A strain of Bacillus Velezii FS-3 and the bacteriocin produced thereby and its application

Abstract

The present invention discloses a strain of Bacillus Velez FS‑3 and the bacteriocin produced and its application, belonging to the field of microbial application technology. The deposit number of the Bacillus Velez FS‑3 is CGMCC NO.30514. The strain has a good antagonistic effect on Gram-negative bacteria such as multidrug-resistant Escherichia coli. The bacteriocin PFS‑3 prepared by fermentation of the strain shows good safety and bactericidal activity, and has a good therapeutic effect on mice infected with Escherichia coli resistant to polymyxins and carbapenems. The bacteriocin PFS‑3 prepared by fermentation of Bacillus Velez FS‑3 of the present invention is expected to enrich the current existing antimicrobial drug library and provide new hope for the treatment of multidrug-resistant Gram-negative bacteria infection.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, in particular to a strain of Bacillus Velezii FS-3 and bacteriocin produced thereby and application thereof. Background Art

[0002] As one of the most important medical discoveries of the 20th century, antibiotics have saved millions of lives and set off a medical revolution. However, in recent years, due to the irrational use of antibiotics, more and more pathogens have become resistant to them. At present, the abuse of antibiotics leading to bacterial resistance has become an international problem. From 2017 to 2024, the World Health Organization has included carbapenem-resistant Gram-negative bacteria, including Enterobacter, Acinetobacter baumannii and Pseudomonas aeruginosa, in the emergency list of antibiotic-resistant "priority pathogens" for seven consecutive years. In order to curb the problem of Gram-negative bacterial resistance, in addition to the rational use of antibiotics, accelerating the research and development of antimicrobial drugs has become an effective strategy to reduce drug resistance.

[0003] Since the discovery of penicillin, bacterial secondary metabolites have become one of the main sources of antibiotics, and these secondary metabolites can be used as drugs or pesticides. Related investigations and studies have shown that Bacillus Velezii can synthesize a variety of bacteriocins against drug-resistant pathogens through non-ribosomal pathways, and is a rich source of new antibacterial drugs. However, the existing Bacillus Velezii that can synthesize bacteriocins, although the bacteriocins synthesized by them can produce resistance to certain drug-resistant pathogens, most of them have broad-spectrum antibacterial properties and can resist a variety of pathogens including Gram-negative bacteria and Gram-positive bacteria. In clinical practice, most pathogens resistant to antibiotics are Gram-negative bacteria. Bacteriocins with broad-spectrum antibacterial effects are not ideal to a certain extent against Gram-negative bacteria, especially super-resistant bacteria resistant to polymyxins and carbapenems. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Bacillus Velez FS-3 and the bacteriocin produced thereby and their application, so as to solve the problems existing in the above-mentioned prior art. The present invention independently isolates a strain of Bacillus Velez FS-3 having good antagonistic effect on Gram-negative bacteria such as multidrug-resistant Escherichia coli. The bacteriocin PFS-3 prepared by fermentation of the bacterium shows good safety and bactericidal activity, which is expected to enrich the current antibacterial drug library and provide new hope for the treatment of multidrug-resistant Gram-negative bacteria infection.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a bacteriocin-producing Bacillus velezensis FS-3, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 7, 2024, with a deposit number of CGMCCNO.30514.

[0007] The present invention also provides a bacteriocin PFS-3 for inhibiting Gram-negative bacteria. The bacteriocin PFS-3 is obtained by fermenting the above-mentioned Bacillus Velezii FS-3, and the amino acid sequence is shown in SEQ ID NO.1.

[0008] The present invention also provides a method for preparing the bacteriocin PFS-3, comprising the steps of inoculating and fermenting the Bacillus velez FS-3, extracting and purifying the fermentation product, and obtaining the bacteriocin PFS-3.

[0009] Furthermore, during the fermentation process, the inoculation amount of the Bacillus Velez FS-3 is 1.5%.

[0010] Furthermore, the initial pH of the fermentation is 7, the fermentation temperature is 37° C., and the fermentation time is 48 hours.

[0011] Furthermore, the fermentation is a tremor fermentation of the Bacillus Velez subtilis FS-3 at a rotation speed of 180 rpm.

[0012] The present invention also provides the use of the Velez subtilis FS-3 or the bacteriocin PFS-3 in preparing a drug for inhibiting Gram-negative bacteria.

[0013] Furthermore, the Gram-negative bacteria are multidrug-resistant Gram-negative bacteria.

[0014] Furthermore, the multi-drug resistant Gram-negative bacteria are polymyxin-resistant and carbapenem-resistant Escherichia coli.

[0015] The invention also provides a medicine for inhibiting Gram-negative bacteria, wherein the effective ingredient is the bacteriocin PFS-3.

[0016] The present invention discloses the following technical effects:

[0017] The present invention independently separated a strain of Bacillus Velezii FS-3 which has good antagonistic effect on Gram-negative bacteria such as multidrug-resistant Escherichia coli, and obtained bacteriocin PFS-3 through stepwise purification and separation. Bacteriocin PFS-3 shows good safety and bactericidal activity, has good therapeutic effect on mice infected with multidrug-resistant Escherichia coli, and can improve the survival rate of mice infected with multidrug-resistant Escherichia coli by 90%. Bacteriocin PFS-3 has the potential to become a small molecule antibacterial drug.

[0018] The present invention uses a strain of Escherichia coli B2 resistant to polymyxin and carbapenems as an indicator bacteria to screen a novel broad-spectrum antibacterial drug against Gram-negative bacteria. Escherichia coli B2 is a super-resistant pathogen resistant to polymyxin and carbapenems, and has stronger drug resistance than other resistant bacteria. The Bacillus Velezii FS-3 screened by using the strain as an indicator bacteria has a stronger antibacterial effect on drug-resistant Gram-negative bacteria and a better effect.

[0019] The bacteriocin PFS-3 prepared by fermenting Bacillus velez FS-3 of the present invention is expected to enrich the current antibacterial drug library and provide new hope for treating infections caused by multidrug-resistant Gram-negative bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The effect of different inoculation amounts of Bacillus Velez fermentation supernatant on the antibacterial activity;

[0022] Figure 2 The effect of different initial pH on the antibacterial activity of the fermentation supernatant of Bacillus velez.

[0023] Figure 3 The effect of different culture temperatures on the antibacterial activity of the fermentation supernatant of Bacillus velez.

[0024] Figure 4 The effect of different culture time on the antibacterial activity of the fermentation supernatant of Bacillus Velez.

[0025] Figure 5 The effect of different rotation speeds on the antibacterial activity of the fermentation supernatant of Bacillus Velez;

[0026] Figure 6 The effect of different liquid volumes on the antibacterial activity of the fermentation supernatant of Bacillus Velez.

[0027] Figure 7 The antibacterial diameters of the crude bacteriocin extract and ethyl acetate at different ratios detected by the liquid-liquid mass transfer filter paper, where the different ratios are the volume ratios of the crude bacteriocin extract and ethyl acetate, and the ratios not indicated are the crude bacteriocin extracts not mixed with ethyl acetate;

[0028] Figure 8The results of purification tests on normal silica gel column and thin layer chromatography preparative silica gel plate, where A is the result of thin layer chromatography of components after silica gel column purification, B is the result of thin layer chromatography preparative silica gel plate, and C is the result of filter paper verification of each part of the substances after purification;

[0029] Fig. 9 This is the first step C18 column elution curve of bacteriocin PFS-3;

[0030] Fig.10 This is the second step C18 column elution curve of bacteriocin PFS-3;

[0031] Fig.11 The results of cytotoxicity assay of Bacillus velez bacteriocin PFS-3 are shown;

[0032] Fig.12 The hemolytic activity of different concentrations of bacteriocin PFS-3 on fresh rabbit blood;

[0033] Fig.13 The survival rate of mice after being treated with different doses of bacteriocin PFS-3;

[0034] Fig.14 The bacterial load in the mouse heart after treatment with bacteriocin PFS-3;

[0035] Fig.15 is the bacterial load in the lungs of mice after treatment with bacteriocin PFS-3;

[0036] Fig.16 is the bacterial load in mouse liver after treatment with bacteriocin PFS-3;

[0037] Fig.17 is the bacterial load in the kidneys of mice after treatment with bacteriocin PFS-3;

[0038] Fig.18 Figure 2 shows the bacterial load in the spleen of mice after treatment with bacteriocin PFS-3. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] The indicator bacteria used for screening strains and determining antibacterial activity in the embodiments of the present invention are all polymyxin-resistant and carbapenem-resistant Escherichia coli B2 (E. coli B2), which is a model strain with multidrug resistance among Gram-negative bacteria and has stronger drug resistance than other drug-resistant bacteria. The strain was donated by China Agricultural University and stored in the Veterinary Pharmacology Laboratory of Jilin Agricultural University. Other clinical isolates were isolated and obtained by the Veterinary Pharmacology Laboratory of Jilin Agricultural University. It has been experimentally verified that clinical isolates show strong resistance to antibiotics such as carbapenems and polymyxins.

[0045] Example 1 Isolation, screening and identification of strain FS-3

[0046] 1. Sample collection and strain isolation and purification

[0047] For 530 strains isolated from the soil environment of Changbai Mountain, the soil samples were resuspended in sterile water to 0.5 g / mL, diluted 10,000 times, streaked on LB plates, and purified until the colony morphology was single.

[0048] 2. Preliminary screening of antagonistic strains

[0049] Using multidrug-resistant Escherichia coli B2 as an indicator bacterium, the plate confrontation method was used for the initial screening of antagonistic bacteria. A single colony of multidrug-resistant Escherichia coli B2 was inoculated into an LB liquid test tube and cultured at 37°C until the logarithmic growth phase. The bacterial solution was appropriately diluted and 10 μl was spread on the LB solid medium. 6 CFU of multidrug-resistant Escherichia coli B2, aspirate 2.5 μL of bacterial solution, add it dropwise to the solid culture medium, and culture it in a 37°C incubator for 12 hours. Fifteen antagonistic strains were screened out, among which the strain numbered FS-3 had the largest inhibition zone diameter against multidrug-resistant Escherichia coli B2, which could reach 20 mm.

[0050] 3. Rescreening of Antagonistic Strains

[0051] The antagonistic bacteria FS-3 obtained in the initial screening was inoculated into LB liquid culture medium at a 1% inoculum, centrifuged at 8000 rpm for 25 min at 4°C, and the supernatant was filtered using a 0.22 μm filter membrane, concentrated 10 times using a rotary evaporator, and filtered through a 0.22 μm microporous filter membrane to obtain the fermentation supernatant concentrate. The bacterial solution concentration was diluted to 10 7 CFU / mL, 100 μL of bacterial solution was spread on the solid culture medium, 200 μL of sterile concentrate was added to the pre-placed Oxford cup wells, and cultured at 37°C for 12 h. The re-screening result showed that the antibacterial diameter was 30 mm.

[0052] 4. 16S rDNA Identification of Antagonistic Strain FS-3

[0053] Using the DNA of strain FS-3 as a template, PCR amplification was performed using 16S rDNA universal primers. The product after PCR verification was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were subjected to BLAST comparison analysis, and the results showed that the antagonistic strain FS-3 had 99.8% homology with Bacillus velezensis NRRLB-41580. The phylogenetic tree was constructed using MEGA software. Strain FS-3 belongs to the same branch as the bacteria and has the closest relationship. Therefore, strain FS-3 was identified as Bacillus velezensis. The strain was deposited in the China General Microbiological Culture Collection Center on May 7, 2024. Its address is Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO: 30514.

[0054] Example 2 Optimization of fermentation conditions for bacteriocin production by Bacillus velez FS-3

[0055] The indicator bacteria used in the following determination of antibacterial activity were all multidrug-resistant Escherichia coli B2.

[0056] The following agar diffusion method was used to determine the antibacterial activity. The specific steps were as follows: streak the multidrug-resistant E. coli B2 stored in a glycerol tube onto solid LB medium and culture it in a 37°C constant temperature incubator for 8h-12h. After the colonies were formed, pick a single colony and inoculate it into liquid LB medium and culture it in a shaking incubator at 37°C and 180rpm until the OD 600 The concentration of the bacterial suspension was 0.6, and the bacterial suspension was diluted 1000 times in sequence. 100 μL of the diluted bacterial suspension was placed in the solid LB medium and evenly spread. A sterile Oxford cup with an inner diameter of 6 mm was placed on the solid LB agar. Then 100 μL of the bacteriocin sample was added to the hole of the Oxford cup, and finally it was placed in a 37°C constant temperature incubator for 12 hours, and the diameter of the inhibition zone was measured and recorded.

[0057] 1. Effect of different inoculum sizes on antibacterial activity

[0058] The activated Bacillus Velez FS-3 bacterial suspension was inoculated into LB medium (pH of LB liquid medium was 6.5) at a volume percentage of 0.5%, 1%, 1.5%, 2%, 2.5% and 3%, respectively, and cultured at 37°C and 180r / min for 48h. The antibacterial activity was detected by agar diffusion method. Figure 1 As shown, the optimal inoculation amount was determined to be 1.5%.

[0059] 2. Effect of different pH medium on antibacterial activity

[0060] The pH of the culture medium was adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 respectively. 1.5% of Bacillus Velezii was inoculated into 300 mL of liquid LB medium and cultured at 37°C and 180 r / min for 48 h. After the fermentation, the inhibition diameter was detected by agar diffusion method. The results are shown in Figure 2 As shown, the optimum pH was determined to be 7.

[0061] 3. Effect of different culture temperatures on antibacterial activity

[0062] 1.5% activated bacterial solution of Bacillus Velezii FS-3 was inoculated into 300 mL of liquid LB medium at pH 7.0, and the culture temperatures were set at 25°C, 28°C, 31°C, 34°C, 37°C and 40°C at 180 r / min for 48 hours. After the fermentation, the inhibition diameter was detected by agar diffusion method. The results are shown in Figure 3 As shown, the optimal culture temperature was determined to be 37°C.

[0063] 4. Effect of different culture times on antibacterial activity

[0064] 1.5% activated bacterial solution of Bacillus Velezii FS-3 was inoculated into 300 mL of liquid LB medium at pH 7.0 and cultured at 37°C and 180 r / min for 12 h, 24 h, 36 h, 48 h, 60 h and 72 h respectively. After fermentation, the inhibition diameter was determined by agar diffusion method. The results are shown in Figure 4 As shown, the optimal culture time was determined to be 48h.

[0065] 5. Effect of different rotation speeds on antibacterial activity

[0066] 1.5% activated bacterial solution of Bacillus Velezii FS-3 was inoculated into 300 mL of liquid LB medium at pH 7.0, and the rotation speed was set to 120 r / min, 150 r / min, 180 r / min, 210 r / min and 240 r / min, respectively, and cultured in a shaker at 37°C for 60 h. After the fermentation, its antibacterial activity was determined by agar diffusion method. The results are shown in Figure 5 As shown, the optimal culture speed was determined to be 180r / min.

[0067] 6. Effect of different volume of culture medium on antibacterial activity

[0068] 50mL, 100mL, 150mL, 200mL, 250mL and 300mL of LB liquid culture medium were respectively placed in 500mL conical flasks, the initial pH value of the culture medium was 6.5 (unadjusted), 1.5% of the activated bacterial solution of Bacillus Velezii FS-3 was inoculated into the culture medium with different liquid volumes, and cultured at 37°C and 180r / min for 48h. After the fermentation, the antibacterial activity was determined by the agar diffusion method. The results are shown in Figure 6 As shown, the optimal culture medium volume was determined to be 300 mL.

[0069] In summary, the optimal culture conditions for bacteriocin production by Bacillus Velez FS-3 were finally determined to be: inoculation size 1.5%, initial pH = 7, culture temperature 37°C, culture time 48h, culture speed 180rpm, and liquid volume 300mL.

[0070] Example 3 Preparation of bacteriocin PFS-3

[0071] 1. Preparation of Fermentation Supernatant

[0072] The activated bacterial solution of Bacillus Velezii FS-3 (concentration of 1×10 8The fermentation was carried out under the optimal fermentation conditions of inoculation amount of 1.5%, initial pH of 7, culture temperature of 37°C, culture time of 48h, shaker speed of 180rpm and liquid volume of 300mL, the fermentation broth was centrifuged at 8000rpm for 25min, and sterilized by filtration through a 0.22μM filter membrane using a diaphragm vacuum pump, and the obtained sterile supernatant was concentrated 10 times by a rotary evaporator to obtain a sterile concentrated supernatant.

[0073] 2. Preparation of crude extract of bacteriocin PFS-3

[0074] The pH of the sterile concentrated supernatant of Bacillus Velez FS-3 was adjusted to 2.0 using 6 mM hydrochloric acid, stirred in a 4°C refrigerator for 12 h, centrifuged at 4°C, 10,000 rpm for 30 min, and the precipitate was collected. The precipitate was repeatedly extracted with methanol to finally obtain a crude extract of bacteriocin PFS-3.

[0075] 3. Purification of Bacteriocin PFS-3

[0076] Liquid-liquid mass transfer: Take the crude extract of bacteriocin and add ethyl acetate (crude extract of bacteriocin: ethyl acetate) in the volume ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 and 1:9 respectively, centrifuge at 10000r / min for 30min, take the precipitate of each ratio and dry it, dissolve it with 1mL methanol respectively, at the same time, dry the supernatant with a rotary evaporator and dissolve it with 1mL methanol, after the precipitate and supernatant are dissolved, use resistant Escherichia coli B2 as indicator bacteria, and use the filter paper method to detect the antibacterial diameter of the supernatant and precipitate of each ratio. The test results are as follows: Figure 7 As shown, the antibacterial activity of the supernatant against multidrug-resistant Escherichia coli B2 gradually began to weaken starting from a crude bacteriocin:ethyl acetate ratio of 1:6, so the condition of a crude bacteriocin:ethyl acetate ratio of 1:6 was selected for the subsequent purification of bacteriocin PFS-3.

[0077] Forward silica gel column chromatography: The crude extract of bacteriocin obtained by hydrochloric acid precipitation and methanol extraction is used as the raw material, and the silica gel column chromatography is used for separation and purification. The silica gel column chromatography adopts wet column loading, and is activated in an oven at 120°C for 4 hours. After cooling to room temperature, dichloromethane is used as the mobile phase to suspend the silica gel, and then the chromatographic column is loaded. Dichloromethane with a volume fraction of 20%, 40%, 60% and 80% is selected, and gradient elution is performed in sequence, with a flow rate of 1mL / min, and the elution volume is not less than 3 each time, and then each gradient component is collected and determined by thin layer chromatography. Using multidrug-resistant Escherichia coli B2 as an indicator bacteria, the antibacterial activity of each component is detected by the filter paper method, and the separated antibacterial components are dried, dissolved in methanol, and then separated and purified.

[0078] Purification by thin layer chromatography preparative silica gel plate: The crude bacteriocin purified in the previous step is sampled by a capillary tube and spotted on a thin layer chromatography preparative silica gel plate, the solvent is evaporated, and the thin layer chromatography preparative silica gel plate is placed in a chromatography cylinder containing a developing agent. The developing agent ratio is ethyl acetate: methanol: water: formic acid = 8: 1.5: 1: 0.5 (v: v: v: v). After the developing agent is developed, the solvent is evaporated, and local color development is performed with a ninhydrin color developer. By observing the color development on the silica gel plate, the different components corresponding to the color development are cut out, crushed, and loaded into a chromatography column and repeatedly eluted with methanol. The obtained eluate is concentrated by a rotary evaporator, and the antibacterial activity of different components against the indicator bacteria is detected by the filter paper method using multidrug-resistant Escherichia coli B2 as the indicator bacteria.

[0079] Thin layer chromatography analysis: Use a capillary to draw an appropriate amount of the component to be tested and spot it on a silica gel thin layer plate, evaporate the solvent, and place the silica gel thin layer plate in a chromatography tank filled with a developing agent. The developing agent ratio is ethyl acetate: methanol: water: formic acid = 8:1.5:1:0.5 (v:v:v:v). After the developing agent is developed, evaporate the solvent and use ninhydrin as a color developer for color development. The purification and detection results are shown in the table below. Figure 8 shown.

[0080] 4. Semi-preparative reversed-phase HPLC

[0081] The purified active components were filtered through a 0.22 μM microporous filter membrane and separated and purified by a high performance liquid phase system equipped with a C18 chromatographic column. A linear gradient elution was performed using 10-90% acetonitrile containing 1% trifluoroacetic acid at a flow rate of 1 mL / min for 60 min. The eluted single peaks were collected at UV 280 nm on a UV detector. The results are shown in FIG. Fig. 9 As shown, two single peaks were separated and the two components were freeze-dried and re-dissolved in ddH2O. The antibacterial activity of the two single peak samples against multi-drug resistant Escherichia coli B2 was detected by agar diffusion method. The results showed that only the 2 peak samples had good antibacterial activity against multi-drug resistant Escherichia coli B2. The 2 peak samples were taken and passed through the C18 column again. The purification results are shown in Fig.10 As shown, a single peak was obtained, and the peak sample had good antibacterial activity against multidrug-resistant Escherichia coli B2.

[0082] 5. LC-MS / MS Analysis of Bacteriocin PFS-3

[0083] MaxQuant (V1.6.6) software was used to search the original peptide sequences in the bacteriocin samples and match the protein database (the database used for the search was a proteome reference database obtained based on the whole genome sequencing of Bacillus velez FS-3). The mass spectrometry analysis results of bacteriocin PFS-3 showed that the molecular weight of bacteriocin PFS-3 was 465.2Da. The secondary mass spectrometry results showed that the amino acid sequence of bacteriocin PFS-3 was STYLFEGL (SEQ ID NO.1).

[0084] Example 4 Determination of the Inhibitory Spectrum and Minimum Inhibitory Concentration (MIC) of Bacillus Velezii Bacteriocin PFS-3

[0085] 1. Antibacterial spectrum of Bacillus velez bacteriocin PFS-3

[0086] Weigh 0.1024g of bacteriocin PFS-3 lyophilized powder, dissolve it in 10mL of sterile water to obtain a bacteriocin PFS-3 solution with a concentration of 1024μg / mL, and filter it through a 0.22μm filter membrane for sterilization. The indicator bacteria in Table 1 were inoculated into LB broth medium, cultured at 37°C with shaking until the logarithmic growth phase, and then evenly spread on solid LB medium after gradient dilution. The antibacterial activity of bacteriocin PFS-3 was detected by the agar powder method, and the results are shown in Table 1.

[0087] Table 1 Antibacterial spectrum of bacteriocin PFS-3

[0088]

[0089]

[0090] Note: “G+” represents Gram-positive bacteria, and “G-” represents Gram-negative bacteria.

[0091] As shown in Table 1, bacteriocin PFS-3 has poor antibacterial activity against positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), but exhibits good antibacterial activity against a variety of Gram-negative bacteria, especially polymyxin-resistant and carbapenem-resistant Escherichia coli B2, and also has a good inhibitory effect on clinically isolated drug-resistant strains, indicating that bacteriocin PFS-3 has a better antibacterial effect on Gram-negative bacteria than on Gram-positive bacteria.

[0092] 2. MIC Determination of Bacillus Velezii Bacteriocin PFS-3

[0093] The MIC of bacteriocin PFS-3 against multidrug-resistant Escherichia coli, Shigella, Salmonella, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa was determined. First, a bacteriocin PFS-3 solution with a concentration of 1024 μg / mL was prepared. Cultured at 37°C with shaking until the logarithmic growth phase, diluted 1000 times for later use. Add 200 μL of LB medium to well 1 as a negative control, and add 200 μL of indicator bacteria to well 2 as a positive control. Add 100 μL of LB liquid culture medium to wells 3-12 of a 96-well plate, add 100 μL of 1024 μL / mL bacteriocin PFS-3 solution to well 3, add 100 μL to well 4 after mixing, dilute to well 12, add 100 μL of indicator bacteria to wells 3-12, mix and place in a 37°C incubator for 16 hours. The results are shown in Table 2.

[0094] Table 2 MIC of bacteriocin PFS-3

[0095]

[0096]

[0097] As shown in Table 2, bacteriocin PFS-3 exhibited good antibacterial activity against Gram-negative bacteria such as multidrug-resistant Escherichia coli B2 and Shigella, and it was speculated that bacteriocin PFS-3 was a broad-spectrum bacteriocin with good antibacterial effect against Gram-negative strains.

[0098] Example 5 Safety Assessment of Bacteriocin PFS-3

[0099] 1. Bacteriocin PFS-3 Cytotoxicity Assay

[0100] The cytotoxicity of different concentrations of bacteriocin PFS-3 on human renal epithelial cells (293T) and mouse macrophages (RAW264.7) was determined. Fig.11 As shown, the cytotoxicity of bacteriocin PFS-3 to the two cell types slightly increased with the increase of concentration, but overall the cell viability of RAW264.7 was above 80%, and the cell viability of 293T was above 90%.

[0101] 2. Determination of hemolytic activity of bacteriocin PFS-3

[0102] Collect fresh blood from healthy rabbits, centrifuge at 3000rpm and 4℃ for 10min, discard the supernatant, and slowly wash with saline three times to prepare a 2% red blood cell suspension. Prepare 11 1.5mL centrifuge tubes, add 100μL of red blood cell suspension to each, take 9 centrifuge tubes and add 100μL of bacteriocin with a concentration of 2-256μg / mL, take the 10th tube as a negative control, and add 100μL of red blood cell suspension. Take the 11th tube as a positive control, add 100μL of 2% Triton X-100, and incubate at 37℃ for 1h. Centrifuge at 4℃ and 3000rpm for 10min, add an equal amount of 150μL of supernatant to a 96-well plate, and measure the OD with an enzyme reader. 570 The hemolytic activity of bacteriocin PFS-3 at different concentrations on fresh rabbit blood was obtained. Fig.12 As shown, when the concentration of bacteriocin PFS-3 was 256 μg / mL, the hemolysis rate was less than 2%.

[0103] This method uses the following formula to calculate the hemolysis rate:

[0104] Hemolysis rate (%) = (OD 样本 -OD 阴性对照 ) / (OD 阳性对照 -OD 阴性对照 )×100%.

[0105] Example 6 Experiment on the therapeutic effect of bacteriocin PFS-3 on mice

[0106] The results of preliminary experiments showed that mice injected intraperitoneally with 1×10 8 CFU of multidrug-resistant E. coli B2 can cause 100% mortality in mice within 48 hours. Therefore, the experiment was divided into 4 groups, each with 10 mice, namely the positive control (PBS) group, the experimental group including the PFS-3 low-dose (5 mg / kg) group, the PFS-3 medium-dose (10 mg / kg) group and the PFS-3 high-dose (20 mg / kg) group.

[0107] Mouse treatment: First, each mouse was injected with 200 μL of multidrug-resistant Escherichia coli B2 solution (5×10 8 CFU / mL), and 1 hour later, the mice in the experimental group were injected with 200 μL of different doses of bacteriocin PFS-3 into the right abdomen for treatment, and the positive control group was injected with 200 μL PBS. The number of mice surviving in each group was recorded every 24 hours. The mice were raised for 7 days, and all the surviving mice were killed by dislocating the cervical vertebra after 7 days. The mouse organs were ground, graded diluted with sterile PBS, and smeared on the plate to calculate the number of bacterial colonies in each organ.

[0108] Compared with the positive control injected with PBS, the 7-day survival rate of the treatment group injected with different doses of bacteriocin PFS-3 was significantly improved. Among them, 20 mg / kg of PFS-3 could increase the survival rate of mice by 90%. Fig.13 After treatment with bacteriocin PFS-3, the bacterial load in various organs (heart, lung, liver, kidney and spleen) of mice was significantly reduced ( Figure 14-18 ), indicating that bacteriocin PFS-3 has a good therapeutic effect on mice infected with multidrug-resistant Escherichia coli B2. The bacteriocin disclosed in the present invention is expected to enrich the current antibacterial drug library and provide new hope for the treatment of multidrug-resistant Gram-negative bacteria infection.

[0109] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A bacteriocin-producing Bacillus velezensis FS-3, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 7, 2024, with the deposit number CGMCCNO.30514.

2. A bacteriocin PFS-3 for inhibiting Gram-negative bacteria, characterized in that: The bacteriocin PFS-3 is obtained by fermenting the Bacillus Velez subtilis FS-3 described in claim 1, and the amino acid sequence is shown in SEQ ID NO.

1.

3. A method for preparing the bacteriocin PFS-3 as claimed in claim 2, characterized in that: The method comprises the steps of inoculating and fermenting the Bacillus Velez subtilis FS-3 described in claim 1, extracting and purifying the fermentation product, and obtaining the bacteriocin PFS-3.

4. The preparation method according to claim 3, characterized in that: During the fermentation process, the inoculation amount of the Bacillus Velez FS-3 was 1.5%.

5. The preparation method according to claim 3, characterized in that: The initial pH of the fermentation was 7, the fermentation temperature was 37° C., and the fermentation time was 48 h.

6. The preparation method according to claim 3, characterized in that: The fermentation is performed by osmotic fermentation of the Bacillus Velez FS-3 at a rotation speed of 180 rpm.

7. Use of the Bacillus Velez FS-3 according to claim 1 or the bacteriocin PFS-3 according to claim 2 in the preparation of a drug for inhibiting multi-drug resistant Gram-negative bacteria, characterized in that: The multi-drug resistant Gram-negative bacteria include Escherichia coli, Shigella, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Salmonella.

8. The use according to claim 7, characterized in that: The multi-drug resistant Gram-negative bacteria are Escherichia coli resistant to polymyxins and carbapenems.

9. A drug for inhibiting Gram-negative bacteria, characterized in that: The active ingredient is the bacteriocin PFS-3 described in claim 2.

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