We monitored adaptation to inhibitory concentrations of fluconazole over 330 generations of experimental evolution in 12 replicate populations founded from a single azole-susceptible cell of C. albicans
(fluconazole MIC, 0.25 μg/ml). Six populations were grown with fluconazole at twice their most recently measured MIC, and six were propagated without drug. There was no change in MIC for any of the six populations grown without drug (Fig. ). Among the six populations grown with fluconazole (Fig. ), two populations (D9 and D11) achieved the highest level of fluconazole resistance (MIC, 64 μg/ml) measured in this standard test and retained this level to generation 330. One population (D8) achieved an intermediate level of resistance (MIC, 4.0 μg/ml) and remained at this level until generation 330. Two populations (D10 and D12) achieved increased resistance (MIC, 16.0 and 64.0 μg/ml, respectively) and then showed a decrease (MIC, 1.0 and 4.0 μg/ml, respectively). One population (D7) achieved a small increase in resistance (MIC, 0.5 μg/ml), sharply increased resistance at generation 260 (MIC, 8.0 μg/ml), and then a decrease at generation 330 to the previous level. In all cases, the level of resistance achieved at generation 330 was stable over three transfers on azole-free solid medium. Resistance, relative to the progenitor, was retained over 50 generations of further experimental evolution in the absence of drug (Fig. ), with one exception (D7-330-M). The increase in fluconazole MIC was accompanied by a corresponding increase in resistance to itraconazole and ketoconazole (data not shown). When MICs were determined at pH 4, the basic relationship of MICs among the populations was not altered, but MICs of fluconazole were slightly higher at pH 4 than at pH 7, as was observed in some cases by Marr et al. (9
). All populations contained ergosterol in their cell membranes and were amphotericin sensitive (data not shown).
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 (more ...)
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 (more ...)
Each population grown in the presence of drug acquired resistance in a different way, overexpressing a unique combination of four genes known to be important in fluconazole resistance (26
): the gene encoding the target enzyme of azoles in the ergosterol biosynthesis pathway, ERG11
; two ATP-binding cassette transporters, CDR1
; and a major facilitator, MDR1
(Fig. ). While the CDR
gene products pump many azoles from the cell, the MDR1
product specifically pumps fluconazole. There was highly significant variation in mRNA levels among the experimental populations for CDR1
(Kruskal-Wallis test, P
< 0.001). CDR2
was strongly expressed in one population (D8-330), was expressed at a low level in another population (D10-200), and was not detected in the remaining nine populations. Four populations (D9-330, D11-330, D12-260, and D12-330) strongly expressed the MDR1
gene, while MDR1
mRNA was not detected in seven populations. Other factors contributing to azole resistance must be operating in the populations overexpressing MDR1
to account for the azole-cross-resistant phenotypes, as the efflux pump encoded by this gene appears to specifically pump fluconazole (26
). No mutations were detected in the nucleotide sequence of ERG11
in the replicate populations. This was confirmed for all populations under selection by functional expression of the ERG11
alleles in Saccharomyces cerevisiae
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 < (more ...)
In addition to four genes known to be important in azole resistance, we also monitored molecular markers with no known relation to drug resistance: polymorphic nucleotide sites in five DNA regions known to be heterozygous in the progenitor genotype and the 27A DNA fingerprint widely used to type clinical isolates of C. albicans
). No changes in neutral markers or DNA fingerprint were detected in any of the populations not exposed to drug, while several changes including loss of heterozygosity in two of the five genes known to be heterozygous in the progenitor were detected in several populations evolved in the presence of fluconazole. The changes were as follows: C15F2
, position 174, AG to AA in D7-260; and PDE1
, position 1046, CT to TT in D7-330 and CT to CC in D10-200 (single-colony isolate only). Changes in the DNA fingerprint were detected in two of nine samples from populations evolved in the presence of drug. The changes were the gain of a band in D7-260 and the loss of a band in D12-260 (single-colony isolate only).
Southern hybridizations of electrophoretic karyotypes revealed numerous chromosomal changes in the evolved populations relative to the ancestral isolate (Fig. ). The most variable chromosome was chromosome R, which contains the genes coding for rDNA. Both probes for chromosome R showed the same hybridization results: the progenitor (T118-0) had one distinct strongly hybridizing band and one weakly hybridizing band of unknown origin, the six populations evolved without drug at generation 330 each had a smear, and the drug populations each had one or two distinct bands of variable size (Fig. ).
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 (more ...)
Further experiments examined the nature of the variation in chromosome R. Hin
dIII liberates the tandem rDNA array from each chromosome R homologue as a single fragment (14
). Southern hybridization of Hin
dIII digests with the intergenic spacer region of the rDNA demonstrated variation in the size of the rDNA arrays among the experimental populations (Fig. ). The variation in size of the rDNA array corresponded to the size variation of chromosome R. Not
I has a single cutting site within the rDNA unit (7
). Southern hybridization of Not
I digests with the intergenic spacer region of the rDNA showed that there were no changes in the rDNA unit size among the experimental populations (data not shown).
In the chromosomes other than chromosome R, there was no variation detected for chromosomes 1, 2, 3, 6, and 7 with either of the probes specific to the terminal Sfi
I fragments for each chromosome. With the exception of chromosome R, the C. albicans
chromosomes are numbered 1 through 7, largest to smallest (3
). For chromosome 4, there was no variation detected with the probe for Sfi
I fragment BB, while hybridization with the probe specific for Sfi
I fragment H showed a change for populations D10-330 and D7-330. The probe hybridized to one distinct band in all other populations, but there were two distinct bands for D10-330, one the same size as that in the other populations and the other, of equal intensity, at the same position as chromosome 5. D7-330 had one strongly hybridizing band of the expected size and also a weakly hybridizing band at the same position as chromosome 2. For chromosome 5, both probes hybridized to one distinct band with no size variation for all population samples except D12-260, in which the band was the same size as chromosome 2. In the sample from D12-260, there was no band corresponding to that of chromosome 5 in the other samples (Fig. ).
Increased doubling time in the absence of drug was detected in samples grown in both RPMI 1640 and yeast-peptone-glucose media from population D7 at generation 260, D9 at 330, D11 at 330, D12 at 260, and D12 at 330 (Fig. ). Variation among population samples was highly significant (Kruskal-Wallis test, P < 0.001).
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 (more ...)