A type of Bacillus mohaiweii VT275 and its application

Abstract

This invention provides a strain of Bacillus mojavensis, belonging to the field of microbial technology. The Bacillus mojavensis VT275 of this invention, with accession number CGMCC No. 29974, can effectively inhibit common skin pathogens and foodborne pathogens, such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Shigella, and Enterococcus faecalis. The Bacillus mojavensis VT275 of this invention can be used to prevent or treat skin diseases such as atopic dermatitis or to control foodborne infections, and has good application prospects in the fields of pharmaceuticals, cosmetics, edible microorganisms, and feed microorganisms.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular, to a type of Bacillus mohaiweii and its application in the prevention and treatment of skin pathogen infections. Background Technology

[0002] As the largest organ in the human body, the skin coordinates many functions essential for survival. The skin microbiome is an ecosystem composed of microorganisms such as bacteria, fungi, and viruses, skin cells and their secretions, and the immune system. Influenced by various external and internal factors, the skin microbiome not only colonizes the skin surface but also interacts with the skin through multiple mechanisms, significantly affecting its barrier function. Notably, the skin microbiota is one of the most diverse microbial communities in the human body and is crucial to the host's defense system. Skin symbiotic microbiota protect the host from harmful pathogens through mechanisms such as competitive inhibition, while also participating in the balance regulation of the immune system between effective protection and destructive inflammation. Furthermore, the skin microbiota can synthesize and release various bioactive substances, such as histamine, glutamate, γ-aminobutyric acid (GABA), and peptides such as α-melanocyte-stimulating hormone (α-melanocyte-stimulating hormone). Therefore, skin symbiotic microbiota play a vital role in maintaining normal skin microecological homeostasis. Imbalance in the skin microbiota can lead to skin microecological disorders, subsequently causing disease.

[0003] Staphylococcus aureus, a transient resident microorganism of the skin, is mainly found in pathological conditions, especially in patients with atopic dermatitis (AD), where it colonizes extensively, and its colonization level is positively correlated with the severity of the disease. Studies have shown that 64% to 82% of neonatal methicillin-resistant Staphylococcus aureus (MRSA) skin infections occur in infants delivered by cesarean section. The virulence factors of Staphylococcus aureus can induce keratinocytes to express endogenous proteases, disrupting the epidermal barrier function. This is a key mechanism in the pathophysiology of AD, further confirming the important role of Staphylococcus aureus in the pathogenesis of AD. Furthermore, AD has a high incidence in children, affecting approximately one-fifth of children worldwide. Currently, AD treatment is mainly drug-based, including topical corticosteroids, anti-infective therapy, topical immunomodulators, oral antihistamines, and biologics. However, long-term use of these drugs may cause adverse reactions. Therefore, treatment from a microecological perspective can not only effectively relieve symptoms but also avoid problems such as the spread of Staphylococcus aureus drug resistance.

[0004] Previous studies have shown that certain skin isolates can inhibit the growth of Staphylococcus aureus and alleviate symptoms in Alzheimer's disease (AD) patients. Therefore, isolating and culturing strains with significant inhibitory effects against Staphylococcus aureus from the commensal flora of healthy children's skin has become a research hotspot and challenge in this field. This research direction is expected to promote the effective application of microecological therapy in skin diseases. Summary of the Invention

[0005] This invention provides a Bacillus mojavensis VT275 strain, which is isolated from the symbiotic flora of the skin in the popliteal fossa of healthy children. This strain has a good inhibitory effect on atopic dermatitis pathogens such as Staphylococcus aureus and has a broad-spectrum antibacterial effect.

[0006] The first objective of this invention is to provide a *Bacillus mojavensis*, with accession number CGMCC No. 29974. This *Bacillus mojavensis* is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on March 8, 2024, with accession number CGMCC NO. 29974.

[0007] The Bacillus mojavensis described in this invention was isolated from the skin of healthy children and identified as belonging to the genus Bacillus, species Bacillus mojavensis, named VT275.

[0008] A second objective of this invention is to provide a microbial preparation containing the aforementioned *Bacillus mohaiweiii* VT275. To further enhance the function of the strain, in some embodiments of this invention, the strain can be modified using conventional methods in the prior art. The modified variants retain the basic functions and properties of the strain, and these modified variants are also considered to be within the scope of protection of this invention. Furthermore, the variants also include strain variants with natural or spontaneous genetic alterations and variant strains obtained through continuous passage. These variants typically have the same or substantially the same 16S rDNA fragment gene sequence as the preserved strain, for example, at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the preserved strain. Similarly, these variants are also considered to be within the scope of protection of this invention.

[0009] In this invention, the aforementioned *Bacillus mohaiweii* strain is stored or used in a suitable formulation, such as a lyophilized formulation, capsule formulation, liquid formulation, tablet formulation, gel formulation, emulsion formulation, etc. In one embodiment of the invention, the aforementioned *Bacillus mohaiweii* strain is a lyophilized liquid formulation.

[0010] A third objective of this invention is to provide a product containing the aforementioned Bacillus mohaiweii or dead bacteria and its metabolites.

[0011] In this invention, the product is a pharmaceutical or cosmetic product.

[0012] A fourth objective of this invention is to provide the use of the above-mentioned Bacillus mohaiweii VT275 or the above-mentioned microbial preparation in the preparation of products that inhibit skin pathogens.

[0013] In this invention, the skin pathogens are one or more of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and / or Staphylococcus epidermidis.

[0014] A fifth object of the present invention is to provide the use of the above-mentioned Bacillus mohaiweii VT275 or the above-mentioned microbial preparation in the preparation of products that inhibit foodborne pathogens, wherein the foodborne pathogens are one or more of Listeria monocytogenes, Shigella and / or Enterococcus faecalis.

[0015] A sixth object of the present invention is to provide the use of the above-mentioned Bacillus mohaiweii VT275 or the above-mentioned microbial preparation in the preparation of a medicine for the prevention and / or treatment of atopic dermatitis.

[0016] The beneficial effects of this invention are:

[0017] (1) The present invention isolates and screens a strain of Bacillus mojavensis VT275 that has a significant inhibitory effect on a variety of skin pathogens from the skin surface of healthy children. This strain and its fermentation supernatant have significant antibacterial effects on Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Shigella, and Enterococcus faecalis, with an inhibition zone diameter of 9-20 mm;

[0018] (2) The source of Bacillus mojavensis VT275 is safe and can be used to prevent and / or treat atopic dermatitis caused by common pathogens such as Staphylococcus aureus. It has broad application prospects in the fields of pharmaceuticals and cosmetics.

[0019] Preservation of biological materials:

[0020] Accession number: CGMCC No. 29974

[0021] Deposit date: March 8, 2024

[0022] Preservation Institution: China General Microbiological Culture Collection Center (CGMCC), Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0023] Classification and nomenclature: Bacillus mohaiweiensis Attached Figure Description

[0024] Figure 1 The colony morphology of strain Mohaiwei Bacillus VT275 on TSB plates.

[0025] Figure 2 The morphology of strain Mohaiwei Bacillus VT275 is shown under a scanning electron microscope.

[0026] Figure 3 The inhibitory effect of cell-free supernatant of Bacillus mohaiweii VT275 on the growth of Staphylococcus aureus ATCC 29213 was investigated.

[0027] Figure 4 A phylogenetic tree for strain Bacillus mohaiweii VT275 constructed based on the 16S rRNA gene.

[0028] Figure 5 The genome sequence diagram of the whole genome of Bacillus mohaiweii VT275.

[0029] Figure 6 This image shows the antagonistic effect of strain Mohaiwei Bacillus VT275 on Staphylococcus aureus ATCC 29213.

[0030] Figure 7 This image shows the antagonistic effect of strain Mohaiwei Bacillus VT275 against methicillin-resistant Staphylococcus aureus (MRSA).

[0031] Figure 8 This image shows the antagonistic effect of strain Mohaiwei Bacillus VT275 against Staphylococcus epidermidis.

[0032] Figure 9 This image shows the antagonistic effect of strain Mohaiwei Bacillus VT275 against Listeria monocytogenes.

[0033] Figure 10 This image shows the antagonistic effect of strain Mohaiwei Bacillus VT275 on Shigella.

[0034] Figure 11 This image shows the antagonistic effect of strain Mohaiwei Bacillus VT275 on Enterococcus faecalis. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0036] The culture media involved in the following examples are as follows:

[0037] The R2A solid culture medium consists of the following components at the following mass concentrations: 0.25 g tryptone, 0.5 g acid-hydrolyzed casein, 0.5 g yeast extract, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.1 g magnesium sulfate, 0.3 g sodium pyruvate, 0.25 g peptone, 0.5 g glucose, and 15 g agar powder. The pH is adjusted to 7.2 ± 0.2 at room temperature and dissolved in 1 L of distilled water.

[0038] DSMZ solid medium: 10g casein hydrolysate, 10g yeast extract, 100g NaCl, 15g agar powder. Adjust the pH to 7.2±0.2 at room temperature and dissolve in 1L distilled water.

[0039] Preparation of culture medium for amplifying bacteria: The culture medium used for skin symbiotic bacteria is TSB broth medium.

[0040] The TSB broth culture medium consists of the following components by mass concentration: 17g tryptone, 3.0g soybean peptone, 5.0g sodium chloride, 2.5g dipotassium hydrogen phosphate, and 2.5g glucose. The pH was adjusted to 7.3±0.2 at room temperature and dissolved in 1L of distilled water.

[0041] Example 1: Isolation, culture and identification of dermal commensal bacteria

[0042] 1. Sample source and sample collection

[0043] Samples were collected from participating children at the First Clinical Medical College of Shanxi Medical University. The purpose of this study was to investigate the commensal flora of healthy children aged 1-12 years with no history of chronic skin diseases or autoimmune diseases. Samples were collected from 30 healthy children. Participants were asked not to shower / bathe for two days prior to sampling and to complete a questionnaire. Informed consent was obtained from all participants and their parents.

[0044] During sample collection, personnel wore disposable sterile gloves and medical masks. Sterile cotton swabs were immersed in a microbial sample preservation solution for 30 seconds, then removed and excess solution squeezed out. Volunteers fully exposed their popliteal fossa, and the swab was placed 2×2cm in the center of the popliteal fossa. 2 Apply consistent friction and pressure to the swab, repeating 100 times within 2 minutes. Immediately break the swab and place it in a sterile cryopreservation tube containing microbial preservation solution. After sample collection, send the sample to the laboratory for processing within 2 hours.

[0045] 2. Sample processing, growth conditions, and strain isolation

[0046] (1) Preparation of culture medium

[0047] Preparation of culture medium for isolation: The culture medium used for culturing skin symbiotic bacteria is blood agar plate solid medium, R2A solid medium, and DSMZ solid medium.

[0048] (2) Bacterial culture

[0049] The skin swab sample was shaken thoroughly using a vortex mixer. 100 μl of the sample was then spread onto a solid culture medium, including blood agar, R2A, and DSMZ media. The plates were incubated at 37°C under aerobic and anaerobic conditions for 3-7 days until obvious colony formation was observed. From each plate, all colonies with different phenotypes were selected and transferred to fresh culture medium for three-zone streaking isolation. The culture was repeated at least twice to isolate individual strains. The purified single colonies were then cultured for bacterial amplification and incubated at 37°C for 24 hours before being stored in 25% glycerol at -80°C.

[0050] (3) Identification of strains in the sample

[0051] The purified single colonies obtained in step (2) were inoculated into TSB broth medium and amplified in a 37 ℃ incubator for 24 h. Bacterial DNA was extracted according to the bacterial DNA extraction kit instructions, and PCR amplification was performed using universal primers for bacterial 16S rDNA. Universal primers: 27F: 5'-AGA GTTTGATCCTGGCTCAG-3', 1492R: 5'-TACGGTTACCTTGTTACGACTT-3'. The PCR amplification system consisted of 20 μL of ddH2O, 10 μL of 2×Taq PCR Mix, 0.8 μL each of forward and reverse primers, and 0.42 μL of DNA template. The PCR reaction system was as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s; 55 ℃ annealing for 45 s; 72 ℃ extension for 90 s, 35 cycles; 72 ℃ extension for 10 min. PCR products were detected by 1% agarose gel electrophoresis, and the bands were observed and analyzed using a gel imaging system. The PCR products with bands were sent to Qingke Biotechnology Co., Ltd. for sequencing. The obtained 16S rRNA gene sequences were compared with the NCBI database using BLAST analysis. Those with a similarity greater than 99% were considered to be the same species, resulting in 74 different bacterial species. Finally, a phylogenetic tree was constructed using MEGA software. The results showed that the isolated commensal bacteria mainly consisted of three phyla: Firmicutes, Actinobacteria, and Proteobacteria. Firmicutes accounted for the largest proportion, with Staphylococcus being the most prevalent, followed by Micrococcus and Bacillus.

[0052] Example 2: Screening of Bacillus mohaiweii VT275

[0053] 1. Screening and identification of Bacillus mohaiweii VT275

[0054] The 74 isolated strains were streaked in three zones on TSB solid medium until single colonies appeared. Single colonies were picked and cultured overnight in TSB liquid medium. The culture was centrifuged at 10,000 rpm for 10 min at 4°C. The precipitate was discarded, and the supernatant was filtered through a 0.22 mm sterile filter membrane to obtain the fermentation supernatant of each isolated strain.

[0055] Staphylococcus aureus ATCC 29213 was cultured overnight and its concentration was adjusted to 10 using a McFarland turbidimeter. 4 CFU / mL, 100 μL of diluted Staphylococcus aureus bacterial suspension was mixed with 100 μL of each of the above fermentation supernatants and placed in a honeycomb culture plate (for use with the Bioscreen fully automated growth curve analyzer). The optical density (OD) was measured every 30 min at 30℃ using the Bioscreen fully automated growth curve analysis system.600 Growth curves of Staphylococcus aureus under the intervention of fermentation supernatants of different isolated bacteria were obtained. The results showed that a VT275 strain had a good inhibitory effect on the growth of Staphylococcus aureus, as shown in the figure below. Figure 1 As shown, it began to exert an inhibitory effect on Staphylococcus aureus after 7 hours of co-culture, and achieved complete inhibition after 9 hours. After 9 hours, the OD600 value of Staphylococcus aureus growth decreased from 0.446±0.002 to 0.376±0.009 (P<0.001).

[0056] The colony morphology of VT275 obtained by screening is as follows: Figure 2 As shown, the colony morphology on TSB plates was as follows: irregularly shaped, round, grayish-white colonies with wrinkled centers. After incubation at 37°C and 150 rpm for 72 hours, the incubated bacterial solution was centrifuged to remove the supernatant bacterial suspension. The cells were washed three times with PBS buffer. The bacterial pellet was then fixed in 2.5% glutaraldehyde (prepared in PBS) at 4°C for 10 hours, followed by washing twice with sterile PBS and sterile water, and then fixed with 1% osmium tetroxide solution for 5 hours after washing in sterile water. The bacterial pellet was then placed in alcohol solutions of various concentrations (30%, 50%, 70%, 80%, 90%, and 100%), and finally treated with isoamyl acetate for 2 hours. The treated bacterial cells were then plated onto a steel plate, dried in a vacuum evaporator, and the morphology of the candidate strain was observed using a scanning electron microscope (SEM) (Hitachi High Technology, Hitachi, Japan). The morphology shown by the scanning electron microscope is as follows. Figure 3 As shown in the figure, VT275 is a short rod-shaped organism with flagella, no capsule, and can form spores. The size of a single cell is 0.7–0.8 μm × 2–3 μm.

[0057] 2. Phylogenetic analysis of Bacillus mohaiweii strain VT275

[0058] Phylogenetic tree of 16S rRNA of strain VT275 as follows Figure 4 As shown, VT275 is most closely related to Bacillus mojavensis (GCF_000245335.1), with a similarity of 100%. Therefore, we identified strain VT275 as Bacillus mojavensis and deposited it at the China General Microbiological Culture Collection Center on March 8, 2024.

[0059] Example 3: Whole genome sequencing of Bacillus mohaiweii VT275 and prediction analysis of its antibacterial substances

[0060] This sequencing work was commissioned to Shanghai Meiji Biotechnology Co., Ltd. After sample quality control, sequencing analysis was performed using second-generation sequencing technology and a third-generation high-throughput sequencing platform. Genome assembly was based on the third-generation sequencing data and completed using Unicycler v 0.4.8 software. GeneMarkS software was used to predict coding sequences in the genome, and the obtained genome sequences, predicted coding genes, and non-coding RNA information were integrated to generate a GBK file. CGView software was used to draw a genome circle map of a single sample to comprehensively display the characteristics of the sequenced genome. The whole genome circle of Bacillus mohaiweii VT275 is shown below. Figure 5 As shown, the results indicate that the genome of strain VT275 is a closed circular DNA molecule with a total length of 3,987,704 bp and an average GC content of 43.79%. No plasmids were detected. A total of 3,926 protein-coding genes, 86 tRNA genes, 30 rRNA genes, and 90 sRNA genes were predicted in the genome.

[0061] The predicted encoded protein sequences were compared and annotated with protein sequences in the COG, GO, KEGG, and CAZyme databases using BLAST. The gene clusters for antimicrobial synthesis in the whole genome of *Bacillus mohaiweii* VT275 were analyzed and predicted using the online software AntiSMASH and Bagel4. The results are shown in Table 1. The prediction results showed that the VT275 strain genome contained nine gene clusters for secondary metabolite synthesis. BLAST comparison of the gene clusters of strain VT275 with known secondary metabolite gene clusters in the database revealed that the functions of six gene clusters were clearly predicted, while the functions of two gene clusters remained unclear, suggesting the possible existence of unknown metabolite synthesis genes. The six predicted secondary metabolites were surfactantin, zwittermicin A, fengycin, bacillibactin, subtilosin A, and bacilysin. Except for zwittermicin A, the similarity of the other metabolites was greater than 70%, with fengycin, bacillibactin, subtilosin A, and bacilysin showing 100% similarity. Surfactin, fengycin, bacillibactin, subtilosin A, and bacilysin are known to be common antibacterial substances secreted by Bacillus, therefore we preliminarily speculate that the antibacterial substances produced by strain VT275 are mainly these five metabolites.

[0062] Table 1 Prediction of secondary metabolite synthesis gene clusters

[0063]

[0064] Example 4: Antibacterial experiment of Bacillus mohaiweii VT275

[0065] 1. Inhibition experiment of Bacillus mohaiweii VT275 against common skin pathogens

[0066] To verify the antibacterial effect of Bacillus mohaiweii VT275 on common skin pathogens, this experiment selected Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus epidermidis as common skin pathogens.

[0067] The inhibitory effect of Bacillus mohaiweii VT275 on the aforementioned skin pathogens was verified using the agar diffusion method. Both VT275 and the aforementioned pathogens were inoculated into TSB liquid medium and incubated in a shaker at 37°C and 170 r / min until the logarithmic growth phase was reached. The concentration of the pathogens was then adjusted to 1 x 10⁻⁶ using TSB liquid. 8 CFU / mL, at a 2% inoculum, the bacterial suspension was added to TSB solid medium containing 0.6% agar and shaken well. The medium temperature was approximately 50℃ at this point. The antibacterial activity was determined using the double-layer agar method. First, approximately 15 ml of TSB solid medium containing 1.5% agar was poured into a sterile Petri dish. After the medium solidified, sterile Oxford cups were evenly placed in the Petri dish, and approximately 20 ml of TSB solid medium containing the bacterial suspension (3-5 mm thick) was poured into the Petri dish. After solidification, the Oxford cups were removed with sterile forceps. The sample name and concentration were marked on the back of the Petri dish. 100 μL of *Bacillus mohaiweii* VT275 bacterial suspension was added to each well. After the addition was complete, the dish was sealed with film and transferred to a 37℃ incubator for 24 hours. Subsequently, the inhibition zone size of *Bacillus mohaiweii* VT275 against three common pathogens was measured. The results are shown in Table 2. Figure 6-8 As shown.

[0068] Table 2: Diameter of the inhibition zone of Bacillus mohaiweii VT275 against three common pathogens

[0069]

[0070] Based on the inhibition zone diameter of *Bacillus mohaiweii* VT275 against three common skin pathogens, the results showed that *Bacillus mohaiweii* VT275 had a good inhibitory effect on all three pathogens, especially against *Staphylococcus epidermidis*, with an inhibition zone diameter of 20.11 mm, demonstrating a very significant effect. Therefore, *Bacillus mohaiweii* VT275 can be used for the prevention and / or treatment of atopic dermatitis.

[0071] 2. Inhibition experiment of Bacillus mohaiweii VT275 against other pathogens.

[0072] The inhibitory activity of Bacillus mohaiweii VT275 against Listeria monocytogenes, Shigella, and Enterococcus faecalis was verified using the agar diffusion method described above. The results are shown in Table 3. Figure 9-11 As shown.

[0073] Table 3: Diameter of inhibition zones of Bacillus mohaiweii VT275 against three common pathogens

[0074]

[0075] Based on the inhibition zone diameter of *Bacillus mohaiweii* VT275 against the three pathogens mentioned above, the results showed that *Bacillus mohaiweii* VT275 had an inhibitory effect on all three pathogens, especially showing the best inhibitory effect against *Listeria monocytogenes*, reaching 18.44 mm. Therefore, *Bacillus mohaiweii* VT275 has a broad-spectrum antibacterial effect.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Bacillus mojavensis VT275, which is classified and named as Bacillus mojavensis (Bacillus mojavensis) VT275, preserved in the China General Microbiological Culture Collection Center on March 8, 2024, and the preservation number is CGMCC No. 29974. Bacillus mojavensis ​ 2. A microbial preparation containing Bacillus mojavensis VT275 as claimed in claim 1.

3. The microbial preparation of claim 2, wherein: The preparation form is a freeze-dried preparation, a capsule preparation, a liquid preparation, a tablet preparation, a gel preparation or an emulsion preparation.

4. A product characterized by: The product contains Bacillus mojavensis as claimed in claim 1; the product is a pharmaceutical product.

5. Use of the Bacillus mojavensis VT275 according to claim 1 or of the microbial preparation according to claim 3 for the manufacture of a product for inhibiting skin pathogenic bacteria, characterized in that: The cutaneous pathogenic bacteria are one or more of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and / or Staphylococcus epidermidis.

6. Use of Bacillus mojavensis VT275 as claimed in claim 1 or a microbial preparation as claimed in claim 3 for the manufacture of a product for inhibiting foodborne pathogenic bacteria, which are one or more of Listeria monocytogenes, Shigella and / or Enterococcus faecalis.

7. Use of Bacillus mojavensis VT275 as claimed in claim 1 or a microbial preparation as claimed in claim 3 for the manufacture of a pharmaceutical product for the prevention and / or treatment of atopic dermatitis.

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