Use of a combination of sanguilutins and gentamicin in the preparation of a medicament for the treatment of MRSA persister infections

Abstract

The present application relates to the use of quercetin in the preparation of methicillin-resistant Staphylococcus aureus (MRSA) biofilm and persister inhibitor. By biofilm formation inhibition experiment, it is found that quercetin can significantly inhibit the formation of MRSA biofilm, and by fluorescence quantitative PCR test, it is found that quercetin significantly reduces the transcription of MRSA biofilm related genes, and by persister killing test, it is found that quercetin enhances the killing ability of gentamicin to MRSA persister, and by lung epithelial cell adhesion test, it is found that quercetin significantly reduces the adhesion of MRSA strain to epithelial cells. By constructing a Galleria mellonella model infected with MRSA, it is found that quercetin shows a systemic protective effect on the Galleria mellonella model infected with MRSA.

Description

Technical Field

[0001] This invention belongs to the field of medical and pharmaceutical technology, specifically relating to the use of acetic acid from *Pseudomonas aeruginosa* in the preparation of MRSA biofilms and inhibitors of persistent bacteria. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (S. aureus) is a Gram-positive, facultative anaerobic pathogen widely distributed in the natural environment and on human surfaces, and is one of the leading pathogens of hospital-acquired infections and community-acquired infections. This bacterium can cause a variety of serious diseases, including skin and soft tissue infections, pneumonia, endocarditis, sepsis, and toxic shock syndrome. In particular, the widespread prevalence of methicillin-resistant Staphylococcus aureus (MRSA) significantly increases the difficulty of clinical treatment and poses a serious threat to global public health security. The resistance of Staphylococcus aureus to multiple antibiotics and its ability to form biofilms on medical device surfaces further complicate infection control, resulting in a huge medical and economic burden.

[0003] In methicillin-resistant Staphylococcus aureus (MRSA) infections, biofilm formation is one of the key reasons for persistent infection and difficulty in eradication. Biofilms not only provide a physical barrier for the bacterial flora, hindering antibiotic penetration and immune cell clearance, but can also induce some bacteria to transform into metabolically dormant, antibiotic-resistant "persistent bacteria," leading to recurrent and chronic infections. Traditional antibiotics primarily target actively growing bacteria, significantly reducing their effectiveness against bacteria within biofilms and persistent bacteria, making the treatment of MRSA-related chronic infections (such as implant infections, chronic wound infections, and pneumonia) extremely difficult.

[0004] Currently, the treatment of MRSA still relies primarily on traditional antibiotics. However, long-term use of these antibiotics not only accelerates the spread of drug resistance but also fails to effectively eliminate biofilms and persistent bacteria, resulting in insufficient clinical efficacy and high recurrence rates. Therefore, exploring novel strategies that can intervene in biofilm formation, eliminate persistent bacteria, or inhibit their key virulence mechanisms has become an important research direction in addressing MRSA infection.

[0005] Pseudolaric acid B (PAB) is a natural diterpenoid compound, mainly isolated from the bark of the traditional Chinese medicine Pseudolaricis, and is commonly used in traditional medicine to treat dermatophyte infections. Studies have shown that Pseudolaric acid B possesses various biological activities, including antifungal, antitumor, anti-inflammatory, and anti-angiogenic effects, with a unique mechanism of action and abundant natural sources. Although previous studies have reported that Pseudolaric acid B inhibits the growth and biofilm formation of some pathogenic microorganisms, there are currently no publicly available results regarding its specific targeting of MRSA biofilms and persistent bacteria, interference with early bacterial adhesion, or reduction of pathogenicity in the host.

[0006] Based on this, exploring the new uses of acetic acid from *Pseudolarix amabilis* as an inhibitor of MRSA biofilms and persistent bacteria not only provides innovative ideas for the development of antiviral drugs, but also opens up sustainable strategies for solving the problem of drug-resistant bacterial infections, which has important scientific value and application prospects. Summary of the Invention

[0007] The technical problem to be solved: The purpose of this invention is to study the use of *Euonymus alatus* bark extract acetic acid in the preparation of MRSA biofilm and persistent bacteria inhibitors. Starting from MRSA biofilms and persistent bacteria, it was found that the natural Chinese herbal monomer compound *Euonymus alatus* bark extract acetic acid reduces the formation of MRSA biofilms and their adhesion to human lung epithelial cells by decreasing the transcription of MRSA biofilm and adhesion-related genes. Furthermore, persistent bacteria can cause persistent recurrent infections, increasing the difficulty of treatment. This study found that *Euonymus alatus* bark extract acetic acid can enhance the bactericidal effect of gentamicin against persistent MRSA bacteria. Based on this, *Euonymus alatus* bark extract acetic acid inhibits the adhesion of MRSA to human lung epithelial cells and shows a protective effect in a *R. RMSA* infection model of *R. RMSA*. The above results indicate that *Euonymus alatus* bark extract acetic acid has good potential for the prevention and treatment of chronic recurrent RMSA infection.

[0008] Technical solution: The use of acetic acid in the preparation of MRSA biofilms and persistent bacteria inhibitors.

[0009] Furthermore, the structural formula of the acetic acid from *Pseudolarix amabilis* bark is as follows: .

[0010] Furthermore, the acetic acid obtained from *Pseudolarix amabilis* bark is extracted from *Pseudolarix amabilis* bark and has a purity of ≥98%.

[0011] Furthermore, the minimum inhibitory concentration of the acetic acid in *Pseudomonas spp.* is >128 µg / mL.

[0012] Furthermore, the use of the acetic acid derived from *Eucommia ulmoides* or its prepared MRSA biofilm and persistent bacteria inhibitor in the preparation of MRSA infection drugs.

[0013] Beneficial effects: This invention found that MRSA exhibited the same growth trend when co-cultured with different concentrations of PAB, indicating that PAB does not affect MRSA growth. In addition, PAB significantly inhibited MRSA biofilm formation by reducing the transcription of MRSA biofilm-related genes.

[0014] This invention, by inducing MRSA to form persistent bacteria and then co-culturing them with gentamicin and PAB, found that PAB can enhance the killing effect of gentamicin on persistent MRSA bacteria.

[0015] This invention found through a lung epithelial cell adhesion assay that PAB can significantly reduce the adhesion of MRSA to epithelial cells; and through an experiment to construct a large wax moth infection model, it was found that PAB treatment significantly improved the survival rate of MRSA-infected large wax moths and alleviated the symptoms of MRSA-infected large wax moths, demonstrating a protective effect. Attached Figure Description

[0016] Figure 1 Figure showing the effect of PAB on MRSA growth; Figure 2 The diagram shows the effect of PAB on MRSA biofilm formation, where A represents the effect of PAB on MRSA biofilm formation and B is a crystal violet staining diagram. Figure 3 Figure showing the effect of PAB on the transcription of MRSA biofilm and adhesion-related genes; Figure 4 The effect of PAB on the killing effect of gentamicin on persistent MRSA bacteria is shown in the figure. Figure 5 Figure showing the effect of PAB on the adhesion of MRSA to human lung epithelial cells; Figure 6 This diagram illustrates the protective effect of PAB against MRSA infection in a large wax moth model. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: The PAB is extracted from the bark of *Pseudolarix amabilis* and has a purity of ≥98%.

[0018] Example 1: Effect of acetic acid on the growth of MRSA (Pseudomonas aeruginosa). MRSA and different concentrations of PAB were co-cultured at 37℃ with shaking. Every 2 hours, an appropriate amount of sample was taken to detect the absorbance at 600 nm to analyze the effect of PAB on MRSA growth.

[0019] Depend on Figure 1 It is evident that MRSA exhibits the same growth trend when co-cultured with different concentrations of PAB, indicating that PAB does not affect the normal growth of MRSA.

[0020] Minimum inhibitory concentration (MIC) assay: LB medium containing a series of gradient concentrations of PAB was prepared in 96-well cell culture plates, and then MRSA strain was added to achieve a final concentration of 5 × 10⁻⁶. 5 The sample was incubated at 37°C for 24 hours with CFUs / mL, and the minimum inhibitory concentration of PAB against MRSA was observed.

[0021] The minimum inhibitory concentration of PAB against MRSA is higher than 128 µg / mL (Table 1), indicating that PAB has no antibacterial activity, does not exert selective pressure on bacterial growth, and does not induce bacterial resistance.

[0022] Table 1. Minimum inhibitory concentration (MIC) values ​​of PAB against MRSA

[0023] Example 2: Effect of PAB on MRSA biofilm formation MRSA was co-cultured with different concentrations of PAB at 37℃ for 24 hours. The culture medium was discarded, and the samples were washed three times with sterile phosphate (PBS). After drying, the samples were stained with crystal violet for 20 minutes. After discarding the excess staining solution, the samples were washed three times with PBS and dried. The samples were then treated with glacial acetic acid, and the absorbance at 570 nm was measured to analyze the effect of PAB on MRSA biofilm formation.

[0024] Depend on Figure 2 It is evident that when MRSA was not treated with PAB, a large amount of biofilm formed. However, when different concentrations of PAB were used to treat MRSA, the amount of biofilm formation gradually decreased with increasing concentration. Figure 2 (A-2B) indicates that PAB can dose-dependently inhibit the formation of MRSA biofilms.

[0025] Example 3: Effects of PAB on MRSA biofilm and transcription of adhesion-related genes MRSA and PAB (32 µg / mL) were co-cultured at 37°C for 8 hours. Bacterial cells were collected and washed three times with sterile PBS. Bacterial RNA was obtained using the trizol method, and cDNA was then obtained using a reverse transcription kit. Quantitative PCR was performed using the cDNA as a template to analyze the effect of PAB on the transcription of biofilm and adhesion-related genes.

[0026] After MRSA was treated with PAB, the transcriptional levels of genes related to biofilm and adhesion, fnbA, fnbB, clfA, and clfB, were significantly reduced. Specifically, in the PAB-treated group, fnbA, fnbB, clfA, and clfB were reduced to 47.33%, 59%, 39.67%, and 53.67% of those in the control group, respectively. Figure 3 This indicates that PAB reduces the transcription of biofilm and adhesion-related genes.

[0027] Example 4: Effect of PAB on the killing of persistent MRSA bacteria by gentamicin MRSA cultured overnight was treated with gentamicin for 3 hours, bacteria were collected and washed three times with sterile PBS, and the bacterial density was adjusted to approximately 1×10⁻⁶. 6CFUs / mL, gentamicin and different concentrations of PAB were added and co-cultured at 37℃ for 3 hours. An appropriate amount of sample was spread on LB agar medium and cultured overnight. The number of colonies was counted and the effect of PAB on the killing of MRSA residual bacteria by gentamicin was analyzed.

[0028] The bacteria that survived overnight cultured MRSA strains treated with gentamicin for 3 hours were considered persistent bacteria. Subsequent gentamicin treatment failed to kill MRSA, further indicating that these bacteria formed persistent bacteria. However, when different concentrations of PAB were added, the logarithmic bacterial density showed a significant decreasing trend. Figure 4 This indicates that PAB can assist gentamicin in killing persistent MRSA bacteria.

[0029] Example 5: Effect of PAB on the adhesion of MRSA to human lung epithelial cells Human lung epithelial cells (A549) were seeded in 24-well cell culture plates and cultured overnight. The following day, MRSA and different concentrations of PAB were added and treated for 1.5 hours. The culture medium was discarded, and the cells were washed three times with sterile PBS, collected, appropriately diluted, and plated on LB medium for overnight culture. Colony counts were performed to analyze the effect of PAB on MRSA adhesion to host cells.

[0030] Depend on Figure 5 It can be seen that the colony count of samples treated with MRSA but not with PAB was recorded as 100. When different concentrations of PAB were used to treat the samples, the adhesion of MRSA to A549 cells decreased to 56.97% and 42.18%, respectively, indicating that PAB can reduce the adhesion of MRSA to human lung epithelial cells.

[0031] Example 6: Protective effect of PAB on a MRSA-infected model of the large wax moth. MRSA was injected into the large wax moth to establish an MRSA infection model. The infected moths were then treated with PAB (50 mg / kg), designated as the PAB-treated group. The infected group received an equal volume of solvent, while the normal group received an equal volume of sterile PBS. The survival rate of the large wax moths was monitored at specific time points, and the protective effect of PAB against MRSA infection was analyzed. The alleviating effect of PAB on the symptoms of MRSA infection was also analyzed by observing different groups of samples.

[0032] Mortality was observed in the MRSA-infected group and the PAB-treated group of large wax moths at 24 and 48 hours, respectively. When the infection time was extended to 120 hours, all large wax moths in the infected group died, with a survival rate of 0%, while the survival rate of large wax moths in the PAB-treated group was 56.67%. Figure 6 This indicates that PAB can delay the death time of MRSA-infected giant wax moth, providing valuable time for clinical treatment, and can significantly improve the survival rate of MRSA-infected giant wax moth.

[0033] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. Use of the combination of acetic acid from *Pseudomonas aeruginosa* bark and gentamicin in the preparation of drugs for treating persistent MRSA infections.

2. The use according to claim 1, characterized in that: The acetic acid obtained from *Pseudolarix amabilis* bark is extracted from *Pseudolarix amabilis* bark and has a purity of ≥98%.

3. The use according to claim 1, characterized in that: The minimum inhibitory concentration of the acetic acid in *Pseudomonas spp.* is >128 µg / mL.

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