CN112322695A - Experimental method for in-vitro antibacterial activity of AR-12 on MABC - Google Patents
Experimental method for in-vitro antibacterial activity of AR-12 on MABC Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Abstract
The invention discloses an experimental method of in-vitro antibacterial activity of AR-12 on MABC, belonging to the field of in-vitro antibacterial activity experiments and comprising the following steps: performing drug sensitivity test, accurately weighing a certain amount of CAMH powder, adding a certain amount of ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and autoclaving the solution at a certain temperature for a period of time; the invention sets a plurality of experimental processes such as drug sensitivity experiment, stability experiment and the like, can carry out comprehensive experiment aiming at the in-vitro antibacterial activity of AR-12 to the MABC, greatly improves the accuracy of the experimental result of the in-vitro antibacterial activity of the AR-12 to the MABC aiming at the influence of the AR-12 to the in-vitro antibacterial activity of the MABC under different environmental conditions, and also sets a control experimental step of controlling variables in the experimental method, controls all the analysis experimental results mutually, can quickly observe the change of the experimental result brought by the variables, thereby effectively exploring the factors of the experimental image.
Description
Technical Field
The invention belongs to the technical field of in-vitro antibacterial activity experiments, and particularly relates to an experimental method for in-vitro antibacterial activity of AR-12 on MABC.
Background
AR-12 is reported to have a broad range of biological activities against different pathogens in addition to its original anti-cancer function. AR-12 exhibits in vitro antiviral effects against a variety of viral infections, including mumps virus, Lassa virus, Zika virus, influenza virus, measles virus, HIV virus (wild type and protease resistant), dengue virus, Ebola virus, and hemorrhagic fever virus, among others. AR-12 is also effective against a variety of fungi including Candida albicans, Bacillus dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides. In addition, AR-12 also has inhibitory activity against parasites such as Leishmania, Dowanol, etc. Most importantly, AR-12 is now found to have antibacterial activity, such as Salmonella enteritidis, Francisella, Escherichia coli, and Neisseria gonorrhoeae, among others, significantly limiting bacterial burden both in vivo and in vitro. To date, the mechanistic basis for the pleiotropic biological effects of AR-12 has been reported to be related to the following factors and pathways, mainly including three aspects: first, the chaperone mechanism: activation of PKR-like endoplasmic reticulum kinase (PERK), upregulation of endoplasmic reticulum stress signals, regulation of various chaperones (GRP78/BIP/HSPA5/DnaK, etc.), a second kinase pathway: inhibition of phosphoinositide-dependent kinase (PDK1) activity, inhibition of the PI3k/Akt pathway, reduction of acetyl-coa synthetase activity, and third, autophagy: strongly induce apoptosis and autophagy, and clear unfolded proteins. However, the exact mechanism by which AR-12 has a broad spectrum of anti-pathogenic and anti-tumor activity remains unclear, and AR-12 has been approved by the U.S. Food and Drug Administration (FDA) as a research drug (IND). At present, AR-12 is designated as an orally available small molecule targeted anticancer drug, and 35 phase I human clinical trials for patients over 35 years of age with advanced or recurrent solid tumors or lymphomas have been successfully completed (clinical trial registration number NCT 00978523). In addition, AR-12 is currently approved by the european commission for combination therapy for clinical cryptococcosis (with fluconazole) and tularemia (with gentamicin).
Recently, AR-12 was found to be effective against certain gram-negative bacteria, which is another strong evidence supporting its broad spectrum antibacterial activity. Thus, the above studies and clinical experience indicate that AR-12 has a number of favorable pharmacological properties, which makes AR-12 a powerful candidate for a new anti-MABC therapeutic, and there is a need for an experimental approach to the in vitro antibacterial activity of AR-12 against clinical strains of MABC.
Disclosure of Invention
The invention aims to: in order to solve the problem of analysis of the in vitro antibacterial activity of AR-12 on MABC clinical strains, the experimental method for the in vitro antibacterial activity of AR-12 on MABC is provided.
In order to achieve the purpose, the invention adopts the following technical scheme: an experimental method for the in vitro antibacterial activity of AR-12 against MABC comprises the following steps;
s1, performing drug sensitivity test, accurately weighing a certain amount of CAMH powder, and adding a certain amount of ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and autoclaving the solution at a certain temperature for a period of time;
s2, accurately weighing a certain amount of AR-12 powder, adding a certain amount of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with an initial concentration, and placing a disposable turbidimetric tube, a turbidimetric apparatus, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for a period of time;
s3, diluting the concentration of the drug in the sample adding groove according to a multiple proportion dilution principle, keeping the diluted concentration in a certain range, taking out the bacterial liquid from the shaking table after liquid culture to logarithmic phase, placing the bacterial liquid in a biological safety cabinet, absorbing a small amount of the bacterial liquid, adding the bacterial liquid into a Mach turbiditube, adding a proper amount of physiological saline to dilute the bacterial liquid to a certain Mach concentration, respectively absorbing the bacterial liquid and 2 xCAMH culture liquid, adding the bacterial liquid and the 2 xCAMH culture liquid into the sample adding groove, and preparing bacterial suspension;
s4, respectively sucking the drug solution and the bacterial suspension with different concentrations and adding the drug solution and the bacterial suspension into a 96-well plate, wherein the drug solution is not added into the twelfth hole of each row and is used as a positive control hole, and the final experimental concentration of the drug after the sample addition is within a certain range;
s5, sealing the well-added 96-well plate, placing the well-sealed 96-well plate in an incubator at a certain temperature for incubation for a certain time, reading the MIC value after a period of time, and reading the MIC value again after a period of time;
s6, performing stability test, accurately weighing a certain amount of CAMH powder, adding a certain amount of ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and autoclaving the solution at a certain temperature for a period of time;
s7, accurately weighing a certain amount of AR-12 powder, adding a certain amount of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with an initial concentration, and placing a disposable turbidimetric tube, a turbidimetric apparatus, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for a period of time;
s8, diluting the concentration of the drug in the sample adding slot according to a multiple proportion dilution principle, keeping the diluted concentration in a certain range, respectively sucking drug solutions with different concentrations, adding the drug solutions into a 96-hole micro-measurement determination plate, using the twelfth hole of each row as a positive control hole without adding the drug solution, and respectively pre-incubating the 96-hole plate added with the drug solution for a certain number of days at a certain temperature;
s9, taking out the bacterial liquid cultured to logarithmic phase from the shaking table, placing the bacterial liquid in a biological safety cabinet, sucking a small amount of bacterial liquid, adding the bacterial liquid into a Mach-Zehnder tube, and adding a proper amount of normal saline to dilute the bacterial liquid to a certain Mach concentration;
s10, sucking the bacterial suspension, adding the bacterial suspension into a 96-well plate, sealing the well-added 96-well plate when the final experiment concentration of the drug after sample addition is within a certain range, placing the well-added 96-well plate in an incubator at a certain temperature for incubation for a period of time, and reading the plate with MIC value after a period of time.
As a further description of the above technical solution:
of the S1 and S6, 8.8g of CAMH powder was weighed and 200ml of ddH was added2O, autoclaving at 121 ℃ for 30 minutes.
As a further description of the above technical solution:
in S2 and S7, 5mg of AR-12 powder was weighed, 1ml of DMSO was added, and a drug solution having an initial concentration of 5000. mu.g/ml was irradiated with ultraviolet rays for 30 minutes.
As a further description of the above technical solution:
in S3, the diluted concentration range is 0.016-32 μ g/mL, and 20 μ l of bacterial liquid and 2000 μ l of 2 xCAMH culture liquid are respectively sucked.
As a further description of the above technical solution:
in the S4, 50 mul of drug solution and bacterial suspension with different concentrations are respectively sucked, and the final experimental concentration range is 0.008-16 mug/mL.
As a further description of the above technical solution:
in the S5, the cells were incubated in an incubator at 37 ℃ for 14 days, and MIC value reading was performed 3 days later and again 14 days later.
As a further description of the above technical solution:
in the S8, 50. mu.l of drug solutions with different concentrations were aspirated and pre-incubated at 30 ℃ for 0, 3, 7 and 14 days, respectively.
As a further description of the above technical solution:
and adding a proper amount of physiological saline to dilute the bacterial liquid to 0.5 McLeod concentration in S3 and S9:
in the S10, the final experiment concentration range is 0.008-16 mu g/mL, the well-added 96-well plate is sealed, the plate is placed in an incubator at 37 ℃ for incubation for 3 days, and MIC value reading is carried out after 3 days.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the method has the advantages that through the arrangement of a plurality of experimental processes such as drug sensitivity experiments, stability experiments and the like, comprehensive experiments can be carried out on the in-vitro antibacterial activity of the AR-12 on the MABC, the accuracy of the experimental results of the in-vitro antibacterial activity of the AR-12 on the MABC is greatly improved for the influence of the AR-12 on the in-vitro antibacterial activity of the MABC under different environmental conditions, the experimental method is also provided with a control experimental step for controlling variables, all analysis experimental results are mutually controlled, the change of the experimental results caused by the variables can be rapidly observed, and therefore the factors for experimental images are effectively explored.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The invention provides a technical scheme that: an experimental method for the in vitro antibacterial activity of AR-12 against MABC, comprising the following steps:
s1, carrying out drug sensitivity test, accurately weighing a certain amount of CAMH powder 8.8g, adding 200ml ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and performing autoclaving treatment on the solution at 121 ℃ for 30 minutes;
s2, accurately weighing 5mg of AR-12 powder, adding 1ml of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with the initial concentration of 5000 mug/ml, and placing a disposable Machilus turbidimeter, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for 30 minutes;
s3, diluting the concentration of the drug in the sample adding groove according to the multiple proportion dilution principle, wherein the diluted concentration is 0.016 mu g/mL, taking out the bacterial liquid from the shaking table after the liquid culture reaches the logarithmic phase, placing the bacterial liquid in a biological safety cabinet, sucking a small amount of bacterial liquid, adding the bacterial liquid into a Mach 'S turbidimetric tube, adding a proper amount of physiological saline to dilute the bacterial liquid to 0.5 Mach' S concentration, sucking 20 mu l of bacterial liquid and 2000 mu l of 2 xCAMH culture liquid respectively, adding the bacterial liquid and the sample adding groove to prepare bacterial suspension;
s4, respectively sucking 50 mu l of drug solutions and bacterial suspensions with different concentrations, adding the drug solutions into a 96-well plate, wherein the drug solution is not added into the twelfth hole of each row and is used as a positive control hole, and the final experimental concentration of the drug after the sample addition is 0.008 mu g/mL;
s5, sealing the well-added 96-well plate, placing the well-sealed 96-well plate in an incubator at 37 ℃ for incubation for 14 days, reading the MIC value after 3 days, and reading the MIC value again after 14 days;
s6, performing stability experiment, performing drug sensitivity experiment, accurately weighing a certain amount of CAMH powder 8.8g, adding 200ml ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and performing autoclaving treatment on the solution at 121 ℃ for 30 minutes;
s7, accurately weighing 5mg of AR-12 powder, adding 1ml of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with the initial concentration of 5000 mug/ml, and placing a disposable Machilus turbidimeter, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for 30 minutes;
s8, diluting the concentration of the drug in the sample adding slot according to a multiple proportion dilution principle, keeping the diluted concentration in a certain range, respectively sucking 50 mu l of drug solutions with different concentrations, adding the drug solutions into a 96-hole micro-measurement plate, using the twelfth hole of each row as a positive control hole without adding the drug solutions, and pre-incubating the 96-hole plate added with the drug solutions at 30 ℃ for 0, 3, 7 and 14 days respectively;
s9, taking out the bacterial liquid cultured to logarithmic phase from the shaking table, placing the bacterial liquid in a biological safety cabinet, sucking a small amount of bacterial liquid, adding the bacterial liquid into a McLeeb tube, and adding a proper amount of normal saline to dilute the bacterial liquid to 0.5 McLeeb concentration;
s10, sucking 50 mu l of bacterial suspension, adding the bacterial suspension into a 96-well plate, sealing the well-added 96-well plate, placing the well-added 96-well plate in an incubator at 37 ℃ for incubation for 3 days, and reading the MIC value after 3 days, wherein the final experiment concentration of the drug after sample addition is 0.008 mu g/mL.
Example 2
The invention provides a technical scheme that: an experimental method for the in vitro antibacterial activity of AR-12 against MABC, comprising the following steps:
s1, carrying out drug sensitivity test, accurately weighing a certain amount of CAMH powder 8.8g, adding 200ml ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and performing autoclaving treatment on the solution at 121 ℃ for 30 minutes;
s2, accurately weighing 5mg of AR-12 powder, adding 1ml of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with the initial concentration of 5000 mug/ml, and placing a disposable Machilus turbidimeter, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for 30 minutes;
s3, diluting the concentration of the drug in the sample adding groove according to a multiple proportion dilution principle, wherein the diluted concentration is 0.025 mu g/mL, taking out the bacterial liquid from the shaking table after the liquid is cultured to the logarithmic phase, placing the bacterial liquid in a biological safety cabinet, sucking a small amount of bacterial liquid, adding the bacterial liquid into a Mach 'S turbidimetric tube, adding a proper amount of physiological saline to dilute the bacterial liquid to 0.5 Mach' S concentration, sucking 20 mu l of bacterial liquid and 2000 mu l of 2 xCAMH culture liquid respectively, and adding the bacterial liquid and the sample adding groove to prepare bacterial suspension;
s4, respectively sucking 50 mu l of drug solutions and bacterial suspensions with different concentrations, adding the drug solutions into a 96-well plate, wherein the drug solution is not added into the twelfth well of each row and is used as a positive control well, and the final experimental concentration of the drug after the sample addition is 0.012 mu g/mL;
s5, sealing the well-added 96-well plate, placing the well-sealed 96-well plate in an incubator at 37 ℃ for incubation for 14 days, reading the MIC value after 3 days, and reading the MIC value again after 14 days;
s6, performing stability experiment, performing drug sensitivity experiment, accurately weighing a certain amount of CAMH powder 8.8g, adding 200ml ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and performing autoclaving treatment on the solution at 121 ℃ for 30 minutes;
s7, accurately weighing 5mg of AR-12 powder, adding 1ml of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with the initial concentration of 5000 mug/ml, and placing a disposable Machilus turbidimeter, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for 30 minutes;
s8, diluting the concentration of the drug in the sample adding slot according to a multiple proportion dilution principle, keeping the diluted concentration in a certain range, respectively sucking 50 mu l of drug solutions with different concentrations, adding the drug solutions into a 96-hole micro-measurement plate, using the twelfth hole of each row as a positive control hole without adding the drug solutions, and pre-incubating the 96-hole plate added with the drug solutions at 30 ℃ for 0, 3, 7 and 14 days respectively;
s9, taking out the bacterial liquid cultured to logarithmic phase from the shaking table, placing the bacterial liquid in a biological safety cabinet, sucking a small amount of bacterial liquid, adding the bacterial liquid into a McLeeb tube, and adding a proper amount of normal saline to dilute the bacterial liquid to 0.5 McLeeb concentration;
s10, sucking 50 mu l of bacterial suspension, adding the bacterial suspension into a 96-well plate, sealing the well-added 96-well plate, placing the well-added 96-well plate in an incubator at 37 ℃ for incubation for 3 days, and reading the MIC value after 3 days, wherein the final experiment concentration of the drug after sample addition is 0.012 mu g/mL.
Example 3
The invention provides a technical scheme that: an experimental method for the in vitro antibacterial activity of AR-12 against MABC, comprising the following steps:
s1, carrying out drug sensitivity test, accurately weighing a certain amount of CAMH powder 8.8g, adding 200ml ddH2Shaking to dissolve completely to obtain 2 × CAMH culture solution, and autoclaving the solution at 121 deg.C for 30 min；
S2, accurately weighing 5mg of AR-12 powder, adding 1ml of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with the initial concentration of 5000 mug/ml, and placing a disposable Machilus turbidimeter, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for 30 minutes;
s3, diluting the concentration of the drug in the sample adding groove according to the multiple ratio dilution principle, wherein the diluted concentration is 0.032 mu g/mL, taking out the bacterial liquid from the shaking table after the liquid is cultured to logarithmic phase, placing the bacterial liquid in a biological safety cabinet, absorbing a small amount of the bacterial liquid, adding the bacterial liquid into a Mach 'S turbidimetric tube, adding a proper amount of physiological saline to dilute the bacterial liquid to 0.5 Mach' S concentration, absorbing 20 mu l of the bacterial liquid and 2000 mu l of 2 xCAMH culture liquid respectively, adding the bacterial liquid into the sample adding groove, and preparing into bacterial suspension;
s4, respectively sucking 50 mu l of drug solutions and bacterial suspensions with different concentrations, adding the drug solutions into a 96-well plate, wherein the drug solution is not added into the twelfth well of each row to serve as a positive control well, and the final experimental concentration of the drug after the sample addition is 0.016 mu g/mL;
s5, sealing the well-added 96-well plate, placing the well-sealed 96-well plate in an incubator at 37 ℃ for incubation for 14 days, reading the MIC value after 3 days, and reading the MIC value again after 14 days;
s6, performing stability experiment, performing drug sensitivity experiment, accurately weighing a certain amount of CAMH powder 8.8g, adding 200ml ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and performing autoclaving treatment on the solution at 121 ℃ for 30 minutes;
s7, accurately weighing 5mg of AR-12 powder, adding 1ml of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with the initial concentration of 5000 mug/ml, and placing a disposable Machilus turbidimeter, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for 30 minutes;
s8, diluting the concentration of the drug in the sample adding slot according to a multiple proportion dilution principle, keeping the diluted concentration in a certain range, respectively sucking 50 mu l of drug solutions with different concentrations, adding the drug solutions into a 96-hole micro-measurement plate, using the twelfth hole of each row as a positive control hole without adding the drug solutions, and pre-incubating the 96-hole plate added with the drug solutions at 30 ℃ for 0, 3, 7 and 14 days respectively;
s9, taking out the bacterial liquid cultured to logarithmic phase from the shaking table, placing the bacterial liquid in a biological safety cabinet, sucking a small amount of bacterial liquid, adding the bacterial liquid into a McLeeb tube, and adding a proper amount of normal saline to dilute the bacterial liquid to 0.5 McLeeb concentration;
s10, sucking 50 mu l of bacterial suspension, adding the bacterial suspension into a 96-well plate, sealing the well-added 96-well plate, placing the well-added 96-well plate in an incubator at 37 ℃ for incubation for 3 days, and reading the MIC value after 3 days, wherein the final experiment concentration of the drug after sample addition is 0.016 mu g/mL.
Examples 1-3 also relate to the Minimum Bactericidal Concentration (MBC) test for AR-12:
a total of 12 MABC strains including 5 Mycobacterium abscessus (type A) and 5 Mycobacterium mosaicus (type M) and the standard strain ATCC19977 were randomly selected and subjected to experiments to determine MBC values.
(1) Accurately weighing CAMH powder 8.8g, adding 200ml ddH2In O, shaking to fully dissolve the mixture to prepare a 2 xCAMH culture solution;
(2) autoclaving the solution at 121 deg.C for 30 min;
(3) accurately weighing 5mg of AR-12 powder, adding the powder into 1ml of DMSO, and oscillating to fully dissolve the powder to obtain a drug solution with the initial concentration of 5000 mug/ml;
(3) placing the disposable turbidimeter tube, the turbidimeter, the 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for 30 minutes;
(4) diluting the concentration of the medicine in the sample adding groove according to a multiple proportion dilution principle, wherein the diluted concentration range is 0.016-32 mu g/mL;
(5) taking out the bacterial liquid cultured to logarithmic phase from the shaking table, and placing in a biological safety cabinet. A small amount of bacteria liquid is sucked and added into a McLeod turbidimetric tube, and a proper amount of normal saline is added to dilute the bacteria liquid to 0.5 McLeod concentration;
(6) respectively sucking 20 mul of bacterial liquid and 2000 mul of 2 xCAMH culture liquid, adding the liquid and the culture liquid into a sample adding groove, and preparing bacterial suspension;
(7) pipetting 100. mu.L of the bacterial suspension, performing a series of ten-fold dilutions, uniformly spreading the dilutions in duplicate on a 7H11 solid culture plate, incubating the dilutions in an incubator at 37 ℃ for 5 days, and counting the initial Colony Forming Units (CFU);
(8) respectively sucking 50 mul of drug solution and bacterial suspension with different concentrations, adding the drug solution and the bacterial suspension into a 96-well plate, wherein the drug solution is not added into the twelfth hole of each row and is used as a positive control hole, and the final experimental concentration range of the drug after the sample addition is 0.008-16 mug/mL;
(9) sealing the well-added 96-well plate, and placing the plate in an incubator at 37 ℃ for incubation for 3 days;
(10) uniformly resuspending the contents of the wells with the drug concentration higher than the MIC;
(11) sucking 100 μ L of the contents in the wells, performing a series of ten-fold dilutions, uniformly spreading the dilutions in duplicate on a 7H11 solid culture plate, incubating the dilutions in an incubator at 37 ℃ for 5 days, and counting the remaining colony-forming units;
(12) the percentage of remaining CFU relative to the initial CFU was calculated, and MBC refers to the minimum drug concentration required to kill 99.9% of the initial CFU.
Interpretation of the results: and calculating the ratio of MBC/MIC, and when the ratio of MBC/MIC is less than or equal to 4, considering that the antibiotic has a bactericidal effect, otherwise, the antibiotic has a bacteriostatic effect.
Standard strain ATCC19977 and clinical strain G72 were selected for experiments to evaluate the time-dependent antibacterial activity of AR-12.
(1) Placing a test tube, a cuvette and the like in a biological safety cabinet for ultraviolet irradiation for 30 minutes;
(2) taking out the bacterial liquid cultured in the liquid to logarithmic phase from the shaking table, placing in a biological safety cabinet, and sucking
Adding a small amount of bacterial liquid into a cuvette, and adding a proper amount of physiological saline to dilute the bacterial liquid until the OD value is 0.245 (about 106CFU/mL concentration);
(3) weighing AR-12 and amikacin powder, and preparing into medicinal solution with concentration increased by 2 times (from 0.5 to 4 xMIC);
(4) 5ml of 7H9 liquid medium was added to the tube;
(5) respectively sucking 100 mu L of bacterial suspension (about 105CFU) and 100 mu L of AR-12 or amikacin solution with different concentrations, adding into test tubes, and arranging test tubes added with 100 mu L of bacterial suspension and 100 mu L of normal saline in each group as positive control;
(6) placing the well-loaded test tube in an orbital shaker (100rpm), and incubating at 37 ℃;
(7) sucking 100 μ L of bacterial suspension, performing 10-fold serial dilution, uniformly spreading on a 7H11 solid culture plate in triplicate, placing in an incubator at 37 ℃ for incubation for 5 days, and calculating Colony Forming Units (CFU) of 0H;
(8) resuspending the contents of the tubes at 24H, 48H, 72H and 120H incubation, respectively, serial 10-fold dilutions were performed, plated evenly in triplicate on a 7H11 solid culture plate, incubated in a 37 ℃ incubator for 5 days, and viable Colony Forming Units (CFU) were counted;
(9) the experiment was repeated 3 times and the TK curve was plotted.
And (4) interpretation of results: sterilization was defined as a reduction in the number of surviving colonies > 3log10cfu/mL during the interaction compared to the 0h time point.
To investigate whether low doses of AR-12 induction would decrease MABC sensitivity, pre-exposure experiments were performed to determine changes in MIC values. 21 MABC isolates, including ATCC19977, were randomly selected for testing. After the bacterial suspension was diluted to an OD600 of 0.05, AR-12 was added at a sub-inhibitory concentration of 1 μ g/ml (1/4 corresponding to MIC 50), and the mixture was incubated overnight, and the bacterial suspension not exposed to AR-12 was used as a negative control. The remaining procedure was the same as for the drug sensitivity test.
In vitro interactions between AR-12 and 5 anti-MABC drugs, clarithromycin, amikacin, cefoxitin, imipenem, and tigecycline, were studied using a microbubs checkerboard method in combination with 7 MABC.
In conclusion, the result analysis is carried out:
AR-12 has strong antibacterial activity to MABC: to investigate whether AR-12 showed similar sensitivity to different subspecies of MABC, 194 clinical isolates of MABC were grouped according to subspecies. For M.abscessus (type A), the MIC ranged from 2-16ug/ml, compared to 2-8ug/ml for M.mosaicensis (type M). The MICs 50 and 90 for AR-12 were identical for both subspecies, 4 and 8 mg/L. Furthermore, the ratio of each MIC value also had a similar distribution between the two subspecies (Table 5.1), and the results above show that there were no significant differences in the response of different subspecies of MABC to AR-12. Furthermore, the MIC at day 3 was consistent with the MIC at day 14, indicating that AR-12 did not exhibit induced drug resistance. For AR-12, there is currently no standard that can account for MIC breakpoints, and therefore sensitivity and resistance are not calculated.
TABLE 5 MIC profiles of MABC strains 1-194 against AR-12
AR-12 may be unstable when stored at 30 ℃: to investigate whether AR-12 can maintain antibacterial activity when stored at 30 ℃ for up to 14 days, a stability experiment was performed. By comparing the MIC values measured at different preincubation times, no difference in MIC values was found between the two subspecies within 3 days. However, when preincubated at 30 ℃ for 7 days, the MIC value increased 2-fold; at 14 days, the MIC values exceeded the upper limit (> 16. mu.g/mL) (Table 5.2). This indicates that AR-12 retains its sensitivity at 30 ℃ for 3 days and gradually loses its antibacterial activity with the lapse of time. In general, storage of AR-12 at 30 ℃ for 7 days affected its drug sensitivity properties.
TABLE 5.2 MIC value Change at different time of Pre-incubation
AR-12 is bactericidal mode for MABC: the MBC/MIC ratio is calculated by measuring the MBC value of AR-12 to MABC in vitro, so as to identify whether the antibacterial mode is bacteriostatic or bactericidal. As shown in Table 5.3, the MBC/MIC ratio of AR-12 for all tested strains ranged from 2 to > 4. For mycobacterium abscessus (type a), the ratio of 1 strain is 2; the ratio of 3 plants is 4; the ratio of another 2 strains is > 4. For M.mosaicensis (type M), the ratio of 3 strains was 4, and the ratio of the remaining 2 strains was 2 and >4, respectively. As previously defined, the MBC/MIC ratio is 4 or less in most strains (8/11) and AR-12 is in bactericidal mode. Thus, the bactericidal pattern of AR-12 dominates in both subspecies.
TABLE 5 MBC values and antibacterial patterns of 3-AR-12 vs. MABC
Pre-exposure to sub-inhibitory concentrations of AR-12 did not induce a decrease in MABC sensitivity: to investigate whether MABCs present any potential or unknown induced phenomena of AR-12 resistance, 11 mycobacterium abscessus (type a) and 10 mycobacterium mosaic (type M) were pre-exposed to 1/4xMIC50AR-12 and MIC values were again determined. As shown in Table 5.4, the MIC values before and after the pre-exposure were identical in 19 strains. Compared to MIC values without prior exposure, 1 mycobacterium abscessus (type a) and mycobacterium mosaic (type M) increased 1-fold on days 3 and 14, respectively. Overall, both the MIC50 and MIC90 were unchanged for both subspecies after pre-exposure to AR-12, indicating that pre-exposure to AR-12 did not induce a decrease in MABC sensitivity.
TABLE 5.4 MIC value Change before and after Pre-Exposure
The combined effect of AR-12 and other drugs appears mostly to be an unrelated effect: little is currently known about the interaction between AR-12 and drugs that have been approved for treatment of MABC infection. The results of the checkerboard combination experiment are shown in Table 5.5. None of the drug combinations showed antagonism in the study, and no independent effect was observed at 97.14% (34/35) in all tested combinations. In 1 mycobacterium abscessus (type a), the interaction of AR-12 with imipenem appears to be synergistic (FICI ═ 0.5). The FICI ranged from 0.5 to 1.5, indicating no antagonism between AR-12 and all tested drugs.
TABLE 5 in vitro combination Effect assay results of 5-AR-12 with other antibacterial drugs
In summary, the following steps: the purpose of this experiment was to evaluate the potential activity of AR-12 as a new antibacterial agent against MABC in vitro. In general, most antibacterial drugs are less sensitive to MABCs and have high variability. But is more effective for AR-12 than most antibacterial drugs. Preliminary experiments were performed to confirm the maximum test concentration of AR-12, and it was found that the final concentration of DMSO of 1% had no significant effect on the growth of MABC, while higher concentrations (. gtoreq.32. mu.g/mL) of AR-12 were insoluble in DMSO of 1%. Therefore, the upper concentration limit for MIC determination in this experiment was determined to be 16. mu.g/mL. In this study, the AR-12MIC of 194 MABCs ranged from 2-16ug/m (lMIC50 ═ 4ug/ml, MIC90 ═ 8ug/ml), which corresponded to the in vitro antifungal MIC values for AR-12 [31 ]. In addition, data from phase I human clinical trials of AR 12 as a targeted anticancer agent showed that serum concentrations of the drug in patients could be well and safely achieved at levels of 8 μ M (i.e., 3.7 μ g/mL) and were well tolerated by patients, which corresponds to the in vitro MIC50 value of AR 12 for MABC. Although there is no current criterion to explain the criticality of MIC, it is reasonable to conclude that AR-12 exhibits good antimicrobial activity against MABC as compared to previously published studies relating to AR-12 antifungal activity. AR-12, as a novel drug for the treatment of MABCs, needs to be integrated into the currently available combination therapy of multiple drugs. The interaction results of AR-12 in combination with existing anti-MABC drugs including clarithromycin, amikacin, imipenem, cefoxitin and tigecycline show that 34 of the 35 drug combinations exhibit unrelated effects, and 1 has synergistic effect and no antagonistic effect. This result suggests that AR-12 can be integrated into current combination treatment regimens without causing antagonism. This experiment was the first time that the time bactericidal kinetics assay for AR-12 was performed in MABC. AR-12 was found to exhibit concentration-dependent bactericidal activity against both ATCC19977 and clinical strain G72, consistent with the results for MBC/MIC.
Numerous desirable properties make AR-12 the most potent competitor of anti-MABC drug candidates. Firstly, AR-12 is an oral preparation, which has the advantages of safety and convenience; second, amikacin has been incorporated as a standard therapeutic in the MABC therapeutic guidelines at present, while AR-12 has a high antibacterial efficacy comparable or superior to that of amikacin; third, AR-12 acts in a bactericidal mode, which is particularly important for the treatment of mycobacterium abscessus infections; fourth, the combined effect of AR-12 with other antibacterial agents has no clear antagonism, indicating that AR-12 can be used in existing combination regimens and do not substantially affect each other's antibacterial activity; finally, the best evidence is the fact that AR-12 has good pharmacological properties and safety, and has been approved for clinical trials in humans and successfully completed. This study also has some limitations. First, AR-12 is highly hydrophobic, with solubility in water <0.1mg/mL (insoluble in water), and higher concentrations of soluble AR-12 were found to be somewhat cytotoxic to mammalian cells [14, 21 ]. Thus, this finding, combined with avoiding the toxicity of the solvent (DMSO), makes it impossible to explore AR-12 in a high concentration range. Second, AR-12 was tested for sensitivity to 30 MABC strains in 7H9, while all other parameters remained unchanged, but the MIC values for all strains exceeded the maximum tested concentration (> 16. mu.g/mL). Therefore, for better comparison, the concentrations of subsequent experiments were purposely designed as multiples of the MIC determined by broth microdilution. Given that the MIC values of AR-12 vary widely among different test media, the implication of this study should be based on the same MIC determination method (i.e., broth microdilution using CAMH). Third, while the time sterilization (TK) kinetic assay provides a more dynamic assessment than the MIC assay, it still does not reflect the actual dynamic concentration of the drug during in vivo interactions. In addition, in time sterilization experiments, the drug was added only at the beginning of the experiment without continuous replenishment, which failed to offset the effects of drug degradation. Therefore, it is not clear whether the bacterial regeneration observed after 3 days is due to instability of the drug, and the efficacy of the drug is likely to be underestimated.
Although this study experimentally demonstrated the effectiveness of AR-12 in vitro on MABC, its clinical use has been a long and arduous task. The next direction of study will be to fully evaluate AR-12 in animal models and clinical trials, including efficacy, safety, tolerability, toxicity, bioavailability, metabolic stability, pharmacokinetics, and the like. Notably, AR-12 has been successful in achieving effective treatment of tularemia in macrophages without causing cytotoxicity [20, 21 ]. At the same time, further optimization of the drug is required, such as some chemical modifications, changes in drug formulation and delivery methods to produce higher drug concentrations in vivo. It has been reported that AR-12 is encapsulated in acetalized dextran (Ace-Dex) microparticles to reduce cytotoxicity while maintaining drug efficacy [22-24 ]. Furthermore, the effective molecular targets for AR-12 to mediate antibacterial activity remain unknown. In summary, this study shows that AR-12 has good in vitro anti-MABC activity, either alone or in combination with other drugs.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. An experimental method for the in vitro antibacterial activity of AR-12 against MABC, characterized in that: the method comprises the following steps:
s1, performing drug sensitivity test, accurately weighing a certain amount of CAMH powder, and adding a certain amount of ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and autoclaving the solution at a certain temperature for a period of time;
s2, accurately weighing a certain amount of AR-12 powder, adding a certain amount of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with an initial concentration, and placing a disposable turbidimetric tube, a turbidimetric apparatus, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for a period of time;
s3, diluting the concentration of the drug in the sample adding groove according to a multiple proportion dilution principle, keeping the diluted concentration in a certain range, taking out the bacterial liquid from the shaking table after liquid culture to logarithmic phase, placing the bacterial liquid in a biological safety cabinet, absorbing a small amount of the bacterial liquid, adding the bacterial liquid into a Mach turbiditube, adding a proper amount of physiological saline to dilute the bacterial liquid to a certain Mach concentration, respectively absorbing the bacterial liquid and 2 xCAMH culture liquid, adding the bacterial liquid and the 2 xCAMH culture liquid into the sample adding groove, and preparing bacterial suspension;
s4, respectively sucking the drug solution and the bacterial suspension with different concentrations and adding the drug solution and the bacterial suspension into a 96-well plate, wherein the drug solution is not added into the twelfth hole of each row and is used as a positive control hole, and the final experimental concentration of the drug after the sample addition is within a certain range;
s5, sealing the well-added 96-well plate, placing the well-sealed 96-well plate in an incubator at a certain temperature for incubation for a certain time, reading the MIC value after a period of time, and reading the MIC value again after a period of time;
s6, performing stability test, accurately weighing a certain amount of CAMH powder, adding a certain amount of ddH2O, shaking to fully dissolve the solution to prepare 2 xCAMH culture solution, and autoclaving the solution at a certain temperature for a period of time;
s7, accurately weighing a certain amount of AR-12 powder, adding a certain amount of DMSO, oscillating to fully dissolve the powder to obtain a drug solution with an initial concentration, and placing a disposable turbidimetric tube, a turbidimetric apparatus, a 96-hole microporous plate frame and the like in a biological safety cabinet for ultraviolet irradiation for a period of time;
s8, diluting the concentration of the drug in the sample adding slot according to a multiple proportion dilution principle, keeping the diluted concentration in a certain range, respectively sucking drug solutions with different concentrations, adding the drug solutions into a 96-hole micro-measurement determination plate, using the twelfth hole of each row as a positive control hole without adding the drug solution, and respectively pre-incubating the 96-hole plate added with the drug solution for a certain number of days at a certain temperature;
s9, taking out the bacterial liquid cultured to logarithmic phase from the shaking table, placing the bacterial liquid in a biological safety cabinet, sucking a small amount of bacterial liquid, adding the bacterial liquid into a Mach-Zehnder tube, and adding a proper amount of normal saline to dilute the bacterial liquid to a certain Mach concentration;
s10, sucking 50 mul of bacterial suspension, adding the bacterial suspension into a 96-well plate, sealing the well-added 96-well plate when the final experiment concentration of the drug after sample addition is within a certain range, placing the well-added 96-well plate in an incubator at a certain temperature for incubation for a period of time, and reading the plate with MIC value after a period of time.
2. An AR-12 to M according to claim 1The method for testing the in vitro antibacterial activity of ABC is characterized in that 8.8g of CAMH powder is weighed in S1 and S6, and 200ml of ddH is added2O, autoclaving at 121 ℃ for 30 minutes.
3. The method of claim 1, wherein in S2 and S7, 5mg of AR-12 powder is weighed, 1ml of DMSO is added, and a drug solution with an initial concentration of 5000 μ g/ml is irradiated with UV light for 30 minutes.
4. The method of claim 1, wherein the diluted concentration of S3 ranges from 0.016 to 32 μ g/mL, and 20 μ l of the culture solution and 2000 μ l of 2 xCAMH culture solution are aspirated respectively.
5. The method of claim 1, wherein 50 μ l of each of the drug solution and the bacterial suspension is aspirated in S4, and the final concentration range is 0.008 μ g/mL to 16 μ g/mL.
6. The method of claim 1, wherein the step of incubating the AR-12 cells in the incubator at 37 ℃ for 14 days is followed by MIC reading after 3 days and then by MIC reading again after 14 days in S5.
7. The method of claim 1, wherein 50 μ l of different concentrations of the drug solution is aspirated in S8, and pre-incubated at 30 ℃ for 0, 3, 7 and 14 days respectively.
8. The method of claim 1, wherein the S3 and S9 are diluted with physiological saline to 0.5 McLee' S concentration.
9. The method of claim 1, wherein in S10, 50 μ l of bacterial suspension is aspirated into 96-well plate, the final concentration range is 0.008-16 μ g/mL, the well sealed 96-well plate is sealed, incubated in 37 ℃ incubator for 3 days, and then MIC reading is performed after 3 days.
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