Bacillus strain H7 and application thereof

Abstract

The invention discloses a bacillus strain H7 and an application thereof. The invention provides bacillus velezensis H7 or a progeny of the bacillus velezensis H7, and the preservation number of the bacillus velezensis H7 in the China Center for Type Culture Collection (CCTCC) is CCTCC NO: M 2025630. The bacillus velezensis H7 disclosed by the invention has a very good antibacterial effect on various common clinical pathogenic bacteria (including klebsiella pneumoniae, escherichia coli, candida albicans, enterobacter cloacae complex, acinetobacter baumannii and the like), and particularly shows a very high inhibition rate on some drug-resistant clinical pathogenic bacteria; therefore, the compound has important application value in development of drug and non-drug antibacterial products (such as medical antibacterial materials and the like), a new choice can be provided for drug and non-drug prevention and control of clinical pathogenic bacterium infection, and particularly, the compound has a supplementary effect in the aspects of reducing drug-resistant bacterium propagation risk and reducing drug dependence.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a Bacillus strain H7 and its applications. Background Technology

[0002] Bacillus is widely found in soil, plants, food, and animal intestines. Its vegetative cells divide and multiply approximately every half hour. Changes in environmental factors such as nutrient status, temperature, pH, oxygen content, and salt concentration can trigger the interconversion between spores and vegetative cells. Bacillus is characterized by its heat resistance, rapid reactivation, and strong enzyme secretion, allowing it to survive under both aerobic and anaerobic conditions. It forms spores under nutrient-deficient or drought conditions and can regerminate into vegetative cells when conditions are suitable.

[0003] Bacillus belesiensis ( Bacillus velezensis ) is a genus of Bacillus ( Bacillus Bacillus vesiliflorus is a Gram-positive, spore-forming rod-shaped bacterium named after its initial isolation from soil in the Belé River estuary region of Spain. It has become a hot topic of research in microbiology and applied sciences in recent years, with its ability to produce various active substances and its strong environmental adaptability making it important in agriculture, ecology, and probiotics. Current research shows that the existing antibacterial spectrum of Bacillus vesiliflorus primarily targets plant pathogens and some environmental pathogens. For example, it exhibits significant inhibitory effects against plant pathogenic fungi (such as Fusarium, Botrytis cinerea, and rice blast fungus); it also shows some inhibitory effects against certain bacteria (such as Staphylococcus aureus, Pseudomonas aeruginosa, and some streptococci); and it has antagonistic activity against a few agricultural-related bacteria (such as Ralstonia solanacearum and Ralstonia solanacearum). However, there are relatively few reports on the antibacterial activity of Bacillus belyi against clinical pathogens. Its effects on a variety of common clinical pathogens (especially Gram-negative bacilli and fungi) are limited, particularly against the following strains: Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Candida albicans, Enterobacter cloacae complex, and drug-resistant strains of these bacteria. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus strain H7 and its applications.

[0005] In a first aspect, the present invention claims protection for a strain of Bacillus belye or its progeny.

[0006] The Bacillus belesiensis claimed in this invention is Bacillus belesiensis ( Bacillus velezensis The strain number is H7, and its accession number at the China Center for Type Culture Collection is CCTCC NO: M 2025630.

[0007] Secondly, the present invention claims protection for compositions containing Bacillus belye or its progeny as described in the first aspect above.

[0008] Further, the composition can be a culture; the culture is a substance obtained by culturing the Bacillus velezensis or its offspring in a microbial culture medium (all substances in the culture vessel, i.e. fermentation product, such as a substance containing the Bacillus velezensis or its offspring and substances secreted into the liquid culture medium, i.e. fermentation broth, or such as a substance containing the Bacillus velezensis or its offspring and substances secreted into the solid culture medium, i.e. solid fermentation product).

[0009] In the above culture, the substance includes the Bacillus velezensis or its offspring (bacterial cells themselves) and metabolites thereof as described in the first aspect above.

[0010] The term "metabolite" refers to primary metabolites and / or secondary metabolites produced in the process of microbial metabolism. Primary metabolism refers to the process in which a microorganism absorbs various nutrients from the outside world, and through catabolism and anabolism, generates substances and energy to maintain life activities. The product of primary metabolism is a primary metabolite, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and other monomers, as well as various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process in which a microorganism, using primary metabolites as precursors, synthesizes substances that have no clear function for the life activities of the microorganism. The product of secondary metabolism is a secondary metabolite, which is mostly a compound with a complex molecular structure. According to its function, it can be classified into antibiotics, hormones, alkaloids, toxins, etc.

[0011] In the above culture, the microbial culture medium is a bacterial culture medium. Further, the bacterial culture medium can be a solid culture medium or a liquid culture medium.

[0012] The term "culture" refers to a general term for a liquid or solid culture medium that has grown a population of microorganisms after being inoculated and cultured by humans. That is, a product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or can contain a certain amount of culture medium, metabolites, or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing microorganisms, which can be a culture of a certain generation, or a mixture of several generations.

[0013] In some embodiments, the composition is a microbial inoculant.

[0014] Further, in the microbial inoculant, the Bacillus velezensis or / and its metabolites can exist in the form of live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0015] In the present application, the metabolite can be obtained from the fermentation broth of the Bacillus velezensis or its progeny. The metabolite can be a sterile metabolite of the Bacillus velezensis or a metabolite containing the Bacillus velezensis. The sterile metabolite (sterile fermentation filtrate) can be prepared by culturing the Bacillus velezensis or its progeny in a liquid culture medium, and filtering the Bacillus velezensis or its progeny in the liquid culture (fermentation broth) to obtain the sterile metabolite of the Bacillus velezensis. The metabolite containing the Bacillus velezensis can be prepared by culturing the Bacillus velezensis or its progeny in a liquid fermentation medium, and collecting the fermentation broth containing the Bacillus velezensis or its progeny and the substances secreted into the liquid culture medium, which is the metabolite containing the Bacillus velezensis or its progeny.

[0016] In the present application, the composition has the function of inhibiting clinical pathogenic bacteria.

[0017] In a third aspect, the present application claims the use of the Bacillus velezensis or its progeny as described in the first aspect above or the composition as described in the second aspect above in any one of the following: (A1) preparing a product for inhibiting clinical pathogenic bacteria; (A2) preparing a medicament for preventing and / or treating diseases caused by infection of clinical pathogenic bacteria.

[0018] In (A1), the product can be a medicament; or a disinfectant / sterilant product or a medical antibacterial material, etc.

[0019] In a fourth aspect, the present application claims a product for inhibiting clinical pathogenic bacteria.

[0020] The product for inhibiting clinical pathogenic bacteria claimed in the present application can have the active ingredient of the Bacillus velezensis or its progeny as described in the first aspect above or the composition as described in the second aspect above.

[0021] The product can be a medicament; or a disinfectant / sterilant product or a medical antibacterial material, etc.

[0022] In a fifth aspect, the present application claims a medicament for preventing and / or treating diseases caused by infection of clinical pathogenic bacteria.

[0023] The medicament for preventing and / or treating diseases caused by infection of clinical pathogenic bacteria claimed in the present application can have the active ingredient of the Bacillus velezensis or its progeny as described in the first aspect above or the composition as described in the second aspect above.

[0024] In the above-mentioned related aspects, the clinical pathogenic bacteria can be drug-resistant clinical pathogenic bacteria or non-drug-resistant clinical pathogenic bacteria.

[0025] In the aforementioned relevant aspects, the clinically pathogenic bacteria may be clinically pathogenic fungi or clinically pathogenic bacteria.

[0026] In some implementation cases, the clinical pathogens are selected from all or some of the following: drug-resistant or non-drug-resistant Klebsiella pneumoniae, drug-resistant or non-drug-resistant Escherichia coli, drug-resistant or non-drug-resistant Candida albicans, drug-resistant or non-drug-resistant Enterobacter cloacae complex, and drug-resistant or non-drug-resistant Acinetobacter baumannii.

[0027] In some embodiments, the Klebsiella pneumoniae is a carbapenem-resistant Klebsiella pneumoniae (such as meropenem, imipenem, ertapenem), such as Klebsiella pneumoniae KP47434. In some embodiments, the Klebsiella pneumoniae is an ampicillin- and / or chloramphenicol-resistant Klebsiella pneumoniae.

[0028] In some implementations, the Escherichia coli is ceftazidime- and / or avibactam- and / or gentamicin-resistant Escherichia coli, such as Escherichia coli 73552.

[0029] In some implementations, the Candida albicans is Candida albicans ATCC 10231.

[0030] In some implementations, the Enterobacter cloacae complex is an Enterobacter cloacae complex resistant to cephalosporins and / or polymyxins (such as cefoperazone and / or polymyxin E) and / or ampicillin, such as Enterobacter cloacae complex. E. bugandensis strain ECC23.

[0031] In some implementations, the Acinetobacter baumannii is chloramphenicol- and / or carbenicillin- and / or cefuroxime sodium-resistant Acinetobacter baumannii, such as Acinetobacter baumannii ATCC 17978.

[0032] This invention isolated and obtained a strain of Bacillus belye ( Bacillus velezensis Bacillus vesiculosus H7, as tested, has been found to have excellent antibacterial effects against a variety of common clinical pathogens (including Klebsiella pneumoniae, Escherichia coli, Candida albicans, Enterobacter cloacae complex, Acinetobacter baumannii, etc.), and has shown a particularly high inhibition rate against some drug-resistant clinical pathogens. The Bacillus vesiculosus H7 provided by this invention has a broad-spectrum antibacterial effect against clinical pathogens, making it of significant application value in the development of pharmaceutical and non-pharmaceutical antibacterial products (such as medical antibacterial materials). It can provide new options for the drug and non-pharmaceutical prevention and control of clinical pathogen infections, especially playing a supplementary role in reducing the risk of drug-resistant bacterial transmission and reducing drug dependence.

[0033] Deposit Description Proposed category naming:Bacillus velezensis H7; Depository: China General Microbiological Culture Collection Center; Abbreviation of the depository: CCTCC; Address: Wuhan, China. Wuhan University; Date of deposit: March 31, 2025; Accession number of the depository: CCTCC NO: M 2025630. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Figure 1 is the colony morphology of Bacillus velezensis H7.

[0035] Figure 2 Figure 2 is the confrontation effect diagram of Bacillus velezensis H7 on Candida albicans ATCC 10231 (Oxford cup method). In the figure, the label "H7" represents Bacillus velezensis H7; the label "YPD" represents YPD liquid medium (negative control); and the label "FIZ" represents fluconazole (positive control).

[0036] Figure 3 Figure 3 is the confrontation effect diagram of Bacillus velezensis H7 on Candida albicans 2310223003 (Oxford cup method). In the figure, the label "H7" represents Bacillus velezensis H7; the label "YPD" represents YPD liquid medium (negative control); and the label "FIZ" represents fluconazole (positive control).

[0037] Figure 4 Figure 4 is the confrontation effect diagram of Bacillus velezensis H7 on Candida albicans ATCC 10231 (double-layer plate method).

[0038] Figure 5 Figure 5 is the confrontation effect diagram of Bacillus velezensis H7 on Klebsiella pneumoniae 1053, Escherichia coli 73552 and Candida albicans 2310223003 (double-layer plate method). In the figure, the label "H7" represents Bacillus velezensis H7; the label "1053" represents Klebsiella pneumoniae 1053; the label "73552" represents Escherichia coli 73552; and the label "2310223003" represents Candida albicans 2310223003. In the small graph of "H7 vs 1053", the left half of the plate is streaked with H7 in advance, and the right half is not streaked with H7 in advance (i.e., negative control); in the small graph of "H7 vs 73552", the left half of the plate is streaked with H7 in advance, and the right half is not streaked with H7 in advance (i.e., negative control); and in the small graph of "H7 vs 2310223003", the right half of the plate is streaked with H7 in advance, and the left half is not streaked with H7 in advance (i.e., negative control). DETAILED DESCRIPTION

[0039] The present application will be further described in details with specific embodiments, the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.

[0040] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0041] The medium formula involved in the following examples is shown in Table 1.

[0042] Table 1, the medium involved in the present application

[0043] The clinical pathogenic bacteria involved in the following examples are as follows: Klebsiella pneumoniae KP47434: resistant to carbapenem antibiotics (such as meropenem, imipenem, ertapenem), recorded in “Zhou Kai, Xue Chun-Xu, Xu Tingting, Shen Ping, Wei Sha, Wyres Kelly L., Lam Margaret M.C., Liu Jinquan, Lin Haoyun, Chen Yunbo, Holt Kathryn E., the BRICS Working Group, Xiao Yonghong * . A point mutation in recC associated with subclonal replacement of carbapenem-resistant Klebsiella pneumoniae ST11 in China .Nature Communications , 2023, 14: 2464.” In the case of meeting the biosafety, the public can obtain from the applicant, only for repeated use of the present application experiments, not for other purposes.

[0044] Escherichia coli 73552: resistant to ceftazidime / avibactam and gentamicin, recorded in “Xu Tingting #Wu Weiyuan, Huang Lili, Liu Bin, Zhang Qiaodong, Song Jingjie, Liu Jialong, Li Bing, Li Zhao, Zhou Kai. Novel plasmid-mediated CMY variant (CMY-192) conferring ceftazidime-avibactam resistance in multidrug-resistant Escherichia coli According to the article "Antimicrobial Agents and Chemotherapy, 2024, 68:e00906-24," the invention is available to the public from the applicant, provided that it complies with biosafety requirements, and may only be used to repeat the experiments of this invention and may not be used for other purposes.

[0045] Candida albicans ATCC 10231: Obtained from ATCC, its product number is 10231.

[0046] Enterobacter cloacae complex ( Enterobacter bugandensis ECC23: Resistance to cephalosporins and polymyxins (such as cefoperazone and polymyxin E) and ampicillin, as described in "Xu Tingting, XueChun-Xu, Chen Yuxin, Huang Junxi, Wu Weiyuan, Lu Yuemei, Huang Qiuhui, Chen Dandan, Zhou Kai. Frequent convergence of mcr-9 and carbapenemase genesin Enterobacter cloacae complex driven by epidemic plasmids and hostincompatibility. Emerging Microbes&Infections Table S2 in the supplementary materials of the article “, 2022, 11(1):1959-1972” indicates that, subject to biosafety requirements, the public may obtain the information from the applicant and use it only for repeating experiments of the present invention, and may not use it for any other purpose.

[0047] Acinetobacter baumannii ATCC17978: resistant to chloramphenicol, carbenicillin and cefuroxime sodium, obtained from ATCC, product number 17978.

[0048] Example 1, Bacillus belye ( Bacillus velezensis Isolation and identification of H7 The purified strain H7 was obtained by dilution plating and streak plating methods on seawater samples collected from the Shijiao Mangrove Nature Reserve in Fangchenggang City, Guangxi Zhuang Autonomous Region (21°36'56''N, 108°13'55''E).

[0049] Strawberry strain H7 was streaked onto YPD solid medium plates and incubated upside down at 37°C for 24 hours. Its colony morphology was observed and recorded. Figure 1 As shown, the colonies are irregularly round, about 3-4 mm in diameter, with root-like spread at the edges; the surface is dry and rough, covered with wrinkles, with particularly dense wrinkles in the central area; the whole is opaque milky white, hard in texture, and not easy to pick up.

[0050] Genomic DNA was extracted from strain H7 using the FastPure Bacteria DNA Isolation Mini Kit. PCR amplification of the 16S rRNA gene was performed using universal bacterial identification primers 27F and 1492R, and the sequence was sent to BGI for sequencing. The obtained 16S rRNA gene sequence is shown in SEQ ID NO:1. This sequence was then compared with existing Bacillus belysinus strains (…) using NCBI BLAST. Bacillus velezensis The sequence coverage was 99%, and the similarity was 99.93%, which is greater than the species differentiation threshold. Therefore, strain H7 was confirmed to be a Bacillus species, specifically Bacillus belesiensis. Bacillus velezensis ).

[0051] Based on the above identification results, strain H7 was identified as Bacillus belye (B. belye). Bacillus velezensis The strain was deposited at the China Center for Type Culture Collection (CCTCC) on March 31, 2025, with the collection number CCTCC NO: M 2025630. Hereinafter referred to as strain H7 or Bacillus bereaves H7.

[0052] Example 2, Bacillus belye ( Bacillus velezensis Inhibitory effects of H7 on various clinical pathogens, including drug-resistant strains. I. Oxford cup method for identifying the antibacterial effect of Bacillus belyssus H7 against Candida albicans Candida albicans ATCC 10231 and Bacillus bereaves H7 were inoculated into YPD liquid medium and cultured overnight at 37°C. After 24 hours, the OD of Candida albicans ATCC 10231 was adjusted. 600=0.5, 100 μL of the adjusted concentration of C. albicans ATCC 10231 liquid was uniformly coated on the surface of the YPD solid culture medium plate, the plate was divided into three zones, and sterilized Oxford cups were placed on the plate. 100 μL of B. bellus H7 liquid was taken in the Oxford cups, YPD liquid medium was used as a negative control, and 128 μg / ml fluconazole was used as a positive control. The culture dish was smoothly sent into the incubator, and the results were observed after 24h of culture at 37°C. The results are shown in Figure 2 It can be seen that the B. bellus H7 liquid produces a clear inhibition zone on C. albicans ATCC 10231, and the negative control (YPD medium) does not detect an inhibition zone.

[0053] The same method was used to determine the inhibition effect of B. bellus H7 on C. albicans 2310223003 using Oxford cups, and the results are shown in Figure 3 It can be seen that the B. bellus H7 liquid produces a clear inhibition zone on C. albicans 2310223003, which is significantly larger than the inhibition zone of the positive control (128 μg / mL fluconazole), and the negative control (YPD medium) does not detect an inhibition zone.

[0054] The above results show that B. bellus H7 has a significant inhibition effect on C. albicans standard strain ATCC 10231 and clinically derived strain 2310223003.

[0055] II. Double-layer plate method for identifying the inhibition effect of B. bellus H7 on various clinically pathogenic bacteria B. bellus H7 was streaked on a YPD plate Figure 4 The upper half of the plate), and cultured overnight at 37°C. C. albicans ATCC 10231 single colonies were inoculated in YPD liquid medium and cultured overnight at 37°C. After 24h, the OD 600 =0.5, 100 mL of melted and cooled YPD solid medium at about 50°C was prepared, 200 μL of the adjusted concentration of C. albicans ATCC 10231 liquid was added, and it was quickly and thoroughly mixed, and poured onto the upper layer of the B. bellus H7 streaked YPD plate Figure 4 The C. albicans ATCC 10231 liquid was poured onto the entire upper layer of the plate, Figure 4 The lower half of the plate was not streaked with strain H7 in advance as a negative control), and the results were observed after 24h of culture at 37°C. As shown in Figure 4 It can be seen that the indicator bacteria (C. albicans ATCC 10231) in the upper layer of the area previously streaked with strain H7 in the lower layer cannot grow Figure 4 The upper half), and the antagonistic effect is obvious, and the negative control (no sterile streaking in the lower layer, Figure 4The upper layer indicates that the bacteria grow uniformly and there is no inhibition phenomenon.

[0056] The same method was used to determine the bacteriostatic effect of strain H7 fermentation broth on C. albicans 2310223003, K. pneumoniae 1053 (a clinical isolate, resistant to ampicillin and chloramphenicol), and E. coli 73552 (resistant to ceftazidime / avibactam and gentamicin) using the double-layer plate method. The results are shown in Table 2. Figure 5 As can be seen, after the lower layer was inoculated with strain H7, the upper layer of C. albicans 2310223003, K. pneumoniae 1053, and E. coli 73552 could not grow.

[0057] The above results show that Bacillus velezensis H7 has obvious bacteriostatic effect on various clinical pathogenic bacteria, including drug-resistant bacteria.

[0058] III. Test tube method for detecting the bacteriostatic rate of Bacillus velezensis H7 on C. albicans 1. Shake the bacteria: Bacillus velezensis H7 and C. albicans ATCC 10231 were shaken in YPD liquid medium and cultured at 37°C overnight.

[0059] 2. Transfer: After 24 h, the bacteria and medium were transferred to a new medium at a ratio of 1:100 (volume ratio) and cultured at 37°C for 24 h.

[0060] ① Experimental group: Bacillus velezensis H7 + C. albicans ATCC 10231; in this group, both bacteria were transferred to the same new medium at a volume ratio of 1:100 for culture.

[0061] ② Control group: C. albicans ATCC 10231; in this group, only C. albicans ATCC 10231 was transferred to a new medium at a volume ratio of 1:100 for culture, i.e., the amount of initial inoculation of C. albicans ATCC 10231 was consistent with the experimental group.

[0062] 3. Spread: The bacteria solution of the experimental and control groups was diluted with PBS to 10 -1 to 10 -6 , 100 μl of the bacteria solution at 10 -3 to 10 -5 gradients was spread on a Sabouraud plate, 100 μl of the bacteria solution at 10 -5 to 10 -6 gradients was spread on a Sabouraud plate, and the process was repeated 3 times. The plates were incubated in a 37°C incubator, and after 24 h, colony counting was performed, and the results were averaged.

[0063] 4. Colony counting: The number of colonies in the two groups was unified as the viable concentration of the original bacterial solution by dilution conversion. The viable concentration of the Candida albicans cultured alone (i.e. the control group) was taken as CK, and the viable concentration of the Bacillus velezensis H7 and Candida albicans ATCC 10231 co-cultured group (i.e. the experimental group) was taken as T. The inhibition rate calculation formula is as follows:

[0064] The results show that the inhibition rate of Bacillus velezensis H7 on Candida albicans ATCC 10231 is 98%.

[0065] Similarly, 12 strains of clinically isolated Candida albicans (as shown in Table 2) were used to repeat the test tube method to verify the inhibition rate of Bacillus velezensis H7, and the results are shown in Table 2.

[0066] Table 2. Inhibition rate of Bacillus velezensis H7 on 12 strains of clinically isolated Candida albicans

[0067] The above results show that Bacillus velezensis H7 has a significant inhibitory effect on 12 strains of clinically derived Candida albicans, with the best inhibitory effect on Candida albicans 47, with an inhibition rate as high as 96.13%, while the inhibitory effect on Candida albicans 53 is moderate.

[0068] Four, detection of the inhibition rate of Bacillus velezensis H7 on various clinically pathogenic bacteria including drug-resistant strains by test tube method Antagonistic bacteria: Bacillus velezensis H7 (indicated as H7 in step 2); Indicator bacteria: Klebsiella pneumoniae KP47434 (indicated as KP47434 in step 2, resistant to carbapenem antibiotics such as meropenem, imipenem, and ertapenem), Acinetobacter baumannii ATCC 17978 (indicated as 17978 in step 2, resistant to chloramphenicol, carbenicillin, and cefuroxime sodium), Escherichia coli 73552 (indicated as 73552 in step 2, resistant to ceftazidime / avibactam and gentamicin), and Enterobacter cloacae complex group ECC23 (indicated as ECC23 in step 2, resistant to cephalosporin antibiotics and polymyxin antibiotics such as cefoperazone and polymyxin E, as well as ampicillin).

[0069] 1. Shake the bacteria: Shake the above antagonistic bacteria and each indicator bacteria with LB liquid medium, and incubate at 37°C overnight.

[0070] 2. Transfer: After 24h, transfer the bacterial solution and medium to a new medium at a ratio of 1:100 (volume ratio) and shake culture at 37°C for 24h.

[0071] ① Experimental group: H7+KP47434, H7+17978, H7+73552, H7+ECC23; in this group, antagonistic bacteria and indicator bacteria were both transferred to the same new culture medium according to the volume ratio of 1:100 for culture.

[0072] ② Control group: KP47434, 17978, 73552, ECC23; in this group, only the indicator bacteria were transferred to a new culture medium according to the volume ratio of 1:100 for culture, that is, the amount of the initial inoculation of the indicator bacteria was consistent with the experimental group.

[0073] 3. Coating: the bacterial liquid of the experimental group and the control group was diluted to 10 -1 to 10 -6 with PBS, 100 μl of the bacterial liquid of H7+KP47434 at 10 -3 to 10 -5 gradient was coated on a meropenem (MEM) plate with a resistance of 4 μg / ml; 100 μl of the bacterial liquid of H7+17978 at 10 -3 to 10 -5 gradient was coated on a chloramphenicol (CHl) plate with a resistance of 25 μg / ml; 100 μl of the bacterial liquid of H7+73552 at 10 -3 to 10 -5 gradient was coated on a gentamicin (GEN) plate with a resistance of 32 μg / ml; 100 μl of the bacterial liquid of H7+ECC23 at 10 -3 to 10 -5 gradient was coated on an ampicillin (AMP) plate with a resistance of 100 μg / ml. 100 μl of the bacterial liquid of the control group KP47434 at 10 -5 to 10 -6 gradient was coated on a MEM plate with a resistance of 4 μg / ml; 100 μl of the bacterial liquid of 17978 at 10 -5 to 10 -6 gradient was coated on a CHl plate with a resistance of 25 μg / ml; 100 μl of the bacterial liquid of 73552 at 10 -5 to 10 -6 gradient was coated on a GEN plate with a resistance of 32 μg / ml. 100 μl of the bacterial liquid of ECC23 at 10 -5 to 10 -6 gradient was coated on an AMP plate with a resistance of 100 μg / ml. Each gradient was repeated 3 times and placed in a 37°C incubator for culture, and colony counting was performed after 24 h, and the results were averaged.

[0074] 4. Colony counting: The number of colonies in two groups was unified as the viable bacterial concentration in the original bacterial solution by dilution factor conversion, and the viable bacterial concentration of the indicator bacteria cultured alone (i.e. the control group) was CK, and the viable bacterial concentration of the indicator bacteria cultured with the antagonistic bacteria (i.e. the experimental group) was T. The inhibition rate calculation formula was as follows:

[0075] The results are shown in Table 3. It can be seen that Bacillus velezensis H7 can inhibit a variety of clinical pathogenic bacteria including drug-resistant strains, and the inhibition effect on Escherichia coli 73552 is the best, and the inhibition effect on Acinetobacter baumannii ATCC 17978 is relatively weak.

[0076] Table 3. Inhibition rate of Bacillus velezensis H7 on a variety of clinical pathogenic bacteria detected by tube method

[0077] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In short, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application.

Claims

1. Bacillus belye or its progeny, characterized in that: The *Bacillus belesii* is *Bacillus belesii* (… Bacillus velezensis The strain number is H7, and its accession number at the China Center for Type Culture Collection is CCTCC NO: M 2025630.

2. A composition containing the Bacillus belye or its progeny as claimed in claim 1.

3. The composition according to claim 2, characterized in that: The composition is a culture; the culture is a substance obtained by culturing the Bacillus belye or its progeny in a microbial culture medium.

4. The composition according to claim 2, characterized in that: The composition is a microbial agent.

5. The composition according to claim 4, characterized in that: The composition has the function of inhibiting clinically pathogenic bacteria.

6. The use of the Bacillus belye or its descendants as claimed in claim 1, or the composition of any one of claims 2-5, in any of the following: (A1) Prepare products for inhibiting clinically pathogenic bacteria; (A2) Prepare medicines for the prevention and / or treatment of diseases caused by clinically pathogenic bacteria.

7. A product for inhibiting clinically pathogenic bacteria, wherein the active ingredient is Bacillus belyssus as described in claim 1 or its descendants, or a composition as described in any one of claims 2-5.

8. A medicament for the prevention and / or treatment of diseases caused by clinically pathogenic bacteria, wherein the active ingredient is Bacillus belyssus as described in claim 1 or its descendants, or a composition as described in any one of claims 2-5.

9. The composition according to claim 5, the application according to claim 6, the product according to claim 7, or the medicine according to claim 8, characterized in that: The clinical pathogens are either drug-resistant or non-drug-resistant clinical pathogens; and / or The clinically pathogenic bacteria are clinically pathogenic fungi or clinically pathogenic bacteria.

10. The composition, application, product, or medicine according to claim 9, characterized in that: The clinical pathogens are selected from all or part of the following: drug-resistant or non-drug-resistant Klebsiella pneumoniae, drug-resistant or non-drug-resistant Escherichia coli, drug-resistant or non-drug-resistant Candida albicans, drug-resistant or non-drug-resistant Enterobacter cloacae complex, and drug-resistant or non-drug-resistant Acinetobacter baumannii. Further, the Klebsiella pneumoniae is a carbapenem-resistant Klebsiella pneumoniae; and / or, the Klebsiella pneumoniae is an ampicillin- and / or chloramphenicol-resistant Klebsiella pneumoniae; and / or Furthermore, the *E. coli* is *E. coli* resistant to ceftazidime and / or avibactam and / or gentamicin; and / or Furthermore, the Enterobacter cloacae complex is an Enterobacter cloacae complex resistant to cephalosporin antibiotics and / or polymyxin antibiotics and / or ampicillin; and / or Furthermore, the Acinetobacter baumannii is a chloramphenicol- and / or carbenicillin- and / or cefuroxime sodium-resistant Acinetobacter baumannii.

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