Determination of azole antifungal drug resistance mechanisms involving Cyp51A gene in clinical isolates of Aspergillus fumigatus and Aspergillus niger

Research output: Contribution to journalArticle

Abstract

Aims: The main aim of this research is to investigate azole resistance mechanisms in Aspergillus fumigatus and Aspergillus niger which involve Cyp51A gene that encodes 14-a sterol demethylase enzyme. Methodology and results: Itraconazole susceptibility was determined through E-test method. A conventional PCR method was used to amplify and sequence Cyp51A gene in fungal DNA, to detect the presence of gene mutations. Real-time PCR method was applied to determine overexpression of Cyp51A gene in A. fumigatus and A. niger isolates. Susceptibility test found that 3/13 (23.1%) A. fumigatus and 7/23 (30.4%) A. niger isolates were resistant to itraconazole, with minimum inhibitory concentrations (MICs) of 2.5 μg/mL to 3.0 μg/mL. Sequencing of A. fumigatus DNA showed presence of L98H mutation in 7/13 (53.8%) and M220 mutation in 3/13 (23%) isolates. Whereas, sequencing of A. niger DNA detected the presence of G427S mutation in 3/23 (13%) isolates. Tandem Repeat mutation was not detected in all A. fumigatus and A. niger isolates. Only M220 mutation showed significant correlation (r(13)=0.041038, p < 0.05) with itraconazole antifungal resistance in A. fumigatus isolates while L98H mutation was not involved. G427S mutation also showed correlation (r(15)=0.038434, p < 0.05) with itraconazole antifungal resistance in A. niger isolates. A higher level of Cyp51A gene expression was detected in 4/8 (50%) A. fumigatus isolates and 7/10 (70%) A. niger isolates. Resistant isolates more often showed higher level of Cyp51A gene expression compared to susceptible isolates, however the difference in level of expression between resistant isolates and susceptible isolates is not significant. Conclusion, significance and impact of study: In conclusion the level of azole resistance in A. fumigatus and A. niger isolates in Malaysia is low and mutations in Cyp51A gene may contribute towards itraconazole antifungal resistance, however other factors may also be involved.

Original languageEnglish
Pages (from-to)205-210
Number of pages6
JournalMalaysian Journal of Microbiology
Volume12
Issue number3
DOIs
Publication statusPublished - 2016

Fingerprint

Fungal Drug Resistance
Azoles
Aspergillus fumigatus
Aspergillus niger
Itraconazole
Mutation
Genes
Sterol 14-Demethylase
Fungal DNA
Gene Expression
Tandem Repeat Sequences
DNA
Malaysia
Microbial Sensitivity Tests
Real-Time Polymerase Chain Reaction

Keywords

  • Aspergillus fumigatus
  • Aspergillus niger
  • Cyp51A
  • Itraconazole
  • Resistance

ASJC Scopus subject areas

  • Microbiology (medical)
  • Infectious Diseases

Cite this

@article{031f00e574fb4254a71b0ac0a35e1ade,
title = "Determination of azole antifungal drug resistance mechanisms involving Cyp51A gene in clinical isolates of Aspergillus fumigatus and Aspergillus niger",
abstract = "Aims: The main aim of this research is to investigate azole resistance mechanisms in Aspergillus fumigatus and Aspergillus niger which involve Cyp51A gene that encodes 14-a sterol demethylase enzyme. Methodology and results: Itraconazole susceptibility was determined through E-test method. A conventional PCR method was used to amplify and sequence Cyp51A gene in fungal DNA, to detect the presence of gene mutations. Real-time PCR method was applied to determine overexpression of Cyp51A gene in A. fumigatus and A. niger isolates. Susceptibility test found that 3/13 (23.1{\%}) A. fumigatus and 7/23 (30.4{\%}) A. niger isolates were resistant to itraconazole, with minimum inhibitory concentrations (MICs) of 2.5 μg/mL to 3.0 μg/mL. Sequencing of A. fumigatus DNA showed presence of L98H mutation in 7/13 (53.8{\%}) and M220 mutation in 3/13 (23{\%}) isolates. Whereas, sequencing of A. niger DNA detected the presence of G427S mutation in 3/23 (13{\%}) isolates. Tandem Repeat mutation was not detected in all A. fumigatus and A. niger isolates. Only M220 mutation showed significant correlation (r(13)=0.041038, p < 0.05) with itraconazole antifungal resistance in A. fumigatus isolates while L98H mutation was not involved. G427S mutation also showed correlation (r(15)=0.038434, p < 0.05) with itraconazole antifungal resistance in A. niger isolates. A higher level of Cyp51A gene expression was detected in 4/8 (50{\%}) A. fumigatus isolates and 7/10 (70{\%}) A. niger isolates. Resistant isolates more often showed higher level of Cyp51A gene expression compared to susceptible isolates, however the difference in level of expression between resistant isolates and susceptible isolates is not significant. Conclusion, significance and impact of study: In conclusion the level of azole resistance in A. fumigatus and A. niger isolates in Malaysia is low and mutations in Cyp51A gene may contribute towards itraconazole antifungal resistance, however other factors may also be involved.",
keywords = "Aspergillus fumigatus, Aspergillus niger, Cyp51A, Itraconazole, Resistance",
author = "Mahindran Rajendran and Khaithir, {Tzar Mohd Nizam} and Jacinta Santhanam",
year = "2016",
doi = "10.21161/mjm.79115",
language = "English",
volume = "12",
pages = "205--210",
journal = "Malaysian Journal of Microbiology",
issn = "1823-8262",
publisher = "Malaysian Society for Microbiology",
number = "3",

}

TY - JOUR

T1 - Determination of azole antifungal drug resistance mechanisms involving Cyp51A gene in clinical isolates of Aspergillus fumigatus and Aspergillus niger

AU - Rajendran, Mahindran

AU - Khaithir, Tzar Mohd Nizam

AU - Santhanam, Jacinta

PY - 2016

Y1 - 2016

N2 - Aims: The main aim of this research is to investigate azole resistance mechanisms in Aspergillus fumigatus and Aspergillus niger which involve Cyp51A gene that encodes 14-a sterol demethylase enzyme. Methodology and results: Itraconazole susceptibility was determined through E-test method. A conventional PCR method was used to amplify and sequence Cyp51A gene in fungal DNA, to detect the presence of gene mutations. Real-time PCR method was applied to determine overexpression of Cyp51A gene in A. fumigatus and A. niger isolates. Susceptibility test found that 3/13 (23.1%) A. fumigatus and 7/23 (30.4%) A. niger isolates were resistant to itraconazole, with minimum inhibitory concentrations (MICs) of 2.5 μg/mL to 3.0 μg/mL. Sequencing of A. fumigatus DNA showed presence of L98H mutation in 7/13 (53.8%) and M220 mutation in 3/13 (23%) isolates. Whereas, sequencing of A. niger DNA detected the presence of G427S mutation in 3/23 (13%) isolates. Tandem Repeat mutation was not detected in all A. fumigatus and A. niger isolates. Only M220 mutation showed significant correlation (r(13)=0.041038, p < 0.05) with itraconazole antifungal resistance in A. fumigatus isolates while L98H mutation was not involved. G427S mutation also showed correlation (r(15)=0.038434, p < 0.05) with itraconazole antifungal resistance in A. niger isolates. A higher level of Cyp51A gene expression was detected in 4/8 (50%) A. fumigatus isolates and 7/10 (70%) A. niger isolates. Resistant isolates more often showed higher level of Cyp51A gene expression compared to susceptible isolates, however the difference in level of expression between resistant isolates and susceptible isolates is not significant. Conclusion, significance and impact of study: In conclusion the level of azole resistance in A. fumigatus and A. niger isolates in Malaysia is low and mutations in Cyp51A gene may contribute towards itraconazole antifungal resistance, however other factors may also be involved.

AB - Aims: The main aim of this research is to investigate azole resistance mechanisms in Aspergillus fumigatus and Aspergillus niger which involve Cyp51A gene that encodes 14-a sterol demethylase enzyme. Methodology and results: Itraconazole susceptibility was determined through E-test method. A conventional PCR method was used to amplify and sequence Cyp51A gene in fungal DNA, to detect the presence of gene mutations. Real-time PCR method was applied to determine overexpression of Cyp51A gene in A. fumigatus and A. niger isolates. Susceptibility test found that 3/13 (23.1%) A. fumigatus and 7/23 (30.4%) A. niger isolates were resistant to itraconazole, with minimum inhibitory concentrations (MICs) of 2.5 μg/mL to 3.0 μg/mL. Sequencing of A. fumigatus DNA showed presence of L98H mutation in 7/13 (53.8%) and M220 mutation in 3/13 (23%) isolates. Whereas, sequencing of A. niger DNA detected the presence of G427S mutation in 3/23 (13%) isolates. Tandem Repeat mutation was not detected in all A. fumigatus and A. niger isolates. Only M220 mutation showed significant correlation (r(13)=0.041038, p < 0.05) with itraconazole antifungal resistance in A. fumigatus isolates while L98H mutation was not involved. G427S mutation also showed correlation (r(15)=0.038434, p < 0.05) with itraconazole antifungal resistance in A. niger isolates. A higher level of Cyp51A gene expression was detected in 4/8 (50%) A. fumigatus isolates and 7/10 (70%) A. niger isolates. Resistant isolates more often showed higher level of Cyp51A gene expression compared to susceptible isolates, however the difference in level of expression between resistant isolates and susceptible isolates is not significant. Conclusion, significance and impact of study: In conclusion the level of azole resistance in A. fumigatus and A. niger isolates in Malaysia is low and mutations in Cyp51A gene may contribute towards itraconazole antifungal resistance, however other factors may also be involved.

KW - Aspergillus fumigatus

KW - Aspergillus niger

KW - Cyp51A

KW - Itraconazole

KW - Resistance

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U2 - 10.21161/mjm.79115

DO - 10.21161/mjm.79115

M3 - Article

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JO - Malaysian Journal of Microbiology

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SN - 1823-8262

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