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Antimicrobial Agents and Chemotherapy, February 2002, p. 531-533, Vol. 46, No. 2
0066-4804/01/$04.00+0     DOI: 10.1128/AAC.46.2.531-533.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Sterol and Fatty Acid Composition of Candida lusitaniae Clinical Isolates

F. Peyron,1 A. Favel,1* R. Calaf,2 A. Michel-Nguyen,3 R. Bonaly,4 and J. Coulon4

Laboratoire de Botanique, Cryptogamie et Biologie Cellulaire,1 Laboratoire de Biochimie Fondamentale et Clinique, Faculté de Pharmacie, 13385 Marseille,2 Laboratoire de Microbiologie, CHU Nord, 13015 Marseille,3 Laboratoire de Biochimie Microbienne, LCPME-UMR 7564, Faculté des Sciences Pharmaceutiques et Biologiques, 54001 Nancy, France4

Received 25 April 2001/ Returned for modification 21 May 2001/ Accepted 13 October 2001


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ABSTRACT
 
The sterol and fatty acid compositions of four amphotericin B-resistant and of two amphotericin B-susceptible Candida lusitaniae clinical isolates were determined. A flow cytofluorometric susceptibility test (FCST) with a membrane potential-sensitive cationic dye was used as a complement to the conventional method for selecting the isolates. Compared to susceptible isolates, resistant ones showed a greatly reduced ergosterol content and changes in sterol composition, consistent with a defect in {Delta}8->7 isomerase. Within each group, no correlation between the sterol or fatty acid pattern or composition and both the degree of in vitro susceptibility and FCST MIC was found.


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TEXT
 
Candida lusitaniae is an emerging opportunistic pathogen now recognized as an important cause of nosocomial infection in severely immunocompromised patients (12, 17). Rapidly acquired resistance to amphotericin B has been frequently reported, and some strains of C. lusitaniae may be intrinsically resistant (9, 12, 16). Resistance to amphotericin B in yeasts has been generally considered of marginal importance (8). However, its incidence among Candida sp. clinical isolates might have been underestimated because susceptibility testing was not routinely performed and testing methods were not reliable (19). Few amphotericin B-resistant strains have been characterized (8). They were mainly laboratory-derived mutants and fluconazole- and amphotericin B-resistant clinical isolates (4, 10, 11, 18, 20). Resistance was most often associated with quantitative and/or qualitative changes in the lipid content of the cells, especially sterol (8, 13). The aim of this study was to investigate the sterol and fatty acid compositions of amphotericin B-resistant and -susceptible isolates of C. lusitaniae. An alternative flow cytofluorometric susceptibility test (FCST) was used as a complement to the still-debated conventional amphotericin B testing methods for selecting the isolates (6, 14). This assay uses 3,3'- dipentyloxacarbocyanine iodide (DiOC5[3]), a cationic dye, to monitor the cellular effects, i.e., changes in plasma membrane potential induced by the interaction of the drug with its membrane target sites, ergosterol, and fatty acids (2).

Forty-eight clinical isolates of C. lusitaniae and 3 reference strains, Candida albicans ATCC 38248 (amphotericin B resistant), ATCC 90029 (amphotericin B susceptible), and C. lusitaniae CBS 6936, were tested for susceptibility to amphotericin B by the DiOC5(3) FCST. All the clinical isolates were identified to the species level by the ID 32C system (BioMérieux, Marcy l’Etoile, France). Their susceptibility status had been previously assigned according to in vivo and in vitro data obtained by Etest (15). Seven isolates were used to represent amphotericin B-resistant strains. Strains were maintained on Sabouraud glucose agar (BioMérieux) slants and stored at 4°C. Before being tested, they were subcultured on Sabouraud glucose agar (BioMérieux) and incubated at 35°C for 24 h. FCST conditions using DiOC5(3) (Molecular Probes, Eugene, Oreg.) and instrumental parameters were as previously described (14). Five thousand yeast cells per sample were analyzed, and data were recorded as histograms of fluorescence. For each isolate, the FCST MIC, defined as the concentration of amphotericin B at which the fluorescence intensity was reduced by 80%, was calculated from the dose-response relationship as previously described (14). The susceptibility of the selected strains to amphotericin B was checked by Etest (AB BIODISK, Solna, Sweden) with RPMI 1640 agar (American Bioorganics, Buffalo, N.Y.), according to the manufacturer’s recommendations (Etest technical guide no. 4, AB BIODISK, 1994). For lipid analysis, strains were grown in antibiotic medium 3 broth (AM3; Difco Laboratories, Detroit, Mich.) supplemented with 2% glucose (Difco Laboratories) for 22 to 24 h (late exponential growth phase). After harvest, the yeast cells were washed twice with water and saponified by methanol containing 30% KOH for 4 h at 100°C under reflux. After cooling and filtration, the residue was washed three times with petroleum ether. The ether extract containing the sterols was washed with water, dried, and evaporated to dryness under vacuum. After acetylation and purification, the acetylated sterols were identified by gas-liquid chromatography according to the method of Belrhiti et al. (1). The total sterol content was measured by the Liebermann-Burchard method described by Burke et al. (5). The aqueous solution containing the fatty acid salts was acidified, and the free fatty acids were extracted with petroleum ether. After methylation, the methylated fatty acids were identified according to the method of Belrhiti et al. (1) by gas-liquid chromatography. Saturated, monounsaturated, and polyunsaturated fatty acids in methyl ester form purchased from Sigma (St. Louis, Mo.) were used as standards.

The DiOC5(3) FCST provided a broad range of MICs and a good discrimination between the susceptible and resistant isolates and also within each susceptibility category (data not shown). Together with the reference strains, six clinical isolates, that is, four amphotericin B-resistant isolates and two amphotericin B-susceptible isolates, were selected for sterol and fatty acid analyses (Table 1). As shown in Table 2, they were chosen within each susceptibility category to represent a variety of FCST and Etest MICs. The same sterol pattern was in found in all strains analyzed, but differences between resistant and susceptible isolates for individual sterols were observed (Table 2). In the susceptible isolates, the predominant sterols were ergosterol (range, 24.2 to 25.5%), zymosterol (range, 27.8 to 28.3%), and lanosterol (range, 16.3 to 18.1%). In resistant isolates, only 7.1 to 9.4% of sterol was ergosterol. The predominant sterols were {Delta}8 sterols: lanosterol (range, 23.3 to 28.1%), zymosterol (range, 20.1 to 23.2%), and fecosterol, whose amount was more than twice those in susceptible isolates (range, 15.1 to 18.6% versus 6.1 to 6.8%). Total sterol contents did not vary. All isolates showed similar fatty acid compositions (data not shown). The major fatty acids were, in descending order, oleic acid (18:1), linoleic acid (18:2), and palmitic acid (16:0).


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  		TABLE 1. Characteristics of the strains analyzed for sterol and fatty acid composition

In this study, yeast cells were grown in AM3, the broth medium most sensitive for detection of amphotericin B-resistant isolates (19). For the two reference C. albicans strains, lipid analysis revealed that the sterol compositions differed from those reported (13, 18). We hypothesized the nonstandardized AM3 has a factor that interferes with the multiple biosynthetic pathways of ergosterol (7). However, the two strains exhibited significant differences in their ergosterol contents and in their {Delta}8 sterol compositions. For C. albicans ATCC 38248, {Delta}8 sterol composition was consistent with the mechanism proposed for amphotericin B resistance, i.e., a defect in {Delta}8->7 isomerase (18). A comparison of the two groups of C. lusitaniae isolates showed that those resistant to amphotericin B had a decreased level of ergosterol. According to {Delta}8 sterol compositions, resistance can be also ascribed to a defective {Delta}8->7 isomerization. Such an enzymatic defect has been previously reported to cause resistance in some Candida sp. mutants and in one clinical isolate of Cryptococcus neoformans (4, 10, 18). Within each group, no correlation between the sterol or the fatty acid pattern or composition and both the degree of in vitro susceptibility and the value of the FCST MIC was observed.

In conclusion, resistance to amphotericin B in C. lusitaniae is associated with changes in the ergosterol biosynthetic pathway, which suggests an Erg2 defect (13). As a decreased ergosterol content alone is not sufficient to account for amphotericin B resistance, additional studies are needed to examine the influence of factors such as phospholipid composition of the plasma membrane, cell wall structure, and catalase activity (2, 3, 8).


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  		TABLE 2. Sterol composition of amphotericin B-susceptible and -resistant isolates of Candida spp.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Botanique, Cryptogamie et Biologie Cellulaire, Faculté de Pharmacie, 27 Blvd. J. Moulin, 13005 Marseille Cedex 5, France. Phone: 33-4-91-83-56-37. Fax: 33-91-80-26-12. Email: Reglip{at}pharmacie.univ-mrs.fr. Back


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Antimicrobial Agents and Chemotherapy, February 2002, p. 531-533, Vol. 46, No. 2
0066-4804/01/$04.00+0     DOI: 10.1128/AAC.46.2.531-533.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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