ID Design Press, Skopje, Republic of Macedonia Open Access Macedonian Journal of Medical Sciences. 2018 May 20; 6(5):747-750. https://doi.org/10.3889/oamjms.2018.171 eISSN: 1857-9655 Basic Science Study of Azole - Resistant and Cyp51a Gene in Aspergillus Fumigatus Majid Zarrin1, 2*, Sama Faramarzi2 1Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 2Department of Medical Mycology, Medical School, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Abstract Citation: Zarrin M, Faramarzi S. Study of Azole - AIM: The main goal of the present study was to find azole-resistant and molecular analysis of cyp51A gene in Resistant and Cyp51a Gene in Aspergillus Fumigatus. Aspergillus fumigatus. Open Access Maced J Med Sci. 2018 May 20; 6(5):747- 750. https://doi.org/10.3889/oamjms.2018.171 MATERIALS AND METHODS: Fifty-eight A. fumigatus strains including environmental, clinical and reference Keywords: Azole-resistant; Cyp51 A gene; Aspergillus fumigatus isolates were assessed in this investigation. Azole susceptibility testing for itraconazole and voriconazole was *Correspondence: Majid Zarrin. Department of Medical carried out for A. fumigatus isolates. PCR was performed based on cyp51A gene sequence for all isolates. Mycology, Medical School, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. E-mail: RESULTS: Susceptibility testing verified the minimum inhibitory concentrations (MICs) for itraconazole (0.125 to 2 [email protected] µg/ml) and voriconazole (0.125 to 4 µg/ml). Nine (15.5%) A. fumigatus isolates were resistant to voriconazole with Received: 31-Jan-2018; Revised: 19-Mar-2018; MIC 4 µg/ml. A 1500 bp DNA fragment was amplified using cyp51A gene for all tested Aspergillus isolates. The Accepted: 23-Mar-2018; Online first: 25-Apr-2018 sequences of the fragments showed 99% identity with A. fumigatus cyp51A gene in the GenBank. No point Copyright: © 2018 Majid Zarrin, Sama Faramarzi. This is an open-access article distributed under the terms of mutation was found at cyp51A gene codons. the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0) CONCLUSION: In the current study, we detected the voriconazile resistant in A. fumigatus isolates. Susceptibility Funding: The present study was supported by the tests should be considered in patients who infected by A. fumigatus. Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (grant no. 93128) Competing Interests: The authors have declared that no competing interests exist Introduction The appearance of azoles resistance in yeast species has encouraged researchers of the mechanisms associated with this resistance. Several Aspergillus fumigatus is one of the most genes encoding 14α-demethylase (ERG11/cyp51) common airborne fungal pathogen which causes have been identified for fluconazole-resistant of the invasive aspergillosis. The spores are capable of clinical isolates of Candida albicans [4] [5]. spreading to air and inhaled and eventually cause The mechanisms for azole drug resistance in infection in a susceptible host. A. fumigatus infections A. fumigatus appear to be extremely dissimilar from occur in excessive morbidity and mortality in that in Candida spp. There are two various but related immunocompromised hosts [1] [2]. The azoles are Cyp51 proteins which are encoded by cyp51A and antifungal drugs that inhibit the ergosterol cyp51B genes [6] [7]. biosynthesis pathway by the inhibition of 14α- demethylase. Azoles, for example, itraconazole, Two models of resistance to azoles have voriconazole, and posaconazole are among the discovered in A. fumigatus. First, the A. fumigatus advised first-line agents in the management and could turn into resistant during exposure to azoles in prophylaxis of aspergillosis [3]. the patient. This model was detected in chronic pulmonary aspergillosis and aspergilloma cases in the United Kingdom [8]. In this pattern, eighteen dissimilar _______________________________________________________________________________________________________________________________ Open Access Maced J Med Sci. 2018 May 20; 6(5):747-750. 747 Basic Science _______________________________________________________________________________________________________________________________ amino acid substitutions were distinguished in the Spore suspension for each isolate was set up cyp51A gene [8]. Second, the A. fumigatus can turn in sterile normal saline and adjusted at a into resistance in the environment during the exposure concentration of 106 spores/ml, consequent to 68 to to azole fungicides which are applied in agriculture 82% transmittance at 530 nm [13]. Broth microdilution and material conservation. susceptibility test was carried out as explained in clinical and laboratory standards institute (CLSI) This model was suggested in the Netherlands method with modifications. The antifungal drugs used [9]. In this pattern, a replacement at codon 98 of the were itraconazole (Sigma-Aldrich, Germany) and cyp51A gene combined with a tandem repeat of 34 bp voriconazole (Sigma-Aldrich, Germany). in the promoter (TR/L98H), was detected in 94% of the resistant strains [10]. Stock solutions were prepared in 100% dimethyl sulfoxide (CinnaGene, Karaj, Iran), then Treatment of invasive aspergillosis is mainly diluted in RPMI 1640 medium and dispensed into 96- limited to therapy by the polyene agent amphotericin well microdilution trays. The final concentration of B, and triazoles such as itraconazole, voriconazole voriconazole and itraconazole in the wells ranged and echinocandin caspofungin. Amphotericin B is very from 0.015 to 8.0 µg/ml. The stock spore suspension toxic and can consequence in nephrotoxicity [11], 6 (10 spores/ml) was diluted to a final concentration of while triazoles are fungistatic and subject to 4 5 × 10 CFU/ml and dispensed into the microdilution development of resistance [12]. wells. The inoculated microdilution trays were kept at Here, we explain the analysis of cyp51A gene 35°C and read after 48 h. The minimum inhibitory A. fumigatus which is responsible for the phenotype of concentration (MIC) for voriconazole and itraconazole A. fumigatus azole-resistance. was described as the lowest concentration that created prominent growth inhibition. Some cyp51A gene amplicons were submitted for direct sequencing (Bioneer Corporation, Material and Methods Daejeon, South Korea). The obtained sequences were searched for in the National Center for Biotechnology Information (NCBI) database A total of 58 A. fumigatus strains were used in (http://www.ncbi.nih.gov/). the study including 45 environmental, 9 clinical and 4 The sequences showed 99% similarity with A. reference isolates. The following strains were used as fumigatus cyp51A gene sequences deposited in NCBI a reference: PTCC 5009, IBRC-M 30033, IBRC-M database. The computer software package MEGA5 30040, IBRC-M 30048. Environmental strains were (http://www.megasoftware.net) was employed for recovered from soil or air. sequences alignment. One hundred ml of yeast extract peptone dextrose (YEPD) medium in Erlenmeyer flasks was inoculated with 1 ml of thick spore suspension and incubated at 37°C for 72 h 200 rpm under agitation to Results obtain mycelium growth. The mycelia were harvested, washed with 0.5 M EDTA and sterile dH2O and using liquid nitrogen and a pestle and mortar ground into a A total of 58 samples were tested including 45 fine powder. The DNA was extracted with vivantis is environmental, 9 clinical and 4 reference isolates, GF-1 plant DNA extraction kit, Malaysia. according to the European committee for antibiotic The primer sets, P450-A1 (5'- susceptibility testing (Eucast) methodology suggestion ATGGTGCCGATGCTATGG-3') and P450-A2 (5'- breakpoints for A. fumigatus, itraconazole and CTGTC-TCACTTGGATGTG-3') was used to amplify voriconazole [14]. For itraconazole and voriconazole, an ~ 1500 bp DNA fragment of the full coding <2 µg/ml (susceptible), 2 µg/ml (intermediate) and >2 sequences of cyp51A gene. PCR reactions were µg/ml (resistant). Susceptibility testing verified the carried out with a volume of 30 μl, comprised of 3 μl MICs for itraconazole (0.125 to 2 µg/ml) and 10X reaction buffer, 200 μM each dNTP, 2.2 mM voriconazole (0.125 to 4 µg/ml) (Table 1). MgCl , 2.5 units of Taq DNA polymerase (CinnaGen, 2 Nine (15.5%) A. fumigatus isolates were Tehran, Iran), 30 ng template DNA and 50 pmol of resistant to voriconazole with MIC 4 µg/ml. Twelve each primer. (20.7%) isolates exhibited intermediate susceptibility Initial denaturation for 5 min at 94°C was to itraconazole with MIC 2 µg/ml. The PCR followed by 30 cycles of denaturation at 94°C for 1 amplification of cyp51A gene with set primers P450- min, annealing at 58˚C for 1 min and extension at A1 and P450-A2 produced a 1500 bp fragment for all 68˚C for 2 min. The PCR product (5 μl) was tested Aspergillus isolates (Fig. 1). electrophoresed on 1% agarose gel in TAE buffer and stained with ethidium bromide. _______________________________________________________________________________________________________________________________ 748 https://www.id-press.eu/mjms/index Zarrin et al. Study of Azole - Resistant and cyp51A Gene in Aspergillus fumigatus _______________________________________________________________________________________________________________________________ Table 1: Results of susceptibility testing