KR102548376B1 - Depression and anxiety disorder diagnosis method using intestinal microbiome - Google Patents

Abstract

The present invention relates to a method for diagnosing depression/anxiety disorder using the gut microbiome.
The method for diagnosing depression/anxiety disorders according to the present invention is non-invasive and analyzes fecal microorganisms, which are causative factors, and can diagnose depression and anxiety disorders according to the composition ratio of specific microorganisms.

Description

Depression and anxiety disorder diagnosis method using intestinal microbiome}

The present invention relates to a method for diagnosing depressive anxiety disorder using the gut microbiome.

BACKGROUND OF THE INVENTION In modern society, cranial nerve diseases including anxiety, depression, schizophrenia, and the like are increasing due to rapidly increasing stress. In particular, the number of patients suffering from mental disorders such as depression and anxiety due to various causes such as social and structural causes in the current era of prevailing individualism is increasing.

Patients suffering from mental disorders can lead to suicidal thoughts in severe cases, and in particular, it has been reported that more than half of depressed patients consider suicide, and it is known that 10 to 15% of patients actually commit suicide.

Mental disorders can have different symptoms for each patient. When mental disorders are suspected, treatment according to accurate diagnosis and examination is required, but proper treatment is not provided due to negative social perception of hospital treatment due to mental disorders. am. In addition, drugs such as antidepressants used to treat mental disorders do not have a significant therapeutic effect, and their use is limited because serious side effects such as cardiovascular disease and suicide may occur.

Anxiety disorders, which are manifested by various diseases such as panic disorder, generalized anxiety disorder, and phobias, refer to diseases in which physical and mental symptoms appear due to chronic worry and anxiety. Anxiety disorders are always in a state of tension, the autonomic nerves become sharp, it is difficult to concentrate on work, and there are disorders in daily life. About 25% of the population suffers from an anxiety disorder, with women twice as likely as men.

Depressive disorder (depression) refers to a disease that causes a decrease in daily function by causing various cognitive and psychosomatic symptoms with low motivation and feeling of depression as the main symptoms. Depressive disorder has a lifetime prevalence of 15%, especially about 25% in women, and is a serious disease that causes changes in emotions, thoughts, physical condition, and behavior. Anxiety disorders can develop into depression, and in many cases, anxiety disorders and depressive disorders coexist.

In anxiety and depressive disorders, it is known that the frontal lobe, limbic system, and basal nerve nuclei of the brain cause anxiety, but there is also a theory that the occipital lobe causes anxiety. Functional disturbances in this area (lack or excess of neurotransmitters) cause anxiety.

If anxiety/depression is suspected by a mental status test, a differential diagnosis of other diseases, including hypothyroidism that can cause anxiety/depression, should be prioritized.

Treatment is slightly different for each symptom, but antidepressants (SSRIs, etc.) and anti-anxiety drugs (benzodiazepines, etc.) are usually used for general drug treatment. However, early detection and prevention are the most desirable.

Diagnosis and examination methods are: ① symptoms of anxiety disorder, such as excessive anxiety and worry, lasting for more than several days at a time for more than six months, ② anxiety is not controlled, ③ diagnosis by methods such as surveys. The diagnostic criteria of the Diagnostic Statistical Manual of Mental Disorders (DSM-IV-TR) of the American Psychiatric Association are as follows.

[Diagnosis method]

(1) Anxiety/depressive disorder test - Hospital Anxiety and Depression Scale-depression (HADS-D), Hospital Anxiety and Depression Scale-anxiety (HADS-A), Beck Anxiety Inventory (BAI) and Beck Depression Inventory (BDI) survey method.

(2) Utilization of NGS analysis of gut microbial community based on 16s ribosomal RNA (16s rRNA) gene sequence

Correlation with depression/anxiety in relation to the gastrointestinal microbial community has been reported, but the microbial types that have been identified in the library for interpreting NGS results are limited.

It is not easy to accurately judge the method of (1) because it varies greatly depending on the mood. In addition, the method (2) has insufficient data to analyze the correlation with the gut microbiome community.

Under this background, the present inventors analyzed fecal microorganisms, which are causative factors, in a non-invasive way, and found that a high composition ratio of specific microorganisms can induce depression and anxiety. In addition, it was confirmed whether these gut bacteria induce depression and anxiety in animals (mouse), and a method for diagnosing depression/anxiety disorders using this was completed.

KR10-2019-0047023

An object of the present invention is to provide a method for diagnosing depressive anxiety disorder by evaluating the microbiome, that is, the microbiome.

As one aspect for carrying out the above object, the present invention

(a) measuring the relative abundance of one or more selected from the group consisting of Lactobacillaceae , Bacteroidaceae , Enterobacteriaceae and Enterococcaceae from a biological sample; ; and

(b) diagnosing depressive anxiety disorder based on the measured relative dominance;

Information providing method for diagnosing depressive anxiety disorder according to the present invention is selected from the group consisting of (a) Lactobacillaceae , Bacteroidaceae , Enterobacteriaceae and Enterococcaceae from biological samples measuring the relative abundance of any one or more of the

In the present invention, relative dominance refers to a numerical value indicating the degree of dominance of each “family”, “genus”, and/or “species” within a colony. These dominance plots can represent differences in the degree of dominance at the level of each of the above “family”, “genus”, and/or “species” according to changes in the occupancy of the “family”, “genus”, and/or “species” in the intestine. there is.

The biological sample in step (a) may be from one person or from several people. For example, biological samples derived from a subject, for example, digestive tract contents such as intestinal fluid and feces, are exemplified, but feces are particularly preferable because they do not burden the subject.

The sample may be a normal person without depressive symptoms or a sample of a group of normal people. Such a sample can be used as a normal group in providing information for diagnosing depressive anxiety disorder. Also, in some cases, the above sample may be a biological sample of a group or group having some diseases but not having depressive anxiety disorder. In addition, it may be a biological sample of a subject, an experimental group, or a group having depressive anxiety symptoms or suspected of having such symptoms.

In any of the above embodiments, depression when the relative abundance of any one or more selected from the group consisting of Lactobacillaceae , Enterobacteriaceae and Enterococcaceae is increased compared to the normal group. Diagnosed as having an anxiety disorder.

In any of the above embodiments, if the relative abundance of Enterococcaceae is increased compared to the normal group and the relative predominance of the Bacteroidaceae is decreased compared to the normal group, it is diagnosed as having a depressive anxiety disorder. .

In any of the above embodiments, if the relative abundance of Lactobacillaceae is increased compared to the normal group and the relative abundance of Bacteroidaceae is decreased compared to the normal group, depression anxiety disorder is diagnosed.

In any of the above embodiments the taxonomic Lactobacillaceae increases in relative abundance in the group with depressive anxiety disorder.

In any of the above embodiments the taxonomic Enterobacteriaceae increases in relative abundance in the group with depressive anxiety disorder.

In any of the above embodiments the taxonomic Enterococcaceae family increases in relative abundance in the group with depressive anxiety disorder.

In any of the above embodiments the taxonomic Bacteroidaceae is reduced in relative abundance in the group with depressive anxiety disorder.

In any of the above embodiments, taxonomic Enterococcaceae increases in relative abundance in the group with depressive anxiety disorder while Bacteroidaceae increases in relative abundance in the group with depressive anxiety disorder. decreases.

In any of the above embodiments, the taxonomic Lactobacillaceae increases in relative abundance in the group with depressive anxiety disorder, while the Bacteroidaceae family increases in relative abundance in the group with depressive anxiety disorder. Decrease.

The relative dominance of the gut microbiome can be determined based on analysis of genera, species, and subspecies below a family or “family” as taxonomic classification levels in the gut microbiome. This relative dominance can be quantified and used to determine or diagnose depressive anxiety disorder.

Specifically, according to one embodiment of the present invention, lactic acid bacteria ( Lactobacillaceae ) increases in relative abundance (relative abundance) compared to normal people (healthy people) in the group with depressive anxiety disorder. The increase in relative dominance may be an increase in Lactobacillaceae of 2%, 3%, 4%, 5% or more based on the total microbiome when compared to normal people (healthy people). This increase in relative dominance is also 1.2, 1.3, 1.4, 1.5, 1.6, 1.6, 1.8, 2.0, 2.2, 2.5, 3.0 or more in the measurement of the fold change level of 16S rRNA when compared to normal people (healthy people). may be an increase

Specifically, according to one embodiment of the present invention, the enterobacteriaceae ( Enterobacteriaceae ) increases in relative predominance (relative abundance) compared to normal people (healthy people) in the group with depressive anxiety disorder. The increase in relative dominance may be an increase in Enterobacteriaceae of 2%, 3%, 4%, 5% or more based on the total microbiome when compared to normal people (healthy people). This increase in relative dominance is also 1.2, 1.3, 1.4, 1.5, 1.6, 1.6, 1.8, 2.0, 2.2, 2.5, 3.0 or more in the measurement of the fold change level of 16S rRNA when compared to normal people (healthy people). may be an increase

Specifically, according to one embodiment of the present invention, Enterococcaceae ( Enterococcaceae ) increases in relative predominance (relative abundance) compared to normal people (healthy people) in the group with depressive anxiety disorder. The increase in relative dominance may be an increase of 2%, 3%, 4%, 5% or more of Enterococcaceae based on the total microbiome compared to normal people (healthy people). This increase in relative dominance is also 1.2, 1.3, 1.4, 1.5, 1.6, 1.6, 1.8, 2.0, 2.2, 2.5, 3.0 or more in the measurement of the fold change level of 16S rRNA when compared to normal people (healthy people). may be an increase

Specifically, according to one embodiment of the present invention, Bacteroidaceae ( Bacteroidaceae ) is reduced in relative predominance (relative abundance) compared to normal people (healthy people) in the group with depressive anxiety disorder. The decrease in relative dominance may be a 2%, 3%, 4%, 5% or more reduction of the Bacteroidaceae family based on the total microbiome when compared to a normal person (healthy person). This decrease in relative dominance is also 1.2, 1.3, 1.4, 1.5, 1.6, 1.6, 1.8, 2.0, 2.2, 2.5, 3.0 or more in the measurement of the fold change level of 16S rRNA when compared to normal people (healthy people). may be a decrease

Due to the number of organisms that make up the microbiome itself and/or the diversity among organisms, the above-mentioned changes in relative dominance at the “family” level can occur alone or in combinations of two or more than three. That is, changes in the “and” community of the gut microbiome may have a correlation between “and”.

According to one specific aspect, the taxonomic enterococci family ( Enterococcaceae ) while the relative predominance (relative abundance) increases in the group with depressive anxiety disorder Bacteroid family ( Bacteroidaceae ) is the relative predominance (relative abundance) in the group with depressive anxiety disorder decreases.

In any of the above embodiments, the taxonomic Lactobacillaceae increases in relative abundance in the group with depressive anxiety disorder, while the Bacteroidaceae family increases in relative abundance in the group with depressive anxiety disorder. Decrease.

According to one specific embodiment, the taxonomic enterobacteriaceae ( Enterobacteriaceae ) while increasing the relative predominance (relative abundance) in the group with depressive anxiety disorder Bacteroids ( Bacteroidaceae ) is the relative predominance (relative abundance) in the group with depressive anxiety disorder decreases.

According to one specific embodiment, the taxonomic enterococci ( Enterococcaceae ), enterobacteriaceae ( Enterobacteriaceae ) and lactic acid bacteria ( Lactobacillaceae ) at least one selected from the group consisting of depressive anxiety disorder relative predominance (relative abundance) As Bacteroidaceae increases, the relative abundance of Bacteroidaceae decreases in the group with depressive anxiety disorder.

Among the species included in this family, especially the following species have a great relevance to changes in the community of the "family" of the intestinal microbiome.

For example, with respect to the Enterococcaceae family,

and Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, Enterococcus faecium, and Enterococcus faecalis. .

The above-mentioned strains greatly increase their occupancy level in the intestine when compared to the normal (healthy) group.

In particular, when considering the sum of strains, in depressed patients, Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, and Enterococcus faecium The number of symps may characterize more than Enterococcus faecalis .

For example, in relation to Enterobacteriaceae , Klebsiella pneumonia, Klebsiella oxytoca, Cronobacter sakazakii, Escherichia coli ) and the like.

The above-mentioned strains greatly increase their occupancy level in the intestine when compared to the normal (healthy) group. In particular, when considering the sum of strains, the sum of Enterobacteriaceae ( Klebsiella pneumonia, Klebsiella oxytoca, and Cronobacter sakazakii) in depressed patients is Escherichia coli. (Escherichia coli) .

That is, Pediococcus acidilactici as an additional single strain greatly increases its occupancy level and/or relative dominance in the intestine when compared to the normal (healthy) group. Accordingly, the method according to the present invention comprises the steps of determining the relative abundance of Pediococcus acidilactici from a biological sample; and (b) diagnosing depressive anxiety disorder based on the measured relative dominance.

In the present invention, depressive anxiety disorder is a concept including depression and anxiety disorders.

Specifically, "depression" refers to a neuropsychiatric disorder in which behaviors such as sadness, tastelessness, loss of emotion, anhedonia (lack of pleasure), or hallucinations or delusions are mainly performed, and psychomotor depression, pessimism and It refers to a disease that leads to despair and suicidal thoughts, leading to suicide attempts. The depression is accompanied by various physical symptoms such as decreased appetite, insomnia, constipation, decreased libido, or increased susceptibility to diseases caused by reduced immune function. More specifically, it means a symptom of a decrease in activity.

In addition, “anxiety disorder” refers to a neuropsychiatric disorder in which physical or behavioral symptoms such as increased heart rate, increased respiratory rate, panic disorder, fear, and sweating due to anxious feelings appear, and more specifically, restlessness and confined spaces. It means to be located only in the corner, but is not limited thereto. Examples of anxiety disorders include insomnia, delusional disorder, phobic disorder, panic disorder, generalized anxiety disorder, and obsessive-compulsive disorder.

The step of measuring/determining/quantifying the relative dominance from the selected taxonomic “family” can select an appropriate methodology. eg the number of gut microbiome, the use of 16s rRNA sequencing or whole genome sequencing.

The measurement of the number of intestinal microbiome in a biological sample includes both determination of the presence or absence of the intestinal microbiome (qualitative) and/or quantitative measurement of the number of intestinal microbiome. The means for determining the presence or absence of the intestinal microbiome is, for example, a culture method in which the intestinal microbiome is cultured in a previously predicted selection medium and colonies of the target intestinal microbiome are confirmed or not, or in a selective liquid medium. It may be a method of culturing the biome and measuring turbidity or absorbance.

16S rRNA sequencing or whole genome sequencing may include FISH method, real-time PCR method (qPCR), RT-PCR method, Southern hybridization method, Northern hybridization method, DNA microarray method, and the like.

The analysis method includes, for example, (1) a step of extracting target intestinal microbiome RNA from a sample, (2) a step of PCR using a nucleic acid fragment (primer) that hybridizes to the extracted RNA, and (3) a step. It can be carried out by a step of detecting the DNA fragment amplified by (2). A DNA fragment (PCR product) specific to the target intestinal microbiome can be obtained by combining the nucleic acid fragment with a template cDNA derived from a specimen and performing an amplification reaction. By observing the PCR product over time and specifying the number of PCR cycles when a certain amount of DNA is reached, it becomes possible to quantify the target intestinal microbiome number in the sample.

Time-lapse observation of the amplified PCR product can be performed by labeling the PCR product with an intercalator-forming dye such as SYBR(R) Green I and measuring the fluorescence intensity at each PCR step. Since the intercalator dye has a property of increasing fluorescence intensity by being inserted into double-stranded nucleic acid, PCR products generated by PCR from the cDNA of a target bacterium can be accurately measured.

By specifying the number of PCR cycles (hereinafter referred to as CT value) when a certain fluorescence intensity (DNA amount) that is arbitrarily set is reached, the target intestinal microbiome in the sample can be quantified. In addition, a TaqMan probe labeled with a fluorescent dye or a Moleculer Beacon may be used. A TaqMan probe or Moleculer Beacon is a probe in which a fluorescent dye and a quencher are combined with an oligonucleotide having homology to the internal arrangement of the region to be amplified by PCR, and is used in conjunction with the PCR reaction.

Since fluorescence is emitted according to the PCR amplification reaction due to the interaction of the fluorescent dye bound to the probe and the quencher, the PCR product to be amplified can be observed over time by measuring the fluorescence intensity at each PCR step.

The target intestinal microbiome in a sample can be quantified by a calibration curve of the logarithmic value of the number of bacteria measured by the DAPI count method or the culture method and the CT value. That is, a calibration curve in which the logarithmic value of the number of target bacteria is plotted on the horizontal axis and the CT value on the vertical axis is prepared in advance, the PCR reaction result can be obtained, and the CT value is applied to the calibration curve to quantify the target intestinal microbiome in the sample. carry out

The information providing method for diagnosing depressive anxiety disorder according to the present invention includes (b) diagnosing depressive anxiety disorder based on the measured relative dominance.

The diagnosis assesses the patient's microbiome to determine if the patient is "healthy" (e.g., has a gut microbiome consistent with another individual considered healthy (e.g., free from a disorder or disease)) or " “has depressive anxiety disorder” (e.g., has a gut microbiome consistent with another individual considered “having depressive anxiety disorder” (e.g., has one or more depressive anxiety disorders or conditions)) may be deciding

In this determination, a normal group without depressive anxiety disorder can be used as a control group to determine the normal group. In addition, the patient group with depressive anxiety disorder can be used as a control group for determining the patient group with depressive anxiety disorder. In addition, the diagnosis of gastric depressive anxiety disorder may be performed based on criteria for relative dominance levels in the intestine of fixed values from multiple sample samples.

Diagnosing depressive anxiety disorder in any of the above embodiments may be an evaluation of the effectiveness of treatment of a patient with depressive anxiety disorder.

The number of organisms that make up the microbiome itself and/or the diversity among organisms allows clinicians to evaluate changes to the microbiome, evaluate/diagnose medical conditions, evaluate medical treatments, or otherwise have clinical significance. Characterizing the microbiome in a way that can correlate the microbiome with symptoms present can be difficult.

In the present invention, a method for providing information on depressive anxiety disorder by analyzing the characteristics of the intestinal microbiome by solving the problems of the above existing technologies is specifically provided. This method allows clinicians to assess medical symptoms for depressive anxiety disorder, diagnose medical symptoms/disorders, evaluate the success of medical treatment, combinations thereof, etc., and/or provide other clinically meaningful information to clinical patients. may be allowed to provide.

Depending on the provision of such information, an auxiliary role for providing information on the presence or absence of depressive anxiety disorder, the success of patient treatment, and the prescription of drugs for depressive anxiety disorder may also be provided.

The method for diagnosing depression/anxiety disorders according to the present invention can non-invasively analyze fecal microorganisms, which are causative factors, to induce depression and anxiety disorders according to the composition ratio of specific microorganisms and provide diagnostic information related thereto.

Figure 1 shows changes in intestinal occupancy of Lactobacillaceae, Bacteroidaceae, Enterobacteriaceae and Enterococcaceae (HC normal (healthy) group, IBD/D- (with colitis but without depression), IBD/D+ (with colitis and depression).
Figure 2 shows the positive correlation between the increase in the intestinal occupancy of Lactobacillaceae, Enterobacteriaceae and Enterococcaceae and the HADS-D score.
Figure 3 is Klebsiella sp. (Klebsiella oxytoca (Ko) and Klebsiella pneumoniae (Kp)) Escherichia coli (Ec), Cronobacter sakazakii (Cs) populations , and Enterococci show a greater increase in patients with depression.
Figure 4 is a result confirming that Escherichia coli, Klebsiella pneumonae, Klebsiella oxytoca, and Enterococcus sp . show high expression levels in depressed patients, and show a reduced tendency in the case of the Bifidobacteria population.
5 shows mortality, open arm, and immobility confirmation results measured from an elevated plus maze (EPM) test and a tail suspension test (TST).
6 shows that Enterobacteriaceae increases the number of BDNF+/NeuN+ cells and NF-κB+/Iba1+ cells in Hippocampus.
7 shows that Enterobacteriaceae increases corticosterone and LPS concentrations.
8 shows that Enterobacteriaceae reduce the length of the large intestine.
9 shows that Enterobacteriaceae increases myeloperoxidase (MPO).

Numerical values described in this specification should be construed as including equivalent ranges unless otherwise specified.

Hereinafter, preferred embodiments and experimental examples are presented to aid understanding of the present invention. However, the following examples and experimental examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited thereby.

Example 1. Analysis of intestinal bacteria in human feces

A patient group having the conditions shown in Table 1 below was recruited. Specifically, a normal person (HC), a patient group with colitis symptoms but no depression, and a patient group with depression and colitis symptoms were recruited and the experiment was performed. The presence or absence of depression was judged through HADS-A and HADS-D level tests.

Depression/anxiety disorder patients and healthy people Group age
(year of birth) Depression/anxiety ²⁾ HADS-A HADS-D Healthy person (HC) 39 (1979) N/A N/A 37 (1982) N/A N/A 41 (1978) N/A N/A 36 (1938) N/A N/A Mean±SD 38.2±2.2 - - Patients with colitis (without depression IBD/D ^{- 3)} UC 42 (1977) 4 5 34 (1984) 2 6 51 (1967) 4 5 48 (1971) 9 5 44 (1976) 8 6 CD 30 (1990) 2 3 14 (2006) 3 2 25 (1994) 5 4 Mean±SD 36.0±12.6 4.6±2.6 4.5±1.4 Depression/colitis patients (IBD/D ⁺⁴⁵⁾ UC 59 (1959) 15 16 57 (1962) 9 13 64 (1955) 11 11 26 (1993) 19 11 CD 26 (1992) 19 14 46 (1972) 7 14 47 (1971) 4 15 Mean±SD 46.4±15.3 12.0±5.9 13.4±1.9

²⁾ Psychiatric disorders were described as HADS (Hospital anxiety and depression scale) A (anxiety) and D (depression) score. ³⁾ Inflammatory bowel disease patients without depression (score, <10 on HADS-D)

⁴⁾ Inflammatory bowel disease patients with depression (score, >10 on HADS-D)

Intestinal microbiome analysis of human feces from the patient group was performed.

(1) Fecal microbial analysis by 16S rRNA gene analysis

The microbial community of feces of healthy people (normal group) and patients was measured through 16S rRNA gene to analyze the composition ratio of enteric bacteria. First, fresh feces were obtained, bacterial DNA was isolated with QIAamp DNA stool mini kit (Qiagen, Germany), and using barcoded primers (V4 part of bacterial 16S rRNA gene), Illumina iSeq 100 (San Diego, CA) 16S rRNA gene was measured.

(2) Analysis of intestinal bacterial community by culture method and identification of enteric bacteria

Fresh feces of humans and laboratory mice were suspended in GAM broth (Nissui Pharmaceutical, Japan) and serially diluted. 0.2 mL of the diluted solution was mixed with DHL KEPCO medium (Nissui Pharmaceutical Inc.), a selective medium for Enerobacteriaceae , m- Enterococcus KEPCO medium (BD, USA), a selective medium for Enteroacteriaceae , and MRS agar medium (BD, USA), a selective medium for lactic acid bacteria ( Lactobacillaceae , etc.) , Transplanted into BL medium (Nissui Pharmaceutical Inc.), a Bifidobaceriaceae selection medium, and cultured in a 37 degree incubator under aerobic conditions for 24 hours, aerobic conditions for 24 hours, aerobic conditions for 48 hours, anaerobic conditions for 72 hours, and anaerobic conditions for 72 hours, respectively. Then, the growing colonies were gram-stained, and bacteria were identified by analyzing the 16S rRNA gene.

(3) qPCR (Quantitative polymerase chain reaction)

qPCR was performed to measure the occupancy of Escherichia coli and Paenalcaligenes hominis in the gut microbial community. First, fecal DNA was isolated using a QIAamp DNA stool mini kit (Qiagen, Germany), and real time PCR was performed using Pimers in Table 2 below. The reaction was analyzed by first treating at 95 °C for 30 s, then repeating the reaction 42 times at 95 °C for 5 s and 72 °C for 30 s.

primer sequence Primer Forward Reverse Klebsiella pneumoniae 5′-GAGGGGGATAACTACTGGAAAC-3
(SEQ ID NO: 1)' 5′-TGAGCCATTACCCCACCTAC-3′
(SEQ ID NO: 2) Klebsiella oxytoca 5'-ACCTTACCCTACTCTTGACATCC-3' (SEQ ID NO: 3) 5'-CCCACCTTCCTCCAGTTTTATC-3'
(SEQ ID NO: 4) Escherichia coli 5'-CAGCCACACTGGAACTGA GA-3' (SEQ ID NO: 5) 5′-GTTAGCCGGTGCTCTCTTC TG-3′
(SEQ ID NO: 6) Cronobacter sakazakii 5'-GTAGCTAATACCGCATAAC GTC-3' (SEQ ID NO: 7) 5′-AACCACAACACCTTCCTC-3′
(SEQ ID NO: 8) Pedioccus acidilactici 5'-ACGCATTAAGTAATCCGCC-3' (SEQ ID NO: 9) 5′-ACCACCTGTCATTCTGTCC-3′
(SEQ ID NO: 10) Enterococcus faecium 5′-CGCATGGTTTTGATTTGAAAGG-3′ (SEQ ID NO: 11) 5′-TGTCTCAGTCCCAATGTGG-3′
(SEQ ID NO: 12) Bifidobacterium sp. 5'-CGCGTCYGGTGTGAAAG-3' (SEQ ID NO: 13) 5'-CCCCACATCCAGCATCCA-3'
(SEQ ID NO: 14) 16s rRNA 5′-TCGTCGGCAGCGTCAGAT GTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA-3′ (SEQ ID NO: 15) 5′-GTCTCGTGGGCTCGGAGAT GTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3′ (SEQ ID NO: 16)

(4) Experimental results

go. Confirmation of changes in intestinal occupancy of Lactobacillaceae, Bacteroidaceae, Enterobacteriaceae and Enterococcaceae

Changes in intestinal occupancy of Lactobacillaceae, Bacteroidaceae, Enterobacteriaceae and Enterococcaceae were confirmed and the results are shown in FIG.

Figure 1 shows changes in intestinal occupancy of Lactobacillaceae, Bacteroidaceae, Enterobacteriaceae and Enterococcaceae . Specifically, Bacteroidaceae was decreased in patients with colitis compared to the normal group. In the case of Lactobacillaceae, Enterobacteriaceae, and Enterococcaceae , all showed a tendency to increase in colitis patients with depression compared to the normal group.

Looking at this trend in detail, the following characteristics were shown.

Unlike healthy subjects, patients with depression and anxiety disorder showed a characteristic that the share of Enterobacteriaceae was more than 1% of the total microbiome.

In addition, unlike healthy subjects, patients with depressive disorder showed a characteristic in that the share of Lactobacillaceae and/or Enterococcaceae was more than 2% of the total microbiome.

In addition, in the case of Bacteroidaceae , Lactobacillaceae, Enterobacteriaceae , and Enterococcaceae tended to decrease in contrast to the increase, and when compared to healthy individuals, the occupancy rate was less than 50% of the total microbiome.

That is, the intestinal occupancy of these Lactobacillaceae, Bacteroidaceae, Enterobacteriaceae , and Enterococcaceae showed a change in terms of the total ratio of the “Family”, and it was confirmed that diagnosis of depression and anxiety disorder was possible through this.

The correlation between changes in intestinal occupancy of the Lactobacillaceae, Enterobacteriaceae and Enterococcaceae and the Hospital Anxiety and Depression Scale-depression (HADS-D) was also confirmed, and the results are shown in FIG. 2 .

As can be seen in Figure 2, the increase in the intestinal occupancy of Lactobacillaceae, Enterobacteriaceae and Enterococcaceae showed a positive correlation with the HADS-D score.

From the above results, it was confirmed that the diagnosis of depression was possible by confirming the increase in the intestinal occupancy of Lactobacillaceae, Enterobacteriaceae , and Enterococcaceae .

me. Analysis of the gut bacterial community

Bifidobacteria, Klebsiella sp. (Klebsiella oxytoca (Ko) and Klebsiella pneumoniae (Kp)) Escherichia coli (Ec) and Cronobacter sakazakii (Cs) populations, Enterococcus sp. and Pediococcus acidilactici (Pediococcus) populations, and Enterococcus were cultured to compare their degree of colonization.

The results are shown in Figure 3.

As can be seen in Figure 3, Klebsiella sp. (Klebsiella oxytoca (Ko) and Klebsiella pneumoniae (Kp)) Escherichia coli (Ec), Cronobacter sakazakii (Cs) populations , and Enterococci were found to increase more in patients with depression.

In addition, the expression level of each strain was confirmed through qPCR.

The results are shown in FIG. 4 . Escherichia coli, Klebsiella pneumonae, Klebsiella oxytoca, and Enterococcus sp . showed high expression levels in depressed patients, while Bifidobacteria population showed a decreased tendency.

On the other hand, by specifically analyzing the number of strains, it was confirmed that depressive anxiety disorder symptoms were high when the number of Klebsiella pneumoniae, Klebsiella oxytoca, and Cronobacter sakazakii in Enterobacteriaceae was greater than the number of Escherichia coli .

all. Enterococcaceae Species Analysis

Changes in expression levels of Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, Enterococcus faecium, and Enterococcus faecalis were confirmed through 16S rRNA, and the results are shown in Table 3 below.

experimental group Fold change (/16S rRNA) HC IBD/D- IBD/D+ Enterococcus faecalis 1 (0.06) 0.8 0.7 Enterococcus faecium 1 (0.04) 1.5 1.2 Enterococcus mundtii 1 (<0.01) 1.2 3.5 Enterococcus gallium 1 (<0.01) 0.8 2.1 Enterococcus durans 1 (<0.01) 1.5 1.5 sum (which is 0.1% of total fecal bacteria) (about 0.1%) (<0.05) (>2%)

As can be seen from the table above, the expression levels of Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, and Enterococcus faecium were significantly increased in the depressed patient group compared to the normal control group.

In addition, when the sum of specific strains was calculated, it was confirmed that the group with depression could be distinguished if the sum of Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, and Enterococcus faecium was greater than the number of Enterococcus faecalis .

Example 2. Confirmation of diagnosis of depression symptoms through mouse experiments

(1) Experimental animal design (cultivation and administration method of isolated enteric bacteria)

Experimental animal - 6 weeks old, male C57BL/6

The culture of the isolated strain is cultured anaerobically in a GAM liquid medium at 37 degrees for 24 hours, collected, diluted appropriately (1x10 ⁶ - 1x10 ¹⁰ CFU / mL), and administered 0.1 mL of this diluted solution once daily for 5 days. did Enteric bacteria were finally administered, and anxiety and depression behavioral tests were measured.

(2) Measurement of anxiety/depressive disorder behavior

go. Elevated plus maze (EPM) test

The mouse was placed facing the open arm with its head at the center of the EPM, and the time and number of times spent in the open and closed arms were measured for 5 minutes. Arm entry was made with all four feet entering. Time spent in open arms [(time spent in open arm)/(time spent in open arm+time spent in enclosed arm)x100)] and open arm entries[(entry into open arm)/(open arm entry+enclosed arm Admission) x 100)] was calculated for each mouse. After each behavioral experiment, the remaining odor was removed with 70% ethanol.

Elevated plus-maze experimental setup (two open arms (30 x 7 cm), two enclosed arms (30 x 7 cm) with walls 20 cm high, each extending 7 cm (7 x 7 cm) from the central platform, , made of black plexiglass and raised 50 cm above the floor) is a device capable of recording the movements of mice in a room with a video camera installed above it at a brightness of 20 lux.

me. Tail Suspension Test (TST)

A fixing device was attached to the tip of the mouse's tail about 1 cm in a tub with a diameter of 35 cm and a height of 50 cm, and then it was suspended 50 cm from the ground. The immobility time of the experimental animals was measured for a total of 6 minutes.

all. Cytokine measurement

IL-6, IL-1β, and TNF-α were measured using an ELISA Kit (Ebioscience, GA, USA).

L. Measurement of Myeloperoxidase (MPO) activity

200 μl of 10 mM potassium phosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammonium bromide was added to 100 mg of colon tissue, homogenized, and centrifuged at 4° C. and 10,000 g for 10 minutes to obtain a supernatant. 50 μl of the supernatant was added to 0.95 ml of a reaction solution (containing 1.6 mM tetramethyl benzidine and 0.1 mM H2O2), and the absorbance was measured over time at 650 nm while reacting at 37°C.

mind. Tissue staining test

The mice after the behavioral experiment were perfused whole body with 4% paraformaldehyde after 3 hours, and the brains were separated, fixed in 4% paraformaldehyde for 4 hours, and then immersed in 30% sucrose solution and sectioned. The sectioned tissue was reacted with antibodies of BDNF (1:200, Santa Cruz), NeuN (1:200, Millipore), Iba1 (1:200, Cell Signaling), and NF-kB (1:200, Santa Cruz) for 24 hours. and then reacted with Alexa Fluor 488 (1:200, Invitrogen) or Alexa Fluor 594 (1:200, Invitrogen) secondary antibody. Nuclear staining of cells was performed with 4',6-diamidino-2-phenylindole, dilactate (DAPI, Sigma) and observed with a confocal laser microscope.

(3) Experimental results

go. Mortality, open arm, and immobility check results

The mortality, open arm, and immobility confirmation results measured from the elevated plus maze (EPM) test and the tail suspension test (TST) are shown in FIG. 5 .

As can be seen in FIG. 5, Klebsiella oxytoca (Ko) and Klebsiella pneumoniae (Kp) imparted a negative effect to mice to the extent of affecting mortality.

In addition, Klebsiella oxytoca (Ko), Klebsiella pneumoniae (Kp), and Escherichia coli (Ec) had a negative effect on depression and anxiety disorders in the open arm test, and Klebsiella oxytoca (Ko), Klebsiella pneumoniae (Kp), Escherichia coli (Ec), Cronobacter sakazakii (Cs), and Pediococcus acidilactici (Pa) had negative effects on depression and anxiety disorders in the immobility test.

That is, Enterobacteriaceae isolated from colitis feces accompanied by depression and anxiety increased mortality, lowered OT in EPM, and increased immobility in TST.

me. Confirmation of changes in molecular biological indicators

The results of confirming the number of BDNF+/NeuN+ cells and NF-κB+/Iba1+ cells of Hippo Campus as molecular biological indicators are shown in FIG. 6 .

As can be seen in Figure 6, Enterobacteriaceae increased the number of BDNF+/NeuN+ cells and NF-κB+/Iba1+ cells of Hippocampus.

In addition, the results of measuring corticosterone and LPS concentrations in the blood are shown in FIG. 7 .

As can be seen in Figure 7, Enterobacteriaceae increased the concentration of corticosterone, LPS.

In addition, as can be seen in Figures 8 and 9, Enterobacteriaceae reduced the length of the colon and increased myeloperoxidase (MPO).

From the above results, it was confirmed that Enterobacteriaceae corresponded to factors highly related to depression and anxiety disorders. In particular, Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, and Cronobacter sakazakii were in the order of induction related to depression.

all. Confirmation of depressive-anxiety-inducing effect of Enterococcus

Changes in EPM, TST, serum corticosterone, and IL-6 associated with the induction of depression and anxiety disorders following the administration of E nterococcus mundtii, Enterococcus gallium, Enterococcus durans, Enterococcus faecium, and Enterococcus faecalis were confirmed.

The results are shown in Table 4 below.

experimental group EPM TST blood IL-6 blood corticosterone Normal group 12.5 22.2 22.0 13.2 Enterococcus faecalis 11.7 24.6 25.9 15.8 Enterococcus faecium 10.7 26.0 26.74 18.1 Enterococcus mundtii 6.2 42.8 49.9 191.5 Enterococcus gallium 9.9 34.5 33.1 75.7 Enterococcus durans 8.9 28.4 33.2 62.1

As can be seen in Table 4, the administration of Enterococcus mundtii, Enterococcus gallium, and Enterococcus durans showed the effect of inducing depressive anxiety disorder, but the administration of Enterococcus faecium and Enterococcus faecalis, when used in combination with enterococcus strains, enterobacteriaceae showed an aggravating effect on depression.

<110> KYUNG HEE UNIVERSITY <120> Depression and anxiety disorder diagnosis method using intestinal microbiome <130> KU1.52P <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> artificial sequence <220> <223> Klebsiella pneumoniae <400> 1 gagggggata actactggaa ac 22 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> Klebsiella pneumoniae Reverse <400> 2 tgagccatta ccccacctac 20 <210> 3 <211> 22 <212> DNA <213> artificial sequence <220> <223> Klebsiella oxytoca Forward <400> 3 accttaccta ctcttgacat cc 22 <210> 4 <211> 21 <212> DNA <213> artificial sequence <220> <223> Klebsiella oxytoca Reverse <400> 4 cccaccttcc tccagtttat c 21 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> Escherichia coli <400> 5 cagccacact ggaactgaga 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> Escherichia coli Reverse <400> 6 gttagccggt gcttcttctg 20 <210> 7 <211> 22 <212> DNA <213> artificial sequence <220> <223> Cronobacter sakazakii Forward <400> 7 gtagctaata ccgcataacg tc 22 <210> 8 <211> 18 <212> DNA <213> artificial sequence <220> <223> Cronobacter sakazakii Reverse <400> 8 aaccacaaca ccttcctc 18 <210> 9 <211> 19 <212> DNA <213> artificial sequence <220> <223> Pedioccus acidilactici Forward <400> 9 acgcattaag taatccgcc 19 <210> 10 <211> 19 <212> DNA <213> artificial sequence <220> <223> Pedioccus acidilactici Reverse <400> 10 accacctgtc attctgtcc 19 <210> 11 <211> 22 <212> DNA <213> artificial sequence <220> <223> Enterococcus faecium Forward <400> 11 cgcatggttt tgatttgaaa gg 22 <210> 12 <211> 19 <212> DNA <213> artificial sequence <220> <223> Enterococcus faecium Reverse <400> 12 tgtctcagtc ccaatgtgg 19 <210> 13 <211> 17 <212> DNA <213> artificial sequence <220> <223> Bifidobacterium sp. Forward <400> 13 cgcgtcyggt gtgaaag 17 <210> 14 <211> 18 <212> DNA <213> artificial sequence <220> <223> Bifidobacterium sp. Reverse <400> 14 ccccacatcc agcatcca 18 <210> 15 <211> 52 <212> DNA <213> artificial sequence <220> <223> 16s rRNA Forward <400> 15 tcgtcggcag cgtcagatgt gtataagaga caggtgccag cmgccgcggt aa 52 <210> 16 <211> 54 <212> DNA <213> artificial sequence <220> <223> 16s rRNA Reverse <400> 16 gtctcgtggg ctcggagatg tgtataagag acagggacta chvgggtwtc taat 54

Claims (12)

(a) measuring the relative abundance of Lactobacillaceae , Bacteroidaceae , Enterobacteriaceae and Enterococcaceae from a biological sample; and
(b) diagnosing depressive anxiety disorder based on the measured relative dominance. According to claim 1, the diagnosis of depressive anxiety disorder is depressive anxiety disorder when the relative abundance of the Lactobacillaceae , Enterobacteriaceae , and Enterococcaceae increases compared to the normal group. A method of providing information for diagnosing depressive anxiety disorder, which is to diagnose with having. The information for diagnosing depressive anxiety disorder according to claim 1, wherein the diagnosis of depressive anxiety disorder is diagnosed as having depressive anxiety disorder when the relative predominance (relative abundance) of the Bacteroidaceae is reduced compared to the normal group. How to provide. According to claim 1, the diagnosis of depressive anxiety disorder is the enterococci ( Enterococcaceae ) relative dominance (relative abundance) is increased compared to the normal group and Bacteroids ( Bacteroidaceae ) relative predominance is decreased compared to the normal group depression anxiety A method of providing information for diagnosing depressive anxiety disorder, which is to diagnose as having a disorder. According to claim 1, Lactobacillus ( Lactobacillaceae ) relative dominance (relative abundance) is increased compared to the normal group Bacteroides ( Bacteroidaceae ) If the relative predominance is decreased compared to the normal group to diagnose as having a depressive anxiety disorder An informational method for the diagnosis of depression and anxiety disorders. According to claim 1, Enterococcus family ( Enterococcaceae ) is Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, and Enterococcus faecium. To include any one or more selected from the group consisting of, information providing method for diagnosing depressive anxiety disorder. The method of claim 1, wherein the Enterobacteriaceae are Klebsiella pneumonia, Klebsiella oxytoca, Cronobacter sakazakii and Escherichia coli To include any one or more selected from the group consisting of, information providing method for diagnosing depressive anxiety disorder. The method of claim 1, comprising: measuring the relative abundance of Pediococcus acidilactici from the biological sample; and
(b) diagnosing depressive anxiety disorder based on the measured relative dominance;
An informational method for diagnosing depressive anxiety disorder. The method for providing information for diagnosing depressive anxiety disorder according to claim 1, wherein the step of measuring the relative abundance is by measuring the number of intestinal microbiome, 16s rRNA sequencing or whole genome sequencing. The method of claim 9, wherein the measurement of the number of intestinal microbiome is performed by culturing the intestinal microbiome in a selective medium and confirming the presence or absence of colonies of the target intestinal microbiome, for diagnosing depressive anxiety disorder. How to Provide Information. The method of claim 9, wherein the 16s rRNA sequencing or whole genome sequencing is performed by any one selected from the group consisting of FISH method, real-time PCR method, RT-PCR method, Southern hybridization method, Northern hybridization method, and DNA microarray method. , An informational method for diagnosing depressive anxiety disorder. The method of claim 1, wherein the depressive anxiety disorder is at least one selected from the group consisting of depression, insomnia, delusional disorder, phobia, panic disorder, generalized anxiety disorder, and obsessive-compulsive disorder.

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