Evolution of drug resistance in experimental populations of Candida albicans - PubMed (original) (raw)

Evolution of drug resistance in experimental populations of Candida albicans

L E Cowen et al. J Bacteriol. 2000 Mar.

Abstract

Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast Candida albicans and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes, CDR1 and CDR2; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway, ERG11; and the major facilitator gene, MDR1. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of C. albicans.

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Figures

FIG. 1

FIG. 1

Adaptation to fluconazole in experimental populations. Twelve populations were established from a single cell of an azole-susceptible strain of C. albicans. The populations were propagated in RPMI 1640 medium for 330 generations. Six populations (D7 to D12) were grown in twice their most recently measured MIC of fluconazole, and six (N1 to N6) were grown without drug.

FIG. 2

FIG. 2

The stability of acquired resistance in 50 generations (∼15 days) in RPMI 1640 medium without fluconazole. Susceptibility to fluconazole was determined at generations 0 (solid), 25 (hatched), and 50 (unfilled). M refers to mass cultures; S refers to a single-colony isolate.

FIG. 3

FIG. 3

Relative mRNA levels of four C. albicans genes involved in azole resistance. Bars represent standard deviations for each sample (n = 6 replicate measurements). Variation among the population samples was highly significant (Kruskal-Wallis test, P < 0.001).

FIG. 4

FIG. 4

Electrophoretic karyotypes. (Top panels) Ethidium-stained gels. The arrow indicates chromosome R in isolate T118-0; the bracket indicates range of chromosome R sizes in other isolates. (Middle panels) Southern hybridization with INO1 (GenBank accession no. L22737), a probe specific for chromosome R. The asterisk indicates the strongly hybridizing band in T118-0; the open arrow indicates the weakly hybridizing band of unknown origin in T118-0. (Bottom panels) rDNA as visualized by Southern hybridization of _Hin_dIII digests with the intergenic spacer region of the rDNA.

FIG. 5

FIG. 5

Doubling times of populations during the exponential growth phase. (A) Doubling times in RPMI 1640 medium. (B) Doubling times in yeast-peptone-glucose medium. Bars represent standard deviation for each sample (n = 2 replicate measurements). Variation among the population samples was highly significant (Kruskal-Wallis test, P < 0.001).

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