of chlorpromazine in the bacterial suspensions were 32 μg / mL and 64 μg / mL, respectively.

[0041] 6. Preparation of Antimicrobial Sensitivity Plates Drug susceptibility testing was performed using the bacterial suspension from step 5. A sterile 96-well reaction plate was used. In well 1 of the first row, 108 μL of freshly prepared bacterial suspension containing chlorpromazine and 12 μL of 2560 μg / mL colistin were added; in wells 2-12, 60 μL of freshly prepared bacterial suspension containing chlorpromazine were added; in well 1 of the second row, 60 μL of freshly prepared bacterial suspension without chlorpromazine was added, and in well 2, 60 μL of MHB broth was added as a blank control. Wells 1-12 in the first row were serially diluted to achieve final colistin concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL, respectively. Well 1 in the second row contained drug-free MHB bacterial suspension as a positive control, and well 8 contained drug-free MHB broth as a negative control. The antimicrobial susceptibility plate was incubated at 37℃ for 16-18 h.

[0042] 7. Determination of MIC value After incubating the 96-well plates containing bacteria at 37 °C for 16–18 h, the MIC (minimum inhibitory concentration) results were read. The MIC value (minimum inhibitory concentration) of the drug is the drug concentration in the preceding clear well in the 96-well plate. All experiments were performed in triplicate. Only when the MIC values ​​could be accurately replicated was the experiment accepted; if the MIC values ​​differed by more than one concentration, the experiment needed to be repeated until the requirements were met. The results are shown in Table 1.

[0043] Table 1. MIC of chlorpromazine combined with colistin against tested bacterial strains

[0044] Note: 1 / 4 MIC chlorpromazine: chlorpromazine dosage is 32 μg / mL; 1 / 2 MIC chlorpromazine: chlorpromazine dosage is 64 μg / mL. The results showed that the MIC value of colistin against Salmonella (F30, 11R, S12) was 32 μg / mL, and the MIC values ​​against Klebsiella pneumoniae (E4, 7G, CTR26) were 32, 64, and 64 μg / mL, respectively. The MIC value of chlorpromazine (CPZ) against Salmonella (F30, 11R, S12) and Klebsiella pneumoniae (E4, 7G, CTR26) was 128 μg / mL. When 1 / 4 MIC (32 μg / mL) of chlorpromazine was added, the MIC values ​​of colistin against Salmonella (F30, 11R, S12) were 4, 2, and 0.125 μg / mL, respectively, and the MIC values ​​against Klebsiella pneumoniae (E4, 7G, CTR26) were 2, 0.5, and 0.03125 μg / mL, respectively. When 1 / 2 MIC (64 μg / mL) of CPZ was added, the MIC values ​​of colistin against Salmonella (F30, 11R, S12) were 1, 0.0625, and 0.03125 μg / mL, respectively, and the MIC values ​​against Klebsiella pneumoniae (E4, 7G, CTR26) were 0.00781, 0.00012, and 0.00391 μg / mL, respectively.

[0045] The results showed that when chlorpromazine was used to enhance the antibacterial effect of colistin, the ratio of chlorpromazine to colistin was 8:1, 16:1, 64:1, 256:1, 1024:1, 2048:1, 8195:1, 16368:1, or 533333:1, which enhanced the antibacterial effect of colistin.

[0046] Compared to the MIC values ​​when colistin was used alone, when 32 μg / mL of chlorpromazine was added, the MICs of colistin against Salmonella (F30, 11R, S12) decreased by 8, 16, and 256 times, respectively, and the MICs against Klebsiella pneumoniae (E4, 7G, CTR26) decreased by 16, 128, and 2048 times, respectively. When 64 μg / mL of chlorpromazine was added, the MICs of colistin against Salmonella (F30, 11R, S12) decreased by 32, 512, and 1024 times, respectively, and the MICs against Klebsiella pneumoniae (E4, 7G, CTR26) decreased by 4097, 533333, and 16368 times, respectively.

[0047] The results showed that chlorpromazine concentrations of 32 μg / mL and 64 μg / mL significantly enhanced the antibacterial activity of colistin against Salmonella (F30, 11R, S12) and Klebsiella pneumoniae (E4, 7G, CTR26).

[0048] Example 2: FICI Test Materials and methods 1. Drug testing: Same as Example 1.

[0049] 2. strain Same as Example 1.

[0050] 3. Culture medium Same as Example 1.

[0051] 4. Instruments Same as Example 1.

[0052] 5. Preparation of bacterial culture Same as Example 1.

[0053] 6. Preparation of Antimicrobial Sensitivity Plates The fractional inhibitory concentration index (FICI) of chlorpromazine and colistin combined with other drugs was determined using the checkerboard dilution method. Specifically, the bacterial culture was diluted to 1.0 × 10⁵ CFU / mL with MHB broth. CPZ was then diluted longitudinally to 8 wells, and COL was diluted laterally to 12 wells. The diluted CPZ (colistin) was then added to the diluted COL (colistin) in columns 1-12 and mixed. The mixture was incubated at 37 °C for 16-18 h, and the optical density (OD600 nm) was measured using a multi-mode microplate reader. The experiment was repeated three times. The fractional inhibitory concentration index (FICI) was used as the evaluation criterion for checkerboard drug susceptibility testing. The FICI values ​​of compounds A and B were defined by the following formula: FICI=FICA+FICB=MICAB / MICA+MICBA / MICB.

[0054] MICA is the MIC of compound A alone, MICAB is the MIC of compounds A and B, MICB is the MIC of compound B, and MICBA is the MIC of compounds B and A. FICI ≤ 0.5 indicates a synergistic effect; 0.5 < FICI ≤ 1 indicates an additive effect; 1 < FICI ≤ 2 indicates an unrelated effect; and FICI > 2 indicates an antagonistic effect.

[0055] Table 2. Combined antibacterial effects of chlorpromazine and colistin

[0056] The results showed that chlorpromazine alone had a weak inhibitory effect on the tested strains, but when used in combination with colistin, it showed a synergistic effect on colistin. The FICI index was 0.1533-0.3691, all <0.5, indicating that the combination of the two showed a synergistic antibacterial effect.

[0057] Example 3: Growth Curve Measurement Materials and methods 1. Drug testing: Same as Example 1.

[0058] 2. strain Same as Example 1.

[0059] 3. Culture medium Same as Example 1.

[0060] 4. Instruments Same as Example 1.

[0061] 5. Preparation of bacterial culture Same as Example 1.

[0062] 6. Preparation of Antimicrobial Sensitivity Plates Klebsiella pneumoniae E4 culture was incubated overnight, and the bacterial suspension was transferred to fresh sterile LB broth at a 1:100 ratio. Chlorpromazine was then added to achieve a final concentration of 64 μg / mL. A combined group of 1 / 2 MIC COL and 64 μg / mL chlorpromazine was also prepared. The mixture was added to 24-well cell culture plates. The bacterial growth curve was monitored using a multi-mode microplate reader at 37°C, with measurements taken every 1 hour for 15 hours. Each sample was tested in triplicate. Results are shown below. Figure 1 As shown.

[0063] Growth curve analysis showed that after treatment with 64 μg / mL chlorpromazine alone, E4 strains maintained normal growth, entering the logarithmic growth phase at approximately 2 hours post-culture and gradually reaching the stationary phase at 8 hours. In contrast, the growth of the strain was significantly inhibited after the combined use of colistin and chlorpromazine.

[0064] Example 4: Determination of time-based sterilization curve Materials and methods 1. Drug testing: Same as Example 1.

[0065] 2. strain Same as Example 1.

[0066] 3. Culture medium Same as Example 1.

[0067] 4. Instruments Same as Example 1.

[0068] 5. Preparation of bacterial culture Same as Example 1.

[0069] 6. Time-sterilization curve Klebsiella pneumoniae E4 was selected for time-kill curve testing. Single colonies were inoculated into fresh LB broth and incubated overnight at 37 ℃ and 180 r / min using a shaker. The bacterial culture was then transferred 1:100 to 20 mL of fresh MHB broth, resulting in four experimental groups. Group A: Control; Group B: 1 / 2 MIC COL; Group C: 64 μg / mL CPZ; Group D: 1 / 2 MIC COL + 64 μg / mL CPZ.

[0070] After grouping, the cells were incubated at 37 ℃ with shaking at 180 r / min. At 0 h, 2 h, 4 h, 8 h, and 12 h, 100 μL of bacterial suspension was taken and serially diluted 10-fold with PBS buffer. 100 μL of the diluted solution was then spread on LB agar plates and incubated at 37 ℃ for 16–18 h. Finally, the colonies on the agar plates were counted. A time-sterilization curve was plotted with time on the x-axis and log10 CFU / mL on the y-axis. The experiment was repeated three times.

[0071] Result Interpretation: Based on the synergistic effect criteria in time-bacterial kinetics experiments, when the viable bacterial count in the combined treatment group decreased by more than or equal to 2 log [amount missing] compared to the single-drug treatment group, the result was considered synergistic. 10 When the concentration is CFU / mL, it is considered to have a synergistic bactericidal effect.

[0072] This study systematically evaluated the antibacterial activity of colistin (COL) and chlorpromazine (CPZ) alone and in combination against Klebsiella pneumoniae E4 using time-knockout kinetic experiments. The results showed that neither 1 / 2 MIC COL nor 64 μg / mL CPZ treatments alone exhibited significant inhibitory or bactericidal effects, and their viable bacterial count trends were largely consistent with the control group. However, compared to single-drug treatments, the combined treatment with COL showed a significant decrease in viable bacterial count from the initial treatment stage, with the combined treatment group showing a reduction of approximately 4.8 log [missing value] compared to the single-drug treatment group. 10 The concentration of CFU / mL showed a synergistic bactericidal effect, indicating that CPZ can significantly enhance the bactericidal activity of COL against drug-resistant Klebsiella pneumoniae E4.

[0073] This study, using a checkerboard dilution assay, showed that the combined use of chlorpromazine (CPZ) and colistin (COL) exhibited a significant synergistic antibacterial effect, with a FICI range of 0.1533–0.3691, all less than 0.5. Growth curve results indicated that 64 μg / mL CPZ alone had no significant inhibitory effect on the overall growth of Klebsiella pneumoniae E4, while its combination with COL significantly inhibited bacterial growth and reproduction. Time-kinetic analysis further confirmed that the combined treatment of 1 / 2 MIC COL and 64 μg / mL CPZ had a significant in vitro synergistic bactericidal effect. Neither 1 / 2 MIC COL nor 64 μg / mL CPZ alone showed significant antibacterial or bactericidal activity, but the combination significantly reduced the number of viable bacteria, decreasing the viable bacterial load by approximately 4.8 log compared to single-drug treatment. 10The concentration of CFU / mL reached the synergistic bactericidal threshold and remained at a low level throughout the detection period. Furthermore, combined drug susceptibility analysis showed that the combination of 1 / 4 MIC or 1 / 2 MIC CPZ with COL significantly reduced the MIC value of COL against drug-resistant bacteria, further supporting the synergistic effect of CPZ on the antibacterial activity of COL.

[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of chlorpromazine in the preparation of a colistin-enhancing agent against Salmonella and Klebsiella pneumoniae, characterized in that, The ratio of chlorpromazine to colistin is between 16:1 and 533333:

1.

2. The application according to claim 1, characterized in that, The ratio of chlorpromazine to colistin is 16:1, 64:1, 256:1, 1024:1, 2048:1, 8195:1, 16368:1, or 533333:

1.

3. The application according to claim 1, characterized in that, The ratio of chlorpromazine to colistin is between 64:1 and 1024:1, or between 1024:1 and 2048:

1.

4. The application according to claim 1, characterized in that, The Klebsiella pneumoniae strains were E4, 7G, and CTR26, and the Salmonella strains were F30, 11R, and S12. The ratio of chlorpromazine to colistin was between 64:1 and 2048:

1.

5. The application of chlorpromazine in the preparation of a colistin synergist against Salmonella F30, 11R, and S12, characterized in that... The ratio of chlorpromazine to colistin is between 16:1 and 2048:

1.

6. The application according to claim 5, characterized in that, The ratio of chlorpromazine to colistin is 16:1 or 2048:

1.

7. The application of chlorpromazine in the preparation of a colistin-enhancing agent against Klebsiella pneumoniae E4, 7G, and CTR26, characterized in that... The ratio of chlorpromazine to colistin is between 8195:1 and 533333:

1.

8. The application according to claim 7, characterized in that, The ratio of chlorpromazine to colistin is 8195:

1.

9. The application of chlorpromazine in the preparation of compound formulations for enhancing the anti-Salmonella F30, 11R, and S12 activity of colistin, characterized in that, The ratio of chlorpromazine to colistin is between 1024:1 and 2048:

1.

10. The application according to claim 9, characterized in that, The ratio of chlorpromazine to colistin is 1024:1 or 2048:1.

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