Method for accelerating detection speed of drug sensitivity of saccharomycetes and application of method

Abstract

The invention belongs to the technical field of drug sensitivity detection, and particularly relates to a method for increasing the drug sensitivity detection speed of saccharomycetes and application of the method. The method comprises the steps of bacterial liquid preparation, sample adding, incubation, thallus enrichment, color developing agent adding and result interpretation, ethylenediamine tetraacetic acid or salt thereof needs to be added before thallus enrichment, and drug action and color developing are carried out separately. The detection method provided by the invention can effectively accelerate the developing speed of resazurin, remarkably shortens the drug sensitivity detection time, and is beneficial to early accurate diagnosis and treatment of clinical fungal diseases.

Description

Technical Field

[0001] This invention belongs to the field of drug susceptibility testing technology, specifically relating to a method for accelerating the drug susceptibility testing of yeast and its application. Background Technology

[0002] With the widespread use of broad-spectrum antibiotics, immunosuppressants, and over-the-counter antifungal drugs, as well as the increasing prevalence of organ transplantation and the growing number of immunocompromised individuals such as those with HIV / AIDS, fungal infections, especially yeast infections, are on the rise. Different yeasts exhibit varying susceptibility to drugs; therefore, drug susceptibility testing can determine the sensitivity of infecting yeasts to medications, guiding clinicians in selecting appropriate antifungal drugs for treatment. This helps improve treatment outcomes, reduce the development of drug resistance, and minimize adverse reactions in patients due to inappropriate drug selection.

[0003] Currently, some commonly used methods for yeast antimicrobial susceptibility testing include the disk diffusion method, the E test, and the broth dilution method. The disk diffusion method involves placing a disk containing a certain amount of drug on a culture medium coated with yeast suspension, and observing the diameter of the inhibition zone to determine drug sensitivity. This method is simple to operate, but it does not provide a direct MIC value, and the test results are affected by various factors. The E test combines the principles of the dilution and diffusion methods, using a test strip with an antimicrobial drug concentration distributed exponentially from high to low, directly quantifying the minimum inhibitory concentration (MIC) of the test drug against the test bacteria. However, this test strip is expensive, and determining the MIC endpoint for some drugs can be difficult. The broth dilution method can be used to quantitatively test the in vitro activity of antimicrobial drugs against specific bacteria and fungi. The dilution method is accurate and has a wide range of applications, but it is cumbersome, time-consuming, and labor-intensive, making it unsuitable for clinical use.

[0004] In addition, commercially available automated systems such as VITEK 2, Microscan Panel, BD Phoenix, and Sensititre have reduced the AST (antimicrobial susceptibility testing) time from several days to about 18 hours. However, a common drawback of these conventional methods is that they all require 24-48 hours to isolate pathogenic microorganisms from patient samples through pure culture, followed by 18-72 hours for antimicrobial incubation and drug susceptibility testing, resulting in a total time of 42 hours to 5 days. Furthermore, they are ineffective for pathogenic microorganisms that are difficult to culture or grow slowly, making it difficult to meet the urgent clinical need for rapid drug administration.

[0005] In summary, while there are various methods for yeast antimicrobial susceptibility testing, they still suffer from limitations such as cumbersome operation, long processing time, and high cost. Therefore, there is an urgent clinical need for rapid antimicrobial susceptibility testing systems to improve the treatment efficacy of infectious diseases. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for accelerating the detection speed of yeast antimicrobial susceptibility testing. The method includes the use of a complexing agent, which can accelerate the color development speed of yeast and thus shorten the detection time.

[0007] On the one hand, this invention provides the application of complexing agents in accelerating the detection speed of yeast antimicrobial susceptibility.

[0008] Specifically, the complexing agent is added after drug incubation and before yeast enrichment.

[0009] Specifically, the complexing agent may be ethylenediaminetetraacetic acid or a salt thereof.

[0010] More specifically, the salt can be a sodium salt, ammonium salt, iron salt, magnesium salt, calcium salt, copper salt, manganese salt, zinc salt, cobalt salt, or aluminum salt of ethylenediaminetetraacetic acid.

[0011] Preferably, the salt can be the sodium salt of ethylenediaminetetraacetic acid.

[0012] Specifically, the final concentration of the complexing agent can be 0.01-2 mM.

[0013] Specifically, the yeasts include, but are not limited to, one or more of the following genera: Candida, Cryptococcus, non-green algae, Geotrichum, Malassezia, Rhodotorula, Yeast, Megasporum, Trichosporium, and Ustilago maydis.

[0014] Specifically, the drugs include, but are not limited to, one or more of the following: azole drugs, pyrimidine drugs, polyenes, echinocandins, and allylamines.

[0015] Specifically, the azole drugs include, but are not limited to, one or more of the following: ketoconazole, miconazole, econazole, clotrimazole, bifonazole, itraconazole, fluconazole, voriconazole, posaconazole, or lavconazole;

[0016] The pyrimidine drugs include, but are not limited to: flucytosine;

[0017] The echinocandins include, but are not limited to, one or more of the following: caspofungin, micafungin, rezafenamicin, eurifenamicin, or anidifungin.

[0018] In another aspect, the present invention provides a method for accelerating the detection speed of yeast antimicrobial susceptibility, comprising the following steps:

[0019] S1. Preparation of bacterial solution: Prepare a bacterial solution with an MCF of 0.5-2.0 using sterile water;

[0020] S2, Adding samples: First add the drug to the deep well plate or drug sensitivity card, then add the diluted bacterial solution from step S1;

[0021] S3, incubation;

[0022] S4. Add a complexing agent to the sample after incubation in step S3, and then enrich the bacteria;

[0023] S5. Add color developer;

[0024] S6. Interpretation result.

[0025] Specifically, the MCF of the bacterial solution in step S1 can be 0.5-1.5.

[0026] Specifically, the diluent used in step S2 is a culture medium; the dilution factor can be 5-100, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or any multiple within that range.

[0027] Specifically, the culture medium may be DMEM medium, RPMI 1640 medium, Mueller-Hinton broth, SDB medium, PDB medium, YM medium, MEM medium, LB medium, SOB medium, SOC medium, TB medium, 2×YT medium and / or YPD medium.

[0028] Preferably, the culture medium may be RPMI 1640.

[0029] Specifically, the incubation conditions in step S3 can be: 25-35℃, 0.5-3h.

[0030] Specifically, the complexing agent in step S4 can be ethylenediaminetetraacetic acid or a salt thereof.

[0031] More specifically, the salt can be a sodium salt, ammonium salt, iron salt, magnesium salt, calcium salt, copper salt, manganese salt, zinc salt, cobalt salt, or aluminum salt of ethylenediaminetetraacetic acid.

[0032] Preferably, the salt can be the sodium salt of ethylenediaminetetraacetic acid.

[0033] Specifically, the final concentration of the complexing agent in step S4 can be 0.01-2 mM, such as 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 1 mM, 2 mM, or any value within the concentration range.

[0034] Specifically, the enrichment methods include filtration, centrifugation, or magnetic bead adsorption.

[0035] Preferably, the enrichment method can be filtration.

[0036] Specifically, the yeasts include, but are not limited to, one or more of the following genera: Candida, Cryptococcus, non-green algae, Geotrichum, Malassezia, Rhodotorula, Yeast, Megasporum, Trichosporium, and Ustilago maydis.

[0037] Specifically, the drugs include, but are not limited to, one or more of the following: azole drugs, pyrimidine drugs, polyenes, echinocandins, and allylamines.

[0038] Specifically, the azole drugs include, but are not limited to, one or more of the following: ketoconazole, miconazole, econazole, clotrimazole, bifonazole, itraconazole, fluconazole, voriconazole, posaconazole, or lavconazole;

[0039] The pyrimidine drugs include, but are not limited to: flucytosine;

[0040] The echinocandins include, but are not limited to, one or more of the following: caspofungin, micafungin, rezafenamicin, eurifenamicin, or anidifungin.

[0041] In another aspect, the present invention provides the application of the aforementioned method in the preparation of a kit for accelerating the detection speed of yeast antimicrobial susceptibility.

[0042] Specifically, the yeasts include, but are not limited to, one or more of the following genera: Candida, Cryptococcus, non-green algae, Geotrichum, Malassezia, Rhodotorula, Yeast, Megasporum, Trichosporium, and Ustilago maydis.

[0043] Specifically, the drugs include, but are not limited to, one or more of the following: azole drugs, pyrimidine drugs, polyenes, echinocandins, and allylamines.

[0044] Specifically, the azole drugs include, but are not limited to, one or more of the following: ketoconazole, miconazole, econazole, clotrimazole, bifonazole, itraconazole, fluconazole, voriconazole, posaconazole, or lavconazole;

[0045] The pyrimidine drugs include, but are not limited to: flucytosine;

[0046] The echinocandins include, but are not limited to, one or more of the following: caspofungin, micafungin, rezafenamicin, eurifenamicin, or anidifungin.

[0047] In another aspect, the present invention provides a kit comprising the reagents or culture medium described in the aforementioned methods.

[0048] On another front, the present invention provides the application of the aforementioned method or kit in accelerating the detection speed of yeast antimicrobial susceptibility.

[0049] Specifically, the yeasts include, but are not limited to, one or more of the following genera: Candida, Cryptococcus, non-green algae, Geotrichum, Malassezia, Rhodotorula, Yeast, Megasporum, Trichosporium, and Ustilago maydis.

[0050] Specifically, the drugs include, but are not limited to, one or more of the following: azole drugs, pyrimidine drugs, polyenes, echinocandins, and allylamines.

[0051] Specifically, the azole drugs include, but are not limited to, one or more of the following: ketoconazole, miconazole, econazole, clotrimazole, bifonazole, itraconazole, fluconazole, voriconazole, posaconazole, or lavconazole;

[0052] The pyrimidine drugs include, but are not limited to: flucytosine;

[0053] The echinocandins include, but are not limited to, one or more of the following: caspofungin, micafungin, rezafenamicin, eurifenamicin, or anidifungin.

[0054] The technical effects achieved by this invention are as follows:

[0055] The method for accelerating yeast antimicrobial susceptibility testing provided by this invention can obtain antimicrobial susceptibility data within hours, significantly improving the speed of antimicrobial susceptibility testing and possessing technical advantages that traditional antimicrobial susceptibility testing methods cannot match. Attached Figure Description

[0056] Figure 1 The images show the colorimetric results of drug sensitivity testing after 3 hours with different chelating agents. The control group is the one without EDTA-2Na. The figures are columns 1-5 from left to right.

[0057] Figure 2 The color development is shown in the control group (without adding a complexing agent) after 7 hours. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0059] Basic Implementation

[0060] rapid antimicrobial susceptibility testing solution for yeast

[0061] (1) Preparation of bacterial culture

[0062] Candida albicans was prepared into a bacterial suspension with an MCF of 1.0 using sterile water.

[0063] (2) Sample addition

[0064] (a) In a 96-well deep plate, wells containing different concentrations of the test drug (drugs were serially diluted) and positive control wells (PC wells) were set up. 450 μL of drug solution of different concentrations was added to each well, and RPMI 1640 medium was added to the positive control wells.

[0065] (b) Dilute the bacterial culture 100 times with RPMI-1640 medium to obtain the diluted bacterial culture. Add 450 μL of the diluted bacterial culture to each well.

[0066] (3) Incubation

[0067] Remove from heat after incubating at 35°C for 2 hours.

[0068] (4) Enrichment

[0069] After adding EDTA-2Na to each well, vacuum filtration is performed to enrich the bacteria on the filter membrane.

[0070] (5) Add color developer

[0071] Add 30 μL of azadirachtin solution to each well and observe the color change at 35°C.

[0072] (6) Result Interpretation

[0073] The drugs mentioned in step (2) include, but are not limited to: azole drugs, pyrimidine drugs, polyenes, echinocandins and allylamines.

[0074] The result interpretation rules in step (6) are as follows: for polyene drugs, the lowest concentration of antifungal drug in the micropores (i.e., blue micropores) where no pink or purple color appears is the MIC; for other drugs, the lowest concentration of antifungal drug in the micropores (i.e., blue or purple micropores) where no pink color appears is the MIC.

[0075] Example 1: Antimicrobial susceptibility testing of Candida albicans

[0076] Candida albicans, purchased from ATCC, strain number: ATCC90028.

[0077] The detection method is as follows:

[0078] (1) Preparation of bacterial culture

[0079] Candida albicans was prepared into a bacterial suspension with an MCF of 1.0 using sterile water.

[0080] (2) Sample addition

[0081] (a) In a 96-well deep plate, wells containing different concentrations of the test drug (flucytosine diluted in a serial manner) and positive control wells (PC wells) were set up. 450 μL of flucytosine drug solution with concentrations of 64, 32, 16, 8, 4, 2, and 1 μg / mL and RPMI 1640 medium were added respectively.

[0082] (b) The bacterial suspension was diluted 100-fold with RPMI-1640 medium to obtain the diluted bacterial suspension. 450 μL of the diluted bacterial suspension was added to each well. The final drug concentrations in the test drug wells were 32, 16, 8, 4, 2, 1, and 0.5 μg / mL, respectively.

[0083] (3) Incubation

[0084] Remove from heat after incubating at 35°C for 2 hours.

[0085] (4) Enrichment

[0086] Add 9 μL of 50 mM EDTA-2Na to each well (the final concentration of EDTA-2Na is 0.5 mM) and then perform vacuum filtration. The bacteria are enriched on the filter membrane by vacuum filtration.

[0087] (5) Add color developer

[0088] Add 30 μL of azadirachtin solution to each well and observe the color change at 35°C.

[0089] (6) Result interpretation: The lowest concentration of antifungal drug in the micropores where no pink color appeared (i.e., blue or purple micropores) is recorded as MIC.

[0090] Results: After 3 hours of adding 50 mM EDTA-2Na (final concentration 0.5 mM), the PC wells showed complete color development, and the MIC values ​​could be read correctly. Figure 1 As shown in column 3.

[0091] Example 2: Antimicrobial susceptibility testing of Candida albicans

[0092] The difference from Example 1 is that the MCF in step (1) is 0.5 and the final concentration of EDTA-2Na in step (4) is 2mM. The rest is the same as in Example 1.

[0093] Results: The PC hole development time was 3 hours, and the MIC could be read correctly.

[0094] Example 3: Antimicrobial susceptibility testing of Candida albicans

[0095] The difference from Example 1 is that the MCF in step (1) is 1.5, and the final concentration of EDTA-2Na in step (4) is 0.01mM. The rest is the same as in Example 1.

[0096] Results: The development time for the PC aperture was 3.5 hours, and the MIC could be read correctly. Figure 1 Column 2 shows the color development results after 3 hours.

[0097] Example 4: Antimicrobial susceptibility testing of Candida albicans

[0098] The difference from Example 1 is that in step (4), EDTA-2Na is replaced with EDTA of equal final concentration, otherwise it is the same as Example 1.

[0099] Results: The development time for the PC aperture was 3.5 hours, and the MIC could be read correctly. Figure 1 Column 4 shows the color development results after 3 hours.

[0100] Example 5: Antimicrobial susceptibility testing of Cryptococcus

[0101] The difference from Example 1 is that the bacteria in step (1) is Cryptococcus (purchased from ATCC, strain number: ATCC MYA-4561), and the rest is the same as in Example 1.

[0102] Results: The chromogenic time for Cryptococcus was 5 hours, and the MIC was correctly read.

[0103] Comparative Example 1

[0104] Referring to Example 1, the comparative example was set up, and the settings and results of the comparative example are as follows:

[0105] Replace EDTA-2Na in step (4) of Example 1 with sodium citrate of equal final concentration, otherwise the same as in Example 1.

[0106] Results: The addition of sodium citrate did not accelerate the color development; after 7 hours, the entire column turned blue, making it impossible to read the MIC value for drug sensitivity. Figure 1 Column 5 shows the color development results after 3 hours.

[0107] Comparative Example 2

[0108] Referring to Example 1, the comparative example was set up, and the settings and results of the comparative example are as follows:

[0109] Replace EDTA-2Na in step (4) of Example 1 with PMS of equal final concentration, otherwise the same as in Example 2.

[0110] Results: Adding PMS did not accelerate the color development; the color development was incomplete after 7 hours, and the MIC value of drug sensitivity could not be read.

[0111] Comparative Example 3

[0112] Referring to Example 1, the comparative example was set up, and the settings and results of the comparative example are as follows:

[0113] Replace EDTA-2Na in step (4) of Example 1 with NADH of equal final concentration, otherwise the same as in Example 1.

[0114] Results: Adding NADH did not accelerate the color development; the color development was incomplete after 7 hours, and the MIC value of drug sensitivity could not be read.

[0115] Comparative Example 4

[0116] Referring to Example 1, the comparative example was set up, and the settings and results of the comparative example are as follows:

[0117] In Example 1, step (4) does not involve the addition of EDTA-2Na, but the rest is the same as in Example 1.

[0118] Results: In the control group (without EDTA-2Na), no significant color change was observed after 3 hours of color development. Figure 1 As shown in column 1. After 7 hours of color development, the control column (without EDTA-2Na) did not show complete color development, as shown in column 1. Figure 2 As shown.

[0119] Comparative Example 5

[0120] Referring to Example 1, the comparative example was set up, and the settings and results of the comparative example are as follows:

[0121] In Example 1, the final concentration of EDTA-2Na in step (4) was 2.5 mM, and the rest was the same as in Example 1.

[0122] Results: After adding EDTA to a final concentration of 2.5 mM, the wells turned completely blue 7 hours later, and the MIC value for drug sensitivity could not be read.

Claims

1. Application of complexing agents in accelerating the detection speed of yeast antimicrobial susceptibility.

2. Use according to claim 1, characterized in that, The complexing agent is added after drug incubation and before yeast enrichment.

3. Use according to claim 2, characterized in that, The complexing agent is ethylenediaminetetraacetic acid or its salt.

4. Use according to claim 3, characterized in that, The final concentration of the complexing agent is 0.01-2 mM.

5. Use according to any one of claims 1 to 4, characterized in that, The yeast species include one or more of the following genera: Candida, Cryptococcus, non-green algae, Geotrichum, Malassezia, Rhodotorula, Yeast, Megasporum, Trichosporium, and Ustilago maydis.

6. Use according to any one of claims 1 to 4, characterized in that, The drugs include one or more of the following: azole drugs, pyrimidine drugs, polyenes, echinocandins, and allylamines.

7. A method for accelerating the speed of yeast drug sensitivity detection, characterized in that, Includes the following steps: S1. Preparation of bacterial solution: Prepare a bacterial solution with an MCF of 0.5-2.0 using sterile water; S2, Adding samples: First add the drug to the deep well plate or drug sensitivity card, then add the diluted bacterial solution from step S1; S3, Incubation; S4. Enrichment: Add a complexing agent to the sample after incubation in step S3, and then enrich the bacteria; S5. Add color developer; S6. Interpretation result.

8. The method of claim 7, wherein, The diluent used in step S2 is a culture medium; the dilution factor is 5-100.

9. The method of claim 8, wherein, The culture medium is DMEM medium, RPMI 1640 medium, Mueller-Hinton broth, SDB medium, PDB medium, YM medium, MEM medium, LB medium, SOB medium, SOC medium, TB medium, 2×YT medium and / or YPD medium; preferably, the culture medium is RPMI 1640.

10. The method of claim 7, wherein, The incubation conditions described in step S3 are: 25-35℃, 0.5-3h.

11. The method of claim 7, wherein, The complexing agent in step S4 is ethylenediaminetetraacetic acid or its salt.

12. The method of claim 11, wherein, The final concentration of the complexing agent is 0.01-2 mM.

13. The method of claim 7, wherein, The enrichment method described in step S4 includes filtration, centrifugation, or magnetic bead adsorption.

14. The use of the method according to any one of claims 7-13 in the preparation of a kit for accelerating the detection speed of yeast antimicrobial susceptibility.

15. A kit comprising, The kit comprises the reagents or culture media described in any one of claims 7-13.

16. The application of the method according to any one of claims 7-13 or the kit according to claim 15 in accelerating the detection speed of yeast antimicrobial susceptibility.

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