CN115044478B - Candida otophylla capable of being used for constructing fluconazole single-drug resistance model and application of candida otophylla in construction of fluconazole single-drug resistance model - Google Patents
Candida otophylla capable of being used for constructing fluconazole single-drug resistance model and application of candida otophylla in construction of fluconazole single-drug resistance model Download PDFInfo
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- 229960004884 fluconazole Drugs 0.000 title claims abstract description 142
- RFHAOTPXVQNOHP-UHFFFAOYSA-N fluconazole Chemical compound C1=NC=NN1CC(C=1C(=CC(F)=CC=1)F)(O)CN1C=NC=N1 RFHAOTPXVQNOHP-UHFFFAOYSA-N 0.000 title claims abstract description 142
- 241000222120 Candida <Saccharomycetales> Species 0.000 title claims abstract description 114
- 206010059866 Drug resistance Diseases 0.000 title claims abstract description 47
- 238000010276 construction Methods 0.000 title claims description 5
- 241000645784 [Candida] auris Species 0.000 claims abstract description 39
- 238000009629 microbiological culture Methods 0.000 claims abstract description 13
- 238000004321 preservation Methods 0.000 claims abstract description 11
- 208000013641 Cerebrofacial arteriovenous metameric syndrome Diseases 0.000 claims description 46
- 229940079593 drug Drugs 0.000 abstract description 32
- 239000003814 drug Substances 0.000 abstract description 32
- 230000006698 induction Effects 0.000 abstract description 7
- 206010048723 Multiple-drug resistance Diseases 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 5
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- RAGOYPUPXAKGKH-XAKZXMRKSA-N posaconazole Chemical compound O=C1N([C@H]([C@H](C)O)CC)N=CN1C1=CC=C(N2CCN(CC2)C=2C=CC(OC[C@H]3C[C@@](CN4N=CN=C4)(OC3)C=3C(=CC(F)=CC=3)F)=CC=2)C=C1 RAGOYPUPXAKGKH-XAKZXMRKSA-N 0.000 description 12
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- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 11
- 229960003942 amphotericin b Drugs 0.000 description 11
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- 239000003429 antifungal agent Substances 0.000 description 9
- 108010020326 Caspofungin Proteins 0.000 description 7
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- JYIKNQVWKBUSNH-WVDDFWQHSA-N caspofungin Chemical compound C1([C@H](O)[C@@H](O)[C@H]2C(=O)N[C@H](C(=O)N3CC[C@H](O)[C@H]3C(=O)N[C@H](NCCN)[C@H](O)C[C@@H](C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N2)[C@@H](C)O)=O)NC(=O)CCCCCCCC[C@@H](C)C[C@@H](C)CC)[C@H](O)CCN)=CC=C(O)C=C1 JYIKNQVWKBUSNH-WVDDFWQHSA-N 0.000 description 7
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- 230000000843 anti-fungal effect Effects 0.000 description 6
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- JHVAMHSQVVQIOT-MFAJLEFUSA-N anidulafungin Chemical compound C1=CC(OCCCCC)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(=O)N[C@@H]2C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N[C@H](C(=O)N[C@H](C(=O)N3C[C@H](C)[C@H](O)[C@H]3C(=O)N[C@H](O)[C@H](O)C2)[C@@H](C)O)[C@H](O)[C@@H](O)C=2C=CC(O)=CC=2)[C@@H](C)O)=O)C=C1 JHVAMHSQVVQIOT-MFAJLEFUSA-N 0.000 description 5
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention relates to candida aurea which can be used for constructing a fluconazole single drug resistance model and application thereof in constructing the fluconazole single drug resistance model. The candida otophylla is one of the following strains: candida auriculata (Candida auris) (1) preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23787; candida auriculata (Candida auris) (2) preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23788; candida aurea (3) deposited in China general microbiological culture Collection center with the collection number of CGMCC No.23789. The invention eliminates the risk of multiple drug resistance, can study the change rules of genes, proteins, signal paths and the like in the pressurizing induction process of the fluconazole drug, and has great significance for the study and development of related drugs.
Description
Technical Field
The invention relates to candida aurea which can be used for constructing a fluconazole single drug resistance model and application thereof in constructing the fluconazole single drug resistance model.
Background
Candida otophylla (Candida auris) was first reported in 2009 by japan (Satoh, k.; makimura, k.; hassumi, y.; nishiyama, y.; uchida, k.; yamaguchi, h. Candida auris sp.nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hot. Microbiol. Immunol.2009, 53, 41-44, strain No. DSM21092 =cbs10913=jcm 15448, clade ii, east asia branch), and has a strong viability, and can survive for a long period of time, and is extremely susceptible to nosocomial explosive infection. The candida otophylla is considered to have five branches of south asia clade (clade I), east asia clade (clade II), south african clade (clade III), south america clade (clade IV) and iran clade (clade V).
Compared with antibiotics, the antifungal drugs have very limited varieties, and fewer safe and effective drugs are available for systemic candida infection. Clinical isolates of candida auriculata have been reported to be resistant to various antifungal agents in various countries, and resistant varieties are not limited to triazole drugs, candida auriculata can be resistant to various antifungal agents such as fluconazole, voriconazole, polyenes (such as amphotericin B), echinocandins (such as caspofungin) and the like, and multi-resistant phenomena exist, which are never seen in other candida species and are similar to super bacteria such as staphylococcus aureus (MRSA) resistant to various antibiotics, so that the candida auriculata is called "super pathogen" (Sarma, s.; upadhyay, s.current perspective on emergence, diagnosis and Drug resistance in Candida auris. Foreign scholars have performed meta analysis on 742 total strains of candida otophyllum from 16 countries of five continents (including japan and korea of east asia) and found 44.29% resistance to fluconazole, 15.46% resistance to amphotericin B, 12.67% resistance to voriconazole, 3.48% resistance to caspofungin, even though the new triazole antifungal agent isaconazole has a resistance of 1.53% (oei, s.j.candida auris: A systematic review and meta-analysis of current updates on an emerging multidrug-resistance pathway microbiology 2018,7, e 578). CDC in the united states examined candida 54 strains from 5 different countries and found 93% resistance to fluconazole, 35% resistance to amphotericin B, 7% resistance to echinocandin, whereas amphotericin B resistance was very rare in clinic; in addition, there are 41% resistance to two classes of antifungals (MDR), 4% resistance to all three classes of antifungals currently in clinical use (azoles, polyenes, and echinocandins) (Lockhart, s.r.; etiene, k.a.; vallabhanni, s.; farooqi, j.; chordhaly, a.; govender, n.p.; colonbo, a.l.; calvo, b.; cuomo, c.a.; desjardins, c.a. et al. Simultaneous Emergence of Multidrug-Resistant Candida auris on 3Continents Confirmed by Whole-Genome Sequencing and Epidemiological analyzers.clin. Infect. Dis.2017, 64, 134-140), making the clinical treatment of such infections more troublesome, leading to a high global concern over candida.
The construction of an in vitro drug resistance model is an important way for researching a drug resistance mechanism. Study of TAC1B mutation versus drug resistance of Candida otophylla fluconazole by Jeffrey M.Rybak et al abroad in 2020, candida otophylla AR0387 (B8441, clone I, south Asia clade) was inoculated directly into YPD medium +8mg/L or 32mg/L Fluconazole (FLU), followed by culture with RPMI medium plate +8mg/L or 32mg/L Fluconazole (FLU), after single colony selection, inoculated into YPD medium +64mg/L or 256mg/L Fluconazole (FLU), followed by culture with RPMI medium plate +64mg/L or 256mg/L Fluconazole (FLU) to give strains: FLU-A (MIC 8 mg/L), FLU-A2 (MIC 32 mg/L), FLU-B (MIC 16 mg/L), FLU-C (MIC 32 mg/L), FLU-C2 (MIC 64 mg/L) (Rybak, J.M.; munoz, J.F.; barker, K.S.; parker, J.E.; esquirol, B.D.; berkow, E.L.; lockhart, S.R.; gade, L.; palmer, G.E.; white, T.C. et al. rotations in TAC1B: a Novel Genetic Determinant of Clinical Fluconazole Resistance in Candida auris. MBio 2020, 11). With reference to CDC (Antifungal Susceptibility Testing and Interpretation) standard, candida otorula fluconazole antifungal drug-resistant strain is judged to be drug-resistant strain when MIC is more than or equal to 32 mug/mL. This document investigated the correlation of TAC1B mutation with drug resistance of candida otophylla fluconazole, selection of candida otophylla south asia clade AR0387 (B8441) fluconazole induction, no study was carried out on the stability of drug resistance of the induced strain, and no study was made as to whether only fluconazole was resistant (the risk of multiple resistance was not excluded).
In 2018, the first clinical case of candida otorhinosis infection was reported in China, the strain (BJCA 001) was sensitive to all the clinically commonly used antifungal drugs, the drug resistance characteristics were not found (Table 1), and the strain was closely related to isolates of India, pakistan and other countries (Wang, X.; bing, J.; zheng, Q.; zhang, F.; liu, J.; yue, H.; tao, L.; du H; wang, Y.; wang, H.et al first isolate of Candida auris in China: clinical and biological aspects Microbe information 2018,7, 93). At present, 18 cases of infection cases confirmed in China exist, the research on candida otophylla belongs to a starting stage, no research on the in vitro induction of the east and west branches of candida otophylla is found at home and abroad, and no report on the construction of an in vitro induction model of candida otophylla only for stable drug resistance of fluconazole is found at home and abroad.
Table 1:
candida otophylla (Candida auris) BJCA001 strain antifungal drug susceptibility test
Note that: MIC (minimal inhibitory concentration, minimum inhibitory concentration) (μg/mL)
Disclosure of Invention
The invention aims to provide candida auriculata capable of being used for constructing a fluconazole single-drug resistance model and application thereof in constructing the fluconazole single-drug resistance model.
The candida otophylla strain which can be used for constructing the fluconazole single drug resistance model is obtained by adopting candida otophylla east Asia branch (CBS 10913) strain (which is also preserved in the center of medical fungus division of the national academy of medical science pathogenic microorganism (toxin) collection center) and is preserved with the preservation number CAMS-CCPM-D50822) preserved by the national academy of royal art and science fungus diversity research center (The Westerdijk Fungal Biodiversity Institute, westerdijk fungus biodiversity research institute), and is obtained after induction by a fluconazole concentration increasing method, and is one of the following strains:
candida auriculata (Candida auris) CAMS-CCPM-D51239 was deposited at China general microbiological culture Collection center, with the deposit number CGMCC No.23787, at national institute of China general microbiological culture Collection center, national institute of control of North Chen West Lu No. 1, beijing, korea, 2021, 11 months and 12 days.
Sequencing the Candida otophylla (Candida auris) CAMS-CCPM-D51239 (fluconazole MIC) 50 32 μg/mL) ITS1/ITS4 fragment sequence was as follows:
GTCGTAACAAGGtTTcCGTAgGTGaACCTGCGGAAGGATCATTATTGATATTTTGCATACACACT GATTTGGATTTTAAAACTAACCCAACGTTAAGTTCAACTAAACTATAAAGAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAGTATGACTTGCAGACG TGAATCATCGAATCTTTGAACGCACATTGCGCCTTGGGGTATTCCCCAAGGCATGCCTGTTTGAGCGTGATGTCTTCTCACCAATCTTCGCGGTGGCGTTGCATTCACAAAATTACAGCTTGCACGAAAAAA ATCTACGCTTTTTTTTCGTTTTGTTGTCGCCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCAT A。
the NL1/NL4 fragment sequence is as follows:
AGGGATTGCCTCAGTAACGGCGAGTGaAGCGGCAAGAGCTCAACTTTGGAATCGCTCCGGCG AGTTGTAGTCTGGAGGTGGCCACCACGAGGTGTTCTAGCAGCAGGCAAGTCCTTTGGAACAAGGCGCCAGCGAGGGTGACAGCCCCGTACCTGCTTTTGCTAGTGCTTCCTGTGGCCCACCGACGAGTCG AGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCGAGAGA CCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGTACGTGAAATTGTTGAAAGGGAAGGGCTTGCACCCAGACACGGTTTCGGCCGGGCCAGCATCAAGTA GAACGGGGTTAAAAGACCTGGGGAATGTAGCTACCTCTTGGTAGTGTTATAGCCCTTGGGTGATGACCCCTGTTTTGCTTGAGGACAGCGGTCTCTAGGATGCTGGCGCAATGGTTGC
candida auriculata (Candida auris) CAMS-CCPM-D51240, which was preserved in China general microbiological culture Collection center, china No. 3, with a preservation number of CGMCC No.23788, in North Chen West Lu No. 1, the region of Beijing, in 2021, 11 months, 12 days.
Sequencing the Candida otophylla (Candida auris) CAMS-CCPM-D51240 [. Sup.2 ] (fluconazole MIC) 50 64μg/mThe ITS1/ITS4 fragment sequence of L) is as follows:
GAAGTAAAAGTCGTAACAAGGTTTCcGTAGgTGAaCCTGCGGaAGGATCATTATTGATATTTTGC ATACACACTGATTTGGATTTTAAAACTAACCCAACGTTAAGTTCAACTAAACTATAAAGAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAGTATGACT TGCAGACGTGAATCATCGAATCTTTGAACGCACATTGCGCCTTGGGGTATTCCCCAAGGCATGCCTGTTTGAGCGTGATGTCTTCTCACCAATCTTCGCGGTGGCGTTGCATTCACAAAATTACAGCTTGCA CGAAAAAAATCTACGCTTTTTTTTCGTTTTGTTGTCGCCTCAAATCAGGTAGgACtACCCGCtGAACTTAAGCATATCA。
the NL1/NL4 fragment sequence is as follows:
CCAACAGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAAGAGCTCAACTTTGGAATCGCTC CGGCGAGTTGTAGTCTGGAGGTGGCCACCACGAGGTGTTCTAGCAGCAGGCAAGTCCTTTGGAACAAGGCGCCAGCGAGGGTGACAGCCCCGTACCTGCTTTTGCTAGTGCTTCCTGTGGCCCACCGACG AGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAA CAGTACGTGAAATTGTTGAAAGGGAAGGGCTTGCACCCAGACACGGTTTCGGCCGGGCCAGCATCAAGTAGAACGGGGTTAAAAGACCTGGGGAATGTAGCTACCTCTTGGTAGTGTTATAGCCCTTGGGT GATGACCCCTGTTTTGC。
candida auriculata (Candida auris) CAMS-CCPM-D51241, which has been preserved in China general microbiological culture Collection center, china No. 3, with a preservation number of CGMCC No.23789, in North Chen West Lu No. 1, the region of Beijing, in 2021, month 11, and 12 days.
Sequencing the Candida otophylla (Candida auris) CAMS-CCPM-D51241 [. Sup.3 ] (fluconazole MIC) 50 128 μg/mL) of the ITS1/ITS4 fragment sequence was as follows:
GGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATTGATATTT TGCATACACACTGATTTGGATTTTAAAACTAACCCAACGTTAAGTTCAACTAAACTATAAAGAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAGTAT GACTTGCAGACGTGAATCATCGAATCTTTGAACGCACATTGCGCCTTGGGGTATTCCCCAAGGCATGCCTGTTTGAGCGTGATGTCTTCTCACCAATCTTCGCGGTGGCGTTGCATTCACAAAATTACAGCTT GCACGAAAAAAATCTACGCTTTTTTTTCGTTTTGTTGTCGCCTCAAATCA。
the NL1/NL4 fragment sequence is as follows:
AACAGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAAGAGCTCAACTTTGGAATCGCTCCG GCGAGTTGTAGTCTGGAGGTGGCCACCACGAGGTGTTCTAGCAGCAGGCAAGTCCTTTGGAACAAGGCGCCAGCGAGGGTGACAGCCCCGTACCTGCTTTTGCTAGTGCTTCCTGTGGCCCACCGACGAG TCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCGAG AGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGTACGTGAAATTGTTGAAAGGGAAGGGCTTGCACCCAGACACGGTTTCGGCCGGGCCAGCATCAA GTAGAACGGGGTTAAAAGACCTGGGGAATGTAGCTACCTCTTGGTAGTGTTATAGCCCTTGGGTGATGACCCCTGTTTTGCTTGAGGACAGCGGTCTCTA。
according to the prior study, whether candida otophylla is sensitive or not to the antifungal drug is usually referred to CDC (Antifungal Susceptibility Testing and Interpretation) standard, and fluconazole resistant strain is adopted when MIC is more than or equal to 32 mug/mL; amphotericin B is a drug-resistant amphotericin B strain when MIC is more than or equal to 2 mug/mL; anidulafungin resistant strain when MIC is more than or equal to 4 mug/mL; caspofungin resistant strains are used when the MIC is more than or equal to 2 mug/mL; the micafungin resistant strain is used when the MIC is more than or equal to 4 mug/mL. Although there is currently no well-established standard in the industry for judging antifungal drug-resistant strains of candida auriculata 5-fluorocytosine, voriconazole, posaconazole and itraconazole, the drug-resistant strain judgment standards disclosed in the literature "EUCAST and CLSI compare 8 antifungal compounds with the MICs values and preliminary epidemiological thresholds measured by candida microdilution (Arendrup, m.c.), prakesh, a., meletiadis, j., sharma, c., chordharry, a. (2017), comparison of EUCAST and CLSI reference microdilution MICs of eight antifungal compounds for Candida auris and associated tentative epidemiological cutoff values.Antimicobal Agents and Chemotherapy,61, e 00485-17) and other azole drugs (voriconazole, posaconazole, itraconazole) except fluconazole are considered insensitive when MIC is not less than 1 μg/mL according to the literature.
In view of the fact that candida otophylla commonly has multiple drug resistance (simultaneously resistant to multiple drugs), the candida otophylla fluconazole single drug resistance model can be obtained only after the influence of the drug resistance of other drugs commonly used in clinic is eliminated.
Based on the prior studies, the applicant carried out the following series of experiments on the 3 candida otophylla, and confirms that the 3 candida otophylla can be used as a candida otophylla fluconazole single drug resistance model:
1. and (3) single drug resistance experiment of candida otophylla.
MIC was measured by microdilution using clinically usual drugs (5-fluorocytosine/5-FC, micafungin/MFG, caspofungin/CAS, voriconazole/VRC, posaconazole/POS, amphotericin B/AMB, itraconazole/ITC, anidulafungin/AFG, etc.) according to CISL M27-E4 drug sensitivity measurement standard 50 Value (MIC) 50 The subscript in the values refers to the mortality, i.e., MIC 50 MIC value at 50% mortality), fluconazole resistant strain with MIC equal to or greater than 32 μg/mL measured by candida otophylla fluconazole with reference to CDC (Antifungal Susceptibility Testing and Interpretation) standard; amphotericin B is a drug-resistant amphotericin B strain when MIC is more than or equal to 2 mug/mL; anidulafungin resistant strain when MIC is more than or equal to 4 mug/mL; caspofungin resistant strains are used when the MIC is more than or equal to 2 mug/mL; the micafungin resistant strain is used when the MIC is more than or equal to 4 mug/mL. While the industry judges antifungal drug-resistant strains of candida otophylla 5-fluorocytosine, voriconazole, posaconazole, itraconazole, and the like, currently mainly references (Arendrup, m.c., prakesh, a., meliteadis, j., sharma, c.,&choddory, a. (2017) Comparison of EUCAST and CLSI reference microdilution MICs of eight antifungal compounds for Candida auris and associated tentative epidemiological cutoff values.Antimicrobial Agents and Chemotherapy,61, e 00485-17) which considers that other azole drugs (voriconazole, posaconazole, itraconazole) than fluconazole have a MIC of not less than 1 μg/mL are not considered sensitive.
2. Drug resistance offspring resistance stability test against fluconazole:
the 3 candida otophylla strains related to the invention are passaged on YPD solid culture medium (flat plate or inclined plane), and after the candida otophylla strains grow out, the candida otophylla strains are activated by YPD liquid culture medium, and passaged for 1 time 24-48 hours and passaged for about 10 times, so as to obtain fluconazole drug-resistant offspring. 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 collectingThe high frequency of occurrence of each strain was used as the final MIC 50 Values.
3. Growth curve before and after drug treatment:
dynamic monitoring System of microbial growth Using Denmark BioSense (subcelloscope) TM ) The growth curves of each strain before and after fluconazole treatment were determined, and the data were analyzed by the bca normalized or sesa normalized or TANormalized algorithm carried by the software to compare the effect of drug treatment on growth.
4. Identification of strain mass spectrum:
the 3 strains to be invented are 1-5×10 6 CFU·mL -1 Inoculating the strain to the inclined plane of YPD solid culture medium, and culturing in an incubator at 28-35 ℃ for 24-72h to grow flora. The flora is picked up and coated on a target plate, 1 mu L of 70% formic acid biosafety cabinet is added for ventilation drying or 50 ℃ metal bath drying for 3-5min, 1 mu L of matrix liquid (HCCA, capronitrile and trifluoroacetic acid mixed liquid) is added for drying, and then the mixture is put on an machine (AUTOF MS1000 mass spectrometer) for detection.
5. The strain was observed under a microscope with photographs.
The invention has the following advantages and advances:
only at home and abroad, carrying out related researches on several strains of fluconazole in vitro induced drug-resistant candida otophylla (south Asia branch), but not carrying out related researches on the drug resistance stability of the induced strain, and not researching whether only fluconazole is resistant (does not exclude whether multiple drug resistance exists or not), wherein the application adopts candida otophylla east Asia branch (CBS 10913), 3 strains with different drug resistance degrees on fluconazole are obtained after induction by a fluconazole concentration increasing method, and the risk of multiple drug resistance is eliminated through experiments, so that a single drug resistance model of the fluconazole of the candida otophylla is obtained;
the candida aureobasidium fluconazole single-drug resistance model comprises 3 strains with gradient increased MIC values, can study the change rules of genes, proteins, signal paths and the like in the pressurizing induction process of fluconazole, searches key factors and corresponding mechanisms of candida aureobasidium fluconazole on fluconazole sensitivity and resistance, and lays a foundation for clinical research subsequently;
the candida aureobasidium fluconazole single drug resistance model eliminates the risk of multiple drug resistance, and key factors and corresponding mechanisms found in subsequent researches are related to fluconazole single drug resistance, so that the interference of other drug paths is eliminated, and the candida aureobasidium fluconazole single drug resistance model has great significance for related drug research and development.
Drawings
FIG. 1 is a graph of growth curves of strains before and after treatment with 64. Mu.g/mL fluconazole, as observed using a BioSense microorganism growth dynamics monitoring system (TAnormal algorithm), wherein:
FIG. 1A uses 1640 medium (A1-A5) without drug and FIG. 1B uses 1640 medium (H1-H5) with 64 μg/mL fluconazole.
In fig. 1:
a1 H1 is a control strain CBS10913 (CAMS-CCPM-D50822, fluconazole MIC) 50 1 μg/mL);
a2 H2 is Candida aurea (Candida auris) CAMS-CCPM-D51239 (CGMCC No.23787, fluconazole MIC) 50 32 μg/mL);
a3 H3 is Candida aurea (Candida auris) CAMS-CCPM-D51240 [. Sup.2 ] (CGMCC No.23788, fluconazole MIC) 50 64 μg/mL);
a4 H4 is Candida aurea (Candida auris) CAMS-CCPM-D51241 [. Sup.3 ] (CGMCC No.23789, fluconazole MIC) 50 128 μg/mL);
a5 H5 is control strain CBS12372 (CAMS-CCPM-D50825, fluconazole MIC) 50 64 μg/mL).
FIG. 2 is a graph of growth of each strain before and after treatment with varying concentrations of fluconazole, as observed using a BioSense microbial growth dynamics monitoring system (TAnormal algorithm), wherein:
A1-H1 in FIG. 2 (1) is the control strain CBS10913 (CAMS-CCPM-D50822, fluconazole MIC) 50 1 μg/mL) of growth curves before and after treatment with fluconazole at different concentrations;
A2-H2 in FIG. 2 (2) is Candida auriculata (Candida auris) CAMS-CCPM-D51239 (CGMCC No.23787, fluconazole MIC) 50 32 μg/mL) of growth curves before and after treatment with fluconazole at different concentrations;
A3-H3 in FIG. 2 (3) is Candida aurea (CGMCC No. 23) CAMS-CCPM-D51240 # (CGMCC No. 23)788, fluconazole MIC 50 64 μg/mL) of growth curves before and after treatment with fluconazole at different concentrations;
A4-H4 in FIG. 2 (4) is Candida auriculata (Candida auris) CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole MIC) 50 128 μg/mL) of the growth curve before and after treatment with fluconazole at different concentrations;
A5-H5 in FIG. 2 (5) is control strain CBS12372 (CAMS-CCPM-D50825, fluconazole MIC) 50 64 μg/mL) of the growth curve before and after treatment with fluconazole at different concentrations.
FIG. 3 is a mass spectrum of each strain, wherein:
FIG. 3 (1) is a control strain CBS10913 (CAMS-CCPM-D50822, fluconazole MIC) 50 1. Mu.g/mL) mass spectrum
FIG. 3 (2) is Candida aurea (Candida auris) CAMS-CCPM-D51239 (CGMCC No.23787, fluconazole MIC) 50 32 μg/mL) mass spectrum
FIG. 3 (3) is Candida aurea (Candida auris) CAMS-CCPM-D51240 [. Sup.2 ] (CGMCC No.23788, fluconazole MIC) 50 64 μg/mL) mass spectrum
FIG. 3 (4) is Candida otophylla (3)7 (Candida auris) CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole MIC) 50 128 μg/mL) mass spectrum
FIG. 3 (5) is a control strain CBS12372 (CAMS-CCPM-D50825, MIC) 50 64 μg/mL) mass spectrum
Figure 4 photomicrographs of the morphology of each strain, wherein:
FIG. 4 (1) is a control strain CBS10913 (CAMS-CCPM-D50822, fluconazole MIC) 50 1 μg/mL) photomicrographs of morphology under a microscope;
FIG. 4 (2) is Candida aurea (Candida auris) CAMS-CCPM-D51239 (CGMCC No.23787, fluconazole MIC) 50 32 μg/mL) photomicrographs of morphology under a microscope;
FIG. 4 (3) is Candida aurea (Candida auris) CAMS-CCPM-D51240 [. Sup.2 ] (CGMCC No.23788, fluconazole MIC) 50 64 μg/mL) photomicrographs of morphology under a microscope;
FIG. 4 (4) is Candida aurea (Candida auris) CAMS-CCPM-D51241 [. Sup.3 ] (CGMCC No.23789, fluconazole MIC) 50 128 μg/mL) photomicrographs of morphology under a microscope；
FIG. 4 (5) is a control strain, strain CBS12372 (CAMS-CCPM-D50825, fluconazole MIC) 50 64 μg/mL) of the sample.
Detailed Description
1. Determination of fluconazole single drug resistance model:
to exclude the ubiquitous multiple drug resistance of candida otophylla, the 3 candida otophylla and the control strain related to the invention are used for measuring MIC by adopting clinical common drugs (5-fluorocytosine, micafungin, caspofungin, voriconazole, posaconazole, amphotericin B, itraconazole and anidulafungin) 50 Values, test results are shown in table 2 below:
table 2:
candida otophylla (Candida auris) antifungal drug susceptibility test
Table 2 the test data shows that:
1) Referring to CDC (Antifungal Susceptibility Testing and Interpretation) standard, 3 strains of Candida otophylla (Candida auris) referred to in this application (1) CAMS-CCPM-D51239 (CGMCC No.23787, fluconazole MIC) 50 32 μg/mL), candida otophylla (Candida auris) (2) CAMS-CCPM-D51240 (CGMCC No.23788, fluconazole MIC) 50 64 μg/mL), candida otophylla (Candida auris) (3) CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole MIC) 50 128 μg/mL) is sensitive to amphotericin B, anidulafungin, caspofungin, micafungin.
2) All 3 candida otophylla referred to in this application are resistant to fluconazole.
3) For voriconazole, posaconazole, itraconazole, references (Arendrup, m.c., prakesh, a., meletiadis, j., sharma, c., & chordhaly, a. (2017) Comparison of EUCAST and CLSI reference microdilution MICs of eight antifungal compounds for Candida auris and associated tentative epidemiological cutoff values, ankicobial Agents and Chemotherapy,61, e 00485-17), the MIC of other azole drugs (voriconazole, posaconazole, itraconazole) than fluconazole was considered insensitive to 1 μg/mL.
Experimental results show that the fluconazole resistant strain CBS12372 (50825) of the control strain is prepared against posaconazole (MIC) 50 2 μg/mL), itraconazole (MIC) 50 1. Mu.g/mL) is insensitive, whereas 3 strains of Candida otophylla according to the invention are insensitive to voriconazole MIC 50 The values are all less than or equal to 0.25 mug/mL, and the MIC of the posaconazole is improved 50 The values are all less than or equal to 0.5 mug/mL, and the MIC of the itraconazole is improved 50 The values are all less than or equal to 0.5 mug/mL,
thus, each induced strain of candida albicans, three strains, involved in the application is sensitive to voriconazole, posaconazole and itraconazole.
2. Drug resistance offspring resistance stability test against fluconazole:
the 3 candida otophylla strains related to the invention are transferred to YPD solid inclined plane, after the candida otophylla strains grow out, the candida otophylla strains are activated by YPD liquid culture medium, the candida otophylla strains are transferred for 1 time of 24-48 hours, the candida otophylla strains are transferred for about 10 times, and the MIC of the strains is determined by referring to a CLSI standard M27-E4 scheme 50 Values, repeated results are shown in Table 3 (rechecked fluconazole 1, rechecked fluconazole 2).
Table 3:
candida otophylla (Candida auris) fluconazole drug sensitive test
3. Growth curves before and after drug treatment.
3.1 dynamic monitoring System for microbial growth Using Biosense, denmark (oCelloscope) TM ) The growth curves of each strain before and after fluconazole treatment were determined, and differences in the growth of the induced strain were observed from the tanormized analysis chart compared with the effect of drug treatment on growth.
As shown in FIG. 1 (A), each strain (including Candida otophylla (1) - (3) and two control strains) grew normally in the 1640 medium without the drug.
As shown in FIG. 1 (B), after 64. Mu.g/mL of fluconazole+1640 medium treatment, 3 candida otophylla (1) - (3) can grow normally compared with fluconazole sensitive strain CBS10913 (50822) and fluconazole drug resistant strain CBS12372 (50825). It is demonstrated that drug-resistant Candida otorhinoca (1) - (3) can grow under the inhibition of 64. Mu.g/mL fluconazole.
3.2 dynamic monitoring System of microbial growth Using Biosense, denmark (oCelloscope) TM ) The growth curve of individual strains after treatment with fluconazole at different concentrations was determined and each strain was analyzed by TANormalized for growth in 1640 medium containing fluconazole at different concentrations.
8 treatments (group A-group H) were performed on each strain, and the treatments were group A: CK group, 1640 medium; group B: 1 μg/mL fluconazole+1640 medium; group C: 2 μg/mL fluconazole+1640 medium; group D: 4 μg/mL fluconazole+1640 medium; group E: 8 μg/mL fluconazole+1640 medium; group F: 16 μg/mL fluconazole+1640 medium; group G: 32 μg/mL fluconazole+1640 medium; group H: 64 μg/mL fluconazole+1640 medium.
The corresponding strains were marked as: strain 1 (A1-H1): control strain CBS10913 (CAMS-CCPM-D50822, fluconazole MIC) 50 1 μg/mL), wherein A1-H1 is A1, B1, C1, D1, E1, F1, G1, H1, A1 is group a strain 1, B1 is group B strain 1, … … H1 is group H strain 1, the same as below; strain 2 (A2-H2): candida otophylla (1) CAMS-CCPM-D51239 (CGMCC No.23787, fluconazole MIC) 50 32 μg/mL); strain 3 (A3-H3): candida otophylla (2) CAMS-CCPM-D51240 (CGMCC No.23788, fluconazole MIC) 50 64 μg/mL); strain 4 (A4-H4): candida otophylla (3) CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole MIC) 50 128 μg/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 otophylla (1) CAMS-CCPM-D51239 (CGMCC No.23787, fluconazole MIC) was compared with fluconazole sensitive strain CBS10913 (50822) and fluconazole resistant strain CBS12372 (50825) 50 32 μg/mL), candida otophylla (2) CAMS-CCPM-D51240 (CGMCC No.23788, fluconazole MIC) 50 64 μg/mL), candida otophylla (3) CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole MIC) 50 128 mug/mL) can grow in each concentration (0-64 mug/mL) of fluconazole,wherein candida auriculata (3) CAMS-CCPM-D51241 (CGMCC No.23789, fluconazole MIC) 50 128 μg/mL) was similar to fluconazole resistant strain CBS12372 (50825), and the growth state was good in each concentration of fluconazole. The induced fluconazole resistant strains (candida otophylla (1) - (3)) can grow in 0-64 mug/mL fluconazole.
4. Mass spectrometry of candida otophylla (1) - (3): each strain was subjected to 1X 10 6 CFU·mL -1 The concentration was inoculated on the inclined surface of YPD solid medium, and the bacterial colony was grown in an incubator at 28℃for 48 hours. Selecting a flora coated target plate, adding 1 mu L of 70% formic acid biosafety cabinet for ventilation drying or 50 ℃ metal bath for 5min for drying, adding 1 mu L of matrix liquid (HCCA, capronitrile and trifluoroacetic acid mixed liquid) for drying, and then detecting by a machine (AUTOF MS1000 mass spectrometer), wherein the identification results show that candida otophylla (1) - (3) are candida otophylla, and meanwhile, the characteristic peaks of all strains are found to be different, and the detail is shown in figure 3.
5. Strain morphology observation: the induced strains were observed by microscopic photographing, as shown in FIG. 4.
Various experiments of the embodiment show that Candida auriculata (Candida auris) (1) [ preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23787; the culture medium is deposited in the center medical fungus division center (CAMS-CCPM-D) of pathogenic microorganism (toxin) of China medical science center, and has the deposit number CAMS-CCPM-D51239 and fluconazole MIC 50 32 mug/mL) Candida auriculata (Candida auris) (2) [ preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23788; the culture medium is deposited in the China medical science center for type culture Collection of pathogenic microorganisms (toxins) and has the accession number CAMS-CCPM-D51240 and fluconazole MIC 50 64. Mu.g/mL); candida auriculata (3) [ preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23789; the culture medium is deposited in the China medical science center for type culture Collection of pathogenic microorganisms (toxins) and has the accession number CAMS-CCPM-D51241 and fluconazole MIC 50 128 mug/mL three candida otophylla strains can be singly or combined for constructing fluconazole single-drug resistanceAnd (5) a model.
SEQUENCE LISTING
<110> dermatology hospital of Chinese medical science, dermatology institute of Chinese medical science
<120> Candida otophylla 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 science, dermatology institute of Chinese medical science
<120> Candida otophylla 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 medical science, dermatology institute of Chinese medical science
<120> Candida otophylla 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 science, dermatology institute of Chinese medical science
<120> Candida otophylla 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 science, dermatology institute of Chinese medical science
<120> Candida otophylla 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 science, dermatology institute of Chinese medical science
<120> Candida otophylla 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 (2)
1. Candida otophylla which can be used for constructing a fluconazole single drug resistance model is one of the following strains:
candida auriculata (Candida auris) CAMS CCPM D51239 (1) is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 23787 in the 11 th month of 2021;
candida auriculata (Candida auris) CAMS CCPM D51240 (2) is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 23788 at 11 and 12 of 2021;
candida otophylla (Candida auris) CAMS CCPM D51241 (3) is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 23789 at 11 and 12 of 2021.
2. The use of candida aurea in the construction of a fluconazole single drug resistance model according to claim 1.
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| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| Rybak JM 等.Mutations in TAC1B: a novel genetic determinant of clinical fluconazole resistance in Candida auris.mBio.2020,第11卷e00365-20. * |
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Address after: No. 12 Jiangwang Temple, Xuanwu District, Nanjing City, Jiangsu Province, 210042 Applicant after: Dermatology Hospital of Chinese Academy of Medical Sciences Address before: 210042 No.14 jiangwangmiao street, taipingmenwai, Xuanwu District, Nanjing City, Jiangsu Province Applicant before: Dermatology Hospital of Chinese Academy of Medical Sciences |
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