CN114540238B - Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof - Google Patents

Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof Download PDF

Info

Publication number
CN114540238B
CN114540238B CN202210231801.3A CN202210231801A CN114540238B CN 114540238 B CN114540238 B CN 114540238B CN 202210231801 A CN202210231801 A CN 202210231801A CN 114540238 B CN114540238 B CN 114540238B
Authority
CN
China
Prior art keywords
culture medium
culture
addition amount
clostridium difficile
amount
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210231801.3A
Other languages
Chinese (zh)
Other versions
CN114540238A (en
Inventor
王玉迎
詹晓北
徐星海
李志涛
高敏杰
蒋芸
张倚菲
张梓健
史诚文
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN202210231801.3A priority Critical patent/CN114540238B/en
Publication of CN114540238A publication Critical patent/CN114540238A/en
Application granted granted Critical
Publication of CN114540238B publication Critical patent/CN114540238B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a culture medium for separating clostridium difficile from intestinal microorganisms and a preparation method thereof, and belongs to the technical field of microorganisms. The culture medium contains glucose, maltose, yeast extract, peptone, L-cysteine, isovaleric acid, butyric acid, acetic acid, KH2PO4, naHCO3, naCl, mgSO4-7H2O, growth factors, pigments, and Clostridium difficile can be developed under the culture medium. The culture medium is combined with the fecal flora of the bionic gastrointestinal reactor to separate and culture, so that the real intestinal environment is simulated, and the survival rate of the drug-resistant intestinal microorganisms can be higher. This approach can be applied to recovery of specific flora, also highlighting the ability to culture recovered low abundance bacteria and reveal diversity. Obtaining a population of cultured human microorganisms will provide detailed functional properties of the bacteria, helping to find their biological activity during host bacteria and bacterial interactions.

Description

Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof
Technical Field
The invention relates to a culture medium for separating clostridium difficile from intestinal microorganisms and a preparation method thereof, and belongs to the technical field of microorganisms.
Background
The gastrointestinal microbiota is a highly diverse community, but most bacteria are considered to be non-culturable. Sequencing-based studies have shown that gastrointestinal microbiota has a greater diversity of microbial populations than previous culture methods. Thus, most studies describing human intestinal microorganisms rely on culture-independent sequencing methods. These studies provide new perspectives for the community composition of the intestinal microbiota of healthy individuals, the altered behavior of the gastrointestinal microbiota with environmental disturbances, and its potential role in various diseases. However, information obtained from molecular approaches alone has limitations, requiring isolation of organisms to determine the role of specific bacteria in causing or maintaining health and disease states. Traditional culture methods determine the number of living cells in a population, while most molecular methods do not distinguish between DNA obtained from living or dead cells. In addition, culture using selective media allows for the growth and detection of lower levels of bacteria that may be missed in molecular-based studies due to insufficient sequencing depth. Recent studies have attempted to characterize the culturable human gut microbiota by a combination of culture and next generation sequencing techniques, but at most 50% of gut microorganisms are culturable as detected by Operational Taxa (OTUs) of fecal sample 16S rRNA gene sequencing.
Clostridium difficile @Clostridium difficile) Is a gram-positive toxin-producing bacterium, first described as a commensal microorganism in the fecal flora of healthy newborns. Clostridium difficile is currently the most common cause of diarrhea and colitis. The symptoms of clostridia infection are not apparent to be the leading cause of increased morbidity and mortality in hospitalized patients. The risk of clostridia infection is associated with the use of broad-spectrum antibiotic therapy and an increase in patient age and hospitalization time. Human gastrointestinal microorganisms protect the host from colonization by exogenous pathogenic organisms, a function known as colonization resistance. The use of broad-spectrum antibiotics theoretically destroys this protective function of the local flora, allowing clostridium difficile to proliferate and colonize the gastrointestinal tract.
The invention provides a preparation method and application of a culture medium beneficial to clostridium difficile in-vitro growth. Most human intestinal flora may be enriched by capture by this culture medium, and clostridium difficile may be developed in this medium. This approach can be applied to recovery of specific flora, also highlighting the ability to culture recovered low abundance bacteria and reveal diversity. Obtaining a population of cultured human microorganisms will provide detailed functional properties of the bacteria, helping to find their biological activity during host bacteria and bacterial interactions.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method and application of clostridium difficile isolation medium. The Clostridium difficile isolation medium contains glucose, maltose, yeast extract, peptone, L-cysteine, isovaleric acid, butyric acid, acetic acid, KH 2 PO 4 、NaHCO 3 、NaCl、MgSO 4 -7H 2 O, growth factors, pigments.
The invention provides a culture medium for separating clostridium difficile in intestinal microorganisms, which comprises the following components in 1L of culture medium: glucose addition of 20-25 g, maltose addition of 8-12 g, yeast extract addition of 8-12 g, peptone addition of 13-17 g, L-cysteine addition of 0.4-0.6 g, isovaleric acid addition of 0.08-0.12 mL, butyric acid addition of 1.8-2.2 mL, acetic acid addition of 1.5-1.9 mL, KH 2 PO 4 The addition amount of the catalyst is 95-105 mL and NaHCO 3 The addition amount of (C) is 0.35-0.45 g, the addition amount of NaCl is 0.07-0.09 g, and MgSO is used 4 ~7H 2 The addition amount of O is 0.001-0.003 g, the addition amount of growth factor is 3.5-4.5 g, and the addition amount of pigment is 1.5-1.7 g.
In one embodiment of the invention, each 1L of medium comprises the following components in the following amounts: glucose addition amount of 20 g, maltose addition amount of 10 g, yeast extract addition amount of 10 g, peptone addition amount of 15 g, L-cysteine addition amount of 0.5 g, isovaleric acid addition amount of 0.1 mL, butyric acid addition amount of 2 mL, acetic acid addition amount of 1.7 mL, KH 2 PO 4 The addition amount of (2) is 100 mL and NaHCO 3 Is added in an amount of 0.4 g, naCl in an amount of 0.08 g, mgSO 4 -7H 2 The addition amount of O is 0.002 g, the addition amount of growth factor is 4 g, and pigmentThe addition amount of (2) was 1.7. 1.7 g.
In one embodiment of the present invention, the concentration of isovaleric acid is 50 mM, the concentration of butyric acid is 50 mM, the concentration of acetic acid is 1M, and the concentration of KH 2 PO 4 Is 5M.
In one embodiment of the invention, the growth factor is: lysine.
In one embodiment of the present invention, the pigment is: methylene blue.
In one embodiment of the present invention, when the culture medium is a solid culture medium, the amount of the agar powder added is 1.0 to 1.4%.
In one embodiment of the present invention, the amount of the agar powder added is 1.2%.
The invention also provides a method for isolating clostridium difficile from gut microorganisms, the method comprising the steps of:
(1) Preparing a fecal suspension;
(2) Freezing the fecal suspension:
(3) Inoculating the frozen fecal suspension obtained in the step (2) into the culture medium, and then statically incubating for 58-62 hours at 35-38 ℃ in an anaerobic incubator;
(4) Uniformly coating the culture obtained in the step (3) on a clostridium difficile separation agar medium, performing anaerobic culture at 35-38 ℃ for 45-50 hours, and then taking the thalli developed under fluorescence, inoculating the thalli into the clostridium difficile separation liquid medium, and performing anaerobic culture at 35-38 ℃ for 45-50 hours.
(5) Injecting the liquid culture obtained in the step (4) into an escherichia coli bioreactor filled with clostridium difficile isolation medium for isolation culture.
In one embodiment of the present invention, the step (1) is to dilute the stool sample in five gradients ten times with 900 μl of pre-reduced brain heart infusion medium and 0.05% cysteine in an anaerobic chamber.
In one embodiment of the present invention, the step (2) is performed by mixing 100. Mu.L of the solution obtained in the step (1)10 3 And 10 5 Dilutions were spread evenly on Brain Heart Infusion (BHI) broth 100 mm agar plates and incubated in an anaerobic chamber at 37 ℃ for 5 days. After incubation, 1 ml of BHI broth was added to the agar plate, the surface was scraped with a cell scraper, colonies were collected, frozen in pre-chilled 10% skim milk, and stored at-80 ℃.
In one embodiment of the present invention, step (3) is to inoculate the frozen fecal suspension obtained in step (2) in the above-mentioned culture medium, and then statically incubate 60 h in an anaerobic incubator at 37 ℃;
in one embodiment of the present invention, in step (4), the culture obtained in step (3) is spread uniformly on Clostridium difficile separated agar medium, and subjected to anaerobic culture at 37℃for 48 h. The cells developed under fluorescence were inoculated into Clostridium difficile isolation liquid medium, and subjected to anaerobic culture at 37℃for 48 h.
In one embodiment of the present invention, the step (5) is static culture: injecting the liquid culture obtained in the step (4) into a large intestine bioreactor filled with 150 mL clostridium difficile isolation medium, and keeping the large intestine bioreactor from peristaltic movement.
In one embodiment of the present invention, the step (5) is a dynamic culture: injecting the liquid culture obtained in the step (4) into a large intestine bioreactor filled with 150 mL clostridium difficile isolation medium, wherein peristaltic frequency is set to 3 times/min, and pH is controlled to be 6.5.
Advantageous effects
Currently, clostridium difficile is the most common cause of neonatal diarrhea and colitis, and the symptoms of infection are not obvious as the main cause of increased morbidity and mortality of hospitalized patients. The clostridium difficile separation medium is combined with the fecal flora of the bionic gastrointestinal reactor to separate and culture, so that the high survival rate of fecal microorganisms can be realized, and the identification and enrichment of harmful clostridium difficile can be realized. This approach can be applied to recovery of specific flora, also highlighting the ability to culture recovered low abundance bacteria and reveal diversity.
Detailed Description
Example 1: fecal microbiota culture
The method comprises the following specific steps:
(1) Preparation of fecal suspension
In an anaerobic chamber (condition: 37 ℃ C.), 0.1 g fecal samples were ten-fold diluted with 900. Mu.L of pre-reduced Brain Heart Infusion (BHI) medium and 0.05% cysteine in-situ, five gradients.
(2) 100 mu L of 10 obtained in the step (1) is added 3 And 10 5 Dilutions were spread evenly on Brain Heart Infusion (BHI) broth 100 mm agar plates and incubated in an anaerobic chamber at 37 ℃ for 5 days. After incubation, 1 ml of BHI broth was added to the agar plate, the surface was scraped with a cell scraper, colonies were collected, frozen in pre-chilled 10% skim milk, and stored at-80 ℃.
Example 2: isolation and identification of Clostridium difficile culture
The method comprises the following specific steps:
(1) Preparation of Clostridium difficile separation agar Medium
Table 1: clostridium difficile isolation Medium Components
The above-described formulation of Table 1 was used to obtain a culture medium for Clostridium difficile isolation, and when the culture medium was in a solid state, the amount of agar added was as follows: 12 g.
(2) Fecal samples stored at-80℃in example 1 were spread evenly on Clostridium difficile isolation agar medium and incubated anaerobically at 37℃for 48 h.
Since clostridium difficile develops color under fluorescent irradiation after growing in clostridium difficile isolation medium, the developed colonies grown on the plates are streaked on brain heart infusion solid medium to obtain purity.
(3) All cells on the plate after the end of the incubation in step (2) were scraped off with 1 mL PBS and then boiled for 15 minutes to lyse the cells. Colony PCR was performed using primers (sequences shown in Table 2) to amplify the 16S rRNA gene.
Table 2: primer(s)
Isolates were classified using an online RDP classifier using a 16S rRNA training set with a confidence threshold of 80%. The 16S rRNA gene sequence of this strain was compared with the Clostridium difficile consensus sequence in the obtained human microbiome project (Human Microbiome Projects, HMP).
The results indicated that all chromogenic colonies grown on the plates were Clostridium difficile.
Example 3: static culture and dynamic culture of infant intestinal flora in large intestine bioreactor
The cells developed under the fluorescence of example 2 were inoculated into a Clostridium difficile isolation liquid medium, and subjected to anaerobic culture at 37℃for 48℃ 48 h, whereby a culture was prepared.
Static culture: the culture was injected into a large intestine bioreactor containing 150 mL clostridium difficile isolation medium, keeping the large intestine bioreactor peristaltic free.
Dynamic culture: the culture was injected into a large intestine bioreactor containing 150 mL clostridium difficile isolation medium, peristaltic frequency was set to 3 times/min, and pH was controlled to 6.5.
The fermentation broth after static culture and dynamic culture needs to be subjected to biomass measurement:
collecting 20 mL fermentation broth 8000 g, centrifuging, collecting precipitate, washing with 75% alcohol for 2 times, centrifuging 8000 g to obtain thallus, oven drying at 80deg.C, and weighing.
The results show that: the OD value of the bacterial liquid after static culture is 2.1, and the dry weight of the bacterial body is 0.9 g; the OD value of the bacterial liquid after dynamic culture is 2.5, the dry weight of the bacterial body is 1.2 g, and the clostridium difficile can be well separated and enriched by utilizing the clostridium difficile separation culture medium based on dynamic culture and static culture of the escherichia coli reactor, so that the method is beneficial to more specifically detecting harmful bacteria in diarrhea and colon inflammation of infants.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (6)

1. A culture medium for isolating clostridium difficile from gut microorganisms, comprising the following components in amounts per 1L of culture medium:
glucose addition amount of 20 g, maltose addition amount of 10 g, yeast extract addition amount of 10 g, peptone addition amount of 15 g, L-cysteine addition amount of 0.5 g, isovaleric acid addition amount of 0.1 mL, butyric acid addition amount of 2 mL, acetic acid addition amount of 1.7 mL, KH 2 PO 4 The addition amount of (2) is 100 mL and NaHCO 3 Is added in an amount of 0.4 g, naCl in an amount of 0.08 g, mgSO 4 -7H 2 The addition amount of O is 0.002 g, the addition amount of growth factors is 4 g, and the addition amount of pigments is 1.7 g; the concentration of the isovaleric acid is 50 mM, and the concentration of the butyric acid is 50 mM; the concentration of the acetic acid is 1M, and the KH 2 PO 4 Is 5M, and the growth factors are: lysine; the pigment is as follows: methylene blue.
2. The culture medium according to claim 1, wherein the amount of the agar powder added is 1.0 to 1.4% when the culture medium is a solid culture medium.
3. The medium according to claim 2, wherein the agar powder is added in an amount of 1.2%.
4. A method for isolating clostridium difficile from an intestinal microorganism, comprising the steps of:
(1) Preparing a fecal suspension;
(2) Freezing the fecal suspension:
(3) Inoculating the frozen fecal suspension obtained in the step (2) in the culture medium according to any one of claims 1 to 3, and then statically incubating for 58 to 62 hours at 35 to 38 ℃ in an anaerobic incubator;
(4) Uniformly coating the culture obtained in the step (3) on the solid culture medium prepared by the culture medium of any one of claims 1-3, performing anaerobic culture at 35-38 ℃ for 45-50 hours, taking the thalli which develop color under fluorescence, inoculating the thalli into the liquid culture medium prepared by the culture medium of any one of claims 1-3, and performing anaerobic culture at 35-38 ℃ for 45-50 hours;
(5) Injecting the liquid culture obtained in the step (4) into a large intestine bioreactor filled with the culture medium according to any one of claims 1-3 for separation culture.
5. The method of claim 4, wherein the step (5) is: injecting the liquid culture obtained in the step (4) into a large intestine bioreactor filled with 150 mL of the culture medium according to any one of claims 1 to 3, and keeping the large intestine bioreactor free from peristaltic movement.
6. The method of claim 4, wherein the step (5) is: injecting the liquid culture obtained in the step (4) into a large intestine bioreactor filled with 150 of the culture medium according to any one of claims 1-3 of mL, wherein peristaltic frequency is set to 3 times/min, and pH is controlled to 6.5.
CN202210231801.3A 2022-03-10 2022-03-10 Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof Active CN114540238B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210231801.3A CN114540238B (en) 2022-03-10 2022-03-10 Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210231801.3A CN114540238B (en) 2022-03-10 2022-03-10 Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof

Publications (2)

Publication Number Publication Date
CN114540238A CN114540238A (en) 2022-05-27
CN114540238B true CN114540238B (en) 2023-08-08

Family

ID=81664233

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210231801.3A Active CN114540238B (en) 2022-03-10 2022-03-10 Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof

Country Status (1)

Country Link
CN (1) CN114540238B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105462900A (en) * 2015-12-30 2016-04-06 武汉艾米森生命科技有限公司 Use method of reagent for isolation and culture of clostridium difficile in faeces
CN105838774A (en) * 2016-05-31 2016-08-10 浙江省疾病预防控制中心 Clostridium difficile chromogenic medium and application thereof
CN107099481A (en) * 2017-06-15 2017-08-29 广西华银医学检验所有限公司 It is a kind of suitable for culture medium of clostridium difficile and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105462900A (en) * 2015-12-30 2016-04-06 武汉艾米森生命科技有限公司 Use method of reagent for isolation and culture of clostridium difficile in faeces
CN105838774A (en) * 2016-05-31 2016-08-10 浙江省疾病预防控制中心 Clostridium difficile chromogenic medium and application thereof
CN107099481A (en) * 2017-06-15 2017-08-29 广西华银医学检验所有限公司 It is a kind of suitable for culture medium of clostridium difficile and preparation method thereof

Also Published As

Publication number Publication date
CN114540238A (en) 2022-05-27

Similar Documents

Publication Publication Date Title
Balch et al. Acetobacterium, a new genus of hydrogen-oxidizing, carbon dioxide-reducing, anaerobic bacteria
CN104178437B (en) A kind of Lactobacillus crispatus and the application in gynaecopathia thereof
Ferrari et al. Isolation and characterization of Methanobrevibacter oralis sp. nov.
CN112574929B (en) Paenibacillus gibilinus YPG26 and medical application thereof
CN107541477B (en) Method for culturing photosynthetic bacteria by using lactobacillus fermentation liquor
CN113444664B (en) Lactobacillus brevis for producing gamma-aminobutyric acid and application thereof
CN111100821A (en) Streptococcus and application thereof
CN113403227A (en) Lactobacillus plantarum and preparation method and application thereof
CN109022313A (en) One lactobacillus plantarum
CN111454863A (en) Lactobacillus fermentum and application thereof in preparation of lactobacillus fermentation liquor with anti-aging function
CN109182181A (en) It is a kind of reduce livestock excrement composting ammonia volatilization high-temperature micro-aerobic compost keeping nitrogen bacterium and its application
CN114540238B (en) Culture medium for separating clostridium difficile in intestinal microorganisms and preparation method thereof
Crombach Caseobacter polymorphus gen. nov., sp. nov., a coryneform bacterium from cheese
Kim et al. Characterization of Bacillus polyfermenticus KJS-2 as a probiotic
Williams et al. Genetic transformation in Methylococcus capsulatus
CN101851664A (en) Method for detecting bifidobacterium in sour milk
CN113215067A (en) VBNC (viable but non-viable) state lactobacillus brevis CSHRR5-3 strain and application thereof
CN112553118A (en) Method for enriching beneficial bacteria by using prebiotics
CN116904332B (en) Propionibacterium acnes with good antioxidant effect and application thereof
CN104946555B (en) A kind of microbacterium of degradable zeranol and its application
CN112300962B (en) Lactobacillus plantarum with specific molecular target and antioxidation function and application thereof
Nuyanzina-Boldareva et al. Characterization of a new strain of a purple nonsulfur bacterium from a thermal spring
Omeonu et al. Phylogenetic Identification and Some Physiological Properties of Lactic Acid Bacteria Isolated from the Oral Cavities of Some Selected Students from University of Ibadan, Nigeria
ROACH et al. Anaerobic Fusiform‐shaped Bacteria Isolated from the Caecum of Conventional Mice
CN116333919A (en) Acid-resistant and bile-salt-resistant marine lactobacillus delbrueckii strain and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant