JP7698983B2 - Agents for preventing or improving intestinal infections - Google Patents

Description

The present invention relates to an agent for preventing or improving intestinal infections.

Salmonella enterica is a typical food poisoning bacterium that is pathogenic to humans and animals (Non-Patent Document 1). In healthy adults, Salmonella infection causes only acute gastroenteritis, but it can become severe in children and the elderly. Gastroenteritis caused by Salmonella infection begins with nausea and vomiting, followed several hours later by abdominal pain and diarrhea. Children can develop impaired consciousness, convulsions, and bacteremia, while the elderly can develop acute dehydration and bacteremia, and recovery tends to be delayed.

In general, the principle of bacterial gastroenteritis is to provide symptomatic treatment, focusing on correcting dehydration caused by fever and diarrhea and relieving gastrointestinal symptoms such as abdominal pain. Antibiotics are generally not used in mild cases, but ampicillin, fosfomycin, and new quinolones are used in severe cases.
However, antibiotics must be administered with caution because they can disrupt the intestinal flora, delay elimination, induce resistant bacteria, and increase susceptibility to Salmonella infection.

On the other hand, Neisseria bacteria such as Neisseria mucosa, Neisseria sicca, Neisseria flava, Neisseria subflava, Neisseria flavescens, and Neisseria elongata are known to exist as non-pathogenic normal bacteria in the oral cavity, but their functions remain unclear.

Shu-Kee Eng, Priyia Pusparajah, Nurul-Syakima Ab Mutalib, Hooi-Leng Ser, Kok-Gan Chan & Learn-Han Lee (2015) Salmonella: A review on pathogenesis, epidemiology and antibiotic resistance, Frontiers in Life Science, 8:3, 284-293

The present invention relates to providing a material that suppresses intestinal infection caused by Salmonella and is useful for preventing or improving intestinal infectious diseases.

The present inventors have found that a bacterial culture of Neisseria mucosa, which is known as a non-pathogenic oral indigenous bacterium, suppresses cell infection by Salmonella and is useful as a preventive or therapeutic agent for intestinal infections.

That is, the present invention relates to the following 1) to 4).
1) A Salmonella infection inhibitor containing a culture of Neisseria mucosa as an active ingredient.
2) A preventive or ameliorative agent for intestinal infections, which contains as an active ingredient a culture of Neisseria mucosa bacteria.
3) A food for suppressing Salmonella infection that contains a culture of Neisseria mucosa as an active ingredient.
4) A food for preventing or improving intestinal infections, containing a culture of Neisseria mucosa as an active ingredient.

The Neisseria mucosa of the present invention is a non-pathogenic oral indigenous bacterium, and therefore can provide an agent for preventing or improving intestinal infections that can be taken over a long period of time not only by healthy individuals but also by the elderly and those recovering from illness.

Inhibitory effect of Neisseria mucosa cell culture on infection with Salmonella enterica.

Neisseria mucosa used in the present invention is a gram-negative cocci belonging to the genus Neisseria in the family Neisseria, and is a non-pathogenic normal inhabitant of the oral cavity.
Neisseria mucosa can be isolated from human saliva, dental plaque, etc. Also, it can be obtained from JCM (Neisseria mucosa JCM12992 strain (ATCC19696 strain)).

In the present invention, examples of the "bacterial cell culture" of Neisseria mucosa include a culture containing medium components, bacterial cells, and extracellular polymeric substances (EPS), as well as a culture containing bacterial cells and EPS from which medium components have been removed, a culture containing medium components and EPS from which bacterial cells have been removed, or a culture containing EPS from which medium components and bacterial cells have been removed.
The "cells" contained in the cell culture of Neisseria mucosa may be live cells, wet cells, or dry cells of Neisseria mucosa. They may be not only live cells but also dead cells. The cells also include cell components such as cytoplasm and cell wall fractions obtained by treating the cells with enzymes or physical means.
The bacterial culture of the present invention can be prepared in any form depending on the purpose of use, such as freeze-dried powder, spray-dried powder, or suspension in liquid, as desired.

The medium for culturing the Neisseria mucosa of the present invention is not particularly limited as long as it contains peptone or the like as a nutrient source, and an example of the medium is BHI medium (manufactured by BD Japan).
The culture method is not particularly limited as long as the conditions are such that the Neisseria mucosa cells grow well. For example, the culture may be performed at 20 to 40° C., preferably 35 to 38° C., under aerobic conditions, more preferably under a _CO2 concentration of 5% or under microaerobic conditions, for 16 to 72 hours.

In the present invention, when a bacterial culture of Neisseria mucosa is used, the culture of Neisseria mucosa may be performed using a bacterial cell culture of a periodontal pathogen (e.g., Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, or the like). The culture supernatant of Porphyromonas gingivalis, P. actinomycetemcomitans, Campylobacter rectus, Selenomonas sputigena, etc., preferably Porphyromonas gingivalis, was added to the culture medium, which was obtained by culturing the culture medium under anaerobism and removing the bacterial cells from the culture medium by centrifugation or the like. The culture method after the addition is not particularly limited as long as the conditions are such that a sufficient amount of EPS is produced, but from the viewpoint of EPS production, the culture temperature is preferably 20 to 40°C, more preferably 35 to 38°C, and is preferably performed under aerobic conditions, more preferably under a _CO2 concentration of 5% or under microaerobic conditions, for 24 hours or more.
From the viewpoint of preventing the Porphyromonas gingivalis cells and cell fragments from being mixed into the EPS, it is more preferable to add the culture supernatant after sterilization by filter sterilization or the like, and to perform the incubation at 35 to 38° C., under a _CO2 concentration of 5% or under microaerobic conditions for 24 hours or more.
The amount of culture supernatant of periodontal pathogens added is preferably 0.05 v/v% or more, more preferably 0.5 v/v% or more, and even more preferably 5 v/v% or more, from the viewpoint of the EPS production inducing effect, and is preferably 50 v/v% or less, more preferably 30 v/v% or less, and even more preferably 10 v/v% or less, from the viewpoint of the EPS recovery amount.

As shown in the Examples below, a culture of Neisseria mucosa cells has an inhibitory effect on infection of HeLa cells by Salmonella.
Therefore, a culture of Neisseria mucosa cells can be used as an agent for suppressing Salmonella infection and an agent for preventing or ameliorating intestinal infections, and a culture of Neisseria mucosa cells can be used to produce an agent for suppressing Salmonella infection and an agent for preventing or ameliorating intestinal infections.
Furthermore, a culture of Neisseria mucosa cells can be used to inhibit Salmonella infection and thus prevent or ameliorate intestinal infections. The use can be administration to humans or non-human animals or in specimens derived therefrom, and can be therapeutic or non-therapeutic. Non-human animals include non-human mammals, amphibians, and cartilaginous fish, and non-human mammals include, for example, apes, other primates, mice, rats, horses, cows, pigs, sheep, dogs, cats, hamsters, and companion animals.

In the present invention, the term "intestinal infection" refers to an intestinal infection mainly caused by Salmonella, and the pathology thereof mainly includes gastroenteritis accompanied by nausea, vomiting, abdominal pain, diarrhea, and the like.
In the present invention, Salmonella means Salmonella enterica, which is a gram-negative facultative anaerobic bacillus, and includes drug-resistant Salmonella.
Furthermore, "suppression of Salmonella infection" means suppression of infection of animal cells by Salmonella, and includes inhibition of infection.

In the present invention, "prevention" refers to preventing or delaying the onset of a disease or condition in an individual, or reducing the risk of an individual developing a disease or condition.
Furthermore, "improvement" means improvement of a disease, symptom or condition, prevention or delay of the worsening of a disease, symptom or condition, or reversal, prevention or delay of the progression of a disease or symptom, and includes the concept of so-called "treatment."

The Salmonella infection inhibitor and the agent for preventing or ameliorating intestinal infection of the present invention can be used by itself as a medicine, quasi-drug, or food for suppressing Salmonella infection or preventing or ameliorating intestinal infection, or can be used as a material or preparation to be incorporated into such medicines, quasi-drugs, or foods.
In addition, foods include foods that are based on the concept of preventing or improving intestinal infections and, where necessary, are labeled as such, functional foods, foods for specified health uses, foods for sick people, and supplements.

When used as a pharmaceutical (including quasi-drug), it may be administered orally or parenterally, but oral administration is preferred, and its dosage form may be, for example, a liquid; a solid preparation such as a tablet, granule, fine granule, powder, tablet, or the like; or a capsule, oral spray, troche, or other form in which the liquid or solid preparation is enclosed. Dosage forms for parenteral administration include preparations for external use, transdermal, transmucosal, patch, nasal, enteral, suppository, injection, inhalation, and the like. These various dosage forms of pharmaceutical preparations may be prepared by appropriately combining other pharma- ceutical acceptable excipients, binders, bulking agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavorings, flavorings, coating agents, carriers, diluents, and the like, within the range that does not interfere with the action of the bacterial culture of Neisseria mucosa.

When used as a food, the form may be fruit or vegetable juice beverages, carbonated beverages, tea beverages, dairy beverages, fermented milk, fermented fruit juice, fermented vegetable juice, alcoholic beverages, soft drinks, and other beverages; jelly-like foods, various snacks, baked goods, cakes, chocolates, jams, breads, gums, candies, soups, pickles, and foods boiled in soy sauce; and supplements in the same form as the oral preparations described above (tablets, capsules, syrups, etc.). The food may be prepared according to a standard method by appropriately combining a culture of Neisseria mucosa with any food material, other active ingredients, or additives acceptable for food (e.g., solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bittering agents, pH adjusters, stabilizers, colorants, UV absorbers, antioxidants, moisturizers, thickeners, adhesives, dispersants, flow improvers, wetting agents, aromatics, seasonings, flavor adjusters, etc.).

The amount of Neisseria mucosa bacterial culture contained in the above-mentioned medicines (including quasi-drugs) or foods is not particularly limited, but may be adjusted appropriately according to the daily dosage, etc.

In the above-mentioned medicines (including quasi-drugs) or foods, the dosage of the Neisseria mucosa bacterial culture can be appropriately determined depending on various conditions such as the patient's weight, age, sex, and symptoms, but for example, the dosage is preferably 1 mg or more, more preferably 100 mg or more, even more preferably 500 mg or more, and preferably 5000 mg or less, more preferably 2500 mg or less, and even more preferably 1000 mg or less, in dry weight, per adult per day.

Those to whom the Salmonella infection inhibitor, food for inhibiting Salmonella infection, agent for preventing or ameliorating intestinal infection, and food for preventing or ameliorating intestinal infection of the present invention are administered or ingested are preferably humans who have developed an intestinal infection, humans who wish to prevent the onset of an intestinal infection, etc.

In relation to the above-described embodiment, the present invention further discloses the following aspects.
<1> A Salmonella infection inhibitor containing a culture of Neisseria mucosa as an active ingredient.
<2> An agent for preventing or improving intestinal infections, comprising a culture of Neisseria mucosa bacteria as an active ingredient.
<3> A food for suppressing Salmonella infection that contains a culture of Neisseria mucosa bacteria as an active ingredient.
<4> A food for preventing or improving intestinal infections, which contains a culture of Neisseria mucosa bacteria as an active ingredient.

<5> Use of a culture of Neisseria mucosa cells for producing an agent for suppressing Salmonella infection.
<6> Use of a culture of Neisseria mucosa cells for producing an agent for preventing or ameliorating intestinal infections.
<7> Use of a culture of Neisseria mucosa cells for producing a food for suppressing Salmonella infection.
<8> Use of a culture of Neisseria mucosa cells for producing a food for preventing or improving intestinal infections.

<9> A culture of Neisseria mucosa cells for use in inhibiting Salmonella infection.
<10> A culture of Neisseria mucosa cells for use in the prevention or amelioration of intestinal infections.

<11> Non-therapeutic use of a culture of Neisseria mucosa cells to inhibit Salmonella infection.
<12> Non-therapeutic use of a culture of Neisseria mucosa cells to prevent or ameliorate intestinal infections.

<13> A method for inhibiting Salmonella infection, comprising administering or ingesting an effective amount of a Neisseria mucosa cell culture to a subject in need thereof.
<14> A method for preventing or ameliorating an intestinal infection, comprising administering or ingesting an effective amount of a culture of Neisseria mucosa cells to a subject in need thereof.

<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 bacterium is a drug-resistant Salmonella bacterium.
<17> In any of <1> to <14>, the bacterial cell culture is preferably a culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa and culturing the mixture, or an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa and culturing the mixture, followed by removing the bacterial cells and medium components.

Example 1 Inhibitory effect of a culture of Neisseria mucosa cells 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 using DMEM/F12 (Gibco, containing 10 v/v % FBS, 1 v/v % Penicillin-Streptomycin).

(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 Research Center (Table 1). For both bacteria, colony culture was performed using Brain Heart Infusion (BHI) Agar medium (Becton Dickinson Japan (hereinafter referred to as BD Japan)), and liquid culture was performed using BHI medium (BD Japan). Salmonella enterica was cultured aerobically at a temperature of 37°C, and Neisseria mucosa was cultured at a temperature of 37°C and at a _CO2 concentration of 5%.
For Salmonella enterica, the bacteria were cultured in liquid for 24 hours, then diluted 1,000 times and cultured in liquid for another 24 hours. For Neisseria mucosa, the bacteria were cultured in liquid for 24 hours, then diluted 100 times and cultured in liquid for another 48 hours.

(3) Preparation of Neisseria mucosa Bacterial Culture Neisseria mucosa was cultured in 300 mL of BHI medium at a temperature of 37° C. and a CO ₂ concentration of 5% for 48 hours to prepare a culture solution.
Porphyromonas gingivalis culture supernatant adjusted to a final concentration of 5 v/v% was added to the Neisseria mucosa culture solution, and the mixture was cultured at a temperature of 37°C and a _CO2 concentration of 5% for 48 hours, after which 20 mL of the culture solution was recovered in a 50 mL tube.
The P. gingivalis culture supernatant was prepared by suspending colonies grown by culturing P. gingivalis (ATCC 33277 strain) obtained from ATCC on an anerobic agar medium (Japan BD) at 37° C. for 24 hours or more under anaerobic conditions, suspending the colonies in GAM bouillon medium (Nissui Pharmaceutical, containing 5.0 μg/mL Hemin, 17.4 μg/ _{mL K2HPO4} , 1.0 μg/mL Vitamin K), and similarly culturing the liquid culture solution at 37° C. for 24 hours under anaerobic conditions, adding the liquid culture solution to a final concentration of 1 v/v% to fresh GAM bouillon medium, and centrifuging the culture solution obtained after culturing at 37° C. for 24 hours under anaerobic conditions at 13,000 g and 4° C. for 15 minutes to remove the bacterial cells, and sterilizing the supernatant fraction by filtration using a 0.22 μm filter.
In order to remove medium components from the culture solution of Neisseria mucosa cultured with the addition of Porphyromonas gingivalis culture supernatant, the culture solution was centrifuged at 16,000 g, 4 ° C, for 30 minutes, the supernatant was discarded, 20 mL of ultrapure water was added, and the mixture was vigorously suspended with a vortex and washed. This washing was repeated six times. After washing, the cultured bacteria including the precipitated bacteria and EPS were dispersed in ultrapure water and subjected to ultrasonic treatment. The ultrasonic treatment was performed using BRANSON SONIFIER 150, and the bacteria were precipitated at 16,000 g, 4 ° C, for 30 minutes under the conditions of power 4, 20 seconds of crushing → 20 seconds of cooling, 5 sets. The supernatant was collected into a new 50 mL tube.
In order to completely remove the bacterial cells, the collected supernatant was centrifuged again at 16,000 g at 4° C. for 30 minutes, and the supernatant was collected.
The collected supernatant was freeze-dried to remove the bacteria and medium components, thereby obtaining a Neisseria mucosa bacteria culture containing EPS.

(4) Reagents Carboxyfluorescein diacetate succinimidyl ester (CFSE), a labeling reagent, was purchased from Dojindo Chemical Industries, Ltd. and dissolved in DMSO to give a final concentration of 10 μM.

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

(6) Infection method After removing the medium from HeLa cells that had become confluent in a 12-well plate, the cells were washed with PBS(-). The Neisseria 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 prepare an evaluation medium. The CFSE-labeled cells were suspended in DMEM/F12 (Gibco) so that the cells were infected with a multiplicity of infection (MOI) of 500, and the cells were infected by co-cultivation for 2 hours with a medium containing the Neisseria mucosa cell culture at each concentration.
The infection-inhibiting effect of the Neisseria mucosa cell culture on Salmonella enterica is shown in FIG.

(7) Analysis of infected cells The medium was removed from HeLa cells infected with CFSE-labeled Salmonella enterica, and the cells were washed twice with PBS(-), and then infected cells were detached using 400 μL 0.25% Trypsin-EDTA (Gibco). 750 μL DMEM/F12 (Gibco, containing 10 v/v% FBS) was added, and the entire solution was collected in a 1.5 mL tube, followed by centrifugation at 4° C. and 400 g for 5 minutes to collect the infected cells. The supernatant was discarded, and 400 μL of 4% paraformaldehyde-phosphate buffer (Wako) was added and pipetted, followed by fixation treatment at 4° C. for 10 minutes. After adding 800 μL of PBS (containing 2 v/v % FBS) and pipetting, the mixture was centrifuged at 600 g at 4° C. for 5 minutes and suspended in 200 μL of PBS (containing 2 v/v % FBS) to prepare an analysis sample.
Flow cytometry (BD, FACSVerse) was used for the analysis. Mean fluorescence intensity (MFI) was calculated from the fluorescence intensity obtained in the FITC channel of 20,000 cells gated based on forward scatter (FSC) and side scatter (SSC), and cell infectivity was evaluated by detecting cells attached and infected with fluorescently labeled Salmonella enterica. The results are shown in Figure 1.

(8) Statistical Analysis The results obtained are shown as dots representing the MFI of each sample, and as a bar graph representing the mean ± standard deviation (SD) of each group. The difference in the mean values was examined using the statistical analysis software SPSS (IBM) by Dunnett's test, and a P value of less than 0.05 (*p<0.05), less than 0.01 (**p<0.01), or less than 0.001 (***p<0.001) was considered to be a statistically significant difference.

(9) Results In the evaluation of cell infectivity, a significant decrease in mean fluorescence intensity (cell infectivity) was observed in the groups in which the Neisseria mucosa cell culture was lysed (final concentrations: 0.00625 w/v% (0.00625%), 0.025 w/v% (0.025%), and 0.1 w/v% (0.1%)) compared with the control (0) in which the Neisseria mucosa cell culture was not lysed ( FIG. 1 ).

Claims (5)

An agent for suppressing Salmonella infection , which contains a bacterial culture of Neisseria mucosa as an active ingredient, wherein the bacterial culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture medium of Neisseria mucosa and culturing the culture medium, and then removing the bacterial cells and medium components . An agent for preventing or improving intestinal infections caused by Salmonella, which contains a bacterial cell culture of Neisseria mucosa as an active ingredient, wherein the bacterial cell culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture medium of Neisseria mucosa and culturing the culture medium, and then removing the bacterial cells and medium components . A food for suppressing Salmonella infection , which contains a bacterial culture of Neisseria mucosa as an active ingredient, wherein the bacterial culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa and culturing the culture solution, and then removing the bacterial cells and medium components . A food for preventing or ameliorating intestinal infections caused by Salmonella , which contains a bacterial culture of Neisseria mucosa as an active ingredient, wherein the bacterial culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa and culturing the culture, and then removing the bacterial cells and medium components . The agent according to claim 1 or 2 , or the food according to claim 3 or 4 , wherein the Salmonella bacterium is a drug-resistant Salmonella bacterium.