CN108485999A - 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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CN108485999A
CN108485999A CN201810187740.9A CN201810187740A CN108485999A CN 108485999 A CN108485999 A CN 108485999A CN 201810187740 A CN201810187740 A CN 201810187740A CN 108485999 A CN108485999 A CN 108485999A
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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, are named as：Streptococcus zengyii sp.nov.HTS25 register preservation in China General Microbiological culture presevation administrative center, and deposit number is CGMCC 15344.The Streptococcus zengyii sp.nov.HTS25 of the present invention are cultivated in a variety of fermentation mediums, stronger Substance can be generated, and there is obvious bacteriostasis to pseudomonas aeruginosa and Salmonella typhi, there is wide development space in the prevention to associated diseases such as pseudomonas aeruginosa and Salmonella typhis, there is good development 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 technology
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 of antibacterials and widely applied climax.
20 middle of century, the antibiotic bulk pharmaceutical chemicals listed are planted up to more than 500, are also up to counted 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.
The medicine general administration in later stage the 1970s, 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 are easy the bacterial infection disease for the treatment of now with new variation, the effective antibiotic of script originally Or synthesising bacteria anti-reflecting medicine no longer can effective infection control.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, it needs to continuously search for new antibacterial approach.
Invention content
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, there is good development prospect in terms of antibacterials.
The first aspect of the invention is to provide a kind of streptococcus, is a novel species in streptococcus, is named as： Streptococcus zengyii sp.nov.HTS25 register preservation in China General Microbiological culture presevation administrative center, Deposit number is CGMCC 15344.
The second aspect of the invention is to provide the streptococcus described in the first aspect of the present invention in antagonism P. aeruginosa Application in bacterium.
The third aspect of the invention is to provide the streptococcus described in the first aspect of the present invention and is preparing prevention verdigris vacation Application in disorder agent caused by monad.
The fourth aspect of the invention is to provide the streptococcus described in the first aspect of the present invention in antagonism Salmonella typhi In application.
The fifth aspect of the invention is to provide the streptococcus described in the first aspect of the present invention and is preparing prevention typhoid fever sand Application in disorder agent caused by door bacterium.
The sixth aspect of the invention is to provide streptococcic zymotic fluid or zymotic fluid described in the first aspect of the present invention Filtered fluid.
The seventh aspect of the invention is to provide streptococcic zymotic fluid or zymotic fluid described in the 6th aspect of the present invention Application of the filtered fluid in antagonism pseudomonas aeruginosa.
The eighth aspect of the invention is to provide streptococcic zymotic fluid or zymotic fluid described in the 6th aspect of the present invention Filtered fluid preparing the application caused by preventing pseudomonas aeruginosa in disorder agent.
The ninth aspect of the invention is to provide streptococcic zymotic fluid or zymotic fluid described in the 6th aspect of the present invention Application of the filtered fluid in antagonism Salmonella typhi.
The tengh aspect of the invention is to provide streptococcic zymotic fluid or zymotic fluid described in the 6th aspect of the present invention Filtered fluid preparing the application caused by preventing Salmonella typhi in disorder agent.
The streptococcus Streptococcus zengyii sp.nov.HTS25 of the present invention are Grain-positive, catalase Feminine gender, arranges catenulate ball bacteria, 16S rRNA gene orders 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 genes of sp.nov.HTS25 are nearest with the similitude of S.marmotae respectively, respectively 94.7% He 91.4%.GroEL sequence analyses show Streptococcus zengyii sp.nov.HTS25's and S.himalayensis Similarity highest is 84.8%.By carrying out Pacbio full genomes to Streptococcus zengyii sp.nov.HTS25 Group sequencing analysis, DNA-DNA molecule hybrid experiments 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 are a streptococcus novel species, Genome Size is for development 2,067,971bp, including 2001 genes, G/C content 42.7mol%.
The present invention streptococcus Streptococcus zengyii sp.nov.HTS25 in a variety of fermentation mediums into Row culture, can generate stronger Substance, and have obvious suppression to pseudomonas aeruginosa and Salmonella typhi Bacterium acts on, and has wide development space in the prevention to associated diseases such as pseudomonas aeruginosa and Salmonella typhis, anti- There is good development prospect in terms of bacterium drug.
Description of the drawings
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 figures.
Fig. 3 is that Streptococcus zengyii sp.nov.HTS25 have an X-rayed electron microscope.
Fig. 4 is the Streptococcus zengyii sp.nov.HTS25 and chain built based on 16SrDNA gene orders The main group's type strain phylogenetic relationship figure of Coccus.
Fig. 5 is the evolution of the Streptococcus zengyii sp.nov.HTS25 built based on conserved genetic sequences Tree.
Fig. 6 is antibacterial examination of the zymotic fluid of Streptococcus zengyii sp.nov.HTS25 to pseudomonas aeruginosa Test result figure.
Bacteriostatic test of the zymotic fluid to Salmonella typhi that Fig. 7 is Streptococcus zengyii sp.nov.HTS25 Result figure.
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated, to more fully understand the present invention.
The present invention provides a kind of streptococcus, are a novel species in streptococcus, are named as：Streptococcus Zengyii sp.nov.HTS25 register preservation, deposit number CGMCC in China General Microbiological culture presevation administrative center 15344, the deposit date is 2018-02-06.The present invention streptococcus Streptococcus zengyii sp.nov.HTS25 from It is detached 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 It is ground (about 20mg) in otter inner surface of trachea tissue merging mill, 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 tubes 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 plates with spreading rod, is placed in 5%CO237 DEG C of trainings of incubator 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 cultures of incubator 24-48h.Streptococcus zengyii sp.nov.HTS25 are 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, moistening, the tiny bacterium colony of the tip-like of neat in edge (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 thalline 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, about 0.5-1.0 μm of diameter, and thalline is pairs of or several short catenations, no brood cell, atrichia (figure 3)。
1.3 Streptococcus zengyii sp.nov.HTS25 sodium chloride tolerances test (table 1)
1 Streptococcus zengyii sp.nov.HTS25 sodium chloride tolerances of table
2.5g/L 3.5g/L 4.5g/L 6.5g/L
HTS25 + - - -
Streptococcus zengyii sp.nov.HTS25 are grown on the BHI culture mediums containing 2.5%NaCl, super It crosses on the BHI of 2.5%NaCl and does not grow.
1.4 Streptococcus zengyii sp.nov.HTS25 temperature tolerance tests (table 2)
2 Streptococcus zengyii sp.nov.HTS25 sodium chloride tolerances of table
4℃ 15℃ 22℃ 35℃ 37℃ 42℃
HTS25 - + (after 48h) + +++ ++++ ++
Streptococcus zengyii sp.nov.HTS25 are grown when being cultivated at 22,30,37,42 DEG C, at 4,15 DEG C It does not grow.
2 biochemical tests are identified
2.1 API Rapid ID 32Strep biochemical reactions are identified
A point pure thalline 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 the enough bacterium colonies of cotton swab or oese picking It in the API suspension medium ampoule bottles 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, be separately added into 55 μ L per hole, slightly shake mixing, covers reaction lid, be placed in CO237 DEG C of trainings of incubator Observation is as a result, be shown in Table 3 after supporting 4-4.5h.
2.2 API 50CH biochemical reactions are identified
A point pure thalline is inoculated into Colombia's blood plate, is placed in 37 DEG C of culture 18-24h of CO2 incubators.Then cotton is used In swab or the enough bacterium colony to 1mL distilled water of oese picking, 4.0 Maxwell turbidity are obtained, are then transferred to the suspension In API50CHB culture medium ampoule bottles, then which is turned to be added in reacting hole by mixing, be separately added into 100 μ L per hole, most Glycerine moisturizing is added dropwise per hole afterwards, covers reaction lid, observation is as a result, be shown in Table 3 after being placed in the 37 DEG C of cultures for 24 hours of CO2 incubators.
2.3 API ZYM biochemical reactions are identified
A point pure thalline is inoculated into Colombia's blood plate, is placed in 37 DEG C of culture 18-24h of CO2 incubators.Then cotton is used In swab or the enough bacterium colony to the API suspension medium ampoule bottles of 2mL of oese picking, it is 6.0 Maxwell turbidity to prepare turbidity, Then the suspension to be moved in API 50CHB culture medium ampoule bottles, then mixing takes 65 μ L bacteria suspensions to be added in each reaction cup, Mixing covers the reaction lid on biochemical identification reagent strip, and observation is as a result, be shown in after being placed in 37 DEG C of culture 4-4.5h of CO2 incubators Table 3.
2.4 Lancefield group specific antigens are identified
Divide group to require extraction bacterium colony antigen according to Lancefield, is grasped according to kit with group's serum A, B, C, D, F and G is divided It explains and does agglutination test, the results are shown in Table 3.
3 Streptococcus zengyii sp.nov.HTS25 of table source streptococcus biochemical reaction result ratios close with other Compared with
3 streptococcus novel species 16SrRNA taxonomic identifications
Extracting genome DNA, 16S rRNA sequence PCR amplifications, pcr amplification product, which directly send Beijing to hold up section's biotechnology, to be had Limit company carries out sequencing after purification through company.
Data statistic analysis：SeqMan softwares, DNA star softwares is used to splice gained sequence first, it is forward and reverse Detection, and remove carrier and primer sequence.6.0 softwares of MAGA are used later, using neighbouring-connection method and maximum likelihood method structure Phyletic evolution tree is built, is finally tested with Bootstrap to the chadogram generated.With enterococcus spp Enterococcus faecalis ATCC 19433TAs outer group.The results are shown in Figure 4, it can be seen that Streptococcus Zengyii sp.nov.HTS25 are in an independent evolutionary branching on clustering tree, and S.cuniculi, S.acidominimus is polymerized to cluster, and Bootstrap supporting rates are 100%, show closer affiliation. The identity values that other known kind of Blast of Streptococcus zengyii sp.nov.HTS25 and streptococcus is compared are equal Less 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 as a result display belongs to the " Suis of streptococcus with the high nearly source kind of its identity value group”.Streptococcus zengyii sp.nov.HTS25 are 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 has closer affiliation with S.cuniculi and S.acidominimus.The result and its 16SrRNA sequence construct Chadogram result is consistent.
5 streptococcus novel species genomics are analyzed
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 has been carried out to other known kind of type strain and streptococcus Phylogenetic analysis.
5.1 genome sequencing 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 genomes are measured, using SMRT Analysis 2.3.0 software packages Quality Control filtering is carried out to sequencing data, splicing is assembled into complete cyclic annular genome sequence.
5.2 genome essential characteristics are analyzed
With GeneMarkS (http://topaz.gatech.edu/) software progress encoding gene CDS predictions, it uses TRNAs and rRNA in tRNAscan-SE and rRNAmmer software prediction genomes.It is carried out with IslandPath-DIOMB softwares Genomic islands are predicted.This research is by three generations microarray dataset Pacific Biosciences (PacBio) to from marmota himalayana The Streptococcus zengyii sp.nov.HTS25 that respiratory tract is separated to carry out genome sequencing. Streptococcus zengyii sp.nov.HTS25 full-length genomes 2,067,971bp, G+C contents 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 softwares (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 libraries GenBank Coccus representative sequence not of the same race carries out online genomic hybridization, and DNA-DNA hybridization similitude is divided into together more than 70% One kind.It the results are shown in Table 4.
This research receives Streptococcus zengyii sp.nov.HTS25 whole genome sequences with DDH databases 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 are 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, the bacteriostasis of zymotic fluid is measured with filter paper agar diffusion method, concrete operation step is such as Under, as a result see that Fig. 6 and Fig. 7, concrete analysis the results are shown in Table 5.
The preparation of 7.1 Streptococcus zengyii sp.nov.HTS25 extractive from fermentative：
With oese similar Streptococcus zengyii of the picking form on blood agar plate respectively Sp.nov.HTS25 single bacterium colonies 10~15 are inoculated in fermentation culture I, fermentation culture II and fermentation culture III respectively In, each fermentation culture each 1500mL, 37 DEG C, 180 turns of shaking table culture 36h.
Taking fermented supernatant fluid by fermentation culture 13, after 000rpm centrifugations 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 cultureds Liquid II) and 67.8mg (fermentation culture III) extractive from fermentative.
It is spare that 3 kinds of extractive from fermentative are dissolved to a concentration of 5mg/50 μ L respectively with methanol.
The preparation of 7.2 test strains (pseudomonas aeruginosa, Salmonella typhi) MH tablets：
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, it is respectively coated on MH tablets and (pays attention to：Coating is uniform, fine and close), it waits doing.
7.3 test methods (filter paper agar diffusion method)：
It is added a concentration of 5mg/50 μ L's of 25 μ L in the aseptic filter paper on piece of every diameter 0.8cm respectively Streptococcus zengyii sp.nov.HTS25 extractive from fermentative waits for that the organic solvent volatilization in extractive from fermentative is clean Afterwards, the scraps of paper being gripped respectively with aseptic nipper, being attached on the MH tablets 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, test result and record data are observed after culture dish being cultivated 12h in 37 DEG C, 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 the Streptococcus zengyii of the present invention from the bacteriostatic activity analysis result of table 5 Sp.nov.HTS25 bacterial strains are cultivated in different fermentation mediums, can generate 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 intended only as example, the present invention is simultaneously unlimited It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and It substitutes also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by impartial conversion and Modification, all should be contained within the scope of the invention.

Claims (10)

1. a kind of streptococcus, which is characterized in that be named as：Streptococcus zengyii sp.nov.HTS25, in China General Microbiological Culture preservation administrative center registers preservation, and deposit number is CGMCC 15344.
2. application of the streptococcus as described in claim 1 in antagonism pseudomonas aeruginosa.
3. streptococcus as described in claim 1 is preparing the application in preventing disorder agent caused by pseudomonas aeruginosa.
4. application of the streptococcus as described in claim 1 in antagonism Salmonella typhi.
5. streptococcus as described in claim 1 is preparing the application in preventing disorder agent caused by Salmonella typhi.
6. the filtered fluid of streptococcic zymotic fluid described in claim 1 or zymotic fluid.
7. filtered fluid the answering in antagonism pseudomonas aeruginosa of streptococcic zymotic fluid as claimed in claim 6 or zymotic fluid With.
8. the filtered fluid of streptococcic zymotic fluid as claimed in claim 6 or zymotic fluid is preparing prevention pseudomonas aeruginosa institute Application in pathogenic disease drug.
9. filtered fluid the answering in antagonism Salmonella typhi of streptococcic zymotic fluid as claimed in claim 6 or zymotic fluid With.
10. the filtered fluid of streptococcic zymotic fluid as claimed in claim 6 or zymotic fluid is preparing prevention Salmonella typhi institute Application in pathogenic disease drug.
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CN111471628A (en) * 2020-06-04 2020-07-31 内蒙古农业大学 New streptococcus and application of preparation thereof in inhibiting oral pathogenic bacteria
CN113088472A (en) * 2021-04-21 2021-07-09 海南医学院 Streptococcus sp.64 and application thereof
CN113186128A (en) * 2021-04-21 2021-07-30 海南医学院 New streptococcus strain and application thereof

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