A strain of protococcus myxobacterium and its application in preparing a drug for removing methicillin-resistant staphylococcus aureus biofilm

Abstract

The application discloses a strain of Archangium sp. myxobacteria and application of the strain in preparation of a methicillin-resistant Staphylococcus aureus biofilm removing medicine. The strain is named as Archangium sp. CY-1, and the preservation number is GDMCC No: 63225. The application adopts an Escherichia coli induction method to separate and purify a strain of Archangium sp. myxobacteria Archangium sp. CY-1 from soil of a pig farm. The strain can prey on methicillin-resistant Staphylococcus aureus (MRSA), and the fermentation liquor and the fermentation supernatant concentrated liquid of the strain can remove MRSA biofilm. The above results show that the Archangium sp. CY-1 and the fermentation liquor thereof have great application potential in preparation of the MRSA biofilm removing medicine.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Protozoa myxobacterium and its application in the preparation of a biofilm removal drug for methicillin-resistant Staphylococcus aureus. Background Technology

[0002] Staphylococcus aureus is a common zoonotic opportunistic pathogen that can cause infection and bacteremia in multiple tissues of the host when the host's immunity is low. It has become a major cause of infection and is one of the most prevalent foodborne pathogens globally. Antibiotics have long been effective treatments for Staphylococcus aureus infections; however, with increased antibiotic use, drug-resistant strains of Staphylococcus aureus have become increasingly prevalent. Methicillin-resistant Staphylococcus aureus (MRSA) is more difficult to cure and has become a significant pathogen causing hospital-acquired infections. The adhesion of Staphylococcus aureus to host cells and the formation of biofilms is one of the key mechanisms by which it causes disease. Once a complete biofilm is formed, antibiotics and other antibacterial drugs become ineffective in killing the bacteria, and bacteria continue to escape from the biofilm, leading to long-term infection. This is one of the main factors contributing to increased drug resistance and persistent infection.

[0003] Currently, there are no specific antimicrobial drugs targeting bacterial biofilms, and antibiotics remain the primary treatment. However, the rate of drug resistance in Staphylococcus aureus has been increasing year by year in recent years, making the development of highly effective and low-toxicity antimicrobial drugs with novel targets an urgent clinical need to combat pathogenic bacterial infections. Common antibiotic-derived bacteria have been overexploited to some extent, necessitating the search for different types of antimicrobial compounds from novel drug-derived microorganisms. Myxobacteria are a widely distributed group of predatory bacteria that kill prey cells through various strategies, including producing a variety of antimicrobial substances, secondary metabolites, and hydrolytic enzymes during predation. Following actinomycetes, they represent another large class of novel strategic drug-derived microbial resources awaiting development. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of *Archangium* sp. CY-1, a myxobacterium belonging to the genus *Archangium*. This strain was deposited on October 10, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China (Guangdong Academy of Sciences Institute of Microbiology), Guangdong Province, China, accession number: GDMCC No. 63225.

[0005] A second objective of this invention is to provide a microbial agent comprising the aforementioned myxobacterium Archangium sp. CY-1.

[0006] Preferably, the microbial agent further comprises excipients that can prolong the activity time of the strain, or other excipients acceptable to microbial agents.

[0007] A third object of the present invention is to provide the use of the aforementioned myxobacterium Archangium sp. CY-1 or its agent in the preparation of products that inhibit the growth of methicillin-resistant Staphylococcus aureus.

[0008] A fourth object of the present invention is to provide the use of the aforementioned myxobacterium Archangium sp. CY-1 or its culture, culture, fermentation broth, or concentrated fermentation supernatant in the preparation of a bacterial biofilm removal drug. Preferably, the bacteria is methicillin-resistant Staphylococcus aureus.

[0009] The fifth objective of this invention is to provide a biofilm removal agent for methicillin-resistant Staphylococcus aureus, the agent containing the aforementioned myxobacterium Archangium sp. CY-1 or its culture, bacterial culture, fermentation broth or fermentation supernatant concentrate as the active ingredient.

[0010] Preferably, the culture, bacterial suspension, fermentation broth, or fermentation supernatant concentrate of the myxobacterium Archangium sp. CY-1 is obtained by fermentation in VY / 4 liquid medium, wherein the formula of VY / 4 liquid medium is: Angel yeast 0.25%, CaCl2·2H2O 0.1%, pH 7.2, and water as solvent.

[0011] A sixth object of the present invention is to provide a method for removing bacterial biofilms, comprising the step of contacting the aforementioned myxobacterium Archangium sp. CY-1 or its culture, bacterial suspension, fermentation broth, or concentrated fermentation supernatant with the bacterial biofilm. Preferably, the bacteria are methicillin-resistant Staphylococcus aureus.

[0012] The present invention has the following beneficial effects:

[0013] This invention uses an E. coli induction method to isolate and purify a strain of *Archangium* sp. CY-1, a myxobacterium belonging to the genus *Archangium*, from soil collected from a pig farm. This strain can prey on methicillin-resistant *Staphylococcus aureus* (MRSA), and its fermentation supernatant concentrate can remove existing MRSA biofilms. These results indicate that the myxobacterium *Archangium* sp. CY-1 involved in this invention can play a role in the treatment of MRSA.

[0014] Archangium sp. CY-1, a myxobacterium of the genus Archangium, was deposited on October 10, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China. The accession number is GDMCC No. 63225. Attached Figure Description

[0015] Figure 1 The morphological characteristics of Archangium sp. CY-1 are as follows: a) Colony morphology of strain CY-1 on VY / 2 solid medium; b) Fruiting body morphology of strain CY-1 under a stereomicroscope; c) Vegetative cell morphology of strain CY-1 under a phase contrast microscope.

[0016] Figure 2 It is Archangium sp. CY-1 preying on MRSA.

[0017] Figure 3 The study investigated the scavenging effect of different concentrations of Archangium sp. CY-1 fermentation supernatant on MRSA biofilm. Detailed Implementation

[0018] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0019] Example 1: Isolation, purification and identification of strains

[0020] Soil samples were collected from a pig farm in Shanwei City and air-dried at room temperature immediately after being transported back to the laboratory. Myxobacteria were isolated using the *E. coli* induction method, with the following steps: Soil samples the size of soybeans, treated with actinomycete ketone, were inoculated onto WCX water agar medium (0.1% CaCl2·2H2O and 1.5% agar, pH 7.2, solvent: water; preparation: dissolve all components in water, stir well, adjust pH, sterilize, and after the agar solidifies, inoculate with *E. coli* suspension by streaking). After culturing at 30℃ for 5 days, fruiting bodies were picked under a stereomicroscope and inoculated onto VY / 2 medium (0.5% *Angelica*, 0.1% CaCl2·2H2O, VB...). 12 0.5 mg / L and 1.5% agar, pH 7.2, solvent is water; preparation: dissolve each component in water, stir and mix well, adjust pH, sterilize to obtain) purification, and obtain purified strain CY-1 by continuously transferring colony edge cells.

[0021] Strain CY-1 exhibits sliding, spreading, deep purple colonies on VY / 2 solid medium, with a distinct radial pattern. Mature fruiting bodies are kidney-shaped, brown, and sessile. Under transmission electron microscopy, vegetative cells are observed to be rod-shaped. Figure 1 Genomic DNA was extracted from fresh cells of strain CY-1 using the CTAB method, and the genome sequence was determined using the Illumina Hiseq platform. The sequencing data were then assembled de novo using SPADES software to construct contigs and scaffolds. The integrity of the assembled genome was assessed using QUAST and CheckM software. The assembled genomes were used to calculate the hybridization value (dDDH) and average nucleotide similarity (ANI) with closely related species. The dDDH values ​​between the CY-1 genome and closely related type species of the genus Archangium were 28.6%–30.3%, far below the species threshold (>70%), and the ANI values ​​(84.91–85.93%) were also below the prokaryotic species threshold (95–96%) (Table 1). Therefore, this strain was identified as a new species of the genus Archangium and named Archangium sp. CY-1. It was deposited on October 10, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No. 63225.

[0022] Table 1. Mean nucleotide similarity (ANI) and hybridization value (dDDH) between strain CY-1 and closely related type species of the same genus.

[0023]

[0024] Example 2: Predation of MRSA by Archangium sp. CY-1

[0025] Single colonies of methicillin-resistant Staphylococcus aureus GDMCC 1.1263 were picked and inoculated into 5 mL of nutrient broth (0.1% peptone, 0.03% beef extract, 0.05% NaCl, pH 7.2, solvent: water; preparation: dissolve all components in water, stir well, and sterilize). The culture was incubated at 30°C with shaking at 180 rpm for 24 h to obtain a seed culture. Then, the seed culture was inoculated into fresh nutrient broth at a volume fraction of 1%, and incubated at 30°C with shaking at 180 rpm for 24 h. The cells were collected by centrifugation at 8000 rpm for 10 min, washed three times with TPM buffer (1 mM KH₂PO₄, 10 mM Tris-HCl, 8 mM MgSO₄·7H₂O, pH 7.6, solvent: water), and resuspended to a cell concentration of 1.0 × 10⁻⁶ cells. 11Cells / mL, 150 μL of bacterial suspension was inoculated into the center of TPM solid medium (KH2PO4 1 mM, Tris-HCl 10 mM, MgSO4·7H2O 8 mM, agar 15 g / L, pH 7.6, solvent: water; preparation: dissolve all components in water, adjust pH, stir well, and sterilize) to form a plaque with a diameter of about 2 cm, and then air-dried for later use. Freshly cultured CY-1 cells were extracted using a 200 μL pipette tip and inoculated into the center of the MRSA plaque, and placed in a 30℃ incubator for incubation. A TPM plate inoculated only with MRSA was set up as a negative control. The experiment was performed in triplicate, and predation was observed after 2 days.

[0026] like Figure 2 As shown, strain CY-1 was able to prey on MRSA plaques and use the nutrients produced by lysis to grow. After 5 days, the MRSA plaques were observed to be completely lysed.

[0027] Example 3: Removal of MRSA biofilm by Archangium sp. CY-1 fermentation supernatant

[0028] Strains of strain CY-1 were picked and cultured in VY / 4 liquid medium (Angel yeast 0.25%, CaCl2·2H2O 0.1%, pH 7.2, solvent: water; preparation: dissolve all components in water, stir well, adjust pH, and sterilize). The medium was then cultured on a shaker at 180 rpm and 30°C for 5 days. The culture was centrifuged at 8000 rpm and 4°C for 10 min, and the fermentation supernatant was collected. The supernatant was filtered through a 0.22 μm pore size filter and then concentrated 100-fold using a 3 kDa ultrafiltration tube. The concentration was replaced with PBS buffer. The concentrated supernatant was aliquoted into 2 mL sterile centrifuge tubes and stored at -80°C for later use.

[0029] MRSA cell suspensions were obtained using the same method as in Implementation Case 2, and the cell concentration was adjusted to approximately 1.0 × 10⁻⁶. 7 Cells / mL were counted, and 200 μL of bacterial suspension was inoculated into 96-well plates. The plates were incubated at 37°C for 48 h to form a mature biofilm. The supernatant was discarded, and the plates were washed three times with PBS buffer to remove unattached bacteria. After air-drying at room temperature, 200 μL of fermentation supernatant concentrate and 2, 5, 10, 20, and 40-fold diluted fermentation supernatant concentrates were added. PBS buffer and fermentation supernatant concentrates heat-treated at 100°C were used as controls. All conditions were performed in triplicate. After incubation at 37°C for 2 h, the supernatant was discarded, and the plates were washed three times with PBS buffer. After air-drying at room temperature, the plates were stained with 0.5% crystal violet for 20 min at room temperature to determine the remaining total biofilm amount. The crystal violet stain was gently washed away with sterile water, and then the biofilm attached to the bottom of the wells was completely dissolved with 30% acetic acid. The absorbance at 550 nm was measured. Results are as follows: Figure 3As shown, the MRSA biofilm in the pores after adding CY-1 fermentation supernatant was significantly removed, and the removal effect weakened with the dilution of the fermentation supernatant concentration, indicating that there is a dose-effect relationship between the active substances in the CY-1 fermentation supernatant. The heat-treated supernatant lost its ability to remove MRSA biofilm, suggesting that the heat-sensitive proteins in the fermentation broth and concentrate may be the functional substances for removing MRSA biofilm.

[0030] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope 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 Protomyxobacterium genus Archangium sp. CY-1, with accession number GDMCC No: 63225.

2. A microbial agent, characterized in that, Contains the slime bacteria of claim 1 Archangium sp. CY-1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent also includes excipients acceptable to the bacterial agent.

4. The myxobacteria according to claim 1 Archangium The use of sp. CY-1 or the bacterial agent of claim 2 in the preparation of products that inhibit the growth of methicillin-resistant Staphylococcus aureus.

5. The slime mold as described in claim 1 Archangium The use of sp. CY-1 or its culture, culture, fermentation broth or fermentation supernatant concentrate in the preparation of methicillin-resistant Staphylococcus aureus biofilm removal drugs.

6. A biofilm removal agent for methicillin-resistant Staphylococcus aureus, characterized in that, Containing the slime bacteria of claim 1 Archangium sp. CY-1 or its culture, bacterial culture, fermentation broth or fermentation supernatant concentrate as the active ingredient.

7. A method for eliminating methicillin-resistant Staphylococcus aureus biofilms for non-disease treatment purposes, characterized in that, Includes the slime bacteria described in claim 1 Archangium The step of contacting sp. CY-1 or its culture, bacterial suspension, fermentation broth or fermentation supernatant concentrate with methicillin-resistant Staphylococcus aureus biofilm.

Images