Bacillus Velez NC-B4 and its application

Abstract

The present invention discloses a Bacillus velezensis NC‑B4 and its application, which relates to the field of medical biotechnology, is classified and named as Bacillus velezensis, and has been preserved in the China General Microbiological Culture Collection Management Center on May 19, 2022, with a preservation number of CGMCC NO.24929. The present invention provides an application of Bacillus velezensis NC‑B4 in human pathogenic fungal infection drugs, especially in the prevention and treatment of Candida auris infection. The present invention aims at the problem that there is a lack of efficient and safe drug treatment for Candida auris infection at present, and Candida auris is prone to drug resistance to antifungal drugs. Through the separation and screening of Candida auris antagonistic bacteria and its prevention and efficacy test, strains with antagonistic effects on Candida auris are separated and screened. On this basis, further separation and identification of antibacterial active substances and clinical trials can be carried out, so as to develop a new type of biocontrol microbial factor, which is of great significance for reducing the use of a large number of antibiotics, reducing environmental pollution and the production of drug-resistant bacteria.

Description

Technical Field

[0001] The invention relates to the field of medical biotechnology, in particular to Bacillus Velez NC-B4 and application thereof. Background Art

[0002] Candida auris, also known as Candida auris, is a new species first reported by Japanese scientists in 2009 and is known as the "super fungus". Candida auris can infect patients of different ages, causing invasive infections such as bloodstream infections in ICU patients, and can colonize in different parts of the human body, such as the skin, respiratory tract, and urinary tract. It survives in the environment for a long time and is difficult to eliminate. In addition, the most prominent feature of Candida auris is drug resistance or even multi-drug resistance. It usually has single or multi-drug resistance to the three main types of antifungal drugs - azole drugs, polyene drugs, and echinocandin drugs, and the degree of drug resistance is not the same. This is also the main reason for the failure of treatment of Candida auris infection and the difficulty in controlling its prevalence.

[0003] Candida auris is highly resistant to broad-spectrum triazole antifungal drugs such as fluconazole and voriconazole. Only echinocandin drugs have therapeutic effects on some strains. Antifungal drug treatment is still the main measure for the treatment of Candida auris infection, but the long-term use of antibiotics has caused more and more concerns about environmental pollution and pathogen resistance. The use of biocontrol strains and their active products as a new type of biocontrol microbial factor to control pathogens has attracted more and more attention. Therefore, screening and identifying biocontrol strains with significant antagonistic effects on Candida auris is of great significance for the treatment of Candida auris infection.

[0004] A large number of studies have shown that Bacillus is widely used in industry, agriculture and pharmaceutical production. The antibacterial substances it produces generally have a wide antibacterial spectrum and can kill bacteria including drug-resistant strains, certain fungi, parasites, some viruses and tumor cells, etc., and have the functions of binding lipopolysaccharide and neutralizing endotoxins, which has attracted widespread attention from scientists and medical workers. The microecological preparations prepared by Bacillus have played an important role in the treatment of intestinal flora imbalance, the treatment of Candida infection, the prevention of ulcer infection and other medical processes. As a probiotic, it has been widely studied in the medical field and is an ideal new biocontrol microbial factor. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide a Bacillus Velezii NC-B4 and its application in preventing and treating Candida auris infection.

[0006] The technical solution of the present invention is as follows:

[0007] A Bacillus velezensis NC-B4, classified and named Bacillus velezensis, named Bacillus velezensis NC-B4, was deposited in the General Microbiology Center of the China Culture Collection Committee of Microorganisms (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​May 19, 2022, with the deposit number CGMCC NO.24929.

[0008] Furthermore, the sequences of the 16S rDNA, gyrA and rpoB genes thereon are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0009] The present invention also discloses an application of the above-mentioned Bacillus Velezii NC-B4 in preparing a medicine for preventing and treating human pathogenic fungal infection.

[0010] Furthermore, the human pathogenic fungi are Candida auris, Candida albicans, Cryptococcus, and filamentous fungi.

[0011] Furthermore, the filamentous fungi are Cunninghamella parvula, Trichophyton schrenkiana, Absidia spp. and Lactodispora spp.

[0012] The invention also discloses a medicine for preventing and treating human pathogenic fungal infection and a medicine for preventing and treating Candida auris infection, and the active ingredients of both medicines are the Bacillus Velezii NC-B4 mentioned above.

[0013] The present invention also discloses a method for screening the above-mentioned Bacillus Velezii NC-B4, comprising the following steps:

[0014] S1: Collect plant rhizosphere soil and use LB medium for separation to obtain isolated strains;

[0015] S2: Then, the isolated strains are subjected to a plate antagonism test to obtain the preliminary screening bacteria that have antagonistic effects on human pathogenic fungi;

[0016] S3: The initially screened bacteria were subjected to a plate punching antagonism test, the size of the inhibition zone was measured, and further screening and identification were performed to obtain the Bacillus Velez NC-B4 bacteria.

[0017] Furthermore, the cells of the strain of Bacillus Velez NC-B4 are rod-shaped, and the colonies are round, milky white, opaque, dry and wrinkled on the surface, with irregular edges and a concave center.

[0018] The beneficial effects of the present invention are as follows: the present invention provides an application of Bacillus velezensis NC-B4 in drugs for human pathogenic fungal infections, especially in the application for preventing and treating Candida auris infections. Aiming at the problem that there is a lack of efficient and safe drug treatment for Candida auris infections at present, and Candida auris is prone to drug resistance to antifungal drugs, the present invention separates and screens strains with antagonistic effects on Candida auris through separation and screening of antagonistic bacteria to Candida auris and its prevention efficacy test, and further separation, identification and clinical trials of antibacterial active substances can be carried out on this basis, thereby developing a new type of biocontrol microbial factor, which is of great significance for reducing the use of a large number of antibiotics, reducing environmental pollution and the generation of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the antagonistic effect of the strain NC-B4 and its fermentation liquid on Candida auris in the present invention;

[0020] Figure 2 is a colony morphology diagram of strain NC-B4 in the present invention;

[0021] Figure 3 It is a microscopic morphology of strain NC-B4 in the present invention;

[0022] Figure 4 is the phylogenetic tree of strain NC-B4 of the present invention;

[0023] Figure 5 This is a graph showing the cytotoxicity of the strain NC-B4 and its fermentation broth in the present invention;

[0024] Figure 6 It is the nucleic acid electrophoresis diagram of 16S rDNA, gyrA and rpoB genes of strain NC-B4 of the present invention. DETAILED DESCRIPTION

[0025] The present invention is described in more detail by the following examples, which are merely illustrative and the present invention is not limited by these examples. The methods in the following examples are conventional methods unless otherwise specified.

[0026] Example 1: Isolation and purification of bacteria:

[0027] 1. Culture medium

[0028] LB medium: Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, agar 15 g, H 2 O1000ml, PH 7.0-7.2, sterilize at 121℃ for 20 minutes.

[0029] 2. Experimental steps

[0030] 2.1 Culture medium preparation

[0031] Prepare the LB agar medium as described in 1 to prepare the bacterial culture medium.

[0032] 2.2 Sample collection

[0033] Plant rhizosphere soil was collected from Meiling Park in Nanchang City, put into bags and recorded.

[0034] 2.3 Grind the soil and prepare a suspension

[0035] The soil was ground into small pieces, chopped and resuspended in sterile water, and the soil dilution (10 -1 ). The supernatant was taken and diluted 10 times in series (10 -2 , 10 -3 , 10 -4 , 10 -5 )spare.

[0036] 2.4 Bacterial isolation

[0037] Select the above 10 -2 , 10 -3 , 10 -4 and 10 -5 0.1 ml of each of the four concentrations was spread onto the prepared LB solid culture medium plates. The inoculated culture plates were inverted and cultured at 35°C with three replicates. Single clones with different morphologies were selected as much as possible.

[0038] 2.5 Results

[0039] 200 bacterial strains were isolated according to the above method.

[0040] Example 2: Plate antagonism test of isolated bacteria against Candida auris

[0041] 1. Culture medium

[0042] 1.1 Solid culture medium

[0043] SDA medium: 10g peptone, 15g agar, 40g glucose, add distilled water, make up to 1L. Sterilize at 121℃ for 20 minutes

[0044] 1.2 Liquid culture medium

[0045] SDA liquid culture medium: no agar is added, and the rest of the ingredients are the same as SDA culture medium.

[0046] 2. Experimental steps

[0047] 2.1 Plate antagonism test - initial screening

[0048] 2.1.1 Bacterial Activation

[0049] Activation of Candida auris and bacteria: Candida auris and isolated bacteria were transferred to SDA medium plates and LB medium plates respectively, cultured at 35°C for 1 day and set aside.

[0050] 2.1.2 Preparation of Candida auris plates

[0051] The activated Candida auris was inoculated into liquid culture medium and cultured in a shake flask at 35°C and 200 rpm / min. The next day, SDA culture medium was poured into plates. After the culture medium was cooled to 45°C, 0.1 ml (OD 600 About 2.0) Candida auris fermentation liquid, mix well, pour into plates, blow dry and set aside.

[0052] Note: OD 600 Refers to the absorbance of a solution at a wavelength of 600nm.

[0053] 2.1.3 Preliminary screening of plate antagonism test

[0054] Use a sterilized toothpick to pick up a single colony of isolated bacteria and spot it on a Candida auris plate. Repeat three times for each bacterial strain and culture at 35°C. Observe the experimental results at 24h, 36h and 48h, mainly to observe the presence or absence of inhibition zone.

[0055] 2.2 Plate antagonism test-rescreening

[0056] 2.2.1 Bacterial Activation

[0057] Same as 2.1.1, activate and select the isolated bacteria with inhibition zone in the initial screening.

[0058] 2.1.2 Preparation of Candida auris plates

[0059] Same as 2.1.2. After the plates are dried, use a 5mm diameter hole puncher to punch holes. Punch one hole in each plate for later use.

[0060] 2.2.3 Isolation of bacteria for liquid fermentation

[0061] While the Candida auris was being cultured in a liquid shake flask, the activated isolated bacteria were transferred to a liquid LB medium for shake flask culture at 35°C, 200 rpm / min, and cultured for 24 hours (OD 600 about 2.0).

[0062] 2.2.4 Plate antagonism test rescreening

[0063] Use a pipette to draw 10 μl of the isolated bacterial fermentation liquid into the wells of the Candida auris plate, blow dry, repeat 3 times, and culture at 35°C. Observe the experimental results at 24h, 36h, and 48h, mainly observing the presence and size of the inhibition zone.

[0064] 3. Results

[0065] Through the initial screening test, bacteria with antagonistic effects were obtained, and then through the plate antagonism test, three bacterial strains with good antagonistic effects were obtained, and strain NC-B4 was one of them. Figure 1 . Figure 1 The test was performed by adding 0.1 ml of Candida auris fermentation liquid to a plate, then punching holes with a hole puncher, placing the fermentation liquid of strain NC-B4 into the hole, blowing it dry, and culturing it at 35°C.

[0066] Depend on Figure 1 It can be seen that the inhibition zone is obvious, indicating that strain NC-B4 has a significant antagonistic effect on Candida auris.

[0067] Example 3: Detection of cytotoxicity of the fermentation broth of strain NC-B4. This experiment was conducted to test the toxicity of strain NC-B4 itself.

[0068] Experimental steps:

[0069] 1.1 Thawing A549 cells

[0070] Take out the cryovials from the liquid nitrogen tank, immerse them directly in 37°C warm water, and shake them from time to time to melt them as quickly as possible; collect the cells by centrifugation at 1000 rpm for 5 minutes, resuspend the cells in DMEM containing 10 (v / v)% FBS, inoculate the culture bottles, and culture them in a 37°C, 5 (v / v)% carbon dioxide incubator; replace the culture medium once the next day and continue culturing.

[0071] 1.2 A549 cell plating

[0072] Prepare A549 cell suspension and count, then plate in 96-well plates, add 100ul 1(v / v)% FBS DMEM containing 1×10 4 The cells were cultured overnight in a 37°C, 5 (v / v)% carbon dioxide incubator.

[0073] 1.3 Preparation of Candida auris antagonistic bacteria fermentation broth

[0074] Pick a single colony of antagonistic bacteria and inoculate it into a liquid shake flask for culture at 35°C, 200 rpm / min, overnight, centrifuge to collect the bacteria, wash twice with PBS, and dilute with 1 (v / v)% FBS DMEM to OD 600 About 1.0 spare.

[0075] 1.4 Antagonistic bacteria toxicity test

[0076] In this experiment, a blank control group with no cells was set up, i.e., DMEM with only 1 (v / v)% FBS; a minimum / maximum LDH release well with cells, i.e., only cells without treatment and wells with lysis solution added; for strain NC-B4 treatment: 100ul of the bacterial solution prepared in 1.3 was added.

[0077] 1.5 Detection of LDH release from cells

[0078] After 12 hours, the supernatant was collected from each group to detect the amount of LDH released.

[0079] 2. Results

[0080] like Figure 5 As shown in the figure, IF-1, IF-3, and IF-4 are three independent experiments conducted on the strains of the present invention. Figure 5 The amount of LDH released by the cells is detected after the cells are treated with the supernatant and the bacteria. The more LDH is released, the more cell death there is. Therefore, the toxicity of a substance to cells can be detected by detecting the amount of LDH released by the cells. In this figure, the amount of LDH released in three independent experiments is very small, indicating that the biocontrol bacteria has very weak toxicity to cells or can even be said to be non-toxic, which means that the strain is safe and reproducible when made into a biological agent.

[0081] Example 4: Identification of isolated bacteria

[0082] 1 Physiological and biochemical identification of strains

[0083] 1.1 Bacillus biochemical identification strips

[0084] Use the Bacillus biochemical identification strips (product number: HBIG14) of Qingdao Haibo Biological Company to detect VP, citrate, propionate, D-xylose, L-arabinose, D-mannitol, gelatin liquefaction, 7 (v / v)% sodium chloride growth, pH 5.7 growth, nitrate reduction, and starch hydrolysis. For specific operation steps, please refer to the Qingdao Haibo-HBIG14 (product number) kit instructions.

[0085] 1.2 Catalase

[0086] Inoculate NC-B4 into LB solid medium, culture at 35℃ for 24 hours, pick a single colony and place it on a clean glass slide, then add 2 drops of 3 (v / v)% hydrogen peroxide and observe whether there are bubbles. If bubbles are generated, it is judged as positive.

[0087] 1.3 Oxidase

[0088] Use a disposable inoculation needle to pick up a single colony and apply it to the oxidase test paper. If it turns blue or blue-purple within 30 seconds, it is positive, and if it does not change color, it is negative.

[0089] 1.4 Gram staining

[0090] Add 10ul of water to the slide, pick up the bacteria with a toothpick, and then blow dry and fix the smear; stain with ammonium oxalate crystal violet for 1 minute; rinse with distilled water; add iodine solution to cover the surface and stain for about 1 minute; rinse with water and absorb the water with absorbent paper; add a few drops of 95 (v / v)% alcohol and shake gently to decolorize, rinse with water after 20 seconds, absorb the water; stain with safranin stain solution (dilute) for 1 minute, rinse with distilled water. Dry and examine under a microscope.

[0091] 1.5 Spore staining

[0092] Add 10ul of water to the slide, pick up the bacteria with a toothpick, and then smear and heat to fix; cover the slide with malachite green and place the slide on a hot plate, heat until steam is generated for 2 to 3 minutes; cool the slide and wash it gently with tap water; re-stain with safranin for 30 seconds and wash it gently with tap water; wipe it dry with absorbent paper or dry it in an oven, and observe it with the oil objective of a microscope.

[0093] 1.6 Motility

[0094] Use an inoculation needle to puncture and inoculate the bacillus onto LB medium containing 0.3 (v / v)% agar, culture at 35°C, and observe the growth visually 18 hours after inoculation. If the bacteria spread from the puncture line to the surrounding areas in a cloud-like manner, it means that the test strain is motile and is judged as positive.

[0095] 1.7 Phenylalanine deaminase

[0096] Spread the sample onto a phenylalanine agar plate (phenylalanine 2g; NaCl 5g; yeast extract 3g; disodium hydrogen phosphate 1g; agar 12g; dilute to 1L, pH 7.3, normal sterilization), incubate at 35°C for 24 hours, drop 10 (v / v)% ferric chloride on the growing bacterial lawn and observe. If it turns green, it is considered positive.

[0097] In summary, based on the morphological, cultural, and physiological and biochemical characteristics of strain NC-B4, and referring to Bergey's Bacterial Identification Manual, strain NC-B4 was identified and preliminarily confirmed to be Bacillus.

[0098] The morphological, cultural, physiological and biochemical characteristics of the deposited strain are as follows:

[0099] Bacillus velezensis NC-B4: Bacillus velezensis; grows well in LB medium, the strain cells are rod-shaped, and the colonies on LB agar plates are round, milky white, opaque, dry and wrinkled on the surface, with irregular edges and a concave center. The summary of biochemical identification results is shown in Table 1;

[0100] Table 1. Physical and chemical test results of strain NC-B4

[0101]

[0102] 2 Molecular identification of strains

[0103] 2.1 Strain genome extraction

[0104] A single colony was picked from the LB plate and inoculated into fresh LB liquid medium, and cultured at 37°C with shaking at 200 rpm until the bacterial solution OD600 grew to 1.0. 5 mL of bacterial solution was taken and centrifuged to collect the bacteria. Genomic DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit (CW0552S, Kangwei Century, China).

[0105] 2.2 16S rDNA gene amplification

[0106] The 16S rDNA, gyrA and rpoB genes were amplified using 2×T5 super PCR mix (product number: TSE005) of Qingke Biotechnology with genomic DNA as template and 27F / 1492R, gyrA-F / R, and rpoB-F / R as primers. The amplification system was: 150 ng genomic DNA, 0.4 μM forward primer, 0.4 μM reverse primer, 25 μL 2×T5 super PCR mix, H 2 O was added to 50 μL. DNA amplification procedure: pre-denaturation at 98°C for 2 minutes, denaturation at 98°C for 10 seconds, annealing at Tm for 10 seconds, extension at 72°C for 15 seconds / Kb, 30 cycles, final extension at 72°C for 2 minutes. The amplified fragments were detected as a single band by 1 (g / ml)% agarose gel electrophoresis (the fragment lengths were approximately 1400 bp, 900 bp, and 500 bp, respectively. The results are as follows Figure 6 The sequence was obtained by sequencing and then analyzed by BLAST (http: / / www.ncbi.nlm.nih.gov) in GenBank to obtain similar strain names and identify the strains.

[0107] Primer list

[0108] Primer name Primer sequence (5'-3') Primer Tm value (annealing temperature °C) 27F AGAGTTTGATCCTGGCTCAG 52 1492R GGTTACCTTGTTACGACTT 52 gyrA-F CAGTCAGGAAATGCGTACGTCCTT 59 gyrA-R CAAGGTAATGCTCCAGGCATTGCT 59 rpoB-F AGGTCAACTAGTTCAGTATGGAC 55 rpoB-R AAGAACCGTAACCGGCAACTT 55

[0109] 2.5 16S rDNA, gyrA, and rpoB gene sequencing and comparative analysis

[0110] The results of the gene sequencing of strain NC-B4 are as follows

[0111] 16S rDNA gene sequence (SEQ ID NO: 1)

[0112] GATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACAC

[0113] GTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGC

[0114] TAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGG

[0115] CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGT

[0116] GAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGG

[0117] GTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGA

[0118] GGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCA

[0119] ACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTA

[0120] GGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCT

[0121] AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACG

[0122] TAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAG

[0123] GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGG

[0124] GTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATT

[0125] CCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGG

[0126] CGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGT

[0127] GGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGA

[0128] TGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAAC

[0129] GCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCA

[0130] AAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT

[0131] TCGAAGCAACGCGAAGAACCTTACCAGGTCTTTGACATCCTCTGACAA

[0132] TCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGC

[0133] ATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCCGCAA

[0134] CGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTA

[0135] AGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAA

[0136] ATCATCATGCCCCTTATGACCTGGGCTACACACCGTGCTACAATGGACA

[0137] GAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCT

[0138] GTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGA

[0139] ATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGG

[0140] CCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAA

[0141] GTCGGTGAGGTAAC

[0142] gyrA gene sequence (SEQ ID NO: 2)

[0143] GATGTGCGTGACGGTCTGAAGCCGGTTCACAGGCGGATTTTGTACGC

[0144] AATGAATGATTTAGGCATGACCAGTGACAAACCATATAAAAAATCTGC

[0145] CCGTATCGTCGGTGAAGTTATCGGTAAGTACCACCCGCACGGTGACTC

[0146] AGCGGTTTACGAATCAATGGTCAGAATGGCGCAGGATTTTAACTACCG

[0147] CTACATGCTTGTTGACGGACACGGCAACTTCGGTTCGGTTGACGGCG

[0148] ACTCAGCGGCCGCGATGCGTTACACAGAAGCGAGAATGTCAAAAATC

[0149] GCAATGGAAATCCTCCGGGACATTACGAAAGATACGATTGATTATCAA

[0150] GATAACTATGACGGCGCAGAAAGAGAACCTGTCGTCATGCCTTCGAG

[0151] ATTTCCGAATCTGCTCGTAAACGGAGCTGCCGGTATTGCGGTCGGAAT

[0152] GGCGACAAATATTCCTCCGCATCAGCTTGGGGAAGTCATTGAAGGCGT

[0153] GCTTGCCGTAAGTGAGAATCCTGAGATTACAAACCAGGAGCTGATGG

[0154] AATACATCCCGGGCCCGGATTTTCCGACTGCAGGTCAGATTTTGGGCC

[0155] GGAGCGGCATCCGCAAGGCATATGAATCCGGACGGGGATCCATTACGA

[0156] TCCGGGCTAAGGCTGAAATCGAAGAGACATCATCGGGAAAAGAAAG

[0157] AATTATTGTCACAGAACTTCCTTATCAGGTGAACAAAGCGAGATTAAT

[0158] TGAAAAAATCGCAGATCTTGTCCGGGACAAAAAAATCGAAGGAATTA

[0159] CCGATCTGCGTGACGAATCCGACCGTAACGGAATGAGAATCGTCATTG

[0160] AGATCCGCCGTGACGCCAATGCTCACGTCATTTTGAATAACCTGTACA

[0161] AACAAACGGCCCTGCAGACGTCTTTCGGAATCAACCT rpoB gene sequence (SEQ ID NO: 3)

[0162] GTTAGAATTACCAAATCTCATTGAAATTCAAACCTCTTCTTATCAGTGG

[0163] TTTCTTGATGAGGGTCTTAGAGAGATGTTTCAAGACATATCACCAATTG

[0164] AGGATTTCACTGGTAACCTCTCTCTAGAGTTCATTGACTACAGTTTAG

[0165] GAGATCCTAAGTATCCCGTTGAAGAGTCAAAAGAACGTGATGTGACTT

[0166] ACTCAGCTCCGCTGAGAGTGAAGGTTCGTTTAATTAACAAAGAAACT

[0167] GGAGAGGTAAAAGATCAGGATGTCTTCATGGGTGATTTCCCTATTATG

[0168] ACAGATACCGGTACTTTTATCATCAACGGTGCAGAACGTGTTATCGTAT

[0169] CTCAGCTTGTTCGGTCTCCAAGTGTATATTTCAGTGGTAAAGTAGACA

[0170] AAAACGTAAAAAAGGTTTTACCGCGACTGTCATTCCAAACCGTGGC

[0171] GCATGGTTAGAATACGAAACTGA

[0172] Blast analysis of the three gene sequences showed that the similarity with Bacillus velezensis was 100%. Its morphological characteristics and physiological and biochemical properties were most similar to Bacillus velezensis. Therefore, based on the determination of 16S rDNA, gyrA, and rpoB gene sequences and the results of morphological characteristics and physiological and biochemical characteristics, the strain can be classified as Bacillus velezensis.

[0173] Example 4: Determination of the antibacterial spectrum of strain NC-B4

[0174] The antibacterial activity of Bacillus against pathogenic fungi was detected in vitro by the dual culture method, and the antibacterial activity of Bacillus against yeast-like pathogenic fungi was detected in vitro by the hole diffusion method. The antibacterial activity of NC-B4 was determined using 4 dermatophytes and 3 yeast-like fungi as targets. The selected fungi and the antibacterial results are shown in Table 2.

[0175] 1.1 Activation of pathogenic fungi

[0176] For filamentous fungi, use a sterilized inoculation loop to pick mycelium from pathogenic fungi stored at 4°C onto a new SDA medium plate, and culture it in a constant temperature incubator at 28°C for 5 days for later use. For yeast-like fungi, directly pick a single colony, streak it onto a new SDA plate, and culture it at 35°C for 2 days for later use.

[0177] 1.2 Activation of pathogenic bacteria

[0178] Streak the NC-B4 glycerol stock frozen at -80°C onto LB plates and culture at 35°C for 24 hours for later use.

[0179] 1.3 Antibacterial experiment

[0180] Double culture method: Select activated pathogenic fungal hyphae and inoculate them onto SDA plates using the three-point method. Inoculate bacteria at equal distances in the middle of the plates. After culturing at 28°C for 6 days, begin measuring the width of the inhibition zone (the distance between the antagonistic bacteria and the edge of the pathogenic fungus).

[0181] Digging and diffusion method: Prepare yeast pathogenic fungal bacterial suspension (grow to logarithmic phase OD600 is approximately equal to 2.0), wait for LB solid medium to cool to 45°C, take 100μL of bacterial suspension and add 15ml medium, mix quickly and pour into the plate. Punch a hole in the middle of the medium (hole diameter 6mm), then add 20μL of the test bacteria NC-B4 bacterial suspension or filtered sterilized bacterial suspension (bacterial suspension OD600 is approximately equal to 2.0) into the hole, repeat 3 times for each culture dish, culture at 37°C, and measure the diameter of the inhibition zone after 1-2 days.

[0182] 1.4 Results and Analysis

[0183] The results of the antibacterial activity of strain NC-B4 against human pathogenic fungi are shown in Table 2.

[0184] Table 2 Summary of the antibacterial spectrum of strain NC-B4

[0185]

[0186] As can be seen from the above table, this strain can have an inhibitory effect on a variety of fungi and can be promoted for use, among which it has the best effect on Candida auris.

[0187] Example 5: Phylogenetic tree construction

[0188] The obtained 16S rDNA, gyrA and rpoB gene original sequences were spliced ​​and verified using DNAStar analysis software, and then submitted to GenBank to obtain the sequence accession number. The Blast program was run in NCBI (http: / / www.ncbi.nlm.nih.gov) to perform sequence homology search. The corresponding gene sequences of the relevant strains were downloaded, and a sequence was assembled according to the order of 16S rDNA, gyrA, and rpoB genes. The phylogenetic tree based on the 16SrDNA, gyrA, and rpoB combined gene sequences was constructed using the neighbor-joining method of MEGA 7.0 software. According to the sequence homology size and phylogenetic relationship, the species of the strain was determined, and it was specifically confirmed to be Bacillus velez. The results are shown in Figure 4 .

[0189] Under the premise that no conflict occurs, those skilled in the art may freely combine and superimpose the above-mentioned additional technical features.

[0190] The above description is only a preferred embodiment of the present invention. Any technical solution that achieves the purpose of the present invention by substantially the same means belongs to the protection scope of the present invention.

Claims

1. A Bacillus Velezii ( Bacillus velezensis )NC-B4, characterized in that The strain was deposited in the General Microbiology Center of China Culture Collection Association on May 19, 2022, with the deposit number CGMCCNO.24929.

2. A use of the Bacillus Velez NC-B4 as claimed in claim 1 in the preparation of a drug for inhibiting human pathogenic fungal infection; characterized in that: The human pathogenic fungi are Candida auris, Candida albicans, Cryptococcus, Cunninghamella parvula, Trichophyton schrenckii, Absidia spp. and Lactodiplodia.

3. A drug for inhibiting pathogenic fungal infection in human body, characterized in that: The active ingredient is the Bacillus Velezii NC-B4 described in claim 1, and the human pathogenic fungi are Candida auris, Candida albicans, Cryptococcus, Cunninghamella parvula, Trichophyton schrenckii, Absidia spp. and Lactodiplodia.

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