CN115044478A - Candida auriculata capable of being used for constructing fluconazole single-drug-resistant model and application thereof in construction of fluconazole single-drug-resistant model - Google Patents

Candida auriculata capable of being used for constructing fluconazole single-drug-resistant model and application thereof in construction of fluconazole single-drug-resistant model Download PDF

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CN115044478A
CN115044478A CN202111573160.1A CN202111573160A CN115044478A CN 115044478 A CN115044478 A CN 115044478A CN 202111573160 A CN202111573160 A CN 202111573160A CN 115044478 A CN115044478 A CN 115044478A
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candida
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CN115044478B (en
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李筱芳
周晓伟
刘维达
王琼
景丹蕤
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Abstract

The invention relates to candida auricula for constructing a fluconazole single-drug-resistant model and application thereof in constructing the fluconazole single-drug-resistant model. The candida auricular is one of the following strains: candida auricular (Candida auris) is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 23787; candida auriculata (Candida auris) is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 23788; candida auricular (Candida auris) is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 23789. The invention eliminates the risk of multiple drug resistance, can research the change rules of genes, proteins, signal channels and the like in the pressure induction process of the fluconazole medicament, and has great significance for the research and development of related medicaments.

Description

Candida auriculata capable of being used for constructing fluconazole single-drug-resistant model and application thereof in construction of fluconazole single-drug-resistant model
Technical Field
The invention relates to candida auricula for constructing a fluconazole single-drug-resistant model and application thereof in constructing the fluconazole single-drug-resistant model.
Background
Candida auricula was first reported in 2009 from Japan (Satoh, K.; Makimura, K.; Hasumi, Y.; Nishiyama, Y.; Uchida, K.; Yamaguchi, H. Candida auris sp. nov., a novel ascorbic acid obtained from the external bacterial company of an antibiotic in a Japanese national fungus family. Microbiol. Immunol.2009, 53, 41-44, strain No. DSM21092 CBS 10913. JCM15448, clean II, east Asia Branch), and was highly viable, able to survive for a long period of time, and very prone to nosocomial infections. Candida auricular is currently believed to have five major branches including the south Asia clade (clade I), the east Asia clade (clade II), the south Africa clade (clade III), the south American clade (clade IV) and the Iran clade (clade V).
Compared with antibiotics, the variety of antifungal drugs is very limited, and fewer safe and effective drugs are available for systemic candida infection. The clinical isolates of the Candida auricula have been reported to have Drug resistance to various antifungal drugs in many countries, the Drug resistant varieties are not limited to triazole drugs, the Candida auricula can have Drug resistance to various antifungal drugs such as azole antifungal drugs, fluconazole, polyene drugs (such as amphotericin B), echinocandin drugs (such as caspofungin), and the like, and the Candida auricula has a phenomenon of multiple Drug resistance, and the Candida auricula is an unprecedented super bacterium similar to staphylococcus aureus (MRSA) resistant to various antibiotics, so the Candida auricula is called as 'super pathogen' (Sarma, S.; Uppaply hyy, S. Current sexual on organism, Diagnosis and Drug resistance in Candida aureus. infection Drug resistance 2017,10, 155-. A overseas student performed meta analysis on 742 Candida albicans in total from 16 countries (including Japan and Korea in east Asia) in five continents, found that 44.29% were resistant to fluconazole, 15.46% were resistant to amphotericin B, 12.67% were resistant to voriconazole, 3.48% were resistant to caspofungin, and even the new triazole antifungal drug, isaconazole, had a resistance rate of 1.53% (Osei, S.J. Candida aureus: A systematic review and meta-analysis of current updates on an embedding multi-drug pathway. biologin 2018, 7, yo 578). The American CDC detects 54 strains of Candida albicans from 5 different countries, and finds that 93 percent of the Candida albicans is resistant to fluconazole, 35 percent of the Candida albicans is resistant to amphotericin B and 7 percent of the Candida albicans is resistant to echinocandin, and the amphotericin B is quite rare in clinic; in addition, 41% of antifungal drug resistance (MDR) to two classes, 4% of XDR to all three classes of currently used antifungal drugs (azoles, polyenes and echinocandins) in clinical use (Lockhart, S.R.; Etienne, K.A.; Vallabhaneni, S.J.; Farooqi, J.; Chowdhary, A.; Goven der, N.P.; Colombo, A.L.; Calvo, B.; Cuomo, C.A.; Desjardinins, C.A.et al.Simultaneous embryo of Multi-Resistant Candida, Inc. 3 filled by fiber-Genome and treatment of Candida infection clinical infection, clinical diagnosis, III, 8.140. A.8.8. A. A.7. 8. A. of the clinical interest in Candida.
The construction of in vitro drug resistance models is an important way for researching drug resistance mechanisms. In 2020, Jeffrey M.Rybak et al studied the relation between TAC1B mutation and Candida auricular fluconazole resistance, Candida auricular AR0387(B8441, clade I, south Asia clade) was directly inoculated into YPD medium +8mg/L or 32mg/L Fluconazole (FLU), then cultured with RPMI medium plate +8mg/L or 32mg/L Fluconazole (FLU), after picking up the single colony, inoculated into YPD medium +64mg/L or 256mg/L Fluconazole (FLU), then cultured with RPMI medium plate +64mg/L or 256mg/L Fluconazole (FLU), the strain was obtained: FLU-A (MIC 8mg/L), FLU-A2(MIC 32mg/L), FLU-B (MIC 16mg/L), FLU-C (MIC 32mg/L), FLU-C2(MIC 64mg/L) (Rybak, J.M.; Munoz, J.F.; Barker, K.S.; Parker, J.E.; Equivel, B.D.; Berkow, E.L.; Lockhart, S.R.; Gade, L.; Palmer, G.E.; White, T.C.et al.Mutations in TAC1B: a Novel Genetic determination of Clinical Flucal fluidity in Candida Resistance, M.11). With reference to CDC (synergistic characterization and interpretation) standards, the Candida auricular fluconazole antifungal drug-resistant strain is a drug-resistant strain when the MIC is more than or equal to 32 mu g/mL. The document researches the correlation between the mutation of TAC1B and the drug resistance of Candida auriculata, selects the south Asian clade AR0387(B8441) of Candida auriculata for induction of fluconazole, does not carry out the correlation research on the drug resistance stability of the induced strain, and does not research whether the strain is only resistant to fluconazole (does not exclude whether the strain has the risk of multiple drug resistance).
In 2018, the first clinical case of Candida albicans infection was reported domestically, and this strain (BJCA001) was sensitive to all clinically used antifungal drugs, no drug resistance characteristics were found (Table 1), and it was relatively closely related to isolates in countries such as India and Pakistan (Wang, X.; Bing, J.; Zheng, Q.; Zhang, F.; Liu, J.; Yue, H.; Tao, L.; Du H.; Wang, Y.; Wang, H.et al. the first isolate of Candida aura in China: clinical and biological assays. Emerg Microbes infections 2018, 7, 93). At present, 18 cases of domestic confirmed infection cases exist, the research on candida auricula still belongs to the initial stage, the related research on the east Asia Branch in candida auricula induced drug resistance is not seen at home and abroad, and the report on the construction of an in vitro induction model of the candida auricula only on the stable drug resistance of fluconazole single drug is not seen at home and abroad.
Table 1:
candida auricula (Candida auris) BJCA001 strain antifungal susceptibility test
Figure BDA0003423923280000021
Note: MIC (minimum inhibitory concentration) (μ g/mL)
Disclosure of Invention
The invention aims to provide candida auricula for constructing a fluconazole single-drug resistance model and application thereof in constructing the fluconazole single-drug resistance model.
The candida auricula system for constructing The fluconazole single-drug resistance model adopts a candida auricula-east Asia (CBS10913) strain deposited by royal Institute of technology and science of The Netherlands (The Westerdijk Fungal Biodiversity Institute, Westerdijk Institute of Fungal Biodiversity) [ The strain is also deposited in The medical Fungal center of The Collection of pathogenic microorganisms (strains of Vibrio) of China medical academy of sciences, and The strain is deposited with a deposit number of CAMS-CCPM-D50822 ], and is obtained by inducing The strain by a fluconazole concentration increasing method, and specifically one of The following strains:
candida auricular (Candida auris) CAMS-CCPM-D51239 (China general microbiological culture Collection center) which has been preserved in China general microbiological culture Collection center (CGMCC) No. 3 of No. 1 Hospital of West Lu, North Chen, China, in Beijing Chaoyang, 11.12 days in 2021, with the preservation number of CGMCC No. 23787.
Sequencing the Candida auricularia (Candida auris) CAMS-CCPM-D51239 (fluconazole MIC) 50 32. mu.g/mL) of the ITS1/ITS4 fragment sequence was as follows:
GTCGTAACAAGGtTTcCGTAgGTGaACCTGCGGAAGGATCATTATTGATATTTTGCATACACACT GATTTGGATTTTAAAACTAACCCAACGTTAAGTTCAACTAAACTATAAAGAAAACTTTCAACAACG GATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAGTATGACTTGCAGACG TGAATCATCGAATCTTTGAACGCACATTGCGCCTTGGGGTATTCCCCAAGGCATGCCTGTTTGAGC GTGATGTCTTCTCACCAATCTTCGCGGTGGCGTTGCATTCACAAAATTACAGCTTGCACGAAAAAA ATCTACGCTTTTTTTTCGTTTTGTTGTCGCCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCAT A。
the NL1/NL4 fragment sequence is as follows:
AGGGATTGCCTCAGTAACGGCGAGTGaAGCGGCAAGAGCTCAACTTTGGAATCGCTCCGGCG AGTTGTAGTCTGGAGGTGGCCACCACGAGGTGTTCTAGCAGCAGGCAAGTCCTTTGGAACAAGGC GCCAGCGAGGGTGACAGCCCCGTACCTGCTTTTGCTAGTGCTTCCTGTGGCCCACCGACGAGTCG AGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCGAGAGA CCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGTA CGTGAAATTGTTGAAAGGGAAGGGCTTGCACCCAGACACGGTTTCGGCCGGGCCAGCATCAAGTA GAACGGGGTTAAAAGACCTGGGGAATGTAGCTACCTCTTGGTAGTGTTATAGCCCTTGGGTGATGA CCCCTGTTTTGCTTGAGGACAGCGGTCTCTAGGATGCTGGCGCAATGGTTGC
candida auricula (Candida auris) CAMS-CCPM-D51240 (CGMCC No. 23788) is preserved in China general microbiological culture Collection center (CGMCC No. 3) located in Xilu No. 1 Hospital, Beijing, Chaoyang, China for 11 months and 12 days in 2021.
Sequencing the Candida auricularia (Candida auris) CAMS-CCPM-D51240 (fluconazole MIC) 50 64 ug/mL) the ITS1/ITS4 fragment sequence was as follows:
GAAGTAAAAGTCGTAACAAGGTTTCcGTAGgTGAaCCTGCGGaAGGATCATTATTGATATTTTGC ATACACACTGATTTGGATTTTAAAACTAACCCAACGTTAAGTTCAACTAAACTATAAAGAAAACTTT CAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAGTATGACT TGCAGACGTGAATCATCGAATCTTTGAACGCACATTGCGCCTTGGGGTATTCCCCAAGGCATGCCT GTTTGAGCGTGATGTCTTCTCACCAATCTTCGCGGTGGCGTTGCATTCACAAAATTACAGCTTGCA CGAAAAAAATCTACGCTTTTTTTTCGTTTTGTTGTCGCCTCAAATCAGGTAGgACtACCCGCtGAACT TAAGCATATCA。
the NL1/NL4 fragment sequence is as follows:
CCAACAGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAAGAGCTCAACTTTGGAATCGCTC CGGCGAGTTGTAGTCTGGAGGTGGCCACCACGAGGTGTTCTAGCAGCAGGCAAGTCCTTTGGAAC AAGGCGCCAGCGAGGGTGACAGCCCCGTACCTGCTTTTGCTAGTGCTTCCTGTGGCCCACCGACG AGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCG AGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAA CAGTACGTGAAATTGTTGAAAGGGAAGGGCTTGCACCCAGACACGGTTTCGGCCGGGCCAGCATC AAGTAGAACGGGGTTAAAAGACCTGGGGAATGTAGCTACCTCTTGGTAGTGTTATAGCCCTTGGGT GATGACCCCTGTTTTGC。
candida auricular (Candida auris) CAMS-CCPM-D51241 (C), which has been deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms No. 3, North Chen Xilu No. 1, located in Beijing Chaoyang, China, 11.12 days in 2021, with the collection number of CGMCC No. 23789.
Sequencing the Candida auricularia (Candida auris) CAMS-CCPM-D51241 (fluconazole MIC) 50 128. mu.g/mL) of the ITS1/ITS4 fragment has the following sequence:
GGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATTGATATTT TGCATACACACTGATTTGGATTTTAAAACTAACCCAACGTTAAGTTCAACTAAACTATAAAGAAAA CTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAGTAT GACTTGCAGACGTGAATCATCGAATCTTTGAACGCACATTGCGCCTTGGGGTATTCCCCAAGGCAT GCCTGTTTGAGCGTGATGTCTTCTCACCAATCTTCGCGGTGGCGTTGCATTCACAAAATTACAGCTT GCACGAAAAAAATCTACGCTTTTTTTTCGTTTTGTTGTCGCCTCAAATCA。
the NL1/NL4 fragment sequence is as follows:
AACAGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAAGAGCTCAACTTTGGAATCGCTCCG GCGAGTTGTAGTCTGGAGGTGGCCACCACGAGGTGTTCTAGCAGCAGGCAAGTCCTTTGGAACAA GGCGCCAGCGAGGGTGACAGCCCCGTACCTGCTTTTGCTAGTGCTTCCTGTGGCCCACCGACGAG TCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCGAG AGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACA GTACGTGAAATTGTTGAAAGGGAAGGGCTTGCACCCAGACACGGTTTCGGCCGGGCCAGCATCAA GTAGAACGGGGTTAAAAGACCTGGGGAATGTAGCTACCTCTTGGTAGTGTTATAGCCCTTGGGTGA TGACCCCTGTTTTGCTTGAGGACAGCGGTCTCTA。
according to the existing research, whether the candida auricula is sensitive or not to antifungal drugs is generally referred to CDC (synergistic characterization and interpretation) standard, and when the candida auricula is used for determining that the MIC is more than or equal to 32 mu g/mL, the candida auricula is a fluconazole drug-resistant strain; when the MIC of amphotericin B is more than or equal to 2 mug/mL, the strain is amphotericin B drug-resistant strain; when the MIC of the anidulafungin is more than or equal to 4 mu g/mL, the anidulafungin is a drug-resistant strain; when the MIC of caspofungin is more than or equal to 2 mug/mL, the caspofungin is a caspofungin drug-resistant strain; when the MIC of the micafungin is more than or equal to 4 mu g/mL, the micafungin is a micafungin drug-resistant strain. Although there are no approved standards for the judgment of antifungal resistant strains of Candida albicans 5-fluorocytosine, voriconazole, posaconazole, itraconazole in the industry at present, the document EUCAST and CLSI compares MICs values of 8 antifungal compounds measured by the Candida albicans microdilution method with preliminary epidemiological cut-off values (Arenrup, M.C., Prakash, A., Meletiadis, J., Sharma, C., & Chowdhary, A. (2017), Comparison of Micropositions of EUCAST and CLSI reference microbiological compositions of intrinsic antibacterial compositions for Candida auricular and assisted pharmaceutical consistency focus, approximate values of antifungal Agents and chemical strains, 61, 00485-17) discloses other important drug resistant strains for which the judgment of antifungal resistant strains of Candida albicans and fluconazole is not sensitive, except for 1. mu. fluconazole, the reference is made for the identification of other drugs, except for the reference of miconazole.
In view of the common multiple drug resistance (multiple drug resistance at the same time) of candida auricula, a single drug resistance model of candida auricula fluconazole can be obtained only after the influence of other drug resistance which is commonly used clinically is eliminated.
Based on the above existing research, the applicant performed the following series of experiments on the above 3 strains of candida auricula, and determined that the 3 strains of candida auricula can be used as a single drug resistance model of candida auricula fluconazole:
1. and C, candida auricula single drug resistance experiment.
According to the drug sensitivity determination standard of CISL M27-E4, MIC is determined by microdilution method by using clinical common drugs (5-flucytosine/5-FC, micafungin/MFG, caspofungin/CAS, voriconazole/VRC, posaconazole/POS, amphotericin B/AMB, itraconazole/ITC, anidulafungin/AFG and the like) 50 Value (MIC) 50 Subscript values in the values refer to lethality, i.e., MIC 50 Values refer to MIC values at 50% lethality), and with reference to CDC (synergistic Suscientific Testing and interpretation) standards, a fluconazole resistant strain is obtained when the MIC of Candida auricular is determined to be more than or equal to 32 mug/mL; when the MIC of the amphotericin B is more than or equal to 2 mug/mL, the amphotericin B is a drug-resistant strain; when the MIC of anidulafungin is more than or equal to 4 mu g/mL, the anidulafungin is a drug-resistant strain; when the MIC of the caspofungin is more than or equal to 2 mug/mL, the caspofungin is a caspofungin drug-resistant strain; when the MIC of the micafungin is more than or equal to 4 mu g/mL, the micafungin is a micafungin drug-resistant strain. Antifungal drug-resistant strains for candida auriculata 5-flucytosine, voriconazole, posaconazole and itraconazole in industriesJudgment, currently mainly referred to in the literature (arendirup, m.c., Prakash, a., meletidis, j., Sharma, c.,&chowdhary, A. (2017), comprehensive of EUCAST and CLSI reference microbiological diagnosis of electron anti-negative compounds for Candida auricular and assisted intrinsic experimental focus values, anti-microbial Agents and chemotherapeutics, 61, e 00485-17), which is considered insensitive when MICs of azole drugs other than fluconazole (voriconazole, posaconazole, itraconazole) are > 1. mu.g/mL.
2. And (3) testing the drug-resistant stability of the drug-resistant filial generation to fluconazole:
the 3 candida auricula strains related by the invention are subcultured on a YPD solid culture medium (a flat plate or a slant), and after the candida auricula strains grow out, the YPD solid culture medium is used for activating, and after the candida auricula strains grow out, the strains are subcultured for 1 time in 24-48 hours and about 10 times, and fluconazole drug-resistant progeny is obtained. Determination of the MIC of the Strain with reference to the CLSI Standard M27-E4 protocol 50 Repeating the whole process for 2-3 times, and taking the value with high occurrence frequency of each strain as the final MIC 50 The value is obtained.
3. Growth curves before and after drug treatment:
dynamic monitoring of microbial growth using the Denmark BioSense (oCelloScope) TM ) And (3) measuring the growth curves of the strains before and after the treatment of the fluconazole, analyzing data through a BCANormalized or SESANormalized or TANormalized algorithm carried by software, and comparing the influence of the drug treatment on the growth condition.
4. And (3) identifying the strain by mass spectrum:
the 3 strains to be referred to in the invention are 1-5 multiplied by 10 6 CFU·mL -1 Inoculating to YPD solid culture medium slant at a concentration, and culturing at 28-35 deg.C in incubator for 24-72h to grow flora. Selecting bacteria, coating on target plate, adding 1 μ L70% formic acid biological safety cabinet, air drying or metal bath at 50 deg.C for 3-5min, adding 1 μ L matrix solution (HCCA, hexanenitrile, and trifluoroacetic acid mixed solution), drying, and detecting with machine (AUTOF MS1000 mass spectrometer).
5. The strain is photographed and observed under a microscope.
The invention has the following advantages and progresses:
the method is characterized in that only a few strains of candida auricula (south Asia Branch) with drug resistance induced by fluconazole in vitro are subjected to relevant research at home and abroad, but relevant research on drug resistance stability of the induced strains is not carried out, and whether the strains only have drug resistance to fluconazole (whether the risk of multiple drug resistance is excluded) is not researched, so that 3 strains with different degrees of drug resistance to fluconazole are obtained by adopting the east Asia Branch (CBS10913) of the candida auricula through induction of a fluconazole concentration increasing method, and the risk of multiple drug resistance is excluded through experiments, so that a candida auricula-fluconazole single drug resistance model is obtained;
the candida auriculata single-drug resistance model comprises 3 strains with increased MIC value gradient, can research the change rules of genes, proteins, signal paths and the like in the pressure induction process of fluconazole drugs, finds key factors and corresponding mechanisms of the candida auriculata sensitive and resistant to the fluconazole, and lays the foundation for clinical research;
the Candida auricula-fluconazole single-drug resistance model eliminates the risk of multiple drug resistance, key factors and corresponding mechanisms found in subsequent research are related to single-drug resistance of fluconazole, interference of other drug approaches is eliminated, and the Candida auricula-fluconazole single-drug resistance model has great significance for research and development of related drugs.
Drawings
FIG. 1 is a graph of growth of each strain before and after 64. mu.g/mL fluconazole treatment using the BioSense microorganism growth dynamics monitoring system (TANormalized algorithm), wherein:
FIG. 1A used non-medicated 1640 medium (A1-A5), and FIG. 1B used 1640 medium supplemented with 64. mu.g/mL fluconazole (H1-H5).
In fig. 1:
a1, H1 is the control strain CBS10913 (CAMS-CCPM-D50822, fluconazole MIC) 50 1 μ g/mL);
a2, H2 is Candida auriculata (Candida auris) CAMS-CCPM-D51239 (CGMCC No.23787, Fluconazole MIC) 50 32 μ g/mL);
a3, H3 is Candida auricularia CAMS-CCPM-D51240 (CGMCC No.23788, fluconazole MIC) 50 64 μ g/mL);
a4, H4 is Candida auriculata (Candida auris) CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole M)IC 50 128 μ g/mL);
a5, H5 is the control strain CBS12372 (CAMS-CCPM-D50825, fluconazole MIC) 50 64 μ g/mL).
FIG. 2 is a graph of growth of each strain before and after treatment with fluconazole at different concentrations, as observed using the BioSense microbial growth dynamics monitoring system (TANormalized algorithm), in which:
A1-H1 in FIG. 2(1) is control strain CBS10913 (CAMS-CCPM-D50822, fluconazole MIC) 50 1 μ g/mL) growth curves before and after treatment with fluconazole of different concentrations;
A2-H2 in FIG. 2(2) is Candida auricular (Candida auris) CAMS-CCPM-D51239 (CGMCC No.23787, Fluconazole MIC) 50 32 μ g/mL) before and after treatment with fluconazole of different concentrations;
A3-H3 in FIG. 2(3) is Candida auriculata (Candida auris) CAMS-CCPM-D51240 (CGMCC No.23788, Fluconazole MIC) 50 64 μ g/mL) before and after treatment with fluconazole of different concentration;
A4-H4 in FIG. 2(4) is Candida auriculata (Candida auris) CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole MIC) 50 128 μ g/mL) growth curves before and after treatment with fluconazole of different concentrations;
A5-H5 in FIG. 2(5) is control strain CBS12372 (CAMS-CCPM-D50825, fluconazole MIC) 50 64 μ g/mL) growth curves before and after treatment with different concentrations of fluconazole.
Fig. 3 is a mass spectrum of each strain, in which:
FIG. 3(1) is a control strain CBS10913 (CAMS-CCPM-D50822, fluconazole MIC) 50 1. mu.g/mL) mass spectrum
FIG. 3(2) is a diagram of Candida auricular (Candida auris) CAMS-CCPM-D51239 (CGMCC No.23787, Fluconazole MIC) 50 32. mu.g/mL) mass spectrum
FIG. 3(3) shows Candida auricula (Candida auris) CAMS-CCPM-D51240 (CGMCC No.23788, Fluconazole MIC) 50 64 μ g/mL) mass spectrum
FIG. 3(4) shows Candida utilis strain 3 (Candida auris) CAMS-CCPM-D51241 (CGMCC No.23789, Fluconazole MIC) 50 128 μ g/mL) mass spectrum
FIG. 3(5) is a control strain CBS12372 (CAMS-CCPM-D50825, MIC) 50 64 μ g/mL) mass spectrum
FIG. 4 microscopic morphology photograph of each strain, wherein:
FIG. 4(1) is a control strain CBS10913 (CAMS-CCPM-D50822, fluconazole MIC) 50 1 μ g/mL) morphological photograph under microscope;
FIG. 4(2) shows Candida auricular (Candida auris) CAMS-CCPM-D51239 (CGMCC No.23787, Fluconazole MIC) 50 32 μ g/mL) morphological photograph under microscope;
FIG. 4(3) shows Candida auricula (Candida auris) CAMS-CCPM-D51240 (CGMCC No.23788, Fluconazole MIC) 50 64 μ g/mL) of the morphological photograph under a microscope;
FIG. 4(4) is Candida auriculata (Candida auris) CAMS-CCPM-D51241 (CGMCC No.23789, Fluconazole MIC) 50 128 μ g/mL) microscopic morphology;
FIG. 4(5) is a control strain CBS12372 (CAMS-CCPM-D50825, fluconazole MIC) 50 64. mu.g/mL) of the morphology under a microscope.
Detailed Description
1. Determination of a fluconazole single-drug resistance model:
in order to eliminate the multiple drug resistance condition commonly existing in candida auricula, for 3 candida auricula strains and a control strain related to the invention, clinical common drugs (5-flucytosine, micafungin, caspofungin, voriconazole, posaconazole, amphotericin B, itraconazole and anidulafungin) are adopted to measure MIC 50 The values, measurements are given in table 2 below:
table 2:
candida auricularia antifungal susceptibility test
Figure BDA0003423923280000091
Table 2 the test data show that:
1) reference is made to the CDC (anti theoretical surgery Testing and interpretation) standard, to which the present application relates3 strains of Candida auriculata (Candida auris) CAMS-CCPM-D51239 (CGMCC No.23787, Fluconazole MIC) 50 32 mug/mL), Candida auricular (Candida auris) CAMS-CCPM-D51240 (CGMCC No.23788, Fluconazole MIC) 50 64 mu g/mL), Candida auricular (Candida auris), CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole MIC) 50 128 μ g/mL) sensitive to amphotericin B, anidulafungin, caspofungin, micafungin.
2) The 3 strains of candida auricula related to the application are all resistant to fluconazole.
3) For voriconazole, posaconazole, itraconazole, references (arengrup, m.c., Prakash, a., Meletiadis, j., Sharma, c., & Chowdhary, a. (2017), company of EUCAST and CLSI reference microbiology of electron anti-ingredient complexes for Candida and assisted surgery experimental information consumption values, anti-microbial Agents and chemotherapeutics, 61, 00485-17), it is considered insensitive when the MIC of other azole drugs (voriconazole, saperconazole, itraconazole) other than fluconazole is not less than 1 μ g/mL.
The experimental results found that the control strain fluconazole-resistant strain CBS12372(50825) was resistant to posaconazole (MIC) 50 2 μ g/mL), itraconazole (MIC) 50 1 ug/mL), while the 3 strains of Candida auriculata to which the present invention relates were insensitive to the MIC of voriconazole 50 The values are all less than or equal to 0.25 mu g/mL, and the MIC of posaconazole is 50 The values are all less than or equal to 0.5 mu g/mL, and the MIC for itraconazole 50 The values are all less than or equal to 0.5 mu g/mL,
therefore, the three candida albicans induced strains are sensitive to voriconazole, posaconazole and itraconazole.
2. And (3) testing the drug resistance stability of the drug-resistant progeny to fluconazole:
the 3 Candida auricula strains related by the invention are passaged on YPD solid inclined planes, after the strains grow out, YPD liquid culture medium is used for activation, the strains are passaged for 1 time in 24-48h and about 10 times, and the MIC of the strains is determined by referring to the CLSI standard M27-E4 scheme 50 Values, duplicate results are shown in table 3 (double-nuclear fluconazole 1, double-nuclear fluconazole 2).
Table 3:
candida auricula fluconazole susceptibility test
Figure BDA0003423923280000101
3. Growth curves before and after drug treatment.
3.1 dynamic monitoring of microbial growth Using the Danish BioSense (oCelloscope) TM ) The growth curves of the strains before and after the treatment of the fluconazole are measured, the influence of the treatment of the drug on the growth condition is compared, and the difference of the growth of the induced strains is observed from a TANormalized analysis chart.
As shown in FIG. 1(A), in 1640 medium without drug, each strain (including Candida auriculata- ③ and two control strains) can grow normally.
As shown in fig. 1(B), after treatment with 64 μ g/mL fluconazole +1640 medium, 3 strains of candida albicans (r) -c) can grow normally compared with fluconazole sensitive strain CBS10913(50822) and fluconazole resistant strain CBS12372 (50825). The growth of drug-resistant candida auricula (III) can be induced under the inhibition effect of 64 mu g/mL fluconazole.
3.2 dynamic monitoring of microbial growth by BioSense, Denmark (oCelloScope) TM ) The growth curve of the single strain after the treatment of fluconazole with different concentrations of fluconazole is determined, and the growth condition of each strain in 1640 culture medium containing fluconazole with different concentrations is analyzed by TANormalized.
Each strain was treated with 8 kinds of bacteria (group A-group H), each of which was group A: CK group, 1640 medium; group B: 1 mu g/mL of fluconazole and 1640 culture medium; group C: 2 microgram/mL fluconazole +1640 medium; group D: 4 mu g/mL fluconazole +1640 culture medium; group E: 8 mug/mL fluconazole +1640 medium; and group F: 16 mu g/mL fluconazole +1640 medium; group G: 32 mu g/mL fluconazole +1640 medium; group H: 64 ug/mL fluconazole +1640 medium.
The corresponding strain markers are: strain 1 (A1-H1): control Strain CBS10913 (CAMS-CCPM-D50822, Fluconazole MIC) 50 1 mug/mL), wherein A1-H1 is A1, B1, C1, D1, E1, F1, G1 and H1, A1 is group A strain 1, B1 is group A strain 1Group B strain 1, … … H1 is group H strain 1, the same as below; strain 2 (A2-H2): candida auricula (CAMS-CCPM-D51239) (CGMCC No.23787, Fluconazole MIC) 50 32. mu.g/mL); strain 3(a 3-H3): candida auriculae (CGMCC No.23788, Fluconazole MIC) 51240 50 64. mu.g/mL); strain 4 (A4-H4): candida auricular (CAMS-CCPM-D51241) (CGMCC No.23789, Fluconazole MIC) 50 128 μ g/mL); strain 5 (A5-H5): control Strain CBS12372 (CAMS-CCPM-D50825, Fluconazole MIC) 50 64μg/mL)。
As shown in FIG. 2, it was found that Candida utilis (CGMCC No.23787, fluconazole MIC) was found to be CAMS-CCPM-D51239 (CGMCC No. 23787) in comparison with fluconazole sensitive strain CBS10913(50822) and fluconazole resistant strain CBS12372(50825) 50 32 mug/mL), Candida auricular (CAMS-CCPM-D51240) (CGMCC No.23788, fluconazole MIC) 50 64 mu g/mL), Candida auricularis (CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole MIC) 50 128 mug/mL) can grow in fluconazole with each concentration (0-64 mug/mL), wherein Candida auriculata (CAMS-CCPM-D51241) (CGMCC No.23789, fluconazole MIC) 50 128 μ g/mL) growth was similar to fluconazole resistant strain CBS12372(50825), with good growth status in each concentration of fluconazole. The induced drug-resistant strains of fluconazole (candida auricula- ③) can grow in 0-64 mu g/mL fluconazole.
4. Carrying out mass spectrum identification on candida auriculata (I) -III): each strain was made to 1X 10 6 CFU·mL -1 Inoculating to the inclined surface of YPD solid culture medium at a concentration, and culturing at 28 deg.C in an incubator for 48h to obtain flora. Picking flora, coating a target plate, adding 1 mu L of 70% formic acid biosafety cabinet, ventilating and drying or drying in a metal bath at 50 ℃ for 5min, adding 1 mu L of matrix liquid (HCCA, hexanenitrile and trifluoroacetic acid mixed liquid), drying, and detecting on an automatic TOF MS1000 mass spectrometer, wherein the identification result shows that the candida auricular (r) -the third generation are all candida auricular, and the characteristic peaks of all strains are found to be different, and are shown in figure 3 in detail.
5. And (3) observing the strain morphology: each induced strain was observed by photographing under a microscope as shown in FIG. 4.
The experiments in this example show that Candida auricula (Candida auris) ("deposited in China Committee for culture Collection of microorganismsThe preservation number of the common microorganism center is CGMCC No. 23787; is deposited in the medical fungus center of China medical academy of sciences (CAMS-CCPM-D) of pathogenic microorganism (toxin) species preservation, and has the deposition number of CAMS-CCPM-D51239 and the MIC of fluconazole 50 32 mu g/mL, Candida auriculata (Candida auris) and China general microbiological culture center (CGMCC No. 23788); is deposited in the medical fungus center of the pathogenic microorganism (virus) species preservation center of the Chinese academy of medical sciences (CAMS-CCPM-D) with the deposition numbers of CAMS-CCPM-D51240 and the MIC of fluconazole 50 64 μ g/mL); candida auriculata (Candida aurantium) is deposited in the China general microbiological culture Collection center with the preservation number of CGMCC No. 23789; is deposited in the medical fungus center of the pathogenic microorganism (virus) species preservation center of the Chinese academy of medical sciences (CAMS-CCPM-D) with the deposition numbers of CAMS-CCPM-D51241 and the MIC of fluconazole 50 128 mu g/mL of the three strains of Candida auricula can be used alone or in combination for constructing a fluconazole single-drug resistance model.
SEQUENCE LISTING
<110> dermatology hospital of Chinese medical academy of sciences (institute of dermatology of Chinese medical academy of sciences)
<120> Candida auriculata capable of being used for constructing fluconazole single-drug resistance model and application thereof in constructing fluconazole single-drug resistance model
<130>
<140> 202111573160.1
<141> 2021-12-21
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 397
<212> DNA
<213> unknown
<400> 1
GTCGTAACAA GGtTTcCGTA gGTGaACCTG CGGAAGGATC ATTATTGATA TTTTGCATAC 60
ACACTGATTT GGATTTTAAA ACTAACCCAA CGTTAAGTTC AACTAAACTA TAAAGAAAAC 120
TTTCAACAAC GGATCTCTTG GTTCTCGCAT CGATGAAGAA CGCAGCGAAA TGCGATACGT 180
AGTATGACTT GCAGACGTGA ATCATCGAAT CTTTGAACGC ACATTGCGCC TTGGGGTATT 240
CCCCAAGGCA TGCCTGTTTG AGCGTGATGT CTTCTCACCA ATCTTCGCGG TGGCGTTGCA 300
TTCACAAAAT TACAGCTTGC ACGAAAAAAA TCTACGCTTT TTTTTCGTTT TGTTGTCGCC 360
TCAAATCAGG TAGGACTACC CGCTGAACTT AAGCATA
397
SEQUENCE LISTING
<110> dermatology hospital of Chinese medical academy of sciences (institute of dermatology of Chinese medical academy of sciences)
<120> Candida auriculata capable of being used for constructing fluconazole single-drug resistance model and application thereof in constructing fluconazole single-drug resistance model
<130>
<140> 202111573160.1
<141> 2021-12-21
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 505
<212> DNA
<213> unknown
<400> 1
AGGGATTGCC TCAGTAACGG CGAGTGaAGC GGCAAGAGCT CAACTTTGGA ATCGCTCCGG 60
CGAGTTGTAG TCTGGAGGTG GCCACCACGA GGTGTTCTAG CAGCAGGCAA GTCCTTTGGA 120
ACAAGGCGCC AGCGAGGGTG ACAGCCCCGT ACCTGCTTTT GCTAGTGCTT CCTGTGGCCC 180
ACCGACGAGT CGAGTTGTTT GGGAATGCAG CTCTAAGTGG GTGGTAAATT CCATCTAAGG 240
CTAAATATTG GCGAGAGACC GATAGCGAAC AAGTACAGTG ATGGAAAGAT GAAAAGCACT
300
TTGAAAAGAG AGTGAAACAG TACGTGAAAT TGTTGAAAGG GAAGGGCTTG CACCCAGACA 360
CGGTTTCGGC CGGGCCAGCA TCAAGTAGAA CGGGGTTAAA AGACCTGGGG AATGTAGCTA
420
CCTCTTGGTA GTGTTATAGC CCTTGGGTGA TGACCCCTGT TTTGCTTGAG GACAGCGGTC
480
TCTAGGATGC TGGCGCAATG GTTGC
505
SEQUENCE LISTING
<110> dermatology hospital of Chinese academy of medical sciences (institute of dermatology of Chinese academy of medical sciences)
<120> Candida auriculata capable of being used for constructing fluconazole single-drug resistance model and application thereof in constructing fluconazole single-drug resistance model
<130>
<140> 202111573160.1
<141> 2021-12-21
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 409
<212> DNA
<213> unknown
<400> 1
GAAGTAAAAG TCGTAACAAG GTTTCcGTAG gTGAaCCTGC GGaAGGATCA TTATTGATAT
60
TTTGCATACA CACTGATTTG GATTTTAAAA CTAACCCAAC GTTAAGTTCA ACTAAACTAT 120
AAAGAAAACT TTCAACAACG GATCTCTTGG TTCTCGCATC GATGAAGAAC GCAGCGAAAT 180
GCGATACGTA GTATGACTTG CAGACGTGAA TCATCGAATC TTTGAACGCA CATTGCGCCT 240
TGGGGTATTC CCCAAGGCAT GCCTGTTTGA GCGTGATGTC TTCTCACCAA TCTTCGCGGT 300
GGCGTTGCAT TCACAAAATT ACAGCTTGCA CGAAAAAAAT CTACGCTTTT TTTTCGTTTT 360
GTTGTCGCCT CAAATCAGGT AGgACtACCC GCtGAACTTA AGCATATCA
409
SEQUENCE LISTING
<110> dermatology hospital of Chinese medical academy of sciences (institute of dermatology of Chinese medical academy of sciences)
<120> Candida auriculata capable of being used for constructing fluconazole single-drug resistance model and application thereof in constructing fluconazole single-drug resistance model
<130>
<140> 202111573160.1
<141> 2021-12-21
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 470
<212> DNA
<213> unknown
<400> 1
CCAACAGGGA TTGCCTCAGT AACGGCGAGT GAAGCGGCAA GAGCTCAACT TTGGAATCGC 60
TCCGGCGAGT TGTAGTCTGG AGGTGGCCAC CACGAGGTGT TCTAGCAGCA GGCAAGTCCT 120
TTGGAACAAG GCGCCAGCGA GGGTGACAGC CCCGTACCTG CTTTTGCTAG TGCTTCCTGT 180
GGCCCACCGA CGAGTCGAGT TGTTTGGGAA TGCAGCTCTA AGTGGGTGGT AAATTCCATC 240
TAAGGCTAAA TATTGGCGAG AGACCGATAG CGAACAAGTA CAGTGATGGA AAGATGAAAA
300
GCACTTTGAA AAGAGAGTGA AACAGTACGT GAAATTGTTG AAAGGGAAGG GCTTGCACCC 360
AGACACGGTT TCGGCCGGGC CAGCATCAAG TAGAACGGGG TTAAAAGACC TGGGGAATGT
420
AGCTACCTCT TGGTAGTGTT ATAGCCCTTG GGTGATGACC CCTGTTTTGC
470
SEQUENCE LISTING
<110> dermatology hospital of Chinese medical academy of sciences (institute of dermatology of Chinese medical academy of sciences)
<120> Candida auriculata capable of being used for constructing fluconazole single-drug resistance model and application thereof in constructing fluconazole single-drug resistance model
<130>
<140> 202111573160.1
<141> 2021-12-21
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 398
<212> DNA
<213> unknown
<400> 1
GGAAGTAAAA GTCGTAACAA GGTTTCCGTA GGTGAACCTG CGGAAGGATC ATTATTGATA
60
TTTTGCATAC ACACTGATTT GGATTTTAAA ACTAACCCAA CGTTAAGTTC AACTAAACTA 120
TAAAGAAAAC TTTCAACAAC GGATCTCTTG GTTCTCGCAT CGATGAAGAA CGCAGCGAAA 180
TGCGATACGT AGTATGACTT GCAGACGTGA ATCATCGAAT CTTTGAACGC ACATTGCGCC 240
TTGGGGTATT CCCCAAGGCA TGCCTGTTTG AGCGTGATGT CTTCTCACCA ATCTTCGCGG 300
TGGCGTTGCA TTCACAAAAT TACAGCTTGC ACGAAAAAAA TCTACGCTTT TTTTTCGTTT 360
TGTTGTCGCC TCAAATCA
378
SEQUENCE LISTING
<110> dermatology hospital of Chinese medical academy of sciences (institute of dermatology of Chinese medical academy of sciences)
<120> Candida auriculata capable of being used for constructing fluconazole single-drug resistance model and application thereof in constructing fluconazole single-drug resistance model
<130>
<140> 202111573160.1
<141> 2021-12-21
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 487
<212> DNA
<213> unknown
<400> 1
AACAGGGATT GCCTCAGTAA CGGCGAGTGA AGCGGCAAGA GCTCAACTTT GGAATCGCTC 60
CGGCGAGTTG TAGTCTGGAG GTGGCCACCA CGAGGTGTTC TAGCAGCAGG CAAGTCCTTT 120
GGAACAAGGC GCCAGCGAGG GTGACAGCCC CGTACCTGCT TTTGCTAGTG CTTCCTGTGG 180
CCCACCGACG AGTCGAGTTG TTTGGGAATG CAGCTCTAAG TGGGTGGTAA ATTCCATCTA 240
AGGCTAAATA TTGGCGAGAG ACCGATAGCG AACAAGTACA GTGATGGAAA GATGAAAAGC
300
ACTTTGAAAA GAGAGTGAAA CAGTACGTGA AATTGTTGAA AGGGAAGGGC TTGCACCCAG 360
ACACGGTTTC GGCCGGGCCA GCATCAAGTA GAACGGGGTT AAAAGACCTG GGGAATGTAG
420
CTACCTCTTG GTAGTGTTAT AGCCCTTGGG TGATGACCCC TGTTTTGCTT GAGGACAGCG
480
GTCTCTA
487

Claims (5)

1. The candida auricula which can be used for constructing a fluconazole single-drug resistance model is one of the following strains:
candida auricular (Candida auris) CAMS-CCPM-D51239 (r), which is preserved in China general microbiological culture Collection center (CGMCC) at 11, 12 months in 2021 with the preservation number of CGMCC No. 23787;
candida auricular (Candida auris) CAMS-CCPM-D51240 (CGMCC No. 23788) is preserved in China general microbiological culture Collection center (CGMCC) at 11, 12 months in 2021;
candida auricular (Candida auris) CAMS-CCPM-D51241, deposited in China general microbiological culture Collection center on 12.11.2021 with the preservation number of CGMCC No. 23789.
2. The Candida auricular useful for constructing a model of fluconazole single drug resistance according to claim 1, wherein the Candida auricular (Candida auris) CAMS-CCPM-D51239 (fluconazole MIC) 50 32. mu.g/mL) of the ITS1/ITS4 fragment sequence was as follows:
GTCGTAACAAGGtTTcCGTAgGTGaACCTGCGGAAGGATCATTATTGATATTTTGCATACACACTGATTTGGATTTTAAAACTAACCCAACGTTAAGTTCAACTAAACTATAAAGAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAGTATGACTTGCAGACGTGAATCATCGAATCTTTGAACGCACATTGCGCCTTGGGGTATTCCCCAAGGCATGCCTGTTTGAGCGTGATGTCTTCTCACCAATCTTCGCGGTGGCGTTGCATTCACAAAATTACAGCTTGCACGAAAAAAATCTACGCTTTTTTTTCGTTTTGTTGTCGCCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATA；
the NL1/NL4 fragment sequence is as follows:
AGGGATTGCCTCAGTAACGGCGAGTGaAGCGGCAAGAGCTCAACTTTGGAATCGCTCCGGCGAGTTGTAGTCTGGAGGTGGCCACCACGAGGTGTTCTAGCAGCAGGCAAGTCCTTTGGAACAAGGCGCCAGCGAGGGTGACAGCCCCGTACCTGCTTTTGCTAGTGCTTCCTGTGGCCCACCGACGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGTACGTGAAATTGTTGAAAGGGAAGGGCTTGCACCCAGACACGGTTTCGGCCGGGCCAGCATCAAGTAGAACGGGGTTAAAAGACCTGGGGAATGTAGCTACCTCTTGGTAGTGTTATAGCCCTTGGGTGATGACCCCTGTTTTGCTTGAGGACAGCGGTCTCTAGGATGCTGGCGCAATGGTTGC。
3. the Candida auricular useful for constructing a model of fluconazole single drug resistance as claimed in claim 1, wherein said Candida auricular (Candida auris) CAMS-CCPM-D51240 (fluconazole MIC) 50 64. mu.g/mL) of the ITS1/ITS4 fragment sequence as follows:
GAAGTAAAAGTCGTAACAAGGTTTCcGTAGgTGAaCCTGCGGaAGGATCATTATTGATATTTTGCATACACACTGATTTGGATTTTAAAACTAACCCAACGTTAAGTTCAACTAAACTATAAAGAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAGTATGACTTGCAGACGTGAATCATCGAATCTTTGAACGCACATTGCGCCTTGGGGTATTCCCCAAGGCATGCCTGTTTGAGCGTGATGTCTTCTCACCAATCTTCGCGGTGGCGTTGCATTCACAAAATTACAGCTTGCACGAAAAAAATCTACGCTTTTTTTTCGTTTTGTTGTCGCCTCAAATCAGGTAGgACtACCCGCtGAACTTAAGCATATCA；
the NL1/NL4 fragment sequence is as follows:
CCAACAGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAAGAGCTCAACTTTGGAATCGCTCCGGCGAGTTGTAGTCTGGAGGTGGCCACCACGAGGTGTTCTAGCAGCAGGCAAGTCCTTTGGAACAAGGCGCCAGCGAGGGTGACAGCCCCGTACCTGCTTTTGCTAGTGCTTCCTGTGGCCCACCGACGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGTACGTGAAATTGTTGAAAGGGAAGGGCTTGCACCCAGACACGGTTTCGGCCGGGCCAGCATCAAGTAGAACGGGGTTAAAAGACCTGGGGAATGTAGCTACCTCTTGGTAGTGTTATAGCCCTTGGGTGATGACCCCTGTTTTGC。
4. the Candida auricular useful for constructing a single drug resistance model of fluconazole of claim 1, wherein the Candida auricular (Candida auris) CAMS-CCPM-D51241 (fluconazole MIC) 50 128. mu.g/mL) of the ITS1/ITS4 fragment has the following sequence:
GGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATTGATATTTTGCATACACACTGATTTGGATTTTAAAACTAACCCAACGTTAAGTTCAACTAAACTATAAAGAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAGTATGACTTGCAGACGTGAATCATCGAATCTTTGAACGCACATTGCGCCTTGGGGTATTCCCCAAGGCATGCCTGTTTGAGCGTGATGTCTTCTCACCAATCTTCGCGGTGGCGTTGCATTCACAAAATTACAGCTTGCACGAAAAAAATCTACGCTTTTTTTTCGTTTTGTTGTCGCCTCAAATCA；
the NL1/NL4 fragment sequence is as follows:
AACAGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAAGAGCTCAACTTTGGAATCGCTCCGGCGAGTTGTAGTCTGGAGGTGGCCACCACGAGGTGTTCTAGCAGCAGGCAAGTCCTTTGGAACAAGGCGCCAGCGAGGGTGACAGCCCCGTACCTGCTTTTGCTAGTGCTTCCTGTGGCCCACCGACGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGTACGTGAAATTGTTGAAAGGGAAGGGCTTGCACCCAGACACGGTTTCGGCCGGGCCAGCATCAAGTAGAACGGGGTTAAAAGACCTGGGGAATGTAGCTACCTCTTGGTAGTGTTATAGCCCTTGGGTGATGACCCCTGTTTTGCTTGAGGACAGCGGTCTCTA。
5. use of the Candida auricular of any one of claims 1 to 4 for the construction of a model of fluconazole single drug resistance.
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Citations (2)

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US20120276577A1 (en) * 2009-06-25 2012-11-01 Assistance Publique - Hopitaux De Paris Method for Determining the Susceptibility of a Cell Strain to Drugs
CN106282299A (en) * 2015-05-14 2017-01-04 北京中医药大学 A kind of method of external artificial acquisition bacterial resistance bacterial strain

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120276577A1 (en) * 2009-06-25 2012-11-01 Assistance Publique - Hopitaux De Paris Method for Determining the Susceptibility of a Cell Strain to Drugs
CN106282299A (en) * 2015-05-14 2017-01-04 北京中医药大学 A kind of method of external artificial acquisition bacterial resistance bacterial strain

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Title
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