Method and apparatus for inspecting ulcerative colitis, and screening method of therapeutic drug

Abstract

To provide a method for inspecting ulcerative colitis which is useful for determining a disease state of ulcerative colitis.SOLUTION: A method for producing a strain sample derived from a subject in which the strain diversity and / or the number of strains in the sample of the subject are maintained includes: 1) a process of adding the sample of the subject to a culture medium; 2) a process of performing an operation to bring pH of the culture medium to 6.2 to 6.7 in starting the cultivation if necessary; and 3) a process of culturing the sample after starting the cultivation without performing an operation intended to maintain pH of the culture medium. Preferably, the method includes A) a process of performing sterilization of the culture medium and aeration to anaerobic gas and / or B) a process of suspending the sample to ascorbic acid-added phosphate buffer, before 1).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method and apparatus for examining ulcerative colitis, and a method for screening a therapeutic agent for ulcerative colitis.

Background Art

[0002] Ulcerative colitis is spreading worldwide, and the number of patients is increasing rapidly even in Japan. The current main treatment method for ulcerative colitis is the administration of mesalazine (5-aminosalicylic acid (5-ASA)), a non-steroidal preparation. However, with this treatment method, the disease is temporarily alleviated, but it does not eliminate the possibility of recurrence and does not achieve remission. Therefore, for the cure of ulcerative colitis, there is a need for an examination for determining the disease state of ulcerative colitis and a therapeutic agent therefor.

[0003] Looking at the intestinal environment of ulcerative colitis patients, the growth of Escherichia coli is increased compared to healthy individuals, indicating an abnormality. Correction of the intestinal environment is considered necessary for the cure of ulcerative colitis.

[0004] By the way, in the human intestinal tract, various bacteria are constantly growing, and these are called the intestinal flora. In the study of the intestinal flora, culture using an intestinal tract mimicking culture device that reproduces the human intestinal environment is utilized. A culture method and apparatus that can culture while maintaining almost the balance of the composition of the intestinal flora have been proposed (Patent Document 1 and Non-Patent Document 1). In this culture method, each intestinal flora is cultured in an anaerobic environment using a medium supplemented with a lower fatty acid while adjusting the pH of the medium to the range of 6.0 to 7.0.

[0005] For the examination of ulcerative colitis and the screening of a therapeutic agent, a culture method and apparatus that mimic the intestinal environment of ulcerative colitis patients are required.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Literature

[0007]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present invention aims to solve the above problems, and its object is to provide a method and apparatus for examining ulcerative colitis and a method for screening therapeutic drugs, which are useful for judging the disease state of ulcerative colitis.

Means for Solving the Problems

[0009] The present invention provides a method for examining ulcerative colitis, which method comprises: a step of performing anaerobic culture using a culture solution containing a fecal sample collected from a subject; a step of obtaining data on an index factor of ulcerative colitis from the culture solution; and a step of comparing the data with a standard of the index factor of a healthy individual and / or an individual suffering from ulcerative colitis; including the anaerobic culture is performed by leaving the culture solution standing, with the pH of the culture solution being 6.2 to 6.7 at the start of the culture.

[0010] In one embodiment, the subject is a subject suffering from or suspected of suffering from ulcerative colitis.

[0011] In one embodiment, the index factor of ulcerative colitis is at least one selected from the group consisting of the amount of butyric acid, the amount of bacteria of the family Lachnospiraceae, and the profile of the pH of the culture solution.

[0012] In one embodiment, the pH of the culture medium is 6.2 to 6.5 at the start of the culture.

[0013] The present invention provides a method for screening a therapeutic agent for ulcerative colitis, the method comprising: a step of performing anaerobic culture using a culture medium containing a fecal sample collected from an individual suffering from ulcerative colitis and administered with a therapeutic agent candidate; a step of obtaining data on an indicator factor for ulcerative colitis from the culture medium; and a step of evaluating the effectiveness of the therapeutic agent candidate by comparing the data with the standard of the indicator factor of a healthy individual and / or an individual suffering from ulcerative colitis; including the anaerobic culture is performed by leaving the culture medium, the pH of which is 6.2 to 6.7 at the start of the culture.

[0014] The present invention provides an inspection apparatus for ulcerative colitis, the apparatus comprising: (a) anaerobic culture means for performing anaerobic culture using a culture medium containing a fecal sample collected from a subject; (b) measuring means for measuring data on an indicator factor for ulcerative colitis from the culture medium of the anaerobic culture means; and (c) comparison means for pre-storing the standard of the indicator factor of a healthy individual and / or an individual suffering from ulcerative colitis and comparing the data on the indicator factor obtained from the measuring means with the standard of the indicator factor; provided with.

Advantages of the Invention

[0015] According to the present invention, a culture method simulating the intestinal environment of ulcerative colitis patients is provided, whereby a method and an apparatus for inspecting ulcerative colitis are provided. Further, the culture method is also useful for screening a therapeutic agent for ulcerative colitis.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

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Figure 7

Modes for Carrying Out the Invention

[0017] (Method for Examining Ulcerative Colitis) The present invention provides a method for examining ulcerative colitis. The method for examining ulcerative colitis of the present invention can be used for determining the affected state of ulcerative colitis. The "affected state" includes the presence or absence of the disease and the severity of the disease. The examination method in the present invention can be used for determining whether a subject has ulcerative colitis, whether the symptoms of ulcerative colitis in the subject are progressing, or whether the symptoms of ulcerative colitis in a subject under treatment have been alleviated.

[0018] The subject is any animal having an intestinal tract, for example, a mammal, for example, a human, a dog, a cat, a cow, a pig, etc., and preferably a human. The subject may be a livestock animal or a pet animal. In one embodiment, the subject is a subject suffering from or suspected of having ulcerative colitis.

[0019] The culture solution can be prepared by adding a fecal sample collected from the subject to a culture medium. Any culture medium that can be used for anaerobic culture may be used. The fecal sample can be prepared in advance as a suspension (for example, suspended in 0.1 M phosphate buffer supplemented with 1% L-ascorbic acid) and added, for example, at 100 μL per 100 mL of the medium. Examples of the culture medium for anaerobic culture include the Gifu University formulated anaerobic medium (GAM medium), for example, GAM agar medium, modified GAM agar medium, GAM semi-solid deep medium, GAM broth, and modified GAM broth (for example, all manufactured by Nissui Pharmaceutical Co., Ltd.). Before culturing, the medium can be sterilized (for example, autoclave sterilization). Liquid culture is preferred, and during the culture, the culture solution can be appropriately stirred. The fecal sample can be stored in a container such as an anaerobic culture swab after collection until the start of the examination.

[0020] The anaerobic environment for culturing can be created by aerating an anaerobic gas into the medium. The anaerobic gas is, for example, nitrogen, nitrogen and carbon dioxide, or nitrogen, carbon dioxide and hydrogen. The aeration of the anaerobic gas is, for example, continuously carried out at a flow rate of 0.1 to 1.0 dL / min or intermittently. Also, the anaerobic gas is preferably a mixed gas composed of nitrogen and carbon dioxide. It is preferable to continuously aerate the anaerobic gas.

[0021] In the present invention, the anaerobic culture is carried out with the pH of the culture solution being 6.2 to 6.7 at the start of the culture and then left to stand. Preferably, the starting pH is 6.2 to 6.5. At the start of the culture (for example, when the culture solution containing the fecal sample is placed in an anaerobic environment), it is sufficient if the pH of the culture solution is within the above range, and if necessary, the pH is adjusted within the above range using a pH adjuster. After the start of the culture, the pH is left unadjusted. During the culture, operations intended to maintain the pH within the above range (for example, pH adjustment by adding a pH adjuster or an alkali) are not performed.

[0022] The culture period is preferably a period during which data on the indicator factors of ulcerative colitis can be obtained. The culture period depends on the indicator factors of ulcerative colitis for which data is to be obtained, but is, for example, 24 hours to 48 hours, preferably 24 hours to 30 hours. The culture temperature is a temperature near the body temperature of the subject, for example, when the subject is a human, for example, 36°C to 38°C, preferably 37°C. The mode of culture is not limited, but preferably it is a one - batch type.

[0023] Data on the indicator factors of ulcerative colitis is obtained from the culture solution. The indicator factors of ulcerative colitis refer to factors that can be used to judge the disease state of ulcerative colitis (for example, the presence or absence of the disease or the severity of the disease). Examples of the indicator factors of ulcerative colitis include the amount of butyric acid, the amount of Lachnospiraceae bacteria, and the profile of the culture solution pH, and a combination of at least two of these indicator factors may also be used. The data on the indicator factors of ulcerative colitis is obtained from the culture solution at the start of the culture, during the culture, or after the end of the culture, depending on the indicator factor.

[0024] The amount of butyric acid can be measured from the culture solution according to a method commonly used by those skilled in the art (for example, high - performance liquid chromatography (HPLC)). The culture solution can be recovered at any time as long as it can measure the butyric acid produced by the intestinal bacteria that may be present in the collected feces, but preferably it is recovered from the latter half to around the end of the culture.

[0025] The pH profile is represented by the variation in pH over the culture period. During the culture period, the pH may be continuously monitored or measured intermittently multiple times.

[0026] The amount of Ruminococcaceae bacteria refers to the amount of bacteria classified as belonging to the family Ruminococcaceae, regardless of the genus distinction. The amount of Ruminococcaceae bacteria may be any value that can represent the abundance of Ruminococcaceae bacteria in the sample, such as the number of bacteria in the sample, the ratio of presence to all gut bacteria in the sample, etc. Preferably, it is the ratio of presence to all gut bacteria. The amount of Ruminococcaceae bacteria can be determined using techniques commonly used by those skilled in the art for the detection, identification, and quantification of gut bacteria (e.g., 16S rRNA gene sequencing, real-time PCR, etc.). The primer set used therein may be prepared based on known sequence information or a commercially available one may be used. As a sample for measuring the amount of Ruminococcaceae bacteria, the culture broth collected at any time point during the culture can be used. For example, the culture broth sample (fecal sample) at the start of the culture, the culture broth sample during or after the culture may be used alone or in combination.

[0027] The data of the indicator factor is compared with the criteria of the same indicator factor of healthy individuals and / or individuals suffering from ulcerative colitis. For example, when the indicator factor of ulcerative colitis is the amount of butyric acid, the data obtained for the amount of butyric acid is compared with the criteria of healthy individuals and / or individuals suffering from ulcerative colitis regarding the amount of butyric acid. In the creation of the criteria, the data obtained from the above culture can be used. The "criteria" can be created depending on the presence or absence and severity of ulcerative colitis.

[0028] An individual can also be an animal similar to the subject described above. A "healthy individual" refers to an individual who has at least not suffered from ulcerative colitis. A "healthy individual" may be an individual who had previously suffered from ulcerative colitis but has been cured. An "individual suffering from ulcerative colitis" refers to an individual who is suffering from ulcerative colitis. An "individual suffering from ulcerative colitis" may be an individual under treatment as long as they are suffering from ulcerative colitis. A "healthy individual" or an "individual suffering from ulcerative colitis" may be the same individual as the subject.

[0029] For the "standard" of the index factor of a healthy individual and / or an individual suffering from ulcerative colitis, it includes the case where a standard created based on data previously obtained from the corresponding individual (for example, a population of individuals) is used, and the case where the data itself previously obtained from the subject is used as the standard. In the latter case, for example, as a means for verifying the treatment effect of the subject, the inspection method of the present invention can be used.

[0030] Regarding the amount of butyric acid, ulcerative colitis patients have lower values compared to healthy individuals. Regarding the amount of Lachnospiraceae bacteria, ulcerative colitis patients have lower values compared to healthy individuals. For the pH p Regarding the profile, when comparing healthy individuals and ulcerative colitis patients, the fluctuations in pH during the culture period are different. For example, as shown in Figure 2, in the pH profile of healthy individuals, the fluctuations in the culture solution pH can reproduce the normal pH in the human large intestine, while ulcerative colitis patients may show abnormalities.

[0031] (Method for screening a therapeutic agent for ulcerative colitis) The present invention also provides a method for screening a therapeutic agent for ulcerative colitis. In this method, a fecal sample is collected from an individual suffering from ulcerative colitis and administered with a therapeutic agent candidate. The culturing step and the data acquisition step are as described above. For evaluating the effectiveness of the therapeutic agent candidate, the acquired data is compared with the criteria of indicator factors of healthy individuals and / or individuals suffering from ulcerative colitis. This comparison step is also as described above. It can be determined whether the acquired data conforms to the criteria of healthy individuals or the criteria of individuals with a lower severity of ulcerative colitis than the criteria of severely affected individuals.

[0032] The screening method of the present invention can determine whether a "therapeutic agent candidate" is effective for the treatment of ulcerative colitis by using criteria created based on data previously obtained from corresponding individuals (for example, a population of individuals) as the "criteria". The screening method of the present invention can also be used to select a therapeutic agent suitable for an individual by using, as the "criteria", an affected individual before administration of the therapeutic agent candidate ("an individual suffering from ulcerative colitis") and comparing the data of the individual after administration of the therapeutic agent candidate ("an individual suffering from ulcerative colitis and administered with the therapeutic agent candidate") with this criteria.

[0033] (Inspection apparatus for ulcerative colitis) The present invention also provides a method for inspecting ulcerative colitis. The inspection apparatus of the present invention will be described with reference to the drawings.

[0034] FIG. 1 is a diagram schematically showing an example of the inspection apparatus for ulcerative colitis of the present invention.

[0035] The inspection apparatus 100 of the present invention includes an anaerobic culturing means 110, a measuring means 140, and a comparing means 170.

[0036] In the inspection apparatus 100 of the present invention, the anaerobic culture means 110 includes a culture tank 112 and a lid 114 capable of sealing the culture tank 112. A culture solution 102 containing fecal material collected from a subject is accommodated in the culture tank 112, and anaerobic culture is performed. In the embodiment shown in FIG. 1, a shaft 118 extends from a motor 116 disposed above the lid 114 to a position where it is immersed in the culture solution 102 of the culture tank 112, and at least one stirring blade 120 is provided at the end of the shaft 118. Then, the motor 116 rotates the shaft 118, and the culture solution 102 is stirred by the stirring blade 120.

[0037] In FIG. 1, the motor 116 is provided above the lid 114, but the present invention is not limited only to such an arrangement. The motor may be provided below the culture tank 112. Alternatively, by forming the stirring blade 120 of a magnetic material, the shaft 118 may be separated from the motor. In this case, a member made of another magnetic material is provided around the axis of the motor, and the stirring blade 120 can also rotate as the member rotates.

[0038] Furthermore, in the anaerobic culture means 110 shown in FIG. 1, an air supply pipe 122 is disposed in the culture tank 112, for example, through above the lid 114. The air supply pipe 122 can introduce an inert gas (for example, nitrogen gas, carbon dioxide gas, and combinations thereof) into the culture tank 112 by opening and closing a valve 124, and make the inside of the culture tank 112 in an anaerobic state. An exhaust pipe 126 preferably equipped with a check valve (not shown) is also provided in the culture tank 112, and unnecessary gas in the culture tank 112 is discharged outside the culture tank 112 through the exhaust pipe 126. In FIG. 1, both the air supply pipe 122 and the exhaust pipe 126 are described as passing through the lid 114 and having their ends disposed inside the culture tank 112, but the present invention is not necessarily limited only to such a configuration. For example, both or either one of the exhaust pipe 122 and the exhaust pipe 126 may be disposed through the culture tank 112.

[0039]

[0039] The anaerobic culture means 110 is also provided with temperature control means 128 such as a thermostat in the culture tank 112. By the temperature control means 128, it is possible to maintain the culture solution 102 in the culture tank 112 at a desired temperature.

[0040] In the present invention, the anaerobic culture means 100 may be, for example, a commercially available jar fermenter.

[0041] In the inspection apparatus 100 of the present invention, the measuring means 140 serves to acquire data corresponding to the temperature of the culture solution 102 in the anaerobic culture means 110 and the ulcerative colitis indicator factor from the culture solution 102.

[0042] In the embodiment shown in FIG. 1, as the measuring means 140, a temperature sensor 142 and a chemical sensor 144 are provided at positions immersed in the culture solution 102 of the culture tank 112. Examples of the chemical sensor 144 include, but are not necessarily limited to, ion sensors such as a hydrogen ion electrode (pH electrode) and an ion selective electrode. Such a chemical sensor can selectively detect ulcerative colitis indicator factors (for example, organic acid content such as butyric acid, pH profile of the culture solution) contained in the culture solution 102. In FIG. 1, an example in which two sensors, a temperature sensor 142 and a chemical sensor 144, are provided as the measuring means 140 is described, but the present invention is not limited thereto. In the detection apparatus of the present invention, at least one measuring means is provided.

[0043] In the inspection apparatus 100 of the present invention, the comparison means 170 serves to compare the data of the indicator factor obtained from the measuring means 140 with the criteria of the indicator factor of a healthy individual and / or an individual suffering from ulcerative colitis stored in advance.

[0044] In the embodiment shown in FIG. 1, the comparison means 170 is composed of a control unit 172, a storage unit 174, and a display unit 176.

[0045] In FIG. 1, the control unit 172 is electrically connected to the valve 124 of the air supply pipe 122, the motor 116, and the temperature control means 128 in the anaerobic culture means 110 via cables 181, 182, and 183. Thereby, the control unit 172 can control the opening and closing of the valve 124 of the air supply pipe 122, the rotation speed of the motor 116, and the set temperature of the temperature control means 128. The control unit 172 is also electrically connected to the measuring means 140 (that is, the temperature sensor 142 and the chemical sensor 144) via cables 184 and 185. Thereby, the temperature data and the data of the index factor detected by the temperature sensor 142 and the chemical sensor 144 can be transmitted to the control unit 172 as electrical signals via the cables 184 and 184.

[0046] Note that in FIG. 1, an example of a wired connection by the cables 181, 182, 183, 184, and 185 between the control unit 172 and the valve 124, the motor 116, the temperature control means 128, the temperature sensor 142, and the chemical sensor 144 is described, but the present invention is not limited thereto. Instead of the wired connection, a wireless connection may be made by means of communication means (not shown) between them.

[0047] The storage unit 174 stores in advance the criteria of the index factors of healthy individuals and / or individuals suffering from ulcerative colitis as information, reads out the criteria according to a command from the control unit 172, and transmits them to the control unit 172 as appropriate. The storage unit 174 can also store the data of the index factors transmitted from the control unit 172. Examples of the storage unit 174 include an external or built-in hard disk drive (HDD), a magnetic disk such as a floppy disk; a CD-ROM, a CD-R, a CD-RW, a DVD-ROM, a DVD±R, a DVD-RW, a DVD-RAM, a Blu-ray disk (for example,

[0048] ​Optical discs such as BD-ROM, BD-R, BD-R BDXL, and BD-RE), LaserDisc (registered trademark); magneto-optical discs; and flash memories such as USB memories, solid state drives (SSD), SD memory cards, mini SD cards, micro SD cards, SDHC memory cards, mini SDHC cards, micro SDHC cards, SDXC memory cards, and micro SDXC cards.

[0049] In FIG. 1, the display unit 176 is electrically connected to the control unit 172 via a cable 188. The display unit 176 displays information such as the open / closed state of the valve 124, the rotation speed of the motor 116, and the set temperature of the temperature control means 128 in the anaerobic culture means 110 controlled by the control unit 172, as well as the temperature of the culture solution 102 obtained from the measuring means 140 and the acquired data of the acquired indicator factors, so that the operator can relatively recognize them. Examples of such a display unit 176 include a display, a printer, etc. In FIG. 1, an example of a wired connection between the display unit 176 and the control unit 172 by a cable 188 is described, but the present invention is not limited thereto. Instead of the wired connection, a wireless connection may be made by means of a communication means (not shown) between them.

[0050] In the inspection apparatus 100 of the present invention, the control unit 172 compares the data of the indicator factors of ulcerative colitis regarding the fecal sample in the culture solution 102 obtained from the chemical sensor 144 with the standards of the indicator factors of healthy individuals and / or individuals suffering from ulcerative colitis stored in advance in the storage unit 174. Then, the comparison result transmitted from the control unit 172 is displayed on the display unit 176. Further, the data of the indicator factors of ulcerative colitis obtained from the chemical sensor 144 and / or the comparison result obtained from the control unit 172 may be separately stored in the storage unit 174 as necessary. In the inspection apparatus 100 of the present invention, the control unit 172 may be an electronic computer such as a personal computer.

[0051] In this way, the inspection apparatus 100 of the present invention can perform an inspection for ulcerative colitis from the fecal sample collected from the subject.

Example

[0052] The present invention will be described in detail below with reference to examples. However, the present invention is not limited thereto.

[0053] (Example 1: Collection and culture of fecal samples) Fecal samples were collected from 13 healthy subjects and 11 patients with ulcerative colitis. The experimental participants had not received any antibiotics for over two months. Written informed consent was obtained from the experimental participants. The disease activity was determined by the Mayo clinical score and the partial Mayo score. For patients who had undergone endoscopy one month before fecal collection, the endoscopic Mayo clinical score was determined (n = 9). At the time of fecal collection, the partial Mayo clinical score was determined (n = 11). This experiment was conducted in accordance with the guidelines of the affiliated hospital of Kobe University School of Medicine and was approved by the ethics review committee of Kobe University. All methods used in this experiment followed the approved guidelines of Kobe University's medical ethics. After collection, the fecal samples were stored in anaerobic culture swabs (212550 BD BBL Culture Swab; manufactured by Becton Dickinson and Company) and sent to the laboratory.

[0054] A single-batch culture device mimicking the human large intestine was fabricated using a multi-channel incubator (Bio Jr.8; manufactured by Abel Co., Ltd.). This in vitro model device consists of eight parallel independent containers. In each container, 100 mL of autoclaved (115 °C for 15 minutes) Gifu University-prescribed anaerobic medium (GAM medium [Code 05422]; manufactured by Nissui Pharmaceutical Co., Ltd.) was placed, and the pH at the start of culture was adjusted to 6.5. Before culture, the anaerobic condition of the culture container was constructed by aerating (15 mL / min) with a mixed gas of nitrogen and carbon dioxide (N2:CO2 = 80:20) filtered and sterilized through a 0.2-μm PTFE membrane (manufactured by Pall Corporation) at 37 °C for 1 hour. For the preparation of the inoculum, each fecal sample was suspended in 2 mL of 0.1 M phosphate buffer (pH 6.5, consisting of a 2:1 mixture of NaH2PO4 and 0.1 M Na2HPO4) to which 1% L-ascorbic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added. 100 μL of the above fecal suspension was inoculated into each medium-containing container to initiate anaerobic culture. During the culture, the anaerobic condition was maintained by aerating the medium with the filtered and sterilized mixed gas.

[0055] At the start of culture, 6 hours later, 9 hours later, 12 hours later, 24 hours later, and 30 hours later, a part of the culture solution was collected from the protruding part on the side of the container. pH data was collected every 5 minutes during the culture period. The samples at the start of culture (fecal samples) and the samples collected 30 hours after culture (culture solutions) were also used for microbiota evaluation. The samples collected 30 hours after culture (culture solutions) were also used for quantification of short-chain fatty acids (SCFAs). The fecal samples and culture solutions were stored at -20 °C until measurement.

[0056] (Example 2: Evaluation of pH profiles of fecal cultures from healthy subjects and patients with ulcerative colitis) Based on the pH data collected for the samples from each of healthy subjects (n = 13) and patients with ulcerative colitis (n = 11) according to Example 1, pH profiles were created.

[0057] Figure 2 is a graph showing the pH profiles of the fecal samples and culture broths of 13 healthy individuals (A) and 11 ulcerative colitis patients (B), respectively. The vertical axis indicates the measured pH values, and the horizontal axis indicates the culture time (hours). As a result, most of the healthy individuals showed fluctuations where the pH decreased between approximately 5.5 and 6.0 after 6 hours of culture and then gradually increased, approaching pH 6.5 (the pH of the large intestine) in the late stage of culture (Figure 1(A)). In contrast, in ulcerative colitis patients, after the pH decreased to approximately 6.0 after 6 hours of culture, the pH rapidly increased near 6.5 and then further decreased, and thereafter either maintained the decreased pH or gradually increased, showing clearly abnormal pH movements compared to healthy individuals (Figure 1(B)). Thus, just by looking at the pH profile, the patient's condition can be roughly predicted.

[0058] (Example 3: Bacterial flora evaluation of fecal culture broths of healthy individuals and ulcerative colitis patients) (3-1: Extraction of microbial DNA) For the samples at the start of culture (fecal samples) and the samples collected 30 hours after culture (culture broths), DNA extraction of the bacteria in the colonic microbiota was performed as follows. For the extract in DNA extraction, 500 μL of Tris-EDTA (TE)-saturated phenol, 250 μL of lysis buffer (1 M Tris pH 8.0, 0.5 M EDTA), 50 μL of 10% SDS, and 0.3 g of glass beads (φ0.1 mm) were used. 200 μL of the extract was added to the sample, and using FastPrep24 (MP), it was vigorously shaken vertically at a power level of 5.0 for 30 seconds to disrupt the bacterial cells. Then, the supernatant was recovered by centrifugation, and after protein denaturation using phenol-chloroform-isoamyl alcohol, DNA was precipitated by isopropanol precipitation. The DNA precipitate was dissolved in 100 μL of TE and used as template DNA. The purified DNA was stored at -20°C until use.

[0059] (3-2: 16S rRNA gene sequencing) Genomic DNA was subjected to amplification of the V3-V4 region of the bacterial 16S rRNA gene using the primer pairs of S-D-Bact-0341-b-S-17 (SEQ ID NO: 1) and S-D-Bact-0785-a-A-21 (SEQ ID NO: 2). The Illumina adapter overhang nucleotide sequence (manufactured by Illumina, Inc.) was added to the gene-specific sequence. The PCR cycling reaction was performed according to the manufacturer's instructions. The confirmed amplicons were purified using AMPure XP DNA purification beads (manufactured by Beckman Coulter, Inc.) and eluted in 25 μl of 10 mM Tris (pH 8.5). The amplicons were quantified using an Agilent Bioanalyzer 2100 DNA 1000 chip (manufactured by Agilent Technologies, Inc.) and pooled at equimolar concentrations. The 16S rRNA gene products (along with the internal control (PhiX Control V3; manufactured by Illumina, Inc.)) were subjected to paired-end sequencing using a MiSeq sequencer (manufactured by Illumina, Inc.) together with the 600-cycle MiSeq reagent kit (manufactured by Illumina, Inc.). The PhiX sequences were trimmed, and paired-end reads with a Q-score of 20 or more were joined using the software package QIIME version 1.9.1. Chimeric sequences were identified and excluded from the library. The paired-end reads were taxonomically classified using the GreenGenes phylogenetic database by the Ribosomal Database Project (RDP) Classifier. The OTUs (Operational Taxonomic Units) that reached 97% similarity were used for diversity comparison of the Shannon-Weiner index and Simpson index and for Chao's species number estimation. Principal coordinate analysis (PCoA) was performed using the OTU information from each sample and calculated based on the unweighted UniFrac distance. All of the raw sequence data generated in this experiment were deposited in MG-RAST and assigned an accession number.

[0060] (3-3: Real-time PCR) Real-time PCR was performed using a TP700 Thermal Cycler Dice Real Time System Lite (manufactured by Takara Bio Inc.). As described in Non-Patent Document 1, amplification was carried out using a primer set targeting all intestinal bacteria.

[0061] (3-4: Bioinformatics and Statistical Analysis) The Shannon-Weiner diversity index, Simpson index, and Chao1 were calculated using the QIIME software package. Non-parametric Kruskal-Wallis tests were used to determine significant differences in the number of OTUs, Shannon-Weiner diversity index, Simpson index, Chao1, and the bacterial 16S rRNA gene and SCFA concentrations. The differences in the microbial community composition described in unweighted UniFrac were tested using a MANOVA test. A P-value of less than 0.05 was considered significant.

[0062] (3-5: Results) For each of the healthy subjects and ulcerative colitis (UC) patients, the 16S rRNA gene sequencing data of the samples at the start of culture (fecal samples) and the samples collected 30 hours after culture (culture broth samples) are shown in Table 1 below.

[0063]

Table 1

[0064] The read (number of decoded sequences) is an index of the number of bacteria, and the number of OTUs is an index of the number of bacterial species. The Shannon-Weiner diversity index ("Shannon") and Simpson index ("Simpson") are indices of species diversity, and Chao1 is an index of species richness.

[0065] The number of OTUs in the fecal samples of healthy subjects (collected at the start of culturing) was maintained in the culture fluid samples (samples collected 30 hours after culturing). Similarly, in patients with ulcerative colitis, the number of OTUs in the fecal samples was maintained in the culture fluid samples. For healthy subjects, there were no significant differences in the Shannon - Wiener diversity index, Simpson index, and Chao1 between fecal samples and culture fluid samples. For patients with ulcerative colitis, there were also no significant differences in all diversity indices of the Shannon - Wiener diversity index, Simpson index, and Chao1 between fecal samples and culture fluid samples. Therefore, it can be seen that the species diversity and the number of bacterial species in the bacterial flora in fecal samples are maintained in culture fluid samples as well.

[0066] There was no significant difference in the number of OTUs in fecal samples between healthy subjects and patients with ulcerative colitis, and this tendency was reproduced in the culture fluid samples. Furthermore, in terms of the Shannon - Wiener diversity index, Simpson index, and Chao1 diversity index, there were no significant differences in fecal samples between healthy subjects and patients with ulcerative colitis, and the tendency of no significant differences in all these indices was also reproduced in the culture fluid samples. Therefore, the relationship between species diversity and the number of bacterial species between healthy subjects and patients with ulcerative colitis was the same in both fecal samples and culture fluid samples.

[0067] Figure 3 is a graph showing the principal coordinate analysis (PCoA) of 16S rRNA gene sequencing data of samples at the start of culturing (fecal samples) and samples collected 30 hours after culturing (culture fluid samples) for each of healthy subjects (HS) and patients with ulcerative colitis (UC). In Figure 3, "HS feces" represents fecal samples of healthy subjects, "UC feces" represents fecal samples of patients with ulcerative colitis, "HS culture fluid" represents culture fluid samples of healthy subjects, and "UC culture fluid" represents culture fluid samples of patients with ulcerative colitis.

[0068] As shown in Fig. 3, the UC fecal plots formed different clusters from the HS fecal plots. This indicates that in fecal samples, the microbiota of patients with ulcerative colitis is distinguishable from that of healthy individuals. According to Fig. 3, it can also be seen that such differences in microbiota between patients with ulcerative colitis and healthy individuals were maintained in the comparison of the cluster formation of the culture fluid samples.

[0069] Fig. 4 is a graph showing the abundance ratios of various intestinal bacterial families to the total intestinal bacteria in samples at the start of culture (fecal samples) and samples collected 30 hours after culture (culture fluid samples) for each of healthy subjects (HS) and patients with ulcerative colitis (UC). In Fig. 4, "HS feces" represents fecal samples from healthy subjects, "UC feces" represents fecal samples from patients with ulcerative colitis, "HS culture fluid" represents culture fluid samples from healthy subjects, and "UC culture fluid" represents culture fluid samples from patients with ulcerative colitis. As shown in Fig. 4, interestingly, the abundance ratio of bacteria of the family Lachnospiraceae was significantly lower in patients with ulcerative colitis than in healthy individuals in both fecal samples and culture fluid samples. This point is shown more clearly in Fig. 5. Fig. 5 is a box-and-whisker plot showing the abundance ratio of Lachnospiraceae bacteria to the total intestinal bacteria in samples at the start of culture (fecal samples) and samples collected 30 hours after culture (culture fluid samples) for each of healthy subjects and patients with ulcerative colitis (UC). The vertical axis in Fig. 5 indicates the abundance ratio (%) of Lachnospiraceae bacteria to the total intestinal bacteria, and the horizontal axis indicates the measured samples. Through this culture, the decrease in the abundance ratio of Lachnospiraceae bacteria in patients with ulcerative colitis compared to healthy individuals observed in fecal samples was also observed in culture fluid samples.

[0070] The abundance ratios of bacterial species (at the genus level) belonging to the family Lachnospiraceae to the total intestinal bacteria in samples at the start of culture (fecal samples) and samples collected 30 hours after culture (culture fluid samples) for each of healthy subjects and patients with ulcerative colitis (UC) were also determined. The results are shown in Table 2 below.

[0071] Regarding the number of bacteria of the bacterial species (at the genus level) belonging to the family Lachnospiraceae, the abundance ratios to the total intestinal bacteria in samples at the start of culture (fecal samples) and samples collected 30 hours after culture (culture fluid samples) for each of healthy subjects and patients with ulcerative colitis (UC) were also determined. The results are shown in Table 2 below.

[0072]

Table 2

[0073] Table 2 shows the results of the top 5 species in terms of abundance ratio among the bacterial species belonging to the family Lachnospiraceae, namely Blautia spp., Coprococcus spp., Dorea spp., Lachnospira spp., and Roseburia spp. In Table 2, the asterisk (*) indicates a significant difference between HS and UC patients in fecal samples and the corresponding culture fluid samples (*P<0.05, **P<0.01). For the Coprococcus spp. bacteria, a significant decrease in the abundance ratio of ulcerative colitis patients compared to healthy subjects was observed in both fecal samples and culture fluid samples.

[0074] (Example 4: SCFA Quantification of Fecal Cultures of Healthy Subjects and Ulcerative Colitis Patients) Regarding the samples (culture fluid) collected after 30 hours of culture, the amounts of various acids (succinic acid, lactic acid, acetic acid, propionic acid, and butyric acid) contained in short-chain fatty acids (SCFAs) in the samples were measured using high-performance liquid chromatography (HPLC) (Shimadzu Corporation) equipped with an Aminex HPX-87H column (manufactured by Bio-Rad Laboratories) and an RID-10A differential refractive index detector (Shimadzu Corporation). HPLC was operated at 65 °C using 5 mM H2SO4 as the mobile phase at a flow rate of 0.6 mL / min.

[0075] FIG. 6 is a box-and-whisker plot showing the amounts of acetic acid, propionic acid, butyric acid, and SCFA (total of succinic acid, lactic acid, acetic acid, propionic acid, and butyric acid) in samples (culture fluid samples) collected after 30 hours of culture for healthy subjects and patients with ulcerative colitis (UC), respectively. The vertical axis of each graph in FIG. 6 indicates the concentration (mM) of each acid in the sample, and the horizontal axis indicates the measured sample. In both healthy subjects and patients with ulcerative colitis, acetic acid, propionic acid, and butyric acid were mainly produced. Little production of lactic acid and succinic acid was observed. Regarding the amount of butyric acid produced, a significant decrease was seen in the culture fluid samples of patients with ulcerative colitis compared to those of healthy subjects.

[0076] FIG. 7A shows the result of plotting the presence ratio (%) (horizontal axis) of Ruminococcaceae bacteria in the culture fluid samples derived from patients with ulcerative colitis against the amount of butyric acid (mM) produced by culture (vertical axis). A high correlation was seen between the presence ratio of Ruminococcaceae bacteria in the culture fluid samples and the amount of butyric acid produced by culture (FIG. 7A). That is, the lower the presence ratio of Ruminococcaceae bacteria in the culture fluid samples, the lower the butyric acid production amount.

[0077] FIG. 7 shows the combined results of plotting the presence ratio (%) (horizontal axis) of Ruminococcaceae bacteria in fecal samples (B) and culture fluid samples (C) derived from patients with ulcerative colitis against the partial Mayo score (vertical axis). Regarding the partial Mayo score, no correlation was seen with the presence ratio of Ruminococcaceae bacteria in either the fecal samples or the culture fluid samples (FIGS. 7B and C).

[0078] Since ulcerative colitis is characterized by repeated exacerbation and remission phases, a low abundance of Lachnospiraceae bacteria and thus a low amount of butyric acid produced thereby can cause recurrence of colitis. Therefore, considering that butyric acid induces the differentiation of regulatory T cells in peripheral tissues and has an anti-inflammatory effect (Furusawa, Y. et al., Nature 10.1038 / nature12721. 2013), increasing butyric acid-producing bacteria in the intestine of patients with ulcerative colitis is important for maintaining remission. Furthermore, according to the above results, it can be seen that this culture model can be used to highly throughput evaluate the roles of prebiotics and probiotics on commensal bacteria and metabolite production in patients with ulcerative colitis.

Explanation of Signs

[0079] 100 Testing device 102 Culture solution 110 Anaerobic culture means 112 Culture tank 114 Lid 116 Motor 118 Shaft 120 Stirring blade 122 Air supply pipe 124 Valve 126 Exhaust pipe 128 Temperature control means 140 Measuring means 142 Temperature sensor 144 Chemical sensor 170 Comparison means 172 Control unit 174 Memory unit 176 Display unit

Claims

【Claim 1】 The invention described in the embodiments.

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