CN113122466B - Enterococcus faecalis and application thereof - Google Patents
Enterococcus faecalis and application thereof Download PDFInfo
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- CN113122466B CN113122466B CN201911419590.0A CN201911419590A CN113122466B CN 113122466 B CN113122466 B CN 113122466B CN 201911419590 A CN201911419590 A CN 201911419590A CN 113122466 B CN113122466 B CN 113122466B
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- enterococcus faecalis
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Abstract
The invention provides Enterococcus faecalis (Enterococcus faecalis) with a preservation number of CGMCC No. 19078. The enterococcus faecalis is a novel probiotic, and can resist the acid environment of gastric juice and the bile salt environment in intestinal tract. It has strong acid and bile salt resistance.
Description
Technical Field
The invention relates to the field of biological products, in particular to enterococcus faecalis and application thereof.
Background
In recent years, with the continuous use of antibiotics, the drug resistance of enterococcus faecalis is improved and becomes a conditional pathogen, mainly occurring in the period of resistance reduction of organism and in the cases of urinary tract infection, wound infection and bacteremia in hospitals. A great deal of research shows that enterococcus faecalis can cause infection and even death of human and animals, and a large part of the reasons are that the incidence rate of the enterococcus faecalis is increased due to the existence of virulence factors, and when flora in organisms is disordered or the resistance of the organisms is reduced, the enterococcus faecalis proliferated in a large quantity, invades the organs of the organisms, adheres to cell epidermis, secretes toxic substances, destroys cell tissues and causes infectious diseases. All virulence factors of enterococcus faecalis are present in the virulence island, while the bacterial chromosomes and plasmids are the major sites in the virulence island.
There are studies showing that some enterococcus faecalis can produce a plasmid-encoded hemolysin that increases infection, which may be a significant cause of septicemia caused by enterococcus faecalis, which adheres to the surface of small intestine villus epithelial cells, urothelial cells and cardiac cardiomyocytes and can cause pathological reactions leading to infection when there is a change in the immune system of the organism (manting, high altitude enterococcus faecalis research progress [ J ] public health, 2012,28(11): 1530-1532.); by analyzing the drug resistance of 487 strains of enterococcus faecalis in the ninth eight O Hospital of the United nations' Jiefang Provisions, the average drug resistance of enterococcus faecalis to erythromycin and tetracycline is more than 70%, the average drug resistance to penicillin, ampicillin and nitrofurantoin is less than 3%, and the drug resistance to levofloxacin and ciprofloxacin is on the rise in different years (West Qian, Zhang Wei, Jingping. enterococcus faecalis and enterococcus faecium drug resistance analysis [ J ] microorganism and infection, 2008(02): 8-10.). With the increasing use of new strains, a few reports of side effects related to lactic acid bacteria have appeared, mainly in terms of safety and drug resistance. The use of large amounts of antibiotics for the purpose of disease prevention or treatment in human medicine, farming, animal farming, has led to the widespread, rapid, continuous development and evolution of antibiotic resistance in bacteria, the prevalence of antibiotic resistance worldwide poses a great threat to the health of humans and animals, and an increasing number of microorganisms are resistant to available antibiotics.
In view of the specificity of existing strains of probiotics, enterococcus faecalis has been classified as a microorganism strain for feeding in 2008 (1126 publications of agriculture), and is generally considered to produce L-lactic acid, decompose part of protein, produce bacteriocin with bacteriostatic activity and the like, and have probiotic functions of improving intestinal microenvironment, promoting absorption of nutrients by hosts and the like. However, not all enterococcus faecalis can be used as a probiotic, and because of the special safety of enterococcus faecalis, the enterococcus faecalis needs to be examined for multiple factors to be used as a candidate for a probiotic. In addition, the probiotic candidate strain is subjected to acid and bile salt environments of the gastrointestinal tract as a necessary condition for exerting beneficial physiological effects, among numerous defense mechanisms of a human body, the strong acid environment provided by gastric acid has the greatest influence on microorganisms, most of the microorganisms are killed by the gastric acid after entering the digestive tract from the oral cavity, and only bacteria with acid resistance can survive; the bile salt is sodium salt or potassium salt formed by combining bile acid secreted by hepatic cells with glycine or taurine, the concentration of the bile salt in small intestine of human body is generally 0.03-0.3%, and high osmotic pressure can be generated outside the cells to influence the somatic cells. The probiotic bacteria must therefore have acid-and bile salt-resistant properties to ensure that a certain amount is maintained in the gastrointestinal tract. In conclusion, safety, no toxic or side effect, acid resistance, bile salt resistance, genetic stability and the like are important prerequisites for screening excellent probiotic enterococcus faecalis, and only the enterococcus faecalis meeting the standards can tolerate the severe digestive tract environment of a host to reach the intestinal tract after being taken, so that the enterococcus faecalis safe and beneficial to a human body.
In addition, some microecological preparations containing enterococcus faecalis as an active ingredient for treating diseases have appeared on the market, for example, a bifidobacterium triple live preparation produced by Shanghai Xinyi pharmaceutical factory Co., Ltd contains enterococcus faecalis and another two probiotics; the lactobacillus acidophilus compound produced by the tombarthite pharmaceutical industry group ltd contains enterococcus faecalis and two strains of lactobacillus acidophilus. The medicine can be used for treating gastrointestinal diseases such as diarrhea and constipation.
However, at present, the variety of enterococcus faecalis which can be used as probiotic bacteria is not large, and new and safe probiotic enterococcus faecalis is urgently to be developed, so that a foundation is laid for the development and application of microbial resources.
Disclosure of Invention
The present invention is directed to solving, at least in part, one of the technical problems in the related art. Therefore, the invention aims to provide a probiotic enterococcus faecalis strain with excellent acid and bile salt resistance.
The present invention has been completed based on the following findings of the inventors:
the inventor of the invention separates a strain of bacteria from the feces of healthy children, identifies the strain as enterococcus faecalis (Streptococcus faecalis), and further identifies through a series of biochemical experiments, and unexpectedly discovers that the strain meets a plurality of indexes of probiotics and has excellent acid and bile salt resistance.
In view of the above, the first aspect of the present invention provides an Enterococcus faecalis (Enterococcus faecalis), which has high acid resistance and high bile salt resistance, and the preservation number of the Enterococcus faecalis is CGMCC No.19078, which is preserved in the china general microbiological culture collection center at 12 months and 4 days in 2019, the preservation address is: beijing, Chaoyang district, Beichen Xilu No.1 institute, institute of microbiology, China academy of sciences. As mentioned above, the inventor of the invention firstly separates the strain from the feces of healthy children, identifies the strain as enterococcus faecalis, and further identifies the biochemical characteristics of the strain to find that the strain meets a plurality of indexes of probiotics, so the enterococcus faecalis can be used as a novel probiotic. Specifically, the inventor finds that the strain is moderately sensitive to norfloxacin, vancomycin and macrolide antibiotics, and is sensitive to common antibiotics (penicillins, cephalosporins, tetracyclines, meropenem and the like); experiments prove that the enterococcus faecalis is not toxic to animals such as mice, the mice can survive healthily after taking the strain, the weight is increased normally, and the quality standard requirements of probiotics are met; the enterococcus faecalis can resist the acid environment of gastric juice (for example, the bacterial strain has good survival condition under the artificial gastric juice environment with the pH value of 2.5-4.5); the enterococcus faecalis can resist the bile salt environment in the intestinal tract (for example, the bacteria survive well under the condition of 0.03-0.3% of bile salt concentration).
According to an embodiment of the present invention, H of said enterococcus faecalis+-atpD of the ATPase gene cluster has the nucleotide sequence shown in SEQ ID NO 1; the cholinesterase hydrolase gene of enterococcus faecalis has a nucleotide sequence shown in SEQ ID NO. 2.
CTAGTAGTTTAATTGTTTCGCTTTTTCGATGGCATCTTCAATTTTACCGACACTACGGAAGGCTTCTTCTGGCAGATTATCATGTTTACCTTCAAGAATTTCTTTAAAGCCACGAACTGTTTCCGCAACTGGTACATAAGAACCAGGTTGACCTGTAAATTGTTCAGCAACGTTAAAGTTTTGAGATAAGAAGAATTGAACACGGCGTGCGCGACCAACAAGTACTTTTTCGTCATCTGATAATTCGTCCATCCCTAAAATAGCGATGATATCTTGTAATTCACGGTAACGTTGTAAAATATGTTGCACTTCGGTAGCCACTTCGTAGTGTTCTTCTCCAACAATTTCAGGAGCCAAGGCACTAGAAGATGAAGCTAACGGATCTACCGCTGGATAAATCCCTTGTTCGGTTAATTTACGTTCCAAGTTAGTTGTTGCATCCAAATGGGCAAACGCTGTTGCTGGCGCTGGATCGGTATAGTCATCGGCTGGAACATAGATTGCTTGAATAGAGGTAATTGATCCTTTTTTCGTTGAAGTAATCCGTTCTTGTAATTGTCCCATTTCAGTCGCTAAGGTTGGTTGGTAACCAACGGCTGACGGCATCCGACCTAAAAGGGCAGAAACTTCTGAACCGGCTTGGGTGAAACGGAAAATGTTATCAATAAATAATAGCACGTCTTGTCCTTCCACATCACGGAAATATTCAGCAATCGTTAACCCAGTTAAGGCCACACGCATCCGTGCACCTGGCGGTTCGTTCATTTGACCAAAAACCATGGCTGTTTTTTCAATAACGCCTGAATCTTTCATTTCATAGTACAGATCGTTCCCTTCACGTGTCCGTTCACCAACACCAGTAAAGACGGAAATCCCTCCATGTTCTTGGGCAATATTATGAATTAATTCTTGAATTAAGACGGTTTTACCAACACCGGCACCACCGAAAAGTCCGACTTTACCACCTTTTAGATAAGGTGCTAATAAGTCAATAACTTTAATCCCTGTTTCTAAAATTTCATTACTGGTACTTAATTCATCAAATGCTGGCGCTTTTTTATGAATCCCACTACGTTCAGCATCTGCAGGGAATGGCGCTTCTAAGTCAATTGTGTCTCCTAAAACGTTAAACACACGACCTAATGTATCTTTACCAACAGGAACTGAAATTGATTTTCCTGTATCGATAACTTCCATTCCACGTTGTAAACCATCTGTCGATTCCATGGCGATAGAACGAATCACTCCATCACCTAGTTCTAAAGCGACTTCAAGTACTACTTTTTGTTTTGCTTCGCCATTTTTATAAACGACTAAAGCGTTGTTAATATCGGGTAAGGATTGATCTAATGAAAATTCCACGTCAACAACGGGACCGATTACTTGAACAATCTTTCCTGAACTCAT(SEQ ID NO:1)。
ATGTGTACAGGCATCAAGATTATTTCCAAAACAAATGATATTTTTTATGGACGTACGATGGATTTTACGTTTGACTTTTTCGGCAACGAAGATCCAATTGCACCAAAAATCCCTACTCTAATTGCTCAATTTCCAAAAGGTACAGTTCTAAATAGCCAACTAAATCCTTGGACGGCAAAATATTCCTTTATGGGACTAGCAATGTCAGGCACAGATCAACCAGCGAACGATGGTAAAACCGTCAGCCTTGCTATCACAGATGGTATCAATGAAGCCGGCTTATCTGGTGATATTCAATATTTAATGGAATCTTCAACAGCACCTGCTGAAAGTTTAGCAGAGCGTGGTTTAACACCTCATATTGCAGAAGAAGTTCTCGCTTATATTTTGAGTAATTTTGAAAGCGTTGACGAAGTAAAAGTAGCTTTTGAAAAAATCGGCCTGTTAGATCAAAAATTCCAACTTGATTCATTGGGGGAAGTTCATTTCACCTTACACTGGACCATTAATGATAAAAATAATAATAGTATCGTTTTGCAACCAACAGATAACGGAGCGTTCGTCATTTATGATTCGATTGGCGTAGTCACAAACAGCCCAGAATACAATTATCATTTAACCAATGCACGAAACTATATCGGAATGCGCAACTACGCTATTAAAGAACCTTACACTTTAAAATCAGGCGCAACACTTGACCCAATTGAAGGTGGGACTTCTTACGGACTATTAGGAATCCCAGGAGACTTCACTTCGCCGTCACGCTTCATTCGTGCGTTATACTATTCTGACAATCTCCAAGAATTTGATAGCTCTGAAGGGATTATGCAACTCTATCGCGCCTTCCAAACAGTGATGATTCCTCGGGGAATTGGCCATTTAGGTCAAAGTAATTCTCTGTCTGATTTCACACATTATTGGTCAGGATATGATGTGACTAATCTAACCATGTATGTCCAACCAGAAAGTACTACCTCATTTACAAAATACACTTTGGATCCAGCTTTAACAGAAGTTACTACTTTTGCTGTTTCTAACGAACTACTACTAACAGATTTAAATCAATAA(SEQ ID NO:2)。
It should be noted that "highly acid-resistant" as used herein means that the strain still grows and survives well in an acidic environment with a pH as low as 2.5, and "bile salt-resistant" as used herein means that the strain still grows and survives well in an environment with a bile salt concentration as high as 0.3%.
According to the embodiment of the invention, the enterococcus faecalis has high acid resistance and high bile salt resistance, and the enterococcus faecalis has a good survival condition and shows high acid resistance and high bile salt resistance under an artificial gastric juice environment with the pH value of 2.5-4.5 or a bile salt concentration of 0.03-0.3%.
The term "antibiotic-sensitive" as used herein means that the resistance of the strain to the antibiotic is weak, and the normal growth of the strain can be affected under the condition of trace administration. According to an embodiment of the invention, the enterococcus faecalis is sensitive to penicillin, cephalosporin, macrolide, tetracycline, carbapenem and chloramphenicol antibiotics.
According to an embodiment of the invention, the penicillin comprises at least one selected from the group consisting of ampicillin, penicillin; the cephalosporins comprise at least one selected from cefoperazone, cefazolin and cefuroxime sodium; the macrolides include at least one selected from the group consisting of erythromycin and clarithromycin; the tetracyclines comprise at least one selected from minocycline and tetracycline; the carbapenems are meropenem.
According to the embodiment of the invention, the enterococcus faecalis is high in safety.
According to an embodiment of the invention, the safety is specified by at least one of the following: is not hemolytic; biofilm formation is weak; the transfer level of the drug-resistant gene is low; acute toxicity and intestinal pathological changes did not appear after the animals were gavaged.
In a second aspect of the invention, the invention proposes a microbial preparation comprising enterococcus faecalis as described above according to an embodiment of the invention. As described above, the present inventors have isolated for the first time a novel enterococcus faecalis having excellent acid-and bile salt-resistant properties, which can be used as a probiotic, and thus, it can be prepared into a microbial preparation for convenient administration to patients.
According to an embodiment of the invention, the microbial preparation of the invention may also have at least one of the following additional technical features:
according to an embodiment of the invention, further comprising a pharmaceutically acceptable excipient.
According to the example of the invention, the number of viable bacteria of enterococcus faecalis is not less than 1 x 106CFU/g。
According to an embodiment of the invention, the microbial preparation further comprises at least one selected from the group consisting of: bifidobacterium animalis, lactobacillus paracasei and bacillus cereus.
According to an embodiment of the invention, the pharmaceutically acceptable excipients are lubricants, fillers, sweeteners, disintegrants.
According to an embodiment of the invention, the microbial preparation is at least one of a tablet, a drop, a powder, a capsule.
In a third aspect of the invention, the invention proposes the use of a microbial preparation as hereinbefore described in the manufacture of a food, health product or pharmaceutical product.
Drawings
FIG. 1 is a graph showing the results that enterococcus faecalis according to the example of the present invention showed stability at pH 2.5-4.5;
FIG. 2 is a graph showing the results that enterococcus faecalis according to the embodiment of the present invention showed stability in artificial intestinal fluids having bile salt concentrations of 0.03% and 0.1%;
FIG. 3 is a diagram of H according to an embodiment of the present invention +-a structure diagram of the ATPase gene cluster;
FIG. 4 is a graph showing the result of electrophoresis of plasmids from enterococcus faecalis according to an embodiment of the present invention;
FIG. 5 is a graph showing hemolysis of bacteria on a blood plate according to an embodiment of the present invention;
FIG. 6 is a graph showing the variation of viable count of the enterococcus faecalis powder composition according to the embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
EXAMPLE 1 isolation, purification and characterization of enterococcus faecalis
5-10g of collected fresh feces of children are picked, put into a glycerin tube and transferred into a refrigerator with a biological ice bag for standby. Transferring 1g of anaerobically preserved children feces sample to sterile physiological saline containing 9mL in a sterile operating platform until the sample is uniform, and sequentially performing gradient dilution of 10 times to 1 × 10-8Sucking 1X 10 by using a pipette-6、1×10-7、1×10-83 gradient dilutions 100 μ L each in solid medium MRS-CaCO 33 plates per dilution. And after anaerobic culture at the constant temperature of 37 ℃ for 48 hours, selecting bacterial colonies with the surface having the characteristics of lactic acid bacteria, inoculating the bacterial colonies into an EC liquid culture medium for amplification culture, and then performing gram staining experiments, catalase experiments and 16SrRNA sequencing to determine that the bacterial colonies are enterococcus faecalis. Wherein, MRS-CaCO 310g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 801 mL of Tween, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of diammonium citrate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate, 1L of distilled water, pH 6.3 +/-0.1, 1.5-2.0% of agar powder and 2% of CaCO3(ii) a The EC culture medium has the formula as follows: casein peptone 10.0g, soybean peptone 5.0g, glucose 5.0g, yeast extract 5.0g, disodium hydrogen phosphate 4.0g, potassium dihydrogen phosphate 4.0g, agar 15.0g, adding water to 1L, pH 7.4 + -0.1, 25 deg.C.
The specific detection is as follows:
1.1 colony and cell morphology
The colony is light yellow, round, neat in edge, and slightly raised.
The results of the mycosis examination are gram-positive, oval cocci, mostly arranged in double or short chain.
1.2 culture characteristics
Good growth under both anaerobic and aerobic conditions
1.3 Biochemical Properties
The biochemical reaction characteristics are shown in table 1.
TABLE 1 Biochemical reaction characteristics of the strains to be tested
1.4 API test strip identification
The strain to be detected is identified by using the API 20Strep reagent strip, a good identification result is obtained, the identification result is Enterococcus faecalis (Enterococcus faecalis), the identification value is 99.2%, the T value is 0.99, no inconsistent item exists, and the reaction result is shown in table 2.
TABLE 2 API response characteristics of the strains to be tested
1.516S rRNA sequence analysis
The 16S rRNA gene sequence of the strain to be detected determined in the experiment is compared with the sequence in GenBank by running BLAST program of NCBI to identify the strain as enterococcus faecalis, and the classification of the enterococcus faecalis is named as: enterococcus faecalis with a preservation number of CGMCC No. 19078.
Various properties of this enterococcus faecalis (CGMCC No.19078) will be studied below.
EXAMPLE 2 study of strains acid and bile salt resistance
2.1 cultivation of the strains to be tested
The EC medium was used for the culture of enterococcus faecalis strains and commercially available enterococcus faecalis strains. Weighing according to the formula, adding distilled water, stirring, adjusting pH to 7.4, and sterilizing at 121 deg.C for 20 min. Enterococcus faecalis was inoculated to sterilized EC medium at an inoculum size of 2% and aerobically cultured at 180rpm at 37 ℃.
2.2 methods for preparing Artificial gastric juice and intestinal juice
Artificial gastric juice: taking 16.4mL of dilute hydrochloric acid, adding about 800mL of water and 10g of pepsin, shaking up, and adding water to dilute into 1000mL to obtain the finished product.
Artificial intestinal juice, i.e. phosphate buffer (pancreatin-containing, pH 6.8): taking 6.8g of monopotassium phosphate, adding 500mL of water for dissolving, and adjusting the pH value to 6.8 by using 0.lmol/L sodium hydroxide solution; dissolving pancreatin 10g in water, mixing the two solutions, and diluting to 1000 mL.
The specific experimental procedures are as follows:
2.3 gastrointestinal survival assay
The effect of pH, bile salt concentration and treatment time on the survival rate of enterococcus faecalis was examined in artificial gastric juice and artificial intestinal juice. Considering that gastric juice is normally at about pH 2.5, the present study simultaneously examined the survival of commercially available enterococcus faecalis at pH 2.5; considering that bile is in a state of about 0.2% in a usual state, the present study simultaneously examined the survival of commercially available enterococcus faecalis in a state of about 0.3% in an extreme bile concentration. The experimental results are as follows:
(1) acid resistance
Enterococcus faecalis shows good simulated gastric juice tolerance.
TABLE 3 acid resistance test of enterococcus faecalis
(Note: enterococcus faecalis of the present invention: EC; commercially available enterococcus faecalis: MEC)
As shown in Table 3 and FIG. 1, the enterococcus faecalis of the present invention showed stable activity at pH 2.5-4.5 within 3 h. Under the condition of pH 2.5, the viable count of the enterococcus faecalis treated for 3 hours under the acidic condition is almost unchanged, and the viable count of the commercially available enterococcus faecalis treated for 3 hours under the acidic condition is reduced by about 6 times compared with the initial viable count. Therefore, the enterococcus faecalis has higher viable count compared with the commercially available enterococcus faecalis, and has higher acid resistance.
(2) Resistance to bile salts
TABLE 4 enterococcus faecalis bile salt resistance test
(Note: enterococcus faecalis: EC; commercially available enterococcus faecalis: MEC)
As shown in table 4 and the results of fig. 2, the survival rate of enterococcus faecalis was not greatly affected in the artificial intestinal fluids with bile salt concentrations of 0.03% and 0.3%; when the concentration of bile salts is increased to 0.2%, the survival rate of thalli is slowly reduced along with the processing time; at a bile salt concentration of 0.3%, the enterococcus faecalis still survived about 50% after 1 h. These results all indicate that enterococcus faecalis has good bile salt resistance. And the decrease trend of the viable count of the commercially available enterococcus faecalis after the enterococcus faecalis is treated for 3 hours under the condition of 0.3 percent of bile salt compared with the initial viable count is obviously higher than that of the enterococcus faecalis. Therefore, the enterococcus faecalis has higher viable count compared with the commercially available enterococcus faecalis, and has higher bile salt resistance.
2.4 related characteristic genes
The genome information of the enterococcus faecalis is obtained by second-generation sequencing, and the comparative genome analysis of the enterococcus faecalis and the reported strains with acid resistance and choline resistance shows that the enterococcus faecalis has complete H+-each subunit of ATPase encodes a gene and two Na' s+/H+The antiporter NhaC, a Na+、K+、Li+And Rb+/H+The reverse transport protein NhaK and the coding gene of the cholyl glycine hydrolase. Design of primers by genomic information to give H +-ATPase and cholic acidA gene encoding glycine hydrolase.
Respectively designing primers: getH-ATPase-f, getH-ATPase-r and getCGH-f, getCGH-r.
Activating and culturing strains, and extracting a genome from a fresh culture to be used as a gene cloning template. Cell lysis was performed by using the easy pure Genomic DNAkit enzymolysis method to extract Genomic DNA. The operation steps are as follows:
[ 1 ] overnight-cultured bacteria were collected in 2mL volume, centrifuged at 10,000 Xg for 1min, and the supernatant was discarded.
② centrifuging again, and sucking the supernatant as clean as possible.
③ adding 200 mu L (20mg/mL) of lysozyme into the thalli for resuspension, shaking and incubating for 60min at 37 ℃, centrifuging for 1min at 10,000 Xg, and discarding the supernatant.
Adding 100 microliter LB11 and 20 microliter proteinase K, and sucking with a gun head until the thallus is completely suspended.
And fifthly, incubating for 15min at 55 ℃.
Sixthly, 20 mu L of RNase A is added, mixed evenly and placed for 2min at room temperature.
Adding 400 μ L of BB11 added with absolute ethyl alcohol, vortexing for 30s, adding the whole solution into a centrifugal column, centrifuging for 1min at 10,000 × g, and discarding the effluent.
Adding 500 μ L CB11, centrifuging for 1min at 10,000 Xg, and discarding the effluent.
Ninthly, repeating the operation once.
500 μ L WB11 added with absolute ethanol is added to the R, and then centrifugation is carried out for 1min at 10,000 Xg, and the effluent is discarded.
Excellent acid resistance of enterococcus faecalis (CGMCC No.19078) and its H+ATPase is closely related, by analysis of H+The ATPase gene cluster (structure shown in fig. 3), was found to have 84% homology to atpD and e.hirae ATCC 9790 atpD. The gene sequence is shown in SEQ ID NO. 1.
The bile acyl glycine hydrolase (EC 3.5.1.24) is a hydrolase of enterococcus, has 1068bp, can catalyze the hydrolysis of glycocholic acid to bile acid and glycine, and can provide carbon and nitrogen sources for thalli. The sequence is shown in SEQ ID NO. 2.
Example 3 enterococcus faecalis Strain safety Studies
3.1 drug resistance analysis
The method is carried out by adopting a drug sensitive paper diffusion method. Diluting fresh cultured enterococcus faecalis with normal saline to 107cfu/ml, uniformly coating on an M17 culture dish, standing at room temperature for 3-5min, and placing drug sensitive paper sheets with circle center spacing not less than 24mm and paper edge distance not less than 15mm from agar edge. The cells were cultured in an inverted state in an incubator at 37 ℃ for 24 hours to measure the diameter of the zone of inhibition.
The resistance of enterococcus faecalis strains and their 30 th generation strains was measured by the paper diffusion method, and the results are shown in tables 5 and 6 below.
TABLE 5 enterococcus faecalis resistance results
Note: r ═ drug resistance; i is intermediary; s is sensitive; -: no bacteriostatic zone is formed;
TABLE 6 enterococcus faecalis passage 30 strain resistance results
Note: r ═ drug resistance; i is intermediary; s is sensitive; -: no bacteriostatic zone is formed;
as can be seen from the above resistance results, enterococcus faecalis and enterococcus faecalis-30 showed sensitivity and moderate sensitivity to almost all common antibiotics. The results of the drug resistance of the enterococcus faecalis and the enterococcus faecalis-30 are consistent, which shows that the drug resistance of the enterococcus faecalis is not influenced in the process of passage. Therefore, the enterococcus faecalis antibiotic disclosed by the invention is low in transfer risk and high in strain safety.
3.2 virulence factor analysis
The hemolysin and the biofilm formation of the enterococcus faecalis are measured by adopting a literature report method, and the safety in the animal body is evaluated according to the method of the current pharmacopoeia.
(1) The results obtained by plasmid extraction of the strains are shown in FIG. 4. The strain does not contain plasmids, the transfer level of the drug-resistant gene is low, and the safety of the strain is high.
(2) Hemolysin detection
Hemolysin secreted by bacteria can cause erythrolysis or other tissue damage. The production of hemolysin is mainly related to virulence genes such as cylA, cylB and the like. Hemolysis of each group of bacteria on blood plates is shown in FIG. 5. According to hemolytic experiments, no hemolytic ring exists around the enterococcus faecalis colony, ATCC 29212 strain (hereinafter referred to as control strain) has slight hemolytic ring, and obvious hemolytic ring exists around staphylococcus aureus. The result shows that the enterococcus faecalis strain disclosed by the invention is negative in hemolysis experiment, has no hemolysis and is high in safety.
(3) Biofilm assay
According to statistics, 80% of bacterial infections are related to the formation of bacterial biofilms (biofilms for short), the bacterial morphology and physiological effects in the biofilms are different from those of free bacteria, the tolerance to antibiotics can be improved by 10-1000 times, and the resistance to host immune defense is strong, which is a main reason for causing bacterial drug resistance. The ability of a biofilm to form has a large impact on bacterial virulence.
The results of the experimental detection of the OD value of the biofilm of enterococcus faecalis are shown in Table 7.
TABLE 7 enterococcus faecalis biofilm assay results
| | Experiment | 1 | |
Mean value | OD value of biological membrane |
| Blank space | 0.090 | 0.084 | 0.087 | / | |
| Enterococcus faecalis | 0.412 | 0.412 | 0.412 | 0.325 | |
| Enterococcus faecalis-30 | 0.298 | 0.282 | 0.290 | 0.203 |
According to the analysis of tetracycline-resistant enterococcus faecalis biomembranes in the literature, the enterococcus biomembrane forming ability is strong positive by OD >2, medium by 1< OD <2, and weak positive by OD < 1. The experimental results show that the biofilm forming abilities of the enterococcus faecalis are weak and positive, and the OD values are lower, which shows that the biofilm forming abilities of the enterococcus faecalis are weak, the results are unchanged after 30 generations of bacteria, and the level of drug resistance generated by the antibiotic of the strain is low.
(4) Evaluation of safety in animals
After the mice are gazed, the health condition is good, compared with the normal saline group, the activity condition of the mice is not different, and the hair state is normal. The body weights of the mice before and after gastric gavage were recorded as shown in table 8.
TABLE 8 weight changes of mice before and after gastric lavage
As a result, the body weight of mice in each group was increased, and the body weight changes in the two groups were not very different (the difference between the two groups was not statistically significant, and p > 0.05).
EXAMPLE 4 preparation of enterococcus faecalis composition
The enterococcus faecalis-containing composition can be prepared into powder, tablets, capsules and drops with pharmaceutically acceptable auxiliary materials, and the embodiment provides a preparation method of the enterococcus faecalis-containing powder composition, which comprises the following steps:
4.1 preparation of raw material bacteria powder: activating the enterococcus faecalis working seeds, culturing the first-stage seed liquid, preparing seeds in a seed tank, fermenting, centrifuging, collecting thalli, adding a freeze-drying protective agent, and carrying out thalli freeze-drying to obtain enterococcus faecalis powder.
4.2 pretreatment of raw materials and auxiliary materials: according to the weight percentage of the product, the enterococcus faecalis, bifidobacterium animalis powder, lactobacillus paracasei powder, bacillus cereus powder and pharmaceutically acceptable auxiliary materials such as lubricant (magnesium stearate, talcum powder, polyethylene glycol 6000, stearic acid, sodium dodecyl sulfate/magnesium, sodium stearyl fumarate and glyceryl behenate) glidant (talcum powder, superfine silica gel powder and silicon dioxide), filler (skim milk powder, maltodextrin, fructo-oligosaccharide, pregelatinized starch, lactose, glucose, sucrose, D-mannitol and starch) sweetening agent (mannitol, glucose, sucrose, D-mannitol, steviosin and white sugar) are pretreated.
4.3 mixing preparation: sieving the auxiliary materials with a sieve of 50-200 meshes, adding into a batch mixer, mixing, adding the bacterial powder, mixing, and discharging after 50 minutes.
4.4, packaging: the mixed raw and auxiliary materials are transferred into a powder packaging machine for packaging, and the viable count of the enterococcus faecalis of the final product is more than or equal to 1 multiplied by 107cfu/g, respectively placing the mixture at the temperature of 20 ℃ and the temperature of 25 ℃ for stability inspection, and determining the stability of the enterococcus faecalis in a finished product. The results are shown in table 9 and fig. 6.
TABLE 9 variation of viable count in powder of enterococcus faecalis composition
As can be seen from Table 9 and FIG. 6, the formulations prepared with the strains of the present invention have better stability at 20 ℃ and 25 ℃ during 12 months of storage.
Conclusion
(1) The enterococcus faecalis has better acid resistance and cholate resistance, and can better exert activity in vivo.
(2) The enterococcus faecalis has better safety, and the strain enterococcus faecalis has weak formation capability without hemolytic biofilms; in the aspect of drug resistance, the enterococcus faecalis is sensitive to almost all common antibiotics (penicillin and the like); meanwhile, plasmid is not detected in the strain, and the transfer level of drug-resistant genes is low. Safety experiments in animal bodies also show that the strain is safe in mouse bodies and does not have acute toxicity and intestinal pathological changes.
(3) The enterococcus faecalis of the present invention has good stability in the preparation of various dosage forms. Experiments have shown that enterococcus faecalis compositions prepared from enterococcus faecalis according to the present invention have a better stability during storage for 12 months.
The experimental results show that the enterococcus faecalis is good in safety, does not have phenotypic characteristics of toxicity, is sensitive to common antibiotics, is safe in acute animal toxicity experiments, has good storage stability, and can be used for preparing a microecological preparation.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Hangzhou Yuanda biopharmaceutical Co., Ltd
<120> enterococcus faecalis and application thereof
<130> PIDC3194542
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1407
<212> DNA
<213> Artificial
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<223> nucleotide sequence of atpD of H + -ATPase Gene cluster of enterococcus faecalis
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ttccgcaact ggtacataag aaccaggttg acctgtaaat tgttcagcaa cgttaaagtt 180
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taattcgtcc atccctaaaa tagcgatgat atcttgtaat tcacggtaac gttgtaaaat 300
atgttgcact tcggtagcca cttcgtagtg ttcttctcca acaatttcag gagccaaggc 360
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ttccaagtta gttgttgcat ccaaatgggc aaacgctgtt gctggcgctg gatcggtata 480
gtcatcggct ggaacataga ttgcttgaat agaggtaatt gatccttttt tcgttgaagt 540
aatccgttct tgtaattgtc ccatttcagt cgctaaggtt ggttggtaac caacggctga 600
cggcatccga cctaaaaggg cagaaacttc tgaaccggct tgggtgaaac ggaaaatgtt 660
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cccagttaag gccacacgca tccgtgcacc tggcggttcg ttcatttgac caaaaaccat 780
ggctgttttt tcaataacgc ctgaatcttt catttcatag tacagatcgt tcccttcacg 840
tgtccgttca ccaacaccag taaagacgga aatccctcca tgttcttggg caatattatg 900
aattaattct tgaattaaga cggttttacc aacaccggca ccaccgaaaa gtccgacttt 960
accacctttt agataaggtg ctaataagtc aataacttta atccctgttt ctaaaatttc 1020
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atctgcaggg aatggcgctt ctaagtcaat tgtgtctcct aaaacgttaa acacacgacc 1140
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acgttgtaaa ccatctgtcg attccatggc gatagaacga atcactccat cacctagttc 1260
taaagcgact tcaagtacta ctttttgttt tgcttcgcca tttttataaa cgactaaagc 1320
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<223> nucleotide sequence of cholinesterase hydrolase gene of enterococcus faecalis
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attgctcaat ttccaaaagg tacagttcta aatagccaac taaatccttg gacggcaaaa 180
tattccttta tgggactagc aatgtcaggc acagatcaac cagcgaacga tggtaaaacc 240
gtcagccttg ctatcacaga tggtatcaat gaagccggct tatctggtga tattcaatat 300
ttaatggaat cttcaacagc acctgctgaa agtttagcag agcgtggttt aacacctcat 360
attgcagaag aagttctcgc ttatattttg agtaattttg aaagcgttga cgaagtaaaa 420
gtagcttttg aaaaaatcgg cctgttagat caaaaattcc aacttgattc attgggggaa 480
gttcatttca ccttacactg gaccattaat gataaaaata ataatagtat cgttttgcaa 540
ccaacagata acggagcgtt cgtcatttat gattcgattg gcgtagtcac aaacagccca 600
gaatacaatt atcatttaac caatgcacga aactatatcg gaatgcgcaa ctacgctatt 660
aaagaacctt acactttaaa atcaggcgca acacttgacc caattgaagg tgggacttct 720
tacggactat taggaatccc aggagacttc acttcgccgt cacgcttcat tcgtgcgtta 780
tactattctg acaatctcca agaatttgat agctctgaag ggattatgca actctatcgc 840
gccttccaaa cagtgatgat tcctcgggga attggccatt taggtcaaag taattctctg 900
tctgatttca cacattattg gtcaggatat gatgtgacta atctaaccat gtatgtccaa 960
ccagaaagta ctacctcatt tacaaaatac actttggatc cagctttaac agaagttact 1020
acttttgctg tttctaacga actactacta acagatttaa atcaataa 1068
Claims (8)
1. A pharmaceutical composition, CHENGCHANGXIAOCOCOCOCOCOCOCCUS (enterococcus faecalis, and/or enterococcus faecalis:Enterococcus faecalis) The preservation number is CGMCC No. 19078.
2. The enterococcus faecalis of claim 1, wherein said enterococcus faecalis has H+ -atpD of the ATPase gene cluster has the nucleotide sequence shown in SEQ ID NO 1;
the cholinesterase hydrolase gene of enterococcus faecalis has a nucleotide sequence shown in SEQ ID NO. 2.
3. Enterococcus faecalis according to claim 1, wherein said enterococcus faecalis is sensitive to penicillin, cephalosporin, macrolide, tetracycline, carbapenem and chloramphenicol antibiotics;
the penicillin comprises at least one selected from ampicillin and penicillin; the cephalosporins comprise at least one selected from cefoperazone, cefazolin and cefuroxime sodium; the macrolides include at least one selected from the group consisting of erythromycin and clarithromycin; the tetracyclines comprise at least one selected from minocycline and tetracycline; the carbapenems are meropenem.
4. A microbial preparation comprising enterococcus faecalis, comprising enterococcus faecalis according to claim 1 and optionally pharmaceutically acceptable adjuvants.
5. A microbial preparation according to claim 4 characterised in that the number of viable bacteria of enterococcus faecalis is not less than 1 x 106CFU/g。
6. The microbial preparation of claim 4, further comprising at least one selected from the group consisting of: bifidobacterium animalis, lactobacillus paracasei and bacillus cereus.
7. The microbial preparation of claim 4, wherein the pharmaceutically acceptable excipients are lubricants, fillers, sweeteners, disintegrants.
8. The microbial preparation of claim 4, wherein the microbial preparation is in at least one of a tablet, a drop, a powder, a capsule.
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| CN118178481A (en) * | 2020-09-30 | 2024-06-14 | 杭州远大生物制药有限公司 | Microbial preparation and preparation method thereof |
| CN114107135A (en) * | 2021-12-22 | 2022-03-01 | 苏州普瑞森生物科技有限公司 | Enterococcus faecalis for treating colitis |
| CN116948919B (en) * | 2023-09-18 | 2023-12-22 | 北京悦得微生物科技有限公司 | Enterococcus hai and application thereof |
| CN117481255B (en) * | 2023-12-06 | 2024-03-19 | 圣道生物(山东)集团有限公司 | Anti-stress growth-promoting preparation for livestock and poultry and application of anti-stress growth-promoting preparation in aspect of improving feed utilization rate |
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