Agent for preventing or improving intestinal infectious diseases

Abstract

To provide a material useful for suppressing intestinal infection caused by Salmonella and preventing or improving intestinal infection disease.SOLUTION: Provided is a salmonella infection inhibitor comprising, as an active ingredient, a Neisseria mucosa cell culture.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an agent for preventing or improving intestinal infectious diseases.

Background Art

[0002] Salmonella enterica is a typical causative bacterium of food poisoning that is pathogenic to humans and animals (Non-Patent Document 1). Infections caused by Salmonella usually result in acute gastroenteritis in healthy adults, but can be severe in children and the elderly. Gastroenteritis caused by Salmonella infection usually starts with nausea and vomiting, followed by abdominal pain and diarrhea several hours later. In children, it can lead to disturbances in consciousness, convulsions, and bacteremia, while in the elderly, it is likely to cause acute dehydration and bacteremia, and tends to become severe and recover slowly.

[0003] Generally, in the case of bacterial gastroenteritis, symptomatic treatment is mainly focused on correcting dehydration caused by fever and diarrhea, and relieving gastroenteritis symptoms such as abdominal pain. In principle, antibacterial drugs are not used in mild cases, but in severe cases, ampicillin, fosfomycin, and new quinolone drugs are used. However, administration of antibacterial drugs requires caution because it can disrupt the intestinal flora, delay sterilization, induce resistant bacteria, and increase susceptibility to Salmonella.

[0004] On the other hand, bacteria of the genus Neisseria such as Neisseria mucosa, Neisseria sicca, Neisseria flava, Neisseria subflava, Neisseria flavescens, and Neisseria elongata are known to exist as non-pathogenic resident bacteria in the oral cavity, but their functions are not clear.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention relates to providing a material useful for suppressing intestinal infection caused by Salmonella and for preventing or improving intestinal infectious diseases.

Means for Solving the Problems

[0007] The present inventors have found that the cell culture of Neisseria mucosa, which is known as a non-pathogenic oral commensal bacterium, suppresses cell infection caused by Salmonella and is useful as a preventive or therapeutic agent for intestinal infectious diseases.

[0008] That is, the present invention relates to the following 1) to 4). 1) An inhibitor for suppressing Salmonella infection containing a cell culture of Neisseria mucosa as an active ingredient. 2) A preventive or ameliorating agent for intestinal infectious diseases containing a cell culture of Neisseria mucosa as an active ingredient. 3) A food for suppressing Salmonella infection containing a cell culture of Neisseria mucosa as an active ingredient. 4) A food for preventing or ameliorating intestinal infectious diseases containing a cell culture of Neisseria mucosa as an active ingredient.

Effects of the Invention

[0009] Since Neisseria mucosa of the present invention is a non-pathogenic oral commensal bacterium, it can provide a preventive or ameliorating agent for intestinal infectious diseases that can be ingested by healthy people as well as the elderly and convalescents over a long period of time.

Brief Description of the Drawings

[0010]

Figure 1

Modes for Carrying Out the Invention

[0011] Neisseria mucosa used in the present invention is a Gram-negative coccus belonging to the genus Neisseria of the family Neisseria, and is a non-pathogenic oral commensal bacterium. Neisseria mucosa can be isolated from human saliva, dental plaque, etc. It can also be obtained from JCM (Neisseria mucosa JCM12992 strain (ATCC19696 strain)).

[0012] In the present invention, the "bacterial culture" of Neisseria mucosa includes, in addition to the culture containing medium components, bacterial cells and extracellular polymeric substances (EPS), etc., the culture containing bacterial cells and EPS from which the medium components have been removed from the culture, the culture containing medium components and EPS from which the bacterial cells have been removed from the culture, or the culture containing EPS from which the medium components and bacterial cells have been removed from the culture, etc. The "bacterial cells" contained in the bacterial culture of Neisseria mucosa may be any of viable Neisseria mucosa bacterial cells, wet bacterial cells, or dried bacterial cells. Also, not only viable bacterial cells but also dead bacterial cells may be used. In addition, the bacterial cells also include bacterial cell components such as cytoplasm and cell wall fractions obtained by treating the bacterial cells with enzymes or physical means. The bacterial culture of the present invention can be prepared in any form according to the purpose of use, such as freeze-dried powder, spray-dried powder, suspension in liquid, etc., if desired.

[0013] The medium for culturing Neisseria mucosa of the present invention is not particularly limited as long as it contains peptone, etc. as a nutrient source, and for example, BHI medium (manufactured by BD Japan) etc. can be exemplified. The culturing method is not particularly limited as long as it is a condition under which the cells of Neisseria mucosa grow well. For example, it can be cultured at 20 to 40°C, preferably 35 to 38°C, under aerobic conditions, more preferably under 5% CO2 concentration or microaerobic conditions for 16 to 72 hours.

[0014] In the present invention, when using a cell culture of Neisseria mucosa, the culture of Neisseria mucosa is carried out by adding a culture supernatant of periodontal pathogenic bacteria (for example, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Selenomonas sputigena, etc.), preferably a culture supernatant obtained by culturing Porphyromonas gingivalis under anaerobic conditions and removing the cells by centrifugation or the like from the obtained culture solution. The culturing method after addition is not particularly limited as long as EPS is sufficiently produced. From the viewpoint of EPS production, 20 to 40°C is preferable, more preferably 35 to 38°C, and it is preferably carried out for 24 hours or more under aerobic conditions, more preferably under 5% CO2 concentration or microaerobic conditions. Also, from the viewpoint of preventing the contamination of Porphyromonas gingivalis cells and cell debris in EPS, it is more preferable to add the culture supernatant after sterilization by filter sterilization or the like and carry out the culturing at 35 to 38°C, under 5% CO2 concentration or microaerobic conditions for 24 hours or more. The addition amount of the culture supernatant of the periodontal pathogenic bacterium is preferably 0.05 v / v% or more, more preferably 0.5 v / v% or more, and still more preferably 5 v / v% or more from the viewpoint of the EPS production induction effect. Further, from the viewpoint of the EPS recovery amount, it is preferably 50 v / v% or less, more preferably 30 v / v% or less, and still more preferably 10 v / v% or less.

[0015] As shown in the following examples, the cell culture of Neisseria mucosa has an inhibitory effect on the infection of Salmonella bacteria to HeLa cells. Therefore, the cell culture of Neisseria mucosa can be a Salmonella infection inhibitor and a prophylactic or ameliorating agent for intestinal infectious diseases, and the cell culture of Neisseria mucosa can be used for producing a Salmonella infection inhibitor and a prophylactic or ameliorating agent for intestinal infectious diseases. In addition, the cell culture of Neisseria mucosa can be used for suppressing Salmonella infection and thus for preventing or ameliorating intestinal infectious diseases. Here, such use can be administration to humans or non-human animals, or use in specimens derived therefrom, and can be either therapeutic use or non-therapeutic use. Here, examples of non-human animals include non-human mammals, amphibians, and cartilaginous fish, and examples of non-human mammals include, for example, anthropoid apes, other primates, mice, rats, horses, cows, pigs, sheep, dogs, cats, hamsters, and companion animals.

[0016] In the present invention, "intestinal infectious disease" mainly means an intestinal infectious disease caused by Salmonella bacteria, and the pathological conditions mainly include gastroenteritis accompanied by nausea, vomiting, abdominal pain, diarrhea, and the like. In the present invention, Salmonella bacteria means Salmonella enterica, which is a Gram-negative facultative anaerobic bacillus, and includes drug-resistant Salmonella bacteria. In addition, "suppression of Salmonella infection" means suppressing the infection of Salmonella bacteria to animal cells, and includes inhibition of infection.

[0017] In the present invention, "prevention" means preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of onset of a disease or symptom in an individual. In addition, "improvement" means the alleviation of a disease, symptom or condition, the prevention or delay of the exacerbation of a disease, symptom or condition, or the reversal, prevention or delay of the progression of a disease or symptom, and includes the so-called concept of "treatment".

[0018] The Salmonella infection inhibitor and the prophylactic or improvement agent for intestinal infectious diseases of the present invention can itself be a pharmaceutical, quasi-drug or food for inhibiting Salmonella infection and for preventing or improving intestinal infectious diseases, and can also be a material or preparation to be formulated and used in said pharmaceutical, quasi-drug or food. Note that foods include foods with the concept of preventing or improving intestinal infectious diseases, and which display the same as necessary, functional display foods, foods for specified health use, foods for patients, and supplements.

[0019] When used as a pharmaceutical (including quasi-drugs), the form may be either oral administration or parenteral administration, but oral administration is preferred. The dosage form may be, for example, a liquid preparation; solid preparations such as tablets, granules, fine granules, powders, tablets, etc.; or capsule preparations encapsulating said liquid or solid preparations, oral sprays, troches and other various forms. Dosage forms for parenteral administration include various preparations such as external use, transdermal, transmucosal, patches, nasal, enteral, suppositories, injections, inhalations, etc. Such various dosage forms of pharmaceutical preparations can be prepared by appropriately combining other pharmaceutically acceptable excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, etc. within the range that does not interfere with the action of the cell culture of Neisseria mucosa.

[0020] When used as a food, the forms include beverages such as fruit juice or vegetable juice drinks, carbonated drinks, tea-based drinks, milk drinks, fermented milk, fermented fruit juice, fermented vegetable juice, alcoholic beverages, soft drinks, etc., jelly-like foods, various snacks, baked confectioneries, cakes, chocolates, jams, breads, gums, candies, soups, pickles, simmered foods, etc. In addition, supplements in the same forms as the above-described oral administration preparations (tablets, capsules, syrups, etc.) are also included. The food can be prepared according to a conventional method by appropriately combining the cell culture of Neisseria mucosa with any food material, or other active ingredient, or an additive acceptable in food (e.g., solvent, softening agent, oil, emulsifier, preservative, acidulant, sweetener, bittering agent, pH adjuster, stabilizer, colorant, ultraviolet absorber, antioxidant, humectant, thickening agent, fixing agent, dispersant, fluidity improver, wetting agent, fragrance, seasoning, flavor modifier, etc.).

[0021] The content of the cell culture of Neisseria mucosa in the above-mentioned pharmaceuticals (including quasi-drugs) or foods is not particularly limited, but it may be appropriately adjusted according to the daily dose, etc.

[0022] In the above-mentioned pharmaceuticals (including quasi-drugs) or foods, the dosage of the cell culture of Neisseria mucosa can be appropriately determined according to various conditions such as the patient's body weight, age, gender, symptoms, etc. For example, for an adult, per day, the dry weight is preferably 1 mg or more, more preferably 100 mg or more, still more preferably 500 mg or more, and preferably 5000 mg or less, more preferably 2500 mg or less, still more preferably 1000 mg or less.

[0023] The administration or ingestion targets of the Salmonella infection inhibitor, food for inhibiting Salmonella infection, agent for preventing or improving intestinal infectious diseases, and food for preventing or improving intestinal infectious diseases of the present invention preferably include humans suffering from intestinal infectious diseases, humans who desire to prevent the onset of intestinal infectious diseases, etc.

[0024] Regarding the above-described embodiments, the following aspects are further disclosed in the present invention. <1>An inhibitor of Salmonella infection containing a cell culture of Neisseria mucosa as an active ingredient. <2>A prophylactic or ameliorating agent for intestinal infectious diseases containing a cell culture of Neisseria mucosa as an active ingredient. <3>A food for inhibiting Salmonella infection containing a cell culture of Neisseria mucosa as an active ingredient. <4>A food for preventing or ameliorating intestinal infectious diseases containing a cell culture of Neisseria mucosa as an active ingredient.

[0025] <5>The use of a cell culture of Neisseria mucosa for producing an inhibitor of Salmonella infection. <6>The use of a cell culture of Neisseria mucosa for producing a prophylactic or ameliorating agent for intestinal infectious diseases. <7>The use of a cell culture of Neisseria mucosa for producing a food for inhibiting Salmonella infection. <8>The use of a cell culture of Neisseria mucosa for producing a food for preventing or ameliorating intestinal infectious diseases.

[0026] <9>A cell culture of Neisseria mucosa for use in inhibiting Salmonella infection. <10>A cell culture of Neisseria mucosa for use in preventing or ameliorating intestinal infectious diseases.

[0027] <11>The non-therapeutic use of a cell culture of Neisseria mucosa for inhibiting Salmonella infection. <12>The non-therapeutic use of a cell culture of Neisseria mucosa for preventing or ameliorating intestinal infectious diseases.

[0028] <13>A method for inhibiting Salmonella infection by administering or ingesting a cell culture of Neisseria mucosa in an effective amount to a subject in need thereof. <14>A method for preventing or ameliorating intestinal infectious diseases by administering or ingesting a cell culture of Neisseria mucosa in an effective amount to a subject in need thereof.

[0029] <15>In <2>, <4>, <6>, <8>, <10>, <12> and <14>, the intestinal infection is an intestinal infection caused by Salmonella. <16>In <1>, <3>, <5>, <7>, <9>, <11>, <13> and <15>, the Salmonella bacteria are drug-resistant Salmonella bacteria. <17>In <1> to <14>, the bacterial culture is preferably a culture obtained by adding the culture supernatant of Porphyromonas gingivalis to the culture solution of Neisseria mucosa and culturing, or a culture containing EPS obtained by adding the culture supernatant of Porphyromonas gingivalis to the culture solution of Neisseria mucosa, culturing, and removing the bacterial cells and medium components.

[0030] Example 1 Inhibitory effect of the bacterial culture of Neisseria mucosa on Salmonella infection (1) Cell culture conditions HeLa cells were obtained from the American Type Culture Collection (ATCC). HeLa cells were cultured at 37 °C and 5% CO2 concentration using DMEM / F12 (Gibco, containing 10 v / v% FBS, 1 v / v% Penicillin - Streptomycin).

[0031] (2) Bacterial culture conditions The Salmonella enterica and Neisseria mucosa used were obtained from the National Institute of Technology and Evaluation, or the RIKEN BioResource Center, National Institutes of Biomedical Innovation, Health and Nutrition (Table 1). For both bacteria, colony culture was performed using Brain Heart Infusion (BHI) Agar medium (Nippon Becton Dickinson (hereinafter, Nippon BD)), and liquid culture was performed using BHI medium (Nippon BD). Salmonella enterica was aerobically cultured at 37 °C, and Neisseria mucosa was cultured at 37 °C and 5% CO2 concentration. After 24 - hour liquid culture of Salmonella enterica, the bacteria cultured for an additional 24 hours after 1000 - fold dilution were used. After 24 - hour liquid culture of Neisseria mucosa, the bacteria cultured for an additional 48 hours after 100 - fold dilution were used.

[0032] [Table 1]

[0033] (3) Preparation of the culture of Neisseria mucosa The culture solution of Neisseria mucosa was prepared by culturing Neisseria mucosa in 300 mL of BHI medium at 37 °C and 5% CO2 concentration for 48 hours. The culture supernatant of Porphyromonas gingivalis prepared to a final concentration of 5 v / v% was added to the Neisseria mucosa culture solution, and after culturing at 37 °C and 5% CO2 concentration for 48 hours, 20 mL of the culture solution was collected in a 50 mL tube. The culture supernatant of Porphyromonas gingivalis was prepared by suspending the colonies grown by culturing Porphyromonas gingivalis (ATCC 33277 strain) obtained from ATCC on an anaerobic Columbia agar medium (BD Japan) at 37 °C under anaerobic conditions for 24 hours or more in GAM broth medium (Nissui Pharmaceutical, containing 5.0 μg / mL Hemin, 17.4 μg / mL K2HPO4, 1.0 μg / mL Vitamin K), culturing in the same manner at 37 °C under anaerobic conditions for 24 hours, adding the obtained liquid culture solution to fresh GAM broth medium at a final concentration of 1 v / v%, centrifuging the culture solution obtained after culturing at 37 °C for 24 hours under anaerobic conditions at 13,000 g, 4 °C for 15 minutes to remove the cells, and filtering and sterilizing the supernatant fraction with a 0.22 μm filter. To remove the medium components from the culture solution of Neisseria mucosa cultured with the addition of the culture supernatant of Porphyromonas gingivalis, after centrifugation at 16,000 g, 4 °C for 30 minutes, the supernatant was discarded, 20 mL of ultrapure water was added, and it was vigorously suspended and washed with a vortex. This washing was repeated 6 times. After washing, centrifugation was performed in the same manner, and the cell culture containing the precipitated cells and EPS was dispersed in ultrapure water and subjected to ultrasonic treatment. The ultrasonic treatment was performed using BRANSON SONIFIER 150 under the conditions of power 4, disruption for 20 seconds → cooling for 20 seconds for 5 sets. After ultrasonic treatment, the cells were precipitated at 16,000 g, 4 °C for 30 minutes, and the supernatant was collected in a new 50 mL tube. To completely remove the cells, the collected supernatant was centrifuged again at 16,000 g for 30 minutes at 4°C, and the supernatant was collected. The collected supernatant was lyophilized to remove the cells and culture medium components, and a cell culture of N. mucosa containing EPS was obtained.

[0034] (4) Reagents The labeling reagent Carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Dojindo Laboratories, dissolved in DMSO, and used at a final concentration of 10 μM.

[0035] (5) Method for fluorescent labeling of cells The Salmonella enterica culture solution was centrifuged at 4,500 g for 10 minutes at 22°C, and the precipitated cells were collected. The collected cells were washed by centrifugation at 4,500 g for 5 minutes at 22°C using PBS(-) and then DMEM / F12 (Gibco), adjusted to OD600 = 2.0, and incubated in a 10 μM CFSE solution (dissolving in DMEM / F12) at 37°C in the dark for 30 minutes for fluorescent labeling. The fluorescently labeled cells were washed twice by centrifugation at 4,500 g for 5 minutes at 22°C using DMEM / F12 (Gibco), and a CFSE-labeled cell solution was prepared by adjusting the turbidity of the cell suspension.

[0036] (6) Infection method After removing the culture medium from the confluent HeLa cells in a 12-well plate, the cells were washed with PBS(-). The N. mucosa cell culture prepared in (3) was dissolved in DMEM / F12 (Gibco) to a final concentration of 0.00625 w / v%, 0.025 w / v%, or 0.1 w / v% at the time of evaluation to obtain an evaluation medium. The CFSE-labeled cells were suspended in DMEM / F12 (Gibco) so as to infect the cells at a Multiplicity of Infection (MOI) = 500, and co-cultured with the medium containing the N. mucosa cell culture at each concentration for 2 hours to infect the cells. The inhibitory effect of the N. mucosa cell culture on Salmonella enterica is shown in Fig. 1.

[0037] (7) Analytical method for infected cells The medium was removed from HeLa cells infected with CFSE-labeled Salmonella enterica, washed twice with PBS(-), and then the infected cells were detached using 400 μL of 0.25% Trypsin-EDTA (Gibco). 750 μL of DMEM / F12 (Gibco, containing 10 v / v% FBS) was added, the total volume of the solution was collected in a 1.5 mL tube, and the infected cells were collected by centrifugation at 4°C, 400 g for 5 minutes. The supernatant was discarded, 400 μL of 4% paraformaldehyde phosphate buffer (Wako) was added and pipetted, and fixation was carried out at 4°C for 10 minutes. 800 μL of PBS (containing 2 v / v% FBS) was added and pipetted, followed by centrifugation at 4°C, 600 g for 5 minutes, and resuspended in 200 μL of PBS (containing 2 v / v% FBS) to obtain an analytical sample. For the analysis, Flow cytometry (BD, FACSVerse) was used. The mean fluorescence intensity (MFI) was calculated from the fluorescence intensity obtained in the FITC channel of 20,000 gated cells based on forward scatter (FSC) and side scatter (SSC), and the cell infectivity was evaluated by detecting the cells adhered to and infected with fluorescently labeled Salmonella enterica. The results are shown in Figure 1.

[0038] (8) Statistical analysis The obtained results were shown as dots for the MFI of each sample, and the mean ± standard deviation (Mean ± standard deviation (SD)) of each group was represented by a bar graph. The test for the difference in means was performed using the statistical analysis software SPSS (IBM) with Dunnett's test. When the P value was less than 0.05 (*p < 0.05), less than 0.01 (**p < 0.01), or less than 0.001 (***p < 0.001), it was considered a statistically significant difference.

[0039] (9) Results In the evaluation of cell infectivity, a significant decrease in Mean fluorescence intensity (cell infectivity) was observed in the group in which the cell culture of *Neisseria mucosa* was lysed (final concentrations of 0.00625 w / v% (0.00625%), 0.025 w / v% (0.025%), and 0.1 w / v% (0.1%)) compared to the control (0) in which the cell culture of *Neisseria mucosa* was not lysed (Figure 1).

Claims

1. A Salmonella infection inhibitor containing a cell culture of Neisseria mucosa as an active ingredient, wherein the cell culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa, culturing the mixture, and removing the cells and medium components.

2. A prophylactic or ameliorating agent for intestinal infectious diseases caused by Salmonella bacteria, containing a cell culture of Neisseria mucosa as an active ingredient, wherein the cell culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa, culturing the mixture, and removing the cells and medium components.

3. A food for inhibiting Salmonella infection, containing a cell culture of Neisseria mucosa as an active ingredient, wherein the cell culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa, culturing the mixture, and removing the cells and medium components.

4. A food for preventing or ameliorating intestinal infectious diseases caused by Salmonella bacteria, containing a cell culture of Neisseria mucosa as an active ingredient, wherein the cell culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa, culturing the mixture, and removing the cells and medium components.

5. The agent according to claim 1 or 2, or the food according to claim 3 or 4, wherein the Salmonella bacteria are drug-resistant Salmonella bacteria.

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