WO2017020783A1 - 脆弱拟杆菌在预防和/或治疗脑膜炎中的应用 - Google Patents

脆弱拟杆菌在预防和/或治疗脑膜炎中的应用 Download PDF

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WO2017020783A1
WO2017020783A1 PCT/CN2016/092380 CN2016092380W WO2017020783A1 WO 2017020783 A1 WO2017020783 A1 WO 2017020783A1 CN 2016092380 W CN2016092380 W CN 2016092380W WO 2017020783 A1 WO2017020783 A1 WO 2017020783A1
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bacteroides fragilis
meningitis
bacteroides
strain
bacterial
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PCT/CN2016/092380
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French (fr)
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智发朝
白杨
王晔
刘洋洋
王从峰
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广州知易生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to the technical field of microorganisms, medicines, health care products, foods and daily chemical products, in particular to the application of Bacteroides fragilis for preventing and/or treating meningitis.
  • Bacterial meningitis is a serious infectious disease of the central nervous system, which is common in adults but is most common in neonates. Because of the low immune function of newborns, imperfect leukocyte chemotaxis, adhesion and movement, complement activity and the concentration of specific and non-specific antibodies in vivo, it is a high risk group of bacterial meningitis.
  • Penicillin treatment of pneumococcal meningitis began in the mid-1940s, and the mortality rate of systemic and intrathecal penicillin was 49%.
  • Enterobacter Sakazakii is a newly discovered pathogen in dairy products. Crosonia sakazaki can cause severe neonatal meningitis, enterocolitis and bacteremia with a mortality rate of 40. %-80%.
  • Cronobacter can also cross the blood-brain barrier to the brain to cause meningitis. If it inhibits the entry of the Crohn's bacteria into the body's intestinal barrier, it can prevent it from entering the blood-brain barrier and prevent it from causing meningitis.
  • HBMEC human brain microvascular endothelial cells
  • Probiotics are a kind of active microorganisms beneficial to the host. They are the general term for active beneficial microorganisms that are colonized in the human intestines and reproductive system and can produce exact health effects to improve the balance of the micro-ecology of the host and play a beneficial role. It is resistant to symptoms, regulates intestinal micro-ecology, and enhances its own immunity. Now more and more researchers are focusing on probiotics and gradually realize their great therapeutic effects.
  • probiotics After ingesting humans and animals, probiotics can colonize the intestinal mucosa, colonize the intestines and prevent harmful microbes from adhering to them, and promote the formation of healthy individuals by maintaining the natural microflora in the intestines.
  • a growable microbial preparation that helps maintain the health of people and animals.
  • Bacteroides fragilis is an obligate anaerobic bacterium that is negative for Gram stain, rod-shaped, blunt and densely stained at both ends, and has capsules, no spores, and no motility. It is divided into enterotoxin-producing and non-productive intestines. Toxin type. Bacteroides fragilis is part of the normal flora of human and animal gut, mainly in the colon. In addition, the respiratory tract and genitourinary tract can also grow. Head Previously, more research has been done on Bacteroides fragilis as a conditional pathogen.
  • the host mucosa When the host mucosa is damaged, it can invade the submucosa, cause infection, and can also flow through the blood, causing the body's organs such as the intestines, abdominal cavity, and liver. Pulmonary and brain tissue purulent infection with abscess and acute and chronic diarrhea; in addition, Bacteroides fragilis also promotes the occurrence of colon and rectal cancer.
  • Bacteroides fragilis A large number of studies have been conducted in the field on Bacteroides fragilis.
  • a Bacteroides strain BF839 is isolated from the well-developed infant or the intestine of a younger animal. It can be used as a live bacterial preparation to increase the growth and development of children, to prevent acute and chronic enteritis, dysbacteriosis, upper respiratory tract infection and nerve. It has good curative effect (see application number "90102847.9", the name is "a beneficial strain and its application” Chinese invention patent application; Zhang Lijie, et al. Clinical application of Bacteroides fragilis (BF839) bacterial liquid. Chinese Journal of Biological Products, 1995, Vol. 8, No. 2, pp. 63-65)).
  • the application number is “201310095126.7”, the name is “Bacteroides fragilis with probiotic characteristics”; the application number is “201310085744.3”, the name is “Application of Bacteroides fragilis in the preparation of acute radiation-induced enteritis composition”; No. 201310085716.1, entitled “Bacteroides fragilis” disclosed in the Chinese Patent Application entitled “Application of Bacteroides fragilis in the preparation of a composition for treating inflammatory bowel disease", which was isolated from infant feces in 2012.
  • the probiotic Bacteroides strain (deposited by CGMCC No. 7280) can be used for the treatment of inflammatory bowel disease, diarrhea and the like.
  • the technical problem to be solved by the present invention is to provide a kind of application of Bacteroides fragilis in the prevention and/or treatment of meningitis by detecting and identifying the action of Bacteroides fragilis in various disease models, and can have good effect on meningitis. Therapeutic and / or preventive effects.
  • the present invention provides a use of a Bacteroides fragilis for the preparation of a medicament for preventing and/or treating meningitis.
  • Bacteroides fragilis application wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312, and the Bacteroides fragilis ZY-312 was deposited on the Chinese General Microorganisms Collection and Management Center on April 2, 2015, and its deposit number For CGMCC No.10685.
  • the present invention also provides a pharmaceutical composition for preventing and/or treating meningitis, wherein the pharmaceutical composition comprises a pharmaceutically effective dose of Bacteroides fragilis ZY-312, which is fragile Bacteroides ZY-312 was deposited with the General Microorganisms Collection and Management Center of China on April 2, 2015, and its deposit number is CGMCC No.10685.
  • composition described above wherein the pharmaceutical composition is a capsule, a solution, a suspoemulsion, a bagged powder or a granule, and each single dose of the Bacteroides fragilis ZY-312 strain is 106-1011 cells.
  • the present invention also provides a food for preventing and/or treating meningitis, wherein the food contains Bacteroides fragilis ZY-312, the Bacteroides fragilis ZY-312 in 2015 It was deposited on April 2nd at the China General Microorganisms Collection and Management Center, and its deposit number is CGMCC No.10685.
  • the present invention also provides a health care product for preventing and/or treating meningitis, wherein the health care product comprises Bacteroides fragilis ZY-312, and the Bacteroides fragilis ZY-312 is It was deposited with the General Microorganisms Collection and Management Center of China on April 2, 2015, and its deposit number is CGMCC No.10685.
  • the present invention also provides a food additive for preventing and/or treating meningitis, wherein the food additive comprises Bacteroides fragilis ZY-312, and the Bacteroides fragilis ZY-312 is It was deposited with the General Microorganisms Collection and Management Center of China on April 2, 2015, and its deposit number is CGMCC No.10685.
  • the Bacteroides fragilis of the present invention has a good effect on preventing and/or treating meningitis, does not produce drug resistance, and is safe and non-toxic, and provides a new choice for treating and/or preventing meningitis.
  • the Bacteroides fragilis ZY-312 of the present invention is a new Bacteroides fragilis which is isolated and purified from well-developed infant feces and is a new strain of Bacteroides fragilis having probiotic characteristics.
  • the isolated Bacteroides fragilis ZY-312 does not contain the enteric toxin gene bft (Bacteroides fragilis toxin). Compared with the existing strains, it has probiotic properties such as bile salt resistance and gastric acid resistance, and has broad application prospects.
  • FIG. 1 is a diagram showing the colony morphology of Bacteroides fragilis ZY-312 after anaerobic culture
  • Figure 2 is a Gram stained microscopic examination map of Bacteroides fragilis ZY-312 of the present invention (1000 ⁇ );
  • Figure 3 is a scanning electron microscope observation view of Bacteroides fragilis ZY-312 of the present invention (30000 ⁇ );
  • Figure 4 is a comparison diagram of gel electrophoresis results of the PCR product of the present invention.
  • Figure 5 is a comparison diagram of gel electrophoresis results of PCR products of the present invention.
  • Figure 6 is a screenshot of a phylogenetic tree established from a genome-wide sequence comparison
  • Fig. 7 is a graph showing the effect of inhibition of ZY-312 on K. sakazakii of the present invention.
  • Bacteroides fragilis named ZY-312
  • CGMCC China General Microorganisms Collection and Management Center
  • the deposit address is Beijing Chaoyang. No. 3, No. 1 Courtyard, Beichen West Road, District.
  • Embodiments of the present invention include: the present invention screens a large number of Bacteroides fragilis strains by screening a large amount of feces from healthy infants, and identifies a new Bacteroides fragilis, named ZY, by physical and chemical experiments. -312, compared with the existing Bacteroides fragilis strains, it has probiotic properties such as bile-tolerant salt and gastric acid resistance, which can make up for some defects of the original beneficial bacteria, and still maintain the bile salts and acidic conditions in the digestive tract. Highly biologically active, it is a preferred strain of probiotic products.
  • the Bacteroides fragilis ZY-312 was deposited with the China General Microorganisms Collection and Management Center (CGMCC) on April 2, 2015, and its deposit number is CGMCC No.10685.
  • the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing. number 3.
  • a pharmaceutical composition can be prepared using Bacteroides fragilis ZY-312.
  • the pharmaceutical composition contains a pharmaceutically effective amount of Bacteroides fragilis ZY-312.
  • the pharmaceutical composition may also contain a suitable pharmaceutical carrier.
  • the pharmaceutical composition may be in the form of a capsule, a solution or a drinkable suspoemulsion, a bagged powder, a granule, or the like, and each single dose is preferably about 106-1011 cells of the Bacteroides fragilis ZY-312 strain.
  • the Bacteroides fragilis ZY-312 of the present invention can also be made into foods, health care products or food additives.
  • the food, health care product or food additive contains Bacteroides fragilis ZY-312. These foods, health supplements or food additives can be used to prevent and treat neonatal meningitis.
  • compositions, foods, health supplements and food additives containing the Bacteroides fragilis for preventing and/or treating meningitis or the Bacteroides fragilis of the present invention are administered to a subject, All of which can be applied to the indications described above and exhibit the functions described above, all dosage forms within the scope of the invention have been tested, hereinafter, merely for illustration, only described in the examples A few of them are not to be construed as limiting the invention.
  • Electrophoresis instrument Beijing Liuyi Instrument Factory, model: DYCP-32B
  • Taq enzyme (TaKaRa, article number: DR100A)
  • Vitamin K1 (Qingdao Rishui Biotechnology Co., Ltd., Item No.: 21005)
  • Enterotoxin-producing strain of Bacteroides fragilis (provided by Sun Yong, Department of Gastroenterology, Southern Hospital, isolated from patients with clinical diarrhea)
  • Bacteroides fragilis standard strain ATCC25285 (purchased from Guangdong Institute of Microbiology)
  • Bacteroides fragilis strain Bd312 (the deposit number is CGMCC No.7280, provided by Guangzhou Zhiguang Biotechnology Co., Ltd.)
  • BF839 strain isolated from totem probiotics.
  • Medium C configuration Weighed 28.1 g of Brucella broth medium, dissolved in 1000 mL of distilled water with heating and stirring, and filled in a triangular flask, and autoclaved at 121 ° C for 15 minutes, and set aside. Add 5% fetal calf serum before use.
  • Brinell broth configuration Weigh 24.1g of Brucella broth culture medium, stir it in 1000mL distilled water with heating and stirring, dispense triangle flask, autoclave at 121 °C for 15 minutes, and set aside.
  • Fresh baby feces 0.5 g were taken and placed in a triangular flask containing 4.5 mL of Brucella broth and shaken for 1 minute. 0.1 mL was dripped on the medium, streaked, placed in an anaerobic tank, and cultured at 37 ° C for 48 hours. A typical colony was picked up in Brucella broth for 24 h for Gram staining. The morphology was observed under a microscope, and the bacterial solution of Gram-negative bacteria was selected, streaked into a blood plate, and cultured for 48 hours under anaerobic conditions.
  • Bacteroides fragilis ZY-312 was cultured on a blood plate for 48 hours, showing a round micro-convex, translucent, white, smooth surface, no hemolysis, colony diameter of 1-3mm, see Figure 1.
  • B. fragilis ZY-312 was subjected to Gram staining microscopy. It was a Gram-negative bacterium with a typical rod shape. Both ends were blunt and densely stained. The uncolored part of the cells was shaped like a vacuole. See Figure 2.
  • the fixative was fixed and observed by scanning electron microscopy. Microscopically, the Bacteroides fragilis ZY-312 is 0.5 to 0.8 ⁇ 1 to 4.5 ⁇ m in size, without flagella, and without spores. See Figure 3.
  • reaction substrate Tryptophan - Urea - glucose + Mannitol - lactose + sucrose + maltose + Salicyl alcohol - Xylose +
  • API20A Biochemical Reaction Identification Plate, French BioMerieux Co., Ltd.
  • Bacteroides fragilis ZY-312 can ferment glucose, lactose, sucrose, maltose, xylose, escin, mannose, raffinose , in line with the characteristics of Bacteroides fragilis.
  • PCR primers Synthesized by Infineon (Shanghai) Trading Co., Ltd. have the following sequence:
  • Reverse primer 5'-GCTTAGAGTTTGATCCTGGCTCAG-3'
  • Reverse primer 5'-CGCCCAGTATATGACCTAGTTCGTG-3'
  • Reverse primer 5'-ATCCCTAAGATTTATTATCCCAAGTA-3'
  • PCR identification is a commonly used method for rapidly amplifying genes
  • the above strain was inoculated on medium A, and cultured at 37 ° C for 48 hours under anaerobic conditions.
  • a single strain was inoculated into a liquid medium, and cultured at 37 ° C for 48 hours.
  • the DNA extraction kit extracts bacterial DNA (Tiangen Biochemical Technology (Beijing) Co., Ltd., article number: DP302-02) as a PCR template DNA.
  • primer pair 1 amplified fragment size is about 531 bp; primer pair 2 amplified fragment size is 518 bp; primer pair 3 amplified fragment size is about 970 bp.
  • a 20 ⁇ L PCR reaction system was used: 10 ⁇ L of Taq enzyme, 2 ⁇ L of template DNA, 1 ⁇ L of each of the forward and reverse primers, and 6 ⁇ L of sterile deionized water.
  • the PCR reaction conditions were: pre-denaturation at 95 ° C for 5 min, denaturation at 95 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 72 ° C for 45 s, 30 cycles, and extension at 72 ° C for 10 min.
  • the PCR product was electrophoresed on a 2% agarose gel under electrophoresis conditions of 100 V for 15 min.
  • lanes 1 and 2 are primer pair 1 and primer pair 2 respectively; lanes 4 and 5 are primer pair 1 and primer pair 2 respectively (result of repeated PCR); 3, 6
  • the lane is the primer pair 3; the lane 8 is the DNA molecular weight standard (DL1000 DNA marker).
  • the isolated strain DNA was amplified by PCR with primer pair 1 and the product size was 531 bp. After PCR amplification with primer pair 2, the product size was 518 bp. After PCR amplification with primer pair 3, the product size was 970 bp, which was in line with expectations.
  • the strain is Bacteroides fragilis.
  • the PCR product was subjected to nucleotide sequence determination (determined by Shenzhen Huada Gene Technology Co., Ltd.), and the sequencing results were subjected to BLAST alignment on Genbank (DNA sequence database established by National Biotechnology Information Center) (http://www). .ncbi.nlm.nih.gov/BLAST/), see Table 4.
  • Table 4 shows the BLAST alignment results of the 16S rRNA sequence (partial)
  • the strains screened by the sequencing were inoculated into the medium C, and cultured at 37 ° C for 48 hours under anaerobic conditions. 2 mL of the culture solution was taken, and DNA was extracted using a bacterial DNA extraction kit as a PCR template DNA.
  • the bft gene is amplified using the bft gene primer, and the amplified fragment size should be 294 bp.
  • a 20 ⁇ L PCR reaction system was used: 10 ⁇ L of Taq enzyme, 2 ⁇ L of template DNA, 1 ⁇ L of each of the upper and lower primers, and 6 ⁇ L of sterile deionized water.
  • the PCR reaction conditions were: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s for 30 cycles and extension at 72 °C for 10 min.
  • the PCR product was subjected to 2% agarose gel electrophoresis, and the electrophoresis conditions were 100 V for 15 min.
  • the results are shown in Fig. 5.
  • the lanes 1, 2, 3 and 4 are the electrophoresis results of the isolated strain ZY-312; the 5, 6, and 7 are the electrophoresis results of the enterotoxin-producing strain Bacteroides; the lane 8 is the DL1000 DNA marker.
  • Lanes 4 and 5 are amplification products of bft gene primer pair; lanes 1 and 7 are primer pair 2 amplification products; 2, 6 are primer pair 1 amplification products; and 3 lanes are primer pair 3 amplification products.
  • ZY-312 was a strain of Bacteroides fragilis and did not contain the enterotoxin bft gene, which was a new avirulent strain.
  • the phylogenetic tree shows (see Figure 6) that Bacteroides fragilis ZY-312 is on the same branch as Bacteroides fragilis standard strain ATCC25285 (NCTC9343), indicating that Bacteroides fragilis ZY-312 is a new Bacteroides fragilis, and ATCC25285 is homologous.
  • Virulence gene analysis was performed on the whole genome sequencing results to verify whether it contains the toxin-producing bft gene. The results showed that the whole genome of Bacteroides fragilis ZY-312 does not contain the bft gene, which is a new Bacteroides fragilis that does not produce enterotoxin.
  • the bacterial liquid was collected, centrifuged, the supernatant was discarded, and the physiological saline was resuspended, centrifuged again, and the supernatant was discarded, and the cells were reserved.
  • the live bacteria count was performed by 10-fold serial dilution method: 100 ⁇ L of bacterial liquid was added to 900 ⁇ L of Brucella broth medium, and gradually Dilute to the appropriate concentration. For example, each plate point has 4 concentration gradients, and each gradient is repeated 3 times, each time 20 ⁇ L is spotted. 37 ° C, anaerobic culture for 48 h, the number of colonies (counting the number of colonies counted from 3-30).
  • Viable count sum of three spotted colonies / 3 ⁇ 50 ⁇ dilution
  • Probiotics must enter the gastrointestinal tract of the human body and reach a certain concentration in order to function. Probiotics must first pass through the stomach in the state of live bacteria to enter the intestines from the oral cavity to the intestinal tract. The time that food (especially fluid) passes through the stomach is typically 1-2 hours (hours). According to the different diet structure, the pH value of human gastric juice fluctuates greatly, usually at pH 3.0, which can reach pH 1.5 when fasting or eating acidic food, and up to pH 4-5 when eating alkaline food. The acidic environment activates pepsinogen, which kills bacteria that enter the stomach with food. If probiotics are to have a probiotic effect in the human body, they must have certain acid resistance and pepsin resistance.
  • Tryptone soy broth (TSB for short, brand: OXOID, article number: CM0129B)
  • TSA Tryptone soy agar
  • Bile powder solution Add bovine bile powder to TSB and set three final concentrations of 10g/L (1% bovine powder), 20g/L (2% bovine powder) and 40g/L (4% bovine). powder). After sterilization, serum (final concentration 50 mL/L) was added for use. At the same time, TSB without bovine powder was used as a control.
  • strain culture and collection strain (ZY-312, Bd312, BF839, ATCC25285) at 37 ° C, anaerobic liquid
  • the cells were statically cultured to the late stage of logarithmic growth (about 14-16 hours), dispensed into a centrifuge tube, and each tube was dispensed with 3 ml of bacterial solution, and centrifuged at 4000 rpm for 5 minutes at room temperature.
  • the bacteria were washed once with 0.01 M PBS (centrifugation at room temperature, 4000 rpm for 5 minutes), the supernatant was discarded, and the precipitate was used.
  • the washed bacteria were resuspended with the above bile powder solution, and the initial bacterial solution concentration was adjusted to 1 ⁇ 108 CFU/mL with the bile powder-containing solution.
  • anaerobic culture for 1, 2, 4 hours the number of viable bacteria was counted by plating, and the number of bacteria at 0 hours was used as a control. The experiment was done 3 times in parallel.
  • Bile salt is a sodium or potassium salt formed by the binding of bile acids secreted by hepatocytes to glycine or taurine, which is involved in bile The main ingredient for digestion and absorption. After the bile salt is discharged into the small intestine, most of it is absorbed into the blood by the small intestinal mucosa, and then enters the liver to form bile.
  • the mass concentration of bile salts in the human small intestine fluctuates in the range of 0.03 to 0.3 g/100 mL.
  • bile salts can destroy cell membranes, so tolerance to bile salts is one of the important indicators for evaluating probiotics.
  • Probiotics produce bile salt hydrolase, which catalyzes the hydrolysis of glycine and taurine-bound bile salts to amino acid residues and free bile salts. Strains with bile salt dissociation ability can lower serum cholesterol levels in high cholesterol populations and prevent hypercholesterolemia in normal people.
  • the concentration of bile salts in the digestive tract is not fixed. At the beginning of the digestion and digestion, the mass concentration is 15-20 g/L, and then the mass concentration is reduced to about 3 g/L.
  • Probiotics must survive normal biliary concentrations during passage through the gastrointestinal tract. To colonize the small intestine, they must be resistant to bile salts. Therefore, ZY-312 has a better application prospect than other strains of Bacteroides fragilis.
  • Colonic adenocarcinoma cancer cell Caco-2 is preserved by the Department of Microbiology, Southern Medical University;
  • K. sakazakii ATCC29544 is a standard strain purchased from ATCC (ie American type culture collection);
  • Bacillus E44 and DH5 ⁇ are preserved by the Department of Microbiology, Southern Medical University.
  • MRS lactic acid bacteria medium
  • BHI brain heart infusion broth medium
  • LB bacterial basic medium
  • MRS lactic acid bacteria medium
  • BHI brain heart infusion broth medium
  • LB bacterial basic medium
  • MRS lactic acid bacteria medium
  • BHI brain heart infusion broth medium
  • LB bacterial basic medium
  • fetal bovine serum Stee Fei Shier Technology Co., Ltd., brand: Gbico
  • sterile degreased sheep blood purchased from Nanjing Buick Biotechnology Co., Ltd.
  • the experiment was divided into four groups, which were control group, competition group, replacement group and exclusion group.
  • the control group Caco-2+ATCC29544 was incubated for 3 h; the competition group: Caco-2+ATCC 29544+ZY-312 was incubated for 3 h; the replacement group: Caco-2+ATCC29544 was incubated for 1 h, then ZY-312 was added, and incubation was continued for 2 h.
  • the rejection group Caco-2+ZY-312 was incubated for 1 h, then ATCC 29544 was added, and incubation was continued for 2 h.
  • the cells were washed 3 times with the medium, and 100 ⁇ L of 0.5% Triton X-100 was added to lyse the cells (this concentration of Triton X-100 did not affect the bacterial activity within half an hour), and after incubation for 8 minutes, 50 ⁇ L of distilled water was added immediately. Monolayer cells remain intact during the incubation and wash phases prior to lysis of the cells.
  • mice SD rats were treated with 12 litters of 2-3 days old, 80 rats, and they were in good mental state. They were purchased from the Experimental Animal Center of Southern Medical University.
  • Table 7 is the grouping and treatment methods of animal experiments, in which PBS is phosphate. Buffer.
  • Table VIII is the prevention and treatment effect of ZY-312 on meningitis caused by Cronobacter sakazakii, in which the definition of pathogenic bacteria on the cerebrospinal fluid plate For meningitis.
  • Bacteroides fragilis of the present invention has a good effect on preventing and/or treating meningitis, does not produce drug resistance, and is safe and non-toxic, and provides a new choice for treating and/or preventing meningitis.
  • Bacteroides fragilis ZY-312 of the present invention is a new Bacteroides fragilis which is isolated and purified from well-developed infant feces and is a new strain of Bacteroides fragilis having probiotic characteristics.
  • the isolated Bacteroides fragilis ZY-312 does not contain the enterotoxin gene bft. Compared with the existing strains, it has probiotic properties such as bile salt resistance and gastric acid resistance, and has broad application prospects.

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Abstract

提供了脆弱拟杆菌在预防和/或治疗脑膜炎中的应用，具体涉及脆弱拟杆菌在制备用于预防和/或治疗脑膜炎的药物、药物组合物、食品、保健品及食品添加剂中的应用，所述脆弱拟杆菌为脆弱拟杆菌ZY-312，该脆弱拟杆菌ZY-312于2015年4月2日保藏于中国普通微生物菌种保藏管理中心，其保藏号为CGMCC No.10685。

Description

脆弱拟杆菌在预防和/或治疗脑膜炎中的应用 技术领域
本发明涉及微生物、药品、保健品、食品及日化产品技术领域，特别是涉及脆弱拟杆菌用于预防和/或治疗脑膜炎的应用。
背景技术
细菌性脑膜炎是中枢神经系统严重的感染性疾病，常见于成人，但以新生儿患者最多。因为新生儿免疫功能低下，白细胞趋化、黏附和移动功能不完善，补体活性以及体内特异性和非特异性抗体的浓度都比较低，所以是细菌性脑膜炎的高危人群。
20世纪30年代，磺胺药的问世将球菌性脑膜炎的病死率降至5％-15％。到1944年，采用磺胺和静脉注射兔抗血清治疗了87例b型流感嗜血杆菌性脑膜炎患儿，将病死率降至22％。20世纪60年代初期，氯霉素(加磺胺嘧啶)将流感嗜血杆菌性脑膜炎的病死率进一步降至5％-10％，使用抗血清治疗从此成为了历史。但磺胺对肺炎球菌性脑膜炎的疗效较差，病死率在45％-95％之间波动。
青霉素治疗肺炎球菌性脑膜炎始于20世纪40年代中期，全身和鞘内应用青霉素的病死率为49％。1949年道林(Dowling)采用大剂量青霉素(每2小时肌肉注射100万单位)治疗了21例肺炎球菌性脑膜炎病人，将病死率降至38％，开创了青霉素未经鞘内给药而达到“脑脊膜剂量”的现代疗法新时代。
在过去15年内，细菌性脑膜炎的治疗方法为静脉注射青霉素(或氨苄青霉素)和/或第3代头孢菌素。此时，脑膜炎球菌性脑膜炎的病死率徘徊于10％左右，流感嗜血杆菌性脑膜炎的病死率已降至5％以下，肺炎球菌性脑膜炎的病死率仍保持在20％左右。贝克(Beek)等通过大量荷兰病例证实了这一结果：脑膜炎球菌性脑膜炎的病死率为7％，肺炎球菌性脑膜炎的病死率为30％。
当脑脊液(CSF)内的细菌成分可引起炎性细胞因子释放的现象被揭示后，人们开始开展地塞米松辅助治疗的研究。在20世纪90年代初期进行的4项研究证明，地塞米松并不能改变流感嗜血杆菌性脑膜炎患儿的病死率，但可降低神经系统后遗症的发生率，主要是感觉神经性耳聋。2002年，荷兰科学家证明，地塞米松辅助治疗可使成年病人的后遗症发生率从25％降至15％，其中以肺炎球菌性脑膜炎的预后最好。
近30年来，尽管在各种新型有效抗生素不断出现以及现代医疗技术飞速发展的背景 下，细菌性脑膜炎急性期的病死率有所下降，但神经系统后遗症的发生率仍居高不下。
在抗生素时代，细菌性脑膜炎病原菌对抗生素耐药性的问题将长期存在。例如，20世纪60年代出现了脑膜炎奈瑟菌对磺胺的耐药，20世纪70年代出现了流感嗜血杆菌对氨苄青霉素的耐药；20世纪90年代从美国脑膜炎病人分离出的肺炎链球菌菌株对青霉素耐药(中度耐药占21％，高度耐药占14％)，因此不得不联合应用第3代头孢菌素和万古霉素进行治疗。
但是抗生素在杀死病原菌的同时，会造成机体菌群失调、抗性的出现及免疫功能的下降。而菌群失调会导致便秘、急慢性腹泻、肠胃炎、肠功能紊乱，以及外来菌感染等疾病。长期下去，还可导致其他疾病并促进衰老，甚至引起癌症。
阪崎克罗诺杆菌(EnterobacterSakazakii)是乳制品中近年新发现的一种致病菌，阪崎克罗诺杆菌能引起严重的新生儿脑膜炎、小肠结肠炎和菌血症，死亡率高达40％-80％。
克罗诺杆菌传播入血需首先穿越肠屏障，进而引起肠菌血症，而肠上皮细胞是该屏障重要组成部分，是克罗诺杆菌侵入机体的门户。克罗诺杆菌还能穿越血脑屏障到达脑部引起脑膜炎，如果通过抑制克罗诺杆菌进入机体的肠屏障，则可以阻止其进入血脑屏障，并进而防止其引发脑膜炎。
阪崎克罗诺杆菌跨越人体血脑屏障最终引发脑膜炎有几个关键性的环节：高浓度的菌血症、细菌黏附、侵袭人脑微血管内皮细胞(HBMEC)、诱导HBMEC的细胞骨架重排及相关信号通路的激活，最终成功穿越HBMEC。HBMEC是人体血脑屏障的主要组成部分，黏附于细胞表面是细菌进入HBMEC的第一步，然后通过“拉链”(Zipper)机制进入细胞，并以空泡的形式穿越HBMEC。
益生菌是一类对宿主有益的活性微生物，是定植于人体肠道、生殖系统内，能产生确切健康功效从而改善宿主微生态平衡、发挥有益作用的活性有益微生物的总称，其具有缓解乳糖不耐症、调整肠道微生态、增强自身免疫力等多种功效，目前越来越多的研究人员聚焦于益生菌，逐渐意识到它们的巨大治疗功效。
益生菌在摄入人和动物体内后，它们能够在肠粘膜上定居，在肠道内建群并且防止有害微生物在其上粘附，能通过保持肠内自然微生物区系，促进生物个体形成健康的可生长的微生物制剂，帮助维持人和动物的健康。
脆弱拟杆菌是一种革兰氏染色阴性、杆状、两端钝圆而浓染，有荚膜、无芽胞、无动力的专性厌氧细菌，其分为产肠毒素型和非产肠毒素型。脆弱拟杆菌作为人及动物肠道正常菌群的一部分，主要存于结肠中，此外，呼吸道胃肠道及泌尿生殖道也可定植生长。目 前，研究较多的是脆弱拟杆菌作为一种条件致病菌，当宿主粘膜受损时，可侵犯粘膜下层，引起感染，也可经血液流动，引起身体其器官如肠道、腹腔、肝、肺、脑组织化脓性感染并伴发脓肿和引起急慢性腹泻等症状；此外，脆弱拟杆菌对结肠、直肠癌的发生也有促进作用。
本领域已经对脆弱拟杆菌进行了大量研究。例如，从发育良好的婴儿或低龄动物肠道中分离出一种拟杆菌菌株BF839，将其制成活菌制剂后能够增加儿童生长发育，对防治急慢性肠炎、菌群失调、上呼吸道感染和神经官能症等具有较好疗效(参见申请号为“90102847.9”，名称为“一株有益菌株及其应用”的中国发明专利申请；张李阶，等.脆弱拟杆菌(BF839)菌液的临床应用研究.中国生物制品学杂志，1995年，第8卷，第2期，第63-65页))。
再如，申请号为“201310095126.7”，名称为“具有益生菌特性的脆弱拟杆菌”；申请号为“201310085744.3”，名称为“脆弱拟杆菌在制备治疗急性放射性肠炎组合物中的应用”；申请号为“201310085716.1”，名称为“脆弱拟杆菌在制备治疗炎症性肠病组合物中的应用”的中国发明专利申请所公开的脆弱拟杆菌，为在2012年从婴儿粪便中分离出一种具有益生菌特性的脆弱拟杆菌菌株(保藏编号为CGMCC NO.7280)，可用于治疗炎症性肠病、腹泻等。此外，通过对该菌株的进一步鉴定，发现该菌株(Bd312)在细菌形态、培养特性、生理生化反应结果与脆弱拟杆菌相似，经BLASTN序列比对，所分离菌株与脆弱拟杆菌标准株ATCC25285同源性达99％，药敏实验提示，菌株Bd312对头孢拉定、阿莫西林、庆大霉素、磺胺甲嗯唑、甲氧苄啶不敏感，急慢性毒性试验提示无毒性(刘洋洋，等.健康婴儿体内的无毒脆弱拟杆菌的分离及鉴定.中华医学杂志，2014年，第94卷，第30期，第2372-2374页)。然而，目前还没有关于利用脆弱拟杆菌治疗脑膜炎的文献记载。
发明内容
本发明所要解决的技术问题是通过在多种疾病模型中对脆弱拟杆菌的作用进行检测和鉴定，提供一种脆弱拟杆菌在预防和/或治疗脑膜炎中的应用，能对脑膜炎具有良好的治疗和/或预防效果。
为了实现上述目的，本发明提供了了一种脆弱拟杆菌在制备用于预防和/或治疗脑膜炎的药物中的应用。
上述的脆弱拟杆菌应用，其中，所述脆弱拟杆菌为脆弱拟杆菌ZY-312，该脆弱拟杆菌ZY-312于2015年4月2日保藏于中国普通微生物菌种保藏管理中心，其保藏号为CGMCC  No.10685。
上述的脆弱拟杆菌应用，其中，所述脑膜炎为新生儿脑膜炎。
上述的脆弱拟杆菌应用，其中，所述脑膜炎为细菌性脑膜炎。
上述的脆弱拟杆菌应用，其中，所述新生儿脑膜炎为细菌性脑膜炎。
为了更好地实现上述目的，本发明还提供了一种用于预防和/或治疗脑膜炎的药物组合物，其中，所述药物组合物包括药学有效剂量的脆弱拟杆菌ZY-312，该脆弱拟杆菌ZY-312于2015年4月2日保藏于中国普通微生物菌种保藏管理中心，其保藏号为CGMCC No.10685。
上述的药物组合物，其中，所述药物组合物为胶囊、溶液、悬乳液、袋装粉剂或颗粒剂，每一单一剂量的所述脆弱拟杆菌ZY-312菌株为106-1011细胞。
为了更好地实现上述目的，本发明还提供了一种用于预防和/或治疗脑膜炎的食品，其中，所述食品含有脆弱拟杆菌ZY-312，该脆弱拟杆菌ZY-312于2015年4月2日保藏于中国普通微生物菌种保藏管理中心，其保藏号为CGMCC No.10685。
为了更好地实现上述目的，本发明还提供了一种用于预防和/或治疗脑膜炎的保健品，其中，所述保健品含有脆弱拟杆菌ZY-312，该脆弱拟杆菌ZY-312于2015年4月2日保藏于中国普通微生物菌种保藏管理中心，其保藏号为CGMCC No.10685。
为了更好地实现上述目的，本发明还提供了一种用于预防和/或治疗脑膜炎的食品添加剂，其中，所述食品添加剂含有脆弱拟杆菌ZY-312，该脆弱拟杆菌ZY-312于2015年4月2日保藏于中国普通微生物菌种保藏管理中心，其保藏号为CGMCC No.10685。
本发明的技术效果在于：
本发明的脆弱拟杆菌对于预防和/或治疗脑膜炎具有很好的效果，不产生耐药性，且安全无毒，为治疗和/或预防脑膜炎提供了新的选择。此外，本发明的脆弱拟杆菌ZY-312是一株新的脆弱拟杆菌，该菌株是从发育良好的婴儿粪便中分离纯化得到的，是具有益生菌特性的脆弱拟杆菌新菌株，实验表明所分离到的脆弱拟杆菌ZY-312不含肠毒素基因bft(Bacteroides fragilis toxin)，与现有菌株相比，具有耐胆盐、耐胃酸等益生特性，具有广阔的应用前景。
以下结合附图和具体实施例对本发明进行详细描述，但不作为对本发明的限定。
附图说明
图1为本发明的脆弱拟杆菌ZY-312厌氧培养后菌落形态图；
图2为本发明的脆弱拟杆菌ZY-312革兰氏染色镜检图(1000×)；
图3为本发明的脆弱拟杆菌ZY-312扫描电镜观察图(30000×)；
图4为本发明的PCR产物凝胶电泳结果对比图；
图5为本发明的PCR产物凝胶电泳结果对比图；
图6为根据全基因组序列比较建立的系统发育树截图；
图7为本发明的ZY-312对阪崎克罗诺杆菌的抑制作用对比效果图。
脆弱拟杆菌(Bacteroides fragilis)，命名为ZY-312，于2015年4月2日保藏于中国普通微生物菌种保藏管理中心(CGMCC)，其保藏编号为CGMCC No.10685，保藏地址为北京市朝阳区北辰西路1号院3号。
具体实施方式
下面结合附图对本发明的结构原理和工作原理作具体的描述：
本发明的实施方式包括：本发明通过对大量来自健康婴儿的粪便进行筛选，筛选出大量脆弱拟杆菌菌株，通过理化实验鉴定，发现了一种新的脆弱拟杆菌(Bacteroides fragilis)，命名为ZY-312，与现有的脆弱拟杆菌菌株相比，其具有耐胆盐、耐胃酸等益生特性，可以弥补原有益生菌的一些缺陷，在消化道的含胆盐、酸性条件下仍然保持较高生物活性，是益生菌制品的优选菌株。该脆弱拟杆菌ZY-312于2015年4月2日保藏于中国普通微生物菌种保藏管理中心(CGMCC)，其保藏编号为CGMCC No.10685，保藏地址为北京市朝阳区北辰西路1号院3号。
利用脆弱拟杆菌ZY-312可以制备成药物组合物。该药物组合物含有药学有效剂量的脆弱拟杆菌ZY-312。此外，所述药物组合物还可以含有合适的药物载体。该药物组合物可以为胶囊、溶液或可饮用悬乳液、袋装粉剂、颗粒剂等形式，每一单一剂量优选为脆弱拟杆菌ZY-312菌株约为106-1011细胞。
本发明的脆弱拟杆菌ZY-312还可以制成食品、保健品或食品添加剂等。所述食品、保健品或食品添加剂均含有脆弱拟杆菌ZY-312。这些食品、保健品或食品添加剂均可用于预防和治疗新生儿脑膜炎。
下面将结合具体实施例对本发明作进一步说明。需要指出的是，由本发明中的用于预防和/或治疗脑膜炎的脆弱拟杆菌或含有本发明的脆弱拟杆菌的药物组合物、食品、保健品和食品添加剂在施用于受试者后，都可以应用于上文所述的适应症并展现出上文所述的功能，在本发明范围内的所有剂型均已测试，下文中，仅仅是为说明，只在实施例中描述了 其中一少部分，然而不应将其理解为对本发明的限制。
除非特殊说明，否则本发明中所使用的试剂都是市售可购买的。
实施例1
脆弱拟杆菌ZY-312的分离、纯化
试剂和仪器
(1)培养基A：类杆菌-胆汁-七叶苷(BBE)琼脂(青岛海博生物科技有限公司，货号：HB7028)基础上加入改良配方，具体成分如下表一：
表一
Figure PCTCN2016092380-appb-000001
(2)培养基B：类杆菌-胆汁-七叶苷(BBE)琼脂(青岛海博生物科技有限公司，货号：HB7028)基础上加入改良配方，具体成分如下表二：
表二
Figure PCTCN2016092380-appb-000002
(3)培养基C：布氏肉汤(青岛海博生物科技有限公司，货号：HB0241)中加入胎 牛血清，加入量为5％(v/v)(四季青，货号：HB0205)。
(4)实验仪器
2.5L密封培养罐(三菱瓦斯化学株式会社，C-31)
恒温培养箱(上海一恒，型号：DHP-9082)
显微镜(尼康仪器有限公司，型号：E100)
PCR仪(ABI，型号：Applied 
Figure PCTCN2016092380-appb-000003
 PCR系统9700)
电泳仪(北京市六一仪器厂，型号：DYCP-32B)
(5)试剂
厌氧产气袋(三菱瓦斯化学株式会社，货号：C-1)
细菌DNA提取试剂盒(Bacterial DNA Kit(50)OMEGA，货号：D3350-01)
Taq酶(TaKaRa公司，货号：DR100A)
琼脂糖(Biowest，货号：91622)
磺胺甲恶唑(sigma，货号：S7507-10G)
甲氧苄啶(sigma，货号：T7883-5G)
维生素K1(青岛日水生物科技有限公司，货号：21005)
DL1000DNA Marker(TaKaRa公司，货号：D526A)
脆弱拟杆菌产肠毒素株(南方医院消化科孙勇老师提供，分离自临床腹泻患者)
脆弱拟杆菌标准株ATCC25285(购自广东省微生物所)
脆弱拟杆菌菌株Bd312(保藏号为CGMCC No.7280，由广州知光生物科技有限公司提供)
BF839菌株(分离自图腾益生液)。
(6)培养基配置
培养基A配置：称取BBE培养基61.5克，加热溶解于1000mL蒸馏水中，121℃高压灭菌15分钟，冷却至50℃左右时，加入过滤除菌的磺胺甲噁唑1g、甲氧苄啶4g和无菌脱纤维羊血50mL，混匀，倾入无菌平皿，备用。
培养基B配置：称取BBE培养基61.5克，加热溶解于1000mL蒸馏水中，121℃高压灭菌15分钟，冷却至50℃左右时，加入过滤除菌的磺胺甲噁唑1g、甲氧苄啶4g，混匀，倾入无菌平皿，备用。
培养基C配置：称取布氏肉汤培养基28.1g，加热搅拌溶解于1000mL蒸馏水中，分装三角瓶，121℃高压灭菌15分钟，备用。使用前，加入5％的胎牛血清。
布氏肉汤配置：称取布氏肉汤培养基28.1g，加热搅拌溶解于1000mL蒸馏水中，分装三角瓶，121℃高压灭菌15分钟，备用。
方法：
1、分离纯化
取新鲜的婴儿粪便0.5g，置于盛有4.5mL布氏肉汤的三角瓶中，振荡1分钟。取0.1mL滴于培养基上，划线后，置于厌氧罐中，37℃、培养48小时。挑取典型菌落于布氏肉汤培养基24h，进行革兰氏染色。显微镜下观察形态，选取革兰氏阴性菌的菌液，划线接种于血平皿，厌氧培养48h。根据平板上菌落形态特征及镜下观察菌体的染色特性、大小、球杆状和分布情况，判断是否纯化。如细菌不纯，则继续以上步骤，反复多次分离传代，直至得到纯化的菌株。
2、菌落特征
脆弱拟杆菌ZY-312在血平皿上培养48h后，呈现圆形微凸、半透明、白色、表面光滑、不溶血，菌落直径在1-3mm，参见图1。
3、显微镜下形态
脆弱拟杆菌ZY-312进行革兰氏染色镜检，为革兰阴性细菌，呈现典型的杆状，两端钝圆而浓染，菌体中间不着色部分形如空泡，参见图2。
4、电镜下形态
固定液固定，扫描电镜观察。镜下可见，脆弱拟杆菌ZY-312大小在0.5～0.8×1～4.5μm，无鞭毛，无芽孢，参见图3。
5、生化鉴定
生化鉴定结果如下表三(表中，+表示阳性，-表示阴性)
表三
测定反应底物 结果
色氨酸 -
脲素 -
葡萄糖 +
甘露醇 -
乳糖 +
蔗糖 +
麦芽糖 +
柳醇 -
木糖 +
阿拉伯糖 -
明胶 -
七叶灵 +
甘油 -
纤维二糖 -/+
甘露糖 +
松叁糖 -
棉子糖 +
山梨醇 -
鼠李糖 -
海藻糖 -
API20A(生化反应鉴定板，法国生物梅里埃股份有限公司)生理生化反应结果显示：脆弱拟杆菌ZY-312可发酵葡萄糖、乳糖、蔗糖、麦芽糖、木糖、七叶灵、甘露糖、棉子糖，符合脆弱拟杆菌的特征。
实施例2
脆弱拟杆菌ZY-312鉴定
聚合酶链式反应(PCR)引物(由英潍捷基(上海)贸易有限公司合成)序列如下：
引物对1：
正向引物：5’-ACGCTTGCACCCTCCGTATTA-3’
反向引物：5’-GCTTAGAGTTTGATCCTGGCTCAG-3’
引物对2：
正向引物：5’-TGGGTGGTTGCTGCCTGGACACA-3’
反向引物：5’-CATCCGGGTATGGATATGAA-3’
引物对3：
正向引物：5’-GATGCTCCAGTTACAGCTTCCATTG-3’
反向引物：5’-CGCCCAGTATATGACCTAGTTCGTG-3’
bft基因引物对：
正向引物：5’-GACGGTGTATGTGATTTGTCTGAGAGA-3’
反向引物：5’-ATCCCTAAGATTTATTATCCCAAGTA-3’
1、PCR鉴定(PCR即聚合酶链式反应，是常用的快速扩增基因的方法)
(1)16S rRNA序列测定
取上述菌株接种于培养基A上，37℃、厌氧培养48h。取单一菌接种于液体培养基中，37℃、厌氧培养48h。DNA提取试剂盒提取细菌DNA(天根生化科技(北京)有限公司，货号：DP302-02)，作为PCR模板DNA。
16S rRNA基因序列的扩增：引物对1扩增片段大小约为531bp；引物对2扩增片段大小为518bp；引物对3扩增片段大小约为970bp。
采用20μL PCR反应体系：Taq酶10μL、模板DNA 2μL、正向反向引物各1μL、无菌去离子水6μL。
PCR反应条件为：95℃预变性5min、95℃变性30s、55℃退火30s、72℃延伸45s、30个循环、72℃延伸10min。
PCR产物在2％的琼脂糖凝胶进行电泳，电泳条件为100V、15min。
PCR产物凝胶电泳结果如图4所示，其中1、2泳道分别为引物对1、引物对2；4、5泳道分别为引物对1、引物对2(重复PCR的结果)；3、6泳道为引物对3；8泳道为DNA分子量标准物(DL1000DNA marker)。分离菌株DNA采用引物对1进行PCR扩增后产物大小为531bp，采用引物对2进行PCR扩增后产物大小为518bp，采用引物对3进行PCR扩增后产物大小为970bp，符合预期，所分离菌株为脆弱拟杆菌。
将PCR产物进行核苷酸序列测定(由深圳华大基因科技有限公司进行测定)，测序结果在Genbank(美国国家生物技术信息中心建立的DNA序列数据库)上进行BLAST比对(http://www.ncbi.nlm.nih.gov/BLAST/)，见表四。
结果表明分离到为一株脆弱拟杆菌。
表四为16S rRNA序列的BLAST比对结果(部分)
表四
Figure PCTCN2016092380-appb-000004
(2)PCR检测bft基因
将测序筛选到的菌株接种于培养基C中，37℃、厌氧培养48小时。取培养菌液2mL，用细菌DNA提取试剂盒提取DNA，作为PCR模板DNA。bft基因的扩增采用bft基因引物，扩增片段大小应为294bp。
采用20μL PCR反应体系：Taq酶10μL、模板DNA 2μL、上下引物各1μL、无菌去离子水6μL。
PCR反应条件为：95℃预变性5min、95℃变性30s，55℃退火30s、72℃延伸45s，共30个循环，72℃延伸10min。PCR产物进行2％的琼脂糖凝胶电泳，电泳条件为100V、15min。
结果参见图5，其中1、2、3、4泳道为分离菌株ZY-312电泳结果；5、6、7为产肠毒素株脆弱拟杆菌电泳结果；8泳道为DL1000DNA marker。4、5泳道为bft基因引物对扩增产物；1、7泳道为引物对2扩增产物；2、6为引物对1扩增产物；3泳道为引物对3扩增产物。
结果表明，ZY-312为一株脆弱拟杆菌，且不含有肠毒素bft基因，为一株新的无毒株。
2、全基因组测序分析鉴定
对脆弱拟杆菌ZY-312进行全基因组测序(深圳华大基因科技有限公司)，测序结果与已发表的菌株序列相互比对，利用treebest软件(可以在sourceforge网站上免费下载http://treesoft.svn.sourceforge.net/viewrc/treesoft/)采用邻接法构建NJ-tree或利用软件PhyML采用最大似然法构建最大似然树。系统发育树显示(参见图6)，脆弱拟杆菌ZY-312与脆弱拟杆菌标准株ATCC25285(即NCTC9343)在同一分支上，表明检脆弱拟杆菌ZY-312为一株新的脆弱拟杆菌，与ATCC25285同源。
对全基因组测序结果进行毒力基因分析，以验证其是否含产毒素bft基因。结果显示，脆弱拟杆菌ZY-312全基因组中不含bft基因，为一株不产肠毒素的新的脆弱拟杆菌。
实施例3
脆弱拟杆菌ZY-312对胃酸的耐受性
1、人工胃液配制(根据2010年《中国药典》人工胃液配制方法)
23.4mL浓HCl溶解于100mL纯化水中，即得稀盐酸。取8.2mL稀盐酸，加入400mL纯化水与5g胃蛋白酶(
Figure PCTCN2016092380-appb-000005
猪源，1：15000)，定容到500mL。于37℃、磁力搅拌过夜，即得人工胃液。
2、菌体准备
收集菌液，离心，弃上清，生理盐水重悬后，再次离心，弃上清，菌体备用。
3、加入人工胃液测定活菌数
向菌体加入人工胃液，重悬，分别测定0、1、2、3h活菌数。
活菌计数采用10倍系列稀释法：取100μL菌液加入900μL布氏肉汤培养基中，逐步 梯度稀释至合适浓度。例如，每个平板点4个浓度梯度，每个梯度重复点样3次，每次点样20μL。37℃、厌氧培养48h，数菌落数(取菌落数为3-30的浓度梯度计数)。
活菌数(CFU/mL)＝三个点样菌落总和/3×50×稀释度
表五位不同菌株胃酸耐受实验结果(其中，数据为活菌浓度log值，h为小时)
表五
Figure PCTCN2016092380-appb-000006
益生菌必须进入人体的胃肠道并达到一定浓度才能发挥其功能。从口腔到肠道过程中，益生菌首先必须以活菌状态通过胃才有可能进入肠道。食物(尤其是流体)通过胃的时间一般为1-2h(小时)。根据饮食结构的不同，人体胃液的pH值波动很大，通常在pH3.0左右，空腹或食用酸性食品时可达pH1.5，食用碱性食品时可达pH 4-5，胃液的这种酸性环境可以激活胃蛋白酶原，从而杀死随食物进入胃内的细菌。益生菌如果要在人体内发挥益生作用，就必须具有一定的耐酸能力和耐胃蛋白酶的能力。
结果表明，与脆弱拟杆菌其他菌株相比，脆弱拟杆菌ZY-312在3h后活菌浓度仍然较高，而其他菌株的活菌浓度随时间降低很快，说明ZY-312对胃酸耐受性较好，具有很好的益生潜力和应用前景。
实施例4
脆弱拟杆菌ZY-312对胆盐耐受实验
1、实验材料
胰蛋白胨大豆肉汤(简称TSB，品牌：OXOID，货号：CM0129B)
胰蛋白胨大豆琼脂(简称TSA，品牌：OXOID，货号：CM0131B)
牛胆粉(生物工程上海(股份)有限公司，货号：ON1210)
胎牛血清(美国MP Biomedicals公司，货号：2916754)
2、菌株和试剂的准备
胆粉溶液：在TSB中加入牛胆粉，设置三个终浓度，分别为10g/L(1％牛胆粉)、20g/L(2％牛胆粉)和40g/L(4％牛胆粉)。灭菌后加入血清(终浓度50mL/L)待用。同时，以不加牛胆粉的TSB作为对照。
菌株培养与收集：菌株(ZY-312、Bd312、BF839、ATCC25285)于37℃、厌氧液体 静态培养至对数生长后期(约14-16小时)，分装至离心管中，每管分装3ml菌液，室温、4000rpm离心5分钟。再用0.01M PBS洗菌1次(室温、4000rpm离心5分钟)，弃上清，沉淀待用。
3、人工胆粉培养基中培养
用上述胆粉溶液将洗涤后的细菌重悬，用含胆粉溶液调整初始菌液浓度为1×108CFU/mL。并在37℃、厌氧培养1、2、4小时，涂板计数活菌数目的变化，0小时细菌数目作为对照。实验平行做3次。
4、计算细菌耐受胆粉情况
将上述三个时间点涂板结果，与对应的0小时结果进行比较，即可得到菌株在人工胆粉溶液中作用不同时间后其耐受胆粉的结果，以均数±标准差及统计结果说明。
表六为SK08菌株耐胆粉实验结果(n＝3)
表六
Figure PCTCN2016092380-appb-000007
结果如表六所示，0-4h观察，ZY-312在1％、2％、4％浓度胆粉中均可正常生长，随着胆粉浓度升高其活菌数越高，ZY-312活菌数显著高于其他菌株组。结果表明ZY-312耐受胆盐，并显著优于其他菌株。
胆盐是肝细胞分泌的胆汁酸与甘氨酸或牛磺酸结合形成的钠盐或钾盐，它是胆汁参与 消化和吸收的主要成分。胆盐排到小肠后，大部分由小肠黏膜吸收入血，再入肝脏组成胆汁。人体小肠中胆盐的质量浓度在0.03～0.3g/100mL的范围波动。
对于活细胞来说，胆盐能破坏细胞膜，因此对胆盐的耐受性是评价益生菌的重要指标之一。益生菌可产生胆盐水解酶，此酶可将甘氨酸和牛磺酸结合的胆盐催化水解为氨基酸残基和游离胆盐。具有胆汁盐解离能力的菌株可以降低高胆固醇人群的血清胆固醇水平和防止正常人高胆固醇血症的发生。消化道中胆盐的浓度不是固定不变的，在进食消化的开始1h，其质量浓度为15～20g/L，之后其质量浓度降为3g/L左右。益生菌必须在通过胃肠过程中，可以在正常的胆盐浓度下存活，如要在小肠中定殖，必须耐受胆盐的抑制作用。因此，相对于脆弱拟杆菌的其他菌株，ZY-312具有更好的应用前景。
实施例5
脆弱拟杆菌ZY-312对阪崎克罗诺杆菌的抑制作用的细胞实验
细胞和菌株：结肠腺癌细胞Caco-2由南方医科大学微生物学系保存；阪崎克罗诺杆菌ATCC29544为购自ATCC(即美国模式培养物集存库American type culture collection)的标准株；大肠杆菌E44、DH5α由南方医科大学微生物学系保存。
培养基：乳酸细菌培养基(简称MRS)、脑心浸液肉汤培养基(简称BHI)、细菌基础培养基(简称LB)，购自广东环凯微生物科技有限公司；胎牛血清(赛默飞世尔科技公司，品牌：Gbico)，购自广州鼎国生物科技有限公司；无菌脱脂纤维绵羊血，购自南京便诊生物科技有限公司。
本实验分为四个组，分别为对照组、竞争组、置换组、排斥组。对照组：Caco-2+ATCC29544共孵育3h；竞争组：Caco-2+ATCC 29544+ZY-312共孵育3h；置换组：Caco-2+ATCC29544共孵育1h，再加ZY-312，继续孵育2h；排斥组：Caco-2+ZY-312共孵育1h，再加ATCC 29544，继续孵育2h。
细胞感染倍数为100(细胞：细菌＝1:100)，ZY-312为108CFU(Colony-Forming Units，菌落形成单位，指单位体积中的活菌个数)。孵育后，细胞用培养基洗3次，加入100μL 0.5％Triton X-100裂解细胞(此浓度Triton X-100在半小时之内不会影响细菌活性)，孵育8min后，立即加入50μL蒸馏水。在溶解细胞之前的孵育和清洗阶段，单层细胞都要保持完整。反复吹打后吸出样品，做梯度稀释(10-l～10-4)后涂血平板计数菌落数。计算侵袭率，侵袭率＝胞内细菌数/孵育细菌数×100％。
共孵育分别采用竞争、排斥、置换方式，参见图7，结果显示：细胞中加入ZY-312后， 克罗诺杆菌侵袭率明显降低(*P＝0.0184<0.05)，竞争组、排斥组、置换组与对照组有显著性差异。实验结果表明脆弱拟杆菌ZY-312可抑制ATCC29544对Caco-2细胞的侵袭。
实施例6
脆弱拟杆菌ZY-312对阪崎克罗诺杆菌的抑制作用的大鼠实验
实验动物：SD大鼠窝鼠2-3日龄12窝，80只，精神状态良好，购自南方医科大学实验动物中心。
分别进行预防和治疗实验，检测ZY-312在动物体内对阪崎克罗诺杆菌的抑制效果，实验分组及方案具体见表七，表七为动物实验分组及处理方法，其中，PBS为磷酸盐缓冲液。
表七
Figure PCTCN2016092380-appb-000008
通过ZY-312对乳鼠的预防和治疗实验，结果显示：血液中均无ZY-312，表明使用ZY-312，不会引起益生菌性菌血症；血液中对照组100％检测到ATCC 29544，预防实验组、治疗实验组检测率分别为60％、70％；脑脊液检测结果，对照组检测率为70％(预防)和75％(治疗)，而实验组为0％(预防)和5％(治疗)。实验组检测到脑膜炎的小鼠数量明显少于对照组，说明ZY-312在体内可抑制阪崎克罗诺杆菌穿越肠屏障，从而减少进入血脑屏障的细菌数量，用ZY-312预防和/或治疗细菌性脑膜炎具有显著效果，见表八，表八为ZY-312对乳鼠阪崎克罗诺杆菌致脑膜炎的预防和治疗效果，其中，脑脊液平板上长有致病菌的即定义为脑膜炎。
表八
Figure PCTCN2016092380-appb-000009
Figure PCTCN2016092380-appb-000010
可见，本发明的脆弱拟杆菌对于预防和/或治疗脑膜炎具有很好的效果，不产生耐药性，且安全无毒，为治疗和/或预防脑膜炎提供了新的选择。此外，本发明的脆弱拟杆菌ZY-312是一株新的脆弱拟杆菌，该菌株是从发育良好的婴儿粪便中分离纯化得到的，是具有益生菌特性的脆弱拟杆菌新菌株，实验表明所分离到的脆弱拟杆菌ZY-312不含肠毒素基因bft，与现有菌株相比，具有耐胆盐、耐胃酸等益生特性，具有广阔的应用前景。
当然，本发明还可有其它多种实施例，在不背离本发明精神及其实质的情况下，熟悉本领域的技术人员当可根据本发明作出各种相应的改变和变形，但这些相应的改变和变形都应属于本发明所附的权利要求的保护范围。

Claims (10)

  1. 脆弱拟杆菌在制备用于预防和/或治疗脑膜炎的药物中的应用。
  2. 如权利要求1所述的脆弱拟杆菌应用，其特征在于，所述脆弱拟杆菌为脆弱拟杆菌ZY-312。
  3. 如权利要求1或2所述的脆弱拟杆菌应用，其特征在于，所述脑膜炎为新生儿脑膜炎。
  4. 如权利要求1或2所述的脆弱拟杆菌应用，其特征在于，所述脑膜炎为细菌性脑膜炎。
  5. 如权利要求3所述的脆弱拟杆菌应用，其特征在于，所述新生儿脑膜炎为细菌性脑膜炎。
  6. 一种用于预防和/或治疗脑膜炎的药物组合物，其特征在于，所述药物组合物包括药学有效剂量的脆弱拟杆菌ZY-312。
  7. 如权利要求6所述的药物组合物，其特征在于，所述药物组合物为胶囊、溶液、悬乳液、袋装粉剂或颗粒剂，每一单一剂量为106-1011个所述脆弱拟杆菌ZY-312菌株细胞。
  8. 一种用于预防和/或治疗脑膜炎的食品，其特征在于，所述食品含有脆弱拟杆菌ZY-312。
  9. 一种用于预防和/或治疗脑膜炎的保健品，其特征在于，所述保健品含有脆弱拟杆菌ZY-312。
  10. 一种用于预防和/或治疗脑膜炎的食品添加剂，其特征在于，所述食品添加剂含有脆弱拟杆菌ZY-312。
PCT/CN2016/092380 2015-07-31 2016-07-29 脆弱拟杆菌在预防和/或治疗脑膜炎中的应用 WO2017020783A1 (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021191274A1 (en) * 2020-03-25 2021-09-30 INSERM (Institut National de la Santé et de la Recherche Médicale) New strategy to treat and prevent diseases caused by enterobacteriae
EP3795676A4 (en) * 2018-05-18 2022-03-09 Revaissant (Shenzhen) Biosciences Co., Ltd. APPLICATION OF B. FRAGILIS OR AKKERMANSIA MUCINIPHILA IN THE PREPARATION OF A MEDICINE INTENDED FOR PREVENTING OR TREATING A TUMOR

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109908184A (zh) * 2017-12-12 2019-06-21 中山大学 脆弱拟杆菌在制备用于诱导γδT细胞增殖和/或积累的药物中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101125151A (zh) * 2006-08-16 2008-02-20 大连森佰澳科技有限公司 类杆菌信号分子微生态制剂及其制备方法
JP2012180289A (ja) * 2011-02-28 2012-09-20 Morinaga Milk Ind Co Ltd 抗菌剤

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101125151A (zh) * 2006-08-16 2008-02-20 大连森佰澳科技有限公司 类杆菌信号分子微生态制剂及其制备方法
JP2012180289A (ja) * 2011-02-28 2012-09-20 Morinaga Milk Ind Co Ltd 抗菌剤

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FENG, SHUZHEN ET AL.: "The Research Progress of Bacteroides Fragilis", MICROBIOLOGY, vol. 42, no. 7, 20 July 2015 (2015-07-20), pages 1366 - 1371, XP055361914, ISSN: 0253-2654, DOI: doi:10.13344/j.microbiol.china.140778 *
OCHOA-REPÁRAZ, J. ET AL.: "A Polysaccharide from the Human Commensal Bacteroides Fragilis Protects against CNS Demyelinating Disease", MUCOSAL IMMUNOLOGY, vol. 3, no. 5, 30 September 2010 (2010-09-30), pages 487 - 495, XP055188873, ISSN: 1933-0219 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3795676A4 (en) * 2018-05-18 2022-03-09 Revaissant (Shenzhen) Biosciences Co., Ltd. APPLICATION OF B. FRAGILIS OR AKKERMANSIA MUCINIPHILA IN THE PREPARATION OF A MEDICINE INTENDED FOR PREVENTING OR TREATING A TUMOR
WO2021191274A1 (en) * 2020-03-25 2021-09-30 INSERM (Institut National de la Santé et de la Recherche Médicale) New strategy to treat and prevent diseases caused by enterobacteriae

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