JP2024065767A - Method for detecting irritable bowel syndrome using enterobacteria and evaluation of therapeutic effect - Google Patents

Abstract

[Problem] To provide a method for detecting irritable bowel syndrome using intestinal bacteria. [Solution] A method for detecting irritable bowel syndrome, comprising detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of a subject. [Selected Figure] Figure 2

Description

The present invention relates to a method for detecting irritable bowel syndrome using intestinal bacteria and for assessing the effectiveness of treatment.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder without organic changes that presents with lower gastrointestinal symptoms (Non-Patent Document 1). IBS is found in 15% of young adults, and many cases go untreated because it cannot be diagnosed by imaging or blood tests.

D A Drossman et al. Dig Dis Sci Sep;38(9):1569-80.

If it were possible to test for IBS based on markers present in a subject's sample, it would be useful to be able to evaluate the subject's condition with a simple test.

The problem to be solved by the present invention is to provide a method for detecting irritable bowel syndrome using intestinal bacteria.

The present invention encompasses the embodiments described below.
Item 1. A method for detecting irritable bowel syndrome, comprising:
1. A method comprising detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of a subject.
Item 2. The method according to Item 1, wherein the detecting the bacterium comprises detecting a bacterium of the genus Lachnospiraceae Blautia.
Item 3. The method according to Item 1, wherein detecting the bacterium comprises measuring the amount of a nucleic acid sequence of 16S rRNA of the bacterium.
Item 4. The method for detecting irritable bowel syndrome includes a method for detecting the onset or predisposition to the onset of irritable bowel syndrome,
A high detection value of bacteria of the genus Lachnospiraceae Blautia compared to the standard value indicates the onset or predisposition to onset of irritable bowel syndrome,
A low detection value of bacteria of the genus Ruminococcaceae Oscillospira compared to the standard value indicates the onset or predisposition to onset of irritable bowel syndrome,
Item 2. The method according to Item 1, wherein a detection value of Faecalibacterium prausnitzii species that is lower than a reference value indicates the onset or predisposition to the onset of irritable bowel syndrome.
Item 5. The method according to Item 4, wherein the reference value is a measurement obtained from a healthy subject or a measurement obtained from the same subject after treatment for irritable bowel syndrome.
Item 6. The detection of the bacteria includes detecting at least one type of bacteria selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the subject's feces on a reference date and on a day after the reference date;
Item 2. The method according to item 1, wherein when the measurement on the subsequent day is at least any one of the following (i) to (iii) compared with the measurement on the reference day, it is highly likely that the symptoms of irritable bowel syndrome are worsening.
(i) the measured value of the bacteria of the genus Lachnospira Blautia on the later day is higher than that on the reference day; (ii) the measured value of the bacteria of the genus Ruminococcus oscillospira on the later day is lower than that on the reference day; (iii) the detected value of the species Faecalibacterium prausnitzii on the later day is lower than that on the reference day. Item 7. Detecting the bacteria includes detecting at least one type of bacteria selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the subject's feces on the reference day and on a day after the reference day;
Item 2. The method according to item 1, wherein the measurement on the subsequent day is at least any one of the following (i) to (iii) when compared with the measurement on the reference day, indicating that the symptoms of irritable bowel syndrome are unlikely to worsen:
(i) the measured value of the bacteria of the genus Lachnospira Blautia on the later day is the same or lower than the measured value on the reference day; (ii) the measured value of the bacteria of the genus Ruminococcus oscillospira on the later day is the same or higher than the measured value on the reference day; (iii) the detected value of the species Faecalibacterium prausnitzii on the later day is the same or higher than the detected value on the reference day. Item 8. The method for detecting irritable bowel syndrome includes a method for evaluating the effectiveness of a measure for treating, preventing or improving irritable bowel syndrome,
Detecting the bacteria includes detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of the treated subject;
Item 2. The method according to Item 1, which indicates that the measure is likely to be effective in treating, preventing, or ameliorating irritable bowel syndrome when the detected value after the measure is at least any one of the following (i) to (iii) compared with the reference value or the value of the same patient before the measure:
(i) the detection value of bacteria of the genus Lachnospiraceae Blautia after the treatment is lower than the standard value or the value before the treatment; (ii) the detection value of bacteria of the genus Ruminococcaceae Oscillospira after the treatment is higher than the standard value or the value before the treatment; (iii) the detection value of the species Faecalibacterium prausnitzii after the treatment is higher than the standard value or the value before the treatment. Item 9. A method for screening for a treatment effective for treating, preventing, or improving irritable bowel syndrome, comprising:
detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of the treated subject;
The method comprises selecting the measure as one that is likely to be effective in treating, preventing, or ameliorating irritable bowel syndrome when the detected value after the treatment is at least any one of the following (i) to (iii) compared with a reference value or the value of the same patient before the treatment.
(i) the detection value of bacteria of the genus Lachnospiraceae Blautia after the treatment is lower than the standard value or the value before the treatment; (ii) the detection value of bacteria of the genus Ruminococcaceae Oscillospira after the treatment is higher than the standard value or the value before the treatment; (iii) the detection value of the species Faecalibacterium prausnitzii after the treatment is higher than the standard value or the value before the treatment. Item 10. The method according to item 7 or 8, wherein the treatment is at least one selected from the group consisting of administration of medicine, administration of food, dietary therapy, and fecal transplantation.
Item 11. The method according to any one of Items 1 to 10, wherein the irritable bowel syndrome is diarrhea-predominant irritable bowel syndrome.
Item 12. Use of the expression level of at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and Faecalibacterium prausnitzii as a biomarker for diagnosing and/or monitoring irritable bowel syndrome in vitro.
Item 13. A diagnostic kit for irritable bowel syndrome, comprising:
(i) a nucleic acid probe capable of hybridizing with a nucleic acid sequence of 16S rRNA or a nucleic acid sequence of 16S rDNA of at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii; or (ii) a kit comprising a pair of nucleic acid primers capable of specifically amplifying a nucleic acid sequence of 16S rRNA or a nucleic acid sequence of 16S rDNA of at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii.

Clinical study timeline. Graph showing the relative abundance of bacteria of the genus Lachnospiraceae Blautia in each group. A graph showing the relative abundance of bacteria of the genus Ruminococcaceae Oscillospira in each group; Graph showing the relative abundance of Faecalibacterium prausnitzii in each group An explanatory diagram of the bacterial species that showed differences between groups.

In this specification, "detection" of irritable bowel syndrome (IBS) includes the determination of IBS, tests to assist in the diagnosis of IBS, and test methods for treating IBS, the determination of IBS patients (responders) who respond to treatment for IBS (e.g., therapeutic drugs), the determination of the preventive effect of IBS, and the determination of the therapeutic effect of IBS. "Determination" of IBS includes not only the determination of IBS, but also the preventive determination of the predisposition or risk of developing IBS, and the determination of the therapeutic effect of IBS treatment after the treatment.

As used herein, "treatment" means curing or ameliorating a disease, or curing, ameliorating, or suppressing a symptom, and includes "prevention." "Prevention" means preventing the onset of a disease or symptom.

As used herein, 16S ribosomal RNA (16S rDNA) refers to the DNA that encodes 16S rRNA.

In this specification, detection of 16S rRNA includes measurement of the amount of the nucleic acid sequence of 16S rRNA, and is detected by amplifying any of the variable regions V1 to V9 of the 16S rRNA gene. The amount of the nucleic acid sequence of 16S rRNA can be detected by measuring the number of sequence data (hereinafter simply referred to as the number of data) of the 16S ribosomal RNA (rRNA) gene, which is a conserved region unique to bacterial DNA.

The inventors found that the relative amounts of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and Faecalibacterium prausnitzii in the feces of subjects to the total number of bacteria (specifically, the number of amplified reads) differed between healthy subjects and IBS patients. These three types of bacteria may be used as markers for diagnosing or determining whether or not a patient has IBS.

The inventors also found that the relative amounts of the bacteria Lachnospira Blautia, Ruminococcus oscillospira, and Faecalibacterium prausnitzii in the feces of subjects vary depending on the condition of the same IBS patient, and also vary before and after treatment in the same IBS patient. These three types of bacteria may be used as markers for diagnosing or determining the condition of IBS patients, or as markers that can quantitatively grasp the condition of IBS patients.

Furthermore, the inventors found that administration of Kampo medicine to IDS patients changes the relative amounts of bacteria of the genus Lachnospira Blautia, bacteria of the genus Ruminococcus oscillospira, and Faecalibacterium prausnitzii. These three types of bacteria may be used to screen for effective measures for the treatment, prevention, or improvement of IBS.

According to one aspect of the present invention, there is provided a method for detecting irritable bowel syndrome, comprising the steps of:
A method is provided that includes detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of a subject. The method for detecting irritable bowel syndrome may also be referred to as a method for providing data for the detection of irritable bowel syndrome.

Irritable bowel syndrome includes diarrhea-predominant irritable bowel syndrome, constipation-predominant irritable bowel syndrome, mixed type irritable bowel syndrome, and unclassifiable type irritable bowel syndrome.

In some embodiments, the irritable bowel syndrome is diarrhea-predominant irritable bowel syndrome.

The subject includes a mammal, preferably a human.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Lachnospira Blautia.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Ruminococcus oscillospira.

In some embodiments, detecting the bacteria includes detecting Faecalibacterium prausnitzii.

In some embodiments, detecting the bacteria includes measuring the amount of bacteria, and measuring the amount of bacteria includes a nucleic acid-based quantification method, such as 16S rRNA gene amplicon sequencing, in which a region of 16S rRNA (16S ribosomal rRNA) is amplified by PCR and sequenced. Methods for the qualitative and quantitative measurement of bacteria in a sample using 16S rRNA gene amplicon sequencing are described in the literature and known to those of skill in the art. Other techniques may include PCR, rtPCR, qPCR, high-throughput sequencing, metatranscriptome sequencing, or 16S rRNA analysis.

In some embodiments, detecting the bacteria includes measuring the number of reads of the at least one bacterium relative to the total number of bacteria (particularly, the total number of amplified reads) in the subject's feces or the number of reads of the operational taxonomic unit (OTU) to which the bacteria belongs. As known in the art, an operational taxonomic unit (OTU) is an operational definition used to classify closely related populations. As used herein, an "OTU" is a group of organisms classified by DNA sequence similarity of specific taxonomic marker genes.

In some embodiments, the Ribosomal Database Project (RDP) classifier is used to assign taxonomy to representative OTU sequences.

For example, the base sequence of SEQ ID NO: 1 is used to classify whether a bacterium belongs to the genus Lachnospira Blautia.
TGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGATAGTGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTGTGGCAAGTCTGATGTGAAAGGCATGGGCTCAACCTGTGGACTGCATTGGAAACTGTCATACTTGAGTGCCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGG (SEQ ID NO: 1)

The base sequence of SEQ ID NO:2 is used to classify whether a bacterium belongs to the genus Ruminococcaceae Oscillospira.
TGGGGAATATTGGGCAATGGGCGCAAGCCTGACCCAGCAACGCCGCGTGAAGGAAGAAGGCTTTCGGGTTGTAAACTTCTTTTGTCGGGGACGAAACAAATGACGGTACCCGACGAATAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGCGTGTAGGCGGGATTGCAAGTCAGATGTGAAAACTGGGGGCTCAACCTCCAGCCTGCATTTGAAACTGTAGTTCTTGAGTGCTGGAGAGGCAATCGGAATTCCGTGTGTAGCGGTGAAATGCGTAGATATACGGAGGAACACCAGTGGCGAAGGCGGATTGCTGGACAGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGG (SEQ ID NO: 2)

The base sequence of SEQ ID NO:3 is used to classify whether a bacterium belongs to the species Faecalibacterium prausnitzii.
TGGGGAATATTGCACAATGGGGGGAACCCTGATGCAGCGACGCCGCGTGGAGGAAGAAGGTCTTCGGATTGTAAACTCCTGTTGTTGAGGAAGATAATGACGGTACTCAACAAGGAAGTGACGGCTAACTACGTGCCAGCAGCCGCGGTAAAACGTAGGTCACAAGCGTTGTCCGGAATTACTGGGTGTAAAGGGAGCGCAGGCGGGAGAACAAGTTGGAAGTGAAATCCATGGGCTCAACCCATGAACTGCTTTCAAAACTGTTTTTCTTGAGTAGTGCGAGAGGTAGGCGGAATTCCCGGTGTAGCGGTGGAATGCGTAGATATCGGGAGGAACACCAGTGGCGAAGGCGGCCTACTGGGCACCAACTGACGCTGAGGCTCGAAAGTGTGGGTAGCAAACAGG (SEQ ID NO: 3)

In some embodiments, detecting the bacteria includes measuring the amount of the at least one type of bacteria in the subject's feces before a therapeutic, preventive, or ameliorative treatment and the amount of the at least one type of bacteria in the subject's feces after the treatment. The amount of bacteria can be measured, for example, from the number of reads of the bacteria relative to the total number of bacteria (particularly, the number of amplified reads) in the subject's feces or the number of reads of the operational taxonomic unit (OTU) to which the bacteria belongs. The treatment is, for example, at least one selected from the group consisting of administration of a medicine, administration of a food, a dietary therapy, and a fecal transplant.

According to one embodiment of the present invention, the method for detecting irritable bowel syndrome includes a method for detecting the onset or predisposition to the onset of irritable bowel syndrome, comprising detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the stool of a subject;
A high detection value of bacteria of the genus Lachnospira Blautia compared to the standard value indicates the onset or predisposition to onset of irritable bowel syndrome,
A lower detection value of the bacteria of the genus Ruminococcus oscillospira compared to the standard value indicates the onset or predisposition to onset of irritable bowel syndrome,
and/or a lower detection level of the species Faecalibacterium prausnitzii compared to a reference level indicates the onset or predisposition to the onset of irritable bowel syndrome.

Irritable bowel syndrome includes diarrhea-predominant irritable bowel syndrome, constipation-predominant irritable bowel syndrome, mixed type irritable bowel syndrome, and unclassifiable type irritable bowel syndrome.

In some embodiments, the irritable bowel syndrome is diarrhea-predominant irritable bowel syndrome.

The subject includes a mammal, preferably a human.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Lachnospira Blautia.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Ruminococcus oscillospira.

In some embodiments, detecting the bacteria includes detecting Faecalibacterium prausnitzii.

In some embodiments, detecting the bacteria includes measuring the amount of bacteria, and measuring the amount of bacteria includes a nucleic acid-based quantification method, such as 16S rRNA gene amplicon sequencing, in which a region of 16S rRNA (16S ribosomal rRNA) is amplified by PCR and sequenced. Methods for the qualitative and quantitative measurement of bacteria in a sample using 16S rRNA gene amplicon sequencing are described in the literature and known to those of skill in the art. Other techniques may include PCR, rtPCR, qPCR, high-throughput sequencing, metatranscriptome sequencing, or 16S rRNA analysis.

In some embodiments, detecting the bacterium includes measuring the number of reads of the bacterium relative to the total number of bacteria (particularly the number of amplified reads) in the subject's feces or the number of reads of the operational taxonomic unit (OTU) to which the bacterium belongs. As is known in the art, an operational taxonomic unit (OTU) is an operational definition used to classify closely related populations.

As used herein, an "OTU" is a group of organisms classified by DNA sequence similarity of specific taxonomic marker genes. In some embodiments, the Ribosomal Database Project (RDP) classifier is used to assign taxonomy to representative OTU sequences.

In some embodiments, the reference value is a measurement value obtained from a healthy subject, or a measurement value obtained from the same subject after treatment for irritable bowel syndrome. The measurement value obtained from a healthy subject is, for example, the average or median of measurements obtained from multiple healthy subjects. In some embodiments, the reference value is a certain value that is higher than the average or median of multiple healthy subjects and lower than the average or median of multiple patient groups (hereinafter simply referred to as "patient groups" in this paragraph) diagnosed by a doctor as suffering from irritable bowel syndrome. In some embodiments, the reference value is a cutoff value that separates the healthy subject group from the patient group. The cutoff value can be determined by various statistical analysis methods well known to those skilled in the art. For example, in some embodiments, the cutoff value is a value at which the P value in the log-rank test is minimum in the measurements of the amount of bacteria in the healthy subject group and the patient group, or a value at which the P value is less than a certain level (e.g., P value < 0.05 or P value < 0.01). In some embodiments, the cutoff value is a value determined based on ROC (Receiver Operating Characteristic) analysis so as to maximize the sum of sensitivity and specificity in the measured values of the amount of bacteria in the healthy subject group and the patient group. In some embodiments, the cutoff value is a value at which the P value in the chi-square test is minimized or the P value is less than a certain level (e.g., P value < 0.05 or P value < 0.01) in the measured values of the amount of bacteria in the healthy subject group and the patient group. The specific examples of the reference values described in this paragraph can be applied in an appropriate manner to each embodiment of the detection method of the present specification.

A high detection value of bacteria of the genus Lachnospira Blautia compared to a standard value indicates the onset or predisposition to onset of irritable bowel syndrome. Therefore, when the measured amount of bacteria of the genus Lachnospira Blautia in the subject's feces is high compared to a standard value, the subject can be diagnosed or determined to have a high possibility of onset of irritable bowel syndrome or a high predisposition to onset. When the measured amount of bacteria of the genus Lachnospira Blautia in the subject's feces is the same as or lower than the standard value, the subject can be diagnosed or determined to have a low possibility of onset of irritable bowel syndrome or a low predisposition to onset.

A lower detection value of bacteria of the genus Ruminococcus oscillospira compared to the standard value indicates the onset or predisposition to onset of irritable bowel syndrome. Therefore, if the measured amount of bacteria of the genus Ruminococcus oscillospira in the subject's feces is lower than the standard value, the subject can be diagnosed or determined to have a high possibility of onset of irritable bowel syndrome or a high predisposition to onset. If the measured amount of bacteria of the genus Ruminococcus oscillospira in the subject's feces is the same as or higher than the standard value, the subject can be diagnosed or determined to have a low possibility of onset of irritable bowel syndrome or a low predisposition to onset.

A lower detection value of Faecalibacterium prausnitzii compared to the standard value indicates the onset or predisposition to onset of irritable bowel syndrome. Therefore, if the measured amount of Faecalibacterium prausnitzii bacteria in the subject's stool is lower compared to the standard value, the subject can be diagnosed or determined to have a high probability of onset of irritable bowel syndrome or a high predisposition to onset. If the measured amount of Faecalibacterium prausnitzii bacteria in the subject's stool is the same as or higher than the standard value, the subject can be diagnosed or determined to have a low probability of onset of irritable bowel syndrome or a low predisposition to onset.

According to one aspect of the present invention, the method for detecting irritable bowel syndrome includes a method for testing for symptoms of irritable bowel syndrome. The method for testing for symptoms of irritable bowel syndrome includes detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the stool of a subject on a reference date and on a day after the reference date.

Irritable bowel syndrome includes diarrhea-predominant irritable bowel syndrome, constipation-predominant irritable bowel syndrome, mixed type irritable bowel syndrome, and unclassifiable type irritable bowel syndrome.

In some embodiments, the irritable bowel syndrome is diarrhea-predominant irritable bowel syndrome.

The subject includes a mammal, preferably a human.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Lachnospira Blautia.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Ruminococcus oscillospira.

In some embodiments, detecting the bacteria includes detecting Faecalibacterium prausnitzii.

In some embodiments, detecting the bacteria includes measuring the amount of bacteria, and measuring the amount of bacteria includes a nucleic acid-based quantification method, such as 16S rRNA gene amplicon sequencing, in which a region of 16S rRNA (16S ribosomal rRNA) is amplified by PCR and sequenced. Methods for the qualitative and quantitative measurement of bacteria in a sample using 16S rRNA gene amplicon sequencing are described in the literature and known to those of skill in the art. Other techniques may include PCR, rtPCR, qPCR, high-throughput sequencing, metatranscriptome sequencing, or 16S rRNA analysis.

In some embodiments, detecting the bacteria includes measuring the number of reads of the at least one bacteria relative to the total number of bacteria (particularly the number of amplified reads) in the subject's feces or the number of reads of an operational taxonomic unit (OTU). As known in the art, an operational taxonomic unit (OTU) is an operational definition used to classify closely related populations. As used herein, an "OTU" is a group of organisms classified by DNA sequence similarity of specific taxonomic marker genes. In some embodiments, the Ribosomal Database Project (RDP) classifier is used to assign taxonomy to representative OTU sequences.

In some embodiments, a higher measurement value of bacteria of the genus Lachnospira Blautia on a later day compared to the measurement value on the reference day indicates an exacerbation of symptoms of irritable bowel syndrome, a lower measurement value of bacteria of the genus Ruminococcus oscillospira on a later day compared to the measurement value on the reference day indicates an exacerbation of symptoms of irritable bowel syndrome, and/or a lower detection value of the species Faecalibacterium prausnitzii on a later day compared to the value on the reference day indicates an exacerbation of symptoms of irritable bowel syndrome. The reference day is, for example, the day (exacerbation day) when the subject notices that their IBS symptoms (e.g., diarrhea) are severe.

Therefore, if the amount of bacteria of the genus Lachnospira Blautia in the subject's feces on a later day is higher than the amount on the reference day, it can be diagnosed or determined that there is a high possibility that the symptoms of irritable bowel syndrome in the subject are worsening. If the amount of bacteria of the genus Lachnospira Blautia in the subject's feces on a later day is the same as or lower than the amount on the reference day, it can be diagnosed or determined that there is a low possibility that the symptoms of irritable bowel syndrome in the subject are worsening.

If the amount of Ruminococcus oscillospira bacteria in the subject's feces on a later day is lower compared to the measurement on the reference day, it can be diagnosed or determined that there is a high possibility that the symptoms of irritable bowel syndrome in the subject are worsening.If the amount of Ruminococcus oscillospira bacteria in the subject's feces on a later day is the same as or higher compared to the measurement on the reference day, it can be diagnosed or determined that there is a low possibility that the symptoms of irritable bowel syndrome in the subject are worsening.

If the amount of Faecalibacterium prausnitzii bacteria in the subject's stool on a later day is lower compared to the baseline measurement, it can be diagnosed or determined that the subject's symptoms of irritable bowel syndrome are likely to be worsening. If the amount of Faecalibacterium prausnitzii bacteria in the subject's stool on a later day is the same or higher compared to the baseline measurement, it can be diagnosed or determined that the subject's symptoms of irritable bowel syndrome are unlikely to be worsening.

In some further embodiments, a lower measurement of the bacteria of the genus Lachnospira Blautia on a later day compared to the measurement on the reference day indicates an improvement in the symptoms of irritable bowel syndrome, a higher measurement of the bacteria of the genus Ruminococcus oscillospira on a later day compared to the measurement on the reference day indicates an improvement in the symptoms of irritable bowel syndrome, and/or a higher detection value of the species Faecalibacterium prausnitzii on a later day compared to the value on the reference day indicates an improvement in the symptoms of irritable bowel syndrome. The reference day is, for example, a day on which the subject feels that he or she has no or only mild IBS symptoms (e.g., diarrhea) (a mild day).

Therefore, if the amount of Lachnospira Blautia bacteria in the subject's feces on a later day is lower than the amount on the reference day, it can be diagnosed or determined that the subject's symptoms of irritable bowel syndrome are likely to have improved. If the amount of Lachnospira Blautia bacteria in the subject's feces on a later day is the same as or higher than the amount on the reference day, it can be diagnosed or determined that the subject's symptoms of irritable bowel syndrome are unlikely to have improved.

If the amount of Ruminococcus oscillospira bacteria in the subject's feces measured on a later day is higher than the amount on the reference day, it can be diagnosed or determined that the subject's symptoms of irritable bowel syndrome are likely to have improved.If the amount of Ruminococcus oscillospira bacteria in the subject's feces measured on a later day is the same as or lower than the amount on the reference day, it can be diagnosed or determined that the subject's symptoms of irritable bowel syndrome are unlikely to have improved.

If the amount of Faecalibacterium prausnitzii bacteria in the subject's stool on a later day is higher than the baseline measurement, it can be diagnosed or determined that the subject's symptoms of irritable bowel syndrome are likely to have improved.If the amount of Faecalibacterium prausnitzii bacteria in the subject's stool on a later day is the same as or lower than the baseline measurement, it can be diagnosed or determined that the subject's symptoms of irritable bowel syndrome are unlikely to have improved.

According to one embodiment of the present invention, the method for detecting irritable bowel syndrome includes a method for evaluating the effectiveness of a measure for treating, preventing, or ameliorating irritable bowel syndrome. The method for evaluating the effectiveness of a measure for treating, preventing, or ameliorating irritable bowel syndrome includes:
detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of the treated subject;
When the detected value after the treatment is at least any one of the following (i) to (iii) compared with the reference value or the value of the same patient before the treatment, this indicates that the treatment is likely to be effective in treating, preventing, or ameliorating irritable bowel syndrome.
(i) The detection value of bacteria of the genus Lachnospiraceae Blautia after the treatment is lower than the standard value or the value before the treatment. (ii) The detection value of bacteria of the genus Ruminococcaceae Oscillospira after the treatment is higher than the standard value or the value before the treatment. (iii) The detection value of the species Faecalibacterium prausnitzii after the treatment is higher than the standard value or the value before the treatment.

In some embodiments, the treatment is at least one selected from the group consisting of administration of a medication, administration of a food, a dietary regimen, and a fecal transplant.

Irritable bowel syndrome includes diarrhea-predominant irritable bowel syndrome, constipation-predominant irritable bowel syndrome, mixed type irritable bowel syndrome, and unclassifiable type irritable bowel syndrome.

In some embodiments, the irritable bowel syndrome is diarrhea-predominant irritable bowel syndrome.

The subject includes a mammal, preferably a human.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Lachnospira Blautia.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Ruminococcus oscillospira.

In some embodiments, detecting the bacteria includes detecting Faecalibacterium prausnitzii.

In some embodiments, detecting the bacteria includes measuring the amount of bacteria, and measuring the amount of bacteria includes a nucleic acid-based quantification method, such as 16S rRNA gene amplicon sequencing, in which a region of 16S rRNA (16S ribosomal rRNA) is amplified by PCR and sequenced. Methods for the qualitative and quantitative measurement of bacteria in a sample using 16S rRNA gene amplicon sequencing are described in the literature and known to those of skill in the art. Other techniques may include PCR, rtPCR, qPCR, high-throughput sequencing, metatranscriptome sequencing, or 16S rRNA analysis.

In some embodiments, detecting the bacteria includes measuring the number of reads of the at least one bacteria relative to the total number of bacteria (particularly the number of amplified reads) in the subject's feces or the number of reads of an operational taxonomic unit (OTU). As known in the art, an operational taxonomic unit (OTU) is an operational definition used to classify closely related populations. As used herein, an "OTU" is a group of organisms classified by DNA sequence similarity of specific taxonomic marker genes. In some embodiments, the Ribosomal Database Project (RDP) classifier is used to assign taxonomy to representative OTU sequences.

If the detection value of bacteria of the genus Lachnospira Blautia after the treatment is lower than the standard value or the value before the treatment, it indicates that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. Therefore, if the measured value of the amount of bacteria of the genus Lachnospira Blautia in the subject's feces after the treatment is lower than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. If the measured value of the amount of bacteria of the genus Lachnospira Blautia in the subject's feces after the treatment is the same as or higher than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be ineffective in treating, preventing, or improving irritable bowel syndrome.

If the detection value of bacteria of the genus Ruminococcus oscillospira after the treatment is higher than the standard value or the value before the treatment, it indicates that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. Therefore, if the measured value of the amount of bacteria of the genus Ruminococcus oscillospira in the subject's feces after the treatment is higher than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. If the measured value of the amount of bacteria of the genus Ruminococcus oscillospira in the subject's feces after the treatment is the same as or lower than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be ineffective in treating, preventing, or improving irritable bowel syndrome.

If the detection value of Faecalibacterium prausnitzii after the treatment is higher than the standard value or the value before the treatment, it indicates that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. Therefore, if the measured value of the amount of Faecalibacterium prausnitzii in the subject's feces after the treatment is higher than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. If the measured value of the amount of Faecalibacterium prausnitzii in the subject's feces after the treatment is the same as or lower than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be ineffective in treating, preventing, or improving irritable bowel syndrome.

According to one aspect of the present invention, there is provided a method for screening for a measure effective for treating, preventing, or ameliorating irritable bowel syndrome, comprising the steps of:
detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of the treated subject;
The method includes selecting the measure as one that is likely to be effective in treating, preventing, or ameliorating irritable bowel syndrome when the detected value after the treatment is at least any one of the following (i) to (iii) compared with a reference value or a value of the same patient before the treatment:
(i) The detection value of bacteria of the genus Lachnospiraceae Blautia after the treatment is lower than the standard value or the value before the treatment. (ii) The detection value of bacteria of the genus Ruminococcaceae Oscillospira after the treatment is higher than the standard value or the value before the treatment. (iii) The detection value of the species Faecalibacterium prausnitzii after the treatment is higher than the standard value or the value before the treatment.

In some embodiments, the treatment is at least one selected from the group consisting of administration of a medication, administration of a food, a dietary regimen, and a fecal transplant.

Irritable bowel syndrome includes diarrhea-predominant irritable bowel syndrome, constipation-predominant irritable bowel syndrome, mixed type irritable bowel syndrome, and unclassifiable type irritable bowel syndrome.

In some embodiments, the irritable bowel syndrome is diarrhea-predominant irritable bowel syndrome.

The subject includes a mammal, preferably a human.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Lachnospira Blautia.

In some embodiments, detecting the bacteria includes detecting bacteria of the genus Ruminococcus oscillospira.

In some embodiments, detecting the bacteria includes detecting Faecalibacterium prausnitzii.

In some embodiments, detecting the bacteria includes measuring the amount of bacteria, and measuring the amount of bacteria includes a nucleic acid-based quantification method, such as 16S rRNA gene amplicon sequencing, in which a region of 16S rRNA (16S ribosomal rRNA) is amplified by PCR and sequenced. Methods for the qualitative and quantitative measurement of bacteria in a sample using 16S rRNA gene amplicon sequencing are described in the literature and known to those of skill in the art. Other techniques may include PCR, rtPCR, qPCR, high-throughput sequencing, metatranscriptome sequencing, or 16S rRNA analysis.

In some embodiments, detecting the bacteria includes measuring the number of reads of the at least one bacteria relative to the total number of bacteria (particularly the number of amplified reads) in the subject's feces or the number of reads of an operational taxonomic unit (OTU). As known in the art, an operational taxonomic unit (OTU) is an operational definition used to classify closely related populations. As used herein, an "OTU" is a group of organisms classified by DNA sequence similarity of specific taxonomic marker genes. In some embodiments, the Ribosomal Database Project (RDP) classifier is used to assign taxonomy to representative OTU sequences.

If the detection value of bacteria of the genus Lachnospira Blautia after the treatment is lower than the standard value or the value before the treatment, it indicates that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. Therefore, if the measured value of the amount of bacteria of the genus Lachnospira Blautia in the subject's feces after the treatment is lower than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. If the measured value of the amount of bacteria of the genus Lachnospira Blautia in the subject's feces after the treatment is the same as or higher than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be ineffective in treating, preventing, or improving irritable bowel syndrome.

If the detection value of bacteria of the genus Ruminococcus oscillospira after the treatment is higher than the standard value or the value before the treatment, it indicates that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. Therefore, if the measured value of the amount of bacteria of the genus Ruminococcus oscillospira in the subject's feces after the treatment is higher than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. If the measured value of the amount of bacteria of the genus Ruminococcus oscillospira in the subject's feces after the treatment is the same as or lower than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be ineffective in treating, preventing, or improving irritable bowel syndrome.

If the detection value of Faecalibacterium prausnitzii after the treatment is higher than the standard value or the value before the treatment, it indicates that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. Therefore, if the measured value of the amount of Faecalibacterium prausnitzii in the subject's feces after the treatment is higher than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be effective in treating, preventing, or improving irritable bowel syndrome. If the measured value of the amount of Faecalibacterium prausnitzii in the subject's feces after the treatment is the same as or lower than the standard value or the value before the treatment, it can be diagnosed or determined that the treatment is likely to be ineffective in treating, preventing, or improving irritable bowel syndrome.

In some embodiments, the method of any of the above aspects preferably has a sensitivity of greater than 40% (e.g., greater than 45%, greater than 50%, or greater than 55%) and a specificity of greater than 90% (e.g., greater than 93% or greater than 95%). The area under the curve (AUC) is preferably 0.60 or greater (e.g., 0.70 or greater, or 0.80 or greater).

According to one aspect of the present invention, there is provided an in vitro use of the expression level of at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii as a biomarker for diagnosing and/or monitoring irritable bowel syndrome.

These bacteria can be used as biomarkers in any of the methods of the present invention described above.

According to one aspect of the present invention, there is provided a kit for diagnosing irritable bowel syndrome, comprising:
(i) a nucleic acid probe capable of hybridizing with a nucleic acid sequence of 16S rRNA or a nucleic acid sequence of 16S rDNA of at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii; or (ii) a pair of nucleic acid primers capable of specifically amplifying a nucleic acid sequence of 16S rRNA or a nucleic acid sequence of 16S rDNA of at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii.

The kit may include a nucleic acid probe or detection reagent for detecting the amplified 16S rRNA or 16S rDNA nucleic acid sequence. The kit may include instructions for use.

The disclosures of all patent applications and literature cited herein are hereby incorporated by reference in their entireties.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these.

I. Research Objectives This research has three objectives:
1. To clarify the differences in physical characteristics and gut microbiota characteristics between patients with diarrhea-predominant irritable bowel syndrome (IBS-D) and healthy individuals
2. To clarify the changes in IBS-D symptoms in IBS-D patients by taking Kampo medicines (Hangeshashinto, Rikkunshito)
3. To clarify the chronological changes in the intestinal flora before, after, and after taking the herbal medicine. To clarify this, we compared the intestinal flora of adult male IBS-D patients with that of healthy subjects, and also conducted a clinical study in which we administered the herbal medicine (Hangeshashinto or Rikkunshito) to IBS-D patients and investigated its clinical effects and changes in the intestinal flora.

II. Research Methods The clinical evaluation portion of this study was conducted at the Department of Traditional Chinese Medicine at Tohoku University Hospital.
1. Study design The study design was a background factor comparison study to compare the characteristics of healthy subjects and IBS-D patients, and an open-label prospective intervention study to compare IBS-D patients before and after Kampo medicine treatment (see Figure 1). The background factor comparison study relates to item 1 in "I. Research Objectives" above, and the open-label prospective intervention study relates to items 2 and 3 in "I. Research Objectives" above.
In the background factor comparison study, IBS-D patients were included as the disease group and healthy subjects as the control group, and background factors, clinical scores, and intestinal flora were compared.

In this open-label, prospective intervention study, patients in the IBS-D group were administered herbal medicines (Hangeshashinto or Rikkunshito; administration method is described below), and symptom improvement was assessed at three time points: before treatment, after treatment, and when the patients were followed up after treatment without further treatment (after follow-up), and clinical scores were compared. Additionally, the intestinal flora was compared at four time points: before treatment when symptoms were mild, when symptoms worsened, after treatment, and after follow-up.

2. Subjects Japanese patients with diarrhea-predominant irritable bowel syndrome (IBS-D) and healthy controls were recruited from Tohoku University.

IBS-D patients are defined as (1) men aged 20 years or older and younger than 60 years, (2) diagnosed with IBS-D according to the ROME III diagnostic criteria, (3) diagnosed with either a substantial or deficiency syndrome by a Chinese medicine specialist, and (4) with no history of notable organic or psychiatric illness and not currently receiving treatment for IBS. Regarding (3), a substantial syndrome is defined as a person with a firm build, active personality, good skin tone, a strong voice, a firm palpable pulse, and abdominal strength as determined by abdominal palpation. In contrast, a deficiency syndrome is defined as a person with a thin build, low activity level, easily fatigued personality, a relatively weak palpable pulse, and reduced abdominal strength as determined by abdominal palpation.

Healthy individuals were defined as (1) men aged 20 years or older and under 60 years old, (2) individuals who did not have any illness requiring treatment or were not currently undergoing treatment, and who were in good physical, mental, and social condition.

In both the IBS-D patient and healthy control groups, subjects who met any of the following criteria (1)-(8) were excluded from the test.
(1) Those with serious complications such as neoplastic disease or inflammatory bowel disease. (2) Those who have taken laxatives, antidiarrheals, or probiotics within one week of the start of the study. (3) Those who have taken antibiotics, anti-inflammatory drugs, or steroids within six weeks of the start of the study. (4) Those receiving treatment with anticholinergics, antimuscarinics, proton pump inhibitors, or minor tranquilizers. (5) Those with a history of gastrointestinal surgery. However, appendectomy is permitted. (6) Those with a serum potassium level of 3.5 mEq/L or less and an aspartate aminotransferase (AST) level of more than twice the normal range in the initial blood test. (7) Those with atopic dermatitis or allergies. (8) Those with depression, anxiety disorder, or psychiatric symptoms that make it difficult for them to participate in the study.

3. How to administer herbal medicine
The IBS-D group underwent a Kampo medical examination by a Kampo medical instructor, and those diagnosed with IBS-D were given 2.5g/packet of Tsumura Hangeshashinto Extract Granules (for medical use), and those diagnosed with IBS-D were given 2.5g/packet of Tsumura Rikkunshito Extract Granules (for medical use), two packs per day, either before breakfast and dinner or between meals, for 28 days.
This study was not randomized or blinded. No intervention was administered to the healthy control group.

4. Study Schedule The study schedule is shown in Figure 1. When collecting data, subjects were contacted periodically by email to prevent them from dropping out of the study.
4-1. IBS-D group Before participating in the study, subjects were interviewed regarding the inclusion and exclusion criteria and IBS, and screening for IBS-D was performed. After obtaining consent, subjects who could be enrolled were examined and evaluated according to each evaluation item (Table 1). A Kampo medical diagnosis was made based on the examination by a Kampo specialist, and those diagnosed with a strong symptom were given Hangeshashinto, and those diagnosed with a weak symptom were given Rikkunshito as an intervention drug. Since the symptoms of IBS-D change depending on the time of year, stool samples were collected in random order twice before taking the Kampo medicine, when the patient's diarrhea symptoms were mild (mild symptoms) and when the symptoms were severe (exacerbation). The subjects brought the samples in a cooler bag containing an ice pack on the day of collection.
Subjects in the IBS-D group who submitted two stool samples took the herbal medicine for 28 days. On the last day of medication, they submitted a third stool sample (after treatment), and each evaluation item was measured and examined. They were then observed for 28 days without taking the herbal medicine, and on the 56th day after starting medication, they submitted a fourth stool sample (after observation), and each evaluation item was measured and examined. There was a one-week grace period before and after the two time points after treatment and observation.

4-2. Healthy subjects Before participating in the study, healthy subjects were screened by interviewing about the inclusion and exclusion criteria, abdominal symptoms, medical history, family history, and allergies. After obtaining consent to participate in the study, subjects who were eligible for inclusion were measured for height, weight, blood pressure, and pulse rate, and evaluated according to each evaluation item (Table 1). In addition, stool samples were collected once at a later date, similar to the IBS-D group.

5. Safety Evaluation To evaluate safety, peripheral blood tests and serological tests (AST, ALT, ALP, γ-GTP, BUN, Cr, Na, K, Cl) were performed, and hypersensitivity to Kampo medicines (skin symptoms and gastrointestinal symptoms) was also measured.

6. Clinical Evaluation Items
6-1. Primary endpoint: Subjects were asked whether symptoms had improved after herbal medicine treatment and were rated as yes/no.

6-2. Secondary Evaluation Items The following evaluation items were set.
(1) IBS-QOL Japanese version (Patric DL et al., Digestive Diseases and Sciences. 1998;43:400-11 and Kanazawa M et al., Biopsychosoc Med. 2007;1:6.)
The test consists of 35 questions about IBS-related symptoms and quality of life, and is self-reported on a five-point scale from 0 to 4, with the overall score ranging from 0 to 140, with higher scores indicating more severe symptoms.
(2) Gastrointestinal Symptom Rating Scale (GSRS) (Svedlund J et al., Digestive Diseases and Sciences. 1988;33:129-34. and Hongo Michi et al., Diagnosis and Treatment. 1999;87:731-6.)
It consists of 15 items regarding gastrointestinal symptoms and a self-report score on a 7-point scale from 1 to 7. The average score of the 15 items is calculated as 1 to 7, with higher scores indicating more severe symptoms.
(3) GSRS diarrhea score (Svedlund J et al., Digestive Diseases and Sciences. 1988;33:129-34. and Hongo Michi et al., Diagnosis and Treatment. 1999;87:731-6.)
The score is calculated by taking the average of the three sub-scores of the GSRS: diarrhea, loose stools, and urgent urge to defecate, and is on a scale of 1 to 7, with higher scores indicating more severe symptoms.
(4) IBS Symptom Severity Scale Japanese version (IBS-SSS) (Shinozaki M et al., J Gastroenterol. 2006;41:491-4.)
The IBS severity index, a self-administered questionnaire on the severity of IBS, was calculated by adding the severity of abdominal pain (0-100), the average number of abdominal pains in 10 days x 10, the severity of abdominal distension (0-100), satisfaction with bowel movements (0-100), and the impact of abdominal symptoms on daily life (0-100). A total score of 0-75 was considered to be no problem, 76-175 was considered to be mild, 176-300 was considered to be moderate, and 301 or more was considered to be severe.
(5) Self-rating Depression Scale (SDS) (Zung WW. Arch Gen Psychiatry. 1965;12:63-70., Fukuda Kazuhiko, Kobayashi Shigeo. Studies on the Self-rating Depression Scale. Journal of Psychiatry and Neurology. 1973;75:673-9.).
There were 20 questions about depression, and the participants were asked to report their symptoms on a four-point scale, from 1 to 4, with scores ranging from 20 to 80. The higher the score, the more severe the symptoms, and a score of 40 or above indicated a tendency to depression.
(6) State-Trait Anxiety Inventory (STAI) (Spielberger CD et al., Mind Garden, Inc. 1970.)
STAI: 40 items related to anxiety, self-reported on a four-point scale from 1 to 4, divided into 20 items for current anxiety (state anxiety) and 20 items for personality-related, innate anxiety (trait anxiety). Each is calculated on a score from 20 to 80, with higher scores indicating more severe symptoms. In the case of the male subject in this study, a state anxiety score of 42 or more and a trait anxiety score of 44 or more were considered to be high anxiety.
(7) Changes in intestinal flora through analysis of stool samples Safety evaluation items included measurements of peripheral blood tests, abnormalities in blood biochemistry tests (AST, ALT, ALP, γ-GTP, BUN, Cr, Na, K, Cl), and herbal medicine-induced hypersensitivity (skin symptoms and gastrointestinal symptoms).

6-3. Analysis method of clinical evaluation items
6-3-a Comparison between IBS-D group and healthy control group For background factors of each group, t-test was performed for continuous variables, and chi-square test was performed for ordinal and dichotomous variables. Body mass index (BMI) was calculated as weight/height2 [kg/m2] and compared.
6-3-b Analysis of the treatment effect in the IBS-D group No statistical analysis was performed on the primary outcome, i.e., whether or not symptoms improved 28 days after taking the herbal medicine, but it was used to examine the association with changes in the bacterial flora in the bacterial flora analysis. For the secondary outcome, analysis of variance (Friedman test) was performed for continuous and ordinal variables, and chi-square test for binary variables at four time points: before treatment (mild symptoms, severe symptoms), 28 days after taking the herbal medicine (post-treatment), and 56 days after follow-up (follow-up). For items with significant differences, Wilcoxon signed rank test or chi-square test was performed at the two time points.

7. Metagenomic analysis of gut microbiota Stool samples collected in clinical studies were frozen at -80℃ and thawed at room temperature before use. Metagenomic analysis of gut microbiota (16S rRNA analysis) was performed using the stool samples.
7-1. DNA extraction DNA was extracted from 300 μL of stool sample solution collected with a cotton swab (Puritan) using a DNeasy PowerSoil Kit (QIAGEN Inc., Germany). For samples from which DNA could not be extracted due to poor sample quality, approximately 1 g of solid stool sample (frozen at -80°C) was substituted, and DNA was extracted using the same method as above.

7-2. Polymerase chain reaction (PCR)
To amplify DNA for sequence analysis, a two-step PCR method was used to amplify the variable region V3-V4 region contained in the DNA coding for 16S ribosomal RNA (rRNA), a conserved region unique to bacterial DNA. Amplification was performed to obtain an amplicon.
7-2-a 1st PCR
The first PCR was performed using specific primers targeting the V3-V4 region, the sequences of which are as follows: (Forward) 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNCCTACGGGNGGCWGCAG-3' (55bp) (SEQ ID NO: 4)
In SEQ ID NO:4, N is A, C, G or T, and W is A or T.
(reverse) 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGACTACHVGGGTATCTAATCC-3' (60bp) (SEQ ID NO:5)
In SEQ ID NO:5, N is A, C, G or T; H is A, T or C; and V is A, C or G.
The protocol for the 1st RCR is as follows:
94℃ 3 min, (94℃ 30 sec, 55℃ 30 sec, 72℃ 30 sec) × 30 cycles, 72℃ 5 min, 8℃∞.
7-2-b 2nd PCR
A second PCR was performed to attach an adapter sequence required for sequence analysis to the amplified amplicon. The primers used in the second PCR had a different index sequence for each sample and an adapter sequence for binding to the flow cell in MiSeq. The region to which the sequencing primer binds is already attached. The reagents and equipment used were the same as those used in the 1st PCR.
7-2-c Bead purification, gel excision
The 2nd PCR products from 229 samples were pooled in a tube (3 μL each). To remove impurities other than amplified DNA from the pooled samples, magnetic beads AMPure XP (Beckman Coulter Inc., USA) were used. The amplified DNA was purified using 100% ethanol. Isopropanol precipitation was performed 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 had been obtained. The solution and the amplified DNA obtained by the 2nd PCR were subjected to electrophoresis to confirm that the band in question (630 bp in the case of the V3-V4 region) matched.

7-3. Amplicon DNA sequencing using MiSeq Amplicon DNA was sequenced from the purified DNA solution using MiSeq (Illumina Inc., USA).
It was confirmed that the base sequences of the V3-V4 regions of the bacteria contained in the stool were properly obtained.

7-4. Phylogenetic analysis of bacterial flora The sequences were analyzed using QIIME 2 (version 2019.10) (Bolyen E et al., Nat Biotechnol. 2019;37:852-7.), an integrated analysis pipeline program for metagenomic analysis. Sequencing errors were removed using a program called DADA2 (Callahan BJ et al., Nat Methods. 2016;13(7):581-3.). After adding the clinical data of the subjects to the noise-removed data, DNA sequences with 97% or more homology were clustered. These sequences are called representative sequences, meaning they represent a single bacterium, and are counted in units called amplicon sequence variants (ASVs), with 1 ASV corresponding to one bacterial species.

7-5. Phylogenetic tree creation A phylogenetic tree was created by clustering the bacterial flora from the common sequences shared by multiple bacteria. This process was carried out in QIIME 2 using MAFFT (Katoh K etl. Nucleic Acids Res. 2002;30:3059-66.).
Using the bacterial phylogenetic data obtained in this way, we carried out the following analyses, as per usual methods.

7-6. Diversity analysis (rarefaction analysis)
Analysis to estimate the number of bacterial species contained in a sample is called diversity analysis (rarefaction analysis), and can be broadly divided into two types. The diversity of the bacterial flora in a single sample is called α diversity, and the diversity of the bacterial flora composition among multiple groups is called β diversity. Before analysis, rare fraction processing was performed to align the sampling depth (number of reads) of the samples (1400 reads).
In this study, we calculated the following three indices of alpha diversity:
(1) Observed ASVs: The number of ASVs that have been observed at least once. A diversity index that takes into account the existence of rare species.
(2) Shannon index: Calculated based on the proportion of species in the total sample. It is widely used but is considered to be sensitive to the presence of predominant species.
(3) Faith's phylogenetic distance: Phylogenetic diversity taking into account the length of the branches of the phylogenetic tree.
For each index, between-group comparisons before treatment between the healthy control group and the IBS-D group were performed using the Wilcoxon rank-sum test, and time-series comparisons at four time points within the IBS-D group were performed using the Wilcoxon signed-rank test.
In addition, we performed UniFrac principal coordinate analysis, which expresses the diversity between groups in two-dimensional coordinates, to analyze β diversity. Two types of analysis were performed: Weighted UniFrac analysis (analysis of quantitative diversity) that takes into account the number of reads, and Unweighted UniFrac analysis (analysis of qualitative diversity) that does not.

7-7. Phylogenetic analysis The representative bacterial sequence obtained by sequencing was compared with the DNA sequence of known bacteria (reference sequence), and sequences with 99% or more homology were identified as the bacterial species, and a phylogenetic analysis of the bacterial species was performed. In this study, Greengenes (release 13_8) (https://docs.qiime2.org/2021.11/data/tutorials/moving-pictures-usage/gg-13-8-99-515-806-nb-classifier.qza) was used as an existing bacterial DNA sequence dataset. All organisms are classified into seven levels: kingdom, phylum, class, order, family, genus, and species. In this study, the proportion of bacterial species was calculated at all levels that could be identified.

7-8. Comparative analysis between each observation time point The relative amount of the bacterial flora data at all levels obtained above was calculated in each sample, and comparisons were made between each group. Comparisons between the healthy and IBS-D groups before treatment were performed using the Wilcoxon rank-sum test. In addition, it has been reported that the ratio of Firmicutes and Bacteroidetes, the two most prevalent bacterial species, in the phylum is increased in IBS patients compared to healthy individuals (Duan R, et al. Clin Transl Gastroenterol 2019;10(2):e00012.). In this study, the F/B ratio was also calculated and compared. Furthermore, since the IBS-D group was divided into herbal medicines used according to constitution, we considered the possibility that the characteristics of the bacterial flora may differ depending on the constitution, and compared the bacterial flora according to the herbal medicine used. Comparisons were made at each classification level using the Wilcoxon rank-sum test, and the F/B ratio was compared for the phylum.
For comparisons within the IBS-D group, the change in the proportion of bacteria in each family at each time point was calculated and a heat map was created. In addition, analysis of variance was performed for the four groups within the IBS-D group: before treatment (mild, severe), after Kampo treatment, and after follow-up, and the Wilcoxon signed rank test was used to compare the IBS group at each time point.

7-9. Correlation between background factors and intestinal flora (family)
The Spearman correlation coefficient between pretreatment physical findings, clinical scores, and the proportion of each bacterial species was calculated, and an analysis of the association between background factors and the intestinal bacterial flora was performed.

7-10. Correlation between changes in clinical scores and changes in gut flora in IBS-D group (genus)
The Spearman correlation coefficient between the change in clinical score before and after treatment in the IBS-D group and the percentage change in each bacterial species was calculated to analyze the relationship between changes in clinical score and changes in the intestinal bacterial flora.

7-11. Functional profile prediction using PICRUSt2
PICRUSt2 has recently been developed as a pipeline for predicting the functional profile of the entire microbiota from the microbiota profile obtained by metagenomic analysis of 16S rRNA (Douglas GM et al., Nat Biotechnol. 2020;38:685-8.). Using this pipeline, the genome sequences of the microbiota data identified in the gut microbiota analysis can be compared with the gene sequence data of more than 40,000 species of bacteria registered in PICRUSt2, and the gene copy number can be calculated by multiplying the abundance ratio of each bacterium in the sample. From the results, the metabolic function that the entire gut microbiota will have can be predicted. In this study, PICRUSt2 was used to predict how the function of the entire microbiota changed between each time point.
All statistical analyses were performed using R (version 3.5.2). The significance level for the analyses performed in this study was set at 5%. Results with a significance level between 5% and 10% were considered to indicate a trend.

III. Results Table 1 shows the patient background factors of each group of subjects classified by a physician as healthy subjects (n=45) and IBS-D patients (n=57). There were significant differences between the healthy subjects and the IBS-D group in the items of IBS-QOL, GSRS, GSRS diarrhea score, and IBSS.

Of 45 healthy subjects and 57 IBS-D patients, those who did not submit samples (healthy subjects N=4, IBS-D patients N=13), those with food poisoning (IBS-D patient N=1), those who discontinued medication (IBS-D patient N=1), and those whose samples were difficult to analyze (IBS-D patient N=1) were excluded, and the fecal bacterial flora of 41 healthy subjects and 42 IBS-D patients was analyzed.

When patients were asked whether their symptoms had improved after taking the herbal medicine for one month, 33 out of 41 patients (81%) reported that their symptoms had improved.

As shown in Figure 2, the relative amount of bacteria of the genus Lachnospiraceae Blautia in the feces of IBS-D patients during exacerbation was elevated, and the relative amount in the feces of healthy subjects, IBS-D patients during mild symptoms, and IBS-D patients after treatment was lower. The relative amount of bacteria of the genus Lachnospiraceae Blautia was significantly different in the comparison between healthy subjects and IBS-D patients (P = 0.006), between IBS-D patients during mild symptoms and exacerbation (P = 0.0002), and between IBS-D patients during exacerbation and after treatment observation (P = 0.004).
A graph showing the above.

As shown in Figure 3, the relative amount of bacteria of the genus Ruminococcaceae Oscillospira in the feces of IBS-D patients during exacerbation was decreased, and the relative amount in the feces of healthy subjects, IBS-D patients during mild symptoms, and IBS-D patients after treatment was higher. The relative amount of bacteria of the genus Ruminococcaceae Oscillospira was significantly different in the comparison between healthy subjects and IBS-D patients (P = 0.03), between IBS-D patients during mild symptoms and exacerbation (P = 0.0005), and between IBS-D patients during exacerbation and after treatment observation (P = 0.0004).

As shown in Figure 4, the relative amount of Faecalibacterium prausnitzii in the feces of IBS-D patients during exacerbation was decreased, and the relative amount in the feces of healthy subjects, IBS-D patients during mild symptoms, and IBS-D patients after treatment was higher. The relative amount of Faecalibacterium prausnitzii showed a difference in fluorescence between healthy subjects and IBS-D patients (P = 0.06), and a significant difference was observed in both the comparison of IBS-D patients during mild symptoms and exacerbation (P = 0.03) and the comparison of IBS-D patients during exacerbation and after treatment (P = 0.04).

As a result of the comparative analysis, as shown in Figure 5, differences were observed between healthy subjects and IBS-D patients, between IBS-D patients during mild symptoms and exacerbation, and between IBS-D patients during exacerbation and after treatment for bacteria of the genus Lachnospiraceae Blautia, Ruminococcaceae Oscillospira, and Faecalibacterium prausnitzii.

Claims (13)

1. A method for detecting irritable bowel syndrome, comprising:
1. A method comprising detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of a subject. The method of claim 1, wherein detecting the bacteria includes detecting bacteria of the genus Lachnospiraceae Blautia. The method of claim 1, wherein detecting the bacterium includes measuring the amount of a 16S rRNA nucleic acid sequence of the bacterium. The method for detecting irritable bowel syndrome includes a method for detecting the onset or predisposition to the onset of irritable bowel syndrome,
A high detection value of bacteria of the genus Lachnospiraceae Blautia compared to the standard value indicates the onset or predisposition to onset of irritable bowel syndrome,
A low detection value of bacteria of the genus Ruminococcaceae Oscillospira compared to the standard value indicates the onset or predisposition to onset of irritable bowel syndrome,
The method according to claim 1, wherein a lower detection value of the Faecalibacterium prausnitzii species compared to a reference value indicates the onset or predisposition to the onset of irritable bowel syndrome. The method according to claim 4, wherein the reference value is a measurement obtained from a healthy subject or a measurement obtained from the same subject after treatment for irritable bowel syndrome. Detecting the bacteria includes detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the subject's feces on a reference date and on a subsequent day after the reference date;
The method of claim 1, wherein the measurement on the subsequent day is at least any one of the following (i) to (iii) when compared to the measurement on the reference day, indicating a high possibility of an exacerbation of irritable bowel syndrome symptoms:
(i) The measured value of bacteria of the genus Lachnospira Blautia on the later day is higher than that on the reference day. (ii) The measured value of bacteria of the genus Ruminococcus oscillospira on the later day is lower than that on the reference day. (iii) The detected value of the species Faecalis bacterium prausnitzii on the later day is lower than that on the reference day. Detecting the bacteria includes detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the subject's feces on a reference date and on a subsequent day after the reference date;
The method of claim 1, wherein the measurement on the subsequent day is at least one of the following (i) to (iii) when compared to the measurement on the reference day, indicating that the symptoms of irritable bowel syndrome are unlikely to worsen.
(i) the measured value of bacteria of the genus Lachnospira Blautia on the later day is the same or lower than the measured value on the reference day; (ii) the measured value of bacteria of the genus Ruminococcus oscillospira on the later day is the same or higher than the measured value on the reference day; (iii) the detected value of the species Faecalibacterium prausnitzii on the later day is the same or higher than the detected value on the reference day. The method for detecting irritable bowel syndrome includes a method for evaluating the effectiveness of measures for treating, preventing, or ameliorating irritable bowel syndrome,
Detecting the bacteria includes detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of the treated subject;
The method according to claim 1, wherein when the detection value after the treatment is at least any one of the following (i) to (iii) compared with the reference value or the value of the same patient before the treatment, the treatment is likely to be effective in treating, preventing, or ameliorating irritable bowel syndrome.
(i) The detection value of bacteria of the genus Lachnospiraceae Blautia after the treatment is lower than the standard value or the value before the treatment. (ii) The detection value of bacteria of the genus Ruminococcaceae Oscillospira after the treatment is higher than the standard value or the value before the treatment. (iii) The detection value of the species Faecalibacterium prausnitzii after the treatment is higher than the standard value or the value before the treatment. A method for screening for a measure effective for treating, preventing, or ameliorating irritable bowel syndrome, comprising:
detecting at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii in the feces of the treated subject;
The method comprises selecting the measure as one that is likely to be effective in treating, preventing, or ameliorating irritable bowel syndrome when the detected value after the treatment is at least any one of the following (i) to (iii) compared with a reference value or the value of the same patient before the treatment.
(i) The detection value of bacteria of the genus Lachnospiraceae Blautia after the treatment is lower than the standard value or the value before the treatment. (ii) The detection value of bacteria of the genus Ruminococcaceae Oscillospira after the treatment is higher than the standard value or the value before the treatment. (iii) The detection value of the species Faecalibacterium prausnitzii after the treatment is higher than the standard value or the value before the treatment. The method according to claim 8 or 9, wherein the treatment is at least one selected from the group consisting of administration of medicine, administration of food, dietary therapy, and fecal transplantation. The method according to any one of claims 1 to 10, wherein the irritable bowel syndrome is diarrhea-predominant irritable bowel syndrome. The in vitro use of the expression level of at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii as a biomarker for diagnosing and/or monitoring irritable bowel syndrome. A diagnostic kit for irritable bowel syndrome, comprising:
(i) a nucleic acid probe capable of hybridizing with a nucleic acid sequence of 16S rRNA or a nucleic acid sequence of 16S rDNA of at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii; or (ii) a kit comprising a pair of nucleic acid primers capable of specifically amplifying a nucleic acid sequence of 16S rRNA or a nucleic acid sequence of 16S rDNA of at least one bacterium selected from the group consisting of bacteria of the genus Lachnospiraceae Blautia, bacteria of the genus Ruminococcaceae Oscillospira, and the species Faecalibacterium prausnitzii.