KR20220114234A - Depression and anxiety disorder diagnosis method using intestinal microbiome - Google Patents

Abstract

The present invention relates to a depression/anxiety disorder diagnosis method using intestinal microbiome. The depression/anxiety disorder diagnosis method according to the present invention can diagnose a depression/anxiety disorder according to the composition ratio of specific microorganisms by non-invasively analyzing fecal microorganisms, which are the causative factors.

Description

Depression and anxiety disorder diagnosis method using intestinal microbiome

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

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

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

Mental disorders may have different symptoms for each patient, so if a mental disorder is suspected, treatment according to an accurate diagnosis and examination is required, but proper treatment is not performed due to the negative social perception of hospital treatment due to mental disorders to be. In addition, drugs such as antidepressants used to treat psychiatric disorders have limited therapeutic effects and may cause serious side effects such as cardiovascular disease and suicide.

Anxiety disorder, which is caused by various diseases such as panic disorder, generalized anxiety disorder, and phobia, refers to a disease in which physical and mental symptoms appear due to chronic worry and anxiety. Anxiety disorder is always in a state of tension, the autonomic nervous system is sharp, it is difficult to concentrate on work, and there are disturbances in daily life. About 25% of the population suffers from an anxiety disorder, and women are twice as likely as men.

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

Anxiety and depressive disorders are known to cause anxiety in the frontal lobe, limbic system, and basal nerve nuclei of the brain, but there is also a theory that the occipital lobe causes anxiety. A dysfunction in this area (a lack or excess of a neurotransmitter) causes anxiety.

If anxiety/depression is suspected by the mental state test, the differential diagnosis for other diseases, including hypothyroidism, which can cause anxiety/depression, should be prioritized.

Treatment varies slightly depending on each symptom, but for general drug treatment, antidepressants (such as SSRIs) and anti-anxiety drugs (such as benzodiazepines) are used. However, early detection and prevention is best.

Diagnosis and testing methods are: ① symptoms of anxiety disorders such as excessive anxiety and worry that persist for more than several days at a time for more than 6 months, ② anxiety is not controlled, ③ questionnaire, etc. 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/depression 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) Analysis and utilization of NGS of the intestinal microbial community based on the 16s ribosomal RNA (16s rRNA) gene sequence

Although correlations with depression/anxiety are reported in relation to the gastrointestinal microbial community, there are still limited types of microorganisms that have been identified in the library for interpreting the results of NGS.

It is not easy to accurately judge the method of (1) because there are many differences depending on mood. In addition, the method of (2) lacks sufficient data to analyze the correlation with the intestinal microbiome community.

Under this background, the present inventors analyzed fecal microorganisms, which are causative factors, by a non-invasive method, and found that if the composition ratio of specific microorganisms is high, depressive anxiety may be induced. In addition, we confirmed whether these gut bacteria induce depressive anxiety in animals (mouse), and completed a method for diagnosing depression/anxiety disorder using this.

KR10-2019-0047023

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

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

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

(b) diagnosing depressive anxiety disorder based on the measured relative dominance; provides an information providing method for diagnosing depressive anxiety disorder, including.

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

In the present invention, the relative dominance refers to a numerical value indicating the degree of dominance of each “family”, “genus”, and/or “species” within a community. These dominance plots may represent differences in the degree of dominance at the level of each of the above “family”, “genus”, and/or “species” depending on changes in the share of “family”, “genus”, and/or “species” in the gut. have.

The biological sample of step (a) may be derived from one or several people. For example, a biological sample derived from a subject, for example, the contents of the digestive tract such as intestinal fluid and feces, may be mentioned, but feces are particularly preferable because they have less burden on the subject.

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

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

In any of the above embodiments, when the relative abundance of Enterococcaceae increases compared to the normal group and the relative abundance of Bacteroidaceae decreases compared to the normal group, it is diagnosed as having depressive anxiety disorder. .

In any of the above embodiments, when the relative abundance of Lactobacillaceae increases compared to the normal group and the relative abundance of Bacteroidaceae decreases compared to the normal group, it is diagnosed as having depressive anxiety disorder.

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

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

In any of the above embodiments the taxonomic Enterococcaceae has an increased relative abundance in the group with depressive anxiety disorder.

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

In any of the above embodiments the taxonomic Enterococcaceae has an increased relative abundance in the group with depressive anxiety disorder while the Bacteroidaceae has a relative abundance in the group with depressive anxiety disorder is decreased

In any of the above embodiments the taxonomic Lactobacillaceae has an increased relative abundance in the group with depressive anxiety disorder, while the Bacteroidaceae has a relative abundance in the group with depressive anxiety disorder. decreases.

In the gut microbiome, the relative dominance of the gut microbiome can be determined based on the analysis of genera, species, and subspecies in the family or sub-family, at the taxonomic classification level. This relative dominance can be quantified and used to determine or diagnose depressive anxiety disorder.

Specifically, according to one embodiment of the present invention, Lactobacillaceae increases the relative abundance in the group with depressive anxiety disorder compared to normal (healthy). This increase in relative dominance may be an increase of 2%, 3%, 4%, 5% or more of the lactic acid bacteria family ( Lactobacillaceae ) based on the entire microbiome when compared to a normal person (healthy person). This increase in relative dominance also showed that 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 measuring the fold change level of 16S rRNA when compared to normal (healthy). may be an increase.

Specifically, according to one embodiment of the present invention, Enterobacteriaceae has an increased relative abundance in the group with depressive anxiety disorder compared to normal (healthy) people. This increase in relative dominance may be an increase in Enterobacteriaceae by 2%, 3%, 4%, 5% or more of the total microbiome compared to a normal person (healthy person). This increase in relative dominance also showed that 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 measuring the fold change level of 16S rRNA when compared to normal (healthy). may be an increase.

Specifically, according to one embodiment of the present invention, Enterococcaceae has an increased relative abundance in the group with depressive anxiety disorder compared to normal (healthy) people. This increase in relative dominance may be 2%, 3%, 4%, 5% or more of Enterococcaceae based on the total microbiome when compared to a normal person (healthy person). This increase in relative dominance also showed that 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 measuring the fold change level of 16S rRNA when compared to normal (healthy). may be an increase.

Specifically, according to one embodiment of the present invention, Bacteroidaceae has a reduced relative abundance in the group with depressive anxiety disorder compared to normal (healthy) people. This increase in relative dominance may be a decrease of 2%, 3%, 4%, 5% or more of the entire microbiome when compared to a normal person ( healthy person). This decrease in relative dominance was also found to be 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 (healthy). may be a decrease.

Due to the number of organisms constituting the microbiome and/or diversity among organisms, the above-mentioned changes in the relative dominance of the “and” level may occur alone, in combination of two or more than three. In other words, such changes in the “and” community of the gut microbiome may have a correlation between “and”.

According to one specific embodiment, the taxonomic Enterococcaceae increases the relative abundance in the group with depressive anxiety disorder, while the Bacteroidaceae shows the relative abundance in the group with depressive anxiety disorder. is decreased

In any of the above embodiments the taxonomic Lactobacillaceae has an increased relative abundance in the group with depressive anxiety disorder, while the Bacteroidaceae has a relative abundance in the group with depressive anxiety disorder. decreases.

According to one specific embodiment, the taxonomic Enterobacteriaceae increased the relative abundance in the group with depressive anxiety disorder, while the Bacteroidaceae had a relative abundance in the group with depressive anxiety disorder. is decreased

According to one specific embodiment, at least one selected from the group consisting of taxonomic enterococcaceae , Enterobacteriaceae and Lactobacillaceae has a relative abundance in the group with depressive anxiety disorder. As Bacteroidaceae increases, the relative abundance of Bacteroidaceae decreases in the group with depressive anxiety disorder.

Among the species included in this family, the following species have a great relevance to changes in the colony of the intestinal microbiome “family”.

For example, with respect to Enterococcaceae ,

Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, Enterococcus faecium, Enterococcus faecalis, etc. may be mentioned. .

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

In particular, when considering the sum of strains, in patients with depression , Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, Enterococcus faecium The number of sums may exhibit more characteristics than that of Enterococcus faecalis .

For example, with respect to Enterobacteriaceae , Klebsiella pneumoniae, Klebsiella oxytoca, Cronobacter sakazakii, Escherichia coli ) and the like.

The above-mentioned strains significantly increase their occupancy level in the intestine when compared to the normal (healthy) group. In particular, when considered in terms of the sum of strains, the consensus number of Enterobacteriaceae ( Klebsiella pneumonia, Klebsiella oxytoca, and Cronobacter sakazakii) in patients with depression is Escherichia coli (Escherichia coli) may exhibit a more detectable feature.

That is, as an additional single strain, Pediococcus acidilactici significantly 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 measuring 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” mainly refers to a neuropsychiatric disorder in which actions such as sadness, tastelessness, loss of emotion, anhedonia (lack of pleasure), or hallucinations, delusions, etc. It refers to a disease that leads to suicidal thoughts and desires to commit suicide. The depression is accompanied by various physical symptoms such as decreased appetite, insomnia, constipation, decreased libido, or increased susceptibility to diseases due to decreased immune function. More specifically, it means a symptom of decreased activity.

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

Measuring/determining/quantifying the relative dominance from a selected taxonomic “family” may select an appropriate methodology. This may include, for example, determination of gut microbiome numbers, the use of 16s rRNA sequencing or whole genome sequencing.

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 of culturing the intestinal microbiome in a previously predicted selective medium and confirming the presence or absence of colonies of the target intestinal microbiome, or the intestinal microbiome in a selective liquid medium. It may be a method of culturing a biome to measure turbidity or absorbance.

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

The analysis method is, for example, (1) a step of extracting RNA of the target intestinal microbiome from a sample, (2) a step of PCR using a nucleic acid fragment (primer) that hybridizes to the extracted RNA, and (3) step (2) can be carried out by the step of detecting the amplified DNA fragment. A DNA fragment (PCR product) specific to the target intestinal microbiome can be obtained by combining the nucleic acid fragment with the template cDNA derived from the 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 number of intestinal microbiome in a sample.

The temporal observation of the PCR product to be amplified can be performed by labeling the PCR product with an intercalator shaping photochromic dye such as SYBR(R) Green I and measuring the fluorescence intensity in each PCR step. Since the intercalator dye has the property of increasing the fluorescence intensity by inserting it into the double-stranded nucleic acid, it is possible to accurately measure the PCR product generated by the PCR reaction in the cDNA of the target bacterium.

By specifying the number of PCR cycles (hereinafter referred to as the CT value) when a arbitrarily set constant fluorescence intensity (DNA amount) is reached, it is possible to quantify the target intestinal microbiome in the specimen. In addition, a TaqMan probe, Moleculer Beacon, etc. labeled with a fluorescent dye can also be used. A TaqMan probe or Moleculer Beacon is a probe that combines a fluorescent dye and a quencher to an oligonucleotide having homology with the internal arrangement of the region amplified by PCR, and is used in the PCR reaction.

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

Quantification of the target intestinal microbiome in the specimen can be obtained from the logarithmic value of the number of bacteria measured by the DAPI count method or the culture method, and a calibration curve of the CT value. That is, a calibration curve in which the logarithmic value of the target bacterial count 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 is an assessment of the patient's microbiome to determine whether the patient is "healthy" (e.g., having a gut microbiome consistent with another individual considered healthy (e.g., free of a disorder or disease)) or " have a depressive-anxiety disorder (e.g., having a gut microbiome consistent with other individuals considered "having depressive-anxiety disorder" (e.g., having 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 for judging the normal group. In addition, the patient group with depressive anxiety disorder can be used as a control group for judging the patient group with depressive anxiety disorder. It is also possible to perform a diagnosis of gastric depressive anxiety disorder based on a criterion for a relative dominance level in the intestine of a fixed value from a plurality of sample samples.

In any of the above embodiments the diagnosis of depressive anxiety disorder may be an evaluation of effectiveness for treatment of a patient having depressive anxiety disorder.

Due to the number of organisms that make up the microbiome and/or the diversity among organisms, clinicians can evaluate changes to the microbiome, evaluate/diagnose medical symptoms, evaluate medical treatment, or otherwise clinically meaningful Characterizing the microbiome in a way that can correlate the symptoms present with the microbiome can be challenging.

The present invention specifically provides a method of providing information on depressive anxiety disorder by analyzing the characteristics of the intestinal microbiome by solving the problems of the existing techniques as described above. This method allows clinicians to evaluate medical symptoms for depressive anxiety disorder, diagnose medical symptoms/disorders, evaluate the success of medical treatment, combinations thereof, and/or other clinically meaningful information. may be allowed to provide.

By providing such information, it can also provide an auxiliary role for providing information on the presence or absence of depressive anxiety disorder, the success of patient treatment, prescription of drugs for depressive anxiety disorder, and the like.

The method for diagnosing depression/anxiety disorder according to the present invention is a non-invasive method that analyzes fecal microorganisms as causative factors to induce depression and anxiety disorders according to the composition ratio of specific microorganisms and provide diagnostic information related thereto.

1 shows changes in intestinal occupancy of Lactobacillaceae, Bacteroidaceae, Enterobacteriaceae and Enterococcaceae (HC normal (healthy) group, IBD/D- (with colitis but no depression), IBD/D+ (with colitis and depression).
Figure 2 shows a positive correlation between Lactobacillaceae, Enterobacteriaceae and Enterococcaceae increased intestinal occupancy and HADS-D score.
3 shows Klebsiella sp. (Klebsiella oxytoca (Ko) and Klebsiella pneumoniae (Kp)) Escherichia coli (Ec), Cronobacter sakazakii (Cs) populations , and Enterococci showed a greater increase in the patients with depression.
4 is a result confirming that Escherichia coli, Klebsiella pneumonae, Klebsiella oxytoca, and Enterococcus sp . show a high expression level in depressed patients, and show a decreased tendency in the case of the Bifidobacteria population.
Figure 5 shows the results of the confirmation of mortality, open arm and immobility measured from the elevated plus maze (elevated plus maze, EPM) test and tail suspension test (Tail Suspension Test, TST).
6 shows that Enterobacteriaceae increases the number of BDNF+/NeuN+ cells and NF-κB+/Iba1+ cells of Hippocampus.
7 shows that Enterobacteriaceae increases corticosterone and LPS concentrations.
8 shows that Enterobacteriaceae decrease the length of the large intestine.
9 shows that Enterobacteriaceae increases myeloperoxidase (MPO).

Numerical values described herein should be construed to include equivalent ranges unless otherwise specified.

Hereinafter, preferred examples and experimental examples are presented to help the understanding of the present invention. However, the following Examples and Experimental Examples are only provided for easier understanding of the present invention, and thus the content of the present invention is not limited thereto.

Example 1. Analysis of enterobacteriaceae in human feces

A patient group having the conditions shown in Table 1 below was recruited. Specifically, the experiment was conducted by recruiting normal persons (HC), a group of patients with colitis symptoms but without depression, and a group of patients with colitis symptoms along with depression. 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 - - Colitis patients (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 was performed from the patient group.

(1) Analysis of fecal microorganisms by 16S rRNA gene analysis

The composition ratio of intestinal bacteria was analyzed by measuring the microbial community in the feces of healthy people (normal group) and patients through the 16S rRNA gene. First, obtain fresh feces, separate bacterial DNA with QIAamp DNA stool mini kit (Qiagen, Germany), use barcoded primer (V4 part of bacterial 16S rRNA gene), and Illumina iSeq 100 (San Diego, CA) The 16S rRNA gene was measured.

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

Fresh feces of human and laboratory animals were suspended in GAM broth (GAM broth; Nissui Pharmaceutical, Japan) and serially diluted. Add 0.2 mL of the diluted solution to the Enerobacteriaceae selective medium (Nissui Pharmaceutical Inc.), Enteroacteriaceae selective medium, m- Enterococcus Korean culture medium (BD, USA), and lactic acid bacteria ( Lactobacillaceae , etc.) MRS agar (BD, USA) , Bifidobaceriaceae were transplanted into BL medium (Nissui Pharmaceutical Inc.), a selective medium, and cultured in a 37 degree incubator at 24 h aerobic, 24 h aerobic, 48 h anaerobic, and 72 h anaerobic conditions, respectively. Then, the grown colonies were Gram-stained and the bacteria were identified by analyzing the 16S rRNA gene.

(3) qPCR (Quantitative polymerase chain reaction)

qPCR was performed to determine the share of Escherichia coli, Paenalcaligenes hominis , etc. in the intestinal microbiota. First, fecal DNA was isolated using the QIAamp DNA stool mini kit (Qiagen, Germany), and real-time PCR was performed using the Pimers in Table 2 below. The reaction was analyzed by first treatment at 95 °C for 30 s, followed by 42 repetitions 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'-ACCTTACCTACTCTTGACATCC-3' (SEQ ID NO: 3) 5'-CCCACCTTCCTCCAGTTTATC-3'
(SEQ ID NO: 4) Escherichia coli 5'-CAGCCACACTGGAACTGA GA-3' (SEQ ID NO: 5) 5'-GTTAGCCGGTGCTTCTTC 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'-CGCATGGTTTTGATTGAAAGG-3' (SEQ ID NO: 11) 5'-TGTCTCAGTCCCAATGTGG-3'
(SEQ ID NO: 12) Bifidobacterium sp. 5'-CCGGTCYGGTGTGAAAG-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. 1 .

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

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

Unlike healthy subjects, patients with depressive anxiety disorder showed that the share of Enterobacteriaceae was greater than 1% of the total microbiome.

In addition, patients with depressive disorder showed a characteristic that, unlike healthy people, the share of Lactobacillaceae and/or Enterococcaceae was 2% or more with respect to the entire microbiome.

In addition, in the case of Bacteroidaceae , Lactobacillaceae, Enterobacteriaceae, and Enterococcaceae showed a tendency to decrease opposite to the increase, and compared with healthy people, the occupancy rate was 50% or less for the entire microbiome.

That is, the intestinal occupancy of these Lactobacillaceae, Bacteroidaceae, Enterobacteriaceae and Enterococcaceae showed changes in the overall ratio of the “Family”, confirming that the diagnosis of depressive anxiety disorder was possible.

The correlation between changes in intestinal occupancy of Lactobacillaceae, Enterobacteriaceae and Enterococcaceae and 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 intestinal occupancy of Lactobacillaceae, Enterobacteriaceae and Enterococcaceae was positively correlated with the HADS-D score.

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

me. Analysis of the intestinal 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, Enterococcus was cultured and the degree of colony formation was compared.

The results are shown in FIG. 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 the 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, and decreased in the Bifidobacteria population.

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

All. Enterococcaceae Species analysis

For Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, Enterococcus faecium, and Enterococcus faecalis , the change in expression level was 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 (0.1% of total bacteria in feces) (about 0.1%) (<0.05) (>2%)

As can be seen from the above table, 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 when the sum of Enterococcus mundtii, Enterococcus gallium, Enterococcus durans, and Enterococcus faecium was greater than the number of Enterococcus faecalis , it was possible to distinguish it as a depressed patient group.

Example 2. Confirmation of Depression Symptom Diagnosis through Mouse Experiment

(1) Experimental animal design (culture and administration of isolate enterobacteriaceae)

Experimental animal - 6 weeks old, male C57BL/6

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

(2) Measurement of anxiety/depressive disorder behavior

go. Elevated plus maze (EPM) test

The mouse was placed with the open arm facing the head so that the head was in the center of the EPM, and the time and number of times spent on the open arm and the closed arm for 5 minutes were measured. Arm entry was made with all four feet. Time spent in open arms [(Time spent in open arm)/(Time spent in open arm+Time spent in closed arm)x100)] and open arm entries[(Entry into open arm)/(Entry into open arm+Enclosed arm) entry) x 100)] for each mouse. At the end of each behavioral experiment, the remaining odor was removed with 70% ethanol.

Elevated plus-maze experimental device (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 50cm above the floor) is a device that can record the movement of a mouse in a room with a video camera installed above it with a brightness of 20 lux.

me. Tail Suspension Test (TST)

In a barrel with a diameter of 35 cm and a height of 50 cm, a fixing device was mounted about 1 cm from the tip of the tail of the mouse, and then hung 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).

D. Myeloperoxidase (MPO) activity measurement

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 experiment

After the behavioral experiment was completed, the mice were systemically perfused with 4% paraformaldehyde 3 hours later, the brain was separated, fixed in 4% paraformaldehyde for 4 hours, and sectioned after soaking in 30% sucrose solution. Sectioned tissues were 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 confirmation results

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

As can be seen in Figure 5, Klebsiella oxytoca (Ko), Klebsiella pneumoniae (Kp) was given a negative effect to the mouse enough to affect mortality.

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

That is, Enterobacteriaceae isolated from colitis feces with depressive 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 Hippocampus as molecular biomarkers are shown in FIG. 6 .

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

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

As can be seen in FIG. 7 , Enterobacteriaceae increased corticosterone and LPS concentrations.

In addition, as can be seen in FIGS. 8 and 9, Enterobacteriaceae decreased the length of the large intestine and increased myeloperoxidase (MPO).

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

All. Confirmation of Depressive Anxiety Inducing Effects of Enterococcus

Changes in EPM, TST, blood corticosterone and IL-6 related to depression and anxiety disorders following administration of Enterococcus 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 was used in combination with enterobacteriaceae and strains. It has been shown to have the effect of exacerbating 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 Forward <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 Forward <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 any one or more selected from the group consisting of Lactobacillaceae , Bacteroidaceae , Enterobacteriaceae and Enterococcaceae from the biological sample ; and
(b) diagnosing depressive anxiety disorder based on the measured relative dominance; information providing method for diagnosing depressive anxiety disorder comprising a. According to claim 1, wherein the diagnosis of depressive anxiety disorder is selected from the group consisting of Lactobacillaceae , Enterobacteriaceae , and Enterococcaceae . The relative abundance of at least one selected from the group is normal. A method of providing information for diagnosing depressive anxiety disorder, which is to diagnose as having depressive anxiety disorder when it increases compared to the present invention. The information for diagnosing depressive anxiety disorder according to claim 1, wherein the diagnosis of depressive anxiety disorder is to diagnose as having depressive anxiety disorder when the relative abundance of the Bacteroidaceae is decreased compared to the normal group. How to provide. According to claim 1, Depressive anxiety disorder diagnosis is when the relative abundance of Enterococcaceae increases compared to the normal group and the relative abundance of Bacteroidaceae decreases compared to the normal group. A method for providing information for diagnosing depressive anxiety disorder, which is to diagnose as having a disorder. The method according to claim 1, wherein the relative abundance of Lactobacillaceae increases compared to the normal group and the relative abundance of Bacteroidaceae decreases compared to the normal group. Diagnosing with depressive anxiety disorder Phosphorus, an informational method for diagnosing depressive anxiety disorder. According to claim 1, Enterococcus family ( Enterococcaceae ) Enterococcus mundtii (Enterococcus mundtii), Enterococcus gallium (Enterococcus gallium), Enterococcus durans (Enterococcus durans) and Enterococcus faecium (Enterococcus faecium) A method of providing information for diagnosing depressive anxiety disorder, comprising any one or more selected from the group consisting of. According to claim 1, Enterobacteriaceae ( Enterobacteriaceae ) Klebsiella pneumoniae ( Klebsiella pneumonia), Klebsiella oxytoca (Klebsiella oxytoca), Chronobacter sakazakii (Cronobacter sakazakii) and Escherichia coli (Escherichia coli) Including any one or more selected from the group consisting of, information providing method for diagnosing depressive anxiety disorder. The method of claim 1, further comprising: measuring the relative abundance of Pediococcus acidilactici from the biological sample; and
(b) diagnosing depressive anxiety disorder based on the measured relative dominance;
Informational Methods for Diagnosing Depressive Anxiety Disorder. The method 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. 10. The method of claim 9, wherein the measurement of the number of intestinal microbiome is a culture method of culturing the intestinal microbiome in a selective medium and confirming the presence or absence of colonies of the target intestinal microbiome, and an information providing method for diagnosing depressive anxiety disorder. The method according to 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, phobias, panic disorder, generalized anxiety disorder and obsessive-compulsive disorder.

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