Intestinal-derived bacteroides variabilis with anti-candida activity and application thereof

Abstract

The present application provides an intestinal-derived variable fusobacterium with anti-candida activity and application thereof. Specifically, the present application provides a variable fusobacterium (Fusobacterium varium) with a preservation number of CGMCC No. 1.18079. The variable fusobacterium of the present application can inhibit and kill pathogenic candida, and has good biosafety.

Description

Technical Field

[0001] This invention relates to a variable fusobacterium and its applications, specifically, an enterogenic variable fusobacterium with anti-Candida activity and its applications. Background Technology

[0002] According to the World Health Organization's list of key pathogenic fungi, Candida albicans, Candida parapsilosis, and Candida glabrata, which colonize the human gut, are among the pathogenic fungi that pose the greatest threat to public health. Currently, the only clinically available drugs for treating infections caused by these pathogens are polyenes, triazoles, and echinocandins. However, with the long-term use of single-drug therapy, drug resistance in these pathogens is becoming increasingly severe, further complicating the treatment of these infections.

[0003] Current antifungal drug development primarily relies on three classes of antifungal drugs: polyenes, triazoles, and echinocandins. This involves developing synthetic formulations by adding additional components to enhance the efficacy of existing antifungal drugs; developing antifungal drugs using compounds with antifungal activity; and developing antifungal drugs using bacterial strains with antifungal activity from the environment, food, skin, and other habitats. However, current antifungal drug development strategies suffer from drawbacks such as poor biosafety, weak antifungal activity, and the inability to prevent fungal infections at their source.

[0004] Therefore, there is an urgent need to develop new antifungal agents. Summary of the Invention

[0005] The inventors in this case discovered an anaerobic bacterium from the gut, isolated from a fecal sample of a healthy volunteer, which can inhibit and kill common intestinal pathogenic fungi such as Candida albicans, Candida parapsilosis, and Candida glabrata. Based on 16S rDNA sequence alignment, the 16S rDNA sequence (SEQ ID No. 1) of this strain has a sequence similarity of 99.71% with that of *Fusobacterium varium* strain NCTC1056, thus identifying it as a different strain of the same species of *Fusobacterium varium*. In this invention, it is named *Fusobacterium varium* DA690. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), deposited on August 5, 2024, with accession number CGMCC No. 1.18079, and classified as *Fusobacterium varium*.

[0006] SEQ ID No. 1:

[0007]

[0008] The anaerobic bacterium *Fusobacterium varium* DA690, derived from the gut and of the present invention (CGMCC No. 1.18079), is capable of effectively killing *Candida*, exhibiting strong antifungal activity. It can be used to develop novel anti-*Candida* preparations and has good biosafety, making it suitable for application in the intestinal environment. It can inhibit infection at its source by preventing *Candida* colonization.

[0009] Thus, on the one hand, the present invention provides a Fusobacterium varium with accession number CGMCC No.1.18079.

[0010] On the other hand, the present invention also provides a bacterial preparation containing the variable fusobacterium described in the present invention (i.e., variable fusobacterium DA690, i.e., the variable fusobacterium with accession number CGMCC No.1.18079).

[0011] According to a specific embodiment of the present invention, the bacterial preparation of the present invention may be in the form of live bacteria or inactivated bacteria, and may be in the form of liquid preparation or solid preparation.

[0012] On the other hand, the present invention also provides a metabiotic, which is a metabiotic containing bacterial cells and metabolites obtained by fermenting and culturing the variable Fusobacterium DA690 described in the present invention in a culture medium, or a metabiotic containing fermentation products.

[0013] According to a specific embodiment of the present invention, when the variable fusobacterium DA690 of the present invention is fermented in a culture medium, the culture medium can be one or more of BHIs medium, YCFA medium, and GAM medium.

[0014] According to a specific embodiment of the present invention, the variable Fusobacterium DA690 of the present invention is cultured under anaerobic conditions during fermentation in a culture medium.

[0015] According to a specific embodiment of the present invention, the variable Fusobacterium DA690 of the present invention is grown in BHIs medium, and its optimal growth temperature is 37°C.

[0016] According to a specific embodiment of the present invention, the variable Fusobacterium DA690 of the present invention was grown in BHIs medium, and after anaerobic static culture for 12 h, the cell density reached 1.03±0.02, and the pH was 6.0.

[0017] According to a specific embodiment of the present invention, the formula of the BHIs culture medium of the present invention is as follows: 10.0g tryptone, 17.5g beef heart powder, 5.0g sodium chloride, 2.0g glucose, 2.5g disodium hydrogen phosphate, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min. Add filtered and sterilized vitamin K3 and heme chloride solution to the above sterilized culture medium to make their final concentrations 1mg / L and 5mg / L, respectively.

[0018] In this invention, the term "post-biotic" refers to the collective term for metabolites obtained from the fermentation culture of the bacterial strain, which may contain bacterial cells or metabolically active substances obtained by further removing bacterial cells.

[0019] According to a specific embodiment of the present invention, the fermentation product obtained by fermenting *Fusobacterium variableum* DA690 in a culture medium is a metabiotic containing bacterial cells and metabolites; or, the fermentation product is further centrifuged and / or filtered to remove bacterial cells, for example, the fermentation broth supernatant is collected by further centrifugation (e.g., centrifugation at 6000-10000 rpm for 10 min) and filtered with a filter membrane (e.g., a 0.22 μm filter membrane) to obtain a metabiotic containing metabolites with removed bacterial cells (which can be stored at -80°C for later use); optionally, the metabiotic can be further dried to prepare a powder.

[0020] On the other hand, the present invention also provides a method for culturing the aforementioned Fusobacterium DA690, the method comprising:

[0021] The aforementioned Fusobacterium variableis DA690 was inoculated into a culture medium for fermentation culture.

[0022] According to a specific embodiment of the present invention, preferably, the culture medium is one or more of BHIs culture medium, YCFA culture medium, and GAM culture medium.

[0023] According to a specific embodiment of the present invention, more preferably, the culture conditions are as follows: 1%-5% of *Fusobacterium variableum* is transferred to fresh BHIs medium and cultured anaerobically at 37°C for 24 hours.

[0024] On the other hand, the present invention also provides the application of the aforementioned Fusobacterium DA690, the aforementioned bacterial preparation, or the aforementioned metabiotic in the fight against pathogenic fungi.

[0025] According to a specific embodiment of the present invention, the anti-pathogenic fungus refers to inhibiting the growth of pathogenic fungi and / or killing pathogenic fungi.

[0026] According to a specific embodiment of the present invention, the pathogenic fungi include one or more of the following enteric pathogenic fungi: Candida albicans, Candida parapsilosis, and Candida glabrata.

[0027] According to a specific embodiment of the present invention, the variable Fusobacterium, the bacterial preparation, or the metabiotic are products used to prepare anti-pathogenic fungi.

[0028] According to a specific embodiment of the present invention, preferably, the antifungal product is a drug (e.g., a microecological drug), a health food, feed, or an in vitro disinfectant.

[0029] On the other hand, the present invention also provides an antifungal preparation containing the *Fusobacterium DA690* described in the present invention, the bacterial preparation described therein, and / or the metabiotic described therein.

[0030] According to a specific embodiment of the present invention, the antifungal preparation may further include one or more additives: excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof.

[0031] According to a specific embodiment of the present invention, the antifungal preparation can be a powder, tablet, granule, suppository, microcapsule, ampoule, or liquid spray.

[0032] In summary, this invention provides an enterogenic Fusobacterium variant with anti-Candida activity and its application in developing into an anti-Candida preparation. This invention addresses the shortcomings of existing antifungal preparations, such as poor biosafety and weak antifungal activity, and can inhibit infection at the source by preventing Candida colonization. Attached Figure Description

[0033] Figure 1 This is a morphological diagram of Fusobacterium varium CGMCC No. 1.18079, which is the present invention.

[0034] Figure 2This image shows the results of detecting the inhibitory activity of the fermentation broth of *Fusobacterium varium* CGMCC No. 1.18079 on pathogenic fungal growth in BHIs medium. Note: BHI-BHIs: BHIs medium supernatant without bacterial inoculation served as a negative control; BHI-PBS: sterile PBS buffer served as a negative control; BHI-Bb: fermentation supernatant containing the probiotic strain *Bifidobacterium bifidum* served as a negative control; BHI-Fv: fermentation supernatant containing *Fusobacterium varium* CGMCC No. 1.18079 (Fv) served as the experimental group.

[0035] Figure 3 This image shows the results of the activity test of Fusobacterium varium CGMCC No. 1.18079 in BHI medium against pathogenic fungi. Note: Initial Candida, the amount of Candida inoculated during co-culture; Candida+Fv, the number of viable Candida cells after co-culturing the fermentation supernatant of Candida and Fusobacterium varium CGMCC No. 1.18079 (Fv) for 48 hours.

[0036] Microbial preservation for patented procedures:

[0037] Fusobacterium varium DA690:

[0038] Date of deposit: August 5, 2024;

[0039] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0040] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing;

[0041] Accession number: CGMCC No.1.18079;

[0042] Classification and nomenclature: Fusobacterium varium. Detailed Implementation

[0043] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0044] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and reagents used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described in the embodiments of this invention may be used to implement this invention.

[0045] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0046] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0047] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment / implementation, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0048] In each embodiment, the experimental method for the test strain to kill pathogenic fungi was as follows: the pathogenic fungi activated overnight in YPD medium were washed once with sterile PBS buffer and resuspended in an equal volume of sterile PBS buffer; the concentration of pathogenic fungal cells was adjusted using a cell counting chamber at 1-5 × 10⁻⁶. 5 The final cell / well concentration of the treated pathogenic fungal cells was inoculated into 96-well plates containing fresh BHI medium. The treated bacterial fermentation broth was added to the 96-well plates at a 1:1 volume ratio, and the plates were incubated at 37°C and 600 rpm for 24 hours with shaking. The growth curve of the pathogenic fungus was recorded. The cultured bacterial solution was diluted and spread onto YPD agar plates, incubated statically at 37°C for 24 hours, and the number of surviving pathogenic fungi was counted. The YPD medium formulation was as follows: 20.0 g tryptone, 10.0 g yeast extract, 20.0 g glucose, 1000 mL distilled water, and 20.0 g agar for solids. The medium was sterilized at 121°C for 15 minutes.

[0049] Example 1: Screening and identification of Fusobacterium varium strains

[0050] Using a sterile sampler, the air-free portion of a healthy volunteer's stool was collected and dissolved in an appropriate amount of sterile water. 100 μL of the stool solution was serially diluted 10-fold and spread onto BHI agar plates. The plates were incubated at 37°C for 48 hours in an anaerobic environment. White, round colonies with well-defined edges were picked and examined under a microscope. These colonies were identified as extremely small, spindle-shaped cells. Repeated streaking was used to confirm pure colonies, and the resulting bacterial strain was named DA690. Its morphology is shown in [image / description missing]. Figure 1 .

[0051] The formula for the BHIs culture medium is as follows: 10.0g tryptone, 17.5g beef heart powder, 5.0g sodium chloride, 2.0g glucose, 2.5g disodium hydrogen phosphate, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min. Add filtered and sterilized vitamin K3 and heme chloride to the sterilized culture medium to make their final concentrations 1mg / L and 5mg / L, respectively.

[0052] The strain was inoculated into BHIs liquid medium, and after 16S rRNA gene sequence analysis, the strain was identified as Fusobacterium varium.

[0053] The above-mentioned Fusobacterium variable DA690 was grown in BHI medium, and its optimal growth temperature was 37℃.

[0054] The aforementioned *Fusobacterium variableum* DA690 was grown in BHIs medium. After 12 hours of anaerobic static culture, the cell density reached 1.03 ± 0.02 cells at a pH of 6.0.

[0055] The aforementioned Fusobacterium varium DA690 strain was deposited on August 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC) (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 1.18079 and classification name: Fusobacterium varium.

[0056] Example 2: Preparation of active fermentation broth against pathogenic fungi using Fusobacterium varium in BHI medium

[0057] A single colony of *Fusobacterium varium* CGMCC No. 1.18079 was inoculated into fresh BHIs medium and cultured in an anaerobic chamber at 37°C for 24 h. The activated *Fusobacterium varium* culture was then transferred to fresh BHIs medium at a volume ratio of 2% and cultured in an anaerobic chamber at 37°C for 24 h. The bacterial culture was collected, centrifuged at 10,000 rpm for 10 min to remove bacterial cells, and the supernatant was filtered through a 0.22 μm filter membrane. The resulting fermentation broth was stored at -80°C for later use.

[0058] The formula for the BHIs culture medium is as follows: 10.0g tryptone, 17.5g beef heart powder, 5.0g sodium chloride, 2.0g glucose, 2.5g disodium hydrogen phosphate, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min. Add filtered and sterilized vitamin K3 and heme chloride to the sterilized culture medium to make their final concentrations 1mg / L and 5mg / L, respectively.

[0059] Example 3: Application of Fusobacterium varium active fermentation broth in inhibiting the growth of pathogenic fungi

[0060] Pathogenic fungi *Candida albicans*, *Candida parapsilosis*, and *Candida glabrata* isolated from the human gut were streaked onto YPD agar plates for resuscitation and incubated at 37°C for 48 h. Single colonies of these pathogenic fungi were picked and cultured in fresh YPD liquid medium at 37°C and 200 rpm for 18 h with shaking. The activated pathogenic fungi were washed once with sterile PBS buffer and resuspended in an equal volume of sterile PBS buffer. The suspension was centrifuged at 3000 rpm for 1 min. The resuspended pathogenic fungal suspension was serially diluted 10-fold twice. 10 μL of the 100-fold diluted suspension was transferred to the cell counting chamber, and the cell concentration was read under a regular optical microscope. The concentration was then determined by adding 1-5 × 10⁻⁵ cells per cell. 5 The final cell / well concentration was inoculated into the diluted pathogenic fungal suspension in a 96-well plate containing fresh BHIs medium; the bacterial active fermentation broth treated in Example 2 was added to the 96-well plate at a volume ratio of 1:1, and the plate was placed in an enzyme-linked immunosorbent assay (ELISA) reader and cultured at 37°C and 600 rpm for 24 h with shaking, and the growth curve of the pathogenic fungus was recorded.

[0061] Among them, according to 1-5×10 5After inoculating the diluted pathogenic fungal suspension with the final cell / well concentration into a 96-well plate containing fresh BHIs medium, fresh BHIs medium, sterile PBS buffer, and fermentation supernatant of the probiotic strain Bifidobacterium bifidum were added to the 96-well plate at a 1:1 volume ratio as a negative control group.

[0062] The YPD culture medium formula is as follows: 20.0g tryptone, 10.0g yeast extract, 20.0g glucose, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min.

[0063] Figure 2 This study demonstrates the inhibitory activity of the fermentation broth of *Fusobacterium varium* CGMCC No. 1.18079 in BHIs medium on the growth of pathogenic fungi. Note: BHI-BHIs, with the addition of BHIs medium supernatant without bacterial inoculation, serves as a negative control; BHI-PBS, with the addition of sterile PBS buffer, serves as a negative control; BHI-Bb, with the addition of fermentation supernatant of the probiotic strain *Bifidobacterium bifidum*, serves as a negative control; BHI-Fv, with the addition of fermentation supernatant of *Fusobacterium varium* CGMCC No. 1.18079 (Fv), serves as the experimental group. It can be seen that the fermentation broth of *Fusobacterium varium* CGMCC No. 1.18079 of this invention exhibits inhibitory activity on the growth of pathogenic fungi.

[0064] Example 4: Application of Fusobacterium varium active fermentation broth in killing pathogenic fungi

[0065] Pathogenic fungi *Candida albicans*, *Candida parapsilosis*, and *Candida glabrata* isolated from the human gut were streaked onto YPD agar plates for resuscitation and incubated at 37°C for 48 h. Single colonies of these pathogenic fungi were picked and cultured in fresh YPD liquid medium at 37°C and 200 rpm for 18 h with shaking. The activated pathogenic fungi were washed once with sterile PBS buffer and resuspended in an equal volume of sterile PBS buffer. The suspension was centrifuged at 3000 rpm for 1 min. The resuspended pathogenic fungal suspension was serially diluted 10-fold twice. 10 μL of the 100-fold diluted suspension was transferred to the cell counting chamber, and the cell concentration was read under a regular optical microscope. The concentration was then determined by adding 1-5 × 10⁻⁵ cells per cell. 5The final cell / well concentration was inoculated into the diluted pathogenic fungal suspension in a 96-well plate containing fresh BHIs medium. The bacterial active fermentation broth treated in Example 2 was added to the 96-well plate at a 1:1 volume ratio, and the plate was incubated in a microplate reader at 37°C and 600 rpm with shaking for 24 h. The cultured bacterial solution was diluted and spread on YPD agar plates, and after incubation at 37°C for 24 h, the number of surviving pathogenic fungi was counted.

[0066] Among them, according to 1-5×10 5 After inoculating the diluted pathogenic fungal suspension into 96-well plates containing fresh BHIs medium at the final cell / well concentration, fresh BHIs medium and sterile PBS buffer were added to the 96-well plates at a 1:1 volume ratio as a negative control group.

[0067] The YPD culture medium formula is as follows: 20.0g tryptone, 10.0g yeast extract, 20.0g glucose, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min.

[0068] Figure 3 This demonstrates the fungicidal activity of the fermentation broth of *Fusobacterium varium* CGMCC No. 1.18079 in BHI medium. Note: Initial Candida, the inoculum of *Candida* during co-culture; Candida+Fv, the viable *Candida* count after 48 hours of co-culturing the fermentation supernatant of *Candida* and *Fusobacterium varium* CGMCC No. 1.18079 (Fv). It can be seen that the fermentation broth of *Fusobacterium varium* CGMCC No. 1.18077 of this invention possesses fungicidal activity.

[0069] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. For example, *Fusobacterium variableum* CGMCC can be used. No. 1.18079 can be used to prepare anti-Candida preparations with other antifungal active substances, such as other strains of *Fusobacterium variableum* (e.g., using *Fusobacterium variableum* of other intestinal origin to prepare anti-Candida microecological drugs, or preparing microecological drugs, health foods, feeds, or in vitro disinfectants based on its fermentation supernatant or metagener). It can also be used to prepare anti-Candida active fermentation broth based on *Fusobacterium variableum* using other culture media (e.g., using YCFA medium, GAM medium, chemically defined medium, or other culture media capable of culturing *Fusobacterium variableum* to prepare anti-Candida microecological drugs, or preparing microecological drugs, health foods, feeds, or in vitro disinfectants based on its fermentation supernatant or metagener). Furthermore, the anti-Candida preparation can be used to inhibit other pathogenic fungi (e.g., to inhibit or kill Candida albicans, *Candida parapsilosis*, and *Candida glabrata*, and other Candida species and pathogenic fungi) to achieve the same purpose. Therefore, all equivalent technical solutions also fall within the scope of protection of this invention.

Claims

1. A type of variable fusobacterium ( Fusobacterium varium It has the accession number CGMCC No. 1.18079.

2. A bacterial preparation comprising the Fusobacterium varium of claim 1.

3. The bacterial preparation of claim 2, which is a liquid or solid preparation in a live or inactivated form.

4. A postbiotic obtained by fermentative cultivation of the Fusobacterium varium of claim 1 in a culture medium, which comprises bacterial cells and metabolites.

5. The postbiotic of claim 4, wherein, The culture medium is BHIs medium.

6. The postbiotic of claim 5, wherein, The postbiotic obtained by fermentative cultivation of the Fusobacterium varium of claim 1 in a culture medium, which comprises bacterial cells and metabolites.

7. The postbiotic of claim 6, wherein, The postbiotic is further dried to prepare a powder.

8. A method for culturing the Fusobacterium varium of claim 1, which comprises: inoculating the Fusobacterium varium of claim 1 in a culture medium for fermentative cultivation.

9. The method of claim 8, wherein, The culture medium is one or more of BHIs medium, YCFA medium and GAM medium.

10. The method of claim 9, wherein, The culture condition is anaerobic culture.

11. Use of the variable F. sulcatus of claim 1, the bacterial preparation of claim 2 or 3, or the postbiotic of claim 4 in the manufacture of a medicament for the treatment of a pathogenic fungus, the pathogenic fungus being one or more of Candida albicans (C. albicans) Candida albicans ), C. parapsilosis (C. parapsilosis) Candida parapsilosis and C. glabrata (C. glabrata) Candida glabrata .​​​ 12. Use according to claim 11, wherein, The antifungal agent is an external antiseptic.

13. An antifungal agent comprising the Fusobacterium varium of claim 1, the bacterial preparation of claim 2 or 3 and / or the postbiotic of claim 4.

14. The antifungal agent of claim 13, which is a powder, a tablet, a suppository, a microcapsule, an ampoule or a liquid spray.

Images