CN109679942A - A kind of method and application based on the reduction antibiotic dosage for inhibiting bacterial virulence release - Google Patents
A kind of method and application based on the reduction antibiotic dosage for inhibiting bacterial virulence release Download PDFInfo
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Abstract
The present invention relates to a kind of methods and application based on the reduction antibiotic dosage for inhibiting bacterial virulence release.A kind of application of QQAM albumen and the drug of antibiotic combination production in the pathogenic bacteria for killing generation envelope, the QQAM albumen are selected from: (i) has albumen of amino acid sequence shown in SEQ ID NO.2.Present invention firstly discovers that when carrying out killing the pathogenic bacteria of generation envelope by QQAM albumen and antibiotic combination, so that antibiotic more easily enters in pathogenic bacteria body, to realize that a small amount of antibiotic plays the effect of inhibition pathogenic bacteria, and the significant effect is better than the albumen of existing known other similar mechanism of action, more surprisingly, when QQAM albumen of the present invention and antibiotic are combined, the antibiotic resistance of relevant pathogenic bacteria can be significantly reduced, this has exceeded the expectation of those skilled in the art completely.
Description
Technical field
The present invention relates to a kind of methods and application based on the reduction antibiotic dosage for inhibiting bacterial virulence release, belong to egg
White matter field of engineering technology.
Background technique
Generally existing between different microorganisms species to have a kind of exchange way, bridge has been erected in its communication between group, and
There is very important influence to the normal physiological metabolism process of microorganism, this kind of exchange way is known as quorum sensing or density sense
Answer (quorum sensing, QS).It refers specifically to microbes and secretes certain chemical signal factors into microenvironment where it, pass through
The regulation of related gene expression is completed to its monitoring, and then changes the physiological metabolism of microorganism.QSA (is induced by AHL
QS) be a kind of microorganism communication mechanism, adjustable gene expression simultaneously coordinates micropopulation behavior, first in several bacterium objects
It is accredited in kind, wherein most of is pathogen.It is a series of studies have shown that the virulence factor and QSA of the secretion of a variety of pathogens are close
Cut phase is closed.
The new strategy that (QQ) interferes QSA system code treatment bacteriosis is quenched by quorum sensing.From theory
On say, QQ can reduce the expression for the virulence factor that any pathogen generates under QSA control.The degradation of AHL signaling molecule is resistance
One of the major way of disconnected QSA process.In research before, it was found that a kind of new QQ enzyme MomL (QQAM) can degrade
Generally existing signaling molecule AHL.QQAM is to identify from ocean oil resistant tool handle bacterium Muricauda olearia Th120
, belong to metal-beta-lactamase family, there is highest with the Aii20J albumen in adhesion bacillus Tenacibaculum sp.
Identity.In addition, QQAM and AiiA has 24.5% identity, AiiA is in bacillus sp.240B1
It finds for the first time, is to study most sufficient AHL lactonase.In vitro test demonstrates the virulence factor table in QQAM inhibition pathogen
The ability reached.Enzyme kinetics experiment shows that QQAM middle chain with higher and long-chain AHL degrading activity are horizontal.The drop of C6-HSL
Solve efficiency kcat/KMValue reaches 2.9 × 105s-1M-1, and degradation capability increases with the increase of signaling molecule chain length；For example,
The k of 3OC10-HSLcat/KMIt is 5.1 × 105s-1M-1.This extensive substrate may selectively assign QQAM generally inhibit QSA according to
Rely the ability of pathogen virulence.
Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) is a kind of conditioned pathogen, is easy to infect
Weak and immune deficiency patient is immunized, is one of main bacterial strain of inside-hospital infection.Clinically, it easily causes postoperative skin
The infection such as skin, subcutaneous tissue, soft tissue, bone, ear, eye, urinary tract, respiratory system and heart valve, is the three big pathogenic bacteria of the mankind
One of.Meanwhile it is also the primary pathogenic bacteria for causing pneumonia.After charrin disease, due to its be actively pumped out system,
Biofilm, outer membrane permeability reduce and bacterium changes the multidrug resistants mechanism such as the target position of antibacterials effect, and resistant rate is high, energy
Enough antibacterial actions for escaping and being resistant to rapidly antibacterials, clinically often cause the obstinate infection for being difficult to cure, at
For the thorny problem of current clinical treatment.
Biofilm refers to that bacterial adhesion in contact surface, secretes polysaccharide matrix, fibrin, lipid protein etc., by it
A large amount of bacterial accumulation film sample objects that itself wrapping is wherein formed.Polysaccharide matrix typically refers to polysaccharide protein complex, also includes
By the organic matter of periphery precipitating and inorganic matter etc..Bacterial biofilm is that bacterium is reform of nature environmental benefits in one kind of existence
Biological phenomena is gathered by microorganism and its secretion and is formed.Microorganism with biofilm is than no unicellular micro- life
Object has stronger environmental suitability, its formation brings serious harm to the mankind.Such as in field of medicaments, table according to the study
Bright, about 65% human bacterial's property infectious disease is related with biofilm, and the bacterium of biofilm package is to almost all of anti-
Raw element shows extremely strong resistance, the immune response of host can be escaped, so as to cause the chronic infection cured is difficult to.Biology
In envelope microorganism to the drug resistance of antibiotic than floating state when it is hundreds and thousands of times high, considerably increase the difficulty of clinical treatment
Degree.
Bacterial drug resistance is that bacterium creates antagonism the insensitive phenomenon of raw element, and producing cause is bacterium in own existence process
One of special representing form.Natural antibiotics are bacteriogenic secondary metabolites, for resisting other microorganisms, are protected
Protect the chemical substance of inherently safe.Micro- life that antibacterials are used to kill infection is made in bacteriogenic this substance by the mankind
Object, microorganism touch antimicrobial, also can resist antibacterials by changing metabolic pathway or producing corresponding inactivating substance.
Selection pressure representated by large-scale use antibiotic propagates the microbial strains with mechanism of drug resistance, thus very much
In the case of hinder suitable clinical treatment.
Traditionally, antibiotic is considered as the effective agent for controlling these bacterial pathogens.However, excessive antibiotic makes
With having caused serious drug resistance and accelerated the appearance of multiple drug resistant bacteria, so that the bacterium with antibiotic resistance is extensive
It propagates, to hinder the treatment of disease in many cases, influences the yield of crops and aquaculture, in some instances it may even be possible to can endanger
And the life of patient.At the same time, developing new antibiotic is a very long and expensive process.This predicament promotes people to seek
New strategy is looked for, makes these pathogen again to antibiotic sensitive, and slows down the differentiation of antibiotic resistance.QQ strategy is to treatment
Infectious diseases is distinguished by, because they do not directly affect the existence of pathogen, but influences the table of its virulence factor
It reaches, they will not generate survival pressure, therefore avoid the appearance of drug resistance.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of based on the reduction antibiotic dosage for inhibiting bacterial virulence release
Method and application.
Technical solution of the present invention is as follows:
A kind of application of QQAM albumen and the drug of antibiotic combination production in the pathogenic bacteria for killing generation envelope, institute
QQAM albumen is stated to be selected from:
(i) albumen with amino acid sequence shown in SEQ ID NO.2；
(ii) amino acid sequence as shown in SEQ ID NO.2 passed through into the substitution of one or several amino acid residues, lacked
Lose or addition and formed, with the albumen as derived from (i) to QSA degradation capability.
Preferred according to the present invention, mass ratio associated with the QQAM albumen and antibiotic is 2:3.
Preferred according to the present invention, the antibiotic is selected from: kanamycins, gentamicin, polymyxins b；
According to the present invention it is further preferred that the antibiotic is selected from: kanamycins, polymyxins b.
Preferred according to the present invention, the pathogenic bacteria of the generation envelope are selected from: pseudomonas aeruginosa, thermophilic aqueous vapor unit cell
Bacterium, Bai Kehuode bacterium, carrot pectin bacillus, pathogenic Erwinia, kerekou pneumonia cypress Salmonella, corn wilt.
According to the present invention it is further preferred that the pathogenic bacteria of generation envelope are selected from: pseudomonas aeruginosa, primary gram of new onion
Hall moral bacterium, carrot soft rot Erwinia, Bacteria erwinia, kerekou pneumonia cypress Salmonella.
Preferred according to the present invention, the dosage form of the drug is solid particle.
Technical effect
1, present invention firstly discovers that when carrying out killing the pathogenic bacteria of generation envelope by QQAM albumen and antibiotic combination,
So that antibiotic more easily enters in pathogenic bacteria body, to realize that a small amount of antibiotic plays the effect for inhibiting pathogenic bacteria, and should
Significant effect is better than the albumen of existing known other similar mechanism of action, more surprisingly, QQAM egg of the present invention
When the white combination with antibiotic, the antibiotic resistance of relevant pathogenic bacteria can be significantly reduced, this has exceeded those skilled in the art completely
Expectation；
2, QQAM albumen of the present invention imitates the killing of specific pathogenic bacteria when being combined with certain antibiotics
Fruit is significantly better than QQAM albumen and other similar pathogenic bacteria are killed in the combination of other antibiotic, and can reduce specific pathogenic bacteria offspring
Biofilm yield.
Detailed description of the invention
Influence of Fig. 1 QQAM albumen to 10 kinds of common pathogen survival rates；
Fig. 2 galacturonic acid standard curve；
Influence of Fig. 3 QQAM albumen to 10 kinds of common causative bacteria pathogenics；
Influence of Fig. 4 QQAM albumen to the infecting potential of Pcc；
The survival rate of Pcc under Fig. 5 Kana after continuous passage measures；
The survival rate of PAO1 under Fig. 6 Kana after continuous passage measures；
The antibiotic resistance (MIC) of Pcc after Fig. 7 Kana or QQAM albumen continuous passage measures；
The antibiotic resistance (MIC) of PAO1 after Fig. 8 Kana or QQAM albumen continuous passage measures；
Pathogenic influence of Fig. 9 QQAM albumen on the Pcc after the passage of continuous Kana or QQAM albumen；
Figure 10 QQAM albumen is to 5 kinds of representative antibiotic resistances (MIC) of pathogenic bacteria and the tolerance (MBC) of antibiotic
Detection；
Influence of Figure 11 QQAM albumen to the evolutionary rate for slowing down antibiotic resistance；
Influence of Figure 12 QQAM albumen to pseudomonas aeruginosa PAO1 biofilm formation.
Specific embodiment
In some embodiments, QQAM albumen can be the albumen with amino acid sequence shown in SEQ ID NO:2.?
In some embodiments, QQAM albumen be can be amino acid sequence shown in SEQ ID NO:2 by one or several amino acid
Replacing, missing or adding for residue and formed, with degradation QSA and inhibit cell membrane Forming ability albumen.In some realities
It applies in example, QQAM albumen can be having for homology >=95% of sequence shown in amino acid sequence and SEQ ID NO:2 and degrade
QSA and the albumen for inhibiting cell membrane Forming ability.Wherein it is preferred to homology >=98%.For example, can be to SEQ ID
Albumen shown in NO:2 is transformed, to improve the albumen of its QSA ability of degrading.
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Bacterial strain and culture medium
Strain pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, pseudomonas aeruginosa (Pseudomonas
Aeruginosa) SM45, Acinetobacter baumannii (Acinetobacter baumannii) YC28, new Burkholderia cepacia
(Burkholderia cenocepacia) BNCC157156, Aeromonas hydrophila (Aeromonas hydrophila) YC57,
Bacteria erwinia (Pectobacterium carotovorum) BNCC138474, Burkholderia glumae
(Burkholderia glumae) BNCC341645, carrot soft rot Erwinia (Erwinia carotovora)
BNCC166445, erwinia amylovora (Erwinia amylovora) ATCC 51855, P.stwartii subsp.stewartii (Pantoea
Stewartii ssp.Stewartii) DSM30176 is ordinary commercial products.
LB solid medium: 10g tryptone, 5g yeast powder, 10g sodium chloride and 1000ml distilled water, pH=7.0.
LB liquid medium: 10g tryptone, 5g yeast powder, 10g sodium chloride, 20g agar powder and 1000ml distillation
Water, pH=7.0.
MH fluid nutrient medium: 2g powdered beef, 1.5g soluble starch, 17.5g acid hydrolyzed casein and 1000ml distillation
Water, pH=7.4.
Influence of the embodiment 1:QQAM to 10 kinds of common pathogen survival rates
By activated 10 pathogenic bacteria (PAO1 (Pseudomonas aeruginosa PAO1), SM45 (Pseudomonas
Aeruginosa SM45), AB (Acinetobacter baumannii YC28), BC (Burkholderia cenocepacia
BNCC157156), AH (Aeromonas hydrophila YC57), Pcc (Pectobacterium carotovorum
BNCC138474), BG (Burkholderia glumae BNCC341645), EC (Erwinia carotovora
BNCC166445), EA (Erwinia amylovora ATCC 51855), PS (Pantoea stewartii
Ssp.Stewartii DSM30176)) it is inoculated in LB liquid medium by 1% inoculum concentration, it mixes well, is sub-packed in parallel
In four sterilizing test tubes, the QQAM enzyme solution of equivalent is added in each test tube, the enzyme stock solution of equivalent is added in control group.It will be every
The pathogenic bacteria bacterium solution of group is placed in 28 DEG C, 170rpm shake culture for 24 hours after, 10 are taken after gradient dilution-4~10-6Diluted bacterium solution again
It is applied on LB plate, 28 DEG C of incubator culture 15h.Single colonie on plate is counted, on each pathogenic bacteria control plate
Clump count is 100%, as a result such as Fig. 1.The experimental results showed that after QQAM and various pathogens co-culture, the survival rate of pathogenic bacteria
It is declined slightly, but effect is not significant, illustrates that QQAM is little for the existence growth effect of pathogenic bacteria, not will cause existence pressure
Power.
Influence of the embodiment 2:QQAM to 10 kinds of common causative bacteria pathogenics
Use DNS measuring method measurement pathogen cell exopolygalacturonase activity.
D- galacturonic acid, is dried to that quality is unchanged, and being configured to concentration is by the preparation of galacturonic acid standard curve
The galacturonic standard acid solution of 1.0mg/ml.Then the galacturonic acid solution of dilution configuration various concentration.In various concentration
In galacturonic acid solution, it is separately added into the mixing of 400 μ l DNS reagents, is cooled to room temperature after boiling water bath 5min.After drawing cooling
50 μ l of liquid to be detected, add 100 μ l distilled water dilute three times, after mixing under 540nm wavelength with microplate reader survey absorbance, every group
Do three in parallel.Using the concentration of galacturonic acid solution as abscissa, OD540As ordinate, galacturonic acid standard is drawn
Curve；As shown in Figure 2.
Firstly, (NaCl of 0.2% polygalacturonase, 0.2M is molten for 0.2% polygalacturonic acid reaction buffer of preparation
Solution is in the 0.05M sodium acetate buffer of pH=5.2).Then, in order to obtain the culture samples for enzymatic determination, will implement
Supernatant is filtered through at 4 DEG C or on ice 0.22 μm of filter mistake with 4,000rpm centrifugation 5 minutes by culture in example 1
Filter out bacterium.0.2% polygalacturonase reaction buffer of the above-mentioned crude enzyme liquid of 200 μ l and 400 μ l is mixed and is immersed in
It is kept for 30 minutes in 48 DEG C of water-baths.The DNS solution of 400 μ l is added in above-mentioned 600 μ l reaction system later, by mixture
Boiling water bath 5 minutes, and with 12,000rpm centrifugation 1 minute, then discard sediment.Three times by supernatant dilution, and in 540nm
Place's measurement absorbance, with the value of each pathogenic bacteria control group for 100%, as a result as shown in Figure 3.QQAM significantly reduce 10 kinds it is general
It is pathogenic all over existing pathogenic bacteria, about all reduce 50% or so, wherein QQAM to disease inhibiting effect caused by Pcc most
Obviously.
Influence of the embodiment 3:QQAM to the infecting potential of Pcc
Pcc can infect the various vegetables such as a variety of hosts, including Cruciferae, Solanaceae, pulse family, Curcurbitaceae, cause economic agriculture
Crop damage.This experiment studies influence of the QQAM to Pcc plant infecting potential inhibitory effect so that Pcc infects Chinese cabbage as an example.
Pcc is inoculated in LB liquid medium 170rpm shaken cultivation at 28 DEG C, until OD600=0.8, to bacterium solution into
Row dilution, makes bacterial density 106cfu/mL.By the QQAM of final concentration of 0.05U/ml, 0.1U/ml, 0.5U/ml, 20 μ g/ml
Kana and boil the QQAM of inactivation and mixed with the Pcc bacterium solution after dilution, for use.Select same Chinese cabbage form, size, growth hair
It educates blade similar in situation to be cleaned, is placed in naturally dry in superclean bench, and spray 70% ethyl alcohol and sterilized.To second
After alcohol volatilizees completely, the wound of 1 long 2cm is cut out in central vane with sterile razor blade, 10 μ L mixed bacteria liquids are injected into wound, with
Pcc bacterium solution is positive control.Finally, aseptic filter paper piece 10mL sterile water is drenched, with the cabbage leaf one for taking over pathogen
It is sealed with being put in freshness protection package, 28 DEG C are cultivated 48 hours.The case where observation cabbage leaf is infected.As a result as shown in Figure 4.
Only there is apparent bacterial soft rot symptom in the cabbage leaves of inoculation Pcc, and tissue is in sticky web rot shape, plant
Cell wall is destroyed, and moisture extravasation forms water stain shape scab.And it is inoculated with the great Bai of the QQAM of Pcc and 0.05U/ml simultaneously
The wound infection symptom that dish is shown slightly is lighter than positive controls, suppression of the Kana of the QQAM of 0.5U/ml and 20 μ g/ml to Pcc
Effect processed is preferably and suitable, and the QQAM after boiling inactivation does not influence the infecting potential of Pcc.
The survival rate of Pcc/PAO1 under embodiment 4:Kana after continuous passage measures
Activated Pcc and PAO1 are inoculated in new LB liquid medium respectively according to 1% inoculum concentration, first from adding
The Kana for entering low concentration starts, and is placed in 28 DEG C, and 170rpm is cultivated for 24 hours, and the training of identical Kana concentration is transferred to according to 1% inoculum concentration
It supports in base, continues to cultivate.Continuous passage bacterium to the Kana for gradually adapting to that higher concentration is added after the concentration antibiotic environment,
And so on, until passage 90 is control group on behalf of Kana continuous passage only, is not added.Every several generations, by the Pcc/PAO1 of passage
Bacterium solution is set to take after gradient dilution and is applied on LB plate, 28 DEG C of incubator culture 15h.Single colonie on plate is counted, with each
It is 100% that pathogenic bacteria, which compare the clump count on plate, as a result as shown in Figure 5 and Figure 6.Pcc and PAO1 is passed under antibiotic pressure
The bacterial strain in generation gradually shows the obvious resistance to Kana.
The antibiotic resistance (MIC) of Pcc/PAO1 after embodiment 5:Kana or QQAM continuous passage measures
The Pcc/PAO1 in 90 generations will be passed in Kana or QQAM respectively in Mueller-Hinton (MH) broth bouillon
It is middle to cultivate 18 hours and be adjusted to OD600=1.0, it then dilutes and uses in 1:1000 suspension, obtain 106Cfu/ml's
Pcc/PAO1 culture solution.It is mixed with the Kana of various concentration, Kana final concentration successively 4,8,12,16,24,32,48,64,
80,96,128,160,192,256,320,384,512,768,1024μg/ml.After 37 DEG C of standings incubate 18 hours, it is measured
Light absorption value at 600nm, every group setting 3 parallel.As a result as shown in Figure 7 and Figure 8.
The minimum inhibitory concentration (MIC) of the Pcc/PAO1 passed in the presence of Kana is increased to from 8/256 μ g/ml respectively
128/1024 μ g/ml shows that Pcc/PAO1 obtains Kana resistance during continuous passage.Meanwhile it being passed in the presence of QQAM
The Pcc/PAO1 in generation does not obtain the resistance to Kana, and it is anti-to illustrate that the continuous use of QQAM not will lead to pathogenic bacteria generation antibiotic
Property.
Pathogenic influence of the embodiment 6:QQAM on the Pcc after continuous Kana or QQAM passage
The Pcc (Pcc-AT/Pcc-QT) for passing on for 90 generations in Kana or QQAM is inoculated in the training of LB liquid according to 1% inoculum concentration
It supports in base, Pcc-AT experimental group is separately added into the QQAM of the Kana and 0.5U/ml of 100 μ g/ml of final concentration, and Pcc-QT tests component
Not Jia Ru 20 μ g/ml of final concentration Kana and 0.5U/ml QQAM, at 28 DEG C 170rpm shaken cultivation for 24 hours, culture with 4,
000rpm is centrifuged 5 minutes, and supernatant is filtered through at 4 DEG C or on ice to 0.22 μm of filter filtration sterilization.By the upper of 200 μ l
The 0.2% polygalacturonase reaction buffer for stating crude enzyme liquid and 400 μ l, which is mixed and is immersed in 48 DEG C of water-baths, keeps 30
Minute.The DNS solution of 400 μ l is added in above-mentioned 600 μ l reaction system later, by mixture boiling water bath 5 minutes, and with
12,000rpm centrifugations 1 minute, then discard sediment.Three times by supernatant dilution, and absorbance is measured at 540nm, with list
The value of only Pcc-AT control group is 100%, as a result as shown in Figure 3.QQAM significantly reduces 10 kinds of generally existing pathogenic bacteria
It is pathogenic, 50% or so is about all reduced, wherein QQAM is most obvious to disease inhibiting effect caused by Pcc.As a result such as Fig. 9 institute
Show.Although the Pcc passed in the presence of Kana obtains antibiotic resistance, Kana is unknown to the inhibiting effect of its infecting potential
It is aobvious, but QQAM still is able to control its infecting potential；I.e. after 90 generations, the QQAM of 0.5U/ml is almost inhibited in antibiotic
In the presence of the infecting potential of strains Pcc-AT that generates.
Tolerance (MBC) inspection of the embodiment 7:QQAM to the antibiotic resistance (MIC) and antibiotic of 5 kinds of representative pathogenic bacteria
It surveys
MIC is measured using 2x dilution technology in 96 hole microtiter plates.By 5 kinds of representative pathogenic bacteria in Mueller-
It is cultivated 18 hours in Hinton (MH) broth bouillon and is adjusted to OD600=1.0, then diluting in 1:1000 suspension makes
With obtaining 106The bacterium solution of cfu/ml.2x dilution is carried out using the antibiotic that original concentration is 1024 μ g/ml.In brief, will
The MH culture medium of 100 μ l is drawn in each hole, and the antibiotic of from the 1024 μ g/ml to 100 μ l of the first row addition, after mixing
100 μ l solution are drawn from first hole with liquid-transfering gun again to the 2nd hole, 100 μ l is drawn after mixing again to the 3rd hole, so connects
Continuous doubling dilution is drawn 100 μ l from the 11st hole and is discarded to the 11st hole, and the 12nd hole is not antibiotic MH broth bouillon
Control.Each hole antibiotic concentration is followed successively by 512,256,128,64,32,16,8,4,2,1,0.5 μ g/ml at this time.Into each hole
The 100 diluted bacterial suspensions of μ l are added, at this time each hole antibiotic concentration be followed successively by 256,128,64,32,16,8,4,2,1,
0.5,0.25 μ g/ml and the QQAM of 10 μ l is added into each hole.After 37 DEG C of standings incubate 18 hours, it is measured in 600nm
The light absorption value at place, the lowest concentration of drug for completely inhibiting bacterial growth is MIC.Continue cultivate 48h after observe bacterium growing state,
Plate observation is applied, lowest concentration of drug contained by asepsis growth hole is MBC, and the results are shown in Figure 10 in parallel for every group of setting 3.
Other than Erwinia carotovora (EC), antibiotic resistance (MIC) can be reduced to by QQAM individually to be made
60%-70% when with antibiotic.The result shows that the application of QQAM- Antibiotic combination can reduce QSA bacterium to Conventional antibiotic
Drug resistance.In the presence of QQAM, compared with antibiotic is used alone, there are five types of test strain to the resistance to of antibiotic (MBC)
It is significantly reduced by property to 40-70%.
Influence of the embodiment 8:QQAM to the evolutionary rate for slowing down antibiotic resistance
Activated PAO1 and Pcc are inoculated in new LB liquid medium respectively according to 1% inoculum concentration, set respectively
3 combinations are set, there is only the QQAM of the QQAM of antibiotic, antibiotic and 0.1U/ml and antibiotic and 0.5U/ml.First from addition
The antibiotic of low concentration starts, and is placed in 28 DEG C, and 170rpm is cultivated for 24 hours, is transferred to according to 1% inoculum concentration dense containing identical antibiotic
In the culture medium of degree, continue to cultivate.Higher concentration is added to the concentration antibiotic environment is gradually adapted in continuous passage bacterium later
Kana, and so on, until passage 90 on behalf of only, with without antibiotic passage bacterium be control.
By the PAO1 and Pcc in each combination 90 generations of passage in Mueller-Hinton (MH) broth bouillon embodiment 7
Method measure MIC value, as a result as shown in figure 11.
After 90 generations, in the presence of single antibiotic, the MIC of PAO1 and Pcc increase to 4.0 Hes horizontally relative to its ancestors
16.0 times.In contrast, the evolution for having significantly slowed antibiotic resistance is used in combination in antibiotic-QQAM.In the QQAM of 0.1U/ml
In the presence of, the antibiotic resistance level of PAO1 and Pcc respectively than no QQAM when it is 2.2 and 3.9 times low.Simultaneously to antibiotic resistance
The inhibition of evolution is directly proportional to QQAM dosage.
In addition, found by using the experiment that the mass ratio of different Q QAM enzyme and kanamycins carries out above-described embodiment,
The optimal use ratio of QQAM enzyme and kanamycins is 2:3 (mass ratio), and only be can be only achieved in the mixing of this ratio
Good result, if ratio is higher than 2:3, restraining and sterilizing bacteria effect is unobvious, and such as when ratio is 1:1, restraining and sterilizing bacteria effect is not
And the 20% of 2:3 ratio effect；If ratio is lower than 2:3, such as when ratio is 2:4, then Antibiotic Resistance delays to imitate
Fruit significantly reduces, not as good as the 50% of 2:3 ratio.
Influence of the embodiment 9:QQAM to pseudomonas aeruginosa PAO1 biofilm formation
By pseudomonas aeruginosa PAO1 at 37 DEG C, 170rpm is cultivated 18 hours, calibrates to OD600=1.0, and use tryptose
Peptone soy broth (TSB) is diluted with 1:1000.Diluted culture is micro according to 96 holes of the every hole equivalent addition of 200 μ l
In titer plate, the QQAM crude extract that 10 μ l are added is experimental group, and the TSB broth bouillon of 10 μ l is added as blank control.?
After 37 DEG C of standings are grown 24 hours, the content in hole is gently discarded, is then washed 3 times with PBS buffer solution (pH=7.2).It will
Biomembrane is dry and fixes 20 minutes with 200 μ l methanol.Attached cell 1% crystal violet solution of 200 μ l is dyed 20 minutes.
It will be re-dissolved 30 minutes with the dyestuff of cell combination with 95% ethyl alcohol of 160 μ l, and measure the light absorption value at 570nm, the reality
It is parallel to test 3 groups of setting.As a result as shown in figure 12, in PAO1, the yield of biomembrane is reduced to 65% by QQAM.
Comparative example 1
The existing known QQA albumen with identity function is AiiA albumen and AttM albumen, respectively according to embodiment 6, reality
The method for applying example 7 and embodiment 8 is detected, by comparing discovery, the disease suppression difference of QQAM albumen described herein
It is 12.3 times of 10.8 times of AiiA albumen and AttM albumen；QQAM albumen is AiiA respectively to antibiotic resistant inhibitory effect
8.9 times of 5.2 times of albumen and AttM albumen；QQAM albumen is AiiA albumen respectively to the inhibitory effect of antibiotic resistance
9.3 times of 7.2 times and AttM albumen, AiiA albumen and AttM albumen only have fainter inhibition antibiotic resistant and tolerance
Effect.QQAM is also significantly better than AiiA albumen and (slow 6.3 times respectively of AttM albumen to antibiotic resistance evolution frequency is slowed down
With 5.5 times), AiiA albumen and AttM albumen only have the faint effect for slowing down antibiotic resistance evolution frequency.
Comparative example 2
Be respectively adopted QQAM and gentamicin, polymyxin B, tetracycline, rifampin, erythromycin, chloramphenicol, cephalo he
Heavy stone used as an anchor, acetyl spiramycin, ampicillin are combined effect analysis by the method for above-described embodiment, the results showed that in identical survey
Under test ring border, the synergy of the above antibiotic and QQAM enzyme is not as good as the synergy of QQAM enzyme and kanamycins；Wherein celebrate
Big mycin is the 60% of kanamycins effect, and polymyxins b is the 80% of kanamycins effect, remaining effects of antibiotics is not
And the 50% of QQAM enzyme and kanamycins synergy.
Sequence table
<110>Chinese Marine University
<120>a kind of method and application based on the reduction antibiotic dosage for inhibiting bacterial virulence release
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 816
<212> DNA
<213>ocean oil resistant tool handle bacterium (Muricauda olearia)
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accacgtaca cgggtcagtc caaggaattt gccgatgctt tttacgtcat cgttcacccc 180
aagggcactt tgatgtggga tgcaggtctt cccgaaagct tggtgggtct tccagagccc 240
tttacatcac ccgatggggc atttaccgtt tcccgaaaag attccgtggc caaccaattg 300
gcaagtatcg acatgactgt tgatgatatt gattttatcg cattgtccca tacccatttt 360
gatcatattg gccatgcgaa cgtgtttgca gggtccacgt ggttggtaca ggaaaaggaa 420
tacgactttg tgacaagcga ggacaaccaa aagagcaatc cggacatcta caattccatt 480
aaagagctta ccaaagtgaa gaaaatcaat ggggattacg atgtgttcgg ggatggaagt 540
gtggtaatga aatttatgcc aggccatacc cctggccatc aagtgttgta cttggatatg 600
gttgagcacg gaccgttgat gctttctggg gacatgtacc atttttacga gaaccgggag 660
ttccgaagag tgcccatttt taattacgat gtggccctca ccaagaaaag tatgggagag 720
tttgaagctt ttgccgaaga aaaaggggca aaagtgtatt tgcaacactc caaggaagat 780
tttgaaaaac tgccccaggc acccaactat ttacaa 816
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<400> 2
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Glu Phe Ala Asp Ala Phe Tyr Val Ile Val His Pro Lys Gly Thr Leu
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Phe Thr Ser Pro Asp Gly Ala Phe Thr Val Ser Arg Lys Asp Ser Val
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Ala Asn Gln Leu Ala Ser Ile Asp Met Thr Val Asp Asp Ile Asp Phe
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Ile Ala Leu Ser His Thr His Phe Asp His Ile Gly His Ala Asn Val
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Phe Ala Gly Ser Thr Trp Leu Val Gln Glu Lys Glu Tyr Asp Phe Val
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Thr Ser Glu Asp Asn Gln Lys Ser Asn Pro Asp Ile Tyr Asn Ser Ile
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Gly Asp Gly Ser Val Val Met Lys Phe Met Pro Gly His Thr Pro Gly
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Phe Glu Ala Phe Ala Glu Glu Lys Gly Ala Lys Val Tyr Leu Gln His
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Claims (7)
1. a kind of application of QQAM albumen and the drug of antibiotic combination production in the pathogenic bacteria for killing generation envelope, special
Sign is that the QQAM albumen is selected from:
(i) albumen with amino acid sequence shown in SEQ ID NO.2；
(ii) by the amino acid sequence as shown in SEQ ID NO.2 by one or several amino acid residues substitution, missing or
Addition and formed, with the albumen as derived from (i) to QSA degradation capability.
2. application as described in claim 1, which is characterized in that mass ratio associated with the QQAM albumen and antibiotic is 2:3.
3. application as described in claim 1, which is characterized in that the antibiotic is selected from: kanamycins, and gentamicin is more
Colistin b.
4. application as claimed in claim 3, which is characterized in that the antibiotic is selected from: kanamycins, polymyxins b.
5. application as described in claim 1, which is characterized in that the pathogenic bacteria of the generation envelope are selected from: P. aeruginosa
Bacterium, Aeromonas hydrophila, Bai Kehuode bacterium, carrot pectin bacillus, pathogenic Erwinia, kerekou pneumonia cypress Salmonella, corn
Wilt.
6. application as claimed in claim 5, which is characterized in that the pathogenic bacteria of the generation envelope are selected from: P. aeruginosa
Bacterium, new Burkholderia cepacia, carrot soft rot Erwinia, Bacteria erwinia, kerekou pneumonia cypress Salmonella.
7. application as described in claim 1, which is characterized in that the dosage form of the drug is solid particle.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111171122A (en) * | 2020-01-07 | 2020-05-19 | 华中农业大学 | Antibacterial peptide PtR946 derived from pinellia ternata and application thereof |
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| CN103981140A (en) * | 2014-06-09 | 2014-08-13 | 中国海洋大学 | Muricauda olearia and application thereof |
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| WO2010053455A1 (en) * | 2008-11-07 | 2010-05-14 | Agency For Science, Technology And Research | Novel polyamino polyketide antibiotics and uses thereof |
| CN103981140A (en) * | 2014-06-09 | 2014-08-13 | 中国海洋大学 | Muricauda olearia and application thereof |
| CN108486138A (en) * | 2018-03-27 | 2018-09-04 | 中国海洋大学 | A kind of construction method of high vigor albumen for disease control and application |
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| CN111171122A (en) * | 2020-01-07 | 2020-05-19 | 华中农业大学 | Antibacterial peptide PtR946 derived from pinellia ternata and application thereof |
| CN111171122B (en) * | 2020-01-07 | 2021-06-01 | 华中农业大学 | Antibacterial peptide PtR946 derived from pinellia ternata and application thereof |
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