Development of MALDI-TOF MS to detect fluconazole resistance in Candida albicans

Name: Chanika Maenchantrarath

keyword: Candida albicans

; Matrix assisted laser desorption ionization-time of flight mass spectrometry

; MALDI-TOF MS

; Fluconazole susceptibility testing

; Overexpression

Abstract: Candida albicans is the most important yeast that caused the infection in humans, the trend of resistance to fluconazole (FLC) was also increased. This study was conducted to develop and evaluate the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for FLC susceptibility testing of C. albicans isolates. Four ATCC standard strains (C. albicans MYA2876, C. albicans ATCC90028, C. albicans MYA4440, and C. albicans ATCC96901), 1 of spontaneous mutational strains (ΔMYA4440) and 27 of clinical strains were performed the FLC susceptibility testing to define the minimal inhibitory concentrations (MIC) level by Sensititre YeastOne (SYO) and broth microdilution (BMD) method. Real-time PCR (qRT-PCR) was used to determine the resistance mechanism of FLC in CDR1, CDR2, MDR1 and ERG11. C. albicans control strains in different range of MIC were exposed at different FLC concentrations (0.25, 1, 4 and 8 µg/mL) and harvested at 2, 4, 6 and 24 hours intervals. The mass spectra were analyzed with MALDI-TOF MS to obtain the reference intensity. After that, all clinical strains of C. albicans were evaluated the suitable procedure. C. albicans MYA2876 (susceptible strain ; MIC = 0.25 µg/mL), mutant C. albicans MYA4440 (ΔMYA4440) (susceptible-dose dependent strain ; MIC = 4 µg/mL), and C. albicans ATCC96901 (resistant strain ; MIC ≥64 µg/mL) are the control different level of MIC. The expression of susceptible-dose dependent strain (ΔMYA4440) and resistant strain (ATCC96901) have statistically significant overexpression in CDR efflux genes (CDR1 and CDR2) when compared to susceptible strain (MYA2876), while MDR1 and ERG11 genes are not show any significant expression among C. albicans susceptible strain, susceptible-dose dependent strain and resistant strain. Protein expression results by MALDI-TOF MS are found that the spectral intensity at 3376-3382 m/z significantly relates to the FLC susceptibility when incubated with FLC at 4 µg/mL and 8 µg/mL for 6 hours. The intensity of resistant strain (ATCC96901) was slightly decreased or not changed in both FLC concentrations, while the intensity of susceptible strain (MYA2876) was increased in both FLC concentrations. But, the intensity of susceptible-dose dependent strain (ΔMYA4440) was decreased for 4 µg/mL of FLC, whereas the intensity was increased for 8 µg/mL of FLC. All of 27 clinical C. albicans strains showed agreement of significant spectral changes at position 3376-3382 m/z after 6 hours incubation with FLC at 4 µg/mL and 8 µg/mL. The suitable condition for FLC susceptibility by MALDI-FOF MS detection is incubation with FLC 4 and 8 µg/mL for 6 hours comparison to without FLC condition. The FLC resistance mechanisms in this study plays role in overexpression of CDR1 and CDR2 via increased efflux of the drug from cells by ABC transporters, and the protein spectrum at position 3376-3382 m/z is related with the FLC resistance of C. albicans. The MALDI-TOF MS could be used as a rapid screening technique for FLC resistance in C. albicans

Thammasat University. Thammasat University Library

Address: BANGKOK

Email: preserv@tu.ac.th

Name: Worada Samosornsuk

Role: advisor

Name: Seksun Samosornsuk

Role: co-advisor

Name: Amornrat Geadkaew Krenc

Role: co-advisor

Created: 2020

Modified: 2023-02-02

Issued: 2023-02-02

วิทยานิพนธ์/Thesis

application/pdf

DOI: 10.14457/TU.the.2020.940

eng

DegreeName: Master of Science

Level: Master's Degree

Descipline: Biomedical Sciences

Grantor: Thammasat University

©copyrights Thammasat University

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