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Antimicrob. Agents Chemother. doi:10.1128/AAC.00088-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Mutations in the fks1 gene in Candida albicans, C. tropicalis and C. krusei correlate with elevated caspofungin MICs uncovered in AM3 medium using the EUCAST method

Institut Pasteur, Centre National de Référence Mycologie et Antifongiques, Unité de Mycologie Moléculaire; CNRS URA3012, 75724 Paris Cedex 15, France; Université Paris Descartes; Faculté de Médecine; AP-HP; Hôpital Européen Georges Pompidou; Unité de Parasitologie – Mycologie, 75015 Paris, France

^* To whom correspondence should be addressed. Email: dannaoui{at}pasteur.fr.

	Abstract

Mutations in two specific regions of the Fks1 subunit of the 1-3--D-glucan synthase are known to confer decreased caspofungin susceptibility in Candida spp. Clinical isolates of Candida spp. (404 C. albicans, 62 C. tropicalis, and 21 C. krusei) sent to the French National Reference Center were prospectively screened for caspofungin in vitro susceptibility by the broth microdilution reference method of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST). Twenty-eight isolates (25 C. albicans, 2 C. tropicalis and 1 C. krusei) with a caspofungin MIC above the MIC that inhibited 90% of the isolates of the corresponding species (MIC₉₀) were subject to molecular analysis in order to identified mutations in fks1 gene. Substitutions in the Fks1 deduced protein sequence were found for 8 isolates and 20 isolates had wild-type sequence. Among the 6 C. albicans harboring mutations, 6 patterns were observed involving amino acid changes at position 641, 645, 649, and 1358. For C. tropicalis, one isolate showed a L644W mutation and for one C. krusei isolate two mutations, L658W and L701M, were found.

Two media, RPMI and AM3, were tested for their ability to discriminate between isolates with wild-type Fks1 and those with mutant Fks1. In RPMI, caspofungin MIC ranged from 0.25 to 2 µg/ml for wild-type isolates and from 1 to 8 µg/ml for mutant isolates. A sharper difference was observed in AM3 as all wild-type isolates were inhibited by 0.25 µg/ml of caspofungin while all mutant isolates had caspofungin MICs of 0.5 µg/ml. These data demonstrate that clinical isolates of C. albicans, C. tropicalis and C. krusei with decreased in vitro susceptibility to caspofungin have diverse mutations in fks1 gene and that AM3 is potentially a better medium than RPMI for discrimination between mutant and wild-type isolates using the AFST-EUCAST method.