CN115645414B - Antibacterial pharmaceutical composition and application thereof - Google Patents

Abstract

The invention provides an antibacterial pharmaceutical composition and application thereof. The invention examines the antibacterial and anti-biofilm activity of tripterine on enterococci and the ability of VRE to restore sensitivity to vancomycin in an auxiliary way. The results of the study show that tripterine has significant antibacterial and anti-biofilm activity against enterococci including VRE. The sub-minimum inhibitory concentration of celastrol restores the activity of vancomycin on VRE in vitro and in vivo. The combination of vancomycin and tripterine produces a synergistic effect, and the tripterine is hopeful to be used as a new antibacterial agent or a sensitizer of the vancomycin, so that a selection scheme is provided for the treatment of VRE.

Description

Antibacterial pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to an antibacterial pharmaceutical composition and application thereof.

Background

Enterococci are commensal bacteria that are predominantly present in the gastrointestinal tract of humans and animals and are considered to be one of the major causes of nosocomial infections (Agudelo Higueta et al, 2014; kim et al, 2019). More than 20 enterococci have been identified, of which enterococcus faecalis (Enterococcus faecalis) and enterococcus faecium (Enterococcus faecium) are the most clinically relevant species, and the most common infections caused thereby are urinary tract infections, bacteremia, endocarditis and surgical site infections. The mortality rate of severe enterococcus infections is as high as 20% -40% (Miro et al, 2013). Studies have shown that nosocomial infections with ampicillin and vancomycin resistant enterococci have increased over the past decade (Haque et al, 2018). The emergence and spread of multi-drug resistant enterococci, particularly Vancomycin Resistant Enterococci (VRE), poses a serious threat to clinical treatment. The world health organization has listed vancomycin-resistant enterococcus faecium in the "high priority pathogen" list (WHO, 2017). Thus, there is a need to develop new antibacterial agents with new backbones or to discover new strategies to treat VRE-induced infections.

The natural products provide abundant medicinal sources for people since ancient times, and active compounds such as ipecac, artemisinin, houttuynin, berberine and the like are representative of plant-derived anti-infective drug lead compounds. The parent nucleus structure and active group of the monomer compound obtained by separation from natural products are formed by long-term natural selection, and the biological activity shown in the screening process has the advantage which is incomparable with that of artificially synthesized compounds. Therefore, the natural products and derivatives thereof with various structures are extremely important in the research of innovative drugs.

Celastrol (CEL) is a major bioactive component of tripterygium wilfordii and has received wide attention for its various promising biological activities, including anticancer, anti-inflammatory, slimming, cardioprotection, antibacterial, antioxidant, antiallergic, neuroprotection, antithrombotic, anti-osteoarthritis, anti-alzheimer's disease, etc. (Hou et al 2020). Tripterine has been reported to have anti-planktonic and anti-biofilm activity against staphylococci (Ooi et al, 2015; woo et al, 2017). At present, related research reports on the anti-enterococcus activity of tripterine are not yet seen.

Disclosure of Invention

The invention aims to provide a novel antibacterial pharmaceutical composition and application thereof.

To achieve the object of the present invention, in a first aspect, the present invention provides the use of celastrol in the preparation of an antibacterial drug or composition; wherein the bacteria are enterococcus and drug-resistant bacteria thereof.

In the present invention, the enterococcus is preferably enterococcus faecalis (E.faecalis) and enterococcus faecium (E.faecium), and their drug-resistant bacteria.

Further, the resistant bacteria may be selected from Vancomycin Resistant Enterococci (VRE) and ampicillin resistant enterococci, such as vancomycin resistant enterococci and vancomycin resistant enterococci faecium.

In a second aspect, the invention provides the use of celastrol in the preparation of an FtsZ protein inhibitor.

Preferably, the FtsZ protein is derived from enterococci and drug resistant bacteria thereof.

In a third aspect, the present invention provides a novel antibacterial pharmaceutical composition comprising tripterine and vancomycin as active ingredients.

Further, the mass ratio of the tripterine to the vancomycin is 1:16-1:2.

In a fourth aspect, the present invention provides the use of said composition for the preparation of an antimicrobial biological; wherein the bacteria are enterococcus and drug-resistant bacteria thereof.

Further, the tripterine and the vancomycin can be administered independently or simultaneously together.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

compared with the prior art, the invention has at least the following advantages:

the invention examines the antibacterial and anti-biofilm activity of tripterine on enterococci and the ability of VRE to restore sensitivity to vancomycin in an auxiliary way. The results of the study show that tripterine has significant antibacterial and anti-biofilm activity against enterococci including VRE. The sub-MIC concentration (sub-minimum inhibitory concentration) of celastrol restored the activity of vancomycin on VRE in vitro and in vivo. The combination of vancomycin and tripterine produces a synergistic effect, and the tripterine is hopeful to be used as a new antibacterial agent or a sensitizer of the vancomycin, so that a selection scheme is provided for the treatment of VRE.

Drawings

FIG. 1 shows the activity of CEL at various concentrations on enterococcus strains in a sterilization curve according to a preferred embodiment of the present invention. A: enterococcus faecalis ATCC700802 (mic=4 μg/mL); b: enterococcus faecalis ATCC51575 (mic=2 μg/mL); c: enterococcus faecium ATCC700221 (mic=2 μg/mL); d: enterococcus faecium ATCC51559 (mic=2 μg/mL).

Fig. 2 shows the concentration-dependent activity of CEL, vancomycin and ampicillin in clearing biofilm (n=3, ×p < 0.001) in a preferred embodiment of the invention.

FIG. 3 shows the activity of CEL and vancomycin alone and in combination at sub-MIC concentrations on VRE strains in a preferred embodiment of the invention. A: enterococcus faecalis ATCC700802,1/8 MIC (0.5 μg/mL) CEL and <1/128MIC (2 μg/mL) vancomycin (CEL MIC = 4mg/mL, MIC = 16 μg/mL); b: enterococcus faecium ATCC700221,1/4 MIC CEL (0.5 μg/mL) and <1/64MIC (16 μg/mL) vancomycin (MIC of CEL = 2mg/mL, MIC of vancomycin >256 μg/mL).

FIG. 4 shows the protective effect of CEL and vancomycin alone and in combination on lethal infection of E.maxima larvae in a preferred embodiment of the invention. A: enterococcus faecalis ATCC 700802; b: enterococcus faecium ATCC 700221. Each group had 10 larvae. The infection dose is 2×10 ⁶ CFU/CFU.

FIG. 5 is a scanning electron micrograph of enterococcus faecalis ATCC700802 and Bacillus subtilis ATCC 21332 without drug treatment and after 4 hours of CEL treatment in a preferred embodiment of the present invention. A1: enterococcus faecalis ATCC700802, control, magnification 5000-fold; a2: enterococcus faecalis ATCC700802, control, magnification 20000 times; b1: enterococcus faecalis ATCC700802,2 mug/mL CEL treatment, magnification is 5000 times; b2: enterococcus faecalis ATCC700802,2 mug/mL CEL treatment, magnification is 20000 times; c: bacillus subtilis ATCC 21332, control; d: bacillus subtilis ATCC 21332 2 μg/mL CEL treatment.

FIG. 6 shows the results of the docking of CEL and FtsZ proteins, including interactions of different amino acids of CEL and FtsZ, in a preferred embodiment of the invention.

FIG. 7 shows the results of surface plasmon resonance measurements obtained on FtsZ coated chips at different concentrations of CEL in a preferred embodiment of the invention.

FIG. 8 is a dose-response curve for CEL inhibiting the GTPase activity of enterococcus faecalis FtsZ in a preferred embodiment of the invention. Each point represents three independent experiments.

Detailed Description

The invention aims to study the antibacterial and anti-biofilm activity of tripterine on enterococci and the auxiliary ability of the tripterine to restore the sensitivity of VRE on vancomycin.

The in vitro antibacterial activity of tripterine is studied by adopting a Minimum Inhibitory Concentration (MIC) measurement, a biological film removal experiment and a time sterilization curve experiment. The synergy between tripterine and vancomycin is determined by a chessboard method and a sterilization curve method. In vivo studies were performed on the larval infection model of Chilo suppressalis. The potential bacteriostatic mechanism of the tripterine is discussed through molecular docking, biomolecular binding interaction and the enzyme inhibition of the tripterine on bacterial mitosis protein FtsZ.

The research result shows that the tripterine inhibits all tested enterococcus faecalis and enterococcus faecium strains, the MIC range is 0.5-4 mug/mL, and in the sterilization curve analysis, the tripterine inhibits the bacterial growth in a concentration-dependent manner. After 24 hours of exposure at a concentration of 16 mug/mL, the tripterine can clear more than 50% of the biofilm. The combined use of tripterine and vancomycin showed a synergistic effect on all 23 strains tested, with a median Fractional Inhibitory Concentration Index (FICI) of 0.25 in the checkerboard experiment. The combined use of sub-MIC levels of tripterine and vancomycin showed a synergistic effect in sterilization curve experiments and a significant protective effect in the larval infection model of Chilo suppressalis compared to either drug alone. Tripterine has strong binding and inhibiting ability to FtsZ, kd and IC ₅₀ 1.75.+ -. 0.06. Mu.M and 1.04.+ -. 0.17. Mu.g/mL, respectively.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

Abbreviations and terms involved in the present invention are as follows:

VRE: vancomycin-resistant enterococci;

CELs: tripterine;

MIC: minimum inhibitory concentration;

CFU: colony forming units;

FICI: fractional inhibition concentration index;

VSE: vancomycin-sensitive enterococci;

MHA: mueller-Hinton agar;

camdb: cation-regulated Mueller-Hinton broth;

BHI: brain heart infusion;

PBS: phosphate buffered saline;

SD: standard deviation;

CLSI: the american society of clinical and laboratory standards;

SEM: scanning electron microscope;

SPR: surface plasmon resonance.

EXAMPLE 1 Leptospira bacteriostasis study

1. Materials and methods

1.1 Strain and growth conditions

allstrainsusedinthepresentinventionwerefromtheChinamedicalsciencecenterfortypecollectionofpathogenicmicroorganisms(CAMS-CCPM-A),including5ATCCstandardVREstrains(2enterococcusfaeciumand3enterococcusfaecalis),18clinicalVREstrains(17enterococcusfaeciumand1enterococcusfaecalis),5ATCCstandardvancomycin-sensitiveenterococcus(VSE)(2enterococcusfaeciumand3enterococcusfaecalis),72clinicallyisolatedVSEstrains(31enterococcusfaeciumand41enterococcusfaecalis). Strains are typically grown in cation-regulated Mueller-Hinton broth (CAMHB) or Mueller-Hinton agar (MHA) at 37 ℃.

1.2 chemicals and reagents

Vancomycin, ampicillin and tripterine were purchased from the national institute of pharmaceutical and biological products (china, beijing). Vancomycin, tripterine and ampicillin were dissolved in distilled water, dimethyl sulfoxide and phosphate buffer (pH 8.0) respectively, and the solutions were stored at-20℃after filtration through a 0.22 μm filter at a concentration of 10mg/mL as stock solutions. CarMB, MHA and Brain Heart Infusion (BHI) were purchased from BD company (Franklin Lakes, NJ, USA) for bacterial culture and antibacterial drug susceptibility experiments.

CytoPhos phosphate assay Biochem kit and recombinant enterococcus faecalis FtsZ protein (FTZ 04-B) were purchased from Cytoskeleton (Denver, colorado, U.S.A.).

1.3 sensitivity test

All strains were stored at-80 ℃, streaked onto MHA plates for overnight incubation prior to testing. Minimal inhibition of celastrol and vancomycin was determined by broth microdilution according to the institute of Clinical and Laboratory Standards (CLSI) guidelines (CLSI, 2021)Concentration (MIC), ampicillin as a quality control antibiotic. The compound at an initial concentration of 256 μg/mL was added to wells of a 96-well microtiter plate and serially double diluted with camdb medium. The final inoculum size was 5X 10 by adding 10. Mu.L of bacterial suspension ⁵ CFU/mL, plates were incubated at 37℃for 24 hours and MIC was read. MIC is defined as the lowest concentration of a compound that inhibits bacterial growth. Vancomycin MIC break points are specified as follows: sensitive, less than or equal to 4 mug/mL; 8-16 mug/mL, intermediary; not less than 32 mug/mL, drug resistance.

1.4 time sterilization curve of Tripterine

The kinetics of bactericidal activity of tripterine was assessed by bactericidal curve experiments according to the method described by CLSI (CLSI, 1999). Experiments were performed on enterococcus faecalis ATCC700802, ATCC51575, enterococcus faecalis ATCC700221, ATCC 51559. Briefly, overnight cultured bacterial fluids were diluted to about 2X 10 with CAPMB ⁶ The final concentration of CFU/mL was aliquoted into sterile glass tubes, 10mL per tube. Different concentrations of tripterine (1/4 MIC, 1/2MIC, 2MIC and 4 MIC) were obtained by adding a volume of tripterine stock solution to each tube, with the tube containing bacteria but no tripterine serving as a growth control. Bacterial counts of the bacterial solutions were determined at 0, 2, 4, 6, 8 and 24 hours of incubation. After 10-fold serial dilution of the bacterial fluid, 10. Mu.L of sample was taken, trickled on MHA plate, triplicated, and incubated at 37℃for 24 hours, viable counts were performed and recorded as log ₁₀ CFU/mL. The bactericidal activity was defined as a reduction of 3log or more in colony count compared to the initial inoculum size ₁₀ CFU/mL (99.9% clearance). The detection limit is 2log ₁₀ CFU/mL。

1.5 biofilm removal experiments

Biofilm removal experiments were performed on the investigated strains as described previously (Wang et al, 2019). Enterococcus faecalis ATCC700802 was inoculated into BHI broth and shaken overnight at 37 ℃. After dilution of the bacterial liquid at 1:100, the bacterial liquid was added to a 96-well plate (100. Mu.L per well) subjected to tissue culture treatment, and the mixture was subjected to stationary culture at 37℃for 24 hours to form a biofilm. The medium was gently removed and 100. Mu.L of fresh culture containing different concentrations of tripterine or antibiotic control was addedMedium (3 biological replicates). After further incubation for 24 hours, the medium was removed, the biofilm was gently rinsed 3 times with Phosphate Buffered Saline (PBS) and thermally fixed at 60 ℃ for 1 hour. To each well was added 50 μl of 0.06% crystal violet staining for 5 minutes, followed by repeated washing with distilled water to remove excess dye. Crystallization violet was eluted from the stained biofilm by adding 200 μl of 30% acetic acid to each well and transferred to another 96-well plate for OD ₅₉₅ And (5) reading.

1.6 statistical analysis

Statistical significance was determined using SPSS 16.0 one-way analysis of variance (ANOVA), with P values <0.05 considered statistically significant.

2. Experimental results

Antibacterial activity of tripterine: MIC values of tripterine and vancomycin on 52 strains of enterococcus faecalis and 48 strains of enterococcus faecium are shown in Table 1. 23% of the strains (23/100) were resistant to vancomycin, including 4 enterococcus faecalis and 19 enterococcus faecium. The tripterine has antibacterial effect on all vancomycin sensitive and resistant strains, and the MIC range is 0.5-4 mug/mL, which possibly shows that the tripterine has antibacterial activity through a mechanism different from the mechanism of action of the vancomycin, and is not easy to generate cross drug resistance with the vancomycin.

As shown in fig. 1, tripterine has a concentration-dependent antibacterial effect on enterococci. For all tested strains, at 24 hours post inoculation, tripterine showed bactericidal effects against enterococcus faecalis ATCC700802, ATCC51575, enterococcus faecium ATCC700221, ATCC51559, respectively, at a concentration of 4 MIC. Tripterine at 2MIC concentration per strain showed bacteriostatic activity for these four strains and lasted for 24 hours. For MIC concentrations, enterococcus faecalis ATCC700802 was still inhibited by tripterine after 24 hours of culture, while bacterial growth was observed in the other three strains. 1/4 and 1/2MIC of tripterine did not show significant antimicrobial activity against all strains.

Biofilms are associated with refractory infections such as endocarditis, osteomyelitis, chronic wound infections, catheter-related infections, and artificial joint infections, and have also been used as models for studying drug resistance. The invention further evaluates the effect of tripterine on VRE biofilm. As a common antibiotic for clinically treating enterococcus infection, ampicillin or vancomycin has little effect on clearing a biofilm. In contrast, more than 50% of enterococcus faecalis ATCC700802 biofilm was cleared by 16 μg/mL of tripterine after 24 hours of exposure (FIG. 2).

Table 1 minimum inhibitory concentration of Tripterygium wilfordii and vancomycin against 100 enterococcus standard strains and clinically isolated strains

Example 2 in vitro synergistic effects of Tripterine and vancomycin

1. Experimental method

The 23 VRE strains (including 5 standard strains and 18 randomly selected clinical isolates) were evaluated for their synergistic effect with vancomycin using a microdilution checkerboard method (Wang et al, 2019). Briefly, the bacterial solution was added to wells of 96-well plates containing serial dilutions of tripterine and vancomycin at a bacterial load of 5×10 ⁵ CFU/mL. The range of the tripterine and vancomycin diluent used in the chessboard method is 0.125-8 mug/mL and 0.25-256 mug/mL respectively. After 24 hours of incubation at 37 ℃, the Fractional Inhibitory Concentration Index (FICI) was calculated to analyze the combined effect of tripterine and vancomycin using the following equation, and the experiment was repeated three times:

fici= (MIC of drug a combination/MIC of drug a alone) + (MIC of drug B combination/MIC of drug B alone)

When FICI is less than or equal to 0.5, the two are cooperated; FICI is not less than 0.5 and not more than 4, and antagonism is realized when FICI is more than 4.

Sterilization curve experiments were performed on enterococcus faecalis ATCC700802 and enterococcus faecium ATCC700221 to evaluate tripterineAnd the bactericidal kinetics of vancomycin. The time sterilization curve study of the combined drug is similar to that of single tripterine. Each strain included sterile liquid as a growth control. Tripterine and vancomycin were tested at sub-MIC concentrations for each drug. Colony counts were determined at 0, 2, 4, 6, 8 and 24 hours. Synergism is defined as a 24-hour reduction of the viable count by ≡2 log% or more in the combination group compared to the more active single group ₁₀ CFU/mL, 24-hour viable count of the simultaneous combination group is reduced by more than or equal to 2log compared with the initial inoculum size ₁₀ CFU/mL。

2. Experimental results

The results of the chessboard method are shown in Table 2. When vancomycin is used in combination with 1/8-1/4MIC (0.25-1. Mu.g/mL) of tripterine, the inhibitory concentration of vancomycin on the strain is significantly reduced to 1/128-1/4MIC (2-16. Mu.g/mL). According to FIC index, tripterine and vancomycin combined show synergistic effect on 23 strains of enterococci.

The results of the sterilization curves when tripterine and vancomycin are combined are shown in figure 3. Vancomycin alone has poor inhibitory activity against bacterial growth at sub-MIC concentrations. The single use of celastrol has a weak inhibitory effect on bacterial growth, and bacterial regeneration is observed 6-24 hours after inoculation. CFU counts of vancomycin and tripterine alone were the same as growth control levels of the respective strains over 24 hours. In contrast, the combination of tripterine and vancomycin showed a strong synergistic effect on all 2 tested strains. After 24 hours of culture, the combined use of the tripterine and vancomycin can obviously reduce the number of living bacteria by more than 3log10 CFU/mL compared with the single use of the tripterine and vancomycin. After 24 hours, inhibition of growth of all strains was still observed. Furthermore, the combination of CEL-vancomycin reduced the viable count by 2log10 CFU/mL compared to the initial inoculum size.

Table 2 results of chessboard method experiments on 23 enterococci with Tripterygium wilfordii and vancomycin combination

EXAMPLE 3 in vivo synergistic action of Tripterine and vancomycin

1. Establishment of larva infection model of Chilo suppressalis

The larvae of Chilo suppressalis were allowed to stand at room temperature for 24 hours. Larvae with a body weight between 270 and 330mg and a size of about 2cm were selected for the test. Enterococcus faecalis ATCC700802 and enterococcus faecium ATCC700221 were streaked on BHI agar plates and grown overnight in BHI. Overnight cultures of both strains were washed with PBS and adjusted to the appropriate density (about 2 x 10 ⁸ CFU/mL), 10 μl of bacterial liquid was injected into the right hind paw of the larvae with a glass microinjector. 1 hour after infection, larvae were injected with vancomycin, tripterine or a combination of vancomycin and tripterine via the left foot, 10 in each group. In addition, 10 larvae were injected with 10 μl PBS as a negative control. Larvae were incubated at 35 ℃ and monitored continuously for 96 hours after infection. When larvae were unresponsive to mechanical stimulus, they were judged to be dead and survival curves were plotted for each group.

Multiple comparison analysis of survival data was performed by log rank test in combination with Bonferroni correction using Kaplan-Meier survival analysis of GraphPad Prism 8.

2. Results

The combination of cel-vancomycin was examined for protection against lethal infection of the VRE strains enterococcus faecalis ATCC700802 and enterococcus faecium ATCC700221 against larvae of Chilo suppressalis. 2X 10 ⁶ After bacterial infection of the larvae by CFU, 10. Mu.l PBS, 0.25mg/kg CEL, 40mg/kg vancomycin or CEL-vancomycin combination were injected. All larvae treated with PBS, CEL or vancomycin alone died within 48 hours after VRE inoculation. CEL or vancomycin alone is insufficient to provide protection, whereas 80% of larvae infected with enterococcus faecalis ATCC700802 and 90% of larvae infected with enterococcus faecium ATCC700221 survive for more than 96 hours after combination, with the combination significantly extending survival time (FIG. 4). Thus, CEL and vancomicThe combined use of the mildews has effective protection effect on the lethal VRE infection.

EXAMPLE 4 cell action mechanism Studies

1. Scanning Electron Microscope (SEM)

Overnight cultures of enterococcus faecalis ATCC700802 and Bacillus subtilis ATCC 21332 were diluted 1:100 in CAMH broth and incubated at 37℃to log phase (OD _600nm =0.4), then bacteria were cultured in a medium with or without sub MIC level CEL for 4 hours, washed with pbs, fixed with 2.5% glutaraldehyde for 24 hours, dehydrated by ethanol gradient, dried, and gold sprayed, and observed by scanning electron microscopy (japanese SU 8020).

2. Docking analysis

Small molecule docking studies were performed using 3D structure (PDB number: 5MN 4) of FtsZ protein (ID in NCBI: 60893384) using Discovery Studio 4.5 software. Proteins were regularized for determination of important amino acids in predicted binding pockets. After energy minimization, libdock is used to dock all conformations of CEL with sites of the selected active cavity. The docking compounds were scored according to their manner of binding at the binding site. 3. Surface Plasmon Resonance (SPR) analysis

SPR analysis was performed on a BIAcore T200 biosensor system (piscataway GE Healthcare Life Sciences, new jersey, usa) using CM5 chips at 25 ℃. CEL (25, 12.5, 6.25, 3.1, 1.6. Mu.M) was combined with FtsZ in 1 XPBS-P+ (GE Healthcare Life Sciences) at a flow rate of 20. Mu.L/min for 120s. After each binding reaction, the signal was returned to baseline with a dissociation time of 60 s. Kd values were calculated using Biacore T200 evaluation software (piscataway GE Healthcare Life sciences version 2.0, new jersey, usa). 4. GTPase Activity assay

The GTPase activity of recombinant enterococcus faecalis FtsZ protein was determined as described in the literature using the Cytophos phosphate assay biochemical kit (Cytoskeleton, USA) (Sun et al, 2017). Enterococcus faecalis FtsZ protein (0.5. Mu.M) was mixed with solvent (1% dimethyl sulfoxide) or CEL (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32. Mu.g/mL) at 20mMIncubation in Tris buffer (pH 7.5) was performed for 10min at room temperature. Then 5mM MgCl is added ₂ And 200mM KCl. The reaction was started by adding 500. Mu.M GTP and was reacted at 37 ℃. After 30 minutes, the reaction was stopped by adding 140. Mu.L of Cytophos reagent and incubated for 10 minutes. Inorganic phosphate was quantified by measuring absorbance at 650nm using a microplate reader (Bio Rad laboratories, inc. In the united kingdom). Determination of relative IC by nonlinear regression using sigmoidal concentration response curves in GraphPad Prism 9 (GraphPad Software, la Jolla, calif.) ₅₀ Values. All experiments were independently analyzed in triplicate.

5. Experimental results

Bacterial cell morphology changes can often provide valuable clues to the mechanism of antimicrobial action and are often used in preliminary mechanism of action studies. The mechanism of the antibacterial action of CEL is first studied deeply by scanning electron microscope observation of bacterial cell morphology. As shown in fig. 5, treatment with CEL did not cause any observable bacterial surface abnormalities compared to untreated cells. However, in the CEL treated group, a significant bacterial growth was observed, in the form of a "sugarcoated haws", indicating inhibition of bacterial division. To further confirm our hypothesis, we performed a cell morphology study with bacillus subtilis. The length of bacillus subtilis increases significantly after CEL addition compared to untreated cells. Thus, we speculate that CEL shows its antibacterial activity by inhibiting bacterial division.

Because of the important role and high conservation of filamentous temperature-sensitive mutant Z (FtsZ) in all bacteria, it has become a target of great interest for new antibacterial drugs (Schaffner Barbero et al 2012). FtsZ recruits other proteins to co-drive bacterial division and the formation of new cell poles through GTP-dependent polymerization into fibrils and further self-assembly into the Z-ring, a key organelle of bacterial cell division.

Based on findings under Scanning Electron Microscopy (SEM) and the important role of FtsZ proteins in bacterial cell division, we propose the hypothesis that CEL may be antibacterial by inhibiting the function of FtsZ and its associated biological activity. We predicted that FtsZ might be one of the targets for the antibacterial action of CEL.

The present invention makes molecular docking between CEL and FtsZ for the first time. Discovery Studio 4.5 software was used for molecular docking. As shown in fig. 6, CEL fits well with the active pocket of FtsZ protein with a docking score of 113.1. The predicted interactions of small molecules with proteins include two hydrogen bonds between the oxygen atom on the x-carbonyl or x-carboxyl group and the ARG143 or THR133 residues, and one Pi anion bond between ring a and GLU 139. Together, these interactions promote binding, suggesting that FtsZ proteins may be direct targets for CEL.

To further confirm the interaction between CEL and FtsZ, the binding between the two was analyzed by SPR. The results showed that CEL can bind to FtsZ dose dependently with Kd values of 2.454 μm (fig. 7), indicating a strong binding capacity to FtsZ.

Since the assembly kinetics of FtsZ are thought to be regulated by its GTPase activity, the inhibition of enterococcus faecalis FtsZ GTPase activity by CEL was assessed. It was found that CEL significantly inhibited the GTPase activity, IC, of the enterococcus faecalis FtsZ protein ₅₀ The value was 1.04.+ -. 0.17. Mu.g/mL (FIG. 8). The GTPase inhibition activity correlates well with antibacterial activity, indicating that the compound interferes with bacterial growth through binding to FtsZ and the GTPase function inhibition mechanism. The antibacterial mechanism of CEL, as opposed to antibiotics, also accounts for its synergistic effect with vancomycin.

In summary, CEL has antibacterial and anti-biofilm activity against enterococci (including VRE strains) and restores vancomycin activity against VRE in vitro and in vivo. In general, CEL is expected to be a new antibacterial agent and antibacterial adjuvant, providing a new therapeutic option for combating VRE.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Reference is made to:

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[3]Miro,J.M.,Pericas,J.M.,del Rio,A.and Hospital Clinic Endocarditis Study,G.,2013.A new era for treating Enterococcus faecalis endocarditis:ampicillin plus short-course gentamicin or ampicillin plus ceftriaxone:that is the question！Circulation 127,1763-1766.

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Claims (1)

1. the application of the antibacterial pharmaceutical composition in preparing antibacterial biological products; wherein the bacteria are selected from vancomycin-resistant enterococcus faecium and vancomycin-resistant enterococcus faecium;

the active ingredients of the antibacterial pharmaceutical composition are tripterine and vancomycin;

the mass ratio of the tripterine to the vancomycin is 1:16-1:2.