Parabacteroides distasonis capable of inhibiting pathogenic bacteria and resisting tumors and application thereof

Abstract

The application discloses Parabacteroides distasonis capable of inhibiting pathogenic bacteria and resisting tumors and an application thereof, relates to the technical field of microorganisms, and has the following technical points: the preservation number of Parabacteroides distasonis NGMCC 1.200926 is CGMCC No: 40687; the preservation address is No. 3, Xili Beichen, Chaoyang District, Beijing; the preservation unit is the General Microbiological Center of China Microorganism Culture Collection Committee; and the preservation time is July 5, 2023. After fermentation culture of Parabacteroides distasonis NGMCC 1.2000926, strong bacteriostatic active substances can be generated, the pathogenic bacteria such as Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa have obvious bacteriostatic effects, and the bacteriostatic active substances have good tumor cell activity, so that the application has a broad development space in the prevention and treatment of diseases caused by the pathogenic bacteria such as Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa and tumors, and has a good development and application prospect in antibacterial drugs and antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to Parabacteroides distasonis capable of inhibiting pathogenic bacteria and resisting tumors and application thereof. BACKGROUND

[0002] There are about 2000 different genera of microorganisms in the gastrointestinal tract of a normal human body, and the total number of bacterial cells is about 3.8x10 13 The intestinal microorganisms of a human body are widely considered as an important organ, a second genome and a second brain of a human being due to their complexity and diversity and important influence on the health and disease process of a human body. Researches have shown that the intestinal microorganisms are closely related to the occurrence of many metabolic diseases (such as obesity and type II diabetes), autoimmune diseases (such as inflammatory bowel disease and type I diabetes), tumors and nervous system related diseases (such as autism). Although the progress of genome sequencing technology, complex metagenomics and phylogenetics greatly changes our understanding of the role of intestinal flora in health and disease, it is of great significance to confirm the cell biology, ecological role and physiological prediction by culture-based experiments to analyze the growth characteristics, metabolism, physiology and other microbial characteristics of the microorganisms, and the research on microbial enrichment or metabolism is helpful to discover new enzymatic reactions and pathways and further reveal the role of human microbiota.

[0003] Antibacterial drugs generally refer to drugs with bactericidal or bacteriostatic activity, including various antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones and other chemical synthetic drugs. Some products obtained by culturing bacteria, actinomycetes, fungi and other microorganisms, or the same or similar substances manufactured by chemical semi-synthesis. Antibacterial drugs are a kind of drugs commonly used in clinical practice and are relatively important. With the long-term use of antibacterial drugs, the environment bacteria continuously develop drug resistance to them, so it is required to develop new antibacterial drugs to cope with this situation. SUMMARY

[0004] The present application aims at solving the above problems, and provides Parabacteroides distasonis capable of inhibiting pathogenic bacteria and resisting tumors and application thereof. The Parabacteroides distasonis has strong bacteriostatic and antitumor activity, and has good development and application prospect in the fields of antibacterial drugs and tumor treatment.

[0005] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0006] The application provides a human source Parabacteroides distasonis, a preservation number of the Parabacteroides distasonis NGMCC 1.200926 is CGMCC No: 40687, a preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, a preservation unit is China General Microbiological Culture Collection Center, a preservation time is July 5, 2023, and a 16s ribosome DNA sequence of the P. distasonis NGMCC 1.200926 is shown in SEQ ID NO. 1.

[0007] SEQ ID NO. 1:

[0008] Parabacteroides distasonis NGMCC 1.200926 16s ribosome DNA sequence

[0009] >NGMCC_1.200926

[0010] ATGCAAGTCGAGGGGCAGCACAGGGTAGCAATACCGCGGTGGCGACCGGCGCACGGGTGAGTAACGCGTATGCAACTTACCTATCAGAGGGGGATAACCCGGCGAAAGTCGGACTAATACCGCATGAAGCAGGGGCCCCGCATGGGGATATTTGCTAAAGATTCATCGCTGATAGATAGGCA

[0011] TGCGTTCCATTAGGCAGTTGGCGGGGTAACGGCCCACCAAACCGAC

[0012] GATGGATAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGTACTGAG

[0013] ACACGGACCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTC

[0014] AATGGGCGTAAGCCTGAACCAGCCAAGTCGCGTGAGGGATGAAGGT

[0015] TCTATGGATCGTAAACCTCTTTTATAAGGGAATAAAGTGCGGGACGT

[0016] GTCCTGTTTT GTATGTACCT TATGAATAAG GATCGGCTAA CTCCGTGC

[0017] CAGCAGCCGC GGTAATACGG AGGATCCGTA GCGTTATCGG ATTTATTGG

[0018] GTTTAAAGGG TGCGTAGGCG GCCTTTTAAG TCAGCGGTGA AAGTCTG

[0019] TGGCTCAACC ATAGAATTGC CGTTGAAACT GGGAGGCTTG AGTATGT

[0020] TTGAGGCAGG CGGAATGCGT GGTGTAGCGG TGAAATGCTT AGATAT

[0021] CACGCAGAAC CCCGATTGCG AAGGCAGCCT GCCAAGCCAT GACTGA

[0022] CGCTGATGCA CGAAAGCGTG GGGATCAAAC AGGATTAGAT ACCCTG

[0023] GTAGTCCACG CAGTAAACGA TGATCACTAG CTGTTTGCGA TACAGTG

[0024] TAAGCGGCAC AGCGAAAGCG TTAAGTGATC CTGGGGAGTA CGCC

[0025] GGCAACGGTG AAACTCAAAG GAATTGACGG GGGCCCGCAC AAGCGG

[0026] AGGAACATGT GGTTTAATTC GATGATACGC GAGGAACCTT ACCCGGG

[0027] TTTGAACGCA TTCGGACCGA GGTGGAAACA CCTTTTCTAG CAATAGC

[0028] CGTTTGCGAG GTGCTGCATG TTGTCGTCAG CTCGTGCCGT GAGGTG

[0029] TCGGCTTAAG TGCCATAACG AGCGCAACCC TTGCCACTAG TTACTAA

[0030] CAGGTAAAGCTGAGGACTCTGGTGGGACTGCCAGCGTAAGCTGCGA

[0031] GGAAGGCGGGGATGACGTCAAATCAGCACGGCCCTTACJCMGGGGC

[0032] GACACACGTGTTACAATGGCGTGGACAAAGGGATGCCACCTGGCGA

[0033] CAGGGAGCGAJCMCCAAACCACGTCTCAGTTCGGATCGGAGTCTGCA

[0034] ACCCGACTCCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCAT

[0035] GGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGC

[0036] CATGGGAGCCGGGGGTACCTGAAGTCCGTAACCGAAAGGATC

[0037] In another aspect, the application also provides uses of Parabacteroides distasonii in antagonizing Staphylococcus aureus, and uses of Parabacteroides distasonii in preparation of a medicine for preventing and treating a disease caused by Staphylococcus aureus.

[0038] In another aspect, the application also provides uses of Parabacteroides distasonii in antagonizing Salmonella typhi, and uses of Parabacteroides distasonii in preparation of a medicine for preventing and treating a disease caused by Salmonella typhi.

[0039] In another aspect, the application also provides uses of Parabacteroides distasonii in antagonizing Pseudomonas aeruginosa, and uses of Parabacteroides distasonii in preparation of a medicine for preventing and treating a disease caused by Pseudomonas aeruginosa.

[0040] In another aspect, the application also provides uses of a fermentation broth of Parabacteroides distasonii, or a supernatant fermentation broth after centrifugation of the fermentation broth, or an extract of the supernatant fermentation broth, in antagonizing Staphylococcus aureus, and uses of the fermentation broth, or the supernatant fermentation broth, or the extract of the supernatant fermentation broth, in preparation of a medicine for preventing and treating a disease caused by Staphylococcus aureus.

[0041] In another aspect, the application also provides uses of a fermentation broth of Parabacteroides distasonii, or a supernatant fermentation broth after centrifugation of the fermentation broth, or an extract of the supernatant fermentation broth, in antagonizing Salmonella typhi, and uses of the fermentation broth, or the supernatant fermentation broth, or the extract of the supernatant fermentation broth, in preparation of a medicine for preventing and treating a disease caused by Salmonella typhi.

[0042] The application further provides application of the fermentation liquor of Parabacteroides distasonis, or the supernatant fermentation liquor after centrifugation of the fermentation liquor, or the extract of the supernatant fermentation liquor in antagonizing Pseudomonas aeruginosa and in preparing a drug for preventing and treating a disease caused by Pseudomonas aeruginosa.

[0043] The application further provides application of Parabacteroides distasonis in preparing a drug, and application of P.distasonis NGMCC 1.200926 or a probiotic preparation containing P.distasonis NGMCC 1.200926 in preparing a product for preventing, relieving or improving infection of pathogenic bacteria such as Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa.

[0044] The application further provides application of Parabacteroides distasonis in preparing a drug, and application of P.distasonis NGMCC 1.200926 or a probiotic preparation containing P.distasonis NGMCC 1.200926 in preparing a product for preventing, relieving or improving infection of pathogenic bacteria such as Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa.

[0045] The application further provides application of Parabacteroides distasonis in preparing a drug, and application of P.distasonis NGMCC 1.200926 or a probiotic preparation containing P.distasonis NGMCC 1.200926 in preparing a product for preventing, relieving or improving infection of pathogenic bacteria such as Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa.

[0046] Compared with the prior art, the application has the following beneficial effects:

[0047] 1、The Parabacteroides distasonis P.distasonis NGMCC 1.200926 provided in the application is gram-negative and short rod-shaped and is an anaerobic bacterium. The 16S rRNA sequence of the Parabacteroides distasonis P.distasonis NGMCC 1.200926 is determined by using a PCR technology, and is compared with nucleotide sequences in a Genbank by using a Blast comparison, and the result shows that the 16S rRNA sequence of the Parabacteroides distasonis P.distasonis NGMCC 1.200926 has the highest similarity with that of P.distasonis JCM 5825 T , and identity values are 98.28%. The whole genome sequencing analysis of the Parabacteroides distasonis P.distasonis NGMCC 1.200926 is carried out, and a DNA-DNA molecular hybridization experiment shows that the Parabacteroides distasonis P.distasonis NGMCC 1.200926 has the highest similarity with P.distasonis JCM 5825 TThe hybridization ratio is 74.30%, which is higher than the threshold value of 70%. The phenotype characteristics and phylogenetic studies show that Parabacteroides sp. NGMCC 1.200926 is identified as Parabacteroides distasonis, the genome size is 5.68 Mbp, contains 4,660 genes, and the GC content is 45.17 mol%.

[0048] 2、The Parabacteroides distasonis NGMCC 1.200926 of the application can produce strong bacteriostatic active substances after fermentation culture, has obvious bacteriostatic effect on pathogenic bacteria such as Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa, has broad development space in the prevention and treatment of diseases caused by pathogenic bacteria such as Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa, and has good development and application prospect in antibacterial drugs and anticancer treatment through antitumor research. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is the mGAM upper plate colony morphology diagram of Parabacteroides distasonis NGMCC 1.200926 in the embodiment of the application;

[0050] Figure 2 is the perspective electron microscope diagram of Parabacteroides distasonis NGMCC 1.200926 in the embodiment of the application;

[0051] Figure 3 is the phylogenetic relationship diagram of Parabacteroides distasonis NGMCC 1.200926 and the main mode species of Parabacteroides genus constructed based on 16S rRNA gene sequence in the embodiment of the application;

[0052] Figure 4 is the genome function gene analysis diagram of Parabacteroides distasonis NGMCC 1.200926 based on KEGG database and COG database in the embodiment of the application;

[0053] Figure 5 is the polar lipid analysis diagram of Parabacteroides distasonis NGMCC 1.200926 in the embodiment of the application;

[0054] Figure 6is a fermentation broth of Parabacteroides distasonis NGMCC 1.200926 in the embodiment of the present application. The figure is the result of the antibacterial test of the fermentation broth of Parabacteroides distasonis NGMCC 1.200926 on Staphylococcus aureus (a1, a2), Salmonella typhi (b1, b2) and Pseudomonas aeruginosa (c1, c2).

[0055] Figure 7 is a fermentation broth of Parabacteroides distasonis NGMCC 1.200926 in the embodiment of the present application. The figure is the result of the antibacterial test of the fermentation broth of Parabacteroides distasonis NGMCC 1.200926 on Staphylococcus aureus (a1, a2), Salmonella typhi (b1, b2) and Pseudomonas aeruginosa (c1, c2). DETAILED DESCRIPTION

[0056] In order to enable persons skilled in the art to better understand the present application, the technical solutions of the present application will be further described in detail below in combination with the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by persons skilled in the art without creative work should belong to the protection scope of the present application.

[0057] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below in combination with the embodiments.

[0058] Embodiment:

[0059] The present application provides a Parabacteroides distasonis NGMCC 1.200926, the preservation number of which is CGMCC No: 40687; the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing; the preservation unit is China General Microbiological Culture Collection Center; and the preservation time is July 5, 2023.

[0060] The intestinal flora refers to the flora colonized in the animal intestine, which mainly lives in the animal intestine. With the help of the intestinal flora, the host can carry out many physiological activities, and a variety of probiotics are distributed in the intestinal flora, which has a maintaining effect on the body microecological balance and releases positive energy. The Parabacteroides distasonis NGMCC 1.200926 in the present application is isolated and screened from the feces of healthy men in Peking Union Medical College Hospital. The bacteria can produce natural substances with antibacterial activity, and the antibacterial spectrum is wide, which plays an important role in maintaining intestinal homeostasis and resisting invasion of external pathogenic bacteria. The study of the bacteria is conducive to obtaining new natural active substance resources of Parabacteroides distasonis from the special environment of human intestine and providing guidance for the treatment of human intestinal inflammation.

[0061] 1. Morphological observation of a new species of *Pseudomonas dilataniae*

[0062] 1.1 Source of strains

[0063] Rectal swab specimens from healthy adult males. Rectal swab specimens were collected from healthy adult males and placed in an anaerobic container, immediately transferred to an anaerobic glove box. Approximately 1g of fecal sample was dissolved in 10ml of sterile anaerobic phosphate-buffered saline (PBS) (pH 7, containing 1% cysteine) and filtered through cell sieves (70μm and 40μm) to remove a large amount of fecal residue. 1ml of each of the prepared stock solutions was transferred and further processed using three different methods: (I) treatment with 70% ethanol, (II) incubation at 65℃ for 30min, and (III) incubation at 80℃ for 10min. (IV) The stock solution was then prepared. The four different treatment solutions were diluted 10-fold with sterile anaerobic PBS to obtain 10... -1 ~10 -7 Diluted fecal solutions were spread onto yeast extract casein hydrolyzed fatty acid medium (YCFA) and modified anaerobic medium (MGAM) agar plates, respectively, with sterile sheep blood (5%) and rumen fluid (10%) added. Strain NGMCC 1.200926 was obtained from 10... -6 Isolates were obtained after a 1:1 dilution on MGAM agar plates for phenotypic, physiological, and phylogenetic analysis. Isolates were stored at -80°C as a 20% w / v glycerol suspension.

[0064] Preparation method of Modified Gibberellin Anaerobic Medium (MGAM) agar plates: 15.0g peptone, 10.0g casein peptone, 3.0g soybean peptone, 5.0g yeast extract, 2.0g beef meal, 13.5g digested serum powder, 1.2g bovine liver extract, 3.0g glucose, 2.5g potassium dihydrogen phosphate, 3.0g sodium chloride, 0.3g soluble starch, 0.3g L-cysteine, 0.15g sodium thioglycolate, and 17.0g agar powder. Add water to adjust the total volume to 1000ml. Autoclave at 121℃ for 15 minutes. Cool to room temperature, add 1ml of sterile 0.1% vitamin K1 solution and 2.5g heme chloride, slowly add 50ml of sterile defibrinated sheep blood and 100ml of clear bovine rumen fluid, and pour into plates to prepare MGAM agar plates.

[0065] Yeast extract casein hydrolyzed fatty acid medium (YCFA) agar plate preparation: Cysteine 1.0 g, Tryptone 10.0 g, Yeast extract 2.5 g, Sodium bicarbonate 4.0 g, Potassium phosphate dibasic 0.45 g, Potassium phosphate monobasic 0.45 g, Sodium chloride 0.9 g, Hemin 0.01 g, Magnesium sulfate heptahydrate 0.09 g, Calcium chloride 0.09 g, Agar powder 15.0 g, add water to a total volume of 1000 ml. Autoclave at 121 °C for 15 min, cool to room temperature, add sterile filtered reagent two (resazurin 1.0 mg, biotin 10.0 μg, cobalamin 10.0 μg, p-aminobenzoic acid 30.0 μg, folic acid 50.0 μg, pyridoxal 150.0 μg) and reagent three (thiamine 0.5 μg, riboflavin 0.5 μg, glucose 25 mM), then slowly add sterile defibrinated sheep blood 50 ml and clarified bovine rumen fluid 100 ml and pour plates to make YCFA agar plates.

[0066] 1.2 Morphological observation

[0067] The isolated and purified Parabacteroides sp. NGMCC 1.200926 was transferred to MGAM agar plates and incubated at 37 °C for 48-72 h in a glove box (90% N2, 5% H2, 5% CO2). Parabacteroides distasonis NGMCC 1.200926 formed 1.0-2.5 mm in diameter, milky white, round, smooth-edged, and central convex colonies on MGAM agar plates after incubation at 37 °C for 72 h Figure 1 The bacteria showed typical characteristics of Parabacteroides distasonis under electron microscopy: single bacterial cells were short rod-shaped ellipses, about 0.35-0.50 μm in diameter and 1.40-1.85 μm in length, without spores or flagella Figure 2

[0068] 1.3 Sodium chloride tolerance experiment of Parabacteroides sp. NGMCC 1.200926

[0069] Table 1. Results of sodium chloride tolerance experiment of Parabacteroides sp. NGMCC 1.200926

[0070]

[0071] Parabacteroides sp. NGMCC 1.200926 grew in culture medium containing 0%-1% sodium chloride, and did not grow in culture medium containing more than and equal to 1.5% sodium chloride.

[0072] 1.4 Temperature tolerance experiment of Parabacteroides sp. NGMCC 1.200926 (Table 2)​

[0073] Table 2 Parabacteroides sp. NGMCC 1.200926 temperature tolerance results

[0074]

[0075] Parabacteroides sp. NGMCC 1.200926 grew at 25°C to 37°C, did not grow at 20°C, 45°C.

[0076] Morphological identification can be initially determined that Parabacteroides sp. NGMCC 1.200926 belongs to the family of Tannerellaceae, Parabacteroides genus. But only from the colony morphology and microscopic observation can not be specific to which species of the family of Tannerellaceae, Parabacteroides genus, in order to further determine its classification position, it is necessary to carry out biochemical and molecular biology analysis.

[0077] 2 Biochemical identification

[0078] 2.1 API VITEK 2 ANC biochemical reaction identification

[0079] The purified bacteria were inoculated into modified Gifu anaerobic medium (MGAM) sheep blood agar plates, and placed in an anaerobic glove box at 37°C for 18-24h. Then use cotton swab or inoculation ring to pick up enough colonies into 3.0mL sterile saline (0.45% to 0.50% NaCl solution, pH 4.5 to 7.0) into a transparent plastic (polystyrene) test tube, using turbidimetric determination, to obtain a McFarland turbidity of 2.70 to 3.30, the bacterial suspension test tube and ANC card were placed in the card holder, and the 2 Compact instrument for detection and results, see Table 3.

[0080] 2.3 API ZYM biochemical reaction identification

[0081] The purified bacteria were inoculated into modified Gifu anaerobic medium (MGAM) sheep blood agar plates, and placed in an anaerobic glove box at 37°C for 18-24h. Then use cotton swab or inoculation ring to pick up enough colonies into 3.0mL sterile saline (0.45% to 0.50% NaCl solution, pH 4.5 to 7.0) into a transparent plastic (polystyrene) test tube, using turbidimetric determination, to obtain a McFarland turbidity of 2.70 to 3.30, the bacterial suspension test tube and ANC card were placed in the card holder, and the

[0082] Table 3 Parabacteroides sp. NGMCC 1.200926 biochemical reaction results

[0083]

[0084]

[0085]

[0086] 3Parabacteroides distasonis NGMCC 1.200926's 16S rRNA phylogenetic analysis

[0087] The single colony of suspected new species (preliminary judgment according to the size, shape, color of the colony) was picked up with a sterile Tip head or sterilized toothpick as the PCR reaction template, and the primer 27F-1492R (27F: 5'-AGAGTTTGATCMTGGCTCAG-3', 1492R: 5'-GGYTACCTTGTTAC GAC TT-3') was used for colony PCR amplification. The amplification product was sent to Guangzhou Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The sequencing results were put into GenBank and compared with the known nucleic acid sequences in GenBank. The results showed that Parabacteroides sp. NGMCC 1.200926 and P. distasonis JCM 5825 T had an identity value of 98.28% for nucleic acid sequences. According to the principle of Identities ≥ 97% for determining the same species, it was preliminarily determined that Parabacteroides sp. NGMCC 1.200926 belonged to P. distasonis.

[0088] The obtained sequences were spliced, forward and reverse detected, and the vector and primer sequences were removed by using SeqMan software and DNA star software. Then, MAGA 7.0 software was used to construct a phylogenetic tree by using the maximum likelihood method, and finally the generated phylogenetic tree was tested by using Bootstrap. Segatella albensis M384 AJ011683 T was used as an outgroup. The results, as shown in Figure 3 , can be seen that Parabacteroides sp. NGMCC 1.200926 is in an independent evolutionary branch on the clustering tree, and P. distasonis JCM 5825 T is clustered together, and the Bootstrap support rate is 100% Figure 3), showing a closer relationship. Parabacteroides sp. NGMCC 1.200926 has an identity value of less than 98% in Blast comparison with other known species of Parabacteroides, showing a distant relationship, and has a closer relationship with the type strain Parabacteroides distasonis JCM 5825, which is greater than 97%. Studies have shown that the classification level of 16S rRNA supports Parabacteroides sp. NGMCC 1.200926 belonging to Parabacteroides distasonis.

[0089] 4. Genomic analysis

[0090] After obtaining the pure culture of Parabacteroides sp. NGMCC 1.200926, the present application carries out whole genome sequencing, carries out prediction analysis on the basic characteristics of the genome, and carries out genomic comparative analysis of Parabacteroides distasonis NGMCC 1.200926 and other known species of Parabacteroides.

[0091] 4.1 Whole genome sequencing and assembly

[0092] The whole genome sequencing of Parabacteroides sp. NGMCC 1.200926 is completed by Beijing Nuoweiziyuan Technology Co., Ltd. The single molecule real-time sequencing technology of the third generation sequencing platform Pacific Biosciences is used to determine the whole genome of Parabacteroides sp. NGMCC 1.200926, and the SMRT Analysis 2.3.0 software package is used to filter the sequencing data for quality control, and the complete circular genome sequence is assembled.

[0093] 4.2 Analysis of basic characteristics of genome

[0094] CDS prediction was performed using GeneMarkS (http: / / topaz.gatech.edu / ) software, tRNAs and rRNAs were predicted using tRNAscan-SE and rRNAmmer software. IslandPath-DIOMB software was used for genomic island prediction. In this study, Parabacteroides sp. NGMCC 1.200926 isolated from healthy adult human intestinal samples was sequenced by the third generation sequencing platform Pacific Biosciences (PacBio). The full-length genome of Parabacteroides sp. NGMCC 1.200926 is 5.68 Mbp, with a G+C content of 45.17 mol%, encoding 4,660 genes Figure 4

[0095] 5Genomic DNA-DNA hybridization

[0096] The DNA-DNA hybridization online software (genome and genome distance calculator, GGDC) was used to perform online genomic hybridization of the whole genome sequence of Parabacteroides sp. NGMCC 1.200926 and the representative sequences of different species of Parabacteroides currently available in the GenBank library. DNA-DNA hybridization similarity greater than 70% is classified into the same species (see Table 4).

[0097] As can be seen from Table 4, the DNA-DNA hybridization similarity between Parabacteroides sp. NGMCC 1.200926 and P. distasonis JCM5825 T The highest hybridization ratio reached 74.30%, and the hybridization ratio of other known close relatives of P. distasonis was between 21.50% and 21.90%, indicating that Parabacteroides sp. NGMCC 1.200926 belongs to Parabacteroides distasonis.

[0098] Table 4 Consistency and sequence hybridization results of Parabacteroides distasonis NGMCC 1.200926 and known Parabacteroides distasonis different species genomic sequences.

[0099]

[0100] 6Antibacterial activity

[0101] ​The Parabacteroides sp. NGMCC 1.200926 was fermented in a modified Gifu anaerobic medium (MGAM) fermentation medium to obtain the corresponding antibacterial active substance. The antibacterial effect of the fermentation extract was determined by the Oxford cup single-layer agar diffusion method. The specific operation steps are as follows, and the results are shown in Figure 6 , and the specific analysis results are shown in Table 5.

[0102] 6.1 Preparation of Parabacteroides sp. NGMCC 1.200926 extract

[0103] 10-15 morphologically similar Parabacteroides sp. NGMCC 1.200926 single colonies were picked up on blood agar plates with a inoculating loop, inoculated into 1000 ml of MGAM fermentation medium (the MGAM fermentation medium includes the following components in the weight ratio: tryptone 15.0 g / L, pancreatic casein peptone 10.0 g / L, soybean peptone 3.0 g / L, yeast extract powder 5.0 g / L, beef powder 2.0 g / L, digested serum powder 13.5 g / L, beef liver extract powder 1.2 g / L, glucose 3.0 g / L, potassium dihydrogen phosphate 2.5 g / L, sodium chloride 3.0 g / L, soluble starch 0.3 g / L, L-cysteine 0.3 g / L, sodium thioglycolate 0.15 g / L; pH 7.0), and the fermentation medium 1000 ml was cultured at 37°C on a shaker at 150 r / min for 60 h. The fermentation medium was centrifuged at 13,000 r / min for 15 min, and the supernatant fermentation broth was freeze-dried with a freeze dryer. The freeze-dried extract was rehydrated to obtain 50 ml of fermentation extract, which was stored at 4°C for standby.

[0104] 6.2 Preparation of LB plates of test strains (Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa)

[0105] 5-10 morphologically similar Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa were picked up on LB agar plates with a inoculating loop, respectively inoculated into LB liquid medium, and after overnight culture, gradient dilution was performed with sterile distilled water. The bacterial liquid dilution gradient was adjusted to 10 -6 . Then 1 ml of Staphylococcus aureus, Salmonella typhi and Pseudomonas aeruginosa bacterial liquid was taken with a pipette and injected into 15-20 ml of LB solid medium in a 90 mm plate, and the plate was gently shaken to mix the bacterial liquid and agar evenly, and then dried.

[0106] 6.3 Test method (Oxford cup single-layer agar diffusion method)

[0107] A sterile forceps was used to pick up the sterilized Oxford cup and place it on the solidified 10 -6LB culture plate (containing the bacterial liquid of the test strain Staphylococcus aureus or the test strains Salmonella typhi and Pseudomonas aeruginosa) in the center of the Oxford cup, 150 μl of the fermentation extract of Parabacteroides sp. NGMCC 1.200926 was added to the hole in the center of the Oxford cup, the plate was covered and placed in a 4°C refrigerator overnight, and the fermentation extract of Parabacteroides sp. NGMCC 1.200926 was allowed to diffuse into the agar, the next day the plate was taken out of the refrigerator and observed for diffusion, and if there was no fermentation extract in the Oxford cup, it was placed in a 37°C incubator. After the above operation, the culture plate was incubated in a 37°C incubator for 8h, and then the size of each inhibition zone was measured with a vernier caliper, and the measurement was repeated three times in parallel, and the average value was calculated.

[0108] Table 5 Parabacteroides sp. NGMCC 1.200926 fermentation extract antibacterial results

[0109]

[0110] From the analysis of the antibacterial activity in Table 5, it can be seen that the Bacteroides sp. NGMCC 1.200926 strain of the present application produces strong antibacterial activity after being cultured in MGAM fermentation broth, and has obvious antibacterial effect on Staphylococcus aureus (A), Salmonella typhi (B) and Pseudomonas aeruginosa (C). Therefore, the strain of the present application has potential application prospects in antibacterial drugs. Figure 6 Figure 6 Figure 6

[0111] 7 Anti-tumor activity

[0112] The anti-tumor activity experiment was carried out using Parabacteroides sp. NGMCC 1.200926 and HT-29 human colon cancer cells and AGS human gastric adenocarcinoma cells at a MOI of 200, and the inhibition effect of the strain Parabacteroides sp. NGMCC 1.200926 on the activity of the two tumor cell lines was determined by CTG detection method based on ATP content, and the specific operation steps are as follows, and the results are shown in Figure 7 .

[0113] 7.1 Preparation of Parabacteroides sp. NGMCC 1.200926 and HT-29 and AGS at a MOI of 200

[0114] ​​​The cells were plated the day before the experiment. Well-grown HT-29 human colon cancer cells and AGS human gastric adenocarcinoma cells were digested and resuspended, and inoculated into 96-well white plates (9000 cells per well for HT-29 cells and 4000 cells per well for AGS cells). According to MOI = 200, the corresponding bacterial inoculum for HT-29 cells was 1.8 x 10^ 6 cells, and the corresponding bacterial inoculum for AGS cells was 8 x 10^ 5 cells. The Parabacteroides sp. NGMCC 1.200926 bacterial solution was centrifuged, washed twice with PBS phosphate buffer, and resuspended in cell culture medium 1640 to prepare a bacterial suspension.

[0115] 7.2 Test method (CTG method)

[0116] The prepared bacterial suspension was added to the 96-well white plate containing the inoculated HT-29 human colon cancer cells and AGS human gastric adenocarcinoma cells, and incubated for 2 h. Then, 60 μl of the growth medium was slowly aspirated from each well of the 96-well plate using a P200 pipette. The remaining 40 μl of the medium should contain the tumor cells in each well, and then 160 μl of 1 x DPBS was slowly added to the side of each well, with the pipette tip tilted at 45° to contact the top of the well. Then, 160 μl of the medium was slowly removed from each well of the 96-well plate. The remaining 40 μl of the medium should contain the tumor cells in each well. The above displacement step was repeated twice, using a p20 pipette to aspirate as much 1 x DPBS as possible without aspirating the tumor cells, and then 200 μl of 1640 medium containing antibiotics was slowly added. The cells were equilibrated overnight, and the next day, CTG detection solution was added to the white plate, which was incubated at 80 rpm for 30 min in the dark. Chemiluminescence detection was performed on a microplate reader.

[0117] The above specific embodiments are merely illustrative of the present application and are not intended to limit the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, and such modifications are within the scope of the present application as long as they are within the scope of the claims.

Claims

1. A type of *Pseudomonas difficile* that can inhibit pathogenic bacteria and has anti-tumor properties (… Parabacteroides distasonis Its characteristics are: The accession number of *Pseudomonas dignitaria* NGMCC 1.200926 is: CGMCC No: 40687; the accession address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the depositary institution is: China General Microbiological Culture Collection Center; the accession date is: July 5, 2023; the 16S ribosomal DNA sequence of *Pseudomonas dignitaria* NGMCC 1.200926 is shown in SEQ ID NO.

1.

2. The *Pseudomonas dilataniae* as described in claim 1 in the preparation of a treatment for Staphylococcus aureus (… Staphylococcus aureus Application of drugs for diseases caused by ) 3. The *Pseudomonas dilataniae* as described in claim 1 in the preparation of preparations for the prevention and treatment of *Salmonella typhi* (…). Salmonela typhi Application of drugs for diseases caused by ) 4. The *Pseudomonas dilataniae* as described in claim 1 in the preparation of a treatment for Pseudomonas aeruginosa (… Pseudomonas aeruginosa Application of drugs for diseases caused by ) 5. The use of the fermentation broth of *Pseudomonas diffusa* as described in claim 1, or the supernatant fermentation broth after centrifugation of the fermentation broth, in the preparation of drugs for the prevention and treatment of diseases caused by *Staphylococcus aureus*.

6. The use of the fermentation broth of *Pseudomonas diffusa* as described in claim 1, or the supernatant fermentation broth after centrifugation, in the preparation of drugs for the prevention and treatment of diseases caused by *Salmonella typhi*.

7. The use of the fermentation broth of *Pseudomonas diffusa* as described in claim 1, or the supernatant fermentation broth after centrifugation of the fermentation broth, in the preparation of drugs for the prevention and treatment of diseases caused by *Pseudomonas aeruginosa*.

8. The use of *Pseudomonas difficile* as described in claim 1 in the preparation of a drug, characterized in that: Application of *Pseudomonas difficile* NGMCC 1.200926 or probiotic preparations containing *Pseudomonas difficile* NGMCC 1.200926 in the preparation of products that alleviate or improve infections caused by pathogens such as Staphylococcus aureus, Salmonella typhi, and Pseudomonas aeruginosa.

9. The use of *Pseudomonas difficile* as described in claim 1 in the preparation of a drug, characterized in that: Application of *Pseudomonas difficile* NGMCC 1.200926 or probiotic preparations containing *Pseudomonas difficile* NGMCC 1.200926 in the preparation of products for the treatment of human colon cancer and human gastric adenocarcinoma.

10. The use of *Pseudomonas dilataniae* as described in claim 1 in the preparation of a drug, characterized in that: Application of *Pseudomonas diffusa* NGMCC 1.200926 or fermentation broth containing *Pseudomonas diffusa* NGMCC 1.200926 in the preparation of therapeutic products for the treatment of Staphylococcus aureus, Salmonella typhi, and Pseudomonas aeruginosa pathogens, or for the relief or improvement of human colon cancer and human gastric adenocarcinoma.

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