CN106318886B - Rhizobium and application thereof - Google Patents
Rhizobium and application thereof Download PDFInfo
- Publication number
- CN106318886B CN106318886B CN201610697617.2A CN201610697617A CN106318886B CN 106318886 B CN106318886 B CN 106318886B CN 201610697617 A CN201610697617 A CN 201610697617A CN 106318886 B CN106318886 B CN 106318886B
- Authority
- CN
- China
- Prior art keywords
- rhizobium
- pathogen
- pseudomonas aeruginosa
- biofilm
- cgmcc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/41—Rhizobium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses rhizobium and application thereof, and wherein the rhizobium (Rhizobium sp.) are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 11st, 2016, and deposit number is CGMCC No.12205.Rhizobium of the invention can effectively inhibit pathogen biofilm to be formed, and inhibit the pathogen virulence factor, and the tunning or metabolin of the rhizobium can act effectively as the active constituent of pathogen biofilm inhibitor, to can effectively inhibit pathogen biofilm to be formed using rhizobium or its tunning, metabolin, control the toxicity of pathogen, and then therapeutic effect can be enhanced effective for bacterial infection treatment to increase pathogen to the neurological susceptibility of common drug.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to rhizobium and application thereof.
Background technique
Bacterium infection is always to threaten and perplex the significant problem of human health, and 20th century are using penicillin as the antibiosis of representative
The discovery of element opens new page for bacteriosis treatment.In past half a century, most of infectious diseases is obtained
Effectively treatment.But the abuse and irrational use for also having caused antibiotic at the same time, cause bacterial drug resistance to increase.Increasingly
More pathogen Antibiotic Sensitivities, which reduces, even to disappear, and the expansion of Antibiotic Resistance and the enhancing of drug resistance come to bacterium treatment zone
Great challenge, while also grave danger is constituted to publilc health.Recently, some superbacterias, such as methicillin-resistant staphylococcus
Staphylococcus (MRSA), vancomycin resistant enterococci (VRE), multi-drug resistant streptococcus pneumonia (MDRSP), multiple drug resistance tuberculosis bar
Bacterium (MDR-TB) and carbapenem enzyme Klebsiella Pneumoniae (KPC) have multi-drug resistant, exempt from completely to Multiple Classes of Antibiotics
Epidemic disease.This undoubtedly proposes challenge to healthy medicine and hygienic biology.Therefore, the substitution drug or new for the treatment of bacterium infection is found
Method has become urgent need.
Studies have shown that bacterium generates the reason of drug resistance mainly from two aspects: being on the one hand because antibiotic is to disease
Opportunistic pathogen generates stress pressure, and pathogen generates adaptive variation under stress；It on the other hand is because under normal circumstances, causing
Germ will form microorganism envelope (Biofilm, BF), provide one layer of protective barrier for it.Biofilm refer to bacterial adsorption in
Inert matter such as medical material or body mucous membrane surface, are colonized as ecological niche, and secrete polysaccharide matrix, fiber egg
The polysaccharide protein complexes such as white, lipoprotein make bacterium stick to each other and the aggregation winding of itself bacterium colony are wherein formed a kind of film class
Structure.Confirm that the microorganism there are about 90% lives in microorganism by the bacteriosis and BF in membranous system, more than 65%
It is related.By the protection of BF, most drugs can only kill the microorganism on micro- surface, and helpless to internal microorganism, and
Internal microorganism also generates variation continuous to be resistant to the attack of antibiotic at this time.Since BF is in terms of pathogenic bacteria resistance to drugs
Unique principle and great potential, weaken its pathogenic has become by interfering or inhibiting cause of disease bacteria microorganism envelope and prevent instantly
Control the new situation of bacteriosis.
Generation, development and the mature adjusting by bacterial community inductive signal (quorum sensing, QS) of BF, microorganism
Envelope inhibitor (Biofilm inhibitor) is main to inhibit BF growth and structure shape by inhibiting the QS between microorganism to reach
At purpose.The action principle of BF inhibitor is embodied in two aspects: firstly, it does not directly kill microorganism, but
For the resistance to film class formation with existence of microorganism, destroys its living environment and achieve the purpose that control its quantity.Secondly, it is not only
It can control the toxicity of pathogen, and can be increased by inhibiting BF that pathogenic microorganism is made to lose protection to common drug
Neurological susceptibility enhances therapeutic effect.Compared with antibiotic, BF inhibitor will not inducing microbial resistance pressure, not will lead to resistance to
The generation of pharmacological property, no quadratic risk function.Therefore, microorganism envelope inhibitor is the Substitutes For Antibiotic of great potential, is expected to become
Fight the effective means of pathogenic bacteria even superbacteria.
However, current microorganism envelope inhibitor research still have it is to be strengthened.
Summary of the invention
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention
It is to propose a kind of effective means for inhibiting and being attenuated for pathogen biofilm.
It should be noted that the present invention is the following work based on inventor and completes:
The effective means for inhibiting and being attenuated to propose a kind of for pathogen biofilm, inventor are intended to find and isolate
A kind of biofilm inhibits and is attenuated relevant bacterium bacterial strain.In turn, inventor has carried out a series of arduous experiments, from various rings
Separation of bacterial in the sample of border, and the inhibition of pathogen biofilm and attenuation correlation research are carried out to it, and finally surprisingly obtain
Obtaining one plant and capable of preparing inhibits pathogen biofilm to be formed and inhibit the bacterium bacterial strain of the pathogen virulence factor --- root nodule
Bacterium (Rhizobium sp.) NA01.
Thus, in the first aspect of the present invention, the present invention provides a kind of rhizobium (Rhizobium sp.).According to this
The embodiment of invention, it is commonly micro- which on March 11st, 2016 is preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers, deposit number are CGMCC No.12205.
According to an embodiment of the invention, the rhizobium (Rhizobium sp.) are Gram-negative bacteria, bacterium colony is at yellowish
Color, surface is smooth, edge infiltrates, and suitable growth conditions is 25-35 DEG C, pH 6.5-7.5, common LB culture medium；Its 16s
The sequence of rDNA is as shown in SEQ ID NO:1.
It is surprisingly found by the inventors that rhizobium of the invention can effectively inhibit pathogen biofilm to be formed, and inhibit
The pathogen virulence factor, and the tunning of the rhizobium or metabolin can act effectively as pathogen biofilm inhibitor
Active constituent, thus, it can effectively inhibit pathogen biofilm to be formed using rhizobium or its tunning, metabolin, control
The toxicity of pathogen processed, and then can be increased effective for bacterial infection treatment with increasing pathogen to the neurological susceptibility of common drug
Strong therapeutic effect.
Wherein, it should be noted that terminology employed herein " tunning ", which refers to, carries out fermented and cultured to rhizobium
The product obtained afterwards.Term " metabolin " is through the following steps that the substance obtained: being centrifuged, takes to above-mentioned tunning
Clear filtering, obtains filtrate, the filtrate is the metabolin.
In the present invention, " microorganism envelope " is also called " biofilm " for short sometimes.Rhizobium of the invention sometimes by
Referred to as " rhizobium (Rhizobium sp.) NA01 " or " rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 ".
In turn, in the second aspect of the present invention, the present invention provides mentioned-above rhizobium to inhibit pathogen biology
Purposes in envelope formation, reduction pathogen virulence.In other words, rhizobium (Rhizobium sp.) NA01 CGMCC
No.12205 has the function of that pathogen biofilm is inhibited to be formed and be attenuated, so as to be effective to inhibit pathogen raw
Object envelope forms, reduces pathogen virulence.
According to an embodiment of the invention, the rhizobium by its metabolin realize inhibit pathogen biofilm formed and
Reduce the purposes of pathogen virulence.But art personnel are appreciated that the tunning of rhizobium of the invention can also be in this field
It is directly used in and pathogen biofilm is inhibited to form and reduce pathogen virulence.
In the third aspect of the present invention, the present invention provides a kind of biological agents.According to an embodiment of the invention, of the invention
Biological agent active constituent be mentioned-above rhizobium metabolin.But be appreciated that can also by art personnel in this field
To provide the metabolin in the form of the tunning of rhizobium of the invention.
The biological agent is " microorganism envelope inhibitor ".According to an embodiment of the invention, utilizing biology of the invention
Preparation can effectively inhibit pathogen biofilm to be formed, and control the toxicity of pathogen.When being used for bacterial infection treatment,
Pathogen can be effectively increased to the neurological susceptibility of common drug, enhancing therapeutic effect namely biological agent of the invention can have
Effectiveness is in prevention and control bacteriosis.Also, compared with antibiotic, biological agent of the invention as microorganism envelope inhibitor not
The resistance pressure of meeting inducing microbial, to will not lead to the generation of drug resistance, therefore no quadratic risk function is great potential
Substitutes For Antibiotic is expected to become the effective means of confrontation pathogenic bacteria even superbacteria.
In the fourth aspect of the present invention, the present invention provides mentioned-above rhizobium or biological agent in preparing product
Purposes.According to an embodiment of the invention, the product is used for: pathogen biofilm being inhibited to be formed；And/or inhibit pathogen
Virulence factor.
It should be noted that term " virulence factor " used herein is the substance for constituting virulence, main includes disease
Opportunistic pathogen elastoser and pathogen carrier.
Some specific examples according to the present invention, the product are used for:
It reduces pathogen biofilm and forms total amount；And/or
Inhibit pathogen elastoser yield；And/or
Inhibit the yield of pathogen carrier；And/or
The killing-efficiency to pathogen is improved with antibiotic collective effect.
According to an embodiment of the invention, the pathogen is pseudomonas aeruginosa, the carrier is siderophore.
In the fifth aspect of the invention, the present invention provides a kind of methods for preparing mentioned-above biological agent.According to
The embodiment of the present invention, method includes the following steps:
Mentioned-above rhizobium are subjected to fermented and cultured, and collect tunning；And
Using the tunning as active constituent, the biological agent is prepared.
According to an embodiment of the invention, the temperature of the fermented and cultured is 28~37 DEG C.
According to some embodiments of the present invention, the time of the fermented and cultured is 24-36 hours.
Some specific example embodiments according to the present invention, the mode of the fermented and cultured are rotation shake culture, preferably
The revolving speed of the rotation shake culture is 200 revs/min~220 revs/min, radius of turn 13.5cm.
According to an embodiment of the invention, carrying out the fermented and cultured using LB liquid medium.It is more according to the present invention
Specific example, in LB liquid medium, specific solute is peptone, yeast extract and sodium chloride, and solvent is water, the egg
The final concentration of 10g/L of white peptone in the medium, the final concentration of 5g/L of the yeast extract in the medium,
The final concentration of 10g/L of the sodium chloride in the medium；The pH of the culture medium is 7.0.
According to an embodiment of the invention, the rhizobium metabolin for including using in the tunning as it is described activity at
Point, it is in turn, further comprising the steps of after collecting the tunning and before preparing the biological agent: by the hair
Ferment product is centrifuged, and supernatant is taken to filter, and obtains filtrate, the filtrate is the metabolin of the rhizobium.According to the present invention
Some specific examples, the revolving speed of the centrifugation is 12000r/min, and the time of the centrifugation is 20min, the centrifugation radius
For 13.5cm, the centrifuging temperature is 4 DEG C；The filter sizes used that filter is 0.22 μm.
According to an embodiment of the invention, rhizobium and biological agent of the invention at least one of have the following advantages that:
(1) rhizobium (Rhizobium sp.) NA01 CGMCC No.12205, which has, inhibits pathogen biofilm to be formed
With the effect of attenuation；
(2) active constituent (rhizobium (Rhizobium sp.) NA01 CGMCC of biological agent of the invention
The metabolin of No.12205) growth that does not influence pathogen, resistance pressure is not generated, is not likely to produce drug resistance；
(3) active constituent of biological agent of the invention not only inhibits the formation of pathogen biofilm, also reduces
The virulence or the relevant Expression element of virulence of pathogen.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Preservation explanation
Strain name: rhizobium
Latin name: Rhizobium sp.
Strain number: NA01
Preservation mechanism: China Microbial Culture Preservation Commission's common micro-organisms center (referred to as: CGMCC)
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Preservation date: on March 11st, 2016
Deposit number: CGMCC No.12205
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 is according to the embodiment of the present invention, rhizobium (Rhizobium sp.) NA01 CGMCC No.12205QSI energy
The detection and screening experiment result of power；
Fig. 2 is according to the embodiment of the present invention, rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 metabolism
The inhibitory effect result figure that object and extract (water phase, organic phase) form pathogen biofilm, wherein abscissa is
Light absorption value under 570nm, ordinate indicate rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 metabolin and its
The dosage of extract.Double asterisk indicates the significance of difference (P < 0.01)；
Fig. 3 is according to the embodiment of the present invention, and rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 is always
The result of pathogen virulence factorial experiment, wherein
Upper figure is rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 metabolin and its extract to cause of disease
The suppression result of bacterium siderophore yield (%), wherein abscissa indicates rhizobium (Rhizobium sp.) NA01 CGMCC
The dosage of No.12205 metabolin and its extract, ordinate indicate siderophore yield (%), and double asterisk indicates significant difference
Property (P < 0.01),
The following figure is for rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 metabolin to pathogen to elastic egg
The suppression result of white enzyme, wherein abscissa indicate rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 metabolin and
The dosage of its extract, ordinate indicate siderophore yield (%), and double asterisk indicates the significance of difference (P < 0.01), single asterisk
It indicates the significance of difference (P < 0.05)；
Fig. 4 is according to the embodiment of the present invention, rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 metabolism
Influence experimental result of the object to pathogen biofilm structure and antibiotics sensitivity, wherein a is pseudomonas aeruginosa M63
Dilution, b are that 5% rhizobium supernatant is added in pseudomonas aeruginosa M63 dilution, and c is that pseudomonas aeruginosa M63 dilution adds
Enter 100 μ g/ml kanamycins, d is that pseudomonas aeruginosa M63 dilution is added on 100 μ g/ml kanamycins and 5% rhizobium
Clear liquid；And
Fig. 5 is according to the embodiment of the present invention, rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 metabolism
Object influences situation experimental result to growth of pathogenic bacteria, wherein triangle solid line is the growth curve of pseudomonas aeruginosa wild type, side
Frame solid line is the growth curve for adding that 5% rhizobium supernatant co-cultures at P. aeruginosa growth initial stage.
Specific embodiment
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Chromobacterium violaceum reporting bacterial strain (Chromobacterium violaceum ATCC 12472) is by the Chinese Academy of Sciences
South Sea institute of oceanography Wang Xiaoxue researcher give.
Pseudomonas aeruginosa (Pseudomonas aeruginosa) from Shanghai Fu Sheng Industrial Co., Ltd., (compile by product
Number: CL5201).The bacterium is pathogenic bacteria, often causes postoperative wound infection, can cause bedsore, abscess, otitis media suppurative etc.；
This microbial infection focus can lead to bacteremia and septicemia.
LB solid medium: peptone 1g, yeast extract 0.5g, sodium chloride 1g, deionized water 100ml, agar 1.5g,
pH7.0。
LB liquid medium: peptone 1g, yeast extract 0.5g, sodium chloride 1g, deionized water 100ml, pH7.0.
M63 fluid nutrient medium: ammonium sulfate 0.2g, potassium dihydrogen phosphate 1.36g, ferrous sulfate heptahydrate 0.5mg, glycerol 0.2g,
Casein hydrolysate 0.5g, epsom salt 0.02g, glucose 0.2g, deionized water 100ml, extremely with potassium hydroxide tune pH
7.0。
Crystal violet dye liquor: weighing crystal violet solid 2.0g into small beaker, and 95% ethyl alcohol 20ml is added and mixes；Weigh oxalic acid
Ammonium 0.8g is added sterile water 80ml, stirs and evenly mixs and mix with above-mentioned solution into another beaker, stands 48h.
Elastin laminin buffer: elastoser 2g, 100mM calcium chloride 1ml, pH7.2Tris-HCl100 μ l, deionization
Water 100ml, pH7.0.
CASCAS detection liquid is prepared: 10mmol/L cetane trimethyl ammonium bromide (HDTMA) solution 6.0ml being taken to be added to
In 100ml cleaning volumetric flask, diluted with distilled water；Again by 1.5ml (l mmol/L) FeCl3Solution and 7.5ml (2mmol/L) sword
After reddish black (CASCAS) solution mixing, it is slowly added to along glass bar to above-mentioned volumetric flask.Weigh the anhydrous double dimethylamine of 4.307g
(piperazine) is dissolved in 30ml distilled water, then is carefully added into 6.25ml (12mmol/L) HCl to get the buffer of pH=5.6.By this
Solution is transferred in aforementioned volumetric flask, is settled to 100ml with distilled water.
The separation and identification of embodiment 1, bacterial strain NA01
One, the separation of bacterial strain NA01
Water sample is acquired in the surface seawater of the North Atlantic Ocean, Preservation in sterile condition takes back (Tsinghua University Shenzhen postgraduate behind laboratory
Institute's marine ecology laboratory) sterile working is coated in 12cm medium size solid medium tablets containing 2216E, 30 DEG C of overnight stands trainings
It supports, until growing high-visible monoclonal.
Then from culture plate, 100 plants of monoclonals of picking are in 2ml EP pipe, LB liquid medium containing 1ml in EP pipe,
This 100 monoclonals are put into constant-temperature table 30 DEG C, 220r/min overnight incubation.It is reported that bacterial strain Chromobacterium
Violaceum ATCC 12472 is detected, and can inhibit the formation of purple circle is positive strain, is by one of Strain Designation
NA01 bacterium.
Two, the identification of bacterial strain NA01 bacterium
The bacterial strain NA01 that step 1 obtains is identified in terms of form and 16s rDNA sequence homology two.
1, Morphological Identification
It will be in logarithmic growth phase, and bacterium colony size is stablized, the isolated bacterial strain NA01 of above-mentioned steps one carries out single bacterium
Fall state description, the main size including bacterium colony, color, transparency, wettability, bacterium colony surface state (whether flat, protrusion,
Fold, recess etc.), colony edge state (whether neat, irregular, radial etc.).
The result shows that: the isolated bacterial strain of above-mentioned steps one is Gram-negative bacteria, and for bacterium colony at faint yellow, surface is wet
Profit, edge are flat.
2,16s rDNA sequence homology analysis
The isolated bacterial strain NA01 of conventional method culture above-mentioned steps one, extracts the total DNA of bacterial strain as gene magnification
Template, using universal primer 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ' (SEQ ID NO:1), 1492r:5 '-
TACGGTTACCTTGTTACGACTT-3 ' (SEQ ID NO:2) expands bacterial 16 S rRNA gene conserved region.25 μ L amplification systems
It include: 1 × PCR reaction buffer, 200 μm of ol/L dNTPs, upstream and downstream primer each 0.2 μm of ol/L, 1U Taq DNA are poly-
Synthase, 1 μ L template DNA.Reaction condition: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 40s, 72 DEG C of extension 30s,
Totally 25 circulations；72 DEG C of extension 10min.1% detected through gel electrophoresis of PCR product, positive findings 27f:5 '-
AGAGTTTGATCCTGGCTCAG-3 ' (SEQ ID NO:1), 1492r:5 '-TACGGTTACCTTGTTACGACTT-3 ' (SEQ
ID NO:2) carry out bidirectional sequencing.Sequence assembly and similarity analysis are completed using DNAStar software, and sequence alignment passes through the U.S.
National Biotechnology Information Center ncbi database (http://www.ncbi.nlm.nih.gov) is completed online, as a result as follows.
The sequence of the 16s rDNA of bacterial strain NA01 are as follows:
In view of above-mentioned morphological feature and 16s rDNA sequence homology analysis as a result, the bacterium that step 1 separation screening is obtained
Strain NA01 is accredited as rhizobium (Rhizobium sp.).Rhizobium (Rhizobium sp.) NA01 is March 11 in 2016
Day is preserved in China Microbial Culture Preservation Commission's common micro-organisms center (abbreviation CGMCC, address are as follows: Chaoyang District, Beijing City
No. 3 Institute of Microorganism, Academia Sinica, institute of North Star West Road 1, postcode: 100101), and preservation registration number CGMCC
No.12205。
Embodiment 2, rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 inhibit pathogen biofilm shape
At experiment
1, the fermented and cultured of rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 and tunning (metabolism
Object) extraction
The rhizobium (Rhizobium sp.) that the separation identification of embodiment one of conservation obtains are taken out from -80 DEG C of refrigerator
NA01 CGMCC No.12205, after thaw at RT, repeatedly scribing line separation, 30 DEG C of stationary cultures in LB solid medium tablets
12h or more, until growing high-visible monoclonal.Therefrom choose 3 plants of monoclonals in the LB liquid medium of 1ml, 30 DEG C,
220r/min shaking table culture is stayed overnight.It takes 1ml bacterium solution that 250ml is added to contain in the conical flask of 100mlLB fluid nutrient medium, is put into and shakes
On bed, 30 DEG C, 220r/min (centrifugal head radius is 13.5cm) culture for 24 hours~36h, until medium nutrient content consumes substantially
To the greatest extent, until cell density is not further added by.
Fermentation liquid is collected, 4 DEG C, 12000r/min, centrifugation 20min (centrifugation radius is 13.5cm) takes supernatant.It will obtain
Supernatant further use 0.22 μm of membrane filtration (remove remaining bacterium solution and impurity), take filtrate (rhizobium Rhizobium
Sp.NA01 CGMCC No.12205), it is stored in sterilization container, 4 DEG C save backup and (save half).
Acetic acid esters, stirring extraction is added in 1:1 ratio in remaining half supernatant；30-50ml aqueous phase liquid is taken, is used
0.22nm filter membrane is crossed spare after degerming.Remaining organic phase removes ethyl acetate with rotary distillation method completely, and is eluted with 5ml methanol,
It is spare to obtain organic phase.
2, the activation culture of pathogen pseudomonas aeruginosa (Pseudomonas aeruginosa)
The pseudomonas aeruginosa (Pseudomonas aeruginosa) of conservation, room temperature solution are taken out from -80 DEG C of refrigerator
After jelly, cross in LB solid medium tablets, 37 DEG C of stationary culture 12h or more, until growing high-visible monoclonal.From
In choose 3 plants of monoclonals in the LB liquid medium of 1ml, 37 DEG C, 220r/min shaking table culture stay overnight.Transfer again into
It is 37 DEG C, 220r/min shaking table culture 8h, spare in 20mlLB fluid nutrient medium.
3, the metabolin and extract for detecting rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 are to verdigris
The inhibitory effect of pseudomonad (Pseudomonas aeruginosa) biofilm
(1) the bacterium solution M63 of the pseudomonas aeruginosa (Pseudomonas aeruginosa) for the activation for obtaining step 2
Fluid nutrient medium is diluted to OD600It is 0.02.
(2) pseudomonas aeruginosa (Pseudomonas aeruginosa) bacterium solution after dilution is added in 96 orifice plates,
100 holes μ l/, then the filtrate supernatant (0,1%, 3%, 5%) that the step 1 of addition different volumes score obtains into each hole, extracting
Object water phase (5%) and organic phase (0.5%), each concentration set 12 multiple holes.Plate lid is covered, stationary culture 48 hours at 37 DEG C,
Pay attention to keeping air humidity.
(3) culture medium is removed, softly cleans plate with sterile water, is added 100 μ l, 1% (1g/100ml) crystal violet dye liquor,
Room temperature dyes 20 minutes, goes dyestuff, thorough and soft cleaning.
(4) crystal violet being attached on wall is dissolved with DMSO, and is moderately diluted, and OD is surveyed570.Experiment in triplicate, as a result takes
Average value.Testing principle is as follows: pseudomonas aeruginosa (Pseudomonas aeruginosa) is cultivated after a period of time, and meeting exists
Biofilm is formed on the side wall of 96 orifice plates and bottom, discards culture medium, after washing away suspended bacterial, the biofilm of side wall and bottom
In bacterium will not wash away, this part bacterium is dyed with crystal violet, then with the dissolving crystallized purple of DMSO, lysate exists
There is maximum absorption band under 570nm wavelength.At this point, absorbance is bigger, illustrate that the concentration of lysate is bigger, the biology of biofilm
It measures also bigger.Conversely, then showing that the biomass of microorganism envelope becomes smaller, biofilm formation is suppressed.
Testing result joined step 1 and obtain as shown in Fig. 2, it can be seen from the figure that compared with control (0 concentration group)
Filtrate and extract (metabolin of rhizobium (Rhizobium sp.) NA01 CGMCC No.12205) each group, to cause of disease
The formation of bacterium pseudomonas aeruginosa (Pseudomonas aeruginosa) biofilm all have apparent inhibiting effect (p <
0.01, difference is extremely significant), and this inhibiting effect enhances with the increase of the concentration of the filtrate of addition.
Embodiment 3, rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 inhibit the pathogen virulence factor real
It tests
1, the metabolin of rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 is detected to pseudomonas aeruginosa
The inhibitory effect of (Pseudomonas aeruginosa) siderophore yield
(1) bacterium of the pseudomonas aeruginosa (Pseudomonas aeruginosa) for the activation for obtaining 2 step 2 of embodiment
Liquid is diluted to OD with LB liquid medium fluid nutrient medium600It is 0.05.
(2) different volumes point are added into pseudomonas aeruginosa (Pseudomonas aeruginosa) bacterium solution after dilution
The filtrate supernatant (0,1%, 3%, 5%) that several 2 steps 1 of embodiment obtains, extract water phase (5%) and organic phase (0.5%),
3 repetitions of each concentration, are put into shaking table, 220r/min, after cultivating 16h under the conditions of 30 DEG C, 4 DEG C, and 12000r/min centrifugation
15min。
(3) three portions of 10ml supernatants are taken, being separately added into three kinds of different hydrolysis substrates that 20 μ l concentration are 5mM, (iron carries
Body), dark in co-culture 9h.Blank control is the supernatant (not plus the obtained filtrate of 2 step 1 of embodiment) boiled and bottom
The product that object co-cultures.
(4) it by the product after co-cultivation 9h, takes out 100 μ l (attention is wrapped with masking foil, is protected from light) in 96 orifice plates, puts
Enter in microplate reader, set exciting light as 365nm, transmitting light is 455nm, detects fluorescent value.Experiment in triplicate, is as a result averaged
Value.Testing principle is as follows: siderophore can will have the substrate hydrolysis of fluorescent marker (MUF), and fluorescent marker is transferred to hydrolysate
On, after the excitation of 365nm, the fluorescence of 455nm is generated, according to fluorescence power, it is known that the concentration of siderophore, it is glimmering
Light value is smaller, is suppressed bigger.
Testing result is as shown in the upper figure of Fig. 3, it can be seen from the figure that joined implementation compared with control (0 concentration group)
The filtrate and extract (metabolin of rhizobium (Rhizobium sp.) NA01 CGMCC No.12205) that 2 step 1 of example obtains
Each group, have to the yield of 3 kinds of pathogen pseudomonas aeruginosa (Pseudomonas aeruginosa) different hydrolases
Have apparent inhibiting effect (p < 0.05, significant difference), and this inhibiting effect is with the increasing of the concentration of the filtrate of addition
Enhance greatly.
2, the metabolin of rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 is to pseudomonas aeruginosa
The inhibitory effect of (Pseudomonas aeruginosa) elastoser
(1) bacterium of the pseudomonas aeruginosa (Pseudomonas aeruginosa) for the activation for obtaining 2 step 2 of embodiment
Liquid is diluted to OD with LB liquid medium600It is 0.05.
(2) different volumes point are added into pseudomonas aeruginosa (Pseudomonas aeruginosa) bacterium solution after dilution
The filtrate supernatant (0,1%, 3%, 5%) that several 2 steps 1 of embodiment obtains, extract water phase (5%) and organic phase (0.5%),
3 repetitions of each concentration, are put into shaking table, 220r/min, after 37 DEG C are cultivated 21 hours, 4 DEG C, 12000g/min, are centrifuged 5 points
Clock.
(3) 1mL elastin laminin buffer is added in the supernatant after taking 500 μ l to be centrifuged, and oscillation co-cultures 6 hours, is added 600
The EDTA that μ l concentration is 0.1M terminates reaction, and precipitating (10000r/min, 2 minutes) is gone in centrifugation, surveys OD495.It tests in triplicate,
Results are averaged.Testing principle is as follows: elastin laminin is slightly soluble in water, and elastoser can hydrolyze elastin laminin, is formed red
Color hydrolysate, this red hydrolysate have maximum absorption band under the wavelength of 495nm.At this point, the concentration of hydrolysate is bigger,
Light energy is stronger, and obtained numerical value is bigger.
Testing result is as shown in the following figure of Fig. 3, it can be seen from the figure that joined implementation compared with control (0 concentration group)
The filtrate and extract (metabolin of rhizobium (Rhizobium sp.) NA01 CGMCC No.12205) that 2 step 1 of example obtains
Each group, the yield of the elastoser of pathogen pseudomonas aeruginosa (Pseudomonas aeruginosa) is all had bright
Aobvious inhibiting effect (p < 0.01, difference are extremely significant), and this inhibiting effect is with the increase of the concentration of the filtrate of addition
And enhance.
Embodiment 4, rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 are to pseudomonas aeruginosa
(Pseudomonas aeruginosa) biofilm structure and antibiotics sensitivity influence experiment
(1) activation pseudomonas aeruginosa (Pseudomonas aeruginosa) bacterium solution obtained 2 step 2 of embodiment is used
M63 fluid nutrient medium is diluted to OD600It is 0.05.Four groups of experimental setup, it is denoted as a, b, c, d respectively.Wherein, a is P. aeruginosa
Bacterium M63 dilution；B is that 5% rhizobium supernatant is added in pseudomonas aeruginosa M63 dilution；C is that pseudomonas aeruginosa M63 is dilute
It releases liquid and 100 μ g/ml kanamycins is added；D is that 100 μ g/ml kanamycins and 5% is added in pseudomonas aeruginosa M63 dilution
Tumor bacterium supernatant.
The characterization of experimental result COMSTAT fluorescence microscopy biofilm biomass, average thickness and maximum thickness
Angle value (PA: pseudomonas aeruginosa；PA+QSI: pseudomonas aeruginosa+5%QSI extract mixing；PA+K: pseudomonas aeruginosa+
The mixing of 100 μ g/ml kanamycins；PA+K+QSI :+100 μ g/ml kanamycins+5%QSI extract of pseudomonas aeruginosa is mixed
It closes).Four figure of top is the biofilm figure obtained after Z axis scans, and four figure of lower section is its 3D rendering.
(2) fluorescent dye (ready-to-use) is prepared, fluorescent dye used is -7012LIVE/DEAD BacLightTM
Bacterial Viability Kit (Molecular Probes Inc), by SYTO9 and PI dye liquor therein in following ratio
Dilution, SYTO9:PI: distilled water=1.5 μ l:1.5 μ l:1ml, oscillation mix (attention is protected from light).
(3) coverslip in 4 samples is taken out, is cleaned with the sodium chloride solution of 0.9% (0.9g/100ml), is dried in the air naturally
It is dry, 200 μ l step (2) prepared fluorescent dyes (attention is protected from light) are added dropwise on each coverslip, dye 30min in dark,
After natural drying, it places and is observed on Laser Scanning Confocal Microscope.Red is dead bacterium, and green is viable bacteria.Testing principle: SYTO9 dyestuff can
To penetrate cell membrane, the DNA of dead bacterium or viable bacteria can be combined, under the exciting light of 488nm wavelength, generate green fluorescence.And
PI cannot penetrate cell membrane, can only be in conjunction with the DNA of dead bacterium, and can replace SYTO9, under the exciting light of 488nm wavelength, generate
Red fluorescence.It when being observed with laser confocal microscope, is successively scanned using Z axis, according to the distance of upper and lower surface up to thick
Degree.
Testing result is as shown in figure 4, it can be seen from the figure that wild type pseudomonas aeruginosa PAO1 can form thickness about
It is 10000nm uniformly with the biofilm of highly structural, and the Bacteria cold shock in biomembrane is good, warp
Whole face is green after dyestuff color, is indicated almost without bacterial death (a)；After 5%QSI supernatant is added, it is apparent that
Biofilm is structural to be destroyed, more discrete, and whole thickness reduces, but almost without bacterial death (b)；Add
Entering biofilm after 100 μ g/ml kanamycins are handled, integral color starts yellowing, this is green and red mixed intermediate state face,
Show that surface bacteria has had begun Mortality, biofilm thickness reduces, but its it is structural compared with wild type almost without
Change (c)；The kanamycins of 100 μ g/ml and the biofilm of 5%QSI supernatant Combined Treatment, can obviously observe its structure
Property significantly destroyed, more discrete, thickness significantly reduces, and it is in show 90% or more thin that red, which is presented, in integral color
Bacterium is dead (d).
Embodiment 5, rhizobium (Rhizobium sp.) NA01 CGMCC No.12205 are to pseudomonas aeruginosa
The influence experiment of (Pseudomonas aeruginosa) growth
(1) pseudomonas aeruginosa (Pseudomonas for the activation that 200 μ l embodiment, 2 step 2 obtains is taken out
Aeruginosa) bacterium solution is divided into 2 parts, every part of 100 μ l.The filtrate that 5 μ l embodiment, 2 step 1 obtains is added in portion, it is another
It is not added in part, carries out the culture of wild type pseudomonas aeruginosa (Pseudomonas aeruginosa).
(2) test combinations control group is put into 30 DEG C of incubators and cultivates 36 hours, does not have the OD of detection in 4 hours600Extinction
Value, the dense situation of change of observation bacterium.
Testing result is as shown in figure 5, can be seen from the chart that two growth curves are almost overlapped, the addition of QSI supernatant
On P. aeruginosa growth almost without influence.This shows the filtrate (rhizobium (Rhizobium that 2 step 1 of embodiment obtains
Sp.) the metabolin of NA01 CGMCC No.12205) have no effect on pathogen pseudomonas aeruginosa (Pseudomonas
Aeruginosa growth).
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this
The range of invention is defined by the claims and their equivalents.
Claims (4)
1. the purposes of rhizobium in medicine preparation, the drug is for inhibiting pathogen biofilm to be formed, reducing pathogen
Virulence, the pathogen are pseudomonas aeruginosa,
Wherein, the rhizobium are preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on March 11st, 2016
Object center, deposit number are CGMCC No.12205.
2. purposes according to claim 1, which is characterized in that the rhizobium are realized by its metabolin inhibits pathogen
Biofilm forms and reduces the purposes of pathogen virulence.
3. rhizobium are used for preparing the purposes in product, the product:
Pseudomonas aeruginosa biofilm is inhibited to be formed；And/or
Inhibit virulence factors production in Pseudomonas aeruginosa,
Wherein, the rhizobium are preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on March 11st, 2016
Object center, deposit number are CGMCC No.12205.
4. purposes according to claim 3, which is characterized in that the product is used for:
It reduces pseudomonas aeruginosa biofilm and forms total amount；And/or
Inhibit elastase coding sequence from pseudomonas aeruginosa yield；And/or
Inhibit the yield of pseudomonas aeruginosa siderophore；And/or
The killing-efficiency to pseudomonas aeruginosa is improved with antibiotic collective effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610697617.2A CN106318886B (en) | 2016-08-19 | 2016-08-19 | Rhizobium and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610697617.2A CN106318886B (en) | 2016-08-19 | 2016-08-19 | Rhizobium and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106318886A CN106318886A (en) | 2017-01-11 |
| CN106318886B true CN106318886B (en) | 2019-09-17 |
Family
ID=57741233
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610697617.2A Active CN106318886B (en) | 2016-08-19 | 2016-08-19 | Rhizobium and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106318886B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359621B (en) * | 2018-03-14 | 2021-05-07 | 中山大学 | Rhizobium capable of efficiently adsorbing low-concentration copper ions and application thereof |
| CN108611306B (en) * | 2018-04-03 | 2021-05-25 | 华南农业大学 | Method for researching gene function of bradyrhizobium |
| US20210051956A1 (en) * | 2019-08-23 | 2021-02-25 | The Hong Kong University Of Science And Technology | Elasnin, a bacteriostatic agent that has potent antibiofilm activities against both mono- and multi-species biofilm |
| CN113832064B (en) * | 2021-09-29 | 2023-05-19 | 清华大学深圳国际研究生院 | Microbial population induction quenching complexing agent and application thereof |
| CN116121147B (en) * | 2023-03-14 | 2023-12-01 | 昆明理工大学 | Chenopodium ambrosioides seed endophytic Larimol agrobacterium and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911899A (en) * | 2012-10-22 | 2013-02-06 | 清华大学深圳研究生院 | Proteus mirabilis and application thereof in suppression of biofilm and detoxification |
-
2016
- 2016-08-19 CN CN201610697617.2A patent/CN106318886B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911899A (en) * | 2012-10-22 | 2013-02-06 | 清华大学深圳研究生院 | Proteus mirabilis and application thereof in suppression of biofilm and detoxification |
Non-Patent Citations (3)
| Title |
|---|
| Huang,J.等.Rhizobium sp. 6001 16S ribosomal RNA gene, partial sequence.《GenBank: JX566662.1》.2013, * |
| Rhizobium sp. 6001 16S ribosomal RNA gene, partial sequence;Huang,J.等;《GenBank: JX566662.1》;20131223;序列说明 * |
| Strain identification and quorum sensing inhibition characterization of marine-derived Rhizobium sp. NAO1;Chang H等;《R. Soc. open sci. 》;20170331;170025：1-12 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106318886A (en) | 2017-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106318886B (en) | Rhizobium and application thereof | |
| US20240093142A1 (en) | Strain for degrading deoxynivalenol and use thereof | |
| Song et al. | Antifungal activity of the lipopeptides produced by Bacillus amyloliquefaciens anti-CA against Candida albicans isolated from clinic | |
| CN101481670A (en) | Copper green pseudomonas with lytic activity and use thereof in blue algae bloom control | |
| CN106906167A (en) | One plant of biological and ecological methods to prevent plant disease, pests, and erosion bacillus amyloliquefaciens and its application | |
| CN102911899B (en) | Proteus mirabilis and application thereof in suppression of biofilm and detoxification | |
| CN108570433A (en) | One plant of acid proof defence pseudomonad CLP-6 and its application | |
| Bhuyan et al. | Optimization and characterization of extracellular cellulase produced by Bacillus pumilus MGB05 isolated from midgut of muga silkworm (Antheraea assamensis Helfer) | |
| Santhi et al. | A novel marine bacterium Isoptericola sp. JS-C42 with the ability to saccharifying the plant biomasses for the aid in cellulosic ethanol production | |
| CN106399156B (en) | Bacillus amyloliquefaciens and application thereof in gulfweed biodegradation | |
| CN109321500B (en) | Bacillus amyloliquefaciens strain and application thereof in prevention and treatment of camellia oleifera anthracnose disease | |
| CN102703420A (en) | Microbial degradation preparation for municipal garbage and sewage and preparation method thereof | |
| Dhara et al. | First report of Exserohilum leaf spot: a unique halophilic pathogen in Cucumis sativus in the South Bengal area of India | |
| CN102787089B (en) | Acinetobacterbeijerinckii and application of acinetobacterbeijerinckii | |
| CN110591927B (en) | Application of trichoderma strain HB20111 in digesting phenolic acid autotoxic substances of crop continuous cropping obstacle factors and microbial inoculum | |
| CN107955795A (en) | One plant degraded cephalosporin analog antibiotic geotrichum candidum CM1 and its application | |
| CN109439582B (en) | Bacillus megaterium grown in chrysanthemum morifolium and application thereof | |
| CN116042452B (en) | Bacillus subtilis and application thereof | |
| CN106085896A (en) | Enterobacter cloacae and application thereof | |
| CN105861356B (en) | A kind of Raoul bacterium pan22x and its application | |
| CN102604868A (en) | Alteromonas and application thereof in inhibiting growth of red-tide algae | |
| CN113046249B (en) | Verticillium lecanii LL-01 and biocontrol application thereof | |
| Rybczyńska-Tkaczyk et al. | Biodecolorization of anthraquinone dyes using immobilised mycelium of Bjerkandera adusta CCBAS930 | |
| CN104109642A (en) | Serratia marcescens, and screening method and application thereof | |
| CN104560818B (en) | Bacillus licheniformis UTM118 for producing high-temperature-resistant acid alpha-amylase and application of bacillus licheniformis UTM118 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |