Alterations of the cyp51A Gene Promoter Contribute to Aspergillus fumigatus Multiple Triazole Resistance.

E. MELLADO1, L. ALCAZAR- FUOLI 1, D. PAJKRT 2, P. E. VERWEIJ 3, W. J. G. MELCHERS 3, M. CUENCA-ESTRELLA 1, J. RODRIGUEZ-TUDELA 1;

Author address: 

1CNM, ISCIII, Madrid, Spain, 2Academic Med. Ctr., Amsterdam, The Netherlands, 3Univ. Ctr. for Infectious Diseases, Nijmegen, The Netherlands.

Abstract: 

Background: We investigated the azole drugs resistance mechanism of one A. fumigatus multiple triazole resistant (MTR) clinical isolate obtained from a patient with chronic granulomatous disease presenting with aspergillus osteomyelitis during azole prophylaxis. Methods: (i) Antifungal susceptibility (M38-A, CLSI standard), (ii) sequencing of cyp51A and cyp51B genes and promoters, (iii) cyp51A mRNA transcription levels (by Real time PCR), (iv) Electroporation was used for A. fumigatus transformation. Results: The MTR strain MIC ranges were: ITC: > 8, VRC: 4-8, and RVZ: 4-8 in mg/L. The MTR strain had no changes in the cyp51 genes. However, sequencing of the cyp51A promoter region revealed the presence of a duplication in tandem of a 53 bp fragment (LTR). Expression analysis showed a 5 fold increase of cyp51A gene expression. A linear DNA fragment including the LTR in the cyp51A promoter was electroporated into an A. fumigatus azole susceptible strain. Transformants that had incorporated the LTR into the cyp51A gene promoter were analyzed. Conclusions: To date azole resistance in A. fumigatus has been matched to Cyp51A point mutations alone or in combination with tandem repeat (34 bp) in the cyp51A gene promoter. Our strain presents a novel cyp51A promoter alteration consisting of the duplication of a 53 bp sequence inserted in tandem (LTR). This alteration was shown to be directly related to the increased cyp51A gene expression. However the presence of the LTR could only explain an slight increased MICs to all azole drugs. It seems clear that the MTR phenotype is the result of a combination of the LTR with another, still unknown, resistance mechanism.
2007

abstract No: 

M-521

Full conference title: 

47th Interscience Conference on Antimicrobial agents and Chemotherapy
    • ICAAC 47th