KR20210087243A - Method for assessmenting microbiome from Patients with inflammatory bowel disease - Google Patents

Abstract

The present invention relates to a technic related to a method for evaluating the microbial community of patients with inflammatory bowel disease by aspirating a colonoscopy lavage fluid using an injector used for intestinal polypectomy as a suction catheter. The method of the present invention can replace an existing feces collection method, which has disadvantages in collection, inspection, analysis, and the like, and in particular, feces is a final product of intestinal microbes and is different from the microbial community actually present in the intestine, so the method has advantages in that it provides an evaluation method.

Description

Method for assessing microbiome from Patients with inflammatory bowel disease

The present invention relates to a method for evaluating the microbial community in patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic bowel disease that includes ulcerative colitis and Crohn's disease. It has been reported that patients with ulcerative colitis (UC) have a bacterial dysbiosis in the gut microbiome. Although the exact mechanism of this disease is not known, it is known that intestinal bacterial imbalance is one of the main causes.

The most common method of assessment of the gut microbiome is collection and analysis of stool. Although the method of collecting feces is standard, according to an analysis of the intestinal microbiome, many people are reluctant to handle feces, and feces collection has disadvantages such as individual side effects and possible contamination, so it is limited. In addition, mucosal tissue collection through endoscopy is the most ideal method, but it is an invasive method and generally requires a lot of work. In addition, rectal smears were found to be significantly different from the intestinal bacteria in the colon. Therefore, there is a need for the development of a new method that can easily evaluate the intestinal microbial imbalance.

Recently, colonoscopy for colorectal cancer screening is rapidly increasing, so that intestinal fluid can be easily aspirated through colonoscopy. In general, it is known that the use of endoscopic laxatives affects the intestinal microbiome. In a small study using colonic collection, colonoscopic laxatives significantly reduced the number of intestinal bacteria, but in most cases, recovery was reported within 14 days. In addition, it has been reported that the results of histological analysis of intestinal fluid through colonoscopy are similar to intestinal bacteria. Accordingly, in the present invention, it is intended to determine the difference in the microbial community in the intestinal flushing fluid in patients with inflammatory bowel disease compared to normal people.

US Patent Publication No. 2017-0159108 (published on June 8, 2017)

It is an object of the present invention to provide a method for evaluating the microbial community in patients with inflammatory bowel disease.

In order to achieve the above object, the present invention provides a method for evaluating the microbial community of a patient with inflammatory bowel disease, comprising the step of predicting the microbial diversity in a sample isolated from the patient with inflammatory bowel disease.

The present invention relates to a method for evaluating the microbial community of a patient with inflammatory bowel disease by aspirating a colonoscopy lavage solution using an injector used for intestinal polypectomy as a suction catheter, and the method of the present invention has disadvantages in collection, examination, analysis, etc. It can replace the existing feces collection method with this, and in particular, feces is the final product of the intestinal microflora, which is different from the microbial community actually present in the intestine, so it has an advantage in that it provides an accurate method for evaluating the microbial community.

1 shows a method for sampling (A, B) colonic endoscopic lavage fluid and (C, D) biopsy.
2 shows the average taxonomic composition of fecal samples, lavage samples and biopsy samples.
Figure 3 shows the alpha diversity analysis of stool samples, wash samples and biopsy samples.
4 shows beta diversity analysis of stool samples, wash samples and biopsy samples.
Figure 5 shows the analysis of Procrustes of a stool sample and a wash solution sample.
6 shows the microbial community of a stool sample and a wash solution sample.
7 shows the analysis of diversity of stool samples, lavage samples and biopsy samples (ulcerative colitis vs. normal control).
8 shows LASSO analysis for disease prediction.
Figure 9 shows the microbial community of the stool sample and the wash sample (ulcerative colitis vs. normal control).

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

The present invention provides a method for evaluating the microbial community of patients with inflammatory bowel disease, comprising the step of predicting microbial diversity in a sample isolated from patients with inflammatory bowel disease.

The sample may be a lavage solution aspirated during colonoscopy, but is not limited thereto.

The washing solution may be aspirated with an injector used for polypectomy, but is not limited thereto.

The microbial diversity can be obtained through an analysis selected from the group consisting of gut microbiome type analysis, alpha diversity analysis, beta diversity analysis, Procrustes analysis, Mantel analysis, substitution multivariate ANOVA, and Lasso analysis, but is not limited thereto. do.

The microbial community evaluation method may further include analyzing the similarity between the predicted intestinal microbial diversity of the inflammatory bowel disease patient and the intestinal microbial diversity of the normal control.

The intestinal microorganisms of the inflammatory bowel disease patient may be selected from the group consisting of Staphylococcus and Leuconostocaceae, and the intestinal microorganisms of the normal control group are Prevotella and Alistipes. ) and disulfovibrio (Desulfovibrio), but may be selected from the group consisting of, but is not limited thereto.

The inflammatory bowel disease may be selected from the group consisting of ulcerative colitis, inflammatory colitis, Crohn's disease, enteric Behcet's disease, hemorrhagic rectal ulcer and ileal capsuleitis, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Ulcerative colitis and normal selection

The patients who visited the hospital were diagnosed with ulcerative colitis (UC) according to the recommendations of the European Crohn's and Colitis Society. Clinical data including medical records, blood tests, biopsy colonoscopy and radiographic examination were comprehensively reviewed, and clinical activity was assessed using the Mayo score of ulcerative colitis. Also, (1) cardiovascular disease, chronic kidney disease or chronic liver disease; (2) patients with mental illness or pregnancy; (3) Patients who had recently (< 3 months) gastrointestinal surgery were excluded.

Subjects without gastrointestinal disease or recurrent abdominal symptoms were selected as a control group, who were normal persons who were matched with a patient with ulcerative colitis and sex and age. Symptomatic questionnaires, blood tests and colonoscopy were completed to rule out recurrent abdominal symptoms and the presence of organic disease.

As shown in Table 1, 16 patients with ulcerative colitis (9 males, 7 females, average age: 52.13 ± 14.09 years old) and 15 normal patients (8 males) visited the hospital from September 2018 to January 2019. , 7 women, mean age: 50.93 ± 14.11 years) participated in this study. Demographic characteristics including age, comorbidities, and body mass index were similar in both groups, and the smoking rate of patients was significantly lower compared to the control group (0 vs. 4; P = 0.043). With the exception of 4 patients with mild to moderate disease activity, most of the patients were in remission.

*: statistically significant, BMI: body mass index, E1, E2, E3: disease localization by Montreal Classification

Example 2: Sample Collection

Colonoscopy was performed at Myongji Hospital for ulcerative colitis patients and normal subjects. Fecal samples were collected 3 days before colonoscopy, and lavage samples and biopsy samples were collected during colonoscopy.

Briefly, stools were collected using a stool sampling kit (ChunLab, Inc), and a total of two pellets (approximately 400 mg each) were obtained from each stool. Without the use of any chemical additives, each pellet of the fecal sample was placed in two sterile sterile tubes and stored at -80°C until used for analysis.

In addition, for colonoscopy, the same intestinal cleansing product, 2 L polyethylene glycol (PEG) + ascorbate solution (Coolprep®; Taejoon Pharmaceutical), was provided to patients and normal subjects participating in the experiment, and all participants' examinations were performed in the evening ( after 14:00). The intake of fiber, seeds, seaweed, and mixed grains was to be avoided for 3 days before the colonoscopy. On the day before the examination, after a light dinner and fasting except for water, they were asked to fast between 8 and 4 hours before the examination. ingestion of intestinal cleansing products.

The study protocol was approved by the Institutional Bioethics Committee of Myongji Hospital in accordance with the ethical guidelines of the Declaration of Helsinki (MJH 2018-08-020), and was registered at http://cris.nih.go.kr (KCT0003352).

All endoscopic procedures were performed using a white light endoscope using a colonoscope (CF-H290; Olympus, Tokyo, Japan). As shown in Fig. 1, during colonoscopy, about 3-5 cmm of lavage fluid from the S colon was collected with a Clear-Hemostat catheter (FM-EH0001, Finemedix Co.), and at the same time endoscopic forceps (FB-24K-1; Olympus, Tokyo, Japan) collected a single mucosal biopsy from the S colon. All samples were stored on ice immediately after collection and then delivered to ChunLab, Inc. on the same day for analysis.

Example 3: Bacterial community analysis

3-1. Extraction of genomic DNA from stool samples

To 400 mg of stool sample , 15 mL of DNA extraction lysis buffer (SDS 4%, Tris-HCL 50 mM, EDTA 50 mM, NaCl 5000 mM) was added and homogenized by vigorous vortexing for 1 minute. After transferring 1.4 mL of the homogenized fecal suspension to a 2 mL tube, bead beating was performed for 50 seconds, followed by centrifugation at 14,000 g for 10 minutes. Then, 200 μL of the supernatant was transferred to a 96-well plate, and 1 μL of the supernatant was dissolved in 29 μL of nuclease-free water. Plates were kept at -25°C until PCR was performed.

The extracted DNA was extracted from 341F (5'-TCGTCGGCAGCGTC-AGATGTGTATAAGAGACACAG- CCTACGGGNGGCWGCAG -3'; the underlined sequence indicates the target site primer) and 805R (5'-GTCTCGTGGGCTCGG-AGATGTGTATAAGAG) targeting the V3-V4 region of the bacterial 16s rRNA gene. - GACTACHVGGGTATCTAATCC -3') was amplified using primers.

PCR conditions were as follows: initial denaturation at 95°C for 3 min; 25 cycles of denaturation at 95°C for 30 seconds, primer annealing at 55°C for 30 seconds, extension at 72 seconds to 30 seconds; Final elongation at 72° C. for 5 min.

Then, for secondary amplification for Illumina NexTera barcode attachment, i5 forward primer (5'-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-3'; X indicates barcode region) and i7 reverse primer (5'-CAAGCAGAAGACGGCATACGAGAT-XXXXXGGGGAGAGTCTGTG-3 ') was used. The PCR conditions for the secondary amplification are the same as the previous conditions except that the amplification cycle is set to 8.

The PCR product was confirmed by 2% agarose gel electrophoresis, and the amplified product was purified using QIAquick PCR purification Kit (Qiagen, Valencia, CA, USA). Purified products at the same concentration were pooled together and short fragments (non-target products) were removed with Ampure beads kit (Agencourt Bioscience, MA, USA). Product size and quality were assessed with a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. Mixed amplicons from various samples were collected and pyrosequencing was performed. The sequencing process was performed by Chunlab, Inc. using an Illumina Miseq sequencing system (Illumina, USA) according to the manufacturer's instructions.

3-2. Sequence data analysis

Reads from the various samples were sorted by the unique barcode of each PCR product. The sequences of barcodes, linkers and primers were then removed from the original reads of the sequences. Among sequence reads, reads containing two or more ambiguous nucleotides, reads with a low quality score (average <25), or reads less than 300 bp were all discarded. Potential chimeric sequences were detected using the Bellerophone method, which compares BLASTN search results between forward and reverse half sequences. After removal of the chimeric sequence, each read was assigned to a taxonomic classification in the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net). To compare the abundance of taxa between different groups, the LDA Effect Size (LEfSe; Linear Discriminant Analysis Effect Size) algorithm was used with the oline interface Galaxy (http://huttenhower.sph.harvard.edu/lefse/). . LEfSe analysis was performed using the Kruskal-Wallis sum-rank test with an alpha value <0.5, followed by the Wilcoxon-rank serum test with an alpha score <0.05 and a one-aginst-all strategy. When effect size > 2 (log scale), it was considered valid.

Experimental Example 1: Comparison of sampling methods

Five biopsy samples out of a total of 93 samples were excluded from analysis due to the low number of reads (<10,000 reads), and the mean taxonomic composition of feces, lavage and biopsies were analyzed ( FIG. 2 ). It was confirmed that microorganisms in close contact with the intestine, including Proteobacteria and Akkermansia (genus of phylru Verrucomicrobia), were increased in the washing fluid and biopsy samples.

Alpha diversity according to the sampling method is shown in FIG. 3 . The number of OTUs in biopsy samples was significantly higher than in biopsy and fecal samples. On the other hand, in Shannon and Simpson's diversity, stool samples were different from biopsy samples and wash samples. The beta diversity between sampling methods is shown in FIG. 4 . All sampling methods of beta diversity differed significantly from each other. The substitution multivariate analysis of variance (PERMANOVA) in the sampling method is shown in Table 2. All sampling methods of substitution multivariate analysis of variance were significantly different from each other.

Another analysis confirmed an important correlation, meaning the correlation between the stool sample and the wash sample, in the Procrustes test (p = 0.001) (FIG. 5) and the Mantel test (p = 0.0001). In the Procrustes test, biopsy samples showed no significant correlation between stool samples (p value = 0.625) and biopsy samples (p value = 0.1417). When the lavage and fecal samples were compared, the microbial properties of the lavage samples were as follows: small intestine [Proteobacteria, Enterobacteriaceae], oral cavity and upper gastrointestinal tract [Granulicatella, Leptotrichia, Porphyromonas gingivalis, Fusobacterium nucleatum group, Parvimonas micra,… ], respiratory [Corynebacterium durum] microbial community (Fig. 6).

Experimental Example 2: Comparison of ulcerative colitis patients and controls

Significant differences in diversity (number of phylogenies) were found in stool samples between patients with ulcerative colitis and normal controls. As a result of analyzing the microbial diversity in the wash sample and the biopsy sample, no significant difference was observed between the ulcerative colitis patient and the normal control group (FIG. 7). However, it was suggested from the taxonomic distribution of the wash samples that the difference in diversity could be seen as the number of samples increased. PERMANOVA analysis confirmed the association between specific factors and overall microbial community variation. Fecal samples showed the presence and higher disease activity of ulcerative colitis, and lavage samples were associated with current smokers, bowel surgery and intestinal symptoms. Colon tissue showed significant association with BMI, surgery, order_number and sampling_date (Table 3).

Modeling confirmed that the microorganisms of each microbial community have the ability to discriminate between diseases. As a result, the fecal sample and the lavage sample predicted the accuracy of AUC 0.85 and 0.81, respectively (FIG. 8). Accuracy increased to AUC 0.88 when using data from stool samples and lavage samples.

In the case of stool samples, functional anaerobes such as Enterococcus and Lactobacillus were found as markers related to ulcerative colitis, and several species of pseudobacilli [Bacteroidetes; Prevotella, Bacteroides, etc.] and Clostridiales were found as normal control markers (FIG. 9).

In the case of the washing solution sample, Staphylococcus and Leuconostocaceae were found as markers related to ulcerative colitis, and Prevotella, Alistipes, and Desulfovibrio were It was found as a normal control marker, the wash sample had fewer markers than the stool sample, and it was confirmed that the stool sample and other microorganisms were found as disease and normal control markers.

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (7)

A method for evaluating the microbial community of patients with inflammatory bowel disease, comprising the step of predicting microbial diversity in a sample isolated from patients with inflammatory bowel disease. The method according to claim 1, wherein the sample is a lavage solution aspirated during colonoscopy. The inflammatory method according to claim 1, wherein the microbial diversity is analyzed by an analysis method selected from the group consisting of intestinal microbial type analysis, alpha diversity analysis, beta diversity analysis, Procrustes analysis, Mantel analysis, substitution multivariate analysis of variance, and Lasso analysis. A method for evaluating the microbiome of patients with intestinal disease. The method according to claim 1, wherein the microbial community evaluation method further comprises the step of analyzing the similarity of the intestinal microbial diversity of the predicted inflammatory bowel disease patient and the intestinal microbial diversity of the normal control microbial community evaluation of patients with inflammatory bowel disease Way. The method according to claim 1, wherein the intestinal microorganism of the inflammatory bowel disease patient is selected from the group consisting of Staphylococcus and Leuconostocaceae. The method according to claim 1, wherein the intestinal microorganisms of the normal control group are selected from the group consisting of Prevotella, Alistipes and Desulfovibrio. . The method according to claim 1, wherein the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, inflammatory colitis, Crohn's disease, intestinal Behcet's disease, hemorrhagic rectal ulcer, and ileal capsulitis.

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