JP2023172408A - PHARMACEUTICAL FOR PREVENTING OR TREATING INFECTIONS CAUSED BY VANCOMYCIN-RESISTANT ENTEROCOCCUS AND AGENT FOR IMPROVING α-DIVERSITY OF INTESTINAL BACTERIAL FLORA - Google Patents

Abstract

To provide a new prophylactic or therapeutic agent for infections caused by vancomycin-resistant Enterococcus.SOLUTION: A pharmaceutical containing hochu-ekki-to is designed to treat or prevent infections caused by vancomycin-resistant Enterococcus or inhibit for inhibiting infections caused by vancomycin-resistant Enterococcus during the administration of antibacterial agents.SELECTED DRAWING: None

Description

Application for application of Article 30, Paragraph 2 of the Patent Act Website publication date May 28, 2021 Website address https://www. nature. com/articles/s41598-021-90890-4

The present invention relates to a medicament for preventing or treating infections caused by vancomycin-resistant enterococci and an agent for improving α-diversity of intestinal flora.

The Enterococcus genus is a Gram-positive coccus that is resident in the intestinal tract and environment. Among enterococci, for example, Enterococcus faecalis, E. faecium, E. gallinarum, and E. casseliflavus are known as bacterial species that are involved in human infections. Enterococci are opportunistic pathogens that can cause bacteremia, endocarditis, enteritis, and urinary tract infections in immunocompromised hosts with reduced ability to defend against infection (e.g., the elderly, diabetics, malignant tumors, patients with heart disease, post-surgery patients, etc.). Causes infections such as abdominal and pelvic infections. Vancomycin is considered an extremely important antibacterial agent for such infectious diseases, particularly for infections caused by enterococci that are resistant to antibacterial drugs such as cephems, carbapenems, and aminoglycosides.

However, some enterococci have developed resistance to vancomycin and are called vancomycin-resistant enterococci (VRE). The transmission route for VRE is to establish itself in the intestinal tract and then spread from person to person.

In healthy people, even if VRE is carried in the intestinal tract due to natural immunity, it is usually harmless and asymptomatic, but in post-operative patients and patients with a weakened infection defense function, VRE may be carried due to weakened immune system. It may cause infections such as peritonitis, surgical wound infection, pneumonia, and sepsis. Similarly, elderly people with weakened immune systems are more likely to develop VRE infections. There are cases where VRE infection occurs in immunocompromised hosts and spreads from person to person, resulting in nosocomial infections, which poses a major problem from the perspective of infection control.

Linezolid, quinupristin, and dalfopristin are indicated as antibiotics for VRE, but VRE that has become resistant to these antibiotics has been reported overseas. Therefore, since there is no other way to deal with VRE infections, there is a strong desire to develop new preventive and therapeutic drugs for VRE infections.

Special re-publication 2013-191225

An object of the present invention is to provide a new preventive or therapeutic agent for infectious diseases caused by vancomycin-resistant enterococci.

Under these circumstances, the present inventors conducted intensive research and found that the above problems could be solved by using Hochuekkito, whose effect on vancomycin-resistant enterococci was not known at all until now. . The present invention is based on this new knowledge. Accordingly, the present invention provides the following items:
Item 1. A medicament containing Hochuekkito for treating or preventing infections caused by vancomycin-resistant enterococci, or for suppressing infections caused by vancomycin-resistant enterococci during administration of antibiotics.
Item 2. Item 2. The medicament according to item 1, which is used in combination with probiotics.
Item 3. Item 3. The medicament according to item 1 or 2, which is an orally administered drug.
Item 4. Agents that increase α-diversity of intestinal flora, including Hochuekkito.
Item 5. Item 4. The α-diversity increasing agent according to item 4, which is an orally administered agent.

According to the present invention, a new prophylactic or therapeutic agent for vancomycin-resistant enterococcal infections can be provided. Hochuekkito, the active ingredient of the present invention, is known to be used as an adjuvant to human papillomavirus (HPV) vaccines (Patent Document 1), but its preventive and therapeutic effects against VRE are not known at all. do not have. Therefore, the present invention produces surprising effects that could not be expected from the prior art.

The results of a retrospective observational study regarding the relationship between the administration of Hochuekkito and VRE infection in Example 1 are shown. FIG. 2 shows a graph of the number of viable VRE bacteria in feces in the second group (test of the therapeutic effect of Hochuekkito) and the control group in Example 2. FIG. The vertical axis of the graph shows the average number of viable VRE bacteria in feces. The horizontal axis of the graph indicates the number of days after inoculation with the VRE bacterial solution (the day of inoculation with the VRE bacterial solution was counted as day 0). 2 shows a graph of the number of viable VRE bacteria in feces in Group 3 (test of preventive and therapeutic effects of Hochuekkito) and control group in Example 2. The vertical axis of the graph shows the average number of viable VRE bacteria in feces. The horizontal axis of the graph indicates the number of days after inoculation with the VRE bacterial solution (the day of inoculation with the VRE bacterial solution was counted as day 0). The results of testing the influence of hochuekkito on α-diversity of intestinal flora in Example 3 are shown. Wilcoxon rank sum test was used for statistics. The vertical axis indicates observed features. The results of testing the influence of hochuekkito on α-diversity of intestinal flora in Example 3 are shown. Wilcoxon rank sum test was used for statistics. The vertical axis shows shannon entropy.

Medicinal The present invention provides a pharmaceutical containing Hochuekkito.
The active ingredient of the present invention is hochuekkito. Hochuekkito contains ingredients derived from the following herbal medicines: ginseng (ginseng), sojutsu (soujutsu), astragalus, dōki (tōki), chinpi (chinpi), turmeric, saiko (saiko), licorice (licorice), Ginger (Ginger), Shoma (Shoma). In this specification, the term "hochuekkito" includes not only the liquid obtained by decoction of the above-mentioned herbal medicines, but also the liquid obtained by decoction of the herbal medicines by drying (for example, freeze-drying), unless specified otherwise. ) is also included. The ratio of each crude drug component can be appropriately set within a range that provides the effects of the present invention. In a preferred embodiment, the ratio of each crude drug component is, for example, 1 to 10 parts by mass of Sandalwood or Soju (preferably 2 to 6 parts by mass, more preferably 4 parts by mass) to 4 parts by mass of ginseng. , 1 to 10 parts by mass of Japanese Astragalus (preferably 2 to 6 parts by mass, more preferably 3 to 4 parts by mass), 1 to 8 parts by mass of Astrax (preferably 2 to 4 parts by mass, more preferably 3 parts by mass) , Chinpi 1 to 4 parts by mass (preferably 1 to 3 parts by mass, more preferably 2 parts by mass), Taisou 1 to 4 parts by mass (preferably 1 to 3 parts by mass, more preferably 2 parts by mass), Saiko 0.1 to 4 parts by weight (preferably 0.5 to 3 parts by weight, more preferably 1 to 2 parts by weight), 0.5 to 4 parts by weight (preferably 1 to 2 parts by weight, more preferably 1.5 parts by mass), 0.1 to 2 parts by mass of ginger or Japanese ginger (preferably 0.1 to 1 part by mass, more preferably 0.5 parts by mass), 0.1 to 3 parts by mass of ginger (preferably (0.1 to 2 parts by mass, more preferably 0.5 to 1 part by mass). Accordingly, in a preferred embodiment, Hochuekkito includes a liquid obtained by decocting a raw material containing each herbal drug component in the above proportions with a liquid such as warm water, an extract obtained by drying the same, and the like. In a typical embodiment, for example, when quantifying Hochuekkito extract , hesperidin 16 to 64mg, Saikosaponin b20.3-1.2mg (Saiko 1g prescription), 0.6-2.4mg (Saiko 2g prescription) and glycyrrhizic acid (C ₄₂ H ₆₂ O ₁₆ : 822.93) 10-30mg. include.

In addition, in the present invention, Hochuekkito itself, which is the active ingredient of the present invention, may be used as a medicine, or various pharmaceutically acceptable carriers (for example, It may be used as a pharmaceutical composition in combination with a tonicity agent, a chelating agent, a stabilizer, a pH adjuster, a preservative, an antioxidant, a solubilizing agent, a thickening agent, etc.).

Examples of tonicity agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, and sodium chloride, potassium chloride, and calcium chloride. Examples include inorganic salts. These tonicity agents can be used alone or in combination of two or more.

Examples of the chelating agent include edetate salts such as disodium edetate, disodium calcium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or its salt, hexamethalin. Examples include acid soda and citric acid. These chelating agents can be used alone or in combination of two or more.

Examples of the stabilizer include sodium hydrogen sulfite.

Examples of the pH adjuster include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate, or hydrogen carbonate. Examples include salts, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, and the like. These pH adjusters can be used alone or in combination of two or more.

Examples of preservatives include paraoxybenzoic acid esters such as sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorhexidine gluconate, benzalkonium chloride, and chloride. Examples include quaternary ammonium salts such as benzethonium and cetylpyridinium chloride, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine. These preservatives can be used alone or in combination of two or more.

Examples of the antioxidant include sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, and the like. These antioxidants can be used alone or in combination of two or more.

Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, and the like. These solubilizing agents can be used alone or in combination of two or more.

Examples of the thickening agent include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, and the like. These thickening agents can be used alone or in combination of two or more.

Further, the above pharmaceutical composition may contain probiotics in addition to Hochuekkito. In the present invention, the term "probiotics", unless explicitly stated otherwise, means live microorganisms that confer a beneficial effect on a subject by improving the balance of the intestinal flora. Therefore, in the present invention, probiotics include bacteria such as lactic acid bacteria, bifidobacteria, butyric acid bacteria, propionic acid bacteria, and Escherichia coli that form bacterial flora; substances that promote the growth of these bacteria; and substances that have beneficial effects on the target. Examples include useful microorganisms such as yeast and fungi that can cause When using probiotics, the amount used is not particularly limited, but for example, 0.001 to 1000 parts by mass of probiotics is preferably added to 1 part by mass of Hochuekkito in terms of Hochuekkito extract. can be used in an amount of 0.01 to 100 parts by mass, more preferably 0.1 to 10 parts by mass. When using multiple types of these probiotics, for example, each probiotic can be used in the above usage ratio.

Furthermore, in addition to the above-mentioned components such as Hochuekkito, the above-mentioned pharmaceutical composition may further contain a compound said to have a preventive or therapeutic effect on VRE infections. Examples of compounds that are said to have preventive or therapeutic effects on VRE infections include linezolid, quinupristin, and dalfopristin. When using these compounds, they can be used alone or in combination of two or more. When using these compounds, the amount used is not particularly limited; It is possible to use 0.001 to 1000 parts by weight, preferably 0.01 to 100 parts by weight, and more preferably 0.1 to 10 parts by weight of a compound that is said to have the same effect. When using multiple types of compounds that are said to have preventive or therapeutic effects on these VRE infections, for example, each compound can be used in the above usage ratio. Moreover, the above-mentioned pharmaceutical composition may contain Chinese herbal medicine components other than Hochuekkito within the range in which the effects of the present invention can be obtained.

In the embodiment of the pharmaceutical composition, the content of Hochuekkito in the composition is not particularly limited, and for example, in terms of Hochuekkito extract, 99% by mass or more, 95% by mass or more, 90% by mass. The above conditions can be set as appropriate from conditions such as 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, and 1% by mass or more.

The formulation form is not particularly limited, and examples include oral preparations such as tablets, pills, capsules, powders, granules, and syrups; injections (intravenous injection, intramuscular injection, local injection, etc.), gargles, and drips. , external preparations (ointments, creams, patches, inhalants), and parenteral preparations such as suppositories. Among the above formulations, preferred examples include oral preparations (granules, tablets, pills, capsules, powders, syrups, etc.).

In the present invention, the dosage of Hochuekkito varies depending on the administration route, patient's age, weight, symptoms, etc., and cannot be unconditionally defined, but it is 7.5g per day for adults in terms of Hochuekkito extract. It may be administered orally in 2 to 3 divided doses. The lower limit of the dosage of Hochuekkito is also not particularly limited, and for example, the daily dosage for adults can be set appropriately within the range of usually 0.1 mg or more, preferably 0.5 mg or more. When administering once a day, this amount may be contained in one preparation, and when administering three times a day, one third of this amount may be contained in one preparation.

The medicament of the present invention is administered to subjects such as mammals and birds. Examples of mammals include humans, monkeys, mice, rats, cats, dogs, pigs, rabbits, cows, horses, and sheep, and examples of birds include chickens.

The medicament of the present invention can be used to prevent or treat VRE infections. In the present invention, the term "prevention or treatment" of VRE infection includes not only use of the medicament of the present invention for either prevention or treatment, but also use for both prevention and treatment. Furthermore, the medicament of the present invention can also be used to suppress vancomycin-resistant enterococcal infection during administration of antibiotics.

Agent for increasing α-diversity of intestinal flora By administering Hochuekkito, which is the active ingredient of the present invention, to subjects (patients, etc.), α-diversity of intestinal flora can be increased. . Accordingly, in one embodiment, the present invention provides an agent for increasing α-diversity of intestinal flora, comprising hochuekkito.

In these embodiments, details of the active ingredient hochuekkito, how to use it, excipients, etc. are the same as described in the above-mentioned "Medicine". The subject of administration is also as described above, and may be a human or a non-human animal.

According to the present invention, by using Hochuekkito, α-diversity of the intestinal flora can be improved, thereby reducing the proportion of VRE in the intestinal flora. In the present invention, it is considered that such a reduction in the proportion of VRE in the intestinal flora contributes to the prevention and treatment of VRE infections. Therefore, unlike conventional antibacterial agents that treat VRE infections by their sterilizing and bactericidal effects against VRE, the present invention is effective because resistant strains are less likely to occur.

The present invention will be described in more detail below with reference to Examples, but the present invention is not limited thereto.

Example 1
We conducted a retrospective observational study to investigate the association between Hochuekkito administration and Vancomycin-resistant enterococcus (VRE) infection. Specifically, it is as follows.
Method
1.1. subject
Patients who tested positive for VRE in a culture test at one facility (tertiary medical institution) from August 2018 to July 2019 were selected. During this period, the facility tested stool cultures of all inpatients weekly, and a total of 122 inpatients confirmed to be positive for VRE were enrolled in the study.
1.2.Evaluation items
Subjects' gender, age, body mass index and serum albumin level at admission, history of diabetes and hypertension, surgical procedures after admission, and positive VRE in the stool, previous antibiotic use, and oral treatment for VRE. Use of probiotics and herbal medicine were retrospectively extracted. The evaluation items were negative VRE and the period from confirmation of VRE positivity in stool to achievement of negative VRE.
1.3. Statistics
Survival analysis was assessed by Kaplan-Meier curves and log-rank test. Multivariate analysis for achieving VRE negativity was assessed by binary logistic regression analysis using specific explanatory variables and additional variables that had a significant effect. Statistical analysis used SPSS Statistics Base 22 software (IBM Corp., Armonk, NY, USA) or MATLAB R2015a (MathWorks, Natick, MA, USA).
Results The combination of probiotics and hochuekkito and the administration of hochuekkito alone promoted VRE negativity in VRE-infected patients. In addition, administration of hochuekkito significantly improved the survival rate of VRE-positive patients. The results regarding survival rate are shown in Figure 1.

Example 2
We investigated the effects of hochuekkito on Vancomycin-resistant enterococcus (VRE) infection using a mouse model.

Method
2.1. Test and control substances
2.1.1.Test substance
Name: Hochuekkito mixed feed Properties: Pellet feed
2.1.2. Control substance
Name: MF control feed Properties: Pellet feed
The MF control feed is a commercially available product from Oriental Yeast Industry Co., Ltd., and the company was asked to create the Hochuekkito mixed feed with a ratio of 97% MF and 3% Hochuekkito extract powder. The MF control feed and Hochuekkito mixed feed were also sterilized by gamma irradiation (30 kGy). Hochuekkito extract powder is provided by Tsumura Co., Ltd., and contains 10 kinds of herbal medicines (Arugis 4.0g, Sojutsu 4.0g, Carrot 4.0g, Angelica 3.0g, Saiko 2.0g, Taisou 2.0g, Chinpi 2.0g, Licorice 1.5 This is a Chinese herbal extract preparation that is made by decocting 1.0g of ginger, 1.0g of ginger, and 0.5g of ginger with only water and making the dried extract into granules using a dry granulation method that does not use any organic solvent or water. .

2.2.Specimen
2.2.1. Preparation method
2.2.1.1. The required amounts of the test substance and control substance as described below were collected and used.

2.3. Pathogenic microorganism Enterococcus faecium (ATCC 700221) (vancomycin-resistant enterococci)
2.3.1. Bacterial strains used
2.3.2.Storage conditions and storage location
It was frozen and stored in an ultra-low temperature freezer (set temperature: -80°C, MDF-394AT, Sanyo Electric Co., Ltd.) at the test facility until use.
2.3.3. Reagents used
(1) Brain Heart Infusion Agar Medium (Eiken Chemical Co., Ltd.)
(2) Brain Heart Infusion Culture Solution (Eiken Chemical Co., Ltd.)
(3) Physiological saline solution (Otsuka Pharmaceutical Factory Co., Ltd.)
2.3.4. Purification of inoculum solution
The preserved bacterial strain was thawed, spread on a brain heart infusion agar medium, and then cultured for 2 days in an incubator (ILE800, Yamato Scientific Co., Ltd.) set at 37°C. Colonies grown on the medium were picked, added to brain heart infusion culture solution, and cultured for 1 day in a thermostat set at 37°C. After culturing, the mixture was centrifuged (2000 rpm, 5 minutes), the supernatant was discarded, and 1 mL of physiological saline was added to the precipitate to suspend it. A bacterial solution was prepared to have a concentration of McFarland 2 (approximately 6.0×10 ⁸ CFU/mL).
2.3.5. Handling of remaining inoculum solution
The remaining inoculum solution after use was autoclaved (LSX-500, Tomy Seiko Co., Ltd.) (121°C, 15 minutes) and then discarded.

2.4.Test system
2.4.1. Animal species and strains Animal species: Mouse (SPF)
Strain: C57BL/6JJmsSlc
Reason for selection: This strain has abundant background data for evaluation in infection experiments.
2.4.2. Sex and age of the animal
Female, 5 weeks old
2.4.3. Environmental conditions and husbandry management
Controlled temperature: 18 to 28°C (actual measurement: 21 to 26°C), Controlled humidity: 30 to 80%RH (actual measurement: 47 to 72%RH), 12 hours of light and darkness (lighting: 6:00 to 18:00) Animals were housed in a vivarium maintained at ). A sterilized plastic cage (W: 175 × D: 245 × H: 125 mm) was lined with a wire mesh drainboard. In addition, in order to take care of the breeding environment, environmental enrichment (nesting sheet, Animec Co., Ltd.) was placed in each cage. After being divided into groups, they were housed individually. The test substance and control substance were placed in a feeder and allowed to be consumed ad libitum. All animals received a control substance during the pre-housing period.
2.4.4.Drinking water
Tap water was available ad libitum using a water bottle. Contaminant concentrations and bacterial counts in the drinking water were analyzed approximately every six months to ensure that they met the test facility's acceptable standards. From after grouping until the day after inoculation with the bacterial solution, the animals were allowed to drink water with ampicillin (0.5 g/L, Fujifilm Wako Pure Chemical Industries, Ltd.) added.
Analysis organization: Tozai Kagaku Sangyo Co., Ltd. and Environmental Pollution Center Co., Ltd.

2.5. Test substance and control substance administration
2.5.1. Administration route, administration method, and administration period Administration route: Mixed feed Administration method: Mixed feed mixed with the test substance was placed in a feeder, and the subjects were allowed to ingest it ad libitum. For the control group, a control substance was placed in the feeder instead of the mixed feed mixed with the test substance, and the animals were allowed to ingest it ad libitum. Feed was replaced with fresh food twice a week, and uneaten feed was discarded.
Administration period: In the second group (testing the therapeutic effect of hochuekkito), the administration start date was the day of bacterial liquid inoculation, and the administration period was 11 days. For the third group (test of preventive and therapeutic effects of Hochuekkito), the date of grouping was the start date of administration, and the administration period was 18 days. In all groups, the start date of each administration was counted as the 1st day of administration.

2.6. Bacterial liquid inoculation
2.6.1. Bacterial solution inoculation method Using a 1 mL disposable syringe (Terumo Corporation) equipped with a mouse sonde (Fuchigami Kikai Co., Ltd.), inoculation was performed on the 8th day, counting the day of grouping as the 1st day. 0.2 mL was given orally.

2.7． Group composition

result
2. 8. Number of viable bacteria in feces Changes over time in the number of viable VRE bacteria in feces in the second group (hochuekkito was administered for treatment) and the control group (the day of inoculation with the bacterial solution was counted as day 0) (The same applies to Figure 3.) is shown in Figure 2. Figure 3 shows the changes over time in the number of viable VRE bacteria in feces in the third group (Hochuekkito was administered for prevention and treatment) and the control group.

In the second group (Hochuekkito was administered for treatment. Hochuekkito was administered for 11 days), the average number of viable bacteria in feces decreased from the 4th day after inoculation with the bacterial solution compared to the control group. A trend was observed, and it decreased significantly on the 9th day after inoculation of the bacterial solution. In the third group (Hochuekkito was administered for prevention and treatment. Hochuekkito was administered for 18 days), the average number of viable bacteria in the feces was lower than that of the control group from the first day after inoculation with the bacterial solution. A decreasing trend was observed, and a significant decrease in the number of viable bacteria was observed on days 2 and 9 after inoculation of the bacterial solution compared to the control group.

Example 3
The effect of hochuekkito on α-diversity of intestinal flora was tested. Specifically, it is as follows.
3.1.DNA extraction
DNA was extracted from mouse stool samples on days 0, 1, 2, 9, and 10 using the DNeasy Power Soil Kit (QIAGEN Inc., Germany). This is a kit that uses beads to destroy cell walls, lyse cells, and salt out proteins, trap the extracted DNA with filter paper, and finally elute and collect it in 30 μL of RNase-free water. However, if the amount of feces contained in the sample solution was small and a sufficient DNA yield could not be obtained, the sample was eluted and collected with 15 μL. The DNA concentration contained in the collected solution was measured using an absorbance meter Qubit 2.0 (Thermo Fisher Scientific Inc., USA) and a dsDNA HS Assay Kit (Thermo Fisher Scientific, Inc., USA).

3.2. 1st Polymerase chain reaction (PCR)
In order to amplify DNA for sequence analysis, we used a two-step PCR method to amplify the variable region V3-V4 contained in DNA encoding 16S ribosomal RNA (rRNA), a conserved region unique to bacterial DNA. was amplified to obtain an amplicon. The 1st PCR used specific primers targeting the V3-V4 region. The arrangement is as follows.
(Forward) 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNCCTACGGGNGGCWGC AG-3' (55bp), (reverse) 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGACTACHVGGGTAT CTAATCC-3' (60bp). The enzyme used for PCR was ExTaq (TaKaRa Bio Inc., Japan), and the PCR device was PCR Thermal Cycler Dice (registered trademark) Touch (TaKaRa Bio Inc., Japan). The protocol for the 1st RCR is as follows: 94°C 3 minutes, (94°C 30 seconds, 55°C 30 seconds, 72°C 30 seconds) x 30 cycles, 72°C 5 minutes, 8°C∞. Thereafter, using a fragment analyzer (Fragment Analyzer, FA, Advanced Analytical Technologies Inc., USA), it was confirmed whether the 1st PCR product (approximately 540 bp) had been amplified. FA is a device that can simultaneously electrophores many PCR products and detect even minute amounts of PCR products. Set 40 ml of Gel + 4 μL of Dye, 35 ml of Capillary Conditioning Solution, and a waste bottle per 96-well plate in this device, and set a tray containing Inlet Buffer, a waste solution tray, a Marker Solution tray, and 10 μL of 1st PCR product in each well tray. Electrophoresis was performed.

3.3. 2nd PCR
2nd PCR was performed to attach an adapter sequence necessary for sequence analysis to the amplified amplicon. The primers used in 2nd PCR are pre-attached with a different index sequence for each sample, an adapter sequence for binding to the MiSeq flow cell, and a region to which the sequencing primer binds. The same reagents and equipment as in the 1st PCR were used.

3.4. Bead purification, gel cutting
3 μL of the 2nd PCR products were combined into one tube to form a pooled sample. In order to remove impurities other than the amplified DNA from this pooled sample, the amplified DNA was purified using magnetic beads AMPure XP (Beckman Coulter Inc., USA). The purified product was electrophoresed on a 1% agarose gel, and the gel containing the purified DNA band was cut out under UV using a transilluminator. The gel was precipitated with isopropanol using MinElute Gel Extraction Kit (QIAGEN Inc., Germany) to obtain a highly purified DNA solution. The DNA concentration in the solution was measured using Qubit to confirm that a sufficient concentration for sequencing was obtained, and this solution and the amplified DNA obtained by 2nd PCR were electrophoresed to identify the band in question (V3-V4 region). 630bp) were confirmed to match.

3.5. Amplicon DNA sequencing using MiSeq Amplicon DNA was sequenced from the purified DNA solution using MiSeq (Illumina Inc., USA).

3.6. Phylogenetic tree analysis of bacterial flora QIIME 2 (version 2019.10), an integrated analysis pipeline program for metagenomic analysis, was used for sequence analysis.
In this study, we calculated the following two indices as α-diversity. (1) Observed features: Number of bacterial species observed at least once. A diversity index that takes into account the presence of rare species. (2) Shannon index: Calculated based on the proportion of species to the total sample. Although it is widely used, it is said to be susceptible to the effects of a large proportion of bacterial species.
Wilcoxon rank sum test was used for comparisons between the three groups. p < 0.05 was considered significant. The statistical software JMP software (version 12.0; SAS Institute Inc., Cary, NC, USA) was used for analysis.
Figure 4 shows the results of observed features on the second day. Furthermore, the results of the Shannon index on the second day are shown in FIG. As shown in Figure 4, observed features indicating α-diversity were significantly increased in the Hochuekki administration group and the Hochuekkito prophylaxis administration group compared to the control group. Furthermore, as shown in Figure 5, shannon entropy, which indicates α-diversity, was significantly increased in the Hochuekkito group and the Hochuekkito prophylaxis group compared to the control group. These results suggest that Hochuekkito may have increased α-diversity and decreased the colonization rate of VRE.

Claims (5)

A medicament containing Hochuekkito for treating or preventing infections caused by vancomycin-resistant enterococci, or for suppressing infections caused by vancomycin-resistant enterococci during administration of antibiotics. The medicament according to claim 1, comprising a combination with probiotics. The medicament according to claim 1, which is an orally administered drug. Agents that increase α-diversity of intestinal flora, including Hochuekkito. The α-diversity increasing agent according to claim 4, which is an orally administered agent.