Use of tegaserod maleate in the preparation of antibacterial drug potentiator

Abstract

The application discloses application of tegaserod maleate in preparation of an antibacterial drug synergist, and proves that the tegaserod maleate can produce a synergistic antibacterial effect with a plurality of commonly used antibacterial drugs (including tylosin, tetracycline, enrofloxacin, ampicillin and the like), can significantly enhance the antibacterial activity of the antibacterial drugs on gram-negative bacteria, and reduce the drug resistance of bacteria to the antibacterial drugs; the antibacterial composition composed of the tegaserod maleate and the antibacterial drugs can be used for preparing a drug for treating escherichia coli infection, and the synergistic antibacterial mechanism is mainly to destroy the inner and outer membrane permeability of bacteria, regulate the proton motive force of bacteria, inhibit the activity of bacterial efflux pumps, interfere with the energy metabolism homeostasis of bacteria and induce the bacteria to produce oxidative stress. The application provides a new technical strategy for the treatment of clinical escherichia coli infection, and has important clinical application value and industrialization potential.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial drug technology, specifically relating to the application of tegaserod maleate in the preparation of antibacterial drug potentiators. Background Technology

[0002] Escherichia coli (E. coli) is a common Gram-negative opportunistic pathogen in clinical and livestock farming. With the widespread use of antibiotics, drug resistance in E. coli has become increasingly prominent, seriously threatening public health. Unlike Gram-positive bacteria, Gram-negative bacteria, due to their unique physiological structure, form a natural osmotic barrier through filtration and chemical repulsion from their outer membrane, broad-spectrum active efflux via efflux pumps, and enzymatic degradation in the periplasmic space. This barrier prevents many large-molecule or hydrophobic antibiotics from entering the bacterial cell, resulting in many drugs with excellent activity against Gram-positive bacteria failing to reach effective inhibitory concentrations within E. coli, thus limiting their clinical application.

[0003] Given the challenges of long development cycles, high costs, and the tendency for cross-resistance to develop novel antimicrobial drugs, screening existing non-antimicrobial drugs as potentiators to assist antimicrobial drugs in overcoming the permeability barrier through mechanisms such as disrupting bacterial outer membrane integrity or inhibiting efflux pumps has become a cutting-edge research topic in anti-infective drug development.

[0004] Tiamulin is a truncated pleurotin antibiotic that exerts its antibacterial effect by inhibiting the 50S subunit of bacterial ribosomes. It is primarily used to treat infections caused by mycoplasma, spirochetes, and Gram-positive bacteria, but its antibacterial efficacy against Gram-negative bacteria such as Escherichia coli is limited. Tegaserod maleate, marketed as Zemarco or Changluoning, is a selective, partial agonist of the 5-HT receptor with favorable pharmacokinetic properties and a good safety record. It is clinically approved for short-term symptom relief in women with constipation-predominant irritable bowel syndrome.

[0005] However, to date, there has been no research on tegaserod maleate as an antibacterial drug potentiator in the existing technology. The present invention studies its combined use with tiamulin for antibacterial application, providing a new technical strategy for the clinical treatment of Escherichia coli infection. Summary of the Invention

[0006] In view of the problems mentioned above, existing truncated pleurotin antibiotics are limited by the dense outer membrane barrier and efflux mechanism of Escherichia coli, which prevents them from effectively entering the bacterial cell to exert their efficacy, resulting in natural drug resistance or low antibacterial activity. Furthermore, the clinical treatment of multidrug-resistant Escherichia coli infections faces challenges such as a lack of available drugs, excessively high doses of single antibiotics causing toxic side effects, and the potential to induce drug-resistant mutations in bacteria. Therefore, this invention provides the application of tegaserod maleate in the preparation of antibacterial drug potentiators.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of tegaserod maleate in the preparation of antimicrobial drug potentiators, wherein the tegaserod maleate, when used in combination with antimicrobial drugs, enhances the antimicrobial activity of the antimicrobial drugs and reduces bacterial resistance to the antimicrobial drugs, wherein the bacteria are Gram-negative bacteria.

[0008] Specifically, the Gram-negative bacteria are selected from one or more of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, and the Escherichia coli includes the standard strain Escherichia coli ATCC 25922 and clinically isolated drug-resistant Escherichia coli strains.

[0009] Furthermore, the antibacterial drug is selected from one or more of the following: tiamulin, tetracycline, quinolone, β-lactam, aminoglycoside, macrolide, lincosamide, and polypeptide antibacterial drugs.

[0010] Specifically, the antibacterial drugs include one or more of the following: tiamulin, tetracycline, enrofloxacin, amoxicillin, ampicillin, ceftiofur, clindamycin, spectinomycin, gentamicin, colistin, florfenicol, vancomycin hydrochloride, erythromycin, and tilmicosin.

[0011] The present invention further provides an antibacterial composition comprising a therapeutically effective dose of tegaserod maleate and a therapeutically effective dose of an antibacterial drug, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0012] Further, the antibacterial drug in the antibacterial composition includes one or more of the following: tiamulin, tetracyclines, quinolones, β-lactams, aminoglycosides, macrolides, lincosamides, and polypeptide antibacterial drugs. Specifically, the antibacterial drug includes one or more of the following: tiamulin, tetracycline, enrofloxacin, amoxicillin, ampicillin, ceftiofur, clindamycin, spectinomycin, gentamicin, colistin, florfenicol, vancomycin hydrochloride, erythromycin, and tilmicosin.

[0013] The present invention further provides the application of the antibacterial composition in the preparation of a drug for treating Escherichia coli infection. The antibacterial composition achieves a synergistic antibacterial effect on bacteria by disrupting bacterial membrane permeability, regulating bacterial proton kinetic potential, inhibiting bacterial efflux pump activity, interfering with bacterial energy metabolism, and inducing bacterial oxidative stress.

[0014] Furthermore, the dosage form of the antibacterial composition is an injection, an oral preparation, or a topical preparation.

[0015] Furthermore, the antibacterial composition is a compound preparation, wherein the tegaserod maleate and the antibacterial drug are mixed in the same unit dosage form; Alternatively, the antimicrobial composition may be a combined packaged formulation comprising individually packaged tegaserod maleate formulation and an antimicrobial drug formulation, and the two are used together in the preparation of a drug for treating Escherichia coli infection, the combined treatment including simultaneous administration or sequential administration.

[0016] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects: 1. Overcoming natural barriers to achieve broadened spectrum of antibacterial drugs and reversal of drug resistance. In existing technologies, truncated pleurotin antibiotics are typically used only for the treatment of Gram-positive bacteria and mycoplasma infections. This invention has discovered that tegaserod maleate can disrupt the integrity of the outer membrane of *E. coli*, increasing cell membrane permeability and / or inhibiting bacterial efflux pump activity. This mechanism successfully assists truncated pleurotin antibiotics, such as tiamulin, in overcoming the natural barrier of Gram-negative bacteria, entering the bacterial cell, and binding to the 50S ribosomal subunit. Experimental data show that, when combined with tegaserod maleate, the MIC value of tiamulin against *E. coli* decreased by more than 256-fold, successfully expanding the antibacterial spectrum of tiamulin to *E. coli* and reversing the resistance phenotype to a susceptible phenotype.

[0017] 2. Significant synergistic effect This invention utilizes the strong synergistic effect of tegaserod maleate combined with antibacterial drugs to effectively inhibit Gram-negative bacteria. It can control the concentration of antibacterial drugs at a concentration with no antibacterial activity or a sub-antibacterial concentration, significantly reducing the clinically effective dose of antibacterial drugs, thereby significantly reducing the dose-dependent toxic side effects that may be caused by antibacterial drugs and improving the safety window for clinical medication.

[0018] 3. Rapid sterilization and inhibition of drug resistance mutations. The antibacterial composition of the present invention has rapid bactericidal ability, significantly reducing bacterial load in a short time. Furthermore, combined use effectively reduces the risk of acquired resistance mutations in Escherichia coli during treatment, extending the lifespan of the antibacterial drugs.

[0019] 4. Possesses promising prospects for clinical translation. This invention utilizes tegaserod maleate as an antibacterial drug synergist, which is a typical example of "repurposing an old drug". Compared with developing entirely new outer membrane permeability agents, tegaserod maleate is a known compound with a mature synthesis process, controllable quality, stable supply chain, and detailed human or animal safety data, which greatly reduces the risk and cost of drug development and has extremely high clinical application value and commercial prospects. Attached Figure Description

[0020] Figure 1 This is a graph showing the determination of the graded inhibition index of combined drug therapy against Escherichia coli provided in Example 1 of the present invention; Figure 2 This is the bactericidal kinetic curve of the combined drug against Escherichia coli provided in Example 2 of the present invention; Figure 3 This is a graph showing the drug resistance development results of Escherichia coli provided in Example 3 of the present invention. Figure 3 A represents the change in MIC values ​​of tegaserod maleate against Escherichia coli ATCC 25922 after 30 consecutive passages. Figure 3 B represents the changes in MIC values ​​of tiamulin alone and in combination with other drugs after 30 consecutive passages of Escherichia coli ATCC 25922. Figure 4 These are the test results of different experimental groups on the membrane permeability of Escherichia coli provided in Example 4 of the present invention. Figure 4 A and Figure 4 B represents the detection results of the outer and inner membrane permeability of E. coli in different experimental groups. Figure 4 C shows the SEM images of different experimental groups after treatment with Escherichia coli; Figure 5 The results show the detection of the effect of the combined drug use provided in Example 5 of this invention on the proton kinetic potential of Escherichia coli. Figure 5 A represents the detection results of bacterial membrane potential under combined administration of tegaserod maleate and tiamulin. Figure 5 B represents the bacterial pH test results under combined administration of tegaserod maleate and tiamulin. Figure 5 C represents the result of bacterial membrane fluidity assay when tegaserod maleate and tiamulin are used in combination. Figure 5 D represents the result of bacterial motility testing under combined administration of tegaserod maleate and tiamulin; Figure 6 The results are the detection results of the effect of the combined drug use provided in Example 6 of this invention on the efflux capacity of Escherichia coli. Figure 6 A represents the change in bacterial fluorescence intensity over 60 minutes without the addition of glucose; Figure 6 B represents the continuous monitoring of bacterial fluorescence intensity changes over 60 minutes after the addition of glucose; Figure 6C represents the detection results of bacterial efflux pump-related gene expression under the combined administration of tegaserod maleate and tiamulin; Figure 7 This is the result of the combined drug use provided in Example 7 of the present invention on the ATP content of Escherichia coli. Figure 7 A represents the ATP content within the Escherichia coli ATCC 25922 cells of different treatment groups. Figure 7 B represents the ATP content in the supernatant of E. coli ATCC25922 from different treatment groups. Figure 7 C represents the total ATP content of Escherichia coli ATCC 25922 in different treatment groups; Figure 8 The results of the detection of ROS content in Escherichia coli (8A) and the detection results of respiratory level (8B) under combined drug use provided in Example 7 of this invention are as follows. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] This invention systematically studies the antibacterial effects, resistance induction effects, and synergistic antibacterial mechanisms of tegaserod maleate when used alone and in combination with antibacterial drugs through seven sets of examples, which are described in detail below.

[0023] The *E. coli* ATCC 25922 used in these examples was purchased from the American Center for Type Culture Collection; clinical isolates of *E. coli* were isolated and preserved at the Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences; Mueller-Hinton broth (MHB) and Luria-Bertani medium (LB) were purchased from Guangdong Huankai Microbial Technology Co., Ltd.; tegaserod maleate, tiamulin, tetracycline, enrofloxacin, amoxicillin, ampicillin, ceftiofur, clindamycin, spectinomycin, gentamicin, colistin, florfenicol, vancomycin hydrochloride, erythromycin, tilmicosin, and LPS were also used. PE and phospholipid mixture were purchased from MedChemexpress Biotechnology, Inc. (Shanghai, China); Enhanced ATP assay kit (S0027), reactive oxygen species assay kit (S0033S), and BCECF-AM probe were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; N-phenyl-1-naphthylamine (NPN), pyridine iodide (PI), 3,3'-dipropylthiocyanocyanine iodide (DiSC3(5)), 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), and ethidium bromide (EBTR) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0024] Example 1 Determination of combined antibacterial activity Tegaserod maleate is used in the preparation of antibacterial synergists for antibacterial drugs. By using tegaserod maleate in combination with antibacterial drugs, the antibacterial activity of antibacterial drugs is enhanced, and the drug resistance of bacteria to antibacterial drugs is reduced. The bacteria are Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii.

[0025] 1. Determination of fractional inhibitory concentration index (FICI) Tiamulin was selected as the antibacterial drug, and the combined effect of tegaserod maleate and tiamulin was determined by the checkerboard broth method.

[0026] First, prepare the stock solutions of tegaserod maleate and tiamulin. The specific method is to dilute tegaserod maleate and tiamulin 2-fold in a horizontal and vertical gradient in a 96-well plate respectively; mix well and set aside. Secondly, inoculate Escherichia coli ATCC 25922 into MHB liquid medium, culture it until the logarithmic growth phase, dilute and resuspend it to 0.5 McFarland turbidity according to the CLSI standard, and inoculate it into MHB medium at a ratio of 1:100 to obtain the test Escherichia coli suspension. Finally, after mixing evenly, add 100 μL of the test Escherichia coli suspension to the above 96-well culture plate, mix well, place it in a constant temperature incubator at 37°C and incubate statically for 18±2 h, then read the results. The test is repeated 3 times, and the fractional inhibitory concentration index (FICI) is calculated to evaluate the combined effect.

[0027] The specific calculation formula is as follows: FICI = MIC AB / MIC A +MIC BA / MIC B ; Among them, MIC A represents the minimum inhibitory concentration of the compound tegaserod maleate; MIC AB represents the minimum inhibitory concentration when tegaserod maleate and tiamulin are combined; MIC B represents the minimum inhibitory concentration of tiamulin; MIC BA represents the minimum inhibitory concentration when tiamulin and tegaserod maleate are combined; The basis for evaluating the combined effect is as follows: When FICI ≤ 0.5, the two compounds have a synergistic effect; When 0.5 < FICI ≤ 1, the two compounds have an additive effect; When 1 < FICI ≤ 4, the two compounds have an irrelevant effect; When FICI > 4, the two compounds have an antagonistic effect.

[0028] The results of the graded inhibition index determination of tegaserod maleate and tiamulin against Escherichia coli are as follows: Figure 1 As shown, its fractionated inhibitory concentration index (FICI) can be as low as 0.266, exhibiting a strong synergistic effect rather than a simple additive effect. This means that even in the presence of extremely low doses of tiamulin, the same bactericidal effect as high-concentration single-drug therapy can be achieved.

[0029] 2. Determination of broad-spectrum synergistic effect To screen for antimicrobial agents with synergistic effects with tegaserod maleate, this example selected several commonly used antimicrobial agents in veterinary clinical practice for combined drug susceptibility testing. The selected antimicrobial agents covered representative drugs with different mechanisms of action, specifically including: tetracycline, enrofloxacin, amoxicillin, ampicillin, ceftiofur, clindamycin, spectinomycin, gentamicin, colistin, florfenicol, vancomycin hydrochloride, erythromycin, and tilmicosin. The checkerboard method was used to determine the fractional inhibitory concentration index (FICI) when the above antimicrobial agents were used in combination with tegaserod maleate. The results are shown in Table 1. Table 1. FICI of tegaserod maleate combined with different antibacterial drugs against Escherichia coli ATCC 25922

[0030] As shown in Table 1, tegaserod maleate, when used in combination with tetracycline, enrofloxacin, ampicillin, gentamicin, colistin, florfenicol, and vancomycin hydrochloride, exhibits synergistic inhibitory effects against Escherichia coli ATCC 25922, indicating that tegaserod maleate has certain potential for broad-spectrum antibacterial enhancement.

[0031] 3. Detection of the antibacterial effect of combined drugs against other bacteria To investigate whether tegaserod maleate combined with other antibacterial drugs provides resistance to other bacteria, tiamulin was selected as the antibacterial drug for related experiments. Gram-negative and Gram-positive bacteria were screened and their inhibitory concentrations were determined. The results are shown in Table 2. Table 2. FICI determination of tegaserod maleate synergistic with tiamulin against different bacteria.

[0032] Table 2 shows that the combined use of tegaserod maleate and tiamulin has a synergistic effect on the tested Gram-negative bacteria, enhancing the antibacterial ability of tiamulin against Klebsiella pneumoniae and Acinetobacter baumannii, but has no synergistic effect on the tested Gram-positive bacteria.

[0033] 4. Detection of the antibacterial effect of tegaserod maleate combined with tiamulin against clinical Escherichia coli strains To investigate whether the combination of tegaserod maleate and tiamulin enhances the antibacterial activity of clinically isolated *Escherichia coli*, a number of drug-resistant strains were screened and their inhibitory concentrations (MICs) were determined. First, the MICs of the drug-resistant strains against tiamulin and tegaserod maleate were measured. Second, the FICIs of the clinical strains were determined. The results are shown in Table 3. Table 3. FICI determination of tegaserod maleate combined with tiamulin against clinical Escherichia coli strains.

[0034] Table 3 shows that clinical isolates exhibited severe resistance to tiamulin, while the drug showed good antibacterial activity against most strains; indicating that tegaserod maleate can enhance the antibacterial ability of tiamulin against most clinical Escherichia coli isolates.

[0035] 5. Detection of the antibacterial effect of tegaserod maleate combined with other antibacterial drugs on clinical Escherichia coli strains. To investigate the antibacterial effects of tegaserod maleate in combination with other antimicrobial agents against clinically resistant strains, different types of antimicrobial agents and corresponding resistant strains were screened, and their MIC and FICI values ​​were determined. The screened antimicrobial agents included enrofloxacin, tetracycline, ampicillin, colistin, and gentamicin. The results are shown in Table 4. The results indicate that tegaserod maleate can enhance the antimicrobial activity of multiple antimicrobial agents against clinically resistant Escherichia coli strains, suggesting its good potential for synergistic antimicrobial effects.

[0036] Table 4. FICI determination of clinical Escherichia coli strains in combination with tegaserod maleate and different antibacterial drugs. .

[0037] Example 2: Combined Antibacterial Synergistic Experiment To evaluate the dynamic bactericidal effect of tegaserod maleate alone and in combination with other antibacterial drugs on Escherichia coli, tiamulin was selected as the antibacterial drug for time-bactericidal curve determination.

[0038] Escherichia coli ATCC 25922 was cultured in MHB liquid medium to the logarithmic growth phase. A certain amount of bacterial suspension was diluted to 1×10⁶ CFU / mL, and four groups were set up: a blank control group (Control), a tegaserod maleate group (Drug 1 / 2 MIC (8 μg / ml), a tiamulin group (Antibiotic 1 / 2 MIC (16 μg / ml)), and a combination group (8 μg / ml tegaserod maleate + 16 μg / ml tiamulin). The bacterial suspensions were treated accordingly according to the grouping. At incubation times of 0, 1, 2, 4, 6, 8, 10, 12, and 24 hours, bacterial suspensions from different treatment groups were collected and serially diluted 10-fold with physiological saline (10⁻¹-10⁻⁸). The different dilutions were then plated onto MHB agar medium and incubated overnight at 37°C. Colony counts were performed, and time-kill curves for different treatment groups of E. coli were plotted. The results are shown below. Figure 2 As shown, for Escherichia coli ATCC 25922, compared with the treatment groups of tegaserod maleate and tiamulin alone, the combination treatment group significantly reduced the number of viable Escherichia coli and showed time-dependent bactericidal efficacy.

[0039] Example 3 Monitoring of Drug Resistance Induction by Combined Drug Use To investigate whether tegaserod maleate alone and in combination with tiamulin would induce drug resistance in *Escherichia coli*, the MIC values ​​of *E. coli* ATCC 25922 were measured after 30 days of continuous passage under the selective pressure of sub-inhibitory concentrations of tegaserod maleate (M / 8), tiamulin (T / 8), and the combination of these drugs (M / 8+T / 8). A blank control group was also included. The results are as follows: Figure 3 As shown, Figure 3 A showed that the MIC value of tegaserod maleate against Escherichia coli ATCC 25922 remained unchanged within 30 days. Monitoring of tiamulin resistance revealed that both the single and combined use of the compound significantly reduced the resistance of Escherichia coli ATCC 25922 to tiamulin (refer to...). Figure 3 (B) indicates that Escherichia coli is less likely to develop resistance to tegaserod maleate, and that it can alleviate the resistance of Escherichia coli to tiamulin.

[0040] Example 4 Membrane permeability detection The effects of tegaserod maleate and tiamulin alone or in combination on the inner and outer membranes of Escherichia coli ATCC 25922 were evaluated by dynamic fluorescence monitoring, scanning electron microscopy observation, and changes in FICI after membrane replenishment.

[0041] 1. Permeability testing of outer and inner membranes First, the blank control group (C), tegaserod maleate group (M / 8, M / 4, M / 2, M) MIC, tiamulin group (T / 2) MIC and combination group: tegaserod maleate (M / 8, M / 4, M / 2, M) MIC + tiamulin (T / 2) MIC were set up respectively, and the corresponding drug solutions were prepared according to the grouping. Secondly, the effects of tegaserod maleate synergistic with tiamulin on the permeability of the outer and inner cell membranes were detected using N-phenyl-1-naphthylamine (NPN) probe and pyridine iodide (PI) probe, respectively.

[0042] The detection method was as follows: *E. coli* ATCC 25922 was inoculated into MHB liquid medium and cultured to the logarithmic growth phase. The bacterial suspension was centrifuged at 4500 × g at room temperature for 10 min, resuspended in sterile PBS until OD 600nm = 0.5, and then probes were added. For outer mold permeability detection, 10 μM NPN probe was added; for inner mold permeability detection, 10 μM PI probe was added. The suspension was then incubated at 37°C and 180 rpm for 30 min. After mixing, 190 μL of the bacterial suspension was transferred to a black 96-well plate, and 10 μL of different grouped drug solutions were added. Fluorescence intensity was then detected.

[0043] During the external membrane permeability test, fluorescence intensity changes were dynamically monitored over 60 minutes at a frequency of 5 times per minute, immediately at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. The results are as follows: Figure 4 As shown in Figure A, treatment with tegaserod maleate alone significantly enhanced the fluorescence intensity of the NPN probe, exhibiting a dose-dependent effect. Compared to the group treated with tiamulin alone, the combined treatment with a series of gradient concentrations of tegaserod maleate and tiamulin resulted in a significant increase in the fluorescence value of the NPN probe, indicating that the compounds have a certain destructive effect on the outer membrane of E. coli.

[0044] During intima permeability testing, fluorescence intensity changes were dynamically monitored over 3 hours at a frequency of 30 min / time at an excitation wavelength of 535 nm and an emission wavelength of 615 nm. The results are as follows: Figure 4 As shown in Figure B, treatment with high concentrations of tegaserod maleate alone enhanced the fluorescence intensity of the PI probe, and the damage to the inner membrane of *E. coli* ATCC 25922 became increasingly severe with increasing drug exposure time. Compared with the tiamulin-only treatment group, the fluorescence value of the PI probe in the high-concentration treatment group was significantly enhanced after treatment with a series of concentrations of tegaserod maleate combined with tiamulin, indicating that the high-concentration compounds had a certain destructive effect on the inner membrane of *E. coli*.

[0045] 2. Bacterial morphology analysis *Escherichia coli* ATCC 25922 was inoculated into MHB medium and cultured to the logarithmic growth phase. After centrifugation at 4500 × g at room temperature for 10 min, the culture was resuspended in sterile PBS (0.01 M, pH 7.4). Four control groups were established: a control group (1 MIC of tegaserod maleate), a drug group (1 MIC of tiamulin), an antibiotic group (1 MIC of tiamulin), and a combination group (1 MIC of tegaserod maleate + 1 MIC of tiamulin). Different compounds were added to the bacterial suspension according to the group. After incubation at 37°C and 180 rpm for 1 h, the samples were washed three times with PBS and fixed with 2.5% (v / v) glutaraldehyde at 4°C for 12 h. Subsequently, the fixed samples were washed three times with PBS, dehydrated, dried, and sputter-coated with gold. Finally, the samples were observed and analyzed under a scanning electron microscope. The results are as follows: Figure 4 As shown in Figure C, the *E. coli* bacteria in the control group (Control) had smooth, plump surfaces and intact morphology. The morphology of bacteria in the tiamulin-only treatment group (Antibiotic) showed no significant difference compared to the control group. In contrast, both the tegaserod maleate monotherapy group (Drug) and the combination therapy group (Combination) resulted in significant membrane damage in *E. coli*, including cell surface shrinkage, collapse, rupture, and pore formation, accompanied by partial bacterial lysis. Notably, the degree of physical membrane damage observed in the tegaserod maleate monotherapy group was more severe than that in the combination group. This phenomenon indicates that in the combination system, tegaserod maleate can alter membrane permeability, disrupting the bacterial physical barrier and thus assisting tiamulin in entering the cell to exert a synergistic bactericidal effect.

[0046] 3. Lipid binding assay First, different bacterial cell membrane components—LPS, PE, and mixtures of different phospholipids—were added to MHB medium to achieve final concentrations of 32 and 64 μg / ml, respectively, which were reserved for subsequent culture media. Following the FICI assay method, *E. coli* ATCC 25922 cultured to the logarithmic growth phase was diluted with the aforementioned medium. Subsequently, tegaserod maleate and tiamulin were serially diluted 2-fold horizontally and vertically in 96-well plates using the medium. 100 μL of the bacterial suspension was added to each well of the 96-well plate, mixed thoroughly, and incubated at 37°C for 18 ± 2 h. Results were then recorded. The experiment was performed in triplicate, and FICI was calculated to evaluate the combined effect of the two compounds after the addition of membrane components. The results are shown in Table 5. Table 5. Determination of FICI in Escherichia coli after exogenous addition of membrane components

[0047] Table 5 shows that LPS, PE, and mixtures of different phospholipids weakened the antibacterial activity of the combined drug against Escherichia coli ATCC 25922, indicating that the combined drug can act on LPS on the outer membrane and phospholipids on the inner membrane of the bacteria, thereby inducing membrane disruption.

[0048] Example 5: Detection of bacterial proton kinetic potential The blank control group (C), tegaserod maleate group (M / 8, M / 4, M / 2, M) MIC, tiamulin group (T / 2) MIC and combination group: tegaserod maleate (M / 8, M / 4, M / 2, M) MIC + tiamulin (T / 2) MIC were set up respectively, and the corresponding drug solutions were prepared according to the grouping.

[0049] 1. Detection of bacterial membrane potential The effect of combined drug administration on bacterial membrane potential was detected using DiSC3(5) dye.

[0050] Escherichia coli ATCC 25922 was cultured in LB liquid medium to the logarithmic growth phase. After washing with sterile PBS and resuspending to OD 600 nm = 0.5, 0.5 μM DiSC3(5) was added. The mixture was incubated at 37°C and 180 rpm in the dark for 30 min. 190 μL of the bacterial suspension was then transferred to a black 96-well plate, and 10 μL of the above-mentioned different drug solutions were added. The plates were incubated at 37°C for 1 h. Finally, the fluorescence intensity was measured using a multi-mode microplate reader at an excitation wavelength of 622 nm and an emission wavelength of 670 nm, continuously monitored for 60 min at a frequency of 5 min / time. Results are as follows: Figure 5 As shown in Figure A, the fluorescence intensity of DiSC3(5) in the combined treatment groups was significantly lower than that in the tiamulin-only group, indicating a hyperpolarization trend in the cell membrane. Furthermore, the fluorescence intensity in the tegaserod maleate-only group was also lower than that in the tiamulin-only group, suggesting that the compound itself can induce slight membrane hyperpolarization. With prolonged treatment time, the fluorescence intensity in each group continued to decrease, and the degree of membrane hyperpolarization continuously increased. This indicates that under the action of tiamulin and its combined drugs, *E. coli* induces membrane potential hyperpolarization through a stress compensation mechanism to maintain membrane functional stability.

[0051] 2. Bacterial ΔpH Detection The effect of combined drug administration on bacterial pH was evaluated using the pH-sensitive fluorescent probe BCECF-AM.

[0052] Escherichia coli ATCC 25922 was cultured in LB liquid medium to the logarithmic growth phase, centrifuged at 4500×g at room temperature for 10 min, washed and resuspended in HEPES buffer (5 mmol / L, pH=7.0) containing 5 mmol / L glucose to OD600 nm=0.5. 2 μM of the BCECF-AM probe was added to the bacterial culture, and the culture was incubated at 37°C for 30 min. 190 μL of the probe-labeled bacterial culture was transferred to a black 96-well plate, followed by the addition of 10 μL of the different drug solutions described above. Immediately, the fluorescence intensity was continuously monitored over 60 min at an excitation wavelength of 488 nm and an emission wavelength of 535 nm at a frequency of 5 min / time. Results are as follows: Figure 5 As shown in Figure B, the results indicated that compared with the tiamulin-only treatment group, the combined treatment group and the tegaserod maleate-only treatment group showed a certain degree of upregulation of E. coli ΔPH, and the fluorescence intensity of the tegaserod maleate-only treatment group was higher than that of the combined treatment group. Furthermore, within a certain concentration range, the ΔPH of E. coli in both the combined treatment group and the tegaserod maleate-only treatment group showed a dose-dependent increase. This suggests that both tegaserod maleate-only and combined tiamulin treatment induced compensatory upregulation of ΔPH in E. coli to maintain the proton gradient.

[0053] 3. Bacterial membrane fluidity test The effect of combined drug administration on bacterial membrane fluidity was detected using the Laurdan fluorescent probe.

[0054] Escherichia coli ATCC 25922 was cultured in LB liquid medium to the logarithmic growth phase. The suspension was diluted with sterile PBS to obtain an OD 600nm = 0.5. The suspension was centrifuged at 4500×g at room temperature for 10 min, and the precipitate was washed 2-3 times with HEPES buffer (5 mmol / L glucose, pH 7.0) and resuspended. Then, 10 µM Laurdan dye was added, and the suspension was incubated at 37°C and 180 rpm in the dark for 30 min. The suspension was washed 2-3 times with HEPES buffer (5 mmol / L glucose) and resuspended. 190 μL of the suspension was transferred to black 96-well plates, and 10 μL of the different drug solutions were added to each well. The plates were incubated at 37°C in the dark for 1 h. The fluorescence values ​​at excitation wavelengths of 350 nm and emission wavelengths of 435 nm and 490 nm were then measured using a multi-mode microplate reader. The calculation formula is as follows: Laurdan GP = (I435 – I490) / (I435 + I490). The result is as follows: Figure 5As shown in Figure C, compared with the tiamulin monotherapy group, the membrane fluidity of E. coli in the combined treatment group and the tegaserod maleate monotherapy group was significantly reduced, and the inhibitory effect of the combined treatment group on membrane fluidity was stronger than that of the tegaserod maleate monotherapy group; indicating that tegaserod maleate inhibits the membrane fluidity of E. coli, and the combined treatment enhances this effect.

[0055] 4. Bacterial motility test Escherichia coli can form a diffuse growth ring in a semi-solid medium of low-concentration agar through the drive of flagella. Detecting changes in the diameter of the growth ring can reflect the effect of combined drug use on bacterial motility to some extent.

[0056] LB medium containing 0.3% agar was prepared and cooled to 50°C. The above-mentioned drug solution was added to the medium at different concentrations, mixed thoroughly, and poured into petri dishes to cool and solidify. A logarithmic growth phase *E. coli* ATCC 25922 bacterial suspension was taken, diluted to OD600 = 0.5, and 10 µL was spot-loaded into the center of the medium using a pipette. After incubation at 37°C for 48 hours, the growth ring formed by bacterial movement was observed, and the bacterial motility was assessed by measuring the diameter of the growth ring. Results are as follows: Figure 5 As shown in Figure D, the horizontal axis represents the grouping, and the vertical axis represents the diameter. The results show that compared with the tiamulin-treated group, the diameter of the *E. coli* growth ring in the combined treatment group was significantly reduced, exhibiting a dose-dependent effect. This indicates that tegaserod maleate significantly reduces *E. coli* motility, while the combined treatment enhances the inhibitory effect on *E. coli* motility.

[0057] Example 6: Detection of bacterial efflux capacity 1. Determination of bacterial efflux capacity Log-growing *E. coli* ATCC 25922 was collected by centrifugation. The cells were washed with sterile PBS (0.01 M, pH 7.2) to remove culture medium components, and resuspended to adjust the bacterial concentration to OD 600 nm = 0.5. Blank control group (C), positive control group (CCCP), tegaserod maleate groups (M / 8, M / 4, M / 2, M) MIC, tiamulin group (T) 1 / 2 MIC, and combination group: tegaserod maleate (M / 8, M / 4, M / 2, M) MIC + tiamulin (T) 1 / 2 MIC were set up. The bacterial suspensions were incubated with the test compounds for 30 min, then centrifuged to collect the cells, and washed and resuspended with PBS (0.01 M, pH 7.2). Subsequently, EBTR was added to a final concentration of 2 μg / mL and mixed well. The fluorescence values ​​were measured using a multi-mode microplate reader at an excitation wavelength of 518 nm and an emission wavelength of 605 nm. The changes in bacterial fluorescence intensity were continuously monitored over 60 minutes at a frequency of 5 min / time. The results are as follows: Figure 6As shown in Figure A, without the addition of glucose, the fluorescence intensity of the combined group was significantly higher than that of the tiamulin monotherapy group, and the fluorescence intensity gradually decreased with the decrease of tegaserod maleate concentration. Subsequently, glucose with a final concentration of 50 mM was immediately added as an energy source, and the fluorescence intensity change curve was continuously monitored and recorded over 60 minutes. The results are shown in Figure A. Figure 6 As shown in Figure B, the efflux pump was activated after the addition of glucose. Although the fluorescence intensity of the combined group decreased, it was still significantly higher than that of the tiamulin monotherapy group. Furthermore, the fluorescence intensity decreased in a gradient manner with decreasing tegaserod maleate concentration. The higher the tegaserod maleate concentration, the stronger the inhibitory effect of the efflux pump, making it impossible for the efflux pump to effectively expel EBTR even when energy was sufficient (refer to Figure B). Figure 6 B). In summary, combined drug therapy can inhibit the efflux pump activity of Escherichia coli ATCC 25922.

[0058] 2. Detection of gene expression related to efflux pumps Log-growing *E. coli* ATCC 25922 was collected by centrifugation. The cells were washed with sterile PBS (0.01 M, pH 7.2) to remove culture medium components, and resuspended to adjust the bacterial concentration to OD 600nm = 0.5. A blank control group (Control), a tegaserod maleate group (1 MIC), a tiamulin group (1 MIC), and a combination group (1 MIC tegaserod maleate + 1 MIC tiamulin) were set up. The bacterial suspensions were incubated with the test compounds for 1 h, then centrifuged at 4500 × g at room temperature for 10 min, and washed three times with PBS (0.01 M, pH 7.2). RNA was extracted from the bacterial samples using an RNA extraction kit and reverse transcribed into cDNA for later use. q-PCR primers for internal control genes and efflux pump-related genes were designed. Using the aforementioned cDNA as a template, q-PCR was performed to relatively quantify efflux pump-related genes in different groups of bacterial samples. The results are shown below. Figure 6 As shown in C, compared with the tiamulin monotherapy group, the combination therapy inhibited the expression of efflux pump-related genes arcA, arcB, TolC, soxS, soxR, marA and marR, indicating that the combination therapy inhibited the efflux of drugs by Escherichia coli at the transcriptional level.

[0059] Example 7 Analysis of bacterial metabolic stress levels 1. Detection of bacterial ATP levels Log-growing *E. coli* ATCC 25922 was collected by centrifugation and resuspended in sterile PBS (0.01 M, pH 7.2) until OD 600 nm = 0.5. A blank control group (Control), a positive control group (CCCP), tegaserod maleate group M (1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, 1 MIC), a tiamulin group T (1 / 2 MIC), and a combination group: tegaserod maleate M (1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, 1 MIC) + tiamulin T (1 / 2 MIC) were set up. The bacterial suspension and the test compound were co-incubated for 1 h, followed by centrifugation at low temperature. The supernatant and bacterial precipitate were collected separately, and the precipitate was washed three times with pre-cooled sterile PBS (0.01 M, pH 7.2). Subsequently, the ATP content of the supernatant and bacterial precipitate was determined using an ATP assay kit (Shanghai Beyotime Biotechnology Co., Ltd.). All experimental steps were performed on ice. Results are as follows: Figure 7 A- Figure 7 As shown in Figure C, 7A represents the ATP content within *E. coli* ATCC 25922 cells in different treatment groups, 7B represents the ATP content in the supernatant of *E. coli* ATCC 25922 cells in different treatment groups, and 7C represents the total ATP content of *E. coli* ATCC 25922 cells in different treatment groups. Compared with the tiamulin-only treatment group, the ATP level in the supernatant of the combined treatment group increased significantly, while the ATP level in the cells decreased significantly. Compared with the compound-only treatment group, within a certain dose range, the ATP level in the supernatant of the combined treatment group decreased, while the ATP level in the cells increased. Furthermore, the total ATP content analysis revealed that, within a certain dose range, the combined treatment reduced the total ATP content of *E. coli* compared with the tiamulin-only group. In summary, the ATP assay results indicate that tegaserod maleate can lead to intracellular ATP leakage and affect energy metabolism homeostasis, thereby synergistically exerting a highly effective bactericidal effect with antibiotics.

[0060] 2. Detection of bacterial ROS and respiratory levels The blank control group (C), tegaserod maleate group (M / 8, M / 4, M / 2, M) MIC, tiamulin group (T / 2) MIC and combination group: tegaserod maleate (M / 8, M / 4, M / 2, M) MIC + tiamulin (T / 2) MIC were set up respectively, and the corresponding drug solutions were prepared according to the grouping.

[0061] Bacterial ROS level detection: Log-growing *E. coli* ATCC 25922 was collected by centrifugation and resuspended in sterile PBS (0.01 M, pH 7.2) until OD 600 nm = 0.5. A final concentration of 10 μM DCFH-DA probe was added to the bacterial suspension, and the mixture was incubated at 37°C in the dark for 30 min. After probe labeling, the bacterial suspension was washed and resuspended in pre-cooled sterile PBS (0.01 M, pH 7.2). 190 μL of the probe-labeled bacterial suspension was transferred to black 96-well plates, and 10 μL of the different groups of reagents prepared above were added. The fluorescence intensity was immediately measured using a multi-mode microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The fluorescence intensity was continuously monitored over 2 h at a frequency of 30 min / time. The results are shown below. Figure 8 As shown in Figure A, compared with the tiamulin monotherapy group, the combined treatment significantly increased the ROS accumulation in E. coli. However, compared with the tegaserod maleate monotherapy group, the ROS in the combined treatment group was relatively lower, indicating that tegaserod maleate increased the accumulation of intracellular ROS, thereby exacerbating bacterial oxidative stress.

[0062] Bacterial respiratory level detection: Log-growing *E. coli* ATCC 25922 was collected by centrifugation and resuspended in sterile PBS (0.01 M, pH 7.2) until OD 600 nm = 0.5. Resazurin was added to the bacterial suspension to a final concentration of 0.1 µg / mL. 190 μL of the resazurin-added bacterial suspension was transferred to black 96-well plates, followed by the addition of 10 μL of different combinations of the above-mentioned drug solutions. Fluorescence changes were dynamically monitored over 1 h at 37℃ using a multi-mode microplate reader with excitation / emission wavelengths of 550 / 590 nm. Results are shown below. Figure 8 As shown in Figure B, compared with the tiamulin group, the respiratory level of E. coli in the combination therapy group was enhanced and comparable to that in the tegaserod maleate monotherapy group, suggesting that tegaserod maleate can affect the energy metabolism process of E. coli, whether used alone or in combination with antibiotics.

[0063] This invention, through multi-dimensional and systematic experiments, investigated the application value, scope of application, and synergistic antibacterial mechanism of tegaserod maleate as an anti-Escherichia coli enhancer for antibiotics (tiamulin). It confirmed that the combined use of tegaserod maleate and antibiotics (tiamulin) can effectively enhance antibacterial activity against Escherichia coli (including drug-resistant strains) and some Gram-negative bacteria, alleviate Escherichia coli resistance, and solve the clinical challenges of treating Escherichia coli infections. This provides reliable experimental evidence and technical support for the development of novel antibacterial drugs and the optimization of clinical antibacterial regimens.

[0064] 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 within the protection scope of the present invention.

Claims

1. The application of tegaserod maleate in the preparation of antibacterial drug potentiators, characterized in that, The tegaserod maleate, when used in combination with an antibacterial drug, enhances the antibacterial activity of the drug and reduces bacterial resistance to the antibacterial drug. The bacteria in question are Gram-negative bacteria.

2. The application as described in claim 1, characterized in that, The Gram-negative bacteria are selected from one or more of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

3. The application as described in claim 1, characterized in that, The antibacterial drug is selected from one or more of the following: tiamulin, tetracycline, quinolone, β-lactam, aminoglycoside, macrolide, lincosamide, and polypeptide antibacterial drugs.

4. The application as described in claim 1, characterized in that, The antibacterial drugs include one or more of the following: tiamulin, tetracycline, enrofloxacin, amoxicillin, ampicillin, ceftiofur, clindamycin, spectinomycin, gentamicin, colistin, florfenicol, vancomycin hydrochloride, erythromycin, and tilmicosin.

5. An antibacterial composition, characterized in that, This includes therapeutically effective doses of tegaserod maleate and therapeutically effective doses of antimicrobial agents, as well as one or more pharmaceutically acceptable carriers, diluents, or excipients.

6. The antibacterial composition according to claim 5, characterized in that, The antibacterial drugs include one or more of the following: tiamulin, tetracyclines, quinolones, β-lactams, aminoglycosides, macrolides, lincosamides, and polypeptide antibacterial drugs.

7. The antibacterial composition according to claim 6, characterized in that, The antibacterial drugs include one or more of the following: tiamulin, tetracycline, enrofloxacin, amoxicillin, ampicillin, ceftiofur, clindamycin, spectinomycin, gentamicin, colistin, florfenicol, vancomycin hydrochloride, erythromycin, and tilmicosin.

8. The use of an antibacterial composition as described in any one of claims 5-7 in the preparation of a medicament for treating Escherichia coli infection.

9. The application as described in claim 8, characterized in that, The antibacterial composition is available in the form of an injection, an oral preparation, or a topical preparation.

10. The application as described in claim 8, characterized in that, The antibacterial composition is a compound preparation, wherein tegaserod maleate and the antibacterial drug are mixed in the same unit dosage form; Alternatively, the antimicrobial composition may be a combined packaged formulation comprising separately packaged tegaserod maleate formulation and an antimicrobial drug formulation, and the two are used in combination in the preparation of a drug for treating Escherichia coli infection.

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