Agent for preventing or improving Listeria infection

Abstract

To provide a material useful for suppressing infection by Listeria monocytogenes and preventing or improving listeria infection disease.SOLUTION: Provided is a Listeria monocytogenes 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 listeriosis.

Background Art

[0002] Listeriosis is an infectious disease caused by Listeria monocytogenes and is a type of food poisoning. Although the number of cases in Japan is small, it is regarded as an important disease in public health due to the severity of symptoms and high fatality rate. In humans, it is orally infected by ingesting infected animals or contaminated food, and it is characteristic that typical acute gastroenteritis symptoms of bacterial food poisoning are not usually shown. Listeria monocytogenes can break through the epithelial barrier of intestinal epithelial cells (Non-Patent Document 1), enter the blood from the intestine, and spread throughout the body. The disease types in humans are mostly meningitis, followed by sepsis, fetal septic granulomatosis, and meningoencephalitis, and infections in the uterus and other parts are also seen in pregnant women.

[0003] In the early stage of listeria infection in humans, penicillin antibiotics, especially ampicillin, are considered effective. Tetracycline antibiotics, gentamicin, etc. are also effective, but cephem antibiotics have no effect.

[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 infection by Listeria monocytogenes and for preventing or improving listeriosis.

Means for Solving the Problems

[0007] The present inventors have found that a cell culture of Neisseria mucosa, known as a non-pathogenic oral commensal bacterium, suppresses cell infection by Listeria monocytogenes and is useful as a prophylactic or therapeutic agent for listeriosis.

[0008] That is, the present invention relates to the following 1) to 4). 1) An inhibitor for suppressing Listeria monocytogenes infection, comprising a cell culture of Neisseria mucosa as an active ingredient. 2) A prophylactic or ameliorating agent for listeriosis, comprising a cell culture of Neisseria mucosa as an active ingredient. 3) A food for suppressing Listeria monocytogenes infection, comprising a cell culture of Neisseria mucosa as an active ingredient. 4) A food for preventing or ameliorating listeriosis, comprising 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 an agent for preventing or improving Listeria infection that can be ingested over a long period of time not only by healthy individuals but also by the elderly and convalescents.

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 "cell culture" of Neisseria mucosa includes, in addition to a culture containing medium components, cells, and extracellular polymeric substances (EPS), etc., a culture containing cells and EPS from which the medium components have been removed from the culture, a culture containing the medium components and EPS from which the cells have been removed from the culture, or a culture containing EPS from which the medium components and cells have been removed from the culture, etc. The "cells" contained in the cell culture of Neisseria mucosa may be any of live cells, wet cells, or dry cells of Neisseria mucosa. Also, not only live cells but also dead cells may be used. In addition, the cells also include cell components such as cytoplasm and cell wall fractions obtained by treating the cells with enzymes or physical means. The cell 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 a liquid, etc.

[0013] The medium for culturing Neisseria mucosa of the present invention is not particularly limited as long as it contains peptone or the like as a nutrient source, and for example, BHI medium (manufactured by BD Japan) can be exemplified. The culturing method is not particularly limited as long as 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 in the 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 Porphyromonas gingivalis is cultured under anaerobic conditions, and the culture supernatant obtained by removing the cells by centrifugation or the like from the obtained culture solution is added. 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 periodontal pathogenic bacteria 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. Also, 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 Listeria monocytogenes to HeLa cells. Therefore, the cell culture of Neisseria mucosa can be a Listeria monocytogenes infection inhibitor, a prophylactic or ameliorating agent for listeriosis, and the cell culture of Neisseria mucosa can be used for producing a Listeria monocytogenes infection inhibitor, a prophylactic or ameliorating agent for listeriosis. In addition, the cell culture of Neisseria mucosa can be used for preventing or ameliorating listeriosis in order to inhibit the infection of Listeria monocytogenes. 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, non-human animals include non-human mammals, amphibians, and cartilaginous fish. Examples of non-human mammals include apes, other primates, mice, rats, horses, cows, pigs, sheep, dogs, cats, hamsters, and companion animals.

[0016] In the present invention, "listeriosis" mainly means an infectious disease caused by Listeria monocytogenes. Its pathological conditions vary depending on the infection site. In the initial stage of the infection, symptoms similar to those of a cold syndrome rather than digestive system symptoms often occur, such as malaise, fever, headache, chills, vomiting and other symptoms. When it becomes severe, meningitis, meningoencephalitis, and septicemia occur. In the present invention, Listeria monocytogenes means Listeria monocytogenes which is a Gram-positive bacillus, and includes drug-resistant Listeria monocytogenes. In addition, "inhibiting Listeria monocytogenes infection" means inhibiting the infection of Listeria monocytogenes into animal cells, and includes inhibiting 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 improvement of a disease, symptom or condition, prevention or delay of deterioration of a disease, symptom or condition, or reversal, prevention or delay of progression of a disease or symptom, and includes the concept of so-called "treatment".

[0018] The Listeria monocytogenes infection inhibitor, the preventive or improving agent for Listeria infection of the present invention can be a pharmaceutical, quasi-drug, or food for inhibiting Listeria monocytogenes infection, preventing or improving Listeria infection by itself, and can also be a material or preparation to be formulated and used in the pharmaceutical, quasi-drug, or food. The food includes foods with the concept of preventing or improving Listeria infection, functional display foods, foods for specified health use, foods for patients, and supplements, which are labeled as such if necessary.

[0019] When used as a pharmaceutical (including quasi-drugs), the form can be either oral administration or parenteral administration, but oral administration is preferred. The dosage form can be, for example, a liquid; a solid such as tablets, granules, fine granules, powders, tablets, etc.; or a capsule encapsulating the liquid or solid, an oral spray, a troche, etc. The dosage forms for parenteral administration include various preparations such as topical, transdermal, transmucosal, patch, nasal, enteral, suppository, injection, inhalation, 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 a range that does not interfere with the action of the cell culture of Neisseria mucosa.

[0020] As forms for use as food, there are 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, there are supplements in the same forms (tablets, capsules, syrups, etc.) as the above-described oral administration preparations. 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 (for example, solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bittering agents, pH adjusters, stabilizers, colorants, ultraviolet absorbers, antioxidants, humectants, thickeners, fixing agents, dispersants, fluidity improvers, wetting agents, fragrances, seasonings, flavor modifiers, 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, in the case of freeze-drying, 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 *Listeria monocytogenes* infection inhibitor, the food for inhibiting *Listeria monocytogenes* infection, the prophylactic or ameliorating agent for listeriosis, and the food for prophylactic or ameliorating listeriosis of the present invention preferably include humans suffering from listeriosis, humans desiring prevention of the onset of listeriosis, etc.

[0024] Regarding the above-described embodiments, the following aspects are further disclosed in the present invention. <1>An inhibitor for suppressing Listeria monocytogenes infection, which contains a bacterial cell culture of Neisseria mucosa as an active ingredient. <2>An agent for preventing or improving Listeria infection, which contains a bacterial cell culture of Neisseria mucosa as an active ingredient. <3>A food for suppressing Listeria monocytogenes infection, which contains a bacterial cell culture of Neisseria mucosa as an active ingredient. <4>A food for preventing or improving Listeria infection, which contains a bacterial cell culture of Neisseria mucosa as an active ingredient.

[0025] <5>The use of a bacterial cell culture of Neisseria mucosa for manufacturing an inhibitor for suppressing Listeria monocytogenes infection. <6>The use of a bacterial cell culture of Neisseria mucosa for manufacturing an agent for preventing or improving Listeria infection. <7>The use of a bacterial cell culture of Neisseria mucosa for manufacturing a food for suppressing Listeria monocytogenes infection. <8>The use of a bacterial cell culture of Neisseria mucosa for manufacturing a food for preventing or improving Listeria infection.

[0026] <9>A bacterial cell culture of Neisseria mucosa for use in suppressing Listeria monocytogenes infection. <10>A bacterial cell culture of Neisseria mucosa for use in preventing or improving Listeria infection.

[0027] <11>The non-therapeutic use of a bacterial cell culture of Neisseria mucosa for suppressing Listeria monocytogenes infection. <12>The non-therapeutic use of a bacterial cell culture of Neisseria mucosa for preventing or improving Listeria infection.

[0028] <13>A method for suppressing Listeria monocytogenes infection, which comprises administering or ingesting a bacterial cell culture of Neisseria mucosa to a subject in need thereof in an effective amount. <14>A method for preventing or improving Listeria infection, which comprises administering or ingesting a bacterial cell culture of Neisseria mucosa to a subject in need thereof in an effective amount.

[0029] <15>In <2>, <4>, <6>, <8>, <10>, <12> and <14>, the Listeria infection is an infection caused by Listeria monocytogenes. <16>In <1>, <3>, <5>, <7>, <9>, <11>, <13> and <15>, Listeria monocytogenes is a drug-resistant Listeria monocytogenes. <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.

Example

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

[0031] (2) Bacterial culture conditions The Listeria monocytogenes and Neisseria mucosa used were obtained from the BioResource Research Center, National Institute of Advanced Industrial Science and Technology (Table 1). For colony culture of any of the bacteria, Brain Heart Infusion (BHI) Agar medium (Nippon Becton Dickinson (hereinafter, Nippon BD)) was used, and for liquid culture, BHI medium (Nippon BD) was used for culturing. Listeria monocytogenes was aerobically cultured at a temperature of 37°C, and Neisseria mucosa was cultured under conditions of a temperature of 37°C and a CO2 concentration of 5%. After culturing Listeria monocytogenes in liquid for 24 hours, the cells were further cultured in liquid at a 1,000-fold dilution for another 24 hours. For Staphylococcus aureus, after culturing in liquid for 24 hours, the cells were further cultured in liquid at a 100-fold dilution for 48 hours.

[0032]

Table 1

[0033] (3) Preparation of Staphylococcus aureus cell culture Staphylococcus aureus was cultured in 300 mL of BHI medium at 37°C and 5% CO2 for 48 hours to prepare a culture solution. To the Staphylococcus aureus culture solution, the culture supernatant of Porphyromonas gingivalis prepared to a final concentration of 5 v / v% was added. After culturing at 37°C and 5% CO2 for 48 hours, 20 mL of the culture solution was collected into a 50 mL tube. The culture supernatant of Porphyromonas gingivalis was prepared by suspending 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 at 37°C under anaerobic conditions for 24 hours in the same way, adding the resulting liquid culture to a new GAM Broth Medium at a final concentration of 1 v / v%, centrifuging the culture obtained after culturing at 37°C under anaerobic conditions for 24 hours at 13,000 g, 4°C for 15 minutes to remove the cells, and filter-sterilizing the supernatant fraction with a 0.22 μm filter. To remove the medium components from the culture solution of *Neisseria mucosa* cultured by adding 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 by vortexing. 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. For the ultrasonic treatment, BRANSON SONIFIER 150 was used, and ultrasonic treatment was performed under the conditions of power 4, crushing 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 into a new 50 mL tube. To completely remove the cells, the collected supernatant was centrifuged again at 16,000 g, 4 °C for 30 minutes, and the supernatant was collected. By lyophilizing the collected supernatant, the cells and medium components were removed, and a cell culture of *Neisseria 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 cells precipitated by centrifuging the *Listeria monocytogenes* culture solution at 22 °C, 4,500 g for 10 minutes were collected. The collected cells were washed by centrifugation at 22 °C, 4,500 g for 5 minutes 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 22 °C, 4,500 g for 5 minutes 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 medium from HeLa cells that had become confluent on a 12-well plate, the cells were washed with PBS(-). The cell culture of *Neisseria mucosa* 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 that the cells were infected with *Listeria monocytogenes* at a multiplicity of infection (MOI) = 500, and the cells were infected by co-culturing them for 2 hours in the medium containing the cell culture of *Neisseria mucosa* at each concentration. The inhibitory effect of the *Neisseria mucosa* cell culture on *Listeria monocytogenes* infection is shown in Fig. 1.

[0037] (7) Method for analyzing infected cells After removing the medium from HeLa cells infected with CFSE-labeled *Listeria monocytogenes* and washing them twice with PBS(-), 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, and the total volume of the solution was collected in a 1.5 mL tube. 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 the cells were fixed 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 the cells were suspended in 200 μL of PBS (containing 2 v / v% FBS) to obtain an analysis sample. Flow cytometry (BD, FACSVerse) was used for the analysis. Based on forward scatter (FSC) and side scatter (SSC), the mean fluorescence intensity (MFI) was calculated from the fluorescence intensity obtained in the FITC channel of 20,000 gated cells, and the cell infectivity was evaluated by detecting the cells infected with fluorescently labeled Listeria monocytogenes. The results are shown in Figure 1.

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

[0039] (9) Results In the evaluation of cell infectivity, a decrease in mean fluorescence intensity (cell infectivity) was observed in the groups 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. An inhibitor for suppressing Listeria monocytogenes infection, comprising 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 cells and medium components.

2. A prophylactic or therapeutic agent for Listeria infection, comprising 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 cells and medium components.

3. A food for suppressing Listeria monocytogenes infection, comprising 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 cells and medium components.

4. A food for preventing or treating Listeria infection, comprising 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 cells and medium components.

5. The agent according to claim 2 or the food according to claim 4, wherein the Listeria bacterium is Listeria monocytogenes.

6. The agent according to claim 1 or 5, or the food according to claim 3 or 5, wherein the Listeria monocytogenes is drug-resistant Listeria monocytogenes.

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