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Antimicrobial Agents and Chemotherapy, June 2007, p. 1897-1904, Vol. 51, No. 6
0066-4804/07/$08.00+0     doi:10.1128/AAC.01092-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A New Aspergillus fumigatus Resistance Mechanism Conferring In Vitro Cross-Resistance to Azole Antifungals Involves a Combination of cyp51A Alterations{triangledown}

E. Mellado,1* G. Garcia-Effron,1 L. Alcázar-Fuoli,1 W. J. G. Melchers,2 P. E. Verweij,2 M. Cuenca-Estrella,1 and J. L. Rodríguez-Tudela1

Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain,1 Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen University Center for Infectious Diseases, Nijmegen, The Netherlands2

Received 30 August 2006/ Returned for modification 23 September 2006/ Accepted 8 March 2007

Fourteen Aspergillus fumigatus clinical isolates that exhibited a pattern of reduced susceptibility to triazole drugs were analyzed. The sequences of the cyp51A gene from all isolates showed the presence of a point mutation at t364a, which led to the substitution of leucine 98 for histidine (L98H), together with the presence of two copies of a 34-bp sequence in tandem in the promoter of the cyp51A gene. Quantitative expression analysis (real-time PCR) showed up to an eightfold increase in the level of expression of the cyp51A gene compared to that by the susceptible strain. Three PCR fragments of one azole-resistant strain (strain CM2627) that included the promoter with the tandem repeat and part of cyp51A with the t364a mutation or PCR fragments with only one of the modifications were used to replace the cyp51A gene of an azole drug-susceptible A. fumigatus wild-type strain (strain CM237). Only transformants which had incorporated the tandem repeat in the promoter of the cyp51A gene and the L98H amino acid substitution exhibited similarly reduced patterns of susceptibility to all triazole agents and similarly increased levels of cyp51A expression, confirming that the combination of both alterations was responsible for the azole-resistant phenotype.

* Corresponding author. Mailing address: Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo Km2, 28220 Madrid, Spain. Phone: 34-91-822 3661. Fax: 34-91-5097034. E-mail: emellado{at}isciii.es

{triangledown} Published ahead of print on 19 March 2007.

Antimicrobial Agents and Chemotherapy, June 2007, p. 1897-1904, Vol. 51, No. 6
0066-4804/07/$08.00+0     doi:10.1128/AAC.01092-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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