CN110755612A - Application and method for killing drug-resistant acinetobacter baumannii by blue light-activated (S) -blebbistatin molecules - Google Patents
Application and method for killing drug-resistant acinetobacter baumannii by blue light-activated (S) -blebbistatin molecules Download PDFInfo
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Abstract
The invention relates to application and a method for killing drug-resistant acinetobacter baumannii by blue light-activated (S) -blebbistatin molecules. The invention uses (S) -blebbistatin molecules to cooperate with blue light, and the (S) -blebbistatin molecules generate hydroxyl free radical with strong oxidizing property by being irradiated under the blue light (·OH), can kill drug-resistant acinetobacter baumannii. The (S) -blebbistatin and the bacterial liquid are uniformly mixed, placed under blue light for irradiation, and then smeared or spotted on the surface of a flat plate for a period of time, and clinical drug resistance acinetobacter baumannii can be observed to be killed. The bactericidal efficiency increases with time without the risk of inducing resistance in the absence of light. The invention is not limited by bacterial drug resistance and is clinical multiple drug resistanceThe treatment of acinetobacter baumannii provides a solid and powerful technical support.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application and a method for killing drug-resistant acinetobacter baumannii by blue light activated (S) -blebbistatin molecules.
Background
Acinetobacter baumannii belongs to non-fermented gram-negative bacteria, is widely existed in natural environment, human skin, digestive system, urinary system and the like, and is mainly used for infected people such as ICU severe patients and old people with low resistance. Acinetobacter baumannii is mainly used for traumatic infection, such as ventilator-associated pneumonia. The acinetobacter baumannii has a prominent drug-resistant phenotype, and particularly shows strong drug resistance to carbapenem antibiotics and quininone antibiotics. With the emergence of multiple drug-resistant acinetobacter baumannii, tigecycline and polymyxin antibiotics become the last line of defense, but the two drugs also have the problems of strong side effect, poor pharmacokinetics and the like. At present, because the research and development of novel antibiotics are seriously lack, an alternative sterilization method needs to be searched to make up the deficiency of the antibiotics. The blue light dependent photoactivation therapy is an attractive therapy with application prospect. The method can not generate gene mutation or induce drug resistance to bacteria under direct regulation and control by light. The blue light synergistic photosensitizer improves the sterilization efficiency with time, and does not have the risk of inducing drug resistance under the condition of no light.
The (S) -blebbistatin molecule is a small molecule compound with left-handed chirality, and is also a selective inhibitor of myosin IIATPase activity. Li (M.D. Li, N-K Wong, et al.J.Am.chem.Soc.140(46):15957-15968(2018)) etc. disclose that (S) -blebbistatin molecules can generate hydroxyl radicals with strong oxidizability under blue light irradiation (·OH), Li and the like previously used human and mouse skin gastric tumor cell models, and the dosage of the (S) -blebbistatin used for tumor cells is 10 to 25 mu M (25 mu M is the upper limit value of the water solubility of the (S) -blebbistatin), so that the efficiency is poor, and the toxic effect of the (S) -blebbistatin on mammalian cells is not obvious, thereby influencing the anti-tumor application of the blebbistatin. There are various methods for generating hydroxyl radicals in chemical systems, such as fenton reaction and ultraviolet irradiation, but they have problems of difficult administration or low safety, and are not suitable for anti-infective therapy.
Disclosure of Invention
The invention aims to provide application and a method for killing drug-resistant acinetobacter baumannii by blue-light activated (S) -blebbistatin molecules, wherein the method has an extremely high-efficient sterilization effect under blue light, avoids bacteria from generating mutation or inducing drug resistance in sterilization, and solves the problems in the prior art.
Application of blue light activated (S) -blebbistatin molecules in killing drug-resistant acinetobacter baumannii.
Preferably, the blue light wavelength is 420 nm.
The drug-resistant acinetobacter baumannii is drug-resistant in three antibiotics of broad-spectrum penicillin, quinolone, β -lactam, monoamide ring, aminoglycoside and polypeptide.
A method for activating (S) -blebbistatin molecules with blue light to kill bed-resistant acinetobacter baumannii mainly comprises the following steps: uniformly mixing 2 times of logarithmic phase acinetobacter baumannii bacterial liquid with 2 times of working concentration of (S) -blebbistatin 1:1, and irradiating for 30-60min by blue light with the wavelength of 420 nm.
The sterilization effect is mainly characterized in that the obtained irradiated bacterial liquid is absorbed by 6-10 mu L and vertically dropped on an agar plate, the plate is inversely cultured for 12h, and then whether the bacterial colony is left or not is observed, so that the sterilization efficiency is identified, the bacterial residue can evaluate the sterilization effect of blue light synergistic with (S) -blebbistatin, the plate has sensitive screening power, if the bacteria have vitality and can grow on the plate, the drug-resistant baumannii can be evaluated whether to kill or not by observing the bacterial colony residue, and the agar plate is an LB (Luria-Bertani) culture plate, an MH (M ü ler-Hinton) culture plate and a Columbia blood agar plate.
OD value measured by Nanodrop on Acinetobacter baumannii bacterial liquid in logarithmic phase is 0.6-1.0.
Preferably, the blue light source is placed at a position 7.2cm above the Acinetobacter baumannii liquid in logarithmic phase.
Preferably, the working density of the Acinetobacter baumannii bacterial liquid in logarithmic phase is 108cfu/mL or less.
Preferably, the working concentration of the (S) -blebbistatin is 2 μ M to 10 μ M.
Preferably, the method for culturing the Acinetobacter baumannii bacterial liquid in logarithmic phase mainly comprises the following steps:
(1) marking drug-resistant acinetobacter baumannii stored at-80 ℃ on an agar plate for multiple times, and performing inverted plate culture for 12h to obtain monoclonal bacteria;
(2) and (3) picking the monoclonal bacteria in an LB culture medium and/or an MH culture medium, and culturing for about 1.5h in a shaking table to obtain the drug-resistant acinetobacter baumannii bacterial liquid in the logarithmic phase. Preferably, 1-4 of the single clones are picked for culture.
Preferably, the preparation method of the (S) -blebbistatin with the 2-fold working concentration comprises the following steps: (S) -blebbistatin is prepared in DMF (dimethyl formamide) solution in advance to prepare mother solution, and then diluted in phosphate buffer PBS to 2 times of working concentration. As acinetobacter baumannii can grow in the culture medium at normal temperature and can not grow in a state of no nutrition in a salt solution, phosphate buffer solution PBS is adopted for dilution after the culture. The phosphate buffer PBS solution comprises NaCl; KCl; na (Na)2HPO4；KH2PO4。
The invention verifies that (S) -blebbistatin has no obvious toxicity after being treated for 1-6h through zebra fish embryo experiments. The (S) -blebbistatin dose needs to be 2 to 10. mu.M, sufficient to completely kill the infection density (10)5cfu/mL) pan-resistant bacteria (many clinical antibiotics are not available). Furthermore, (S) -blebbistatin did not induce the development of drug resistance (no activity in the absence of blue light).
Compared with the prior art, the (S) -blebbistatin molecules are used for cooperating with blue light, and hydroxyl radicals (OH) with strong oxidation polarity are generated by irradiation under the blue light, so that the drug-resistant acinetobacter baumannii can be killed. The (S) -blebbistatin and the bacterial liquid are uniformly mixed, placed under blue light for irradiation, and then smeared or spotted on the surface of a flat plate for a period of time, and clinical drug resistance acinetobacter baumannii can be observed to be killed. The sterilization efficiency is improved along with the increase of time, and the risk of inducing drug resistance does not exist. The invention is not limited by bacterial drug resistance, and provides solid and powerful technical support for the treatment of clinical multi-drug resistant acinetobacter baumannii.
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FIG. 1 is a blue light device platform of the present invention.
FIG. 2 is an electron microscope morphology of the strain (GD0302) used in the present invention: after fixation, observation was performed under an electron microscope (10000 times optical microscope).
FIG. 3 shows that (S) -blebbistatin molecules in example 1 synergize with blue light killing 107Panels of cfu/mL bacteria on MH agar plates and Columbia blood agar plates.
FIG. 4 shows that (S) -blebbistatin molecules in example 2 cooperate with blue light killing 107-103A histogram of the cases of cfu/mL bacteria on MH agar plates and a corresponding histogram of the residual bacterial mass.
FIG. 5 shows that in example 3, (S) -blebbistatin molecules synergize with different blue light wavelengths to kill 108Panels of cfu/mL bacteria on MH agar plates.
FIG. 6 is a microscopic image of the toxicity effect of (S) -blebbistatin molecules on live zebrafish embryos in the absence of blue light: 20X observation under an optical microscope.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings. The equipment and reagents involved in the examples, except for drug-resistant A.baumannii, are commercially available.
Example 1
An application and a method (concentration gradient sterilization) of blue light activated (S) -blebbistatin molecules for killing drug-resistant acinetobacter baumannii comprise the following steps:
1. and (3) carrying out streak culture on the clinical drug-resistant acinetobacter baumannii on a culture plate to obtain monoclonal bacteria.
1.1) taking out clinical drug-resistant acinetobacter baumannii stored at-80 ℃ (number: GD0302) (see FIG. 2), after dissolving on ice, 1. mu.L of the solution was dipped with a ring-shaped inoculating stick and streaked onto LB agar plates.
1.2) inversely buckling the plate and culturing for 12h in an incubator to obtain the monoclonal bacteria.
2. Inoculating into culture medium to make clinical drug-resistant Acinetobacter baumannii reach logarithmic phase.
2.1) 1-4 monoclonal bacteria were picked and cultured overnight in a shaker at 220rpm in a shaker tube containing 3mL LB medium.
2.2) taking 1/10 overnight bacterial liquid, putting the overnight bacterial liquid into a shaking tube filled with a new 3mL LB culture medium, and culturing for 1-1.5h at 220rpm of a shaking table.
2.3) measurement of OD of bacterial liquid Using Nanodrop apparatus600nmValue of OD600nmObtaining logarithmic phase bacterial liquid as 0.6-1.0.
2.4) transfer 3mL of the cell suspension to a 15mL centrifuge tube, and centrifuge at 4000g for 10min to collect the cells as much as possible.
2.5) phosphate buffered saline PBS was prepared, the cells were washed 2 times with 3mL of PBS, and centrifuged at 4000g for 5 min.
2.6) resuspending the cells in 3mL PBS, using Nanodrop, zeroing PBS and reacquiring the correct OD600nmThus, pure log phase bacteria liquid is obtained.
3. And (S) -blebbistatin molecular solution and the bacterial solution are uniformly mixed by adopting a 2-time dilution method, and then are subjected to blue light illumination at a certain distance to obtain sterilized bacterial solution.
3.1) conversion to 2 times the operating density, i.e. 2X 10, from the OD obtained in step 27cfu/mL。
3.2) weighing 5mg of (S) -blebbistatin in DMF solution to prepare 100mM mother liquor, and storing at-20 ℃; the preparation was carried out at 2X working concentration, 2X 2. mu.M, 2X 10. mu.M.
3.3) aspirate 500. mu.L of 2 Xbacterial suspension and 500. mu.L of 2X (S) -blebbistatin into a 1.5mL Eppendorf tube and mix well.
3.4) equipment blue light platform (see fig. 1, blue light platform device is the circular perforation of box cavity of cuboid just can put down lamp pearl position, has the radiator to dispel the heat outward and reduces the error that the heat brought), places the Eppendorf pipe in and opens the lid on the transparent shelf, makes liquid level and lamp pearl distance be 7.2 cm.
3.5) irradiating for 60min by blue light at 420nm to obtain sterilized bacterial liquid, immediately placing on ice and keeping out of the sun to finish the reaction.
4. The bactericidal effect was identified and after a period of time on the plate, it was determined whether there were residual bacteria.
4.1) dripping 6 mu L of stock solution on an MH flat plate according to the sterilized bacterial solution obtained in the step 3.
4.2) diluting the sterilized bacterial liquid obtained in the step 3 by 10000 times, and uniformly coating 100 mu L of the diluted bacterial liquid on a Columbia blood agar culture plate by using a coating rod.
4.3) culturing the culture plate in a reversed incubator for 12h, and observing the sterilization effect.
5. Analysis of results
In this example, drug-resistant A.baumannii strain (No. GD0302) was used at a density of 107cfu/mL, after mixing with 2. mu.M and 10. mu.M of (S) -blebbistatin, it was irradiated at 420nm for 60 min. It can be seen by observation that 2. mu.M of (S) -blebbistatin can kill a large amount of drug-resistant A.baumannii, and 10. mu.M of (S) -blebbistatin can completely kill drug-resistant A.baumannii (see FIG. 3)
The drug sensitivity of the drug-resistant acinetobacter baumannii GD0302 used in this example is shown in table 1.
TABLE 1 drug susceptibility of drug-resistant Acinetobacter baumannii GD0302
Remarking:
i, penicillins; II, cephalosporins; III: penems; IV: quinolones; v: aminoglycosides; VI: chloramphenicol species; VII: compound preparation; VIII: tetracyclines; IX: lipopeptides
Example 2
Application and method for killing drug-resistant acinetobacter baumannii by blue light activated (S) -blebbistatin molecules (density gradient sterilization)
The culture materials, apparatus and process of this example are basically the same as those of example 1, except that:
(1) different from 3.1 in example 1), example 2 improves the upper limit of the bacterial density and relaxes the lower linear density on the basis of example 1, namely 108cfu/mL-103cfu/mL. Bacteria were serially diluted in PBS with a 10-fold density gradient to 2 × working density, i.e., 2 × 107cfu/mL,2×106cfu/mL,2×105cfu/mL,2×104cfu/mL,2×103cfu/mL。
(2) Analysis of results
In this example, drug-resistant A.baumannii strain (No. GD0302) was used at a density of 107cfu/mL,106cfu/mL,105cfu/mL,104cfu/mL,103cfu/mL, 2. mu.M and 10. mu.M of (S) -blebbistatin, and then irradiated for 60min at 420 nm. It can be observed that 2. mu.M of (S) -blebbistatin is at a very high infection density of 107The drug-resistant acinetobacter baumannii cannot be completely eliminated under cfu/mL, and compared with 10 mu M, the drug-resistant acinetobacter baumannii can be completely eliminated with sufficient efficiency under blue light (see figure 4).
Example 3
Application and method for killing drug-resistant acinetobacter baumannii by blue light activated (S) -blebbistatin molecules (with different blue light wavelengths)
The culture materials, apparatus and process of this example are basically the same as those of example 1, except that:
(1) different from 3.1 in example 1), the density of the drug-resistant acinetobacter baumannii adopted in example 3 is 108cfu/mL, i.e. converted to 2-fold working densityIs 2 x 108cfu/mL。
(2) Different from 3.2 in example 1), the (S) -blebbistatin concentration used in example 3 was 0. mu.M, 2. mu.M, that is, 2 Xworking concentration was prepared: 0. mu.M, 2X 2. mu.M.
(3) Different from 3.5 in embodiment 1), embodiment 3 adopts blue light sources of 2 different wavelengths, namely, blue light sources of 420nm and 460 nm.
(4) Different from 4.2 in example 1), the dilution factor used in example 3 was 20,000 times, and 100. mu.L of the suspension was spread evenly on LB agar plates using a spreading bar.
(5) Analysis of results
In this example, drug-resistant A.baumannii strain (No. GD0302) was used at a density of 108cfu/mL, and then is uniformly mixed with 0 mu M and 2 mu M of (S) -blebbistatin and respectively irradiated for 60min at 420nm and 460 nm. The observation shows that the 460nm synergistic (S) -blebbistatin has weak sterilization efficiency, and the 420nm synergistic (S) -blebbistatin can completely kill drug-resistant acinetobacter baumannii strains (see figure 5).
Example 4
Application and method for killing drug-resistant acinetobacter baumannii by blue light activated (S) -blebbistatin molecules (sterilization at different distances)
The culture materials, apparatus and process of this example are basically the same as those of example 1, except that:
(1) different from 3.4 in example 1), example 4 in 3.4) assembling the blue light platform (see fig. 1), the Eppendorf tube was placed on a transparent shelf and the lid was opened, so that the distances between the liquid surface and the bead were 8.2, 10 and 12cm, respectively.
(2) Analysis of results
When the distances between the liquid level and the lamp beads are respectively 8.2 cm, 10 cm and 12cm, the sterilization effect is very weak.
Example 5
Application and method for killing drug-resistant acinetobacter baumannii by blue light activated (S) -blebbistatin molecules (toxicity test)
(1) In this example, 0. mu.M, 5. mu.M, 10. mu.M (S) -blebbistatin was added to zebrafish embryos cultured on day 0, and after 1 hour and 6 hours of treatment without blue light, the zebrafish embryos were observed by 20X light microscope to see whether their developmental characteristics were altered.
(2) Analysis of results
Under the condition of no blue light, the zebra fish embryo treatment group has no toxic effect on the development of the zebra fish embryo in 1h, 6h, 5 mu M and 10 mu M of (S) -blebbistatin treatment group, which indicates that the treatment for 60min by adopting (S) -blebbistatin molecules in living bodies is safe.
Comparative example 1
The drug-resistant A.baumannii in example 1 was replaced with K.pneumoniae under the same conditions. The experimental results show that klebsiella pneumoniae is completely unable to be killed by this method.
Claims (8)
1. Application of blue light activated (S) -blebbistatin molecules in killing drug-resistant acinetobacter baumannii.
2. The use according to claim 1, wherein the blue light wavelength is 420 nm.
3. A method for activating (S) -blebbistatin molecules to kill drug-resistant acinetobacter baumannii by blue light is characterized by mainly comprising the following steps: uniformly mixing 2 times of logarithmic phase acinetobacter baumannii bacterial liquid with 2 times of working concentration of (S) -blebbistatin 1:1, and irradiating for 30-60min by blue light with the wavelength of 420 nm.
4. The method of claim 3, wherein the blue light source is placed 7.2cm above the Acinetobacter baumannii strain solution in logarithmic phase.
5. The method of claim 3, wherein the Acinetobacter baumannii strain liquid in logarithmic phase has a working density of 108cfu/mL or less.
6. The method of claim 3, wherein the working concentration of (S) -blebbistatin is 2 μ M to 10 μ M.
7. The method of claim 3, wherein the method for culturing Acinetobacter baumannii liquid in logarithmic phase mainly comprises the following steps:
(1) marking drug-resistant acinetobacter baumannii stored at-80 ℃ on an agar plate for multiple times, and performing inverted plate culture for 12h to obtain monoclonal bacteria;
(2) and (3) selecting the monoclonal bacteria in an LB culture medium and/or an MH culture medium, and culturing for 1.5h in a shaking table to obtain the drug-resistant acinetobacter baumannii bacterial liquid in the logarithmic phase.
8. The method as claimed in claim 3, wherein the 2-fold working concentration of (S) -blebbistatin is formulated as follows: the (S) -blebbistatin is prepared in advance in DMF solution to prepare mother solution, and then diluted in phosphate buffer PBS to 2 times of working concentration.
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| CN114522230A (en) * | 2022-01-11 | 2022-05-24 | 上海市伤骨科研究所 | Blue light and p-benzoquinone combined sterilization method |
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| CN110812481A (en) * | 2019-11-01 | 2020-02-21 | 深圳市第三人民医院 | Application and method for killing drug-resistant bacteria by blue light activated blebbistatin analogue |
| WO2021082512A1 (en) * | 2019-11-01 | 2021-05-06 | Shenzhen Third People's Hospital | Use of blue-light activated blebbistatin analogues in killing drug-resistant bacteria |
| CN110812481B (en) * | 2019-11-01 | 2021-11-23 | 深圳市第三人民医院 | Application and method for killing drug-resistant bacteria by blue light activated blebbistatin analogue |
| CN114522230A (en) * | 2022-01-11 | 2022-05-24 | 上海市伤骨科研究所 | Blue light and p-benzoquinone combined sterilization method |
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