Constipation-causing bacteria and their uses

Abstract

The present invention relates to novel constipation-causing bacteria and a screening method and use thereof, and also relates to a composition containing the constipation-causing bacteria, a reagent for identifying the constipation-causing bacteria and uses thereof.

Description

Technical Field

[0001] The invention relates to bacteria and uses thereof, in particular to constipation-causing bacteria and uses thereof. Background Art

[0002] With the change of dietary structure, accelerated pace of life and the influence of social and psychological factors, chronic constipation has become a high-incidence chronic disease. The prevalence of chronic constipation in adults in my country is about 4-10%, and the global average prevalence is as high as 16%. Since chronic constipation has a huge impact on the patient's quality of life, physical and mental health, and even life safety, and also greatly increases the social medical burden, the study of chronic constipation has become a major topic in chronic disease research. Although people have conducted extensive basic and clinical research on chronic constipation and have made some important progress, 72.9% of patients are still dissatisfied with the current treatment of constipation, especially refractory chronic constipation, which is almost impossible to treat except for extremely traumatic surgical treatment. Current studies believe that the occurrence of chronic constipation is very complicated, but the key cause of the disease is still unclear, resulting in the slow development of new treatment strategies.

[0003] Constipation is a symptom characterized by difficulty in defecation (or) decreased frequency of defecation (less than 3 times per week), hard and dry stools, and the course of chronic constipation is at least 6 months. Functional diseases associated with chronic constipation include functional constipation, opioid-induced constipation, constipation-type irritable bowel syndrome, and functional defecation disorder. The first three are functional bowel diseases, and the latter is anorectal disease (Rome IV criteria). According to the pathological characteristics of chronic constipation, it can be divided into slow-transit constipation (STC), outlet obstruction constipation (rectal evacuation disorders), and mixed constipation. The formation of STC is due to gastrointestinal transit dysfunction, that is, the passage time of intestinal contents from the proximal colon to the distal colorectum is slowed down. Its main clinical manifestations are decreased frequency of defecation, no urge to defecate, and difficulty in defecation. It is the main type of chronic constipation. Current research has found that there are many causes of STC, which are mainly divided into functional and organic causes. Organic causes include gastrointestinal lesions, systemic diseases involving the digestive tract, sclerosis, and nervous system diseases; functional causes include drug-related, neuropsychological, etc. Interestingly, the cause of many STC patients is unclear and is primary. This suggests that chronic constipation may have unknown causes that have not been discovered.

[0004] The treatment of chronic constipation mainly includes 1. General treatment: defecation physiology education, change of eating habits, etc.; 2. Drug treatment: laxatives, bulking agents, osmotic laxatives, etc.; 3. Psychological and biofeedback therapy: cognitive therapy, tension relief, etc. Many patients can relieve or cure chronic constipation by eliminating the cause and the above comprehensive treatment, but there are still a large number of patients who still have no effect or relapse after the above treatment, showing refractory symptoms. This functional STC is intractable chronic constipation (ICC). Since this type of constipation has a great impact on the patient's physical and mental health, surgical treatment can be considered, but surgical treatment is very harmful, and clinical indications are selected with great caution. ICC is a recurrent and refractory chronic constipation, and its pathogenesis will also be more complicated. The refractory nature of ICC also suggests that the disease may be caused by an unknown cause, and clarifying its cause may improve the treatment effect.

[0005] Like other STCs, the basic feature of ICC is also a slow intestinal transit. As for the cause of slow transit, it is currently believed to be closely related to the abnormal function of gastrointestinal nerve-pacemaker cells-smooth muscle, but it is impossible to confirm whether this abnormal function is the initial cause or a secondary lesion. Through a large number of clinical sample studies, it was found that in constipation, the enteric nervous system (ENS) (such as intestinal plexus, nerve ganglion, intermuscular Cajal cells, etc.) underwent degenerative changes, and the ability of intestinal cells to secrete active molecules also changed significantly. Therefore, some people have proposed that degenerative lesions of the ENS or abnormal secretion of intestinal active molecules are important mechanisms for slow intestinal transit. Since the causes of ENS degenerative lesions and abnormal secretion of active molecules are unknown, the above-mentioned abnormal changes in the ENS may be a secondary process of constipation. There is also a lot of evidence to support this view, such as: Cajal cell network destruction, ENS and other lesions are secondary lesions of abnormal intestinal contraction; Cajal cell-deficient (C-Kit gene mutation) mice do not cause obvious constipation.

[0006] In recent years, it has been found that intestinal microorganisms are correlated with chronic constipation, but the causal relationship with chronic constipation cannot be determined. Studies have found that when chronic constipation occurs, intestinal microorganisms are not only accompanied by dysbacteriosis, but also that changing the composition of intestinal microorganisms in patients can often improve constipation symptoms. This phenomenon suggests that chronic constipation is related to dysbacteriosis, but unfortunately it is still not certain that it is the initial cause of constipation. It is well known that the defecation process or intestinal motility is the most important mechanism for intestinal microbial replacement, and constipation can lead to weakened intestinal microbial replacement and even dysbacteriosis. Therefore, dysbacteriosis is very likely to be a secondary change of chronic constipation. There is still a long way to go in the study of the mechanism of slow intestinal transit, and there is an urgent need to seek new research ideas. Therefore, studying the key causes and mechanisms of the disease and exploring new therapies are key topics in the prevention and treatment of chronic constipation. Summary of the invention

[0007] The inventors discovered the bacterium PIB (peristalsis inhibition bacteria) that inhibits intestinal peristalsis. The bacterium was deposited in the China General Microbiology Center (CGMCC) under the deposit number CGMCC 20603 on September 7, 2020.

[0008] Therefore, the present invention provides an Enterobacteriaceae bacterium, wherein

[0009] (1) the genome of the bacterium has corresponding nucleotides at one or more positions selected from the group consisting of: PIB2013.g.331A, PIB2324.g.562G, PIB2324.g.601A, PIB2629.g.1126T, PIB3156.g.2396T,

[0010] (2) The bacterial genome has one or more characteristics selected from the following:

[0011] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 9 and 10 as primers is A;

[0012] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 11 and 12 as primers is G;

[0013] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 13 and 14 as primers is A;

[0014] The 20th nucleotide of the genomic amplification fragment using SEQ ID NO: 15 and 16 as primers is T;

[0015] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 17 and 18 as primers is T;

[0016] (3) the genome of the bacterium has any one or more sequences selected from SEQ ID NO: 2-6 or a fragment thereof having the nucleotide sequence described in (2) or a variant thereof having at least 98% sequence identity therewith, or

[0017] (4) The 16S rDNA of the bacterium has the sequence shown in SEQ ID NO: 1 or a sequence having at least 98% sequence identity thereto.

[0018] In one or more embodiments, the bacterium is Shigella sp.

[0019] In one or more embodiments, the bacteria is a bacterial strain with deposit accession number CGMCC 20603.

[0020] In one or more embodiments, the bacteria inhibit intestinal motility, including reducing the amplitude, tension and / or frequency of intestinal motility of the animal. In one or more embodiments, the bacteria reduce the amplitude of intestinal motility of the animal by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%. In one or more embodiments, the bacteria reduce the tension of intestinal motility of the animal by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%. In one or more embodiments, the bacteria reduce the frequency of intestinal motility of the animal by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0021] In one or more embodiments, the bacteria reduce intestinal transit rate. In one or more embodiments, the bacteria reduce intestinal transit rate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%, preferably by 20-30%, more preferably by about 25%.

[0022] In one or more embodiments, the bacteria reduces the water content of animal feces by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%.

[0023] In one or more embodiments, the animal is a mammal, preferably a human.

[0024] In one or more embodiments, the intestinal tract is the small intestine and / or the large intestine. In one or more embodiments, the small intestine includes the duodenum, jejunum, and ileum. In one or more embodiments, the large intestine includes the cecum, colon, and rectum.

[0025] The present invention also provides a composition comprising the bacterium described in any embodiment herein or a cell-free extract, filtrate, supernatant or lysate thereof.

[0026] In one or more embodiments, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable excipient.

[0027] In one or more embodiments, the composition is a bacterial culture, and the composition further comprises a culture medium. In one or more embodiments, the culture medium comprises peptone, beef extract, NaCl, agar, and sterile defibrinated sheep blood. In one or more embodiments, the culture medium comprises peptone, yeast extract, and NaCl.

[0028] In one or more embodiments, the composition further comprises other bacteria that inhibit intestinal motility in animals or bacteria-free extracts, filtrates, supernatants or lysates thereof.

[0029] The present invention also provides an isolated nucleic acid molecule, wherein:

[0030] (1) having corresponding nucleotides at positions corresponding to one or more positions selected from the following positions in the genome of the bacterial strain with accession number CGMCC 20603: PIB2013.g.331A, PIB2324.g.562G, PIB2324.g.601A, PIB2629.g.1126T, PIB3156.g.2396T,

[0031] (2) Having one or more characteristics selected from the following:

[0032] The 21st nucleotide of the amplified fragment using SEQ ID NO: 9 and 10 as primers is A;

[0033] The 21st nucleotide of the amplified fragment using SEQ ID NO: 11 and 12 as primers is G;

[0034] The 21st nucleotide of the amplified fragment using SEQ ID NO: 13 and 14 as primers is A;

[0035] The 20th nucleotide of the amplified fragment using SEQ ID NO: 15 and 16 as primers is T;

[0036] The 21st nucleotide of the amplified fragment using SEQ ID NO: 17 and 18 as primers is T;

[0037] (3) having any one or more sequences selected from SEQ ID NO: 2-6 or a fragment thereof having the nucleotide sequence described in (2) or a variant thereof having at least 98% sequence identity therewith, or

[0038] (4) A sequence having the sequence shown in SEQ ID NO: 1 or a sequence having at least 98% sequence identity thereto.

[0039] (5) A sequence having a complementary sequence to any one of (1) to (4).

[0040] The present invention also provides a nucleic acid construct comprising the nucleic acid molecule described herein.

[0041] The present invention also provides a host cell comprising the nucleic acid molecule or nucleic acid construct described herein.

[0042] The present invention also provides an oligonucleotide primer, wherein the product of amplifying the bacterial genome by the oligonucleotide primer comprises:

[0043] (1) a nucleotide in the genome of the bacterium located at a position corresponding to one or more positions selected from the following positions in the genome of the bacterial strain with accession number CGMCC 20603: PIB2013.g.331, PIB2324.g.562, PIB2324.g.601, PIB2629.g.1126, PIB3156.g.2396,

[0044] (2) Nucleotides at one or more positions selected from the group consisting of:

[0045] The 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 9 and 10 as primers;

[0046] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 11 and 12 as primers;

[0047] The 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 13 and 14 as primers;

[0048] The 20th nucleotide of the genomic amplification fragment using SEQ ID NOs: 15 and 16 as primers;

[0049] The 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 17 and 18 as primers;

[0050] (3) any one or more sequences selected from SEQ ID NO: 2-6 or fragments thereof having the nucleotide sequence described in (2) or variants thereof having at least 98% sequence identity therewith, or

[0051] (4) The 16S rDNA sequence shown in SEQ ID NO: 1, or a sequence having at least 98% sequence identity thereto.

[0052] In one or more embodiments, the bacterium is a Shigella bacterium. Preferably, the deposit number of the bacterium is CGMCC 20603.

[0053] In one or more embodiments, the oligonucleotide primer recognizes a nucleic acid molecule described herein.

[0054] In one or more embodiments, the oligonucleotide primer recognizes any one of SEQ ID NOs: 1-6, preferably any one of SEQ ID NOs: 2-6.

[0055] In one or more embodiments, the amplification product of the oligonucleotide primer comprises any one of the sequences in SEQ ID NOs: 1-6, preferably any one of the sequences in SEQ ID NOs: 2-6.

[0056] In one or more embodiments, the oligonucleotide primer is selected from any one or more of SEQ ID NOs: 7 to 18. Preferably, the oligonucleotide primer is SEQ ID NOs: 9 and / or 10.

[0057] In one or more embodiments, the oligonucleotide primers are used to amplify a bacterial genome.

[0058] The present invention also provides an oligonucleotide probe, which recognizes a fragment of a bacterial genome, wherein the fragment comprises

[0059] (1) a nucleotide in the genome of the bacterium located at a position corresponding to one or more positions selected from the following positions in the genome of the bacterial strain with accession number CGMCC 20603: PIB2013.g.331, PIB2324.g.562, PIB2324.g.601, PIB2629.g.1126, PIB3156.g.2396,

[0060] (2) Nucleotides at one or more positions selected from the following positions of the bacterium:

[0061] The 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 9 and 10 as primers;

[0062] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 11 and 12 as primers;

[0063] The 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 13 and 14 as primers;

[0064] The 20th nucleotide of the genomic amplification fragment using SEQ ID NOs: 15 and 16 as primers;

[0065] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 17 and 18 as primers; or

[0066] (3) any one or more sequences selected from SEQ ID NO: 2-6 or fragments thereof having the nucleotide sequence described in (2) or variants thereof having at least 98% sequence identity therewith, or

[0067] The probe recognizes the 16S rDNA sequence shown in SEQ ID NO: 1 or a sequence having at least 98% sequence identity thereto.

[0068] In one or more embodiments, the bacterium is a Shigella bacterium. Preferably, the deposit number of the bacterium is CGMCC 20603.

[0069] In one or more embodiments, the probe recognizes any one of SEQ ID NOs: 1-6, preferably any one of SEQ ID NOs: 2-6.

[0070] In one or more embodiments, the probe recognizes a nucleic acid molecule described herein.

[0071] In one or more embodiments, the probe is used to hybridize to the bacterial genome or a fragment thereof or an amplification product of the primers described herein.

[0072] The present invention also provides a kit for identifying bacteria or their genome, comprising a reagent for detecting SNPs or point mutations in the bacterial genome,

[0073] In one or more embodiments, the kit comprises oligonucleotide primers and / or probes as described herein and optional nucleic acid molecules, bacteria or compositions as described herein as a control. The kit also comprises other reagents required for using the primers and / or probes and optional nucleic acid molecules to identify bacteria, such as buffer, nucleic acid polymerase, dNTP, fluorescent dye, etc. In one or more embodiments, the bacterium is Shigella sp. Preferably, the bacterium is a bacterial strain with a deposit registration number of CGMCC 20603.

[0074] The present invention also provides a method for screening bacteria that inhibit intestinal motility, comprising: contacting the bacteria or an extract thereof with the intestinal tract or its cells or tissues, and measuring intestinal motility parameters.

[0075] In one or more embodiments, the bacterium is Shigella sp.

[0076] In one or more embodiments, the cells are selected from intestinal smooth muscle cells, epithelial cells, and neural cells.

[0077] In one or more embodiments, the intestinal tissue includes smooth muscle tissue, epithelial tissue, and neural tissue.

[0078] In one or more embodiments, the intestinal tissue is at least 5 mm in length and is preferably tubular.

[0079] In one or more embodiments, the bacteria are from the intestines or feces of humans or animals.

[0080] In one or more embodiments, the parameters are intestinal peristalsis amplitude, tension, frequency, intestinal contraction response after depolarization or ligand stimulation, and intestinal relaxation effect.

[0081] In one or more embodiments, the intestinal tract is a mammalian intestinal tract, preferably an 8-10 week old mouse intestinal tract.

[0082] In one or more embodiments, the intestinal tract is the jejunum and / or the colon.

[0083] In one or more embodiments, the method comprises: contacting the bacteria or an extract thereof with the jejunum and measuring a jejunal motility parameter, and contacting the bacteria with the colon and measuring a colon motility parameter.

[0084] In one or more embodiments, bacteria that reduce the amplitude, tone and / or frequency of intestinal motility by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% are intestinal motility-inhibiting bacteria.

[0085] In one or more embodiments, the measurement is performed by a force sensor (eg, MLT0202 from ADInstruments).

[0086] In one or more embodiments, the method further comprises balancing the intestinal tract or its cells or tissues with a buffer before contacting the bacteria with the intestinal tract or its cells or tissues. Preferably, the balancing solution is a HEPES-Tyrode (HT) buffer without Ca2+. More preferably, the balancing solution comprises 140.6mmol / L NaCl, 2.7mmol / L KCl, 1.0mmol / L MgCl2, 10mmol / L HEPES and 5.6mmol / L glucose, and the pH is 7.4. In one or more embodiments, the balance lasts for about 20-30 minutes.

[0087] The present invention also provides a bacterial culture method, comprising incubating the bacteria or composition described herein with a culture medium suitable for culturing bacteria.

[0088] In one or more embodiments, the culture medium comprises peptone, beef extract, NaCl, agar, and sterile defibrinated sheep blood.

[0089] In one or more embodiments, the culture medium comprises peptone, yeast extract, and NaCl.

[0090] In one or more embodiments, the pH of the incubation is 6-8, preferably 7.2-7.4.

[0091] In one or more embodiments, the incubation temperature is 35-40°C, preferably 37°C.

[0092] The present invention also provides a method for identifying bacteria, which comprises detecting SNPs or point mutations in the bacterial genome.

[0093] In one or more embodiments, the bacterium is Shigella sp. Preferably, the bacterium is a bacterial strain with a deposit registration number of CGMCC 20603.

[0094] In one or more embodiments, the method comprises identifying:

[0095] (1) the genome of the bacterium has corresponding nucleotides at positions corresponding to one or more positions selected from the following positions of the genome of the bacterial strain with accession number CGMCC 20603: PIB2013.g.331A, PIB2324.g.562G, PIB2324.g.601A, PIB2629.g.1126T, PIB3156.g.2396T,

[0096] (2) The bacterial genome has one or more characteristics selected from the following:

[0097] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 9 and 10 as primers is A;

[0098] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 11 and 12 as primers is G;

[0099] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 13 and 14 as primers is A;

[0100] The 20th nucleotide of the genomic amplification fragment using SEQ ID NO: 15 and 16 as primers is T;

[0101] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 17 and 18 as primers is T;

[0102] (3) the genome of the bacterium has any one or more sequences selected from SEQ ID NO: 2-6 or a fragment thereof having the nucleotide sequence described in (2) or a variant thereof having at least 98% sequence identity therewith, or

[0103] (4) The 16S rDNA of the bacterium has the sequence shown in SEQ ID NO: 1 or a sequence having at least 98% sequence identity thereto.

[0104] In one or more embodiments, the method comprises identifying a bacterium having a nucleic acid molecule described herein or a sequence thereof.

[0105] In one or more embodiments, the strain having one or more of the corresponding nucleotides, nucleic acid molecules or sequences is identified as CGMCC 20603.

[0106] In one or more embodiments, the method comprises the step of detecting the bacterial genome using the primers and / or probes described in any embodiment herein.

[0107] In one or more embodiments, the method includes the step of amplifying the genome of the bacteria using oligonucleotide primers that recognize any one or more of SEQ ID NOs: 1-6.

[0108] In one or more embodiments, the method comprises the step of amplifying the genome of the bacterium using the oligonucleotide primers of any one of SEQ ID NOs: 9-18.

[0109] In one or more embodiments, the method includes the step of amplifying the genome of the bacterium with a primer of 16S rDNA. In one or more embodiments, the primer of 16S rDNA is a primer for amplifying the 16S rDNA of a bacterium of the genus Shigella. In one or more embodiments, the primer has a sequence shown in SEQ ID NO:7-8.

[0110] In one or more embodiments, the method includes the step of amplifying the genome of the bacterium using one or more groups of oligonucleotide primers selected from the following: (1) SEQ ID NOs: 7 and 8, (2) SEQ ID NOs: 9 and 10, (3) SEQ ID NOs: 11 and 12, (4) SEQ ID NOs: 13 and 14, (5) SEQ ID NOs: 15 and 16, (6) SEQ ID NOs: 17 and 18.

[0111] In one or more embodiments, the method is a non-diagnostic or therapeutic method.

[0112] The present invention also provides bacteria identified by the method for identifying bacteria described herein.

[0113] The present invention also provides the use of the bacteria or composition described in any embodiment of the present invention in the preparation of an agent for inhibiting animal intestinal motility, reducing intestinal transmission speed, reducing the water content in animal feces, or treating diseases that benefit from decreased intestinal motility, such as irritable bowel syndrome, functional gastrointestinal disease, diarrhea, intestinal spasm, enteritis, indigestion, etc.

[0114] The present invention also provides an in vitro method for inhibiting intestinal motility or reducing intestinal transit rate of an animal, comprising contacting the intestinal tract with the bacteria or composition described in any embodiment of the present invention.

[0115] The present invention also provides the use of the bacteria or composition described in any embodiment of the present invention for inhibiting animal intestinal motility, reducing intestinal transmission rate, reducing the water content in animal feces, or treating diseases that benefit from decreased intestinal motility, such as irritable bowel syndrome, functional gastrointestinal disease, diarrhea, etc.

[0116] The present invention also provides a method for inhibiting intestinal motility of animals, reducing intestinal transit rate, reducing water content in animal feces, or treating diseases that benefit from decreased intestinal motility, comprising administering the bacteria or composition described in any embodiment of the present invention to the animal.

[0117] The present invention also provides oligonucleotide primers and / or probes as described herein, and the use of nucleic acid molecules, bacteria and / or compositions as optional controls in identifying bacteria. In one or more embodiments, the bacteria are Shigella bacteria. Preferably, the preservation number of the bacteria is CGMCC 20603.

[0118] The present invention also provides the use of a reagent for detecting SNPs or point mutations in a bacterial genome in the preparation of a kit for identifying a bacterial genome, identifying decreased intestinal motility, or diagnosing an intestinal disease caused by decreased intestinal motility, wherein the reagent is an oligonucleotide primer and / or probe, preferably, the reagent is an oligonucleotide primer and / or probe as described in any embodiment of the present invention, and optionally a nucleic acid molecule, bacteria and / or composition as a control.

[0119] In one or more embodiments, the nucleic acid molecule, bacterium and / or composition is a nucleic acid molecule, bacterium, composition described herein.

[0120] In one or more embodiments, intestinal diseases caused by decreased intestinal motility include chronic constipation, functional constipation, opioid-induced constipation, constipation-type irritable bowel syndrome, and functional defecation disorder.

[0121] In one or more embodiments, chronic constipation includes slow-transmission constipation (STC), outlet obstruction constipation, and mixed constipation.

[0122] In one or more embodiments, slow-transit constipation (STC) is refractory constipation (ICC).

[0123] The present invention further provides a device, characterized in that the device comprises a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that when the processor executes the program, the following steps are implemented:

[0124] (a) identifying a bacterium or whether a gut sample contains a bacterium, wherein the bacterium:

[0125] (1) the genome of the bacterium has corresponding nucleotides at positions corresponding to one or more positions selected from the following positions of the genome of the bacterial strain with accession number CGMCC 20603: PIB2013.g.331A, PIB2324.g.562G, PIB2324.g.601A, PIB2629.g.1126T, PIB3156.g.2396T,

[0126] (2) The bacterial genome has one or more characteristics selected from the following:

[0127] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 9 and 10 as primers is A;

[0128] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 11 and 12 as primers is G;

[0129] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 13 and 14 as primers is A;

[0130] The 20th nucleotide of the genomic amplification fragment using SEQ ID NO: 15 and 16 as primers is T;

[0131] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 17 and 18 as primers is T;

[0132] (3) the genome of the bacterium has any one or more sequences selected from SEQ ID NO: 2-6 or a fragment thereof having the nucleotide sequence described in (2) or a variant thereof having at least 98% sequence identity therewith, or

[0133] (4) the 16S rDNA of the bacterium has the sequence shown in SEQ ID NO: 1 or a sequence having at least 98% sequence identity thereto,

[0134] (b) identifying the strain, identifying decreased intestinal motility, or diagnosing an intestinal disease caused by decreased intestinal motility based on the result of (a).

[0135] In one or more embodiments, (a) is to identify whether a bacterial or intestinal sample has a nucleic acid molecule or sequence thereof described herein.

[0136] In one or more embodiments, the result of (a) shows the presence of one or more of the nucleotides, nucleic acid molecules or sequences, thereby identifying the strain as CGMCC 20603, or identifying decreased intestinal motility, or diagnosing an intestinal disease caused by decreased intestinal motility.

[0137] In one or more embodiments, (a) comprises the step of detecting the bacterial genome using the primers and / or probes described in any embodiment herein.

[0138] In one or more embodiments, (a) comprises the step of amplifying the genome of the bacteria using oligonucleotide primers that recognize any one or more sequences of SEQ ID NOs: 1-6.

[0139] In one or more embodiments, (a) comprises the step of amplifying the genome of the bacterium using the oligonucleotide primers described in any one of SEQ ID NOs: 9-18.

[0140] In one or more embodiments, (a) includes the step of amplifying the genome of the bacteria with primers of 16S rDNA. In one or more embodiments, the primers of 16S rDNA are primers for amplifying 16S rDNA of Shigella bacteria. In one or more embodiments, the primers have the sequences shown in SEQ ID NO:7-8.

[0141] In one or more embodiments, (a) includes the step of amplifying the genome of the bacterium using one or more groups of oligonucleotide primers selected from the following: (1) SEQ ID NOs: 7 and 8, (2) SEQ ID NOs: 9 and 10, (3) SEQ ID NOs: 11 and 12, (4) SEQ ID NOs: 13 and 14, (5) SEQ ID NOs: 15 and 16, (6) SEQ ID NOs: 17 and 18.

[0142] The present invention further provides a computer-readable storage medium, characterized in that a computer program is stored on the storage medium, and when the computer program is executed by a processor, the following steps are implemented:

[0143] (a) identifying a bacterium or whether a gut sample contains a bacterium, wherein the bacterium:

[0144] (1) the genome of the bacterium has corresponding nucleotides at positions corresponding to one or more positions selected from the following positions of the genome of the bacterial strain with accession number CGMCC 20603: PIB2013.g.331A, PIB2324.g.562G, PIB2324.g.601A, PIB2629.g.1126T, PIB3156.g.2396T,

[0145] (2) The bacterial genome has one or more characteristics selected from the following:

[0146] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 9 and 10 as primers is A;

[0147] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 11 and 12 as primers is G;

[0148] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 13 and 14 as primers is A;

[0149] The 20th nucleotide of the genomic amplification fragment using SEQ ID NO: 15 and 16 as primers is T;

[0150] The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 17 and 18 as primers is T;

[0151] (3) the genome of the bacterium has any one or more sequences selected from SEQ ID NO: 2-6 or a fragment thereof having the nucleotide sequence described in (2) or a variant thereof having at least 98% sequence identity therewith, or

[0152] (4) the 16S rDNA of the bacterium has the sequence shown in SEQ ID NO: 1 or a sequence having at least 98% sequence identity thereto,

[0153] (b) identifying the strain, identifying decreased intestinal motility, or diagnosing an intestinal disease caused by decreased intestinal motility based on the result of (a).

[0154] In one or more embodiments, (a) is to identify whether a bacterial or intestinal sample has a nucleic acid molecule or sequence thereof described herein.

[0155] In one or more embodiments, the result of (a) shows the presence of one or more of the nucleotides, nucleic acid molecules or sequences, thereby identifying the strain as CGMCC 20603, or identifying decreased intestinal motility, or diagnosing an intestinal disease caused by decreased intestinal motility.

[0156] In one or more embodiments, (a) comprises the step of detecting the bacterial genome using the primers and / or probes described in any embodiment herein.

[0157] In one or more embodiments, (a) comprises the step of amplifying the genome of the bacteria using oligonucleotide primers that recognize any one or more sequences of SEQ ID NOs: 1-6.

[0158] In one or more embodiments, (a) comprises the step of amplifying the genome of the bacterium using the oligonucleotide primers described in any one of SEQ ID NOs: 9-18.

[0159] In one or more embodiments, (a) includes the step of amplifying the genome of the bacteria with primers of 16S rDNA. In one or more embodiments, the primers of 16S rDNA are primers for amplifying 16S rDNA of Shigella bacteria. In one or more embodiments, the primers have the sequences shown in SEQ ID NO:7-8.

[0160] In one or more embodiments, (a) includes the step of amplifying the genome of the bacterium using one or more groups of oligonucleotide primers selected from the following: (1) SEQ ID NOs: 7 and 8, (2) SEQ ID NOs: 9 and 10, (3) SEQ ID NOs: 11 and 12, (4) SEQ ID NOs: 13 and 14, (5) SEQ ID NOs: 15 and 16, (6) SEQ ID NOs: 17 and 18.

[0161] The present invention also provides a method for screening a substance that can increase or decrease intestinal motility or kill the bacteria described herein, comprising:

[0162] (1) contacting a candidate substance with the bacteria described herein or an extract thereof, or a model containing the bacteria described herein,

[0163] (2) After contact, the number, activity, and / or intestinal contraction of the bacteria described herein are indicative of the candidate substance being a target substance.

[0164] In one or more embodiments, the substance is an organism or a chemical compound.

[0165] In one or more embodiments, the substance is a bacterium, a bacteriophage, an antibiotic, a small molecule compound, or a macromolecular compound.

[0166] In one or more embodiments, the model is an animal or its intestinal tract, tissue or cells.

[0167] In one or more embodiments, the model is a mouse.

[0168] The present invention also includes substances screened by the method for screening substances that can increase or decrease intestinal motility or kill the bacteria described herein, and the substances are preferably bacteriophages.

[0169] Advantages of the present invention:

[0170] 1. This article Figure 1 and Figure 4 The direct and indirect results in vivo and in vitro confirm that PIB affects intestinal transit. Therefore, the present invention is the first to discover the pathogenic bacteria PIB of intractable constipation, the first to determine the causal relationship between bacteria and chronic constipation, and propose that intractable constipation may be an infectious disease. This is completely different from the current research suggesting that chronic constipation may be related to intestinal bacteria.

[0171] 2. The first detection method of PIB was established: It is very difficult to detect a single bacterium in complex fecal bacteria because it is difficult to find a unique sequence. The present invention successfully established a detection method for specific bacteria (PIB) through single nucleotide polymorphism (SNP).

[0172] 3. The present invention can be used for rapid diagnosis, early warning and development of therapeutic drugs for chronic constipation, and has broad industrial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figure 1 Results showing inhibition of intestinal motility by PIB. A, Representative colon motility recordings before or after treatment with PIB cultures. B and C, Quantification of spontaneous tone and frequency of colon before or after treatment with PIB. The values ​​are representative of 6 independent experiments. *P<0.05; **P<0.01.

[0174] Figure 2 The genetic localization of PIB based on 16S rRNA coding sequence sequencing is shown. A phylogenetic tree was constructed using the PIB 16S ribosomal RNA gene sequence (rrs, 1464 bp), and the distance was calculated as the number of base substitutions per site by the maximum likelihood method. Bootstrap values ​​based on 1000 replicates (>80%) are listed at the branch nodes, and the branch length of the phylogenetic tree is proportional to the evolutionary distance. The 16Srrs of related bacteria were extracted from GeneBank. Phylogenetic analysis was performed using the Molecular Evolution Genetic Analysis software version 7.0.

[0175] Figure 3 Shows specific detection of PIB in feces by PCR. A, Fecal samples were collected from healthy individuals (20-29 years old) and analyzed by PCR2013. B, Healthy feces were compared with 1 to 10 6 PIB2013 analysis of PIB cell mixture. M, nucleic acid molecular weight marker; PIB, positive control.

[0176] Figure 4 Implantation and constipation induction in wild-type mice are shown. Adult C57BL / 6 mice were orally administered 1x10 9 PIB bacteria (0.1 ml), and the constipation phenotype was assessed at week 8 after dosing. A, The presence of PIB in feces was measured by PCR2013 at the end of the first week after dosing. B, Total intestinal transit time (CTR, n = 26; PIB, n = 25). C, Fecal water content (CTR, n = 26; PIB, n = 25). D, Body weight (CTR, n = 15; PIB, n = 15). E, Fecal weight (CTR, n = 21; PIB, n = 20). Mice treated with PIB medium were used as controls (CTR). F, Representative colonic peristalsis recordings of mice. G and H, Quantification of colonic spontaneous tension and frequency in mice treated with PIB and CTR mice (CTR, n = 15; PIB, n = 13). I, Representative histological sections of the colon of PIB-treated and CTR mice. **P < 0.01. DETAILED DESCRIPTION

[0177] Current studies suggest that chronic constipation may be related to intestinal bacteria. The main evidence is that chronic constipation causes changes in the intestinal flora, transplanting intestinal bacteria of chronic constipation can lead to the occurrence of constipation phenotypes in animals, and fecal microbiota transplantation has a certain therapeutic effect. However, these studies cannot determine the causal relationship between bacteria and chronic constipation.

[0178] The present invention designs a pathogen screening strategy, isolates bacteria from the ICC mucosa, cultures the bacteria under aerobic conditions to obtain a large number of bacterial clones, and then identifies whether each clone has the ability to inhibit intestinal peristalsis. After 5 years of searching, the inventors found a bacterial clone that inhibits intestinal peristalsis, which they call PIB (peristalsis inhibition bacteria). By discovering PIB bacteria, the present invention has determined for the first time the causal relationship between bacteria and chronic constipation, and proposed that intractable constipation may be an infectious disease.

[0179] PIB

[0180] PIB bacteria enter the intestines from the mouth or other routes, multiply in the intestinal mucosa, produce pathogenic substances, and inhibit normal intestinal peristalsis, resulting in a slowdown in intestinal transmission and constipation. Due to the formation of constipation, the renewal rate of intestinal bacteria slows down, which indirectly aggravates the reproduction of PIB in the local intestine, causing the intestinal transmission rate to slow down further. This cycle leads to continuous constipation. Because PIB has the characteristics of mucosal bacteria, it is extremely difficult to eliminate, which leads to the occurrence of stubborn constipation.

[0181] The present invention provides an Enterobacteriaceae bacterium, preferably a Shigella sp. bacterium, wherein the genome of the bacterium has a SNP or point mutation selected from the following: PIB2013 (g.331G>A), PIB2324 (g.562A>G), PIB2324 (g.601G>A), PIB2629 (g.1126C>T), PIB3156 (g.2396C>T). Specifically, in the bacteria, (1) the genome of the bacteria has corresponding nucleotides at one or more positions selected from the following: PIB2013.g.331A, PIB2324.g.562G, PIB2324.g.601A, PIB2629.g.1126T, PIB3156.g.2396T, (2) the genome of the bacteria has one or more characteristics selected from the following: the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 9 and 10 as primers is A; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 11 and 12 as primers is G; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 13 and 14 as primers is A; the 20th nucleotide of the genomic amplification fragment using SEQ ID NOs: 15 and 16 as primers is T; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 17 and 18 as primers is T; (3) the genome of the bacteria has a nucleotide selected from the following: Any one or more sequences of NO:2-6 or a fragment thereof having the nucleotide sequence described in (2) or a variant thereof having at least 98% sequence identity therewith, or (4) a 16S rDNA of a bacterium having the sequence shown in SEQ ID NO:1 or a sequence having at least 98% sequence identity therewith. The Shigella bacterium described herein is a bacterial strain with a deposit registration number of CGMCC 20603.

[0182] The SNP of the term "PIB2013 (g.331G>A)" indicates that the 331st nucleotide of the 2013th gene of the PIB bacterium changes from G at the corresponding position of the corresponding gene of other bacteria (e.g., other Shigella bacteria) to A. The term "PIB2013.g.331" indicates the 331st nucleotide of the 2013th gene of the PIB bacterium. Therefore, the term "PIB2013.g.331A" indicates that the 331st nucleotide of the 2013th gene of the PIB bacterium is A. And so on. The "point mutation" described herein mainly refers to the base mutation at the corresponding position of the genome of the PIB bacterium relative to the genome of other bacteria (e.g., other Shigella bacteria).

[0183] CGMCC 20603 can be cultured in a culture medium suitable for bacteria known in the art. Exemplary culture media include peptone, beef extract, NaCl, agar, and sterile defibrinated sheep blood, or peptone, yeast extract, and NaCl.

[0184] Nucleic Acids, Nucleic Acid Constructs and Cells

[0185] The present invention also provides an isolated nucleic acid molecule, which (1) has corresponding nucleotides at positions selected from the following one or more positions of the genome of the bacterial strain with accession number CGMCC 20603: PIB2013.g.331A, PIB2324.g.562G, PIB2324.g.601A, PIB2629.g.1126T, PIB3156.g.2396T, (2) has one or more characteristics selected from the following: the 21st nucleotide of the amplified fragment using SEQ ID NO: 9 and 10 as primers is A; the 21st nucleotide of the amplified fragment using SEQ ID NO: 11 and 12 as primers is G; the 21st nucleotide of the amplified fragment using SEQ ID NO: 13 and 14 as primers is A; the 20th nucleotide of the amplified fragment using SEQ ID NO: 15 and 16 as primers is T; the 21st nucleotide of the amplified fragment using SEQ ID NO: 17 and 18 as primers is T; (3) has one or more characteristics selected from the following: Any one or more sequences of ID NO: 2-6 or a fragment thereof having the nucleotides described in (2) or a variant thereof having at least 98% sequence identity therewith, (4) a sequence having the sequence shown in SEQ ID NO: 1 or a sequence having at least 98% sequence identity therewith, or (5) a complementary sequence of any one of (1)-(4). The present invention also provides a nucleic acid construct of the nucleic acid molecule and a host cell comprising the nucleic acid molecule or the nucleic acid construct.

[0186] The present invention also provides oligonucleotide primers or probes that recognize the above-mentioned nucleic acid molecules. When discussing primers or probes, "recognition" or "hybridization" as described herein refers to hybridization of the probe with the template sequence under stringent or highly stringent conditions, and the stringent or highly stringent conditions are well known in the art, for example, the highly stringent conditions can be hybridization at 65°C and washing the membrane in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS.

[0187] The oligonucleotide primers of the present invention are used to amplify the bacterial genome, and the amplified product comprises: (1) nucleotides located at positions in the genome of the bacteria corresponding to one or more positions selected from the following positions of the genome of the bacterial strain with the accession number of CGMCC 20603: PIB2013.g.331, PIB2324.g.562, PIB2324.g.601, PIB2629.g.1126, PIB3156.g.2396, (2) nucleotides selected from the following one or more positions: the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 9 and 10 as primers; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 11 and 12 as primers; the 212th nucleotide of the genomic amplification fragment using SEQ ID NOs: 13 and 14 as primers; the 20th nucleotide of the genomic amplification fragment using SEQ ID NOs: 15 and 16 as primers; NO:17 and 18 as the 21st nucleotide of the genomic amplification fragment of the primer; (3) any one or more sequences selected from SEQ ID NO:2-6 or fragments thereof having the nucleotides described in (2) or variants thereof having at least 98% sequence identity therewith, or (4) the 16S rDNA sequence shown in SEQ ID NO:1 or a sequence having at least 98% sequence identity therewith. The oligonucleotide primers recognize the 16S rDNA shown in SEQ ID NO:1, or recognize the sequences described in SEQ ID NO:2-6. These oligonucleotide primers can be used to identify Shigella bacteria, such as the bacteria described herein. Preferably, the amplification product of the oligonucleotide primers comprises the 16S rDNA of the bacteria described herein or any of the sequences described in SEQ ID NO:2-6. More preferably, the oligonucleotide primers are selected from any one or more of SEQ ID NO:7-18.

[0188] The probe of the present invention is an oligonucleotide probe, which recognizes a fragment of the genome of a bacterium (e.g., CGMCC 20603), wherein the fragment comprises (1) a nucleotide located at a position in the genome of the bacterium corresponding to one or more positions selected from the following positions of the genome of the bacterial strain with accession number CGMCC 20603: PIB2013.g.331, PIB2324.g.562, PIB2324.g.601, PIB2629.g.1126, PIB3156.g.2396, (2) a nucleotide at one or more positions of the bacterium selected from the following positions: the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 9 and 10 as primers; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 11 and 12 as primers; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 13 and 14 as primers; the 20th nucleotide of the genomic amplification fragment using SEQ ID NOs: 15 and 16 as primers; NO:17 and 18 as the 21st nucleotide of the genomic amplification fragment of the primer; (3) any one or more sequences selected from SEQ ID NO:2-6 or fragments thereof having the nucleotides described in (2) or variants thereof having at least 98% sequence identity, or (4) the 16S rDNA sequence shown in SEQ ID NO:1 or a sequence having at least 98% sequence identity therewith. Specifically, the probe is used to hybridize with the genome of bacteria (e.g., Shigella bacteria) or fragments thereof or the amplification products of the primers described herein. The probe recognizes the 16S rDNA of the bacteria described herein and any of the sequences described in SEQ ID NO:2-6.

[0189] Intestinal motility

[0190] The bacteria described herein can inhibit intestinal motility, including reducing the amplitude, tension and / or frequency of intestinal motility of animals. For example, the bacteria reduce the amplitude of intestinal motility of animals by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%; the bacteria reduce the tension of intestinal motility of animals by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%; the bacteria reduce the frequency of intestinal motility of animals by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. The bacteria described herein can reduce the intestinal transit rate, for example, reduce the intestinal transit rate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%, preferably by 20-30, more preferably by about 25%. In addition, the bacteria reduce the water content in animal feces by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%. In one or more embodiments, the animal is a mammal, preferably a human. In one or more embodiments, the intestinal tract is the small intestine and / or the large intestine. In one or more embodiments, the small intestine includes the duodenum, jejunum, and ileum. In one or more embodiments, the large intestine includes the cecum, colon, and rectum. Methods for measuring the amplitude, tension, and / or frequency of intestinal peristalsis of animals are well known in the art, such as full intestinal transit analysis (defecation time) and defecation frequency measurement, intestinal peristalsis (including amplitude, tension, frequency) measurement by force sensor, charcoal transit test, steel ball excretion test, full intestinal transit rate measurement, etc. An exemplary method for measuring whole-gut transit rate involves fasting the animal overnight and feeding it by gavage 100 μL of a test meal - 5% Evans blue and 1.5% methylcellulose, and recording the time to first excretion of blue feces.

[0191] Screening PIB

[0192] The present invention includes a method for screening bacteria that inhibit intestinal motility, comprising: contacting the bacteria described herein or an extract thereof with the intestine or intestinal tissue or intestinal cells, and measuring intestinal motility parameters. The bacteria are from the intestine or feces of an animal. The intestinal motility parameters described herein are intestinal motility amplitude, tension and / or frequency, the contraction response of the intestine after depolarization or ligand stimulation, and the relaxation effect of the intestine. The intestine is a mammalian intestine, preferably the jejunum and / or colon. Intestinal tissue and cells can be any tissue or part thereof and cells derived from the mammalian intestine, including epithelial tissue, smooth muscle tissue, and nervous tissue. Intestinal tissue is mainly tissue containing the inner surface of the intestine, etc. The intestine or intestinal tissue or intestinal cells may also include a pretreatment step before contacting the bacteria. The pretreatment steps of samples used to measure intestinal motility parameters are known in the art, such as balancing the intestine using a HEPES-Tyrode (HT) buffer.

[0193] According to the measurement results, bacteria that reduce the amplitude, tension and / or frequency of intestinal motility by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% are bacteria that inhibit intestinal motility. The measurement can be performed by a force sensor.

[0194] Bacterial culture

[0195] The present invention also provides a bacterial culture method, comprising incubating the bacteria or composition described herein with a culture medium suitable for culturing bacteria. The culture medium may comprise peptone, beef extract, NaCl, agar and sterile defibrinated sheep blood, or peptone, yeast extract and NaCl. The pH of the incubation is 6-8, preferably 7.2-7.4. The temperature of the incubation is 35-40°C, preferably 37°C.

[0196] Composition

[0197] The present invention provides a composition comprising the bacterium described in any embodiment herein or a cell-free extract, filtrate, supernatant or lysate thereof.

[0198] The composition of the present invention may be a pharmaceutical composition, further comprising a pharmaceutically acceptable adjuvant. The composition may also comprise other bacteria or extracts, filtrates, extracts, and lysates that inhibit intestinal motility of animals. The pharmaceutical composition may inhibit intestinal motility of animals, reduce intestinal transmission speed, reduce the water content in animal feces, or treat diseases that benefit from decreased intestinal motility, such as diarrhea. Herein, pharmaceutically acceptable adjuvants refer to carriers, diluents, and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, including but not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, and preservatives. More specifically, suitable pharmaceutically acceptable adjuvants may be adjuvants commonly used in the art for hepatocyte administration. Typically, a therapeutically effective amount of bacteria or filtrates, extracts, and lysates thereof are contained in a pharmaceutical composition. A therapeutically effective amount refers to a dose that can achieve treatment, prevention, alleviation, and / or relief of a disease or condition in a subject. The therapeutically effective amount may be determined based on factors such as the patient's age, sex, the disease and its severity, and other physical conditions of the patient. Herein, a subject or patient generally refers to a mammal, particularly a human.

[0199] The composition of the present invention can also be a cell culture comprising the bacteria described herein, and the composition further comprises a culture medium. The culture medium comprises peptone, beef extract, NaCl, agar and sterile defibrinated sheep blood, or comprises peptone, yeast extract and NaCl. The composition can also comprise other bacteria that inhibit animal intestinal motility.

[0200] Bacterial identification

[0201] The inventors found that, in addition to 16S rDNA, bacterial identification can be achieved by identifying characteristic SNPs or point mutations of different species of bacteria. Therefore, the present invention includes a method for identifying bacteria by determining SNPs or point mutations in bacterial genomes, a kit containing reagents for detecting the SNPs or point mutations, and the use of the reagents in identifying bacteria. This method is used herein to identify Shigella sp. bacteria. Specifically, CGMCC 20603 can be identified by identifying the 16S rDNA or SNPs of CGMCC 20603 bacteria. The SNPs include PIB2013 (g.331G>A), PIB2324 (g.562A>G), PIB2324 (g.601G>A), PIB2629 (g.1126C>T), PIB3156 (g.2396C>T) of CGMCC 20603. Herein, the method for identifying bacteria is generally a non-diagnostic or therapeutic method. Specifically, the method for identifying bacteria comprises identifying (1) the genome of the bacteria has a corresponding nucleotide at a position selected from the following one or more positions of the genome corresponding to the bacterial strain with accession number CGMCC 20603: PIB2013.g.331A, PIB2324.g.562G, PIB2324.g.601A, PIB2629.g.1126T, PIB3156.g.2396T, (2) the genome of the bacteria has one or more characteristics selected from the following: the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 9 and 10 as primers is A; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 11 and 12 as primers is G; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 13 and 14 as primers is A; the 20th nucleotide of the genomic amplification fragment using SEQ ID NOs: 15 and 16 as primers is T; The 21st nucleotide of the genomic amplification fragment using primers NO:17 and 18 is T; (3) the genome of the bacterium has any one or more sequences selected from SEQ ID NO:2-6 or fragments thereof having the nucleotides described in (2) or variants thereof having at least 98% sequence identity therewith, or (4) the 16S rDNA of the bacterium has the sequence shown in SEQ ID NO:1 or a sequence having at least 98% sequence identity therewith.

[0202] Conventional methods and steps and reagents used to achieve the identification are known in the art. For example, the identification result can be obtained by whole genome sequencing and comparison of the above-mentioned SNP or PCR amplification of fragments containing bacteria-specific sequences (such as 16SrDNA or SNP described herein) and optional probe recognition. Therefore, in certain embodiments, the method may include the step of detecting the bacterial genome with primers and / or probes described in any embodiment of the present invention. In one or more embodiments, the method includes the step of amplifying the bacterial genome with oligonucleotide primers that recognize any one or more sequences in SEQ ID NO: 1-6, and the primers are exemplarily shown in SEQ ID NO: 9-18. The amplified product (1) is a nucleotide located at a position in the genome of the bacterium corresponding to one or more positions selected from the following positions in the genome of the bacterial strain with registration number CGMCC 20603: PIB2013.g.331, PIB2324.g.562, PIB2324.g.601, PIB2629.g.1126, PIB3156.g.2396; (2) is a nucleotide selected from the following one or more positions: the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 9 and 10 as primers; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 11 and 12 as primers; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 13 and 14 as primers; the 20th nucleotide of the genomic amplification fragment using SEQ ID NOs: 15 and 16 as primers; the 21st nucleotide of the genomic amplification fragment using SEQ ID NOs: 17 and 18 as primers; (3) is a nucleotide selected from the following Any one or more sequences of NO:2-6 or a fragment thereof having the nucleotides described in (2) or a variant thereof having at least 98% sequence identity therewith, or (4) the 16S rDNA sequence shown in SEQ ID NO:1 or a sequence having at least 98% sequence identity therewith. Specifically, the method comprises the step of amplifying the genome of the bacterium using one or more groups of oligonucleotide primers selected from the following: (1) SEQ ID NO:9 and 10, (2) SEQ ID NO:11 and 12, (3) SEQ ID NO:13 and 14, (4) SEQ ID NO:15 and 16, (5) SEQ ID NO:17 and 18. While or after amplifying the above sequences, whether the sequences contain the above specific SNPs can be determined by sequencing or hybridizing the amplified sequences with probes. The strain having the above SNPs was identified as CGMCC 20603.

[0203] Alternatively, the method for identifying CGMCC 20603 comprises the step of amplifying the genome of the bacterium using primers for amplifying 16S rDNA of Shigella bacteria. Exemplary primers for amplifying 16S rDNA of Shigella bacteria have sequences shown in SEQ ID NOs: 7-8.

[0204] The above-mentioned sequencing, PCR amplification, or probe hybridization steps and conventional reagents other than primers and probes, such as dNTPs, polymerase, ligase, buffer, NaCl, KCl, etc. are known in the art.

[0205] Identification and diagnosis of decreased intestinal motility

[0206] By identifying whether the intestinal sample contains the bacteria described herein, it is possible to identify decreased intestinal motility or diagnose intestinal diseases caused by decreased intestinal motility. Therefore, the oligonucleotide primers and probes described in any embodiment of the present invention can be used to prepare a kit for identifying bacteria or their genomes, identifying decreased intestinal motility, or diagnosing intestinal diseases caused by decreased intestinal motility. The nucleic acid molecules, bacteria, and compositions described herein can be used as positive controls for the kit.

[0207] Herein, intestinal diseases caused by decreased intestinal motility include chronic constipation, functional constipation, constipation caused by opioids, constipation-type irritable bowel syndrome, and functional defecation disorders. Chronic constipation includes slow-transmission constipation (STC), outlet obstruction constipation, and mixed constipation. Exemplarily, slow-transmission constipation (STC) is intractable constipation (ICC).

[0208] Device

[0209] In addition, the present invention also discloses a computer-readable storage medium storing a computer program, and the computer program stored on the storage medium is executed after running to perform the method for diagnosing intestinal diseases caused by decreased intestinal motility described herein. The steps of the method or algorithm described in conjunction with the embodiments disclosed herein can be directly embodied in hardware, in a software module executed by a processor, or in a combination of the two. If implemented as a computer program product in software, each function can be stored on a computer-readable medium or transmitted therefrom as one or more instructions or codes. Computer-readable media include both computer storage media and communication media, including any medium that facilitates the transfer of a computer program from one place to another. The storage medium can be any available medium that can be accessed by a computer. As an example and not limitation, such a computer-readable medium may include RAM, ROM, EEPROM, CD-ROM or other optical disk storage, disk storage or other magnetic storage device, or any other medium that can be used to carry or store a desired program code in the form of an instruction or data structure and can be accessed by a computer. An exemplary storage medium is coupled to a processor so that the processor can read and write information from / to the storage medium. In an alternative, the storage medium can be integrated into the processor. The processor and storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and storage medium may reside in a user terminal as discrete components.

[0210] Treatments and uses

[0211] The present invention provides an in vitro method for inhibiting intestinal motility or reducing intestinal transit speed in animals, comprising contacting the intestinal tract with the bacteria or composition described in any embodiment herein. The present invention also provides the use of the bacteria or composition described in any embodiment herein for inhibiting intestinal motility in animals, reducing intestinal transit speed, reducing the water content in animal feces, or treating diseases that benefit from decreased intestinal motility, such as diarrhea. The present invention also provides a method for inhibiting intestinal motility in animals, reducing intestinal transit speed, reducing the water content in animal feces, or treating diseases that benefit from decreased intestinal motility, comprising administering the bacteria or composition described in any embodiment herein to the animal.

[0212] The present invention also provides the use of the bacteria or composition described in any embodiment of the present invention in the preparation of an agent for inhibiting animal intestinal motility, reducing intestinal transmission speed, reducing the water content in animal feces, or treating diseases that benefit from decreased intestinal motility, such as irritable bowel syndrome, functional gastrointestinal disease, diarrhea, intestinal spasm, enteritis, indigestion, etc.

[0213] Screening substances

[0214] The bacteria found herein can also be used to screen for substances that can increase or decrease intestinal motility or kill bacteria described herein. By contacting the candidate substance with the bacteria described herein or a model (e.g., mouse) containing the bacteria described herein, and detecting the quantity and / or activity and / or intestinal contraction of the bacteria described herein after contact, the candidate substance can be indicated as the target substance. The screened substance can be an organism or a compound. For example, bacteria, bacteriophages, antibiotics, small molecules, or macromolecules. "Model" described herein can be any model with a disease or condition of interest derived from an animal, including the model animal itself or its organs, tissues, or cells. For example, mammals or their intestinal tracts, intestinal tissues, or cells. An exemplary disease model is a mouse.

[0215] Example

[0216] Embodiment 1, experimental method:

[0217] Specimens and stool cultures

[0218] Fresh colon segments were collected from ICC patients who underwent subtotal colectomy plus modified Duhamel procedure at Jinling Hospital (China). The specimens were washed with a common solution and the bacterial precipitate was smeared on a blood plate (1% peptone, 0.3% beef extract, 0.5% NaCl, 1.5% agar and 5% sterile defibrinated sheep blood, pH 7.3 ± 0.1) and then cultured at 37°C for 12-16 hours. Monoclonal bacteria were inoculated into a culture tube containing 3 ml of LB medium (1% peptone, 0.5% yeast extract and 1% NaCl, pH 7.3 ± 0.1). The culture supernatant was collected and screened.

[0219] Intestinal motility testing and bacterial screening

[0220] Colonic tension measurements were performed on bacterial culture supernatants from ICC colons. Briefly, colon segments (approximately 5 mm in length) were isolated from 8-10 week old C57BL / 6 mice and cultured with Ca-free 2+ The colon was equilibrated with HEPES-Tyrode (HT) buffer (140.6mmol / L NaCl, 2.7mmol / L KCl, 1.0mmol / L MgCl2, 10mmol / L HEPES, and 5.6mmol / L glucose, pH 7.4) and then mounted on a force transducer (MLT0202; ADInstruments) connected to a PowerLab (ADInstruments, Shanghai, China) recording device. The amplitude and frequency of colonic peristalsis were recorded with or without the addition of 100uL bacterial supernatant.

[0221] Whole-gut transit analysis

[0222] Mice were fasted overnight and gavaged with 100 μL of the test meal (5% Evans blue and 1.5% methylcellulose). The time of the first excretion of blue feces was recorded.

[0223] Stool frequency and water content

[0224] Free-feeding mice were placed in a quiet environment for 12 h, and feces were collected in the last 2 h. The number of feces represented the frequency of defecation. The water content in feces was determined by comparing the weight of fresh feces with the weight after drying.

[0225] Phylogenetic tree analysis

[0226] The PIB 16S ribosomal RNA gene (rrs) was amplified by PCR using a pair of primers: PIB 16S-F, AGAGTTTGATCCTGGCTCAG (SEQ ID NO: 36) and PIB 16S-R, GGTTACCTTGTTACGACTT (SEQ ID NO: 37). The resulting gene sequence (SEQ ID NO: 1) with high similarity was compared with the rrs sequence (bacteria and archaea) and clustered using ClustalW software. A phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis Software (version 7.0.14). The results are shown in Figure 2. Figure 2 shown.

[0227] Genome sequencing and assembly

[0228] PIB genomic DNA was sequenced using a combination of next-generation sequencing platforms (Illumina HiSeq 4000) and SMRT sequencing (PacBio RS II) at the Beijing Genomics Institute (Shenzhen, China). Library construction, self-correction, and data analysis were performed according to the manufacturer’s protocol.

[0229] Genome component prediction and annotation

[0230] Protein coding sequences were predicted using the HiddenMarkov model by GLIMMER3 (http: / / www.cbcb.umd.edu / software / glimmer / ). tRNA, rRNA and sRNA were analyzed using three databases (tRNAscan-SE, RNAmmer and Rfam). Tandem repeat sequences were identified by Tandem Repeat Finder (http: / / tandem.bu.edu / trf / trf.html), and minisatellite DNA and microsatellite DNA were identified according to the number and length of repeat units. The Genomic Island Suite of Tools (GIST, http: / / www5.esu.edu / cpsc / bioinfo / software / GIST / ), PHAge Search Tool (PHAST, http: / / phast.wishartlab.com / ) and CRISPRFinder were used to predict genomic islands, Prophage regions and CRISPR regions.

[0231] CDS annotation was based on BLASTP, using seven databases (KEGG, Kyoto Encyclopedia of Genes and Genomes; COG, Clusters of Orthologous Groups; NR, Non-Redundant Protein Database databases; Swiss-Prot; GO, Gene Ontology; TrEMBL and EggNOG). Virulence factors and resistance genes were identified according to the VFDB (Virulence Factors of Pathogenic Bacteria) and ARDB (Antibiotic Resistance Genes Database) databases, respectively. Type III secretion system effector proteins were identified by EffectiveT3.

[0232] Detection of fecal PIB bacteria

[0233] Based on the specific region of the PIB genome, the synthetic primers were used for PCR assay. Exemplary primers are PIB2013 (g.331G>A)-F (SEQ ID NO:9) and -R (SEQ ID NO:10), PIB2324A (g.562G>A)-F (SEQ ID NO:11) and -R (SEQ ID NO:12), PIB2324B (g.601G>A)-F (SEQ ID NO:13) and -R (SEQ ID NO:14), PIB2629 (g.1126G>A)-F (SEQ ID NO:15) and -R (SEQ ID NO:16), PIB3156 (g.2396C>T)-F (SEQ ID NO:17) and R (SEQ ID NO:18).

[0234] The collected fresh feces or colon segments were incubated at 37°C overnight and then washed with LB.

[0235] Study Approval

[0236] All animal experiments were approved by the Animal Care and Use Committee of the Model Animal Research Center of Nanjing University, a member of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

[0237] statistics

[0238] All data are presented as mean ± SEM. The differences between the two groups were determined by two-tailed Student's t-test analysis using GraphPad Prism version 7. P < 0.05 was considered to be significantly different.

[0239] Example 2, Identification of motility-inhibiting bacteria

[0240] Four colon biopsy samples were collected from four patients with intractable chronic constipation, and bacterial monoclonal cultures were performed in blood plates. Then, among the resulting colonies, thousands of monoclones were transferred to 3 mL of LB medium without antibiotics and cultured overnight at 37 ° C. The effect of bacterial culture on intestinal peristalsis was detected. Since the jejunum has a faster contraction frequency than the colon, the effect of the culture on jejunal peristalsis was first measured, and then the effect on the colon was confirmed. After a large number of screenings, three colonies with obvious inhibitory effects were obtained, and these colonies maintained the same inhibitory effect even after being passed to the sixth generation. These colonies all showed similar morphology and similar inhibitory activity, especially the rrs sequence similarity of up to 99.7%. Therefore, these colonies are considered to be the same strain (Shigella sp.PIB, deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee (CGMCC) with the deposit number CGMCC 20603 on September 7, 2020). Functionally, the strain was named peristalsis-inhibiting bacteria (PIB). Figure 1 The changes in colonic motility before and after the addition of PIB are directly detected by force sensing experiments. The typical inhibitory effect of PIB culture on colonic motility is shown in Figure 2. Figure 1 As shown in A, the amplitude and frequency of peristalsis changed significantly ( Figure 1 B and C). The inhibitory effect on jejunal motility was similar to that in the colon.

[0241] Example 3, Genetic Characterization of PIB

[0242] The PIB 16S ribosomal RNA gene (1464 bp) was sequenced. Phylogenetic tree analysis showed that PIB 16S rrs was closest to Shigella genus with a high bootstrap value (97%). Figure 2 ), so PIB is a new strain of Shigella. Compared with the reported Shigella strains, the whole genome sequence of PIB contains more non-coding RNA and fewer genes (Table 1). In addition, the genome size between these strains is also different. Therefore, PIB is identified as a new strain of Shigella.

[0243] Table 1. PIB genomic characteristics

[0244]

[0245]

[0246] Example 4, Methodological Establishment of PIB Detection

[0247] In order to establish an effective PIB detection method, the PIB genome sequence was analyzed, but no unique genes or regions unique to PIB were found. Then the single nucleotide polymorphism (SNP) or point mutation of PIB DNA was analyzed. After substantial screening and optimization, specific sites were found and PCR assays for PIB detection were developed. In order to evaluate the sensitivity of these site determinations, 1 to 1000000 PIB bacteria were mixed with healthy feces, and then PCR assays were performed. The exemplary five candidate sites were g.331G>A mutations in the sorbitol dehydrogenase coding region, g.562A>G mutations in the NADPH-dependent curcumin reductase coding region, g.601G>A mutations, g.1126C>T mutations in the 3-oxyacyl-(acyl carrier protein) synthase II coding region, and g.2396C>T mutations in the enterobacterial synthetase coding region, and the primers were as described in Example 1. In order to evaluate the specificity of these sites, fecal bacteria of 24 healthy adults were selected and PCR reactions were performed. The results showed that PCR2013 could be detected from fecal bacteria even if only one PIB was present, showing high sensitivity for PIB detection ( Figure 3 A); PCR2013 did not produce a positive signal in fecal bacteria of 24 healthy adults ( Figure 3 B). The results of the remaining four candidate sites were similar (results not shown).

[0248] Example 5, PIB implantation induced constipation phenotype

[0249] To test whether PIB induces a constipation phenotype, C57BL / 6 wild-type mice not treated with antibiotics were given PIB (1 × 10 9 At the end of the first week after administration, PIB bacteria were detected in the feces, indicating successful engraftment in mice ( Figure 4 A). Eight weeks after administration, the intestinal motility, stool properties, and colon histology of the mice were examined. The gastrointestinal transit time (GTT) of mice treated with PIB increased by 126% compared with the control (CTR: 114 ± 5 ​​minutes vs PIB: 144 ± 6 minutes, p < 0.01, Figure 4 B), the water content in feces was significantly reduced (CTR: 30±2% vs PIB: 18±2%, p<0.01, Figure 4 C). Body weight and fecal dry weight were comparable among the groups ( Figure 4 D and E). Ex vivo tension measurements showed that colonic motility amplitude and frequency from PIB-treated mice were suppressed (reduced by 30%-100%) ( Figure 4 FH), but no obvious histological changes were observed ( Figure 4 , I). The above in vitro and in vivo results collectively indicate that PIB can be effectively implanted into wild-type mice and is sufficient to induce a constipation phenotype.

[0250] Example 6. Presence of fecal PIB in ICC patients

[0251] Fecal bacteria were collected from three groups of people, namely healthy adults (n=17), ICC patients (n=19) and non-ICC patients (n=47) (Table 2). The male / female ratio of healthy adults = 10 / 7. All ICC patients were diagnosed and received surgical treatment at Jinling Hospital. All 47 non-ICC patients were hospitalized in the Department of Gastroenterology of Jinling Hospital, of which 40 were diagnosed with ulcerative colitis. The 7 patients who received treatment for self-diagnosed chronic constipation were not clinically diagnosed with ICC. PCR2013 analysis showed that all healthy adults and non-ICC patients did not show fecal PIB, while 13 / 19 ICC patients showed fecal PIB positive. It is worth noting that ICC patients with positive PIB were randomly distributed in all age groups and were not related to age. In addition, the occurrence of PIB was not significantly correlated with disease history, treatment history and severity of syndrome (Table 3).

[0252] Table 2. ICC patient information

[0253]

[0254] Table 3. Demographic characteristics of the population

[0255]

[0256] The present invention identifies a novel bacterial strain Shigella sp.nov.PIB from the colonic mucosa of ICC patients. PIB significantly inhibits gastrointestinal motility both in vitro and in vivo. Oral administration of PIB can induce a constipation phenotype in wild-type mice. By establishing a sensitive PCR method, it was found that about 68% of ICC patients were PIB positive in their feces, while all control groups were PIB negative, indicating that PIB is closely related to ICC, and therefore PIB is considered to be a pathogenic bacterium of some ICC.

[0257] The identification of PIB in humans has important clinical significance. Since all control groups showed negative fecal PIB, PIB-positive individuals can be considered as ICC or potential ICC patients. The detection of fecal PIB will become an important diagnostic method for ICC. By using isolated PIB bacteria, suitable substances can be screened to kill or modify the bacteria, thereby providing new therapeutic drugs for ICC. Sequence Listing <110> Shaanxi Anning Yunsheng Biotechnology Co., Ltd. <120> Constipation-causing bacteria and their uses <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 4392 <212> DNA <213> Unknown <400> 1 attgaacgct ggcggcaggc ccacacatg caagtcgaac ggtaacaga atcagcttgc 60 tgatttgctg acgagtggcg gacggggtgag taatgtctgg gaactgcct gatggagggg 120 gataactact ggaaacggta gctaataccg cataacgtcg caaccaaa gagggggacc 180 ttcggggcctc ttgccatcgg atgtgcccag atgggattag ctagtaggtg gggtaacggc 240 tcacctaggc gacgatccct agctggtctg agagatgac cagccacact ggaactgaga 300 cacggtccag actcctacgg gaggcagcag tgggaat tgcacaatgg gcgcaagcct 360 gatgcagcca tgccgcgtgt atgagaagg ccttcggtt gtaagtact ttcagcgggg 420 aggaagggag taaagttaat accttgctc attgacgtta cccgcagaag aagcaccggc 480 taactccgtg cccagcagccg cggtaatacg gaggtgcaa gcgttaatcg gatttactgg 540 gcgtaaagcg cacgcaggcg gtttgttaag tcagatgtga atccccgggg ctcaacctgg 600 gaactgcatc tgatactggc aagcttgagt ctcgtagagg ggggtagaat tccaggtgta 660 gcggtgaaat gcgtagagat ctggaggaat accggtggcg aaggcggccc cctggacgaa 720 gactgacgct caggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780 cgccgtaaac gatgtcgact tggaggttgt gcccttgagg cgtggcttcc ggagctaacg 840 cgttaagtcg accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg 900 ggcccgcaca agcggtggag catgtggttt aattcgatgc aacgcgaaga accttacctg 960 gtcttgacat ccacagaact ttccagagat ggattggtgc cttcgggaac tgtgagacag 1020 gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg ggttaagtcc cgcaacgagc 1080 gcaaccctta tcctttgttg ccagcggtcc ggccgggaac tcaaaggaga ctgccagtga 1140 taaactggag gaaggtgggg atgacgtcaa gtcatcatgg cccttacgac cagggctaca 1200 cacgtgctac aatggcgcat acaaagagaa gcgacctcgc gagagcaagc ggacctcata 1260 aagtgcgtcg tagtccggat tggagtctgc aactcgactc catgaagtcg gaatcgctag 1320 taatcgtgga tcagaatgcc acggtgaata cgttcccggg ccttgtacac accgcccgtc 1380 acaccatggg agtgggttgc aaaagaagta ggtagcttaa ccttcgggag ggcgcttacc 1440 actttgtgat tcatgactgg ggtgattgaa cgctggcggc aggcctaaca catgcaagtc 1500 gaacggtaac agaatcagc ttgctgattt gctgacgagt ggcggacggg tgagtaatgt 1560 ctgggaaact gcctgatgga gggggataac tactggaaac ggtagctaat accgcataac 1620 gtcgcaagac caaagagggg gaccttcgg cctcttgcca tcggatgtgc ccagatggga 1680 ttagctagta ggtggggtaa cggctcacct aggcgacgat ccctagctgg tctgagagga 1740 tgaccagcca cactggaact gagacacggt ccagactcct acgggaggca gcagtggga 1800 atattgcaca atgggcgcaa gcctgatgca gccatgccgc gtgtatgaag aaggccttcg 1860 ggttgttaaag tactttcagc ggggaggaag ggagtaaagt taataccttt gctcattgac 1920 gttacccgca gaagaagcac cggctactc cgtgccagca gccgcggtaa tacggagggt 1980 gcaagcgtta atcggaatta ctgggcgtaa agcgcacgca ggcggtttgt taagtcagat 2040 gtgaaatccc cgggctcaac ctgggaactg catctgatac tggcaagctt gagtctcgta 2100 gaggggggta gaattccagg tgtagcggtg aaatgcgtag agatctggag gaataccggt 2160 ggcgaaggcg gccccctgga cgaagactga cgctcaggtg cgaaagcgtg gggagcaaac 2220 aggattagat accctggtag tccacgccgt aaacgatgtc gacttggagg ttgtgccctt 2280 gaggcgtggc ttccggagct aacgcgttaa gtcgaccgcc tggggagtac ggccgcaagg 2340 ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 2400 atgcaacgcg aagaacctta cctggtcttg acatccacag aactttccag agatggattg 2460 gtgccttcgg gaactgtgag acaggtgctg catggctgtc gtcagctcgt gttgtgaaat 2520 gttgggttaa gtcccgcaac gagcgcaacc cttatccttt gttgccagcg gtccggccgg 2580 gaactcaaag gagactgcca gtgataaact ggaggaaggt ggggatgacg tcaagtcatc 2640 atggccctta cgaccagggc tacacacgtg ctacaatggc gcatacaaag agaagcgacc 2700 tcgcgagagc aagcggacct cataaagtgc gtcgtagtcc ggattggagt ctgcaactcg 2760 actccatgaa gtcggaatcg ctagtaatcg tggatcagaa tgccacggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca tggggagtggg ttgcaaaaga tagtagc 2880 ttaaccttcg ggagggcgct taccactttg tgattcatga ctggggtgat tgaacgctgg cggcaggcct aacacatgca agtcgaacgg they are cagcttgctg atttgctgac gagtggcgga cgggtgagta atgtctggga aactgcctga tggaggggga taactactgg 3060 aaacggtagc fathercgtcgc aagaccaaaga gggggacctt cggggcctctt gccatcggat gtgcccagat gggattagct agtaggtggg gtaacggctc acctaggcga 3180 cgatccctag ctggtctgag aggatgacca gccacactgg aactgagac cggtccagac 3240 tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga tgcagccatg ccgcgtgtat gaagaggcc ttcgggttgt aaagtacttt cagcggggag gaagggagta 3360 aagttaatac ctttgctcat tgacgttacc cgcagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga gggtgcaagc gttaatcgga attactgggc gtaaagcgca cgcaggcggt ttgttaagtc agatgtgaaa tccccgggct caacctggga actgcatctg 3540 atactggcaa gcttgagtct cgtagagggg ggtagaattc caggtgtagc ggtgaaatgc 3600 gtagagatct ggaggaatac cggtggcgaa ggcggccccc tggacgaaga ctgacgctca 3660 ggtgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga 3720 tgtcgacttg gaggttgtgc ccttgaggcg tggcttccgg agctaacgcg ttaagtcgac 3780 cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag 3840 cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac cttacctggt cttgacatcc 3900 acagaacttt ccagagatgg attggtgcct tcgggaactg tgagacaggt gctgcatggc 3960 tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg caacgagcgc aacccttatc 4020 ctttgttgcc agcggtccgg ccgggaactc aaaggagact gccagtgata aactggagga 4080 aggtggggat gacgtcaagt catcatggcc cttacgacca gggctacaca cgtgctacaa 4140 tggcgcatac aaagagaagc gacctcgcga gagcaagcgg acctcataaa gtgcgtcgta 4200 gtccggattg gagtctgcaa ctcgactcca tgaagtcgga atcgctagta atcgtggatc 4260 agaatgccac ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgggag 4320 tgggttgcaa aagaagtagg tagcttaacc ttcgggaggg cgcttaccac tttgtgattc 4380 atgactgggg tg 4392 <210> 2 <211> 250 <212> DNA <213> Artificial Sequence <400> 2 gcaaatataa catctgcccg aacgttgatt ttatggcgac acaacctaac taccgcggcg 60 cattaacgca ctatctgtgt catccggaga gctttactta caaactgccc gacaatatgg 120 acacgatgga aggggcgctg gtggagcctg ccgcagtcgg gatgcatgcc gcgatgctgg 180 cagatgttaa accgggtaag aagataatta ttctgggagc gggttgtatt ggtttgatga 240 cgttgcaagc 250 <210> 3 <211> 393 <212> DNA <213> Artificial Sequence <400> 3 aaggttgcag ggtggtgggg gttgccggtg gcgcggaaaa atgccgccat gctaccgaga 60 tgttaggctt cgatgtatgc cttgatcacc acgcggatga ttttgccgaa caactggcga 120 aagcgtgccc gaaaggcgtt gatatctatt atgaaaacgt tggcggtaag gtattcgatg 180 cggtgctacc gttacttaat acatctgcgc gcattcccgt ctgcggctta gtgagcagct 240 ataacgctac agagctacca cccggcccgg atcgattacc tctgttgatg gctacggtgc 300 tgaaaaaacg tattcgcctg caaggattta ttatcgctca ggattatggt caccgcatcc 360 atgagtttca gaaggagatg gggcaatggg tga 393 <210> 4 <211> 351 <212> DNA <213> Artificial Sequence <400> 4 aatgccgcca tgctaccgag atgttaggct tcgatgtatg ccttgatcac cacgcggatg 60 attttgccga acaactggcg aaagcgtgcc cgaaaggcgt tgatatctat tatgaaaacg 120 ttggcggtaa ggtattcgat gcggtgctac cgttacttaa tacatctgcg cgcattcccg 180 tctgcggctt agtgagcagc tataacgcta cagagctacc acccggcccg gatcgattac 240 ctctgttgat ggctacggtg ctgaaaaaac gtattcgcct gcaaggattt attatcgctc 300 aggattatgg tcaccgcatc catgagtttc agaaggagat ggggcaatgg g 351 <210> 5 <211> 468 <212> DNA <213> Artificial Sequence <400> 5 gatggtgccg gtatgctggt acttgaagag tacgaacacg cgaaaaaacg cggtgcgaaa 60 atttacgctg aactcgtcgg ctttggtatg agcagcgatg cttatcatat gacgtcaccg 120 ccagaaaatg gcgcaggcgc agctctggca atggcaaatg ctttgcgtga tgcaggcatt 180 gaagcgagtc agattggcta cgttaacgca cacggtactt ctactccggc tggcgataaa 240 gctgaagcgc aggcggtgaa aaccatcttc ggtgaagctg caagccgtgt gttggtaagc 300 tccacgaaat ctatgaccgg tcacctgtta ggtgcggcgg gtgcagtaga atctatctac 360 tccatcctgg cgctgcgcga tcaggctgtt ccgccaacca ttaacctgga taacccggat 420 gaaggttgcg atctggattt cgtaccgtac gaagcgcgtc aggtcagc 468 <210> 6 <211> 426 <212> DNA <213> Artificial Sequence <400> 6 atgcatccgg tgccgccagg tgtggtgggt gatctctatc tcaccggtat tcaactggcg 60 caggggtatc tgggacgacc cgatctgacc gccagtcgct ttattgccga tccttttgcc 120 ccaggtgaac ggatgtaccg taccggagac gttgcccgct ggctggataa cggcgcagtg 180 gagtacctcg ggcgcagtga cgatcagcta aaaattcgcg ggcagcgtat cgaactaggc 240 gaaattgatc gcgtgatgca ggcgctgccg gatgtcgaac aggccgttac ccacgcctgt 300 gtgattaacc aggcggcagc cactggtggt gatgcgcgtc agttggtggg ctatctggtg 360 tcgcaatcgg gcctgccgtt ggataccact gcattgcagg cgcagttgcg cgaaacattg 420 ccaccg 426 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 agagtttgat cctggctcag 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 ggttaccttg ttacgactt 19 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <400> 9 gcaaatataa catctgcccg a 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 gcttgcaacg tcatcaaacc 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <400> 11 aaggttgcag ggtggtgggg g 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 tcacccattg ccccatctcc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <400> 13 aatgccgcca tgctaccgag a 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 cccattgccc catctccttc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 gatggtgccg gtatgctggt 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <400> 16 gctgacctga cgcgcttcgt a 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <400> 17 atgcatccgg tgccgccagg t 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 cggtggcaat gtttcgcgca 20

Claims

1. An Enterobacteriaceae bacterium, characterized in that Its deposit registration number is CGMCC 20603.

2. A composition comprising the bacterium according to claim 1 or its culture supernatant or lysate.

3. The composition according to claim 2, characterized in that The composition is a pharmaceutical composition, and further comprises a pharmaceutically acceptable excipient.

4. The composition according to claim 2, characterized in that The composition is a bacterial culture, and the composition further comprises a culture medium.

5. An oligonucleotide primer for identifying the bacterium with the deposit number CGMCC 20603, characterized in that: The sequences of the oligonucleotide primers are shown in SEQ ID NOs: 9 and 10.

6. The oligonucleotide primer according to claim 5, characterized in that The sequence of the oligonucleotide primer also includes any one or more groups selected from the following: (1) sequences as shown in SEQ ID NOs: 11 and 12, or (2) sequences as shown in SEQ ID NOs: 13 and 14, or (3) sequences as shown in SEQ ID NOs: 15 and 16, or (4) sequences as shown in SEQ ID NOs: 17 and 18.

7. An oligonucleotide probe for identifying the bacterium with a deposit number of CGMCC 20603, wherein the fragment of the bacterial genome recognized by the oligonucleotide probe comprises the nucleotide at the following position of the bacterium: the 21st nucleotide of the genome amplification fragment using SEQ ID NO: 9 and 10 as primers.

8. The oligonucleotide probe according to claim 7, characterized in that The fragment of the bacterial genome recognized by the oligonucleotide probe further comprises nucleotides at one or more positions of the bacteria selected from the following: The 20th nucleotide of the genomic amplification fragment using SEQ ID NO: 15 and 16 as primers, The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 17 and 18 as primers.

9. A kit for identifying the bacterium with a deposit number of CGMCC 20603 or its genome, comprising the oligonucleotide primers according to claim 5 or 6 or the oligonucleotide probe according to claim 7 or 8.

10. A method for identifying bacteria with a deposit number of CGMCC 20603, the method comprising identifying a SNP or point mutation in the bacterial genome, the method being a non-diagnostic or therapeutic method, the method comprising identifying that the bacterial genome has the following characteristics: the 21st nucleotide of the genome amplification fragment using SEQ ID NO: 9 and 10 as primers is A.

11. The method according to claim 10, characterized in that The bacterial genome also has one or more of the following characteristics: The 20th nucleotide of the genomic amplification fragment using SEQ ID NO: 15 and 16 as primers is T, The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 17 and 18 as primers is T.

12. Use of a reagent for detecting SNP or point mutation in the bacterial genome with the accession number CGMCC 20603 in the preparation of a kit for identifying the bacterial genome with the accession number CGMCC 20603 or for identifying decreased intestinal motility, The SNP or point mutation includes: The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 9 and 10 as primers is A, The reagent is the oligonucleotide primer according to claim 5 or 6, or the oligonucleotide probe according to claim 7 or 8.

13. The use according to claim 12, characterized in that The SNP or point mutation also includes one or more selected from the following: The 20th nucleotide of the genomic amplification fragment using SEQ ID NO: 15 and 16 as primers is T, The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 17 and 18 as primers is T.

14. A device, characterized in that: The device comprises a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the program, the following steps are implemented: (a) identifying bacteria or identifying whether the intestinal sample contains the bacteria by detecting SNPs or point mutations in the genome of the bacteria with the deposit number of CGMCC 20603, and (b) Based on the results of (a), the intestinal motility is reduced. The SNP or point mutation includes: the 21st nucleotide of the genome amplification fragment using SEQ ID NO: 9 and 10 as primers is A.

15. The device according to claim 14, characterized in that The SNP or point mutation is also selected from one or more of the following: The 20th nucleotide of the genomic amplification fragment using SEQ ID NO: 15 and 16 as primers is T, The 21st nucleotide of the genomic amplification fragment using SEQ ID NO: 17 and 18 as primers is T.

16. A method for screening a substance capable of killing the bacteria of claim 1, comprising: (1) contacting a candidate substance with the bacterium according to claim 1 or a culture thereof or a model containing said bacterium, (2) After contact, the number, activity, and / or intestinal motility of the bacteria are enhanced, which is an indication that the candidate substance is a target substance.

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