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Caspofungin resistance in clinical Aspergillus flavus isolates
Zeynab Yassin1, Ensieh Lotfali2, Mohammad Rafi Khourgami3
1Antimicrobial Resistance Research Center, Iran University of Medical Sciences, Tehran, Iran.
Introduction And Aims:
The present study was conducted to determine the candidate genes involved in caspofungin (CAS) resistance in clinical isolates of Aspergillus flavus (A. flavus).
Materials And Methods:
The antifungal susceptibility assay of the CAS was performed on 14 clinical isolates of A. flavus using the CLSI-M-38-A2 broth micro-dilution protocol. Since CAS had various potencies, the minimum effective concentration (MEC) of anidulafungin (AND) was also evaluated in the present study. The FKS1 gene sequencing was conducted to assess whether mutations occurred in the whole FKS1 gene as well as hot spot regions of the FKS1 gene of the two resistant isolates. A complementary DNA-amplified fragment length polymorphism (CDNA-AFLP) method was performed to investigate differential gene expression between the two resistant and two sensitive clinical isolates in the presence of CAS. Furthermore, quantitative real-time PCR (QRT-PCR) was utilized to determine the relative expression levels of the identified genes.
Results:
No mutations were observed in the whole FKS1 gene hot spot regions of the FKS1 genes in the resistant isolates. A subset of two genes with known biological functions and four genes with unknown biological functions were identified in the CAS-resistant isolates using the CDNA-AFLP. The QRT-PCR revealed the down-regulation of the P-type ATPase and ubiquinone biosynthesis methyltransferase COQ5 in the CAS-resistant isolates, compared to the susceptible isolates.
Conclusion:
The findings showed that P-type ATPase and ubiquinone biosynthesis methyltransferase COQ5 might be involved in the CAS-resistance A. flavus clinical isolates. Moreover, a subset of genes was differentially expressed to enhance fungi survival in CAS exposure. Further studies are recommended to highlight the gene overexpression and knock-out experiments in A. flavus or surrogate organisms to confirm that these mentioned genes confer the CAS resistant A. flavus.
Insights
Caspofungin resistance in Aspergillus flavus may involve P-type ATPase and COQ5 genes. Differential gene expression was observed in resistant fungal isolates, suggesting a role in survival against antifungal drugs.
Area of Science:
- Mycology
- Antifungal Resistance
- Molecular Biology
Background:
- Aspergillus flavus is an opportunistic fungal pathogen.
- Caspofungin (CAS) is a key antifungal agent used to treat invasive fungal infections.
- Emerging CAS resistance in A. flavus poses a significant clinical challenge.
Purpose of the Study:
- To identify candidate genes contributing to caspofungin resistance in clinical A. flavus isolates.
- To investigate the genetic mechanisms underlying CAS resistance in A. flavus.
Main Methods:
- Antifungal susceptibility testing of CAS on 14 clinical A. flavus isolates.
- FKS1 gene sequencing to detect mutations in resistant isolates.
- Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) to identify differentially expressed genes.
- Quantitative real-time PCR (qRT-PCR) to validate gene expression levels.
Main Results:
- No mutations were found in the FKS1 gene hot spot regions of resistant isolates.
- cDNA-AFLP identified several differentially expressed genes in CAS-resistant isolates.
- qRT-PCR revealed down-regulation of P-type ATPase and ubiquinone biosynthesis methyltransferase COQ5 in resistant isolates.
Conclusions:
- P-type ATPase and COQ5 may play a role in CAS resistance in A. flavus.
- Differential gene expression contributes to fungal survival under CAS exposure.
- Further studies, including gene overexpression and knockout experiments, are recommended to confirm the role of these genes in CAS resistance.
