Infection-resistant enteric bacterial strains and their uses

Abstract

To provide an infection-resistant enterobacterial strain and use thereof, more particularly, an infection-resistant enterobacterial strain that provides a preventive or therapeutic effect against infectious diseases caused by pathogenic microorganisms and uses thereof.SOLUTION: The infection-resistant enterobacterial strain according to the present invention is characterized in that it is selected from the group consisting of Oribacterium sp. JBO3-101 strain (Accession No. KACC81250BP) and Ruminococcus sp. JBR5-501 strain (Accession No. KACC81249BP). The infection-resistant enterobacterial strain according to the present invention has the effect of providing the host with resistance to infectious diseases caused by pathogenic microorganisms when ingested.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] The present invention relates to an infection-resistant intestinal strain and uses thereof, more particularly to an infection-resistant intestinal strain that confers a preventive or therapeutic effect against infectious diseases caused by pathogenic microorganisms, and uses thereof. [Background technology]

[0002] Since the dawn of history, humanity has been in a constant battle for survival against pathogens. However, with the rapid development of antibiotics, antivirals, and vaccines, the problem of infectious diseases has been largely resolved. However, in recent years, new, more deadly pathogens have emerged due to climate and environmental changes such as global warming, changes in living environments and behavior, and the evolutionary characteristics of microorganisms. In particular, not only tuberculosis, Ebola virus, MERS, and SARS, but also the SARS-CoV-2 virus that emerged at the end of 2019, have caused enormous damage to humanity worldwide. Due to the severity of the novel coronavirus disease (COVID-19), various preventive vaccines have been developed. In addition to traditional vaccines, mRNA vaccines are also being developed. As of December 2022, more than 70% of the world's population will have received at least one dose of a COVID-19 vaccine. However, South Korea, where 87% of the population has received the mRNA vaccine, has the highest per capita COVID-19 incidence rate in the world, more than 10 times higher than third-world countries, where vaccination rates are at 10-30%.Therefore, there is an urgent need to develop a groundbreaking vaccine that, unlike existing vaccine technology, is effective in preventing infectious diseases regardless of the type or mutation of the pathogen.

[0003] The intestinal flora, or microbiome, has a significant impact on the overall health of the host, including various diseases such as obesity, diabetes, dementia, cancer, and cardiovascular disease. Active research over the past 20 years has shown that the intestinal bacterial strains that form communities in the intestine, an environment exposed to various pathogens, affect the host's resistance to infectious diseases. As a result, it is believed that host resistance varies, with some individuals experiencing no infection, asymptomatic symptoms, or severe, even when exposed to the same pathogen.

[0004] For the above reasons, the following techniques have been developed to develop infection-resistant intestinal bacteria that can prevent or treat infectious diseases caused by pathogenic microorganisms.

[0005] U.S. Patent Registration No. 11,471,495 provides C. scindens, C. hiranonis, C. hylemonae, C. perfringens, C. sordelli, Proteocatella sphenisci, Lachnospiraceae 5_1_57FAA, Barnesiella intestihominis, Blautia hansenii, and Pseudoflavonifractor strains as intestinal bacterial strains for preventing infection with the pathogenic bacterium Clostridium difficile.

[0006] WO2021066585 and US20220347229A provide Staphylococcus epidermidis strains as intestinal strains that have immune-enhancing effects against some pathogenic bacteria.

[0007] WO2016086161 provides Ruminococcus obeum, C. hathewayi, Eubacterium desmolans, Dorea longicatena, R. lactaris (Blautia producta), Eubacterium contorum, R. faecis, Holdemania filiformis, and C. sordelli strains as intestinal strains for the treatment of prophylaxis.

[0008] US010195273B provides Akkermansia or Faecalibacterium strains as intestinal strains for PD-1 inhibitor or PD-L1 inhibitor function.

[0009] WO2019028402, US20200376044A, JP2020529478A, EP03661525A provide Roseburia hominis, Eubacterium eligens strains for treating metabolic diseases.

[0010] WO2018117263 and EP03559209A are anti-cancer therapeutic effects of Phascolarctobacterium faecium LN998073, Fusobacterium ulcerans KR822463, Bacteroides dorei CP011531, B. uniformis NR_112945, Subdoligranulum sp. 4_3_54A2FAA, Paraprevotella xylaniphila AB331897, Parabacteroides johnsonii AB261128, Alistipes sp.JC136 NZ-CAEG00000000, P. gordonii AB470343, Eubacterium limosum AB595134, P. distasonis HE974920, B. cellulosilyticus_NR_112933, B. clarus_AB490801, B. salyersiae_AY608696, B. fragilis_CR626927, B. uniformis_AB247141, and B. eggerthii_NR_112935 strains are provided.

[0011] WO2021194281 provides Akkermansia muciniphila microorganisms or products thereof for the prevention and treatment of inflammatory diseases.

[0012] WO2013032744, JP2014528925A, and EP02797606A provide compositions that increase the proportion of Firmicutes relative to the proportion of Bacteroidetes phylum in the intestinal bacterial flora.

[0013] WO2016172658 and US20200009168A provide sugars, sugar alcohols, amino acids, peptides, micronutrients, fatty acids or polyphenols as microbiome regulator compositions.

[0014] US20210332323A provides the L. plantarum CJLP475 strain as a feed additive, which increases cytokine secretion and has antiviral and immunomodulatory effects.

[0015] However, these technologies are not effective in preventing infection, and there are no successful examples of intestinal strains that can prevent or treat serious infectious diseases caused by pathogenic microorganisms. In the case of COVID-19, despite numerous attempts, no intestinal strains resistant to the SARS-CoV-2 virus have been identified at the species level. Therefore, there is an urgent need to develop intestinal strains that can prevent or treat infectious diseases caused by various pathogenic bacteria and viruses. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] U.S. Patent Registration No. 11,471,495 [Patent Document 2] WO2021066585 [Patent Document 3] US20220347229A [Patent Document 4] WO2016086161 [Patent Document 5] US010195273B [Patent Document 6] WO2019028402 [Patent Document 7] US20200376044A [Patent Document 8] JP2020529478A [Patent Document 9] EP03661525A [Patent Document 10] WO2018117263 [Patent Document 11] EP03559209A [Patent Document 12] WO2021194281 [Patent Document 13] WO2013032744 [Patent Document 14] JP2014528925A [Patent Document 15] EP02797606A [Patent Document 16] WO2016172658 [Patent Document 17] US20200009168A [Patent Document 18] US20210332323A Summary of the Invention [Problem to be solved by the invention]

[0017] The present invention relates to an infection-resistant intestinal strain and uses thereof. The present invention also relates to an infection-resistant intestinal strain that confers a preventive or therapeutic effect on infectious diseases caused by pathogenic microorganisms and uses thereof.

[0018] The technical problems that the present invention aims to solve are not limited to the above-mentioned technical problems, and other technical problems not mentioned above will be clearly understood by those skilled in the art to which the present invention pertains from the following description.

[0019] An object of the present invention is to provide an infection-resistant enteric bacterial strain.

[0020] It is still another object of the present invention to provide an infection-resistant enteric bacterial strain that has a preventive or therapeutic effect against infectious diseases caused by pathogenic microorganisms, and uses thereof. [Means for solving the problem]

[0021] To achieve the above-mentioned object, the present invention provides an infection-resistant intestinal strain that confers a preventive or therapeutic effect against infectious diseases caused by pathogenic microorganisms, the infection-resistant intestinal strain being selected from the group consisting of Oribacterium sp. JBO3-101 strain (accession number KACC81250BP) and Ruminococcus sp. JBR5-501 strain (accession number KACC81249BP).

[0022] The present invention also provides a composition for preventing or treating an infectious disease, comprising as an active ingredient one or more infection-resistant enteric strains selected from the group consisting of Oribacterium sp. JBO3-101 strain (accession number KACC81250BP) and Ruminococcus sp. JBR5-501 strain (accession number KACC81249BP), or a culture thereof.

[0023] The present invention also provides a method for preventing or treating an infectious disease, which comprises administering to a mammal one or more infection-resistant enteric strains selected from the group consisting of Oribacterium sp. JBO3-101 strain (accession number KACC81250BP) and Ruminococcus sp. JBR5-501 strain (accession number KACC81249BP), or a culture thereof. [Effects of the Invention]

[0024] The infection-resistant intestinal bacterial strain of the present invention has the effect of providing the host with resistance to infectious diseases caused by pathogenic microorganisms when ingested. [Brief explanation of the drawings]

[0025] [Figure 1] This is an image (top) and graph (bottom) of the temperature measurement results of groups divided according to the degree of infection in experimental animals that were given an infection-resistant human intestinal bacterial strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus (A: control group, B: severely infected group, C: mildly infected group, D: uninfected group). [Figure 2] These are photographs of lungs and H&E stained lung tissues of experimental animals categorized by the degree of infection in which they were administered an infection-resistant human intestinal strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus (A: control group, B: severely infected group, C: mildly infected group, D: uninfected group). [Figure 3] This shows the results of an analysis of the relative abundance of the gut microbiome at the phylum and order levels in experimental animals that were given an infection-resistant human intestinal bacterial strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus, divided into a severely infected group (SI), a mildly infected group (MI), and a non-infected group (NI). [Figure 4] This shows the alpha diversity of the intestinal microbiome in experimental animals that were given an infection-resistant human intestinal bacterial strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus, divided into a severely infected group (SI), a mildly infected group (MI), and a non-infected group (NI). [Figure 5] This is the result of comparing the co-occurrence network analysis of the intestinal microbiomes of the severely infected group (SI), mildly infected group (MI), and non-infected group (NI) in experimental animals that were given an infection-resistant human intestinal bacterial strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus, using the ReBoot algorithm. [Figure 6] This is a heat map comparing the composition of the intestinal microbiome in each group of severely infected (SI), mildly infected (MI), and non-infected (NI) experimental animals that were given an infection-resistant human intestinal bacterial strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus. [Figure 7]This is a non-metric multidimensional scaling (NMDS) plot comparing the distribution of intestinal microbiomes in the severely infected (SI), mildly infected (MI), and non-infected (NI) groups in experimental animals that were given an infection-resistant human intestinal bacterial strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus. [Figure 8] Experimental animals were fed an infection-resistant human intestinal bacterial strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus. These animals were divided into a severely infected group (SI), a mildly infected group (MI), and a non-infected group (NI). (A) Principal component analysis (PCoA) and (B) Distance to group centroid were performed on the intestinal microbiome of these groups. [Figure 9] In experimental animals that were administered an infection-resistant human intestinal bacterial strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus, the differential abundance of the intestinal microbiome between the severely infected (SI) and non-infected (NI) groups was analyzed at the species level, i.e., the log2 fold change for each OTU was calculated using DESeq2 analysis to analyze the differentially increased microorganisms between the groups. [Figure 10] Experimental animals were given an infection-resistant human intestinal bacterial strain according to Example 1 of the present invention, and then infected with the SARS-CoV-2 virus. The differential abundance of the intestinal microbiome between the severely infected (SI) and non-infected (NI) groups, i.e., the microorganisms that were differentially increased between the groups, was analyzed at the species level using DESeq2 analysis, and the results are shown in a clustered heatmap for each OTU. [Figure 11] This shows the differential abundance between the severely infected (SI) and non-infected (NI) groups in experimental animals that were given an infection-resistant human intestinal strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus, i.e., the relative abundance of JBO3-101, a microorganism that was differentially increased between the groups. [Figure 12]This shows the differential abundance between the severely infected (SI) and non-infected (NI) groups in experimental animals that were given an infection-resistant human intestinal strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus, i.e., the relative abundance of JBR5-501, a microorganism that was differentially increased between the groups. [Figure 13] 1 shows photographs of Gram staining of the "infection-resistant strains" JBO3-101 (A) and JBR5-501 (B) isolated from infection-resistant human intestinal strains according to Example 1 of the present invention. [Figure 14] Experimental animals were fed with the "infection-resistant strains" JBO3-101 (A), JBR5-501 (B), JBO3-101 / JBR5-501 (C), or a control strain ((D), Lactobacillus sp.) according to Example 2 of the present invention for one week, and then infected with the SARS-CoV-2 virus. The results show that the body weights were measured daily for seven days, compared to the infected control group (Control). [Figure 15] Experimental animals were fed with the "infection-resistant strains" JBO3-101 (A), JBR5-501 (B), JBO3-101 / JBR5-501 (C), or a control strain ((D), Lactobacillus sp.) according to Example 2 of the present invention for one week, and then infected with the SARS-CoV-2 virus. The survival rates were measured daily for seven days compared to the infected control group (Control). [Figure 16] These are morphological photographs of lung tissue on day 8 after experimental animals were infected with SARS-CoV-2 virus and fed with the "infection-resistant strains" JBO3-101 (A), JBR5-501 (B), JBO3-101 / JBR5-501 (C), or a control strain ((D), Lactobacillus sp.) according to Example 2 of the present invention for one week, compared with an infected control group (Control). [Figure 17]These are photographs of H&E stained lung tissue sections on day 8 after experimental animals were infected with SARS-CoV-2 virus and fed with the "infection-resistant strains" JBO3-101 (A), JBR5-501 (B), JBO3-101 / JBR5-501 (C), or a control strain ((D), Lactobacillus sp.) according to Example 2 of the present invention for one week, compared with an infected control group (Control). [Figure 18] This shows the results of viral RT-PCR of lung tissue on the 8th day after infection with SARS-CoV-2 virus in experimental animals that had been fed with the "infection-resistant strains" JBO3-101 (A), JBR5-501 (B), JBO3-101 / JBR5-501 (C), or a control strain ((D), Lactobacillus sp.) according to Example 2 of the present invention for one week, compared with the infected control group (Control). [Figure 19] 1 shows the results of RT-PCR of influenza virus in lung tissues of experimental animals infected with influenza virus according to Example 3 of the present invention, fed with "infection-resistant strains" JBO3-101 (A), JBR5-501 (B), JBO3-101 / JBR5-501 (C), or a control strain ((D), Lactobacillus sp.) for one week, and then compared with an infected control group (Control). [Figure 20] This shows the results of Mycobacterium tuberculosis CFU counts in lung tissues compared to the infected control group (Control) after experimental animals were infected with Mycobacterium tuberculosis after being fed the "infection-resistant strains" JBO3-101 (A), JBR5-501 (B), JBO3-101 / JBR5-501 (C), or a control strain ((D), Lactobacillus sp.) for one week according to Example 4 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The gut flora, or microbiome, has a significant impact on the overall health of the host, including various diseases such as obesity, diabetes, dementia, cancer, and cardiovascular disease. The gut flora, which form communities in the intestines, an environment exposed to various pathogens, is known to affect the host's resistance to infectious diseases. As a result, it is believed that host resistance varies, with some individuals experiencing no infection, asymptomatic symptoms, or severe symptoms even when exposed to the same pathogen.

[0027] The present inventors have completed the present invention by focusing on the fact that infection-resistant intestinal bacterial strains should form colonies in the intestines of individuals who are not infected even when exposed to pathogenic microorganisms.

[0028] Therefore, the inventors prepared feces from "infection-resistant humans" who had been exposed to the virus during the COVID-19 pandemic but had not contracted COVID-19. Next, COVID-19 model animals were treated with an antibiotic / antibacterial combination, and then ingested the feces samples from the "infection-resistant humans" and infected with the SARS-CoV-2 virus. Through screening, they successfully isolated "infection-resistant animals" that showed no symptoms of infection even after SARS-CoV-2 infection. Through gut microbiome analysis of these animals, they identified two "infection-resistant strains," JBO3-101 and JBR5-501. Furthermore, 16sRNA sequencing of these strains confirmed that they were new species.

[0029] The inventors confirmed the preventive effect against SARS-CoV-2 virus infection in experimental animals in which a de novo microbiome was formed using the JBO3-101 and JBR5-501 strains, which were found to be "infection-resistant strains."

[0030] The present inventors also confirmed the therapeutic effect on viral infections by treating experimental animals infected with influenza virus, the most common viral infectious disease microorganism, with infection-resistant strains JBO3-101 and JBR5-501 strains.

[0031] To confirm the preventive effect against Mycobacterium tuberculosis, which causes the most serious bacterial infectious disease, the inventors formed de novo microbiomes in experimental animals using JBO3-101 and JBR5-501 strains, which were found to be "infection-resistant strains," and then confirmed the preventive effect against Mycobacterium tuberculosis infection.

[0032] In one embodiment of the present invention, there is provided an infection-resistant enteric bacterial strain that confers a preventive or therapeutic effect against infectious diseases caused by pathogenic microorganisms, and uses thereof.

[0033] Therefore, in one aspect, the present invention relates to an infection-resistant intestinal strain selected from the group consisting of Oribacterium sp. JBO3-101 strain (accession number KACC81250BP) and Ruminococcus sp. JBR5-501 strain (accession number KACC81249BP).

[0034] The present invention also relates to a composition for preventing or treating an infectious disease, comprising, as an active ingredient, one or more infection-resistant intestinal strains selected from the group consisting of Oribacterium sp. JBO3-101 strain (accession number KACC81250BP) and Ruminococcus sp. JBR5-501 strain (accession number KACC81249BP), or a culture thereof.

[0035] In the present invention, the infectious disease means a disease caused by infection with a pathogenic virus, pathogenic bacteria, or pathogenic fungus.

[0036] The viral infection includes all infections caused by pathogenic viruses, and specific examples of the pathogenic viruses include, but are not limited to, severe acute respiratory syndrome coronavirus (SARS-CoV), hepatitis B virus, hepatitis C virus, human papillomavirus, influenza virus, human immunodeficiency virus (HIV), Ebola virus, dengue virus, measles virus, Hantan virus, rubella virus, rotavirus, and norovirus.

[0037] The bacterial infection includes all infections caused by pathogenic bacteria, and specific examples of the pathogenic bacteria include, but are not limited to, Mycobacterium tuberculosis, Streptococcus pneumoniae, Vibrio cholerae, diphtheria bacillus, Mycobacterium leprae, Treponema pallidum, tetanus bacillus, and Salmonella typhi.

[0038] The fungal infection includes all infections caused by pathogenic fungi, and specific examples of the pathogenic fungi include, but are not limited to, Aspergillus species, Candida species, yeast, Histoplasma species, Coccidioides species, and Sporothrix species.

[0039] The composition for preventing or treating an infectious disease is not particularly limited, but may be any of the following: 3 ~10 12It is desirable to contain 1000 cfu / g of the strain.

[0040] In the present invention, the culture may be the culture itself obtained by culturing the strain, a culture supernatant obtained by removing the strain from the culture, or a concentrate or lyophilized product of the culture supernatant.

[0041] The composition for preventing or treating infectious diseases of the present invention can be administered to mammals, including humans, via various routes, including any commonly used route, such as oral, cutaneous, intravenous, intramuscular, or subcutaneous, and preferably oral.

[0042] The composition may include a pharmaceutically acceptable excipient or carrier. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field. Suitable carriers include, for example, lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Suitable diluents include, for example, ethanol, glycerol, and water. The choice of pharmaceutical carrier, excipient, or diluent can be selected based on the intended route of administration and standard pharmaceutical practice. The composition may include, or in addition to, any suitable binder, lubricant, suspending agent, coating agent, or solubilizer.

[0043] When the composition is formulated or shaped, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0044] Oral liquid formulations include suspensions, tolerant solutions, oils, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavorings, and preservatives may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, etc. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. The above-mentioned ingredients may be added in combination with the active ingredient, i.e., the killed bacterial mixture, its culture, its extract, or its bacterial component.

[0045] The appropriate dosage of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, administration method, the patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity. It can also be administered once or several times daily at regular intervals, as determined by a physician or pharmacist. For example, the daily dosage may be 0.1 to 10,000 mg / kg, preferably 1 to 2,000 mg / kg, based on the active ingredient content. The above dosage amounts are examples of average cases, and the optimal dosage can be determined by those skilled in the art and can be adjusted depending on various factors, including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, the patient's age, weight, general health condition, sex, and diet, administration time, administration route, excretion rate of the composition, treatment period, and concomitant medications.

[0046] The present invention also relates to a method for preventing or treating an infectious disease, which comprises administering to a mammal one or more infection-resistant enteric strains selected from the group consisting of Oribacterium sp. JBO3-101 strain (accession number KACC81250BP) and Ruminococcus sp. JBR5-501 strain (accession number KACC81249BP), or a culture thereof. [Example]

[0047] The present invention will be described in more detail below with reference to specific examples. However, these examples are merely for the purpose of explaining the present invention in more detail, and the scope of the present invention is not limited thereto.

[0048] Example 1: Detection of infection-resistant bacterial strains To identify infection-resistant gut strains, the inventors screened for gut strains of "infection-resistant humans" that do not become infected even when exposed to pathogenic microorganisms. To this end, 6-week-old Roborovski hamster SH101 experimental animals were administered an antibiotic / antibacterial combination consisting of azithromycin (15 mg / kg), neomycin (25 mg / kg), ciprofloxacin (20 mg / kg), and miconazole (30 mg / kg). Next, feces from "infection-resistant humans" who had not contracted COVID-19 were sampled and freshly prepared into 0.1 g to 0.5 g samples. These samples were then administered to experimental animals whose gut bacteria had been depleted by the antibiotic / antibacterial agent. One week later, 100 mg of feces were administered through the nose of the experimental animals in which a de novo microbiome had been formed. 5 TCID 50 The animals were infected with 50 μl of SARS-CoV-2 virus. Body temperature was measured daily for 7 days (Figure 1), and on the 8th day after infection, the lungs of each test group were examined macroscopically and H / E stained to confirm pathological changes (Figure 2). Based on these results, the animals were divided into severe infection, mild infection, and no infection groups according to fever, a typical symptom of SARS-CoV-2 infection, and the degree of lung tissue infection (Table 1).

[0049] [Table 1]

[0050] To analyze the gut microbiome of the severely infected, mildly infected, and uninfected SARS-CoV-2 virus groups, intestinal contents were collected from each group of test animals and analyzed for 16S rRNA hypervariable regions V3 and V4 using 16S metagenomic sequencing. The total number of operational taxonomic units (OTUs) constituting the gut microbiome of each experimental group was 2,070, which were assigned to 22 phyla, 153 families, and 278 genera.

[0051] Experimental animals were fed a resistant human intestinal strain and then infected with SARS-CoV-2 virus. To analyze the gut microbiomes of the severely infected (SI), mildly infected (MI), and non-infected (NI) groups, the derived OTUs were used to compare the relative abundance, alpha diversity, and co-occurrence network analysis of each group (Figures 3-5). The total analyzed OTUs were n = 974 for the severely infected (SI), mildly infected (MI), and non-infected (NI) groups, respectively, and n = 802 for the non-infected (NI). The gut microbiome composition of each group was compared using a heat map (Figure 6). The distributions of each group were also compared using nonmetric multidimensional scaling (NMDS) plots (Figure 7). The distributions of each group were also compared using (A) principal component analysis (PCoA) and (B) distance to the group centroid (Figure 8).

[0052] Furthermore, in experimental animals that were administered the infection-resistant human intestinal strain according to Example 1 of the present invention and then infected with the SARS-CoV-2 virus, the differential abundance of the intestinal microbiome between the severely infected (SI) and non-infected (NI) groups was analyzed by log2-fold change for each OTU using DESeq2 analysis (Figure 9). Also, a clustered heat map for each OTU was analyzed using DESeq2 analysis (Figure 10).

[0053] As described above, in Example 1 of the present invention, experimental animals with de novo microbiomes derived from "infection-resistant humans" were infected with the SARS-CoV-2 virus, and then the abundance, diversity, composition, and distribution of 2,070 OTUs in the intestinal microbiome were compared and analyzed between the severely infected (SI), mildly infected (MI), and uninfected (NI) groups (Figures 3 to 10). 12 OTUs specific to the uninfected group were identified (Table 2).

[0054] [Table 2]

[0055] Among the six microorganisms that make up the 12 non-infected group OTUs, strains JBO3-101 and JBR5-501, a novel strain belonging to the Oribacterium species and a novel strain belonging to the Ruminococcus species, respectively, exhibited non-infected group (NI) specificity. Indeed, JBO3-101 (Figure 11) and JBR5-501 (Figure 12) showed significantly increased differential abundance in the non-infected group (NI) compared with the severely infected group (SI), demonstrating that these strains are "infection-resistant strains" derived from "infection-resistant humans."

[0056] As shown in Table 2, the morphology of the "infection-resistant strains" JBO3-101 and JBR5-501 extracted from the 12 specific OTUs of the uninfected group was confirmed by Gram staining (Figure 13). For accurate identification, 16S rRNA gene sequence analysis was performed. Genomic DNA was obtained from each strain, and then amplified by PCR using the general primer set 27F-AGA GTT TGA TCC TGG CTC AG (SEQ ID NO: 1) and 149R-GGT TAC CTT GTT ACG ACTT (SEQ ID NO: 2) to obtain the 16S rRNA sequence (SEQ ID NOs: 3-4). The 16S rRNA sequence of each experimental strain was identified using the results of a Blast search of the NCBI database. As a result, the "infection-resistant strains" JBO3-101 and JBR5-501 were confirmed to be new strains belonging to the Oribacterium species, JBO3-101 (accession number KACC81250BP), and Ruminococcus species, JBR5-501 (accession number KACC81249BP), respectively, and were deposited at the Biological Resources Center of the National Academy of Agricultural Sciences on December 20, 2022.

[0057] Example 2. Preventive effect of infection-resistant enteric bacterial strains against COVID-19 Using the JBO3-101 and JBR5-501 strains, which were discovered to be "infection-resistant strains," animal experiments were conducted to confirm their effectiveness against SARS-CoV-2, the COVID-19 virus that causes COVID-19, the most serious viral infectious disease.

[0058] For this purpose, SARS-CoV-2 strain HB-01 was obtained from the National Culture Collection for Pathogens (NCCP) of the Korea Illness Control and Anticipation Organization (KDCA). SARS-CoV-2 cultures were prepared by culturing Vero E6 cells in DMEM medium supplemented with 10% FBS, 100 IU / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2.

[0059] Experimental animals were Roborovskii hamsters (Phodopus roborovskii) strain SH101 (Alphabio Chemicals Co.), an animal model highly susceptible to pathogenic microbial infection. They were fed diet (D12450B; Research Diets Inc.) and water ad libitum. After a one-week adaptation period in the ABL3 Laboratory at Chonbuk National University, experiments began. First, an antibiotic mixture consisting of azithromycin (15 mg / kg), neomycin (25 mg / kg), ciprofloxacin (20 mg / kg), and miconazole (30 mg / kg) was administered to deplete the intestinal flora. After two days, the hamsters were randomly divided into groups, and each group (n = 8–10) was inoculated with 1 x 10 of the relevant microorganism (Gut Microbe Culture Collection). 9 After one week of ingestion of the microorganisms, SARS-CoV-2 virus was administered orally at a dose of 10 CFU / 100 μL PBS / day. 5 TCID 50 SH101 hamsters were infected intranasally with 50 μl of the virus. Body weights were recorded daily after infection (FIG. 14), and the mortality / survival rate on day 8 after infection was recorded (Table 3, FIG. 15). All test animals were sacrificed and lung tissue morphological observations were performed (FIG. 16), and H&E staining of lung sections was performed (FIG. 17). RT-PCR was performed to quantify the virus in the lungs (FIG. 18).

[0060] [Table 3]

[0061] As shown in Figures 14 to 18, the intestinal microbiome of experimental animals was formed using "infection-resistant strains" derived from "infection-resistant humans," and then quantitative analysis of survival rate, weight change, morphological and pathological observations of the lungs, and virus counts upon infection with the SARS-CoV-2 virus was performed. As a result, it was confirmed that the JBO3-101 and JBR5-501 strains are infection-resistant strains against SARS-CoV-2 virus infection.

[0062] Example 3. Therapeutic effect of infection-resistant intestinal bacterial strains on flu Using the JBO3-101 and JBR5-501 strains, which were found to be "infection-resistant strains," animal experiments were conducted to confirm their therapeutic effects against the flu virus, which causes influenza, the most common viral infectious disease.

[0063] For this purpose, influenza A virus H1N1 (NCCP43021) and bacterial strain cultures for viral propagation were prepared by culturing MDCK cells in MEM medium supplemented with 10% FBS, 100 IU / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2.

[0064] Seven-week-old Roborovski hamsters, SH101 (Alpha Biochemicals Co.), were randomly assigned to groups and injected with 2 × 10 influenza virus. 3 Seven days after infection, each group (n=5) was infected with 1×10 9 The animals were orally administered at a dose of CFU / 100 μL PBS / day. After monitoring for the presence or absence of clinical symptoms and recording survival rates (Table 4), all test animals were sacrificed, and lung tissue samples were taken and subjected to RT-PCR for virus quantification (Figure 19).

[0065] [Table 4]

[0066] Based on the above results, experimental animals infected with influenza virus were fed the infection-resistant strains, and the survival rate and virus were quantitatively analyzed. As a result, it was confirmed that JBO3-101 and JBR5-501 were resistant strains to influenza virus infection, unlike the control group.

[0067] Example 4. Preventive effect of infection-resistant enteric bacterial strains on tuberculosis Using JBR5-501 and JBO3-101 strains, which were found to be "infection-resistant strains," we conducted animal experiments to confirm their effectiveness against Mycobacterium tuberculosis, which causes the most serious bacterial infectious disease. Mycobacterium tuberculosis (NCCP15986) was inoculated onto 7H10 agar medium containing 50g of albumin, 20g of dextrose, and 8.5g of NaCl per liter, and cultured at 37°C for 10 days. Experimental animals were prepared using Robinski hamsters whose intestinal bacteria had been depleted in the same manner as in Example 2, and then the microorganisms JBO3-101 (accession number KACC81250BP) and JBR5-501 (accession number KACC81249BP) were inoculated into groups (n=3) at 1x10 9 The animals were orally administered with 1 × 10 CFU / 100 μL PBS / day for one week. 4 The animals were infected intranasally at a level of CFU / 50 μl, and the presence or absence of clinical symptoms was monitored for 14 days after infection, while the survival rate was recorded (Table 5). All test animals were then sacrificed and the CFU of the infectious bacteria in the lung tissue was measured (Figure 20).

[0068] [Table 5]

[0069] Based on the above results, we fed the "infection-resistant strains" to experimental animals infected with tuberculosis bacteria, and then analyzed the survival rate and the quantity of tuberculosis bacteria. As a result, we confirmed that JBO3-101 and JBR5-501 are strains that are resistant to tuberculosis infection. [Accession number]

[0070] Depository institution: Microbial Bank of the National Academy of Agricultural Sciences (KACC), Rural Development Administration Accession number: KACC81249BP Entrustment date: December 20, 2022 Depository institution: Microbial Bank of the National Academy of Agricultural Sciences (KACC), Rural Development Administration Accession number: KACC81250BP Entrustment date: December 20, 2022

Claims

1. An infection-resistant intestinal strain selected from the group consisting of the Oribacterium sp. JBO3-101 strain deposited under accession number KACC81250BP and the Ruminococcus sp. JBR5-501 strain deposited under accession number KACC81249BP.

2. The infection-resistant intestinal bacterial strain according to claim 1, which has a preventive or therapeutic effect against infectious diseases caused by pathogenic microorganisms.

3. A composition for preventing or treating an infectious disease, comprising as an active ingredient one or more infection-resistant intestinal strains or cultures thereof selected from the group consisting of the Oribacterium sp. JBO3-101 strain deposited under accession number KACC81250BP and the Ruminococcus sp. JBR5-501 strain deposited under accession number KACC81249BP, wherein the infectious disease is a disease caused by infection with a coronavirus, influenza virus, or tuberculosis virus.

Images