CN104928220B - The water Rhein sea of one plant of ocean sludge source is write from memory Salmonella bacterial strain and its application - Google Patents
The water Rhein sea of one plant of ocean sludge source is write from memory Salmonella bacterial strain and its application Download PDFInfo
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- CN104928220B CN104928220B CN201510393657.3A CN201510393657A CN104928220B CN 104928220 B CN104928220 B CN 104928220B CN 201510393657 A CN201510393657 A CN 201510393657A CN 104928220 B CN104928220 B CN 104928220B
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Abstract
It writes from memory Salmonella bacterial strain in water Rhein sea the present invention relates to one plant of ocean sludge source（Rheinheimera aquimaris）QSI02 and its application in terms of quorum-quenching screening.The bacterial strain is preserved in China typical culture collection center（Abbreviation CCTCC）, preservation date be on April 23rd, 2015, preserving number CCTCCNO:M2015245.The present invention is to report for the first timeRheinheimeraThe bacterial strain of category has quorum-quenching activity.There is double inhibition effect to chromabacterium biolaceum and pseudomonas aeruginosa intervention school-based, and inhibiting effect is not generated to the generation of the thalline of screening model chromabacterium biolaceum CV026 in Valid concentration by metabolin prepared by the bacterial strain fermentation liquor.The bacterial strain extract can significantly reduce the yield of chromabacterium biolaceum purpurin and the crowd hazards of pseudomonas aeruginosa, weaken the drug resistance of pathogenic bacteria, so as to have broad application prospects in terms of bacterial resistance sex chromosome mosaicism, research and development novel antibacterial drug is solved.
Description
Technical field
The invention belongs to marine microorganism fields, and in particular to the water Rhein sea of one plant of ocean sludge source is write from memory Salmonella
(Rheinheimera aquimaris) QSI02 bacterial strains and its metabolite answering in terms of quorum-quenching screening
With.
Background technology
Quorum sensing (Quorum sensing, QS) is a kind of information interchange phenomenon being widely present between bacterium.In recent years
The research come finds that bacterium can realize phase by the chemical signal molecule for producing, identifying, sensing one or more solubilities
Mutually between information transmission with exchange.Although early in the 1960s just in a kind of ocean Fermi operator (Vibrio
Fischeri quorum sensing phenomenon is found that in), but is just suggested until bacterial community in 1994 senses this concept
(J.Bacteriol.,1994,176:269-275).Many bacteriums can synthesize and discharge a kind of referred to as self-induction substance (auto
Inducer, AI) signaling molecule, when the quantity of bacterium in a specific environment sharply increases, secreted by signaling molecule
Concentration can also increase accordingly, will be adjusted by this signaling molecule between bacterium and bacterium when obtaining certain threshold value
The expression of gene is controlled, for example bioluminescence, biofilm are formed, antibiotic generates, toxin generates, produce spore etc..
Bacterial resistance sex chromosome mosaicism became increasingly conspicuous in recent years, and traditional antibacterials are easy to make due to generating survival pressure
Bacterium generates drug resistance, therefore, bacterium can not be fundamentally solved with the antibacterials that traditional target spot and screening technique obtain
Sexuality dye disease problems.In recent years the study found that bacterium QS systems make, as monadic bacterium, have part class
The function of multicellular organism is similar to, allows bacterium when response environment is challenged, can be harmonious, bacterium is made to form a kind of group behavior
Effectively to resist ambient pressure environment, attack host etc..Using the antibacterials that bacterium QS systems are sieved as target, resist with tradition
The mechanism of action of raw element is entirely different, it does not kill bacterium or inhibits the growth of single bacterium, only inhibits bacteria the cause of QS regulation and control
Sick behavior is not easy inducible resistance mutation.
The complex ecosystems such as the relatively unique with high salt, high pressure in ocean, oligotrophic, low illumination create marine microorganism not
The unique physiological function and metabolic mechanism of terrestrial microorganism are same as, substantially increases structure diversity and the multifarious elder generation of activity
Lead the probability of detection of compound.At present, being found that from marine microorganism largely has chemical constitution novelty, bioactivity
Special compound, some of which have been used as drug to be widely used in clinic.Meanwhile Marine Microbial Kinds are more, are easy to train
It supports, acquisition active metabolite can be cultivated by artificial fermentation, and therefore Marine Microorganisms have become a kind of important environment friend
Good living resources.
Invention content
The present invention is intended to provide one plant of marine bacteria with quorum-quenching activity, Metabolite tool
There is the activity for inhibiting chromabacterium biolaceum and pseudomonas aeruginosa intervention school-based, and the production of pathogenic bacteria pathogenic behavior can be reduced
It is raw, weaken the drug resistance of pathogenic bacteria.
Present invention separating marine bacterium from the mud sample of Huanghai Sea Qingdao sea area ocean is screened using chromabacterium biolaceum CV026
Model carries out isolated bacterial strain the screening of quorum-quenching activity, obtains one plant with bacterial flora efficiently is quenched
Body-sensing answers the bacterial strain of activity.By 16S rDNA sequence analysis combining forms and physiological and biochemical property, it is identified as
Rheinheimera aquimaris QSI02 reach 99% with the write from memory similitude of Salmonella of one plant of water Rhein sea in GenBank.
Marine bacteria of the present invention with quorum-quenching activity is that water Rhein sea is write from memory Salmonella
(Rheinheimera aquimaris) QSI02, be preserved on April 23rd, 2015 China typical culture collection center (in
State Wuhan, Wuhan University), deposit number is CCTCC NO:M2015245.
The preparation method of active metabolite of the present invention mainly includes point of marine bacteria R.aquimaris QSI02
From, culture and two steps of preparation of extract.The marine bacteria R.aquimaris QSI02 after activation are taken, are inoculated into fermentation
Culture medium carries out shaking table culture, and after fermentation, centrifugation, vacuum and low temperature are concentrated into paste, and methanolic extract is extracted to obtain with methanol,
It is detected after methanolic extract is filtered with 0.22 μm of organic phase filter for bioactivity.
Metabolin prepared by marine bacteria R.aquimaris QSI02 according to the present invention have inhibit chromabacterium biolaceum and
The effect of pseudomonas aeruginosa intervention school-based；Chromabacterium biolaceum CV026 is not inhibited adding in the metabolin in the range of 0-300 μ l
Thalli growth, but can significantly reduce the generation of chromabacterium biolaceum purpurin, its inhibiting rate reaches when adding in 100 μ l metabolins
50%.The crowd hazards of pseudomonas aeruginosa can be obviously reduced when adding in 200 μ l metabolins.
Compared with prior art, the present invention with the letter of marine bacteria R.aquimaris QSI02 strain fermentations condition of culture
Single, the preparation process of active metabolite is simple and easy to control, and the metabolin of preparation, which has, is significantly quenched bacterial community sensing
The features such as active.
Description of the drawings
Fig. 1 active bacterial strain R.aquimaris QSI02 colonial morphologies.
The phylogenetic tree of Fig. 2 active bacterial strain R.aquimaris QSI02.
Fig. 3 bacterial strain R.aquimaris QSI02 inhibit the Biological Detection of QS activity.
Fig. 4 bacterial strain R.aquimaris QSI02 fermentation broth extracts inhibit chromabacterium biolaceum purpurin volume analysis.
Influence of Fig. 5 bacterial strain R.aquimaris QSI02 fermentation broth extracts to pseudomonas aeruginosa PA01 crowd hazards.
Specific embodiment
The chromabacterium biolaceum Chromobacterium violaceum (1511C0001ACCC10486) arrived involved in embodiment
Chinese agriculture Microbiological Culture Collection administrative center is purchased from, pseudomonas aeruginosa Pseudomonas aeruginosa PA01 are
The preservation of laboratory institute.
Different culture media formula is as follows used in embodiment：
(1) enriched medium：Denitrifying bacteria enriched medium：KNO32g,K2HPO40.5g,MgSO45g, sodium acetate
9.5g, H2O 1L, pH7.0-7.4.
(2) screening and fermentation medium：LB culture mediums：Peptone 1%；Yeast extract 0.5%；NaCl 0.5%.
Embodiment 1：The enrichment isolation of bacterial strain R.aquimaris QSI02.
1.1 sample collection：In September, 2010 acquires seawater mud sample from Huanghai Sea Qingdao sea area, deposits in aseptic bottle.
1.2 enrichment culture：Sample 2g is taken to be added to 150mL denitrifying bacteria enriched mediums (KNO32g, K2HPO40.5g,
MgSO45g, sodium acetate 9.5g, H2O 1L, pH7.0-7.4) 250mL triangular flasks in, 30 DEG C, cultivate in 150r/min shaking tables, often
3~4d samplings examine NO with Griess reagent2 -Situation of change.Choose NO2 -Change big denitrifying bacteria access fresh cultured
Base.After repetitive operation several times, bacteria suspension 1mL is taken to be placed in the test tube for filling 9mL sterile waters, be uniformly mixed and dilution is made is
10-1Bacteria suspension, same method does 5 dilutions again.
1.3 strain isolations purify：It is 10 to take dilution-5、10-6Each 100 μ L of bacteria suspension be coated on LB culture mediums, 30
DEG C culture 2~5d.The larger bacterium colony scribing line separation of well-grown, bacterium colony is selected, dominant colony inoculation is selected after repeating 2~3 times
In slant preservation culture medium, 4 DEG C of preservations of refrigerator.
Embodiment 2：Bacterial strain R.aquimaris QSI02 are identified and preservation.
2.1 morphological feature：Bacterial strain R.aquimaris QSI02 are creamy white on LB culture mediums, neat in edge, gram
It dyes as negative bacterium.It being shown (Fig. 1) by transmission electron microscopy result, thalline is in rod-short, and size is about 1.5-2.0 μm wide,
7.0-9.0 μm long, containing many flagellums, and intracellular has transparent content.
2.2 bacterial strain 16S rDNA are identified and preservation：16S rDNA sequence analyses use bacterium colony PCR methods, and primer is respectively
fD1:27F 5'-AGAGTTTGATCMTGGCTCAG-3' and rD1:1492R 5'-TACGGYTACCTTGTTACGACTT-3',
PCR reaction conditions are as follows：94 DEG C of pre-degeneration 3min, 94 DEG C of 30s, 55 DEG C of 1min, 72 DEG C of 90s, 30 cycles；72 DEG C of extensions
10min.After PCR amplification, PCR product is subjected to 1.0% agarose gel electrophoresis analysis.It will be through glue recycling column purification
PCR product is ligated and transformed into E.coli DH5a competent cells with carrier pMD-18T.Picking positive transformant carries out sequence survey
It is fixed.By the 16S rDNA sequence inputtings GenBank of bacterium to be measured with Blast programs carry out similarity system design analysis, find with it is known
Strain sequence similitude is more than 99%, therefore bacterial strain of the present invention is accredited as Rheinheimera aquimaris kinds, and names
For Rheinheimera aquimaris QSI02.According to the phylogenetic tree of the 16S rDNA sequence constructs of similar standard bacterial strain
See Fig. 2.Bacterial strain Rheinheimer aaquimaris QSI02 of the present invention have been preserved in Chinese Typical Representative training on April 23rd, 2015
Object collection is supported, deposit number is CCTCC NO:M2015245.
2.3 bacterial strain physiological and biochemical properties：It is removed from office using the rich inspection that Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd produces blue
Family name's negative bacteria identification systems write from memory to the water Rhein sea sieved Salmonella carry out Physiology and biochemistry identification.The results are shown in Table 1.
1 water Rhein sea of table is write from memory the part Physiology and biochemistry qualification result of Salmonella
* "+" represents positive reaction, and "-" represents negative reaction.
Identified, Salmonella is write from memory as Gram-negative bacteria in water Rhein sea.The optimum growing condition and marine environment of the bacterial strain are very
Unanimously, it is shown that stronger marine features.
Embodiment 3：The quorum sensing inhibitory activity detection of bacterial strain R.aquimaris QSI02 metabolins.
The preparation of 3.1 bacterial strain R.aquimaris QSI02 metabolins：Take the marine bacteria R.aquimaris after activation
QSI02 is inoculated into fermentation medium and carries out shaking table culture, and after fermentation, centrifugation, vacuum and low temperature are concentrated into paste, use methanol
Methanolic extract is extracted to obtain, is detected after methanolic extract is filtered with 0.22 μm of organic phase filter for bioactivity.
3.2 quorum sensing inhibitory activity detect：The C.violaceum fermentations that 200 μ l dilute 10 times are coated on LB tablets
Liquid pastes a filter paper for being moistened with 0.5%DMSO aqueous solutions in middle position and compares, sticked successively around and be moistened with bacterium to be measured
The filter paper of extract observes around filter paper whether long bacterium does not occur purpurin but.The result shows that bacterial strain R.aquimaris
Purpurin secretion reduces (Fig. 3) compared with the control around QSI02, shows that the bacterium has QS rejection abilities.In order to further prove to treat
Surveying bacterium has QS rejection abilities, is separately added into and treats in the triangular flask that 7 are equipped with 10ml LB culture mediums and 100 μ l chromobacterium violaceums
Survey fungus extract 0 μ l, 50 μ l, 100 μ l, 150 μ l, 200 μ l, 250 μ l, 300 μ l；It is cultivated on 28 DEG C, the shaking table of 150r/min
48h.Every bottle takes 1ml, centrifuges (4000r/min, 30min), outwells supernatant, precipitation is re-dissolved with 1ml DMSO, centrifuges
(4000r/min, 30min), supernatant is poured into respectively in marked good centrifuge tube, and 200 μ l supernatants is then taken to add successively
Enter into 96 hole microwell plates, the absorbance A of 585nm is surveyed with microplate reader585, the variation of purpurin yield is characterized with this.It uses simultaneously
1ml sterile waters suspension thalline again, then takes 200 μ l bacteria suspensions to be added in 96 hole microwell plates successively, and 600nm is surveyed with microplate reader
Absorbance OD600, the variation of thalli growth density is represented with this.The result shows that add in the supernatant of the fungus extract, purple
The yield of element significantly reduces, but the stand density of thalline is basically unchanged, and shows that the fungus extract has QS rejection abilities (figure
4)。
It is simultaneously model using pseudomonas aeruginosa PA01, analyzes crowd hazards of the fungus extract to PA01
(swarming) influence.The swarming soft agar mediums that 14.75ml heat is taken to melt, are cooled to 30 DEG C or so, Xiang Qi
Middle to add in the 200 μ l extracts, gently mixing, is poured into tablet, after to be solidified, is punched with card punch, and 2 μ l are incubated overnight
Pseudomonas aeruginosa PA01 (OD600nm=0.1) in bacterium solution adding hole, 37 DEG C of constant temperature incubations for 24 hours, observe phenomenon.The result shows that
PA01 shows crowd hazards decrease (Fig. 5) on agar plate, this just illustrates that the fungus extract can be by inhibiting quorum sensing
The crowd hazards of mediation, and then inhibit the expression of virulence factor.
Claims (2)
1. the water Rhein sea of ocean sludge source is write from memory, Salmonella (Rheinheimera aquimaris) QSI02 is in microbial fermentation system
The purposes of the crowd hazards active material of standby inhibition pseudomonas aeruginosa PA01, the bacterial strain is in preservation on April 23 in 2015
In China typical culture collection center, deposit number is CCTCCNO:M2015245.
The method that 2. the water Rhein sea of ocean sludge source is write from memory prepared by Salmonella QSI02 strain fermentations product, it is characterised in that will send out
Zymotic fluid centrifuges, and vacuum and low temperature is concentrated into paste, is extracted with methanol, and methanolic extract is obtained after 0.22 μm of organic phase filter filtering,
The bacterial strain is preserved in China typical culture collection center on April 23rd, 2015, and deposit number is CCTCCNO:
M2015245。
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| CN110358800A (en) * | 2019-07-26 | 2019-10-22 | 福建师范大学 | A kind of small peptide class tunning and its preparation method and application |
| CN110468078B (en) * | 2019-08-27 | 2022-03-29 | 暨南大学 | Rheinheimer HNAD-02 and application thereof in wastewater denitrification |
| CN111264400B (en) * | 2020-02-28 | 2022-02-08 | 贵州绿尚鲜农业股份有限公司 | Fermentation bed for pig breeding and fermentation method thereof |
| CN113215058B (en) * | 2021-06-08 | 2022-05-03 | 山东大学 | Strain capable of degrading collagen and application thereof |
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| CN101974456A (en) * | 2010-09-29 | 2011-02-16 | 中国海洋大学 | Marine streptomyces strain and application thereof |
| CN103695358A (en) * | 2013-12-24 | 2014-04-02 | 扬州大学 | Oceanic streptomyces parvulus and application thereof on aspect of quorum sensing inhibition |
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| CN101974456A (en) * | 2010-09-29 | 2011-02-16 | 中国海洋大学 | Marine streptomyces strain and application thereof |
| CN103695358A (en) * | 2013-12-24 | 2014-04-02 | 扬州大学 | Oceanic streptomyces parvulus and application thereof on aspect of quorum sensing inhibition |
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