CN108410762B - A kind of streptococcus novel species HTS20 and its application - Google Patents
A kind of streptococcus novel species HTS20 and its application Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/10—Antimycotics
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- C12N1/20—Bacteria; Culture media therefor
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Abstract
The present invention provides a kind of streptococcus, are a novel species in streptococcus, name are as follows: Streptococcus zengyii sp.nov.HTS20 registers preservation in China General Microbiological culture presevation administrative center, and deposit number is CGMCC 15343.Streptococcus zengyii sp.nov.HTS20 of the invention is cultivated in a variety of fermentation mediums, it can produce stronger Substance, and there is obvious bacteriostasis to staphylococcus aureus and candida albicans bacterium, there is wide development space in the prevention and treatment to the associated diseases such as staphylococcus aureus and candida albicans bacterium, there is good development and application prospect in terms of antibacterials.
Description
Technical field
The invention belongs to microorganism fields, and in particular to a kind of streptococcus novel species HTS20 and its application.
Background technique
The invention of antibiotic and synthesising bacteria anti-reflecting medicine and application are 20th century one of the greatest achievements of field of medicaments.The mankind
All kinds of serious bacterial infection diseases have effectively been cured using antibiotic and synthesising bacteria anti-reflecting medicine, have fruitfully been reduced
The death rate of various severe bacterial infections sexually transmitted diseases, and then started the research and development and widely applied climax of antibacterials.
20 middle of century, the antibiotic bulk pharmaceutical chemicals listed up to more than 500, also up to count in clinical general types
Over one hundred kind.The mankind start the danger for despising infectious diseases, to antibiosis in face of the obtained great achievement of antibacterials development and application
The application of element and synthesising bacteria anti-reflecting medicine also becomes to do at will.
Later period the 1970s, the medicine general administration of a superpower once claimed to " research of infectious disease accuses one at long last
Paragraph.Present most important thing is exactly to study cancer and cardiovascular disease ".But this confidence is beaten by the harsh fact soon
It is broken, it has been found that some scripts are easy the bacterial infection disease for the treatment of now with new variation, the effective antibiotic of script
Or synthesising bacteria anti-reflecting medicine no longer can be effectively controlled infection.The fact that pathogenic bacteria generates drug resistance to antibiotic, soon
It is proved, and the impetus that some pathogenic bacteria drug resistances occur and propagate makes us staring.Therefore, the same of abuse of antibiotics is controlled
When, need to continuously search for new antibacterial approach.
Summary of the invention
It is an object of the invention to overcome deficiency in the prior art, a kind of streptococcus is provided, there is stronger antibacterial work
Property, in terms of antibacterials with good development and application prospects.
The first aspect of the invention is to provide a kind of streptococcus, is a novel species in streptococcus, name are as follows:
Streptococcus zengyii sp.nov.HTS20 registers preservation in China General Microbiological culture presevation administrative center,
Deposit number is CGMCC 15343.
The second aspect of the invention is to provide streptococcus described in first aspect of the present invention and is preparing antagonism golden yellow
Application in staphylococcus drug.
It is golden yellow in preparation prevention and treatment that the third aspect of the invention is to provide streptococcus described in first aspect of the present invention
Application in disorder agent caused by staphylococcus.
The fourth aspect of the invention is to provide streptococcus described in first aspect of the present invention and is preparing the white false silk of antagonism
Application in saccharomycete drug.
The fifth aspect of the invention is to provide streptococcus described in first aspect of the present invention in the white false silk of preparation prevention and treatment
Application in disorder agent caused by saccharomycete.
The sixth aspect of the invention is to provide streptococcic fermentation liquid or fermentation liquid described in first aspect of the present invention
Filtered fluid.
The seventh aspect of the invention is to provide streptococcic fermentation liquid or fermentation liquid described in the 6th aspect of the present invention
Application of the filtered fluid in plant antagonism staphylococcus aureus drug.
The eighth aspect of the invention is to provide streptococcic fermentation liquid or fermentation liquid described in the 6th aspect of the present invention
Filtered fluid preparation prevention and treatment caused by Staphylococcus aureus disorder agent in application.
The ninth aspect of the invention is to provide streptococcic fermentation liquid or fermentation liquid described in the 6th aspect of the present invention
Filtered fluid preparing the application in antagonism candida albicans bacterium drug.
The tengh aspect of the invention is to provide streptococcic fermentation liquid or fermentation liquid described in the 6th aspect of the present invention
Filtered fluid prevent and treat the application caused by candida albicans bacterium in disorder agent in preparation.
Streptococcus Streptococcus zengyii sp.nov.HTS20 of the invention is Grain-positive, catalase
Feminine gender, arranges catenulate ball bacteria, tentatively represents streptococcic newcomer.16S rRNA gene order studies have shown that
Streptococcus zengyii sp.nov.HTS20 and S.cuniculi, the phase of S.acidominimus and S.marmotae
It is respectively 98.1%, 97.5% and 97.2% like property.Conserved sequence sodA gene sequencing shows Streptococcus
The similitude of zengyii sp.nov.HTS20 and S.marmotae are nearest, are 94.7%;RpoB gene sequencing shows
The similitude of Streptococcus zengyii sp.nov.HTS20 and S.cuniculi are nearest, are 94.6%;GroEL sequence
Analysis shows the similarity highest of Streptococcus zengyii sp.nov.HTS20 and S.himalayensis, are
85.5%.By carrying out the analysis of Pacbio genome sequencing to Streptococcus zengyii sp.nov.HTS20,
DNA-DNA molecule hybrid experiment shows that Streptococcus zengyii sp.nov.HTS20 is known streptococcic miscellaneous with other
Double ratio value is below 70% between 19.9%~29.3%.On the basis of its phenotypic characteristic and systematic growth research, mention
The novel species that the bacterial strain is classified as streptococcus, Streptococcus zengyii sp.nov.HTS20 genome are gone out
Size is 1,941,976bp, includes 1865 genes, G/C content 42.9mol%.
Streptococcus Streptococcus zengyii sp.nov.HTS20 of the invention in a variety of fermentation mediums into
Row culture, can produce stronger Substance, and have to staphylococcus aureus and candida albicans bacterium more apparent
Bacteriostasis, it is empty with wide development in the prevention and treatment to the associated diseases such as staphylococcus aureus and candida albicans bacterium
Between, there is good development and application prospect in terms of antibacterials.
Detailed description of the invention
Fig. 1 is Streptococcus zengyii sp.nov.HTS20 in Colombia's blood plate colonial morphology figure (37
℃,36h)。
There is α haemolysis in Colombia's blood plate for Streptococcus zengyii sp.nov.HTS20 in Fig. 2
(37℃,48h)。
Fig. 3 is Streptococcus zengyii sp.nov.HTS20 Grain stain figure.
Fig. 4 is Streptococcus zengyii sp.nov.HTS20 scanning electron microscope (SEM) photograph.
Fig. 5 is the Streptococcus zengyii sp.nov.HTS20 and chain constructed based on 16SrDNA gene order
The main group's type strain phylogenetic relationship figure of Coccus.
Fig. 6 is the evolution of the Streptococcus zengyii sp.nov.HTS20 constructed based on conserved genetic sequences
Tree.
Fig. 7 is the fermentation liquid of Streptococcus zengyii sp.nov.HTS20 to the antibacterial of staphylococcus aureus
Test result figure.
Fig. 8 is the antibacterial examination of the fermentation liquid dialogue candidiasis of Streptococcus zengyii sp.nov.HTS20
Test result figure.
Specific embodiment
Below with reference to specific embodiment, the present invention is further illustrated, to better understand the invention.
The present invention provides a kind of streptococcus, are a novel species in streptococcus, name are as follows: Streptococcus
Zengyii sp.nov.HTS20 registers preservation, deposit number CGMCC in China General Microbiological culture presevation administrative center
15343, the deposit date is 2018-02-06.Streptococcus Streptococcus zengyii sp.nov.HTS20 of the invention from
It is separated in marmota himalayana respiratory tract specimens, screening obtains.
1 streptococcus novel species morphological observation
1.1 bacterium source
Marmota himalayana respiratory tract specimens.Healthy adult marmot tunica mucosa tracheae sample is taken, using sterile working clip drought
(about 20mg) is ground in otter inner surface of trachea tissue merging dismembyator, the mucous membrane after grinding is homogenized 300 μ L brain heart infusions
(BHI) it dilutes, then transfer has been loaded with high speed in the 2mL sterile centrifugation tube of 1.5mL PBS and is vortexed, later low-speed centrifugal, carefully
About two hectolambda supernatants are drawn, is spread evenly across on BHI sheep blood plate with spreading rod, is placed in 5%CO237 DEG C of incubator trainings
It supports, in triplicate, for screening aerobic bacteria.
1.2 morphological observation
Streptococcus novel species through isolating and purifying is transferred on Colombia's blood plate, 5%CO is set237 DEG C of incubator cultures
24-48h.Streptococcus zengyii sp.nov.HTS20 is cultivated for 24 hours for 37 DEG C on Columbia Blood Agar culture medium
Diameter 0.5-0.9mm is formed afterwards, and canescence, rounded protuberance, wet, neat in edge the tiny bacterium colony of tip-like (Fig. 1) are lasting to train
After supporting 48h, there is the grass green zone of hemolysis (Fig. 2) of 0.9mm or so in its periphery of bacterial colonies.Bacterium leather is blue under the microscope for optical microphotograph
The positive, single thallus is spherical in shape, pairs of or several short catenations (Fig. 3).Electric bacterium under the microscope is in streptococcus characteristic feature:
Single thallus is presented spherical or oval, and about 0.5-0.9 μm of diameter, thallus is pairs of or several short catenations, no brood cell, without whip
Hair (Fig. 4).
1.3 Streptococcus zengyii sp.nov.HTS20 sodium chloride tolerances test (table 1)
1 Streptococcus zengyii sp.nov.HTS20 sodium chloride tolerance of table
2.5g/L | 3.5g/L | 4.5g/L | 6.5g/L | |
HTS20 | + | ? | ? | ? |
Streptococcus zengyii sp.nov.HTS20 is grown on the BHI culture medium containing 2.5%NaCl, super
It crosses on the BHI of 2.5%NaCl and does not grow.
1.4 Streptococcus zengyii sp.nov.HTS20 temperature tolerance tests (table 2)
The tolerance of 2 Streptococcus zengyii sp.nov.HTS20 temperature of table
Streptococcus zengyii sp.nov.HTS20 is grown in 22,30,37,42 DEG C of cultures, at 4,15 DEG C
It does not grow.
The identification of 2 biochemical tests
The identification of 2.1 API Rapid ID 32Strep biochemical reactions
A point pure thallus is inoculated into Colombia's blood plate, is placed in CO237 DEG C of culture 18-24h of incubator.It records first
Haemolysis type (α or β) and chromogenic plain situation (being tested as supplement).Then it is arrived with cotton swab or the enough bacterium colonies of oese picking
It in the API suspension medium ampoule bottle of 2mL, is measured using nephometer, 4.0 Maxwell turbidity bacteria suspensions is obtained, then by the suspension
Turn to be added in reacting hole, every hole is separately added into 55 μ L, and slight concussion mixes, and covers reaction lid, is placed in CO237 DEG C of incubator trainings
Observation is as a result, be shown in Table 3 after supporting 4-4.5h.
The identification of 2.2 API, 50 CH biochemical reaction
A point pure thallus is inoculated into Colombia's blood plate, is placed in 37 DEG C of culture 18-24h of CO2 incubator.Then cotton is used
Swab or the enough bacterium colonies of oese picking obtain 4.0 Maxwell turbidity, are then transferred to the suspension into 1mL distilled water
It in API50CHB culture medium ampoule bottle, mixes, then turns to be added in reacting hole by the suspension, every hole is separately added into 100 μ L, most
Glycerol moisturizing is added dropwise in every hole afterwards, covers reaction lid, and observation is as a result, be shown in Table 3 after being placed in 37 DEG C of CO2 incubator cultures for 24 hours.
The identification of 2.3 API ZYM biochemical reactions
A point pure thallus is inoculated into Colombia's blood plate, is placed in 37 DEG C of culture 18-24h of CO2 incubator.Then cotton is used
In swab or the enough bacterium colonies of oese picking to the API suspension medium ampoule bottle of 2mL, preparing turbidity is 6.0 Maxwell turbidity,
Then the suspension is moved in API 50CHB culture medium ampoule bottle, mixes, 65 μ L bacteria suspensions is then taken to be added in each reaction cup,
It mixes, covers the reaction lid on biochemical identification reagent strip, observation is as a result, be shown in after being placed in 37 DEG C of culture 4-4.5h of CO2 incubator
Table 3.
3 Streptococcus zengyii sp.nov.HTS20 of table source streptococcus biochemical reaction result ratio close with other
Compared with
3 streptococcus novel species 16SrRNA taxonomic identifications
Extracting genome DNA, 16S rRNA sequence PCR amplification, pcr amplification product, which directly send Beijing to hold up section's biotechnology, to be had
Limit company carries out sequencing through company after purification.
Data statistic analysis: first splicing gained sequence using SeqMan software, DNA star software, forward and reverse
Detection, and remove carrier and primer sequence.MAGA6.0 software is used later, is constructed using neighbouring-connection method and maximum likelihood method
Phyletic evolution tree finally tests to chadogram generated with Bootstrap.With enterococcus spp
Enterococcus faecalis ATCC 19433TAs outer group.As a result as shown in Figure 5, it can be seen that Streptococcus
Zengyii sp.nov.HTS20 is on an independent evolutionary branching on clustering tree, and S.cuniculi,
The similitude of S.acidominimus is respectively 98.1%, 97.5%, shows the affiliation relative close between their threes.
The identity value that other known kind of Blast of Streptococcus zengyii sp.nov.HTS20 and streptococcus is compared is equal
Lower than 97%, affiliation is farther out.Research shows that supporting Streptococcus zengyii from 16SrRNA categorization levels
Sp.nov.HTS20 belongs to a novel species in streptococcus.
4 streptococcus novel species conservative gene taxonomic identifications
In order to further identify the classification position of streptococcus novel species, it is remote with known streptococcic affiliation to study them
Closely, this project has carried out taxonomic identification research for its conservative gene sodA, rpoB and groEL.
SodA, rpoB and groEL extension increasing sequence and Genbank of Streptococcus zengyii sp.nov.HTS20
Middle sequence carries out Blast and compares analysis, and the nearly source kind high with its identity value belongs to the " Suis of streptococcus as the result is shown
group".Wherein sodA gene Blast compare analysis, Streptococcus zengyii sp.nov.HTS20 with
The similitude of S.marmotae is nearest, is 94.7%;RpoB gene Blast compares analysis, Streptococcus zengyii
The similitude of sp.nov.HTS20 and S.cuniculi is nearest, is 94.6%.Streptococcus as seen from Figure 6
Zengyii sp.nov.HTS20 gets together as an independent branch, the parent with S.cuniculi and S.acidominimus
Edge relationship relative close, research shows that being supported from three conservative gene (sodA, rpoB and groEL) categorization levels
Streptococcus zengyii sp.nov.HTS20 belongs to a novel species in streptococcus, the bacterium and S.cuniculi and
S.acidominimus has closer affiliation.The result matches with the chadogram result of its 16SrRNA sequence construct.
The analysis of 5 streptococcus novel species genomics
This project has carried out full genome after the pure culture for obtaining Streptococcus zengyii sp.nov.HTS20
Group sequencing has carried out forecast analysis to its genome essential characteristic, and kind carries out known to other to type strain and streptococcus
Phylogenetic analysis.
5.1 genome sequencings and assembling
The genome sequencing work of Streptococcus zengyii sp.nov.HTS20 is had by Beijing promise and biology
Limit company completes.Using the real-time sequencing technologies pair of unimolecule of three generations microarray dataset Pacific Biosciences
Streptococcus zengyii sp.nov.HTS20 full-length genome is measured, soft using SMRT Analysis 2.3.0
Part packet carries out Quality Control filtering to sequencing data, and splicing is assembled into complete cyclic annular genome sequence.
The analysis of 5.2 genome essential characteristics
Encoding gene CDS prediction is carried out with GeneMarkS (http://topaz.gatech.edu/) software, is used
TRNAs and rRNA in tRNAscan-SE and rRNAmmer software prediction genome.It is carried out with IslandPath-DIOMB software
Genomic islands prediction.This research is by three generations's microarray dataset Pacific Biosciences (PacBio) to from marmota himalayana
The Streptococcus zengyii sp.nov.HTS20 that respiratory tract is separated to carries out genome sequencing.
Streptococcus zengyii sp.nov.HTS20 full-length genome 1,941,976bp, G+C content are 42.9mol%, are compiled
1,865 genes of code.
6 genomic DNAs-DNA hybridization
It is right using DNA-DNA hybridization online software (genome and genome distance calculator, GGDC)
Can be retrieved in the whole genome sequence of Streptococcus zengyii sp.nov.HTS20 and the current library GenBank 65
A streptococcus representative sequence not of the same race carries out online genomic hybridization, and DNA-DNA hybridization similitude is greater than 70% and is divided into
Same kind.It the results are shown in Table 4.
This research will receive in Streptococcus zengyii sp.nov.HTS20 whole genome sequence and DDH database
The representative genome sequence that known kind of the streptococcus of record carries out DNA-DNA and hybridizes online, from table 4, it can be seen that
Streptococcus zengyii sp.nov.HTS20 with other it is known it is streptococcic hybridize ratio between 19.9%~
Between 29.3%, it is below 70% threshold value, it is any to illustrate that Streptococcus zengyii sp.nov.HTS20 is not belonging to
Known kind, be a novel species of streptococcus.
Table different from known streptococcus kinds of genomes of 4. Streptococcus zengyii sp.nov.HTS20 streptococcus
Results of hybridization
7 bacteriostatic activities
Fermented and cultured is carried out to Streptococcus zengyii sp.nov.HTS20 using different fermentation mediums
Corresponding antibacterial substance is obtained, with the bacteriostasis of filter paper agar diffusion method measurement fermentation liquid, concrete operation step is such as
Under, as a result see Fig. 7 and Fig. 8, concrete analysis the results are shown in Table 5.
The preparation of 7.1 Streptococcus zengyii sp.nov.HTS20 extractive from fermentative:
With the oese similar Streptococcus zengyii of the picking form on blood agar plate respectively
Sp.nov.HTS20 single colonie 10~15, it is inoculated in fermentation culture I, fermentation culture II and fermentation culture III respectively
In, every kind of fermentation culture each 1500mL, 37 DEG C, 180 turns of shaking table culture 36h.
Taking fermented supernatant fluid by fermentation culture 13, after 000rpm centrifugation 20min, (if after centrifugation, bacteria-eliminating efficacy is bad, can
With miillpore filter filtration sterilization again).
It is extracted using ethyl acetate extraction, respectively obtains 81.3mg (fermentation culture I), 74mg (fermented and cultured
Liquid II) and 65.4mg (fermentation culture III) extractive from fermentative.
It by 3 kinds of extractive from fermentative are diluted to concentration is respectively that 5mg/50 μ L is spare with methanol.
The preparation of 7.2 test strains (staphylococcus aureus, candida albicans bacterium) MH plate:
With the oese similar staphylococcus aureus of picking form, candida albicans bacterium 5 on blood agar plate respectively
~10, culture transferring is in meat soup pipe respectively.
37 DEG C of incubator 4~6h of culture if bacterial concentration is too dense, can be used compared with standard opacity tube (0.5 Maxwell turbidity)
Meat soup or normal saline dilution are extremely identical as standard opacity tube turbidity.
Pick staphylococcus aureus, candida albicans bacterium bacterium solution with aseptic cotton carrier respectively, and on tube wall squeeze go it is extra
Bacterium solution after, be respectively coated on MH plate (note: coating will uniformly, it is fine and close), to dry.
7.3 test methods (filter paper agar diffusion method):
It is 5mg/50 μ L's that 25 μ L concentration, which are added, in the aseptic filter paper on piece of every diameter 0.8cm respectively
Streptococcus zengyii sp.nov.HTS20 extractive from fermentative is clean to the organic solvent volatilization in extractive from fermentative
Afterwards, the scraps of paper being clamped respectively with aseptic nipper, being attached on the MH plate of coated test strain bacterium solution, each plate pastes 3 samples
Product filter paper (being fermentation culture I, the extract of fermentation culture II and fermentation culture III respectively).Complete aforesaid operations
Afterwards, culture dish is observed to test result and record data after 37 DEG C of culture 12h, with vernier caliper measurement inhibition zone size.Often
A sample does 3, and calculating takes its average value.
The antibacterial result of 5 Streptococcus zengyii sp.nov.HTS20 of table
Indicate bacterium culture medium | Staphylococcus aureus | Candida albicans bacterium |
Fermentation medium I (LB) | 8-10mm | 11-13mm |
Fermentation medium II (nutrient broth) | 8-10mm | 9-11mm |
Fermentation medium III (BHI) | 5-7mm | 9-10mm |
Fermentation medium I in upper table includes the weight proportion of following component: tryptone 10.0g/L;Yeast extract
5.0g/L;NaCl10.0g/L;pH7.0.Fermentation medium II includes the weight proportion of following component: peptone 10.0g/L;Ox
Digested tankage 3.0g/L;Sodium chloride 5.0g/L;pH7.0.Fermentation medium III includes the weight proportion of following component: peptone 10.0g/
L;It is dehydrated small bovine brain leaching powder 12.5g/L;It is dehydrated beef heart infusion 5.0g/L;Sodium chloride 5.0g/L;Glucose 2.0g/L;Phosphoric acid hydrogen two
Sodium 2.5g/L;pH7.4.
It can be seen that Streptococcus zengyii of the invention from the bacteriostatic activity of table 5 analysis result
Sp.nov.HTS20 bacterial strain is cultivated in different fermentation mediums, can produce stronger Substance, and right
Staphylococcus aureus and candida albicans bacterium have obvious bacteriostasis.Therefore, bacterial strain of the invention is in antibacterials
Aspect has potential application prospect.
Specific embodiments of the present invention are described in detail above, but it is merely an example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, any couple of present invention carries out equivalent modifications and
Substitution is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and
Modification, all should be contained within the scope of the invention.
Claims (10)
1. a kind of streptococcus, which is characterized in that name are as follows: Streptococcus zengyii sp.nov.HTS20, in China
General Microbiological Culture preservation administrative center registers preservation, and deposit number is CGMCC 15343.
2. streptococcus as described in claim 1 is preparing the application in antagonism staphylococcus aureus drug.
3. application of the streptococcus as described in claim 1 in preparation prevention and treatment caused by Staphylococcus aureus disorder agent.
4. streptococcus as described in claim 1 is preparing the application in antagonism candida albicans bacterium drug.
5. streptococcus as described in claim 1 prevents and treats the application in disorder agent caused by candida albicans bacterium in preparation.
6. the filtered fluid of streptococcic fermentation liquid described in claim 1 or fermentation liquid.
7. the filtered fluid of streptococcic fermentation liquid as claimed in claim 6 or fermentation liquid is preparing antagonism staphylococcus aureus
Application in drug.
8. the filtered fluid of streptococcic fermentation liquid as claimed in claim 6 or fermentation liquid prevents and treats staphylococcus aureus in preparation
Application in caused disorder agent.
9. the filtered fluid of streptococcic fermentation liquid as claimed in claim 6 or fermentation liquid is preparing antagonism candida albicans bacterium medicine
Application in object.
10. the filtered fluid of streptococcic fermentation liquid as claimed in claim 6 or fermentation liquid prevents and treats candida albicans bacterium in preparation
Application in caused disorder agent.
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Streptococcus marmotae sp. nov.,isolated from the respiratory tract of Marmota himalayana;Lina Niu et al.;《International Journal of Systematic and Evolutionary Microbiology》;20161231;第66卷;全文 * |
三个动物源性链球菌新种的发现、基因组及毒力基因分析;牛莉娜;《中国博士学位文论全文数据库(电子期刊)农业科技辑》;20180215;全文 * |
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