A bacteriocin and its use in clearing methicillin-resistant staphylococcus aureus biofilm

Abstract

The application discloses a bacteriocin and application thereof in removing methicillin-resistant Staphylococcus aureus biofilm. A novel bacteriocin PFB252 is separated and identified, the bacteriocin is composed of 102 amino acids, and the relative molecular weight is 11267.34 Da. Research shows that the bacteriocin PFB252 has good thermal stability and pH stability and is acid-resistant, and the antibacterial activity still remains at 76.23%-95.20% after treatment of different proteases. The bacteriocin PFB252 can inhibit the growth of methicillin-resistant Staphylococcus aureus, reduce the biofilm formation amount of bacteria, the metabolic activity in the biofilm and the content of extracellular polysaccharide, and also can reduce the expression of biofilm-related genes, can effectively remove the methicillin-resistant Staphylococcus aureus biofilm, overcome the drug resistance of the methicillin-resistant Staphylococcus aureus, and has important significance for preventing and treating the methicillin-resistant Staphylococcus aureus in clinic.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a bacteriocin and its application in eliminating methicillin-resistant Staphylococcus aureus biofilms. Background Technology

[0002] Staphylococcus aureus ( Staphylococcusaureu Staphylococcus aureus is a Gram-positive bacterium and a major pathogen causing mastitis in dairy cows, posing a significant threat to livestock health. Furthermore, Staphylococcus aureus is considered a prevalent and widespread foodborne pathogen that can spread through contaminated food and produce toxins. The widespread use of antibiotics has led to the emergence of drug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA). Staphylococcusaureus Methicillin-resistant Staphylococcus aureus (MRSA) poses a persistent challenge to livestock farming, the food industry, and human health. Biofilms are bacterial aggregates that adhere to the surface of living or non-living solids and encapsulate themselves within the extracellular matrix, enhancing bacterial resistance to antibiotics. Due to the complex structure of MRSA biofilms and their high resistance to traditional antibiotics, current research has explored novel antibiotics and combination therapies to synergistically enhance the inhibition of MRSA and the clearance of biofilms. However, the use of kinase inhibitors and combinations of paroxetine (an antidepressant) and oxacillin still presents some adverse reactions. Therefore, exploring new natural, safe, and effective anti-biofilm drugs to prevent and treat methicillin-resistant Staphylococcus aureus is of great significance.

[0003] Bacteriocins are proteins or polypeptides synthesized by bacterial ribosomes that can effectively inhibit or kill pathogenic bacteria. Many bacteriocins have been shown to inhibit pathogenic bacteria by suppressing biofilm formation. For example, bacteriocin Z057 effectively reduces the biomass and cell viability of Vibrio parahaemolyticus biofilms, and bacteriocin HW01 produced by Pediococcus lactis inhibits the formation of Pseudomonas aeruginosa biofilms and the production of virulence factors. However, bacteriocins that can inhibit methicillin-resistant Staphylococcus aureus and clear its biofilms are still lacking. This invention is proposed based on this deficiency. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of existing bacteriocins that inhibit methicillin-resistant Staphylococcus aureus and can remove its biofilm, and to provide a bacteriocin and its application in removing methicillin-resistant Staphylococcus aureus biofilm.

[0005] The first objective of this invention is to provide a bacteriocin PFB252.

[0006] A second objective of this invention is to provide a method for preparing bacteriocin PFB252.

[0007] A third objective of this invention is to provide the application of bacteriocin PFB252.

[0008] The fourth objective of this invention is to provide a product.

[0009] The fifth objective of this invention is to provide a strain of Bacillus belyssus and its applications.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention provides a bacteriocin PFB252, the amino acid sequence of which is: KALDKEVSEGSVDQSSWSREFELDENDGAQVNFWISNNGSNDIKISIDGKVSRTIAPGKQGNISAEVKNGWFGWTYNYTFTASPTAQGGSVKMKYKIAQRNN.

[0012] This invention purified and identified a novel bacteriocin, PFB252, from the cell-free supernatant of *Bacillus belye* isolated from seawater. This bacteriocin consists of 102 amino acids with a relative molecular weight of 11267.34 Da. Sequencing homology analysis showed that PFB252 is not homologous to other class II bacteriocins, classifying it as a novel bacteriocin. Studies showed that PFB252 exhibits good thermal stability, retaining 67.07% of its antibacterial activity after treatment at 100℃ for 1 h. It also demonstrates good pH stability and acid resistance, showing little change in antibacterial activity after overnight storage at 4℃ under pH 2-6 conditions. Furthermore, its antibacterial activity remained at 76.23%-95.20% after treatment with pepsin, proteinase K, papain, and trypsin, exhibiting good stability and tolerance. Studies have shown that the bacteriocin PFB252 can inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA), reduce the amount of bacterial biofilm formation, the metabolic activity of bacteria within the biofilm, and the content of extracellular polysaccharides. It can also reduce the expression of biofilm-related genes, clear MRSA biofilms, and overcome the drug resistance of MRSA. The bacteriocin provided by this invention can effectively curb the spread of drug-resistant bacteria and reduce public health risks, and has important clinical and public health significance.

[0013] This invention provides a method for preparing bacteriocin PFB252, which involves using Bacillus belye (… Bacillus velezensis The culture medium was inoculated, and after centrifugation, the supernatant was collected, filtered through a filter membrane to remove bacteria, and then purified to obtain cell-free supernatant.

[0014] Preferably, the purification steps consist of sequential acid precipitation, gel column chromatography, anion exchange chromatography, and reversed-phase high-performance liquid chromatography.

[0015] Preferably, the Bacillus belyss is strain FB25-2, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 9, 2025, with accession number GDMCC No: 66485.

[0016] As a more preferred embodiment, the present invention provides a specific method for preparing bacteriocin PFB252: *Bacillus belyeis* FB25-2 is inoculated into LB liquid medium and cultured overnight at 37°C. The bacterial culture is then inoculated into fermentation medium at a concentration of 2% and cultured at 30°C and 180 r / min for 24 h in a shaking incubator. The supernatant is then collected by centrifugation at 4°C for 30 min and filtered through a 0.22 μm filter membrane to remove bacteria, yielding a cell-free supernatant. Acid precipitation is then performed to obtain crude bacteriocin: the pH of the cell-free supernatant is adjusted to 2.0 using a 6 mol / L hydrochloric acid solution and allowed to stand overnight at 4°C. The precipitate is collected by centrifugation (4°C, 8000 rpm, 30 min), then redissolved in phosphate-buffered saline (PBS) at room temperature and filtered again through a 0.22 μm membrane membrane to remove bacterial cells. Subsequently, bacteriocins were purified by gel column chromatography: the crude bacteriocin extract was purified using TA-GF75 gel column chromatography. 1 mL of the crude extract was added to a TA-GF75 gel column pre-equilibrated with deionized water at a flow rate of 1 mL / min, and the fraction with antibacterial activity was collected using an automated collector. The bacteriocins were then purified by anion exchange chromatography: the fraction was added to a Tiderose Q HP anion exchange column equilibrated with 0.05 mol / L sodium chloride at a flow rate of 3 mL / min, and the fraction with antibacterial activity was collected again. The bacteriocin was then purified by reversed-phase high-performance liquid chromatography (RP-HPLC): the fraction with antibacterial activity was purified by RP-HPLC using solvent A [0.1% (v / v) trifluoroacetic acid dissolved in water] and solvent B [0.1% (v / v) trifluoroacetic acid dissolved in acetonitrile] as eluents. A C18 column (4.6 × 150 mm, 5 μm) was prepared, and the elution was performed at a flow rate of 1 mL / min. The elution process included: 0-10 min, 5% solvent B; 11-20 min, 5%-50% solvent B; 21-30 min, 50%-70% solvent B; 31-38 min, 70%-95% solvent B. Finally, the antibacterial active fraction was collected, concentrated, and filtered for sterilization using a pore size of 0.22 μm. The final sample obtained was bacteriocin PFB252.

[0017] This invention provides the application of bacteriocin PFB252 in the inhibition of methicillin-resistant Staphylococcus aureus for non-disease treatment and diagnostic purposes.

[0018] This invention provides the application of bacteriocin PFB252 in the elimination of biofilms from methicillin-resistant Staphylococcus aureus.

[0019] This invention provides the application of bacteriocin PFB252 in the preparation of antibacterial products or biofilm removal products.

[0020] This invention provides a product containing bacteriocin PFB252.

[0021] The present invention also provides a strain of Bacillus belyssus FB25-2, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 9, 2025, with accession number GDMCC No: 66485.

[0022] This invention provides the application of the above-mentioned Bacillus berleis FB25-2 strain in the preparation of bacteriocins.

[0023] The present invention has the following beneficial effects:

[0024] This invention yielded a novel bacteriocin, PFB252, composed of 102 amino acids with a relative molecular weight of 11267.34 Da. Sequencing homology analysis showed that PFB252 is not homologous to other class II bacteriocins, classifying it as a novel bacteriocin. Studies have shown that PFB252 exhibits good thermal stability and protease and pH tolerance. Furthermore, PFB252 inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA), reduces biofilm formation, metabolic activity within the biofilm, and extracellular polysaccharide content. It also lowers the expression of biofilm-related genes, clearing MRSA biofilms and overcoming methicillin-resistant Staphylococcus aureus resistance. The bacteriocin provided by this invention can effectively curb the spread of drug-resistant bacteria and reduce public health risks, possessing significant clinical and public health implications. Attached Figure Description

[0025] Figure 1 This is a phylogenetic tree diagram of strain FB25-2.

[0026] Figure 2 The purification results of bacteriocin PFB252 are shown in the figure (A: TA-GF75 gel chromatography; B: Tiderose Q HP anion exchange chromatography; C: reversed-phase high performance liquid chromatography (RP-HPLC)).

[0027] Figure 3 This is a graph showing the SDS-PAGE analysis results of bacteriocin PFB252.

[0028] Figure 4The image shows the homology comparison results of the amino acid sequences of bacteriocin PFB252 with those of other bacteriocins (A: comparison of bacteriocin PFB252 with bacteriocin Cerecyclin (AP03198); B: comparison of bacteriocin PFB252 with bacteriocin Sil (AP02399); C: comparison of bacteriocin PFB252 with bacteriocin Microcin PDI (AP02015); D: comparison of bacteriocin PFB252 with bacteriocin Microcin S (AP02014); E: comparison of bacteriocin PFB252 with bacteriocin BM1122 (AP03290)).

[0029] Figure 5 The structure prediction diagram of bacteriocin PFB252 is shown in Figure A (secondary structure prediction; B (tertiary structure prediction)).

[0030] Figure 6 Stability results of bacteriocin PFB252 (A: temperature; B: pH; C: enzyme).

[0031] Figure 7 The effect of different concentrations of bacteriocins on the growth of methicillin-resistant Staphylococcus aureus.

[0032] Figure 8 The effect of bacteriocin PFB252 on the amount of biofilm formation in methicillin-resistant Staphylococcus aureus.

[0033] Figure 9 The effect of bacteriocin PFB252 on the metabolic activity of bacteria within the biofilm of methicillin-resistant Staphylococcus aureus.

[0034] Figure 10 The effect of bacteriocin PFB252 on extracellular polysaccharides in biofilms.

[0035] Figure 11 To investigate the effects of different concentrations of bacteriocin PFB252 on genes related to biofilm formation in methicillin-resistant Staphylococcus aureus.

[0036] Figure 12 The effect of different concentrations of bacteriocin PFB252 on methicillin-resistant Staphylococcus aureus biofilm (A: Control; B: 1 / 32 MIC; C: 1 / 16 MIC).

[0037] Note: In the chart P value(* P <0.05,** P <0.01, *** P <0.001) indicates a statistically significant difference. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] The methicillin-resistant Staphylococcus aureus CICC25138 (MRSA) used in the examples was purchased from the China Industrial Microbial Culture Collection Center. The strain was cultured overnight at 37°C in tryptone soybean agar (TSA) liquid medium.

[0041] The fermentation medium used was prepared by dissolving glucose (4.0 g), beef extract (0.5 g), yeast extract (0.5 g), peptone (1.5 g), NaH2PO4·2H2O (0.02 g), Na2HPO4·12H2O (0.05 g), MgSO4·7H2O (0.05 g), CaCl2·6H2O (0.05 g), and MnSO4 (0.02 g) in distilled water, with a volume of 100 mL.

[0042] LB liquid and LB solid media were purchased from Beijing Luqiao Technology Co., Ltd.; TSA was purchased from Beijing Solarbio Technology Co., Ltd.

[0043] The results in this example were statistically analyzed using GraphPad Prism8, employing one-way ANOVA followed by Tukey's test.

[0044] Example 1: Identification of the strain and preparation of CFS

[0045] 1. Screening and identification of strains

[0046] In this embodiment, a bacterium FB25-2 was isolated from seawater. After extracting bacterial DNA, the 16S rRNA gene sequence was amplified, and the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for subsequent testing.

[0047] The predicted sequences were compared with the NCBI database, and a phylogenetic tree was constructed, such as... Figure 1 As shown, strain FB25-2 and Bacillus velezensis Based on the high homology and morphological identification, the taxonomic classification of this strain was determined to be *Bacillus belyesense* (…). Bacillus velezensisIt was named strain FB25-2, GenBank accession number: PRJNA1242529, and deposited on June 9, 2025 at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 66485, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0048] 2. Preparation of cell-free supernatant (CFS)

[0049] Bacillus berberis ( Bacillus velezensis FB25-2 was inoculated into LB liquid medium and cultured overnight in a constant temperature incubator at 37°C. The bacterial culture was then inoculated into fermentation medium at a concentration of 2% and cultured in a shaking incubator at 30°C and 180 r / min for 24 h. Subsequently, the supernatant was collected by centrifugation at 4°C for 30 min and filtered through a 0.22 μm filter membrane to remove bacteria, resulting in cell-free supernatant.

[0050] Example 2 Purification of bacteriocin FFB252

[0051] Crude bacteriocins were obtained by acid precipitation, followed by a series of purification steps including TA-GF75 gel column chromatography, Tiderose Q HP anion exchange chromatography, and reversed-phase high-performance liquid chromatography. Fractions from each purification step were collected, and the activity against methicillin-resistant Staphylococcus aureus was determined by the solid agar perforation method.

[0052] 1. Obtaining crude bacteriocin samples by acid precipitation method

[0053] The pH of the cell-free supernatant was adjusted to 2.0 using 6 mol / L hydrochloric acid solution and allowed to stand overnight at 4°C. The precipitate was collected by centrifugation (4°C, 8000 rpm, 30 min), then redissolved in phosphate-buffered saline (PBS) at room temperature, and the bacterial cells were removed again by filtration through a 0.22 μm membrane to obtain a crude bacteriocin extract.

[0054] 2. Purification of bacteriocins by TA-GF75 gel column chromatography

[0055] The crude bacteriocin extract obtained by acid precipitation was purified using TA-GF75 gel column chromatography. The purification process included adding 1 mL of the crude extract to a TA-GF75 gel column pre-equilibrated with deionized water at a flow rate of 1 mL / min. The fraction was then collected using an automated collector. Four peaks were obtained by TA-GF75 gel column chromatography, collected, and their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) was determined.

[0056] The results are as follows Figure 2 As shown in Figure A, only peak A3 exhibits significant antibacterial activity.

[0057] 3. Purification of bacteriocins using Tiderose Q HP anion exchange chromatography

[0058] Fractions with antibacterial activity obtained from TA-GF75 gel chromatography were added to a Tiderose Q HP anion exchange column equilibrated with 0.05 mol / L sodium chloride at a flow rate of 3 mL / min. These fractions were then collected and their antibacterial activity was evaluated.

[0059] The A3 peak sample obtained above was further eluted using a Tiderose Q HP anion exchange column, yielding 5 peaks, among which peak B5 showed antibacterial effect, such as Figure 2 As shown in B.

[0060] 4. Purification of bacteriocins by reversed-phase high-performance liquid chromatography (RP-HPLC)

[0061] After purification by Tiderose Q HP anion exchange chromatography, the fraction with antibacterial activity was collected and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Solvent A [0.1% (v / v) trifluoroacetic acid dissolved in water] and solvent B [0.1% (v / v) trifluoroacetic acid dissolved in acetonitrile] were used as eluents. A C18 column (4.6 × 150 mm, 5 μm) was prepared, and elution was performed at a flow rate of 1 mL / min. The elution process included: 0–10 min, 5% solvent B; 11–20 min, 5%–50% solvent B; 21–30 min, 50%–70% solvent B; 31–38 min, 70%–95% solvent B. After collection and concentration, the fraction was filtered through a 0.22 μm filter membrane for sterilization, and its antibacterial activity was subsequently evaluated.

[0062] The B5 fraction obtained above was purified by RP-HPLC, yielding six peaks, among which the highest peak showed antibacterial effect, such as... Figure 2 As shown in C, the fraction from this peak was collected and named bacteriocin PFB252 for subsequent identification.

[0063] Example 3 Identification of bacteriocins

[0064] 1. Determination of molecular weight of bacteriocin PFB252 and analysis by liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0065] Gels were prepared using the Tris-Tricine-SDS-PAGE gel kit. Pre-stained ultra-low molecular weight protein marker (3.3 kD~31.0 kD) and bacteriocin PFB252 obtained after final purification in Example 2 were added to the pores and electrophoresis was performed. After electrophoresis, the gels were stained and destained, and the target bands were cut off. The bacteriocin was analyzed by LC-MS / MS and de novo sequencing by Beijing Biotechnology Co., Ltd.

[0066] Electrophoresis results as follows Figure 3 As shown, the estimated molecular weight of bacteriocins is 6.5-14.4 kDa. LC-MS / MS analysis revealed the amino acid sequence of bacteriocin PFB252 to be KALDKEVSEGSVDQSSWSREFELDENDGAQVNFWISNNGSNDIKISIDGKVSRTIAPGKQGNISAEVKNGWFGWTYNYTFTASPTAQGGSVKMKYKIAQRNN. The calculated precise molecular weight of the bacteriocin is 11267.34 Da, consistent with the SDS-PAGE results. Comparison with other Class II bacteriocins yields the following results: Figure 4 As shown, no homology was found, indicating that it belongs to a novel bacteriocin.

[0067] 2. Physicochemical properties and protein structure prediction of bacteriocin PFB252

[0068] The physicochemical properties of bacteriocin PFB252 were analyzed using the ExPASy-ProtParam tool (https: / / web.expasy.org / protparam / ). The SOPMA online website (https: / / npsa.lyon.inserm.fr / cgi-bin / npsa.cgi / ) for protein secondary structure determination was used to predict its secondary structure, while its tertiary structure was predicted using the SWISS-MODEL platform (https: / / swissmodel.expasy.org / ).

[0069] The physicochemical parameters of bacteriocin PFB252 are shown in Table 1, indicating that the amino acid count of bacteriocin PFB252 is 102, the molecular weight is 11267.34 Da, the theoretical isoelectric point is 6.45, and the molecular formula is C. 494 H 757 N 139 O 162 S1. The structural prediction results of bacteriocin PFB252 are as follows: Figure 5 As shown.

[0070] Table 1 Physicochemical parameters of bacteriocin PFB252

[0071]

[0072] Example 4: Determination of the stability and antibacterial properties of bacteriocins

[0073] 1. Stability of bacteriocin PFB252

[0074] To assess temperature stability, bacteriocin PFB252 was treated at different temperatures (40℃, 60℃, 80℃, 100℃, 120℃) for 20 min, and then cooled to room temperature.

[0075] To assess the effect of pH on bacteriocins, bacteriocin PFB252 samples were adjusted to pH levels of 2, 4, 6, 8, 10, and 12, incubated at 37°C for 3 h, and then their original pH values ​​were readjusted.

[0076] To assess the effects of different proteases on bacteriocin activity, proteinase K, pepsin, trypsin, and papain were added to bacteriocin PFB252 to a final concentration of 1 mg / mL. The mixture was incubated at 37°C for 1 h.

[0077] All of the above experiments used untreated bacteriocins as a control, and the antibacterial activity of bacteriocins against methicillin-resistant Staphylococcus aureus was determined by the solid agar perforation method.

[0078] The effects of different temperatures, pH levels, and enzymes on the activity of bacteriocin PFB252 are as follows: Figure 6 As shown, the study revealed that bacteriocin PFB252 exhibits good thermal stability, retaining 67.07% of its antibacterial activity after treatment at 100℃ for 1 h; it also demonstrates good pH stability and acid resistance, with virtually no change in antibacterial activity after overnight storage at 4℃ under pH 2-6 conditions; and its antibacterial activity remained at 76.23%-95.20% after treatment with pepsin, proteinase K, papain, and trypsin, indicating good stability and tolerance.

[0079] 2. Determination of minimum inhibitory concentration (MIC)

[0080] The minimum inhibitory concentration (MIC) of bacteriocin PFB252 against methicillin-resistant Staphylococcus aureus (MRSA) was determined using the microbroth dilution method. Overnight cultured MRSA was inoculated into TSA liquid medium at a concentration of 1% (v / v). Final bacteriocin concentrations were added at 2×MIC, MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC, 1 / 16MIC, and 1 / 32MIC. The medium was incubated at 37°C for 24 h, and OD values ​​were recorded at different time points. 600 value.

[0081] The results showed that the MIC of bacteriocin PFB252 against methicillin-resistant Staphylococcus aureus (MRSA) was 14.61 μg / mL. The growth curves of MRSA after treatment with bacteriocin PFB252 are shown below. Figure 7 As shown, bacterial growth ceased at concentrations of 2×MIC and 1×MIC, and was inhibited at concentrations of 1 / 2×MIC, 1 / 4×MIC, and 1 / 8×MIC. Concentrations of 1 / 16×MIC and 1 / 32×MIC had no effect on bacterial growth; therefore, concentrations of 1 / 16×MIC and 1 / 32×MIC were selected for subsequent studies.

[0082] Example 5: Anti-biofilm effect of bacteriocin PFB252

[0083] 1. Determination of anti-biofilm activity

[0084] The effect of bacteriocinol PFB252 on biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) was evaluated using crystal violet staining. Overnight cultured MRSA was inoculated into TSA liquid medium at a concentration of 1% (v / v). PFB252 was added to the bacterial suspension to a final concentration of 1 / 32×MIC and 1 / 16×MIC. 200 μL of each suspension was transferred to 96-well plates and incubated at 37°C for 24 h. After incubation, the supernatant was discarded, the plates were washed three times with PBS buffer, dried, and stained with 100 μL of 1% (v / v) crystal violet for 30 min. The staining solution was discarded, and the plates were washed three more times with PBS. After drying, 200 μL of 33% (v / v) acetic acid solution was added, and the plates were incubated at 37°C for 20 min. The OD was measured using a microplate reader. 595 The absorbance value was measured. The treatment group without bacteriocin was used as a positive control.

[0085] The measurement results are as follows Figure 8 As shown, at concentrations of 1 / 32×MIC and 1 / 16×MIC, the amount of biofilm formed decreased by 36.13% and 52.51%, respectively, indicating that bacteriocin PFB252 can significantly affect biofilm formation in a concentration-dependent manner.

[0086] 2. Determination of bacterial activity within biofilms

[0087] The activity of bacteria within the biofilm was determined using the CCK8 reagent kit. Methicillin-resistant Staphylococcus aureus cultured overnight was inoculated into TSA liquid medium at a 1% (v / v) inoculation rate. Bacteriocin PFB252 was added to the bacterial suspension to a final concentration of 1 / 32×MIC and 1 / 16×MIC. 200 μL of each solution was transferred to a 96-well plate, and the plate was incubated at 37°C for 24 hours. The supernatant was removed, and the plate was washed three times with PBS buffer. 100 μL of PBS and 10 μL of CCK8 reagent were added, and the plate was incubated at 37°C for 1 h. OD was measured using a microplate reader. 450 The absorbance value was measured. The treatment group without bacteriocin was used as a positive control.

[0088] The results of the effect of bacteriocin PFB252 on the metabolic activity of bacteria in biofilms are as follows: Figure 9 As shown, at concentrations of 1 / 32×MIC and 1 / 16×MIC, the metabolic activity of bacteria in biofilm decreased by 27.37% and 37.92%, respectively, indicating that bacteriocin PFB252 can significantly affect the formation of metabolic activity of bacteria in biofilm in a concentration-dependent manner.

[0089] 3. Determination of extracellular polysaccharide and protein content in biological membranes

[0090] The effect of bacteriocinol PFB252 on extracellular polysaccharides was investigated using the phenol-sulfuric acid method. Methicillin-resistant Staphylococcus aureus (MRSA) cultured overnight was inoculated into LB broth at a concentration of 1% (v / v). Bacteriocinol PFB252 was added to the bacterial suspension to final concentrations of 1 / 32×MIC and 1 / 16×MIC. Sterile glass slides (18 mm) were placed in 24-well plates, and 1 mL of sample was added to each well. The plates were incubated at 37°C for 24 h. The supernatant was removed, and the plates were washed three times with PBS. 100 μL of PBS buffer was added, followed by freezing for 1 min, sonication for 1 min, and finally, phenol-sulfuric acid solution was added. The plates were then heated in water at 100°C for 20 min, and the OD (exponential growth factor) was measured. 490 Value. The treatment group without bacteriocin was used as the control.

[0091] The effects of bacteriocin PFB252 on extracellular polysaccharides (EPS) and proteins in biomembranes are as follows: Figure 10 As shown, extracellular polysaccharides (EPS) are an important component of extracellular polymers in biomembranes. The results showed that at concentrations of 1 / 32×MIC and 1 / 16×MIC, the EPS content decreased by 30.26% and 39.32%, respectively, indicating that bacteriocin PFB252 can significantly affect the content of extracellular polysaccharides in biomembranes.

[0092] 4. RT-qPCR

[0093] The effects of bacteriocin PFB252 on biofilm-related genes were assessed using reverse transcription quantitative PCR (RT-qPCR). Methicillin-resistant Staphylococcus aureus (MRSA) cultured overnight was re-inoculated into TSA medium at a concentration of 1% (v / v). After 8 h of culture, bacteriocin PFB252 was added to the bacterial suspension to final concentrations of 1 / 16×MIC and 1 / 32×MIC, followed by incubation at 37°C and 180 rpm / min for 16 h. The control group received no bacteriocin. Total RNA was extracted using RNAiso Plus reagent and then reverse transcribed into cDNA using the PrimeScript™ RT kit. (Based on TB Green...) ® The PCR amplification reaction system was prepared according to the Premix Ex Taq™ II instructions, using 16S rRNA as an internal reference gene. Primers used for gene detection are shown in the table. Relative gene expression levels were determined by comparing Ct values ​​(2-). ΔΔ The expression level was determined using the Ct method.

[0094] The effects of bacteriocin PFB252 on genes related to biofilm formation are as follows: Figure 11 As shown, at concentrations of 1 / 32×MIC and 1 / 16×MIC, bacteriocin PFB252 significantly affected biofilm-related genes, significantly reducing the expression of these genes, including quorum sensing genes and virulence genes, in a concentration-dependent manner. The results indicate that bacteriocin PFB252 can significantly reduce the expression of biofilm-related genes in methicillin-resistant Staphylococcus aureus.

[0095] 5. Scanning electron microscope (SEM)

[0096] The effect of bacteriocinolone PFB252 on biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) was investigated using scanning electron microscopy (SEM). A sterile coverslip (9 mm) was placed in a 48-well plate, and 1 mL of MRSA culture medium containing different concentrations of bacteriocinolone PFB252 (1 / 32×MIC, 1 / 16×MIC) was added to each well. After incubation at 37°C for 48 h, the supernatant was discarded, and the samples were washed three times with PBS. Fixation was then performed overnight at 4°C with 2.5% glutaraldehyde. The samples were washed three more times with PBS, then dehydrated sequentially with different concentrations of ethanol solutions (30%, 50%, 70%, 85%, 100%), dried with carbon dioxide, and plated with gold. Finally, the samples were observed using a scanning electron microscope (TESCAN MIRA3 LMU, Czech Republic).

[0097] Scanning electron microscopy results as follows Figure 12As shown, the control group showed that the bacteria were aggregated, with a large number of extracellular polymers encapsulating the bacteria. The bacterial aggregation was significantly reduced in the treatment group with 1 / 32×MIC concentration of bacteriocin PFB252, and only a small number of bacteria were aggregated in the treatment group with 1 / 16×MIC concentration, with most of them scattered. This indicates that bacteriocin PFB252 can effectively clear the biofilm of methicillin-resistant Staphylococcus aureus.

[0098] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A bacteriocin PFB252, characterized in that, The amino acid sequence of the bacteriocin is: KALDKEVSEGSVDQSSWSREFELDENDGAQVNFWISNNGSNDIKISIDGKVSRTIAPGKQGNISAEVKNGWFGWTYNYTFTASPTAQGGSVKMKYKIAQRNN.

2. The method of producing bacteriocin PFB252 according to claim 1, characterized in that, Bacillus berberis ( Bacillus velezensis The culture medium was inoculated, and after centrifugation, the supernatant was collected and filtered through a filter membrane to remove bacteria, resulting in a cell-free supernatant. The supernatant was then purified. The Bacillus belyssus strain was FB25-2, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on June 9, 2025, with accession number GDMCC No: 66485.

3. The preparation method according to claim 2, characterized in that, The purification steps are acid precipitation, gel column chromatography, anion exchange chromatography and reverse phase high performance liquid chromatography in sequence.

4. The bacteriocin PFB252 of claim 1 is used for inhibiting methicillin-resistant Staphylococcus aureus for non-disease treatment and diagnosis purposes.

5. The bacteriocin PFB252 of claim 1 is used for preparing a product for inhibiting or eliminating the biofilm of methicillin-resistant Staphylococcus aureus.

6. A product characterized by, The bacteriocin PFB252 of claim 1.

7. A strain of Bacillus belye ( Bacillus velezensis FB25-2 strain, characterized in that, The strain has been preserved in Guangdong Microbial Culture Collection Center on June 9, 2025, and the preservation number is GDMCC No: 66485.

8. The FB25-2 strain of claim 7 is used for preparing the bacteriocin of claim 1.

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