A method for identifying drug-resistant Candida albicans and a kit for identifying the same

Abstract

The present invention relates to a method for identifying drug-resistant Candida albicans and an identification kit. Candida albicans is a common pathogen. Clinical treatment for fungal infections should first be carried out on the strains for typing detection, and appropriate antifungal drug treatment should be selected based on the drug sensitivity results. Currently, drug sensitivity culture requires a long time. To address this shortcoming, the present invention provides a method for identifying drug-resistant Candida albicans. The strain to be identified is incubated with MKT077 and benomyl, and the degree of rightward shift of the red fluorescence of the strain after incubation is detected. The strain showing a significant rightward shift is Mdr1-type Candida albicans. This identification method has good specificity and fast detection speed. Its application in clinical testing can effectively shorten the time required for detection and has promotional value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for identifying drug-resistant Candida albicans, and also includes the use of MKT077, benomyl and a combination thereof in preparing a drug-resistant Candida albicans identification product. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] In recent years, the incidence of infections caused by Candida species has increased annually, primarily due to the prevalence of HIV / AIDS and the development of modern medical technologies, including organ transplantation, invasive surgical procedures, immunosuppressive therapy, large-scale chemotherapy, and the widespread use of implantable medical devices. Among them, Candida albicans is the main cause of candidiasis. As an opportunistic pathogen, it can coexist with the host under normal conditions. However, when the body's tissues are damaged or the immune system is weakened, it can cause a variety of diseases ranging from superficial mucosal infections to systemic infections. In addition, other Candida species, such as Candida glabrata, Candida parapsilosis, and Candida tropicalis, are also frequently identified as human pathogens.

[0004] The continuous improvement and development of detection technology has enabled more pathogens to be discovered, thus laying the foundation for the effective treatment of more diseases. Traditional methods for detecting Candida albicans mainly include fungal selective culture, direct microscopy, colony PCR, and gene sequencing, which are essential diagnostic procedures for clinical testing of microbial pathogens. In addition, highly specific and sensitive detection methods such as the combined detection of mannan and anti-mannan antibodies and β-1,3-D-glucan detection (BDG) are also widely used in clinical testing. At present, accurate detection of pathogen infection in clinical practice often takes 1 day or even a week to obtain identification results. Clinical data show that the timing of antifungal treatment has a significant impact on the incidence and mortality of the disease. Therefore, it is very important to quickly detect the infectious pathogen and start antifungal treatment as early as possible. Summary of the Invention

[0005] Based on the above technical background, the present invention aims to provide a method for rapidly identifying drug-resistant strains, and specifically, to provide a method for rapidly identifying Mdr1-resistant Candida albicans.

[0006] Based on the above technical objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a method for identifying drug-resistant Candida albicans. The method comprises incubating a strain to be identified with MKT077, and detecting the fluorescence intensity value of the strain before and after incubation.

[0008] The present invention has verified that MKT077, as a sulfur-containing cyanine dye anticancer drug, has specific affinity for Mdr1-overexpressing and resistant Candida albicans strains, particularly accumulating in large quantities in strains that overexpress the efflux pump Mdr1, while its content is relatively low in strains that underexpress it. Therefore, MKT077 can be used as a fluorescent probe to specifically identify strains that overexpress the efflux pump Mdr1 and are resistant.

[0009] The present invention also confirms that benomyl (Benomyl) can upregulate the expression of the MDR1 gene of the MFS transporter of Candida albicans. After benomyl is added to the Candida albicans to be tested, the fluorescence intensity value of MKT077 is significantly enhanced compared to the fluorescence value of the blank reagent, and a significant right shift has occurred from the fluorescence detection spectrum; after incubation of other types of Candida, the above phenomenon can also be observed, but the degree is relatively weak; this phenomenon has not been observed in other bacteria or mammalian cells. Based on the above phenomenon, in the scheme provided by the application, by comparing the degree of right shift of the fluorescence intensity value after incubation with a single MKT077 or incubation with benomyl and MKT077, it can be known whether the strain to be identified is Candida, and by further combining the degree of increase in the fluorescence intensity value, it can be judged whether the strain is Candida albicans. After clarifying Candida albicans, after incubating the strain with MKT077, by comparing the changes in the fluorescence intensity of the wild strain and the strain to be tested, it can be confirmed whether the strain to be tested is Mdr1-resistant Candida albicans.

[0010] The drug resistance type of a strain directly affects the treatment medication and the adjustment of the treatment plan. The identification method provided by the present invention takes less than 2 hours to determine whether it is Mdr1-resistant Candida albicans, which can effectively shorten the preclinical waiting time for test results and gain more treatment opportunities.

[0011] In addition to the above technical solutions, the present invention also provides the use of benomyl and MKT077 in preparing a product for identifying drug-resistant Candida, and the use of a combination of the above ingredients in preparing a kit for identifying drug-resistant Candida.

[0012] The beneficial effects of one or more of the above technical solutions are:

[0013] The primary technical contribution of this invention lies in providing specific affinity for MKT077 against Mdr1-resistant Candida albicans. Based on this specific affinity, the red fluorescence of Candida albicans that ingests MKT077 exhibits a significant rightward shift. This identification method offers the advantages of simple operation, rapid detection, easy result determination, high sensitivity, and strong specificity. This method may offer insights and assistance to hospital laboratory departments and companies developing microbial diagnostic products, and is expected to have significant clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0015] Figure 1 This is the peak spectrum of the wild-type Candida albicans SC5314 strain described in Example 1, obtained by flow cytometry;

[0016] Figure 2 This is the peak spectrum of the Candida albicans clinical strain 28A described in Example 1, obtained by flow cytometry;

[0017] Figure 3 This is the peak spectrum of the Candida albicans clinical strain 28D described in Example 1, obtained by flow cytometry;

[0018] Figure 4 This is the peak spectrum of the clinical Candida albicans strain Gu5 described in Example 1, obtained by flow cytometry detection;

[0019] Figure 5 This is the peak spectrum of the Candida albicans clinical strain DSY296 described in Example 1, obtained by flow cytometry detection;

[0020] Figure 6 This is the peak spectrum of the clinical Candida albicans F5 strain described in Example 1, obtained by flow cytometry;

[0021] Figure 7 This is the peak spectrum of the Candida tropicalis CT2 strain described in Example 1, obtained by flow cytometry;

[0022] Figure 8 This is the peak spectrum of the Candida parapsilosis CP001 strain described in Example 1, obtained by flow cytometry;

[0023] Figure 9 This is the peak spectrum of the Candida krusei CK1 strain described in Example 1, obtained by flow cytometry;

[0024] Figure 10 This is the peak spectrum of the Candida glabrata CG1 strain described in Example 1, obtained by flow cytometry;

[0025] Figure 11 This is the peak spectrum of the Saccharomyces cerevisiae strain 4742 described in Example 1, obtained by flow cytometry;

[0026] Figure 12 This is the peak spectrum of Cryptococcus neoformans H99 strain described in Example 1, obtained by flow cytometry detection;

[0027] Figure 13 This is the peak spectrum of the Aspergillus fumigatus strain AF293 described in Example 1, obtained by flow cytometry;

[0028] Figure 14 This is the peak spectrum of the Staphylococcus aureus strain described in Example 1, obtained by flow cytometry;

[0029] Figure 15 This is the peak spectrum obtained by flow cytometry detection of the Bacillus subtilis strain described in Example 1;

[0030] Figure 16 This is the peak spectrum of the Escherichia coli strain described in Example 1 detected by flow cytometry;

[0031] Figure 17 This is the peak spectrum of the human non-small cell lung cancer cell line A549 described in Example 1, detected by flow cytometry;

[0032] above Figures 1-17 In the figure, peak A is the blank group, peak B is the MKT077 group, and peak C is the MKT077+Benomyl group; the left figure shows a final concentration of MKT077 of 1 μg / mL, and the right figure shows a final concentration of MKT077 of 2 μg / mL.

[0033] Figure 18 This is the peak spectrum obtained by flow cytometry analysis of the Candida albicans YEM12 and YEM13 strains described in Example 2;

[0034] Peak A represents the YEM12 strain treated with MKT077, and peak B represents the YEM13 strain treated with MKT077. The final concentration of MKT077 was 4 μg / mL.

[0035] Figure 19 This is the peak spectrum obtained by flow cytometry detection of the Candida albicans NPC227 and NPC233 strains described in Example 2;

[0036] Peak A represents the NPC227 strain treated with MKT077, and peak B represents the NPC233 strain treated with MKT077. The final concentration of MKT077 was 4 μg / mL.

[0037] Figure 20 The peak spectra of the Candida albicans CAF2-1 and DSY465 strains described in Example 2 were obtained by flow cytometry;

[0038] Peak A represents the CAF2-1 strain treated with MKT077, and peak B represents the DSY465 strain treated with MKT077. The final concentration of MKT077 was 4 μg / mL.

[0039] Figure 21 The peak spectra of the Candida albicans DSY465 and NPC379 strains described in Example 2 were obtained by flow cytometry;

[0040] Peak A represents the DSY465 strain treated with MKT077, and peak B represents the NPC379 strain treated with MKT077. The final concentration of MKT077 was 4 μg / mL. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0043] As described in the background art, Candida albicans can invade multiple organs of the human body and cause disease, with complex clinical symptoms that vary in severity. Currently, drug susceptibility testing is the primary method for typing and identifying Candida albicans. However, determining the strain's drug resistance requires approximately two days, potentially delaying optimal treatment. The present invention provides a method for rapidly identifying drug-resistant Candida species. This method effectively reduces the time required for typing and enables specific detection of Candida albicans and its efflux pump Mdr1 resistance, effectively shortening the time required for clinical treatment.

[0044] In a first aspect, the present invention provides a method for identifying drug-resistant Candida species, comprising incubating a strain to be identified with MKT077, and detecting the fluorescence intensity values ​​of the strain before and after incubation.

[0045] MKT077 described in the above scheme is compound C 21 H 22 The structure of ClN3OS2 (CAS: 147366-41-4) is shown in Formula I below:

[0046]

[0047] Preferably, the drug-resistant type is a drug-resistant strain with high expression of the efflux pump gene MDR1.

[0048] Preferably, the Candida is one including but not limited to Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei or Candida glabrata; in a better solution, the Candida is Candida albicans.

[0049] Existing research results show that the types of drug resistance of Candida albicans mainly include resistance regulated by efflux pump-related resistance genes, resistance regulated by ergosterol biosynthesis-related resistance genes, and resistance regulated by biofilm-related resistance genes. Among them, resistance regulated by efflux pump-related resistance genes is one of the main mechanisms that lead to Candida albicans resistance to azole drugs, and specific regulatory genes include CDR1, CDR2, MDR1 or FLU1. It has been verified that the above-mentioned MKT077 has good affinity for resistant strains with high expression of the MDR1 gene, and the gene may play a major transport role in regulating the strain's uptake of MKT077. Therefore, in one embodiment of the above-mentioned technical solution, the resistant Candida albicans is a multidrug-resistant (Mdr1) Candida albicans.

[0050] Therefore, in a preferred embodiment of the present invention, the identification method further comprises adding benomyl to the strain to be identified and co-incubating the strain. By comparing the degree of red shift in fluorescence intensity after incubation with MKT077 alone or after co-incubation with benomyl and MKT077, it is determined whether the strain to be identified is a drug-resistant Candida albicans that overexpresses the efflux pump Mdr1.

[0051] The benomyl described in the above scheme is the compound 1-n-butylaminoformyl-benzimidazole-2-carbamic acid methyl ester, CAS: 17804-35-2, and its specific structure is shown in the following formula II:

[0052]

[0053] Furthermore, the specific steps of the identification method are as follows: MKT077 and benomyl are added to the activated test strain and incubated together for 0.5 to 1.5 hours, the concentration of the MKT077 is 0.8 to 2.5 μg / mL, and the concentration of the benomyl is 4 to 6 μg / mL. After the incubation is completed, the cells are collected for fluorescence spectrum detection.

[0054] It should be noted that the concentrations of MKT077 and benomyl in the above-mentioned detection method are the concentrations of these substances in the incubation system at the start of incubation. As incubation progresses, the content of these substances may change due to the influence of the strain. Because the strain's uptake of these small molecule compounds is affected by the incubation temperature and incubation time, in the embodiment provided by the present invention, the incubation temperature is 28-32°C and the incubation time is 0.8-1.2 hours.

[0055] Preferably, the detection of the fluorescence intensity value is not limited to a specific detection method, and any device that can realize fluorescence spectrum detection in the art can be used, such as a fluorescence spectrophotometer, a fluorescence detector or a flow cytometer.

[0056] Furthermore, in one embodiment of the method for identifying drug-resistant Candida according to the first aspect, the identification method comprises the following steps: adjusting the cell number of the strain to be tested in the culture medium to 1.0×10 6 MKT077 and benomyl were added to the culture medium to make the final concentration of MKT077 2 μg / mL and the concentration of benomyl 5 μg / mL, and the incubation system was placed at 30°C for 1 hour; after the incubation was completed, the culture medium was discarded and the strain cells were washed and the fluorescence intensity value was detected. After incubation, the strain with obvious red shift in fluorescence intensity was measured at the position of 100, and the movement distance was measured, with the horizontal axis changing by one order of magnitude as 1 unit. When the red shift distance exceeded 0.5 units (with the addition of Figure 1 For example), it can be identified as Candida albicans; when a red shift occurs but the degree is not obvious (about 0.15-0.5 units), the strain belongs to the genus Candida; if no red shift occurs, the strain does not belong to Candida.

[0057] In another embodiment, the identification method comprises the following steps: adjusting the cell number of the strain to be tested in the culture medium to 1.0×10 6MKT077, MKT077 and benomyl were added to the culture medium respectively, so that the final concentration of MKT077 was 2 μg / mL and the concentration of benomyl was 5 μg / mL, and the incubation system was incubated at 30°C for 1 hour; after the incubation was completed, the culture medium was discarded and the strain cells were washed, and the fluorescence intensity value was detected. If the red shift degree was increased and more obvious in the co-incubation with benomyl and MKT077 compared with the MKT077 single incubation, it was determined to be Candida albicans; when the final concentration of MKT077 was incubated at 4 μg / mL, the fluorescence value of the test strain showed a red shift compared with the wild strain of Candida albicans (non-resistant bacteria), it was Mdr1-resistant Candida albicans; if the strain showed a red shift after incubation with MKT077 and incubation with MKT077 and benomyl but to a similar extent, the strain could be determined to be Candida; if no red shift was shown, the strain did not belong to Candida.

[0058] The scheme described in the above embodiment is obtained by screening the following steps:

[0059] (1) The minimum inhibitory concentration of MKT077 against Candida albicans was measured by microdilution method (Table 1) and the appropriate concentration of MKT077 was selected.

[0060] (2) The final concentration of Benomyl was determined to be 5 μg / mL using fluorescence quantitative PCR.

[0061] (3) Different incubation times (30 min, 1 h, 2 h) and different MKT077 concentrations (0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL) were set, and experimental screening was performed in pairs. The optimal experimental conditions were finally determined: incubation time 1 h, final MKT077 concentrations of 1 μg / mL and 2 μg / mL, and Benomyl final concentration of 5 μg / mL.

[0062] (4) The MIC of MKT077 against Candida albicans with different gene mutation types was measured by microdilution method. It was found that MKT077 was more active against MDR1 high-expressing strains than its parent strain, showing strain selectivity.

[0063] (5) The intracellular content of MKT077 in Candida albicans with different gene mutation types was detected by flow cytometry, and it was found that the intracellular drug content of the MDR1 high-expression strain was significantly higher than that of its parent strain.

[0064] In a second aspect, the present invention provides a composition comprising MKT077 and benomyl.

[0065] The third aspect of the present invention provides the use of MKT077, benomyl or the composition described in the second aspect in the preparation of a product for identifying drug-resistant Candida species.

[0066] In a fourth aspect, the present invention provides a kit for identifying drug-resistant Candida species, wherein the kit comprises MKT077, benomyl or the composition according to the second aspect.

[0067] Preferably, the kit is used according to the method for identifying drug-resistant Candida albicans described in the first aspect.

[0068] Preferably, the kit further includes a strain culture medium and a cleaning reagent; wherein MKT077, benomyl or the composition can be prepared using the culture medium as a solvent; the cleaning reagent is a buffer solution that can be used for cell cleaning in the art and does not damage the cell structure, such as a PBS solution.

[0069] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0070] Example 1 Rapid Identification of Candida albicans and Identification of Its Mdr1 Resistance Type

[0071] 1. Drug testing

[0072] Benomyl

[0073] MKT077

[0074] Dimethyl sulfoxide (DMSO)

[0075] All the above compounds were prepared into 10 mg / mL stock solutions in dimethyl sulfoxide and stored at -20°C. Before the experiment, the test drug was taken out and thawed at room temperature, thoroughly mixed using a rotary shaker, and then the pharmacodynamic test was performed.

[0076] 2. Strains

[0077] Candida albicans wild type SC5314, clinically resistant Candida albicans (28A, 28D, Gu5, DSY296 and F5), Candida parapsilosis CP001, Candida tropicalis CT2, Candida krusei CK1, Candida glabrata CG1, Aspergillus fumigatus AF293, Saccharomyces cerevisiae 4742, Cryptococcus neoformans H99, Staphylococcus aureus ATCC6538, Bacillus subtilis ATCC9372, Escherichia coli, and human non-small cell lung cancer cells A549.

[0078] 3. Culture medium

[0079] (1) YPD liquid medium

[0080] Dissolve 8 g of peptone, 8 g of glucose, and 4 g of yeast starch in 350 ml of triple-distilled water, then add triple-distilled water to make up to 400 ml. Sterilize at high temperature and high pressure (121°C, 20 min) and store at 4°C until use.

[0081] (2) RPMI-1640 liquid culture medium

[0082] RPMI-1640 solid powder (Gibco BRL) 10 g, morpholinepropanesulfonic acid (Sigma) 34.5 g, were dissolved in 900 ml of triple-distilled water, the pH was adjusted to 7.0 (25°C) with 1 M NaOH, and the volume was made up to 1000 ml with triple-distilled water. The solution was sterilized by filtration through a 0.22 μm microporous filter, and stored at 4°C after aliquoting.

[0083] (3) LB liquid culture medium

[0084] Dissolve 4 g of peptone, 4 g of NaCl, and 2 g of yeast starch in 350 ml of triple-distilled water, then add triple-distilled water to make up to 400 ml. Sterilize under high temperature and high pressure (121°C, 20 min) and store at 4°C until use.

[0085] (4) MHB liquid culture medium

[0086] Weigh 24 g of MHB powder (Solarbio) and add it to 1000 ml of distilled water. Heat and boil to dissolve. Aliquot and sterilize under high pressure at 121°C for 15 min.

[0087] 4. Instruments and Equipment

[0088] SW-CJ-JC double-sided clean bench (Suzhou Purification Equipment Co., Ltd.)

[0089] NBS Innova 42 constant temperature shaker (Eppendorf, Germany)

[0090] BD FACSCalibur flow cytometer

[0091] 5. Experimental Methods

[0092] (1) Candida

[0093] a. Take the activated Candida strain plate, pick a single colony, inoculate it into YPD liquid medium, and culture it in a constant temperature shaker at 30°C overnight.

[0094] b. Take 50 μl of overnight activated bacterial solution and centrifuge at 12000 rpm for 1 min to collect bacterial cells. Discard the supernatant and resuspend the bacterial cells in RPMI-1640 medium and adjust the concentration to 1×10 6 / mL.

[0095] c. Set up a blank group, an MKT077 group, and an MKT077 + Benomyl group. Adjust the MKT077 group to a final concentration of 1 μg / mL and 2 μg / mL, respectively, and adjust the Benomyl concentration to 5 μg / mL.

[0096] d. Incubate at 30°C for 1 h.

[0097] e. After the bacterial incubation is complete, centrifuge at 15,000 rpm for 1 minute to collect the bacterial cells, wash them twice with PBS, mix thoroughly with 200 μl of PBS, and then transfer them to the flow tube.

[0098] f. Flow cytometry was used for detection and Flowjo software was used for analysis.

[0099] (2) Aspergillus fumigatus

[0100] a. Take the activated Aspergillus fumigatus AF293 strain plate, pick a single colony, inoculate it into YPD liquid medium, and culture it in a constant temperature shaker at 30°C overnight.

[0101] b. Take 50 μl of overnight activated bacterial solution and centrifuge at 12000 rpm for 1 min to collect bacterial cells. Discard the supernatant and resuspend the bacterial cells in RPMI-1640 medium and adjust the concentration to 1×10 6 / mL.

[0102] c. Set up a blank group, an MKT077 group, and an MKT077 + Benomyl group. Adjust the MKT077 group to a final concentration of 1 μg / mL and 2 μg / mL, respectively, and adjust the Benomyl concentration to 5 μg / mL.

[0103] d. Incubate at 30°C for 1 h.

[0104] e. After the bacterial incubation is complete, centrifuge at 15,000 rpm for 1 minute to collect the bacterial cells, wash them twice with PBS, mix thoroughly with 200 μl of PBS, and then transfer them to the flow tube.

[0105] f. Flow cytometry was used for detection and Flowjo software was used for analysis.

[0106] (3) Saccharomyces cerevisiae

[0107] a. Take the activated Saccharomyces cerevisiae 4742 strain plate, pick a single colony, inoculate it into YPD liquid medium, and culture it in a constant temperature shaker at 30°C overnight.

[0108] b. Take 50 μl of overnight activated bacterial solution and centrifuge at 12000 rpm for 1 min to collect bacterial cells. Discard the supernatant and resuspend the bacterial cells in 1640 medium and adjust the concentration to 1×10 6 / mL.

[0109] c. Set up a blank group, an MKT077 group, and an MKT077 + Benomyl group. Adjust the MKT077 group to a final concentration of 1 μg / mL and 2 μg / mL, respectively, and adjust the Benomyl concentration to 5 μg / mL.

[0110] d. Incubate at 30°C for 1 h.

[0111] e. After the bacterial solution incubation is completed, centrifuge at 15000 rpm for 1 min to collect the bacterial cells, wash them twice with PBS, mix thoroughly with 200 μl PBS and then transfer them into the flow tube.

[0112] f. Flow cytometry was used for detection and Flowjo software was used for analysis.

[0113] (4) Cryptococcus neoformans

[0114] a. Take the activated Cryptococcus neoformans H99 strain plate, pick a single colony, inoculate it into YPD liquid medium, and culture it in a constant temperature shaker at 30°C overnight.

[0115] b. Take 50 μl of overnight activated bacterial solution and centrifuge at 12000 rpm for 1 min to collect bacterial cells. Discard the supernatant and resuspend the bacterial cells in 1640 medium and adjust the concentration to 1×10 6 / mL.

[0116] c. Set up a blank group, an MKT077 group, and an MKT077 + Benomyl group. Adjust the MKT077 group to a final concentration of 1 μg / mL and 2 μg / mL, respectively, and adjust the Benomyl concentration to 5 μg / mL.

[0117] d. Incubate at 30°C for 1 h.

[0118] e. After the bacterial incubation is complete, centrifuge at 15,000 rpm for 1 minute to collect the bacterial cells, wash them twice with PBS, mix thoroughly with 200 μl of PBS, and then transfer them to the flow tube.

[0119] f. Flow cytometry was used for detection and Flowjo software was used for analysis.

[0120] (5) Bacteria

[0121] a. Take the activated bacterial strain plate, pick a single colony, inoculate it into LB liquid medium, and culture it in a constant temperature shaker at 37°C overnight.

[0122] b. Take 50 μl of overnight activated bacterial solution and centrifuge at 12000 rpm for 1 min to collect bacterial cells. Discard the supernatant and resuspend the bacterial cells in MHB medium and adjust the concentration to 1×10 6 / mL.

[0123] c. Set up a blank group, an MKT077 group, and an MKT077 + Benomyl group. Adjust the MKT077 group to a final concentration of 1 μg / mL and 2 μg / mL, respectively, and adjust the Benomyl concentration to 5 μg / mL.

[0124] d. Incubate at 37°C for 1 hour.

[0125] e. After the bacterial incubation is complete, centrifuge at 15,000 rpm for 1 minute to collect the bacterial cells, wash them twice with PBS, mix thoroughly with 200 μl of PBS, and then transfer them to the flow tube.

[0126] f. Flow cytometry was used for detection and Flowjo software was used for analysis.

[0127] (6) Tumor cells

[0128] a. When the revived cells grow to 80%-90% density, remove the original culture medium and add 1 ml of trypsin. Incubate for about 3 minutes and observe under a microscope. When most cells become round, add 1 ml of fresh culture medium to terminate digestion.

[0129] b. Use the tip of the gun to gently blow down the cells to mix them and transfer them to an EP tube.

[0130] c. Set up a blank group, an MKT077 group, and an MKT077 + Benomyl group. Adjust the MKT077 group to a final concentration of 1 μg / mL and 2 μg / mL, respectively, and adjust the Benomyl concentration to 5 μg / mL.

[0131] d. Culture in a cell incubator at 37°C and 5% carbon dioxide for 1 hour.

[0132] e. After incubation, the tumor cells were collected by centrifugation at 3000 rpm for 4 min, washed twice with PBS, and thoroughly mixed with 200 μl of PBS before loading into the flow cytometry tube.

[0133] f. Flow cytometry was used for detection and Flowjo software was used for analysis.

[0134] 6. Experimental Results

[0135] Flow cytometric analysis revealed that after incubation with Benomyl, the fluorescence intensity Geomean value of Candida albicans SC5314 and its clinically resistant strains significantly increased, with a significant rightward shift in peak value. This phenomenon was observed only slightly in other Candida species, but not in other fungi (including Aspergillus fumigatus, Saccharomyces cerevisiae, and Cryptococcus neoformans), bacteria, or tumor cells. Therefore, the combination of MKT077 and Benomyl can rapidly identify Candida species, particularly C. albicans.

[0136] Example 2

[0137] 1. MIC determination of MKT077 against Candida albicans with different gene mutation types

[0138] 1. Drug testing

[0139] MKT077

[0140] Fluconazole (FLC)

[0141] Dimethyl sulfoxide (DMSO)

[0142] All compounds were prepared in dimethyl sulfoxide to a stock solution of 10 mg / mL and stored at -20°C. Before the experiment, the test drug was taken out and thawed at room temperature, thoroughly shaken and mixed using a rotary shaker, and pharmacodynamics tests were performed.

[0143] 2. Strains

[0144] YEM12 strain (YEM13 parent strain), YEM13 strain (MDR1 high expression strain caused by strong promoter), NPC227 (NPC233 parent strain), NPC233 (MRR1 P683S The strains included CAF2-1 (mother strain of DSY465), DSY465 (MDR1 knockout strain), NPC379 (DSY465 strain complemented with MDR1 gene), YEM14 strain (mother strain of YEM15), YEM15 (CDR1 and CDR2 high expression strain caused by strong promoter), DSY448 (CDR1 knockout strain), and DSY653 (CDR2 knockout strain).

[0145] 3. Culture medium

[0146] (1) YPD liquid medium

[0147] Dissolve 8 g of peptone, 8 g of glucose, and 4 g of yeast starch in 350 ml of triple-distilled water, then add triple-distilled water to make up to 400 ml. Sterilize at high temperature and high pressure (121°C, 20 min) and store at 4°C until use.

[0148] (2) RPMI-1640 liquid culture medium

[0149] RPMI-1640 solid powder (Gibco BRL) 10 g, morpholinepropanesulfonic acid (Sigma) 34.5 g, were dissolved in 900 ml of triple-distilled water, the pH was adjusted to 7.0 (25°C) with 1 M NaOH, and the volume was made up to 1000 ml with triple-distilled water. The solution was sterilized by filtration through a 0.22 μm microporous filter, and stored at 4°C after aliquoting.

[0150] 4. Instruments and Equipment

[0151] SW-CJ-JC double-sided clean bench (Suzhou Purification Equipment Co., Ltd.)

[0152] NBS Innova 42 constant temperature shaker (Eppendorf, Germany)

[0153] 5. Experimental Methods

[0154] (1) Take the activated Candida albicans strain plate, pick a single clone, mix it in YPD liquid medium, and culture it in a constant temperature shaker at 30℃ overnight.

[0155] (2) Take 50 μl of the overnight activated bacterial solution, centrifuge at 12000 rpm for 1 min to collect the bacterial cells, discard the supernatant, and resuspend the bacterial cells in RPMI-1640 medium and adjust the concentration to 1×10 3 / mL.

[0156] (3) Different concentrations of FLC and MKT077 were added. The concentrations of MKT077 were set at 0, 1, 2, 4, 8, 16, and 32 μg / mL, and the concentrations of FLC were set at 0, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / mL.

[0157] (4) Incubate at 30°C for 24 hours.

[0158] (5) Determine the minimum inhibitory concentration by visual inspection.

[0159] 6. Experimental Results

[0160] The results showed that the MIC of MKT077 against strains with high MDR1 expression was lower than that of its parent strain. As shown in Table 1, the MIC of MKT077 against YEM13 was 2 μg / mL, significantly lower than the 16 μg / mL of MKT077 against YEM12. MKT077 also showed selectivity against strains with high MDR1 expression caused by MRR1 gene mutations. The MIC of MKT077 against NPC379 was 8 μg / mL, lower than the MIC against DSY465, indicating that the MDR1 gene affects the antimicrobial susceptibility of MKT077. Furthermore, the antibacterial activity of MKT077 against Candida albicans was independent of the CDR1 and CDR2 genes.

[0161] 2. Intracellular accumulation experiment of MKT077 in Candida albicans with different gene mutation types

[0162] 1. Drug testing

[0163] MKT077

[0164] Dimethyl sulfoxide (DMSO)

[0165] Compound MKT077 was prepared in dimethyl sulfoxide to a stock solution at a concentration of 10 mg / mL and stored at -20°C. Prior to the experiment, the test drug was taken out and thawed at room temperature, thoroughly shaken and mixed using a rotary shaker, and pharmacodynamic testing was performed.

[0166] 2. Strains

[0167] YEM12 strain (YEM13 parent strain), YEM13 strain (MDR1 high expression strain caused by strong promoter), NPC227 (NPC233 parent strain), NPC233 (MRR1 P683S MDR1 overexpression strain caused by mutation), CAF2-1 (DSY465 parent strain), DSY465 (MDR1 gene knockout strain), and NPC379 (DSY465 strain complemented with MDR1 gene).

[0168] 3. Culture medium

[0169] (1) YPD liquid medium

[0170] Dissolve 8 g of peptone, 8 g of glucose, and 4 g of yeast starch in 350 ml of triple-distilled water, then add triple-distilled water to make up to 400 ml. Sterilize at high temperature and high pressure (121°C, 20 min) and store at 4°C until use.

[0171] (2) RPMI-1640 liquid culture medium

[0172] RPMI-1640 solid powder (Gibco BRL) 10 g, morpholinepropanesulfonic acid (Sigma) 34.5 g, were dissolved in 900 ml of triple-distilled water, the pH was adjusted to 7.0 (25°C) with 1 M NaOH, and the volume was made up to 1000 ml with triple-distilled water. The solution was sterilized by filtration through a 0.22 μm microporous filter, and stored at 4°C after aliquoting.

[0173] 4. Instruments and Equipment

[0174] SW-CJ-JC double-sided clean bench (Suzhou Purification Equipment Co., Ltd.)

[0175] NBS Innova 42 constant temperature shaker (Eppendorf, Germany)

[0176] BD FACSCalibur flow cytometer

[0177] 5. Experimental Methods

[0178] (1) Take the activated Candida albicans strain plate, pick a single clone, mix it in YPD liquid medium, and culture it in a constant temperature shaker at 30℃ overnight.

[0179] (2) Take 50 μl of the overnight activated bacterial solution, centrifuge at 12000 rpm for 1 min to collect the bacterial cells, discard the supernatant, and resuspend the bacterial cells in RPMI-1640 medium and adjust the concentration to 1×10 6 / mL.

[0180] (3) Set up a blank group and an MKT077 group. The final concentrations of the MKT077 group were adjusted to 2 μg / mL and 4 μg / mL, respectively.

[0181] (4) Incubate at 30°C for 1 hour.

[0182] (5) After the bacterial solution incubation is completed, the bacterial cells are collected by centrifugation at 15000 rpm for 1 min, washed twice with PBS, and thoroughly mixed with 200 μl PBS before being loaded into the flow tube.

[0183] (6) Flow cytometry was used for detection, and Flowjo software was used for image analysis.

[0184] 6. Experimental Results

[0185] YEM13 is a drug-resistant strain with a strong promoter that highly expresses MDR1. Compared with its parent strain YEM12, after treatment with MKT077, the peak shifted significantly to the right, and the intracellular drug increased significantly. MKT077 also showed selectivity for strains with high MDR1 expression caused by mutations in the MRR1 gene. In addition, compared with DSY465, the NPC379 strain showed a significant rightward shift in peak and a significant increase in intracellular drug, indicating that high expression of the MDR1 gene affects the intracellular accumulation of MKT077. Therefore, under treatment with the same dose of MKT077, the intracellular drug content of strains with high MDR1 expression increased significantly, and the flow cytometry peak shifted to the right, which can be used to identify strains with MDR1 resistance.

[0186] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A MDR1 A method for identifying drug-resistant Candida albicans with high gene expression, characterized in that: The identification method comprises incubating the strain to be identified with MKT077 and detecting the fluorescence emission spectrum of the strain after incubation.

2. as claimed in claim 1 MDR1 A method for identifying drug-resistant Candida albicans with high gene expression, characterized in that: The identification method further comprises adding benomyl to the strain to be identified and incubating the strains together.

3. As claimed in claim 2 MDR1 A method for identifying drug-resistant Candida albicans with high gene expression, characterized in that: The specific steps of the identification method are as follows: MKT077 and benomyl are added to the activated test strain and incubated together for 0.5 to 1.5 hours, the concentration of the MKT077 is 0.8 to 2.5 μg / mL, and the concentration of the benomyl is 4 to 6 μg / mL. After the incubation is completed, the cells are collected for fluorescence spectrum detection; the incubation temperature is 28 to 32°C, and the incubation time is 0.8 to 1.2 hours.

4. As claimed in claim 3 MDR1 A method for identifying drug-resistant Candida albicans with high gene expression, characterized in that: The identification method steps are as follows: adjust the cell number of the strain to be tested in the culture medium to 1.0×10 6 MKT077 and benomyl were added to the culture medium to make the final concentration of MKT077 2 μg / mL and the concentration of benomyl 5 μg / mL, and the incubation system was incubated at 30° C. for 1 hour. After the incubation was completed, the culture medium was discarded and the strain cells were washed before fluorescence spectrum detection.

5. As claimed in claim 3 MDR1 A method for identifying drug-resistant Candida albicans with high gene expression, characterized in that: The identification method steps are as follows: adjust the cell number of the strain to be tested in the culture medium to 1.0×10 6 MKT077, MKT077 and benomyl were added to the culture medium respectively, so that the final concentration of MKT077 was 2 μg / mL and the concentration of benomyl was 5 μg / mL, and the incubation system was placed at 30°C for 1 hour; after the incubation was completed, the culture medium was discarded and the strain cells were washed and the fluorescence intensity value was detected. When the red shift degree of benomyl and MKT077 co-incubation was increased and more obvious compared with the MKT077 single incubation, it was determined to be Candida albicans; when the final concentration of MKT077 was incubated at 4 μg / mL, the fluorescence value of the test strain showed a red shift compared with the wild strain of Candida albicans, it was MDR1 Drug-resistant Candida albicans with high gene expression; If the strain shows a red shift after incubation with MKT077 and after incubation with MKT077 and benomyl, but the degree is similar, the strain can be determined to be Candida; if no red shift occurs, the strain does not belong to Candida.

6. As claimed in claim 1 MDR1 A method for identifying drug-resistant Candida albicans with high gene expression, characterized in that: The fluorescence emission spectrum can be detected using a fluorescence spectrophotometer, a fluorescence detector or a flow cytometer.

7. MKT077 in preparation MDR1 Application of products in the identification of drug-resistant Candida albicans with high gene expression.

8. A composition in preparation MDR1 The application of the product for identifying drug-resistant Candida albicans with high gene expression is characterized in that: The composition includes MKT077 and benomyl.

9. A kind of identification MDR1 A kit for treating drug-resistant Candida albicans with high gene expression, characterized in that: MKT077 is included in the kit.

10. The kit according to claim 9, wherein The kit also includes benomyl.

11. The kit according to claim 9, wherein The use of the kit is achieved according to the identification method according to any one of claims 1 or 6.

12. The kit according to claim 10, wherein The kit is used according to the identification method according to any one of claims 2 to 5.

13. The kit according to claim 9, wherein The kit also includes a strain culture medium and a cleaning reagent; the cleaning reagent is a PBS solution.

14. The kit according to claim 10, wherein MKT077 and benomyl were prepared using culture medium as solvent.

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