CN108485999B - A kind of streptococcus novel species HTS25 and its application - Google Patents

A kind of streptococcus novel species HTS25 and its application Download PDF

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CN108485999B
CN108485999B CN201810187740.9A CN201810187740A CN108485999B CN 108485999 B CN108485999 B CN 108485999B CN 201810187740 A CN201810187740 A CN 201810187740A CN 108485999 B CN108485999 B CN 108485999B
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牛莉娜
吕刚
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Hainan Medical College
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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.HTS25 registers preservation in China General Microbiological culture presevation administrative center, and deposit number is CGMCC 15344.Streptococcus zengyii sp.nov.HTS25 of the invention is cultivated in a variety of fermentation mediums, it can produce stronger Substance, and there is obvious bacteriostasis to pseudomonas aeruginosa and Salmonella typhi, there is wide development space in the prevention and treatment to associated diseases such as pseudomonas aeruginosa and Salmonella typhis, there is good development and application prospect in terms of antibacterials.

Description

A kind of streptococcus novel species HTS25 and its application
Technical field
The invention belongs to microorganism fields, and in particular to a kind of streptococcus novel species HTS25 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.HTS25 registers preservation in China General Microbiological culture presevation administrative center, Deposit number is CGMCC 15344.
The second aspect of the invention is to provide streptococcus described in first aspect of the present invention and is preparing antagonism verdigris vacation Application in monad drug.
It is false in preparation prevention and treatment verdigris 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 monad.
The fourth aspect of the invention is to provide streptococcus described in first aspect of the present invention and is preparing antagonism typhoid fever sand Application in door bacterium drug.
It is husky in preparation prevention and treatment typhoid fever that the fifth aspect of the invention is to provide streptococcus described in first aspect of the present invention Application in disorder agent caused by door bacterium.
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 Filtered fluid preparing the application in antagonism pseudomonas aeruginosa 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 prevent and treat the application caused by pseudomonas aeruginosa in disorder agent in preparation.
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 Salmonella typhi 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 Salmonella typhi in disorder agent in preparation.
Streptococcus Streptococcus zengyii sp.nov.HTS25 of the invention is Grain-positive, catalase Feminine gender, arranges catenulate ball bacteria, 16S rRNA gene order studies have shown that Streptococcus zengyii Sp.nov.HTS25 and S.cuniculi, S.acidominimus, the similitude of S.marmotae and S.himalayensis Respectively 98%, 97.4%, 97.2% and 97.1%.Conserved sequence analysis shows, Streptococcus zengyii SodA the and rpoB gene of sp.nov.HTS25 is nearest with the similitude of S.marmotae respectively, respectively 94.7% He 91.4%.GroEL sequence analysis shows, Streptococcus zengyii sp.nov.HTS25's and S.himalayensis Similarity highest is 84.8%.By carrying out Pacbio full genome to Streptococcus zengyii sp.nov.HTS25 Group sequencing analysis, DNA-DNA molecule hybrid experiment show Streptococcus zengyii sp.nov.HTS25 with other Know that streptococcic hybridization ratio between 16.4%~30.9%, is below 70% threshold value.To its phenotypic characteristic and system Research shows that Streptococcus zengyii sp.nov.HTS25 is a streptococcus novel species, Genome Size is for development 2,067,971bp, it include 2001 genes, G/C content 42.7mol%.
Streptococcus Streptococcus zengyii sp.nov.HTS25 of the invention in a variety of fermentation mediums into Row culture, can produce stronger Substance, and have obvious suppression to pseudomonas aeruginosa and Salmonella typhi Bacterium effect, has wide development space in the prevention and treatment to associated diseases such as pseudomonas aeruginosa and Salmonella typhis, anti- There is good development and application prospect in terms of bacterium drug.
Detailed description of the invention
Fig. 1 is Streptococcus zengyii sp.nov.HTS25 in Colombia's blood plate colonial morphology figure (37 ℃,36h)。
Fig. 2 is Streptococcus zengyii sp.nov.HTS25 Grain stain figure.
Fig. 3 is that Streptococcus zengyii sp.nov.HTS25 has an X-rayed electron microscope.
Fig. 4 is the Streptococcus zengyii sp.nov.HTS25 and chain constructed based on 16SrDNA gene order The main group's type strain phylogenetic relationship figure of Coccus.
Fig. 5 is the evolution of the Streptococcus zengyii sp.nov.HTS25 constructed based on conserved genetic sequences Tree.
Fig. 6 is antibacterial examination of the fermentation liquid of Streptococcus zengyii sp.nov.HTS25 to pseudomonas aeruginosa Test result figure.
Bacteriostatic test of the fermentation liquid to Salmonella typhi that Fig. 7 is Streptococcus zengyii sp.nov.HTS25 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.HTS25 registers preservation, deposit number CGMCC in China General Microbiological culture presevation administrative center 15344, the deposit date is 2018-02-06.Streptococcus Streptococcus zengyii sp.nov.HTS25 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.HTS25 is cultivated for 24 hours for 37 DEG C on Columbia Blood Agar culture medium Diameter 0.5-1.0mm 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 of 1mm or so in its periphery of bacterial colonies.The optical microphotograph microscopic observation bacterium Grain-positive, it is single A thallus is spherical in shape, pairs of or several short catenations (Fig. 2).Electric bacterium under the microscope is in streptococcus characteristic feature: single bacterium Body is presented spherical or oval, and about 0.5-1.0 μm of diameter, thallus is pairs of or several short catenations, no brood cell, atrichia (figure 3)。
1.3Streptococcus zengyii sp.nov.HTS25 sodium chloride tolerance tests (table 1)
Table 1Streptococcus zengyii sp.nov.HTS25 sodium chloride tolerance
2.5g/L 3.5g/L 4.5g/L 6.5g/L
HTS25 + ? ? ?
Streptococcus zengyii sp.nov.HTS25 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.
1.4Streptococcus zengyii sp.nov.HTS25 temperature tolerance test (table 2)
Table 2Streptococcus zengyii sp.nov.HTS25 sodium chloride tolerance
4℃ 15℃ 22℃ 35℃ 37℃ 42℃
HTS25 ? + (after 48h) + +++ ++++ ++
Streptococcus zengyii sp.nov.HTS25 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 32 Strep biochemical reaction of 2.1API Rapid ID
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 50 CH biochemical reaction of 2.2API
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 API50 CHB culture medium ampoule bottle, mixing, then turns to be added in reacting hole by the suspension, every hole is separately added into 100 μ L, Glycerol moisturizing is added dropwise in last every hole, 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 2.3API ZYM 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 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.
The identification of 2.4Lancefield group specific antigen
Group is divided to require to extract bacterium colony antigen according to Lancefield, with dividing group's serum A, B, C, D, F and G to grasp according to kit It explains and does agglutination test, the results are shown in Table 3.
Compared with table 3Streptococcus zengyii sp.nov.HTS25 source streptococcus biochemical reaction result close with other
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.6.0 software of MAGA is used later, using neighbouring-connection method and maximum likelihood method structure Phyletic evolution tree is built, is finally tested to chadogram generated with Bootstrap.With enterococcus spp Enterococcus faecalis ATCC 19433TAs outer group.As a result as shown in Figure 4, it can be seen that Streptococcus Zengyii sp.nov.HTS25 is in an independent evolutionary branching on clustering tree, and S.cuniculi, S.acidominimus is polymerized to cluster, and Bootstrap supporting rate is 100%, shows closer affiliation. The identity value that other known kind of Blast of Streptococcus zengyii sp.nov.HTS25 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.HTS25 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.HTS25 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".Streptococcus zengyii sp.nov.HTS25 is as an independent branch as seen from Figure 5, with The affiliation relative close of S.cuniculi and S.acidominimus, research shows that from three conservative genes (sodA, rpoB And groEL) categorization levels support Streptococcus zengyii sp.nov.HTS25 belong to a novel species in streptococcus, The bacterium and S.cuniculi and S.acidominimus have closer affiliation.The result and its 16SrRNA sequence construct Chadogram result is consistent.
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.HTS25 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.HTS25 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.25 full-length genome is measured, using SMRT Analysis2.3.0 software package Quality Control filtering is carried out to sequencing data, 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.HTS25 that respiratory tract is separated to carries out genome sequencing. Streptococcus zengyii sp.nov.HTS25 full-length genome 2,067,971bp, G+C content are 42.7mol%, are compiled 2,001 gene of code, 1 plasmid (26,370bp), 8 genomic islands.
6 genomic DNAs-DNA hybridization
It is right using DNA-DNA hybridization online software (genome and genome distance calculator, GGDC) The chain that can be retrieved in the whole genome sequence of Streptococcus zengyii sp.nov.HTS25 and the current library GenBank Coccus 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 together One kind.It the results are shown in Table 4.
This research will receive in Streptococcus zengyii sp.nov.HTS25 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.HTS25 with other it is known it is streptococcic hybridize ratio between 16.4%~ Between 30.9%, it is below 70% threshold value, it is any to illustrate that Streptococcus zengyii sp.nov.HTS25 is not belonging to Known kind, be a novel species of streptococcus.
Different from known streptococcus kinds of genomes of table 4.Streptococcus zengyii sp.nov.HTS25 streptococcus Results of hybridization
7 bacteriostatic activities
Fermented and cultured is carried out to Streptococcus zengyii sp.nov.HTS25 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. 6 and Fig. 7, concrete analysis the results are shown in Table 5.
The preparation of 7.1Streptococcus zengyii sp.nov.HTS25 extractive from fermentative:
With the oese similar Streptococcus zengyii of the picking form on blood agar plate respectively Sp.nov.HTS25 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 85.2mg (fermentation culture I), 79mg (fermented and cultured Liquid II) and 67.8mg (fermentation culture III) extractive from fermentative.
It is that 5mg/50 μ L is spare that 3 kinds of extractive from fermentative, which are dissolved to concentration, respectively with methanol.
The preparation of 7.2 test strains (pseudomonas aeruginosa, Salmonella typhi) MH plate:
With the oese similar pseudomonas aeruginosa of picking form, Salmonella typhi 5~10 on blood agar plate respectively A, 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.
Pseudomonas aeruginosa, Salmonella typhi bacterium solution are picked with aseptic cotton carrier respectively, is squeezed on tube wall and goes extra bacterium solution Afterwards, (note: coating is uniform, fine and close) is respectively coated on MH plate, 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.HTS25 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.HTS25 of table
Indicate bacterium culture medium Pseudomonas aeruginosa Salmonella typhi
Fermentation medium I (LB) 13-15mm 15-16mm
Fermentation medium II (nutrient broth) 11-13mm 11-12mm
Fermentation medium III (BHI) 6-7mm 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.HTS25 bacterial strain is cultivated in different fermentation mediums, can produce stronger Substance, and right Pseudomonas aeruginosa and Salmonella typhi have obvious bacteriostasis.Therefore, bacterial strain of the invention is in terms of antibacterials With 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.HTS25, in China General Microbiological Culture preservation administrative center registers preservation, and deposit number is CGMCC 15344.
2. streptococcus as described in claim 1 is preparing the application in antagonism pseudomonas aeruginosa drug.
3. streptococcus as described in claim 1 prevents and treats the application in disorder agent caused by pseudomonas aeruginosa in preparation.
4. streptococcus as described in claim 1 is preparing the application in antagonism Salmonella typhi drug.
5. streptococcus as described in claim 1 prevents and treats the application in disorder agent caused by Salmonella typhi 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 pseudomonas aeruginosa medicine Application in object.
8. the filtered fluid of streptococcic fermentation liquid as claimed in claim 6 or fermentation liquid prevents and treats pseudomonas aeruginosa institute in preparation Application in pathogenic disease drug.
9. the filtered fluid of streptococcic fermentation liquid as claimed in claim 6 or fermentation liquid is preparing antagonism Salmonella typhi drug In application.
10. the filtered fluid of streptococcic fermentation liquid as claimed in claim 6 or fermentation liquid prevents and treats Salmonella typhi institute in preparation Application in pathogenic disease drug.
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