CN106420734B - A kind of scavenger for killing staphylococcus aureus persister rest cell - Google Patents
A kind of scavenger for killing staphylococcus aureus persister rest cell Download PDFInfo
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- CN106420734B CN106420734B CN201610840995.1A CN201610840995A CN106420734B CN 106420734 B CN106420734 B CN 106420734B CN 201610840995 A CN201610840995 A CN 201610840995A CN 106420734 B CN106420734 B CN 106420734B
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- staphylococcus aureus
- scavenger
- ampicillin
- cinnamic acid
- persister
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/429—Thiazoles condensed with heterocyclic ring systems
- A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/11—Aldehydes
Abstract
The present invention relates to microorganisms technical fields, disclose a kind of scavenger for killing staphylococcus aureus persister rest cell.Scavenger of the present invention includes ampicillin and cinnamic acid, it is a kind of scavenger for staphylococcus aureus persister rest cell, pass through the synergistic function of both ampicillin and cinnamic acid, realize effective removing to target bacteria, it is more obvious compared to the effect that each component is used alone, new therapy approach is provided for clinical treatment related disease.
Description
Technical field
The present invention relates to microorganisms technical fields, particularly relate to a kind of killing staphylococcus aureus
The scavenger of persister rest cell.
Background technique
Food-borne original staphylococcus aureus (Staphylococcus aureus, Sa) belong to Gram-positive ball
Bacterium, can cause the important pathogenic bacteria of human and animal's suppurative infection, and one of common pathogen for causing human foods to be poisoned.
The Center for Disease Control report points out that the infection as caused by staphylococcus aureus accounts for second, is only second to Escherichia coli；?
Disease caused by China staphylococcus aureus also happens occasionally.In addition, staphylococcus aureus is to cause skin infection most
One of common cause.
Some bacteriums will form genetic resistance, and other suspend mode that then can be referred to as " persister " by formation are thin
Born of the same parents' (in this cell the enzyme target of antibiotic be inactivation) and become with tolerance, can be deposited in the presence of antibiotic
It is living.Test result shows that staphylococcus aureus is readily formed persister, even if ampicillin is its 20 times of MIC's
It is still helpless to these obstinate cells under concentration.Even if Joseph Bigger finds that staphylococcus aureus highly concentrated
Still there is minority that can survive under the action of the ampicillin of degree, existence ratio is 1:106~1:104.This few portion
Divide cell resistant to ampicillin and survives, but this resistance does not have heredity, after removing antibiotic
This part cell will continue to growth and breeding, and this cell to survive is referred to as persister.
Staphylococcus aureus can form persister and be difficult thoroughly to be eradicated, this is just clinical treatment correlation disease
Disease causes great challenge, and what invention disclosed or documents and materials studied more before is inhibition to staphylococcus aureus
Effect, and to the persister rest cell of anti-biotic resistance without effect.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of killing staphylococcus aureus persister rest cells
Scavenger, enable the scavenger to kill staphylococcus aureus persister rest cell completely, have apparent
Synergistic effect.
To achieve the goals above, the invention provides the following technical scheme:
A kind of scavenger for killing staphylococcus aureus persister rest cell, including ampicillin and cortex cinnamomi
Aldehyde.
Ampicillin is a kind of semi-synthetic penbritin, although having inhibiting effect to staphylococcus aureus,
But it is invalid to staphylococcus aureus persister rest cell.For this problem, the present invention selects cinnamic acid and ammonia
Parasiticin forms scavenger, generates synergistic effect between the two, has effectively killed staphylococcus aureus persister suspend mode
Cell.
Wherein, the concentration of ampicillin described in scavenger is at least 200 μ g/mL, and the concentration of the cinnamic acid is at least
For 3mM.More preferably, the concentration of the ampicillin is 200-400 μ g/mL, and the concentration of the cinnamic acid is 3-4mM.
In specific implementation process of the present invention, it can be used by the concentration combination of both:
(1) 200 μ g/mL of ampicillin, cinnamic acid 3mM；
(2) 300 μ g/mL of ampicillin, cinnamic acid 3mM；
(3) 400 μ g/mL of ampicillin, cinnamic acid 3mM；
(4) 200 μ g/mL of ampicillin, cinnamic acid 4mM；
(5) 300 μ g/mL of ampicillin, cinnamic acid 4mM；
(6) 400 μ g/mL of ampicillin, cinnamic acid 4mM；
Scavenger of the present invention can effectively kill (80% or more) or kill completely under the premise of using low concentration
Go out staphylococcus aureus persister rest cell, especially under above-mentioned 6 concentration combination situations, can be realized best
Effect, scavenger integrally has apparent synergistic function.
Preferably, scavenger of the present invention further includes solvent, the solvent is not influence ampicillin and cortex cinnamomi
The active suitable solvent of aldehyde is then mixed to form scavenger by the concentration of both solvent adjustment.It was embodied in the present invention
Cheng Zhong, can be used one or both of DMSO, water as solvent, more specifically with water as the solvent of ampicillin,
Use DMSO as the solvent of cinnamic acid.
In the test of ampicillin and cinnamic acid independent role staphylococcus aureus persister rest cell,
As ampicillin concentration increases (from 5 times of MIC to 20 times of MIC), there is part aureus cells always not
It can be killed, this part cell still is able to be killed in renewal cultivation, therefore is not belonging to resisting cell, meets this field
Definition (Maisonneuve E, the Gerdes K. Molecular mechanisms underlying of persister
Bacterial persisters [J] Cell, 2014,157 (3): 539-548.), as a result illustrate that ampicillin cannot
Effectively remove staphylococcus aureus persister；And cinnamic acid individually use 0,1,2,3,4,5mM concentration handle respectively it is golden yellow
Staphylococcus aureus, as the result is shown as the colony count that cinnamic acid concentration for the treatment of increases staphylococcus aureus survival gradually subtracts
It is few, staphylococcus aureus could be killed completely when concentration reaches 5mM, i.e., cinnamic acid could be completely clear under 5mM concentration
Except staphylococcus aureus persister.
When ampicillin and cinnamic acid handle staphylococcus aureus simultaneously, the persister of staphylococcus aureus
Cell quantity is significantly reduced compared to the cinnamic acid with concentration, and with the increase of cortex cinnamomi aldehyde concentration, the removing of persister is imitated
Rate is improved significantly.It is minimum for the clearance rate of persister cell to have reached when cinnamic acid concentration for the treatment of reaches 3mM
50%, highest is more than 80%；When cortex cinnamomi aldehyde concentration reaches 4mM, 15 times and 20 times of MIC ampicillins can be removed all
It is killed concentration reduction 1mM with cinnamic acid processing staphylococcus aureus is used alone by persister cell completely, these
Test result shows that the two is applied in combination to have and acts synergistically.
Based on the technical effect of aforementioned present invention scavenger, the invention proposes the scavengers to kill golden yellow grape
The application in staphylococcus aureus persister rest cell product is killed in coccus persister rest cell and preparation.
Meanwhile the present invention also provides the preparation methods of the scavenger, and ampicillin and cinnamic acid is taken to prepare respectively
At solution, then the two is uniformly mixed, obtains scavenger.More specifically, taking ampicillin that water is added to be configured to solution, draw
Cinnamic acid is configured to solution with DMSO, is then uniformly mixed the two, obtains scavenger.
From the above technical scheme, the present invention forms ampicillin and cinnamic acid compounding a kind of for golden yellow Portugal
The scavenger of grape coccus persister rest cell is realized by the synergistic function of the two to the effective of target bacteria
It removes, it is more obvious compared to the effect that each component is used alone, new therapy approach is provided for clinical treatment related disease.
Detailed description of the invention
Fig. 1 show cinnamic acid to the fungistatic effect of staphylococcus aureus；Wherein a is that cinnamic acid handles golden yellow grape
Coccus for 24 hours with OD when 48h600The line chart of value, b are the growth shape that cinnamic acid handles staphylococcus aureus 48h in 96 orifice plates
Condition；
Fig. 2 show ampicillin to the fungistatic effect of staphylococcus aureus；Wherein a is ampicillin processing gold
Staphylococcus aureus for 24 hours with OD when 48h600The line chart of value, b are that ampicillin handles staphylococcus aureus in 96 orifice plates
The upgrowth situation of 48h；
Fig. 3 show the lower staphylococcus aureus persister cell survived of ampicillin processing；
Fig. 4 show cinnamic acid to the inhibitory effect of staphylococcus aureus persister cell；
Fig. 5 show various concentration cinnamic acid collaboration ampicillin to the plated growth situation after persister effect；
Fig. 6 show various concentration cinnamic acid collaboration ampicillin to the line chart after persister effect, wherein scheming
Example indicates the concentration of cinnamic acid.
Specific embodiment
The embodiment of the invention discloses a kind of scavengers for killing staphylococcus aureus persister rest cell, originally
Field technical staff can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar
Replace and change apparent to those skilled in the art, they are considered as being included in the present invention.The present invention
The scavenger is described by preferred embodiment, related personnel obviously can not depart from the content of present invention, spirit and
Scavenger as described herein is modified in range or appropriate changes and combinations, carrys out implementation and application the technology of the present invention.
In a specific embodiment, if not otherwise specified, the usage amount of the ampicillin under each concentration and cinnamic acid
It is identical, the comparability of guarantee test.
It needs first to carry out activation culture, method to staphylococcus aureus during the test are as follows:
By strain cross cultivate, picking staphylococcus aureus monoclonal 37 DEG C in 50ml LB liquid medium,
150r/min shakes training overnight, then draws 200 μ l of culture solution again and continue to shake in 50ml culture medium after training is used for OD600=1.0
Phase test bacteria liquid.
Wherein, LB culture medium preparation method is to weigh yeast extract 5.0g, peptone 10.0g, sodium chloride 5.0g, agar
15.0g adds water 1000ml in sufficiently dissolving on magnetic stirring apparatus, and adjusting pH value is 7.4-7.6, dispenses triangular flask in 121 DEG C of height
20 min of pressure sterilizing.
Meanwhile scavenger of the present invention using when so that it is connected with staphylococcus aureus persister rest cell
Touching, such as scavenger is connected in a manner of spraying, wiping, dropwise addition with staphylococcus aureus persister rest cell
Touching.
Just a kind of scavenger for killing staphylococcus aureus persister rest cell provided by the present invention is done below
It further illustrates.
Embodiment 1: scavenger of the present invention is prepared
Cinnamic acid uses DMSO for solvent, draws a certain amount of cinnamic acid in DMSO, configures suitable concentration in 4 DEG C of items
It is kept in dark place under part spare；It weighs suitable ampicillin powder to dissolve in distilled water, be filtered with sterilised membrane filter and in -20
It is kept in dark place under the conditions of DEG C spare；Both mixing are required to obtain scavenger according to the final concentration of following any one:
(1) 200 μ g/mL of ampicillin, cinnamic acid 3mM；
(2) 300 μ g/mL of ampicillin, cinnamic acid 3mM；
(3) 400 μ g/mL of ampicillin, cinnamic acid 3mM；
(4) 200 μ g/mL of ampicillin, cinnamic acid 4mM；
(5) 300 μ g/mL of ampicillin, cinnamic acid 4mM；
(6) 400 μ g/mL of ampicillin, cinnamic acid 4mM.
Embodiment 2: the inhibiting effect of cinnamic acid and ampicillin to staphylococcus aureus growth
1, inhibition of the cinnamic acid to staphylococcus aureus
The staphylococcus aureus culture solution prepared in right amount is taken, is diluted to OD with LB liquid medium600=0.02, in 96
In orifice plate, 200 μ l of bacterium solution is added in every hole, is handled with cinnamic acid, and final concentration is respectively 0,0.5,1.0,1.5,2.0,2.5,3.0,
3.5,4.0,4.5,5.0mM, each processing do three it is parallel, 37 DEG C stand and are protected from light culture for 24 hours and 48h, survey it with microplate reader
OD600Value, obtains cinnamic acid to the fungistatic effect of staphylococcus aureus and to staphylococcus aureus with the variation of light absorption value
Minimum inhibitory concentration MIC value, the result is shown in Figure 1.
As seen from Figure 1, it grows by different degrees of after the cinnamic acid processing staphylococcus aureus of various concentration
Inhibition and all show concentration effect, i.e., with concentration for the treatment of increase inhibiting effect enhance.When cinnamic acid concentration for the treatment of
OD value no longer changes substantially when reaching 3mM.Therefore, cinnamic acid is to the minimum inhibitory concentration MIC value of staphylococcus aureus
3mM。
2, inhibition of the ampicillin to staphylococcus aureus
The staphylococcus aureus culture solution prepared in right amount is taken, is diluted to OD with LB liquid medium600=0.02, in 96
In orifice plate, 200 μ l of bacterium solution is added in every hole, is handled respectively with ampicillin, and final concentration is divided into 0,2.5,5,10,15,20,25,
30 μ g/ml, each processing do three it is parallel, 37 DEG C stand and are protected from light culture for 24 hours and 48h, survey its OD with microplate reader600Value, with extinction
The variation of value show that ampicillin to the MIC value of staphylococcus aureus growth, is as a result shown in Fig. 2.
As seen from Figure 2, the ampicillin of various concentration handles staphylococcus aureus, as ampicillin is dense
The increase inhibiting effect of degree enhances, and when concentration reaches 20 μ g/ml, OD value substantially no longer changes, therefore ampicillin is to gold
The 20 μ g/ml of minimum inhibitory concentration MIC value of staphylococcus aureus.
Embodiment 3: the acquisition and detection of staphylococcus aureus persister
Draw the OD prepared600=1.0 staphylococcus aureus culture solution, 100,200,300,400 μ g/ml(5 times
MIC to 20 times of MIC) ampicillin processing, training is shaken under the conditions of 37 DEG C of 150r/min for 24 hours and 48h.5000r/min centrifugation
5min is resuspended with physiological saline, and gradient dilution drips plate, and each gradient does three parallel tests.Plate will have been dripped at 37 DEG C
Under the conditions of cultivate for 24 hours, statistical counting.
Embodiment 4: inhibitory effect of the ampicillin to staphylococcus aureus persister
Draw OD600=1.0 staphylococcus aureus culture solution, at 100,200,300,400 μ g/ml ampicillins
Reason shakes training for 24 hours under the conditions of 37 DEG C of 150r/min, and 5000r/min is centrifuged 5min, is resuspended with physiological saline, and gradient dilution drips
Plate, each gradient do three in parallel.Plate will be dripped to cultivate under the conditions of 37 DEG C for 24 hours, as a result statistical counting is shown in Fig. 3.
Fig. 3 is the results show that as ampicillin concentration increases, and there is part aureus cells always not
It can be killed, since this part cell still is able to be killed in renewal cultivation, be not belonging to resisting cell, meet ability
Definition (Maisonneuve E, the Gerdes K. Molecular mechanisms underlying of domain persister
Bacterial persisters [J] Cell, 2014,157 (3): 539-548.).Ampicillin not can effectively clear
Staphylococcus aureus persister.
Embodiment 5: inhibitory effect of the cinnamic acid to staphylococcus aureus persister
Individually with 0,1,2,3,4,5mM the golden yellow staphylococcus aureus of cinnamic acid processing, as the result is shown with cinnamic acid at
The colony count that reason concentration increases staphylococcus aureus survival gradually decreases, could be by golden yellow Portugal when concentration reaches 5mM
Grape coccus kills completely, as shown in Figure 4.
Embodiment 6: ampicillin cooperates with cinnamic acid to remove persister
When ampicillin and cinnamic acid handle staphylococcus aureus simultaneously, the persister of staphylococcus aureus
Cell quantity significantly reduces, and sees Fig. 5 and Fig. 6, and with the increase of cortex cinnamomi aldehyde concentration, the elimination efficiency of persister is obtained
It significantly improves.It is minimum for the clearance rate of persister cell to have reached 50% when cinnamic acid concentration for the treatment of reaches 3mM, most
Height is more than 80%；When cortex cinnamomi aldehyde concentration reaches 4mM, 15 times and 20 times of MIC ampicillins can be removed all
It is killed concentration reduction 1mM with cinnamic acid processing staphylococcus aureus is used alone by persister cell completely, due to
Ampicillin is invalid to persister cell, then above-mentioned technical effect shows that the two compounding has synergistic function.
The above is only intended to understand method and its core concept of the invention, it is noted that for the art
Those of ordinary skill for, without departing from the principle of the present invention, can with several improvements and modifications are made to the present invention,
These improvement and modification also fall into the protection scope of right of the present invention.
Claims (6)
1. it is a kind of kill staphylococcus aureus persister rest cell scavenger, which is characterized in that its active constituent by
Ampicillin and cinnamic acid composition, the concentration of the ampicillin is 200-400 μ g/mL, and the concentration of the cinnamic acid is
3-4mM。
2. scavenger according to claim 1, which is characterized in that further include solvent.
3. scavenger according to claim 2, which is characterized in that the solvent is one or both of DMSO, water.
4. scavenger described in claim 1-3 any one kills staphylococcus aureus persister rest cell in preparation
Application in product.
5. the preparation method of scavenger described in claim 1, which is characterized in that ampicillin and cinnamic acid is taken to be configured to respectively
Then the two is uniformly mixed by solution, obtain scavenger.
6. preparation method according to claim 5, which is characterized in that take ampicillin that water is added to be configured to solution, draw meat
Cinnamic aldehyde is configured to solution with DMSO, is then uniformly mixed the two, obtains scavenger.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0689842A1 (en) * | 1994-06-27 | 1996-01-03 | DIMOTECH Ltd. | Use of an organic extract of cinnamon plant to inhibit growth of helicobacter pylori |
| CN105595151A (en) * | 2015-08-31 | 2016-05-25 | 北京工商大学 | Staphylococcus aureus inhibitor |
-
2016
- 2016-09-22 CN CN201610840995.1A patent/CN106420734B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0689842A1 (en) * | 1994-06-27 | 1996-01-03 | DIMOTECH Ltd. | Use of an organic extract of cinnamon plant to inhibit growth of helicobacter pylori |
| CN105595151A (en) * | 2015-08-31 | 2016-05-25 | 北京工商大学 | Staphylococcus aureus inhibitor |
Non-Patent Citations (3)
| Title |
|---|
| Anti-biofilm agents: recent breakthrough against multi-drug resistant Staphylococcus aureus;Pooi Y. Chung等;《Pathogens and Disease》;20140224;第70卷;第231-239页，尤其是第232页右栏第1段，第236页右栏第1段 |
| Use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria;Kavitha Palaniappan等;《International Journal of Food Microbiology》;20101231;第140卷;第164-168页，尤其是第166页表3，右栏第2段，165页左栏第4段，右栏第2段 |
| 牛至油、香芹酚、柠檬醛和肉桂醛抑菌作用研究;王新伟等;《食品工业》;20101231(第5期);第13-16页 |
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