KR20200095308A - Prediction of atopic dermatitis development and persistence using changes in the gut microbial community - Google Patents

Abstract

The present invention relates to prediction of atopic dermatitis persistence by using a change in microbiome in feces. More particularly, by detecting the strain of Streptococcus, the strain of Clostridium, or the strain of the genus Akkermansia in the fecal sample of atopic dermatitis patients, it is possible to objectively and easily diagnose the persistence or severity of atopic dermatitis. Accordingly, the present invention can contribute to choosing a treatment method for atopic dermatitis.

Description

Prediction of the occurrence and persistence of atopic dermatitis using changes in intestinal microbial community {PREDICTION OF ATOPIC DERMATITIS DEVELOPMENT AND PERSISTENCE USING CHANGES IN THE GUT MICROBIAL COMMUNITY}

The present invention relates to compositions and uses thereof for predicting the persistence of atopic dermatitis.

Atopic Dermatitis (AD) is a chronic inflammatory skin disease and is associated with skin hypersensitivity to environmental stimuli, and can affect more than 15% of children. In general, atopic dermatitis appears in infancy and childhood, but can persist into adolescence and adulthood. Symptoms of atopic dermatitis change depending on various factors, and the severity of atopic dermatitis may become severe or intractable depending on specific factors, and the treatment situation also greatly affects the course of the treatment.

For the treatment of atopic dermatitis, it is sufficient to use moisturizers to compensate for dryness and deterioration of the skin barrier function and to prevent inflammation, but in severe cases, steroids are used to suppress the inflammation of atopic dermatitis. Since side effects can occur if steroids are used extensively for a long period of time, it is important to apply different treatment policies, such as appropriately selecting drugs according to the severity of atopic dermatitis.

Meanwhile, the judgment of the severity of atopic dermatitis depends on the findings by the naked eye, and the doctor relies on the judgment of the severity according to the findings to determine the patient's treatment guidelines. Severity Scoring of Atopic Dermatitis (SCORAD), which is frequently used worldwide to classify the severity of atopic dermatitis, also has limitations in that it requires professional skills by an experienced doctor and takes a long time. In particular, in infants and toddlers, which account for about 89% of atopic dermatitis patients, since atopic dermatitis worsens in a short period of time, an index that sensitively reflects the condition is required, but the standard index does not sensitively reflect the condition or has a high value in infants and toddlers. There are problems such as showing.

With the recent development of gene sequencing technology, it has become possible to investigate microbiome in various parts of the human body, and various studies have shown that the microbial group of infants suffering from atopic dermatitis is different from that of infants without atopic dermatitis. The facts were found (Fujimura KE, Nat Med 2016; 22: 1187-91). However, so far, there are no studies to determine the relationship between the intestinal microbiome and the phenotype or severity of atopic dermatitis, and to use it for the selection of atopic dermatitis treatment direction and determination of the treatment course.

The present inventors have completed the present invention by characterizing intestinal microorganisms according to the phenotype of atopic dermatitis, and confirming a colony of intestinal microorganisms predicting the onset and persistence of atopic dermatitis using gene sequence analysis.

In the present invention, by analyzing the intestinal microbiome of the normal control group, transient type atopic dermatitis group, and persistent type atopic dermatitis group, by discovering predictive markers suggesting the possibility and persistence of atopic dermatitis occurrence, early prevention of the occurrence of atopic dermatitis in infants or We intend to contribute to choosing a treatment method after dermatitis has occurred.

In one aspect, the present invention is one or more strains selected from the group consisting of strains of the genus Streptococcus , strains of the genus Clostridium and strains of the genus Akkermansia in a fecal sample of atopic dermatitis patient. It provides a composition for predicting the persistence of atopic dermatitis comprising a formulation capable of detecting.

In another aspect, the present invention provides a kit for predicting persistence of atopic dermatitis, comprising the composition.

In another aspect, the present invention is one selected from the group consisting of: i) a strain of the genus Streptococcus , a strain of the genus Clostridium , and a strain of the genus Akkermansia in a fecal sample of atopic dermatitis patient. Measuring the amount of the amplification product of the above strain DNA; Ii) comparing the amount of the amplified product with the amplified product of genomic DNA obtained from a fecal sample of a healthy person who does not have atopic dermatitis; comprising, providing a method of providing information necessary for predicting persistence of atopic dermatitis .

In yet another aspect, the present invention is characterized in that: i) one or more strains of DNA selected from the group consisting of strains of the genus Streptococcus and strains of the genus Clostridium in a fecal sample of a patient undergoing treatment for atopic dermatitis Measuring the amount of the amplification product; And ii) comparing the amount of the amplified product with the amplification product of genomic DNA obtained from a fecal sample of a healthy person who does not have atopic dermatitis; comprising, a method for providing information necessary for determining a treatment policy for atopic dermatitis Provides.

Hereinafter, the present invention will be described in more detail.

In the present invention, as a result of analyzing the effect of the composition of the microbial community on the determination of the persistence and severity of atopic dermatitis in feces of a 6-month-old infant diagnosed as having atopic dermatitis, the increase in strains of Streptococcus genus It was confirmed that the increase of the ( Clostridium ) sp. strain and the decrease or non-detection of the Akkermansia sp. strain affect the persistence and severity of atopic dermatitis.

Therefore, the present invention provides a composition, kit, and method for detecting a strain of the genus Streptococcus , a strain of the genus Clostridium , or a strain of the genus Akkermansia in a fecal sample of a patient with atopic dermatitis. In the present invention, the term "microbiome" refers to the entire genetic information of microorganisms coexisting in the human body, and in the present invention, the intestinal microbiome refers to the entire genetic information of microorganisms present in the intestine. do.

In the present invention, the term "transient AD" refers to a phenotype in which atopic dermatitis occurs at 6 months of age and disappears at 1 year of age, and "persistent AD" develops at 6 months of age and continues until 2 years of age. It means the phenotype that becomes.

In the present invention, the term "predicting persistence of atopic dermatitis" is to determine whether the possibility of treatment for atopic dermatitis is relatively low, or whether the severity of atopic dermatitis becomes severe or refractory for infants and toddlers diagnosed as atopic dermatitis under 24 months of age. Say what to do. The present invention can be used clinically to select the most appropriate treatment method by early diagnosis of infants and toddlers of 24 months or younger with atopic dermatitis with persistent atopic dermatitis or high severity of atopic dermatitis.

In the present specification, "Streptococcus" refers to a microorganism or a community thereof composed of species belonging to the genus Streptococcus taxonomically. In the present invention, Streptococcus is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more when comparing the previously reported Streptococcus and 16S rRNA sequence as well as the previously reported strains. , Most preferably all microorganisms having a sequence homology of 95% or more are included in the scope of the present invention.

In the present invention, "Clostridium" refers to a microorganism or a community thereof composed of species belonging to the genus Clostridium taxonomically. Clostridium in the present invention is preferably 70% or more, more preferably 80% or more, more preferably 90% when comparing the previously reported Clostridium and 16S rRNA sequence as well as the previously reported strains. All microorganisms having a sequence homology of at least 95%, most preferably at least 95% are included in the scope of the present invention.

In the present invention, "Ackermancia" refers to a microorganism or a colony thereof composed of species belonging to the genus Akermancia taxonomically. In the present invention, Akermansia is preferably 70% or more, more preferably 80% or more, and even more preferably 90% when comparing the previously reported Akermancia and 16S rRNA sequence as well as the previously reported strains. All microorganisms having a sequence homology of at least 95%, most preferably at least 95% are included in the scope of the present invention.

In the present invention, the term "detectable agent" means a substance that can be used to detect the presence of streptococcus, clostridial and/or akermansia, which are diagnostic markers for persistent atopic dermatitis in feces. For example, specific organic biomolecules such as proteins, nucleic acids, lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) present specifically in Streptococcus, Clostridium and/or Akermancia It may be a primer, a probe, an antisense oligonucleotide, an aptamer, or an antibody that can be detected.

Preferably, the agent detectable in the present invention may be a primer capable of detecting Streptococcus, Clostridium and/or Akermansia. It is preferable that the primer specifically detects the genomic sequence of Streptococcus, Clostridium and/or Akermancia and does not specifically bind to the genomic sequence of other microorganisms. More preferably, it may be a primer capable of amplifying 16S rRNA of one or more microorganisms selected from the group consisting of Streptococcus, Clostridium and/or Acermancia.

In the present invention, the term "primer" refers to 7 to 50 nucleic acid sequences that can form a base pair complementary to a template strand and function as a starting point for template strand copying. Primers are usually synthetic but can also be used on naturally occurring nucleic acids. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, and only needs to be sufficiently complementary so that it can hybridize with the template. Additional features can be incorporated that do not change the basic properties of the primer. Examples of additional features that may be incorporated include, but are not limited to, methylation, encapsulation, substitution of one or more nucleic acids with homologs, and modifications between nucleic acids. Preferably, the primer of the present invention may be a primer capable of amplifying 16S rRNA of Streptococcus, Clostridium and/or Akermansia microorganisms.

As used herein, the term "16S rRNA" is an rRNA constituting the 30S subunit of a prokaryotic ribosome, a conserved region common to all species and a hypervariable region capable of classifying a specific species Since this exists, microorganisms can be identified through sequencing. In particular, since there is little diversity between homogeneous species, diversity appears between other species, and thus prokaryotes can be usefully identified by comparing the sequence of 16S rRNA. In addition, 16S rDNA is a gene that encodes 16S rRNA, so 16S rDNA can be used to identify microorganisms.

In a preferred embodiment, in the present invention, the primer can be used to amplify a 16S rRNA (or 16S rDNA) sequence specific to the genus Streptococcus, Clostridium and/or Akermancia, and the sequence amplification results in the production of a desired product. Whether or not it is possible to detect the presence of the genus Streptococcus, genus Clostridium, and/or genus Akermansia. A method of amplifying a sequence using a primer may use various methods known in the art. For example, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR, touchdown PCR, hot start PCR, nested PCR, Booster PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction , Vectorette PCR, TAIL-PCR (thermal asymmetric interlaced PCR), ligase chain reaction, repair chain reaction, transcription-mediated amplification, self-maintaining sequence replication, or selective amplification of the target sequence may be used. , The scope of the present invention is not limited thereto.

In addition, the microorganism detection agent in the present invention may be an antibody, and the microorganism may be detected using an immunological method based on an antigen-antibody reaction. Analysis methods for this include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, and rocket immunoelectricity. Electrophoresis, tissue immunostaining, immunoprecipitation assay, Complement Fixation Assay, FACS (Fluorescence activated cell sorter), or protein chip, etc., but are not limited thereto.

In addition, molecular and immunological methods widely used in the art can be used to detect the microorganism of the present invention.

The diagnostic composition including the microorganism detection agent of the present invention may be provided in the form of a diagnostic kit. The kit of the present invention not only includes detection agents such as primers, probes, antisense oligonucleotides, aptamers, or antibodies for detecting the microorganism, but also one or more other constituent compositions, solutions, or devices suitable for analysis methods. May be included.

As a specific example, in the present invention, a kit including a primer specific to the genus Streptococcus, genus Clostridium, and/or genus Akermancia may be a kit including essential elements for performing an amplification reaction such as PCR. . For example, the kit for PCR is a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water ), or sterilized water.

In another aspect, the present invention provides a method of providing information necessary for predicting the persistence of atopic dermatitis in a patient by detecting a strain of Streptococcus sp. do.

In other words, the present invention provides a method of detecting a strain of the genus Streptococcus, a strain of the genus Clostridium and/or a microorganism of the genus Akermansia from feces of a patient with atopic dermatitis in order to provide information necessary for predicting the persistence of atopic dermatitis.

As a preferred example, the method may be implemented including the following steps:

(a) extracting genomic DNA from feces of atopic dermatitis patient,

(b) reacting the extracted genomic DNA with a primer specific to a strain of the genus Streptococcus, a strain of genus Clostridium and/or a microorganism of the genus Akermancia, and

(c) amplifying the reaction.

The extraction of genomic DNA from feces of atopic dermatitis patient in the step (b) can be performed by applying a general technique known in the art, and the strain of the genus Streptococcus, the strain of the genus Clostridium, and/or the microorganism of the genus Akermansia Specific primers are as described above.

The method of amplifying the reactant in step (c) includes general amplification techniques known in the art, for example, polymerase chain reaction, reverse transcription-polymerase chain reaction, multiplex PCR, touchdown PCR, hot start PCR, nested. PCR, booster PCR, real-time PCR, fractional display PCR, rapid amplification of cDNA ends, inverse PCR, vectoret PCR, TAIL-PCR, ligase chain reaction, recovery chain reaction, transcription-mediated amplification, self-maintained sequence replication, target Although the selective amplification reaction of the nucleotide sequence may be used, the scope of the present invention is not limited thereto.

In the step (c), a step of comparing the amount of the amplification product of the reactant with the amplification product or cut-off of the feces of a healthy person without atopic dermatitis may be further performed.

As a preferred example, the method of providing information necessary for predicting the persistence of atopic dermatitis of the present invention includes: i) Streptococcus sp. strain, Clostridium sp. strain and Akkermansia in fecal samples of atopic dermatitis patient. ) Measuring the amount of the amplification product of one or more strains of DNA selected from the group consisting of the genus strains, and ii) the amount of the amplified product was determined from the fecal sample of a healthy person without It may be implemented including the step of comparing with the amplification product.

If the result of the comparison in step ii) corresponds to the following a) or b), it may be evaluated as persistent atopic dermatitis.

a) increase in DNA amplification products of strains of the genus Streptococcus;

b) Reduction of DNA amplification products of strains of the genus Clostridium.

In addition, when the strain of the genus Akermansia is not amplified in step i), and as a result of comparison in step ii), the amplification product of DNA of the strain of genus Streptococcus increases or the amplification product of DNA of the strain of genus Clostridium decreases. It can be evaluated as persistent atopic dermatitis.

If the result of the comparison in step ii) corresponds to one or more of the following c) to e), it may be evaluated as transient atopic dermatitis.

c) reduction of DNA amplification products of strains of the genus Streptococcus;

d) an increase in the DNA amplification product of the strain of the genus Clostridium;

e) Increase in DNA amplification products of strains of the genus Akermansia.

In yet another aspect, the present invention is a stool sample of a patient undergoing treatment for atopic dermatitis. In another aspect, the present invention comprises one or more strains of DNA selected from the group consisting of a strain of the genus Streptococcus and a strain of the genus Clostridium . Determination of a treatment policy for atopic dermatitis comprising the step of measuring the amount of the amplified product, and ii) comparing the amount of the amplified product with the amplified product of genomic DNA obtained from a fecal sample of a healthy person who does not have atopic dermatitis. Provides a way to provide the necessary information.

As a result of the comparison in step ii), if the DNA amplification product of the strain of the genus Streptococcus increases or the DNA amplification product of the strain of the genus Clostridium decreases, the severity of atopic dermatitis is considered severe and the treatment policy can be determined. have.

As a result of the comparison in step ii), when the DNA amplification product of the strain of the genus Streptococcus decreases or the DNA amplification product of the strain of the genus Clostridium increases, the severity of atopic dermatitis is low and the treatment policy can be determined. have.

In the present invention, the proportion of microorganisms means that the amount of 16s rRNA sequence amplification products of the microbiome in the sample occupies the amount of 16s rRNA sequence amplification products of the microorganism, for example, the weight% of the amplification product representing the amount of 16s rRNA sequence amplification /w%), or the amplification product copy number.

The present invention provides a microbial biomarker capable of early diagnosing the persistence of atopic dermatitis, and using this can contribute to determining a treatment method through determination of the persistence or severity of atopic dermatitis through simple fecal collection.

1 is a diagram schematically showing changes in the intestinal microbial community according to the atopic dermatitis phenotype and the atopic dermatitis phenotype of the present invention.
Figure 2 shows the diversity of the intestinal microbial community of the normal control, transient AD and long-acting AD: (AB) OTUs of the intestinal microbial community and the box viscosity of Shannon diversity were compared between groups. The importance of differences between groups was confirmed using the Mann-whitney test.
Figure 3 shows the difference between the intestinal microbial populations of the normal control, transient AD, and long-acting AD at the phyl and genus levels: the most frequently detected microbial phylogenies (A) and It was expressed as mean diameter ± standard deviation (SD) and P value as genus (B). Blue indicates normal control, yellow indicates transient AD group, and red indicates long-acting AD group. (C) Comparison of the composition of the strain of the genus Streptococcus , the strain of the genus Clostridium , and the strain of the genus Akkermansia in the intestinal microbial community. The significance of the differences between groups was confirmed using the Mann-whitney test (* P ≤ 0.05, ** P ≤ 0.01).
Figure 4 shows the analysis of the intestinal microbial levels in (A) normal control versus sustained AD, (B) transient AD versus sustained AD, and (C) normal control versus transient AD. The bar graph represents the LDA score.
Figure 5 is a graph showing the correlation between the intestinal Streptococcus genus strain or Clostridium genus strain and IgE, SCORAD and eosinophil: (A) Intestinal Streptococcus genus strain relative The relationship between the abundance and SCORAD in the AD group, (B) the relationship between the relative abundance of the strain of the genus Streptococcus and the IgE (IU/ml) of the AD group, (C) the genus Streptococcus in the intestine Relationship between relative abundance of strains and eosinophils (%) of AD group, (D) relationship between relative abundance of strains of the genus Clostridium and SCORAD of AD group, (E) intestinal Clostridium ( The relationship between the relative abundance of the strain of the genus Clostridium and IgE (IU/ml) of the AD group, (F) the relationship between the relative abundance of the strain of the genus Clostridium in the intestine and the eosinophil (%) of the AD group.
Figure 6 is a graph showing the relative abundance of metagenomic functional genes related to the KEGG pathway in the (A) 1st and (B) 2nd KO categories: green is a normal control group, yellow is a transient AD group, and red is continuous. It represents the type AD group.
7 is a comparison of metabolic pathways using metagenomic analysis of intestinal microbial communities of normal control, transient AD, and long-acting AD, as a result of comparing functional genes related to oxidative phosphorylation and species included in the group Is (sky blue arrow: Akkermansia muciniphila , P <0.094).

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Example]

Example 1. Selection of study subjects and collection of samples

84 normal control subjects, 22 transient atopic dermatitis (transient AD, transient AD) groups, and 26 persistent atopic dermatitis (persistent AD, persistence AD) groups were recruited through the cohort. Transient AD is defined as a phenotype in which atopic dermatitis occurs at 6 months of age and disappears at 1 year of age, and persistent AD is defined as a phenotype that develops at 6 months of age and persists until 2 years of age. The basic characteristics of are shown in the following [Table 1].

Specifically, patients with atopic dermatitis were diagnosed based on Hanifin and Rajka's criteria, and the normal control group (healthy infants) did not have a history of visible signs of skin eczema, and steroid therapy was not used for skin identified by a pediatric allergy expert. Infants and infants who did not have been selected were selected. At the same time, the severity of atopic dermatitis was evaluated using the SCORAD index at the time of collecting feces at 6 and 12 months in infants and toddlers who had not previously taken antibiotics. This study was conducted by the Institutional Review Board (IRB) of Asan Hospital (IRB no. 2008-0616), Samsung Medical Center (IRB no. 2009-02-021), Severance Hospital (IRB no. 4-2008-0588), and Cha Hospital (IRB no. 2010-010), Seoul National University Hospital (IRB no. H-1401-086-550). It was conducted with the approval of the Asan Hospital Ethics Committee, and written consent was submitted to the participants or parents (if the participants were under the age of 17).

Subjects with no history of antibiotic use were included prior to stool sampling to control external factors that could alter the composition of the microbiota. Parents collected feces from infants in diapers at 6 months and immediately placed them at -20°C. Samples were quickly transferred to the Life Science Research Institute and stored at -70°C until DNA was extracted.

Example 2. Intestinal microorganism, serum IgE level and eosinophil measurement

Total IgE was measured at 12 months as serum immunoglobulin E (IgE) and the percentage of eosinophils in the blood. Total serum IgE levels were measured using the ImmunoCAP-CAP 1000 system (Phadia AB, Uppsala, Sweden). The detection limit was 2 kU/L. Eosinophils were counted using an automatic hemocytometer (XE-100, Sysmex Co., Kobe, Japan). The measurement results were analyzed through correlation analysis with intestinal microorganisms to derive significant results.

Example 3. DNA extraction and pyrosequencing

Metagenomic DNA was extracted from the fecal sample of Example 1 according to the manufacturer's protocol using the Power Microbiome RNA/DNA Isolation Kit (MOBIO/Qiagen, Carlsbad, Calif). The extracted DNA was stored at -20°C. The concentration and purity of DNA were quantified with a spectrophotometer.

A barcode primer targeting the V1-V3 region of the 16S rRNA gene from the DNA extracted for pyrosequencing [Forward: 5'-barcode-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1), Reverse: 5'-ATTACCGCGGCTGCTGG- 3'(SEQ ID NO: 2)] was used to amplify. DNA amplification was performed at 94° C. for 5 minutes, then 30 cycles of 94° C. for 30 seconds, annealing at 55° C. for 30 seconds, stretching at 72° C. for 30 seconds, and the last extension at 72° C. for 7 minutes. The amplification product was purified using the AMPure XP bead kit (Agencout Bioscience, Beverly, MA, USA). Sequencing was performed using a Roche/454 FLX Titanium system (Roche, Mannheim, Germany).

Example 4. Whole gene sequencing and sequence analysis

From the fecal sample of Example 1, 48 fecal samples (20 normal controls, 12 transient AD groups, 16 samples in the persistent AD group) were randomly selected, and the metagenomic DNA of the selected samples was Covaris M220 sonicator (Covaris , Woburn, Mass), and a metagenome library was prepared using TruSeq DNA sample preparation kit (Illumina, San Diego, Calif). Each library (2nM) was calculated through quantitative real-time PCR using Illumina adapter sequence primers [Forward: 5'-ATACGGCGACCACCGAGATC-3' (SEQ ID NO: 3), Reverse: 5'-CAAGCAGAAGACGGCATACGAG-3' (SEQ ID NO: 4)] I did.

A whole-genome sequence was obtained using Illumina HiSeq2000 (Illumina HiSeq 2000). By creating a DNA library, 250-bp paired ends reads were obtained, and sequencing data analysis toolset (ver. 0.127, http://www.vicbioinformatics.com/software.nesoni.shtml ) was used. Illumina adapter sequences (150 bp), short length reads (150 bp), and low Q scores (Q <15) were removed by using. Contaminated genes were removed using BBMap ( http://sourceforge.net/project/bbmap ), and UniRef50 IDs were modified to KO (KEGG Orthology) to match K-numbers, and then KO categories according to groups were set. Analyzed.

[Experiment result]

General characteristics of the research group

General characteristics of the study group are shown in [Table 1] below.

[Table 1]

The study subjects were divided into three groups: normal, transient AD, and persistent AD phenotype. There was no significant difference in sex (P = 0.937). The history of allergic diseases, including asthma, atopic dermatitis and allergic rhinitis, is normal control (P = 0.003), atopic dermatitis (defined as ≥1 positive response, ≥0.35 IU/ml, specific IgE) normal control and transient AD It was increased in persistent AD than phenotype (P <0.001). In addition, the proportion of eosinophils was highest in the persistent AD phenotype (mean ± standard deviation; 6.34 ± 0.94%; P <0.001) than in the normal control group (2.90 ± 0.20%) or transient AD phenotype (4.39 ± 0.83%). When the AD symptom scoring system was 6 months, SCORAD was highest in persistent AD (mean ± standard deviation 29.30 ± 3.43, P = 0.005) than transient AD (16.74 ± 2.07). Overall IgE levels were increased in sustained AD compared to normal control and transient AD (P = 0.013).

Comparison of composition according to diversity and phenotype of intestinal microbial community

Embodiment was analyzed to read the results of a total of 2,507,762 leads from the stool sample of the selected 132 neonates in Example 1. Alpha diversity was checked to test the similarity of the three samples. The alpha diversity results showed that the operational taxonomic unit (observed diversity, OTU) and Shannon diversity index did not differ between the three groups (P>0.05, Fig. 2).

The composition of the intestinal microbial community between the three groups was confirmed at the phylum and genus level. At the phylum level, three phyla, Actinobacteria, Firmicutes and Proteobacteria, were dominant among the three groups. The proportion of Actinobacteria was higher in transient AD than in the normal control group (P=0.029). However, the difference between Permycutis and Proteobacteria was not significant between groups (P>0.05; Fig. 3A).

Next, detailed differences in microbial group composition were confirmed at the genus level. Analysis using LEfSe was significantly differentiated between AD phenotypes with an LDA effect size score> 4 (P <0.05, AC in FIG. 4). In infants with persistent AD, Streptococcus strains (7.93 ± 2.47%) were significantly higher than those of the normal control and transient AD (3.59 ± 0.91% in the control group, P =0.012, 1.09 ± 0.39%, P = 0.004 in the transient AD). ; Fig. 3B, C). The strain of the genus Clostridium was the dominant genus afterwards (6.45 ± 1.24% in the control group, 1.12 ± 0.74% in the long-acting AD, P = 0.015; Figs. 3B and C). However, Clostridium did not differ between the normal control group and transient AD (temporary 6.97 ± 2.93%, P> 0.05, Fig. 3C), and transient AD and persistent AD (P>0.05, Fig. 3C). . In addition, the strain of the genus Akermancia was lower in the long-acting AD (0.01 ± 0.01%) compared to the transient AD (5.61 ± 3.98, P = 0.010, B, C in Fig. 3). Moreover, the composition of the strains of the genus Akermancia in transient AD was increased compared to the normal control group (3.27 ± 1.18%, P = 0.024, B and C of Fig. 3). However, the strains of the genus Akermancia did not have a statistical difference between the normal control and persistent AD (P>0.05, C of FIG. 2). In addition to the samples dominated by Permicutis, the strains of the genus Streptococcus and strains of the genus Clostridium were isolated according to the dominance. E. coli was higher in all groups, but the difference in bacterial composition was not significant (P>0.05; FIG. 3B). Further, Bifidobacterium (Bifidobacterium) was higher than the control group at a time type AD (P = 0.027, B in Fig. 3).

Relationship between total IgG, SCORAD, eosinophils and intestinal microbial composition

Total IgE (IU/ml), SCORAD, eosinophils (%) and Streptococcus strains and Clostridium sp. in infants with atopic dermatitis to investigate the role of strains of the genus Streptococcus and strains of genus Clostridium on atopic dermatitis. The relative abundance of the strain was analyzed.

As a result, as shown in [Fig. 5], the amount of strain of Streptococcus genus showed a clear correlation with SCORAD (ρ = 0.484, P = 0.002; Fig. 5A), but significant in total IgE and eosinophils (%) Did not (P>0.05; Fig. 5B, C). In addition, the amount of strain in Clostridium showed a negative correlation with SCORAD (rho = -0.556, P = 0.013; Fig. 5D), but there was no correlation with total IgE and eosinophils (P> 0.05; E of Fig. 5). ,F).

Various functional profiles of intestinal microflora according to the persistence of atopic dermatitis

Differences in functional genes between each group were compared using the KO item, and human and plant gene sequences were removed and analyzed. KO items with significant differences were selected with a P value <0.05. Among the identified KO categories, 6 paths of the first KO category were shown, including 44 paths of the second KO category.

As a result, as shown in [Fig. 6], compared to the normal control group (P <0.05; Fig. 6B), the immune system and developmental pathway are reduced in the long-acting AD, and the metabolism of the cofactor and the vitamin pathway is transient AD It was confirmed that the increase in persistent AD compared to (P <0.05).

Next, as a result of confirming the third KO category of the group, the relative presence of genes related to oxidative phosphorylation was decreased in long-acting AD compared to the normal control group (P = 0.001; Fig. 7). Akermancia muciniphila weakly contributed to the difference in the expression of oxidative phosphorylation between long-acting AD and normal controls (P = 0.094).

<110> THE ASAN FOUNDATION INDUSTRY ACADEMIC COOPERATION FOUNDATION OF UlSAN UNIVERSITY <120> PREDICTION OF ATOPIC DERMATITIS DEVELOPMENT AND PERSISTENCE USING CHANGES IN THE GUT MICROBIAL COMMUNITY <130> 1065331 <150> KR 2019/0013118 <151> 2019-01-31 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer-F <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer-R <400> 2 attaccgcgg ctgctgg 17 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Illumina adapter sequence primer-F <400> 3 atacggcgac caccgagatc 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Illumina adapter sequence primer-R <400> 4 caagcagaag acggcatacg ag 22

Claims (11)

Contains an agent capable of detecting one or more strains selected from the group consisting of strains of the genus Streptococcus , strains of the genus Clostridium , and strains of the genus Akkermansia in fecal samples of atopic dermatitis patients and,
The agent is any one or more selected from the group consisting of strain-specific primers, probes, antisense oligonucleotides, aptamers and antibodies, a composition for predicting persistence of atopic dermatitis.
The method of claim 1,
The primer is a primer capable of amplifying the 16S rRNA of the strain, composition.
The method of claim 1,
The atopic dermatitis patient is an infant and toddler who is being treated for atopic dermatitis, the composition.
A kit for predicting persistence of atopic dermatitis, comprising the composition of any one of claims 1 to 3.
Ⅰ) The amount of DNA amplification products of one or more strains selected from the group consisting of Streptococcus sp. strain, Clostridium sp. strain, and Akkermansia sp. strain in fecal samples of atopic dermatitis patient Measuring a;
Ii) comparing the amount of the amplified product with the amplified product of genomic DNA obtained from a fecal sample of a healthy person who does not have atopic dermatitis; comprising, providing information necessary for predicting persistence of atopic dermatitis.
The method of claim 5,
If the result of the comparison in step ii) corresponds to the following a) or b), the method is evaluated as persistent atopic dermatitis:
a) increase in DNA amplification products of strains of the genus Streptococcus;
b) Reduction of DNA amplification products of strains of the genus Clostridium.
The method of claim 5,
When the strain of the genus Akermancia is not detected in step i), and as a result of comparison in step ii), the DNA amplification product of the strain of genus Streptococcus increases or the DNA amplification product of the strain of genus Clostridium decreases. The method of evaluating as atopic dermatitis.
The method of claim 5,
A method of evaluating as transient atopic dermatitis when the result of the comparison in step ii) corresponds to any one or more of the following c) to e):
c) reduction of DNA amplification products of strains of the genus Streptococcus;
d) an increase in the DNA amplification product of the strain of the genus Clostridium;
e) Increase in DNA amplification products of strains of the genus Akermansia.
Ⅰ) Measuring the amount of DNA amplification products of one or more strains selected from the group consisting of strains of the genus Streptococcus and strains of the genus Clostridium in a fecal sample of a patient undergoing atopic dermatitis treatment ;
Ii) comparing the amount of the amplified product with the amplification product of genomic DNA obtained from a fecal sample of a healthy person who does not have atopic dermatitis; comprising, providing information necessary for determining a treatment policy for atopic dermatitis.
The method of claim 9,
As a result of the comparison in step ii), when the DNA amplification product of the strain of the genus Streptococcus increases or the DNA amplification product of the strain of the genus Clostridium decreases, the severity of atopic dermatitis is considered severe and the treatment policy is determined. Way.
The method of claim 9,
As a result of the comparison in step ii), when the DNA amplification product of the strain of the genus Streptococcus decreases or the DNA amplification product of the strain of the genus Clostridium increases, the severity of atopic dermatitis is low and the treatment policy is determined. Way.

Images