Journal of Bacteriology, May 2001, p. 2971-2978, Vol. 183, No. 10
Department of Botany, University of Toronto,
Mississauga, Ontario, Canada L5L 1C6
Received 4 December 2000/Accepted 26 February 2001
The dissemination and persistence of drug-resistant organisms in
nature depends on the relative fitness of sensitive and resistant genotypes. While resistant genotypes are expected to be at an advantage
compared to less resistant genotypes in the presence of drug,
resistance may incur a cost; resistant genotypes may be at a
disadvantage in the absence of drug. We measured the fitness of
replicate experimental populations of the pathogenic yeast Candida albicans founded from a single progenitor cell in a
previous study (L. E. Cowen, D. Sanglard, D. Calabrese, C. Sirjusingh, J. B. Anderson, and L. M. Kohn, J. Bacteriol.
182:1515-1522, 2000) and evolved in the presence, and in the absence,
of the antifungal agent fluconazole. Fitness was measured both in the
presence and in the absence of fluconazole by placing each evolved
population in direct competition with the drug-sensitive ancestor and
measuring the reproductive output of each competitor in the mixture.
Populations evolved in the presence of drug diverged in fitness. Any
significant cost of resistance, indicated by reduced fitness in the
absence of drug, was eliminated with further evolution. Populations
evolved in the absence of drug showed more uniform increases in fitness under both conditions. Fitness in the competition assays was not predicted by measurements of the MICs, doubling times, or
stationary-phase cell densities of the competitors in isolation,
suggesting the importance of interactions between mixed genotypes in competitions.
The emergence and spread of drug
resistance among viruses (8, 13, 30), bacteria (4,
24, 26, 32), and fungal pathogens (16, 42) has
followed the widespread use of antimicrobial agents in recent years and
now poses a growing public health problem (26). Whether
drug-resistant organisms persist in nature depends on their fitness
relative to drug-sensitive organisms. Correlations between resistance
to fungicides and fitness traits such as latency, sporulation, and
survival of plant-pathogenic fungi have provided mixed results: that
there is no cost of resistance (34), that there is a cost
for survival of resistant propagules but not somatic cells
(35); and that there is a cost not correlated with any specific trait (15, 18). The evolutionary dynamics of drug resistance and its fitness costs have been studied in viruses (8,
31) and bacteria (2, 6, 7, 25, 38) but not in a
comparable way in fungi, despite their increasing importance as
opportunistic pathogens of humans (1, 14, 17).
Experimental populations of the pathogenic fungus Candida
albicans from a previous study (12) provided the
opportunity here to measure relative fitness by comparing the
reproductive rates of populations that had evolved resistance to the
antifungal drug fluconazole with that of their drug-sensitive ancestor
in a common environment with, and without, fluconazole. Our primary
goal in this study was to determine whether replicate populations
evolved in the presence of fluconazole diverged in fitness and then to
measure any cost of resistance. Our expectation was that fluconazole
resistance would carry a significant fitness cost in the absence of the drug.
In the presence of drug, a resistant genotype is expected to be at an
advantage compared to less resistant genotypes. In the absence of drug,
however, resistant genotypes may be at a disadvantage compared to their
sensitive counterparts. Strategies to control the dissemination of drug
resistance by restricting the use of antimicrobial agents
(27) implicitly assume that resistant genotypes have
reduced fitness relative to their sensitive counterparts in the absence
of drug (38). A fitness cost of drug resistance has been
demonstrated in a number of experiments when resistance genes are
introduced into bacteria (20). The composite of different mutations conferring resistance among different genetic backgrounds may
result in variation in the magnitude of the cost of resistance, as was
the case with resistance to rifampin in Bacillus subtilis (11). Costs of resistance are anticipated to decline
during subsequent evolution as natural selection continues to favor
genotypes with a fitness advantage. For example, compensatory mutations reducing the cost of resistance have been identified in recent experimental studies with diverse microbes, including human
immunodeficiency virus, Salmonella enterica serovar
Typhimurium, and Escherichia coli during evolution both in
the presence and in the absence of drug selection (reviewed by Levin et
al. [25]). In several studies, the evolved, resistant
microbes actually achieved superior fitness relative to that of the
parental drug-sensitive strain even in the absence of drug (9,
31). Adaptations reducing the cost of resistance may create a
genetic background where reversion to the ancestral drug-sensitive
state is virtually precluded due to a selective disadvantage conferred
on sensitive alleles in that background (38).
The focus of this study was the fungus C. albicans. The 12 initially identical experimental populations of C. albicans whose fitness we examine here were established in a
previous study (12) in which we monitored adaptation to
inhibitory concentrations of fluconazole over 330 generations. Each
population propagated in the presence of drug evolved resistance as
measured by an increase in the MIC of fluconazole during the course of
the experiment. These increases in resistance followed strikingly
different trajectories among populations. These populations also showed
various patterns of overexpression of four genes known to be associated
with azole resistance, accompanied in some cases by changes in the
genome likely to have been selectively neutral. The different
trajectories were attributed to the randomness of various kinds of
resistance mutations occurring under the specified conditions of
population size and culture regime, as well as the temporal sequence in
which these mutations may have occurred.
The specific objectives of the present study were (i) to measure the
change in fitness among the six experimental populations of C. albicans evolved in the presence of fluconazole and to determine any cost of resistance in the absence of fluconazole, (ii) to measure
the change in fitness among the six control populations evolved in the
absence of fluconazole, and (iii) to characterize the relationships
among the level of drug resistance, as measured by standard laboratory
MIC tests, standard growth parameters, and fitness in the presence of
drug. We found that fitness diverged among the initially identical,
replicate populations and that any apparent cost of resistance tended
to decrease with further evolution. Furthermore, there were no simple,
direct relationships among fitness in competition assays, MICs of
fluconazole, and various cell growth parameters.
Strains, culture conditions, and determination of MICs.
The
evolutionary history of the experimental populations of C. albicans characterized in this study has been previously described (12). Briefly, 12 populations of C. albicans
were founded from a single colony and were serially propagated for 330 generations (~100 days) in RPMI 1640 medium (29). One
milliliter from each overnight culture was serially transferred into 9 ml of fresh medium daily, and cultures were grown at 35°C with
constant agitation. Six populations were grown in the absence of drug
(N1 to N6) and six populations (D7 to D12) were grown in fluconazole
(Roerig-Pfizer Inc., New York, N.Y.) at a concentration twice the most
recently measured MIC for that population. Despite the drop in MIC that occurred with three populations (D7, D10, and D12) during the experiment, the drug concentrations were never reduced. MIC tests for
populations D8 and D10 from generation 260 showed significant growth
above the MIC to 64 µg/ml (this phenotype is referred to as a
trailing endpoint [28]); these populations were grown
with fluconazole at 128 µg/ml. Population samples were archived in 1 ml of 40% (vol/vol) glycerol containing 3% (wt/vol) trisodium citrate
at Resistance to MPA.
Resistance to mycophenolic acid (MPA) was
used as a marker to distinguish the progenitor from the evolved
populations in competition assays designed to measure relative fitness
(see below). We encountered a spontaneous mutant that was resistant to
MPA while attempting to transform the progenitor strain to resistance
to MPA with a cloned putative inosine 5-monophosphate dehydrogenase
(IMH3) allele (kindly provided by P. T. Magee and J. Beckerman, University of Minnesota; materials and conditions are
available from us upon request), which has been demonstrated to confer
resistance to MPA in C. albicans (5). This
mutant satisfied our requirements for (i) stability during growth in
the absence of MPA and (ii) the absence of any detectable impact on
fitness, the MIC of fluconazole, doubling time, or stationary-phase
cell density. While we do not know the nature of the MPA resistance
mutation, we did not detect any differences between the MPA-resistant
mutant and the progenitor in their nucleotide sequences, mRNA
expression levels, or copy numbers of the IMH3 gene (data
not shown).
Measurement of relative fitness.
The fitness of the 12 populations at generation 330 and of the generation associated with the
MIC peak for the three populations that subsequently dropped in MIC was
determined by placing each of the evolved populations in direct
competition with the progenitor, genetically marked with resistance to
MPA. Competition experiments were conducted in triplicate both in the
presence and in the absence of fluconazole, under the same culture
conditions used in the experimental evolution study. All competition
experiments were conducted in RPMI 1640 at 35°C. For the competition
experiments with populations evolved with drug that were conducted in
the presence of drug, the concentration of fluconazole used was the concentration most recently experienced by the evolved competitor during the evolution experiment (see Fig. 1). For the competition experiments with populations evolved without drug and with the unmarked
progenitor that were conducted in the presence of drug, the
concentration of fluconazole used was twice the MIC for these populations (see Fig. 3). The competing populations were first conditioned by growing each competitor from the frozen archive separately for one complete growth cycle (24 h) in RPMI 1640 medium. Cell counts of the overnight cultures, performed with a hemocytometer, were used to prepare a competition mix containing approximately equal
concentrations of the two competitors (~107 CFU/ml). One
hundred microliters of the competition mix was used to inoculate 9.9 ml
of fresh medium, and the competitors were allowed to grow together
during one standard daily growth cycle. Initial and final densities of
each replicate competition culture were determined by colony counts
from dilution plates on minimal medium (0.667% yeast nitrogen base
without amino acids, 2% D-glucose, 1.5% agar). The
initial and final densities of each competitor were determined by
transferring by means of applicators (Puritan; Hardwood Products
Company L.P., Guilford, Maine) 200 colonies from the dilution plates
from each replicate competition to minimal medium containing 10 µg of
MPA (Sigma, St. Louis, Mo.) per ml in a grid pattern. Under these
selective conditions, the genetically marked progenitor is able to grow
while growth of the other competitor is inhibited.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.2971-2978.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Divergence in Fitness and Evolution of Drug
Resistance in Experimental Populations of Candida
albicans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C. MICs were determined by the broth microdilution method
using approved standard M27-A of the National Committee for Clinical
Laboratory Standards (29).
Measurement of growth parameters. Doubling times during the exponential growth phase and stationary-phase cell densities were determined for the progenitor, the 12 populations at generation 330, and the 3 drug-resistant populations at their MIC peak, with the same culture conditions used in the competition assays. These growth parameters were determined for the progenitor at all four concentrations of fluconazole used in the competition experiments (0.5, 16, 32, and 128 µg/ml). One hundred microliters of an overnight RPMI 1640 culture of each population sample recovered from the frozen archive was inoculated in triplicate into 9.9 ml of medium both in the absence of drug and in the presence of fluconazole at the same concentration used in the competition assays. The concentration of cells was monitored with a spectrophotometer at 530 nm (Du-64 spectrophotometer; Beckman Instruments, Inc.) at 0, 2, 4, 6, 8, 10, 12, and 24 h.
Statistics. Analyses of variance (SYSTAT 5.2.1) were performed on fitness, doubling time, and stationary-phase cell density data obtained both in the presence and in the absence of drug to test for the significance of variation among populations. The significance of differences between pairs of means was evaluated using Tukey tests (SYSTAT 5.2.1) in order to identify which populations differed significantly from others. The strengths of associations between doubling time and fitness and between stationary-phase cell density and fitness were evaluated with Pearson correlation and Bonferroni-adjusted probabilities to account for multiple comparisons. Paired t tests on the number of doublings of each competitor were used to evaluate the significance of the outcome of each competition experiment.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Variation in fitness, doubling time, and stationary-phase cell
density among the experimental populations of C. albicans.
We measured fitness and growth parameters of the
12 replicate experimental populations of C. albicans founded
from a single azole-susceptible cell and reared over 330 generations,
as previously described by Cowen et al. (12). Fitness was
measured by placing each population in direct competition with the
progenitor (T118-0) genetically marked with resistance to MPA. The 16 population samples characterized included the unmarked progenitor,
generation 330 of the 6 replicate populations evolved in the presence
of inhibitory concentrations of fluconazole (D7 to D12), the generation
of the MIC peak for the 3 drug populations that subsequently dropped in
MIC (D7 at generation 260, D10 at generation 200, and D12 at generation
260), and generation 330 of the 6 replicate populations evolved in the
absence of drug (N1 to N6). The experimental populations diverged in
fitness, measured as the difference in the numbers of doublings of the
two competitors standardized by the total number of doublings in the
competition assay (Fig. 1A,
2A, 3A, and
4A). In an analysis of variance, the
variation in fitness among the 16 population samples was highly
significant (P < 0.0001) when it was measured both in
the presence of drug (F15,32 = 8.64 [the
numbers associated with the value of the F distribution are the degrees of freedom associated with variances in the numerator and
denominator, respectively]) and in the absence of drug
(F15,32 = 15.48). Doubling times of 16 population samples were determined under the same conditions used for
competition assays (Fig. 1B, 2B, 3B, and 4B). Doubling times for the
progenitor were determined at all four concentrations of fluconazole
used in the competition experiments (0.5, 16, 32, and 128 µg/ml),
providing a total of 19 population samples for growth parameter
estimates in the presence of drug. Variation in doubling time among the
population samples was highly significant (P < 0.0001), both in the presence of drug (F18,38 = 51.16) and in the absence of drug
(F15,32 = 190.71). Variation in the
stationary-phase cell density among population samples (Fig. 1C, 2C,
3C, and 4C) was also highly significant (P < 0.0001),
both in the presence of drug (F18,38 = 5.65) and in the absence of drug (F15,32 = 5.65). There was no significant correlation between doubling time and
fitness or stationary-phase cell density and fitness for populations
evolved in the presence of drug or for populations evolved in the
absence of drug when these parameters where measured in either
environment (P > 0.25 in all cases).
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Selective neutrality of the genetic marker. Selective neutrality of the genetic marker was confirmed, as there was no significant fitness difference between the progenitor (T118-0) and the genetically marked progenitor either in the presence of fluconazole (P > 0.6) (Fig. 1A) or in the absence of drug (P > 0.9) (Fig. 2A). While doubling times of the progenitor were longer and stationary-phase cell densities were lower in the presence of drug (Fig. 1B and 1C), compared to those in the absence of drug (Fig. 2B and 2C), there was no significant difference among doubling times or stationary-phase cell densities measured at the different drug concentrations (data not shown) (P > 0.2, Tukey test).
Populations evolved with drug. The populations evolved in the presence of drug diverged in fitness relative to the ancestral drug-sensitive state (fluconazole MIC, 0.25 µg/ml). In population D7 at generation 260 (D7-260; MIC, 8 µg/ml), the lack of significant difference in fitness compared to the progenitor in the presence of drug at 16 µg/ml (Fig. 1A) coupled with the fitness disadvantage in the absence of drug (Fig. 2A) indicated a cost of drug resistance. By generation 330, however, compensatory changes in population D7 eliminated the cost of resistance; D7-330 increased in fitness in the presence of drug at 16 µg/ml (Fig. 1A), with no significant difference in fitness compared to the progenitor in the absence of drug (Fig. 2A). The significant improvement in fitness in the presence of drug in population D7 between generations 260 and 330 (P < 0.05, Tukey test) was accompanied by a drop in the fluconazole MIC from 8 to 0.5 µg/ml. Neither doubling times nor stationary-phase cell densities of D7-260 and D7-330 differed significantly from each other either in the presence (Fig. 1B and 1C) or in the absence (Fig. 2B and 2C) of drug.
Population sample D8-330 significantly increased in fitness in the presence of fluconazole at 128 µg/ml (Fig. 1A). The marked progenitor actually decreased in numbers during the competition experiment with drug. D8-330 also maintained a significant fitness advantage in the absence of drug (Fig. 2A). Due to the trailing endpoint of this population (i.e., consistent growth in the MIC test plate at increasing drug concentrations above the MIC), D8 was grown at 128 µg/ml during the evolution experiment from generation 260 rather than at twice the MIC of 4 µg/ml. D8-330 had a significantly shorter doubling time in the presence of drug (Fig. 1B) (P < 0.001, Tukey test), but there was no significant difference in doubling time between the progenitor and D8-330 in the absence of drug (Fig. 2B). There was no significant difference in stationary-phase cell density between D8-330 and the progenitor when they were grown either with or without drug (Fig. 1C and 2C). D9-330 (MIC, 64 µg/ml) significantly increased in fitness at 128 µg of fluconazole per ml (Fig. 1A) but did not change in fitness compared to the progenitor in the absence of drug (Fig. 2A). D9-330 had a significantly longer doubling time than that of the progenitor (P < 0.001, Tukey test) both in the presence (Fig. 1B) and in the absence (Fig. 2B) of drug. There was no significant difference in stationary-phase cell density between the two population samples (Fig. 1C and 2C). Population D10 at generation 200 (MIC, 16 µg/ml) significantly increased in fitness at 32 µg of fluconazole per ml (Fig. 1A) but did not increase in fitness in the absence of drug (Fig. 2A). By generation 330, the fitness of population D10 had improved significantly (P < 0.005, Tukey test) in the absence of drug. D10-330 maintained a significant fitness advantage relative to the progenitor both with 128 µg of fluconazole per ml (Fig. 1A) and without drug (Fig. 2A). Accompanying this improvement in fitness, the MIC of population D10 dropped to 1 µg/ml by generation 330, although this population had been growing at 128 µg of fluconazole per ml since generation 260. There were no significant differences in doubling time or stationary-phase cell density between D10-200 and D10-330 either in the presence or in the absence of drug (Fig. 1B and C and 2B and C). Population sample D11-330 (MIC, 64 µg/ml) did not differ significantly in fitness from the progenitor either with 128 µg of fluconazole per ml (Fig. 1A) or without drug (Fig. 2A). D11-330 had a significantly longer doubling time than the progenitor (P < 0.001, Tukey test) both in the presence (Fig. 1B) and in the absence (Fig. 2B) of drug. The stationary-phase cell density of D11-330 was significantly lower than that of the progenitor only in the absence of drug (Fig. 2C) (P < 0.005, Tukey test). D12-260 (MIC, 64 µg/ml) did not differ significantly in fitness from the progenitor at 128 µg of fluconazole per ml (Fig. 1A) and was less fit in the absence of drug (Fig. 2A). By generation 330, however, the cost of resistance was eliminated, accompanied by a decrease in MIC to 4 µg/ml. D12-330 did not differ in fitness from the progenitor either in the presence of drug at 128 µg/ml (Fig. 1A) or in the absence of drug (Fig. 2A). While D12-260 and D12-330 did not differ significantly in doubling time in the presence of drug (Fig. 1B), D12-330 had a significantly longer doubling time in the absence of drug (Fig. 2B) (P < 0.001, Tukey test). There were no significant differences in the stationary-phase cell densities of the two population samples (Fig. 1C and 2C).Populations evolved without drug. In contrast to the divergence in fitness detected among the populations evolved under the selective pressure imposed by the presence of drug, the replicate populations evolved without drug showed a more uniform trend. In the presence of drug at 0.5 µg/ml (twice the fluconazole MIC for the progenitor and all of the populations evolved in the absence of drug), there was a trend toward increased fitness but the trend was not significant (Fig. 3A). In the absence of drug, four population samples (N1-330, N3-330, N4-330, and N5-330) increased in fitness (Fig. 4A) while the remaining two (N2-330 and N6-330) did not increase in fitness compared to the progenitor. There were no significant differences in doubling times or stationary-phase cell densities detected among populations N1 to N6 at generation 330, either in the presence (Fig. 3B and 3C) or in the absence (Fig. 4B and 4C) of drug. In contrast, at generation 330, N1 to N6 all had shorter doubling times relative to that of the progenitor, both in the presence of drug (P < 0.005 in all cases, Tukey test) and in the absence of drug (P < 0.05 in all cases, Tukey test).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study was designed to identify changes in fitness accompanying the evolution of drug resistance in replicate experimental populations of the pathogenic yeast C. albicans. These changes were identified by placing evolved populations and a genetically marked version of the progenitor in a common environment and measuring the reproductive output of each competitor in the mixture. Among the populations evolved in the presence of drug, the change in fitness relative to the progenitor was highly divergent, ranging from a small decrease to a large increase, when measured both in the presence and in the absence of drug. In these populations, any detectable cost of resistance was mitigated with further evolution; by the end of the evolution experiment, none of the populations evolved with drug showed a significant cost of drug resistance. Among the populations evolved in the absence of drug, the change in fitness was more uniform in both environments, with increases in most. Fitness in the competition assays was not predicted by measurements of the MICs, doubling times, or stationary-phase cell densities of the competitors in isolation. Fitness could be explained only by complex interaction between competitors.
Fitness of populations evolved in the presence of drug. In the presence of drug, a resistant genotype is expected to be at an advantage. Of the nine population samples from the lineages evolved in the presence of drug, five (D7-330, D8-330, D9-330, D10-200, and D10-330) increased in fitness over the progenitor in the presence of drug (Fig. 1A) while four (D7-260, D11-330, D12-260, and D12-330) did not. None of the five population samples that achieved this fitness advantage incurred a cost of resistance, i.e., reduced fitness in the absence of drug (Fig. 2A). Two (D8-330 and D10-330) of the five population samples maintained a significant fitness advantage over the progenitor even in the absence of drug, while three (D7-330, D9-330, and D10-200) did not. In the two populations at generation 260 (D7 and D12) that showed a significant cost of resistance, the cost was eliminated by generation 330. The compensatory changes between generations 260 and 330 were mediated by selective sweeps, i.e., mutations conferring a selective advantage increased in frequency. Two selective sweeps in population D7, one associated with the MIC peak at generation 260 and one with the endpoint at generation 330, were identified by changes in markers with no known relation to drug resistance, including loss of heterozygosity and changes in DNA fingerprints (12).
The diversification in fitness of the populations evolved with inhibitory concentrations of fluconazole is consistent with our previous interpretation (12) that the populations gained altitude by taking different routes on the adaptive landscape (43, 44). While different molecular mechanisms were implicated in azole resistance among the replicate populations, there was no direct association between molecular mechanism and fitness consequence. For example, although overexpression of the gene MDR1, encoding an efflux pump of the major facilitator family (42), was detected in population samples D9-330, D11-330, D12-260, and D12-330 (12), these population samples differed significantly in fitness (Fig. 1A and 2A). Changes in addition to the change in the level of expression of MDR1 must affect fitness in these evolved populations. While most populations evolved with drug increased in fitness relative to the progenitor in the presence of drug, populations D11 and D12 did not. How could D11 and D12 adapt to the presence of drug without showing an increase in fitness by generation 330? The successive nature of selective sweeps in the experimental populations might offer an answer. For example, if genotype B has a fitness advantage over the ancestral genotype A and genotype C has a fitness advantage over genotype B, this does not necessarily imply that genotype C will have a fitness advantage over genotype A. Epistatic interactions between adaptive mutations were offered as an explanation of a comparable successive decrease in mean population fitness detected in asexual evolving populations of the yeast Saccharomyces cerevisiae (33). Alternatively, it is possible that the selective sweeps observed in the experimental populations were the consequence of competition between more than just two competitors, as new genetic variability is continually introduced by mutation.Fitness of populations evolved without drug. In contrast to the divergence in fitness associated with the evolution of azole resistance, the increase in fitness among the populations evolved in the absence of drug was relatively uniform. In the absence of drug, adaptation to culture conditions was detected at generation 330 in four of the six populations (N1 to N6) evolved in the absence of drug (Fig. 4A). Adaptation to culture conditions may also be responsible for the nonsignificant trend towards improved fitness in five of the six populations determined in competition assays with drug (Fig. 3A). Adaptation to general environmental conditions has been well documented in a number of experimental studies with bacteria (19, 21-23, 40, 41).
The relationship between MIC and fitness.
The relationship
between drug resistance, as measured by standard laboratory MIC tests,
and fitness in the presence of drug has important implications for
predicting the response of a genotype to drug treatment. MICs
determined according to the National Committee for Clinical Laboratory
Standards protocol (29) have been used to establish
breakpoints for clinical interpretation of antifungal susceptibility
(36). The breakpoints for fluconazole MICs are as follows:
<8 µg/ml, sensitive; 8 to 32 µg/ml, susceptible dose dependent;
and 
64 µg/ml, resistant. While correlation has been observed
between fluconazole MIC and clinical outcome (reviewed by White et al.
[42]), there have been failures in treating patients
with susceptible strains and successes in treating patients with
resistant strains (37). Our results, in a simple
controlled laboratory experiment, demonstrate that MIC is not an
adequate measure of fitness in the presence of drug. There were three
clear examples of discordance between MIC and fitness. First, in
population D7, the decrease in MIC from 8 µg/ml at generation 260 to
0.5 µg/ml at generation 330 was accompanied by a significant increase in fitness measured with 16 µg of fluconazole, per ml. Second, in
population D10, the decrease in MIC from 16 µg/ml at generation 200 to 1 µg/ml at generation 330 was accompanied by no significant change
in fitness (even though the competition assay for generation 200 was in
32 µg of fluconazole per ml and the competition assay for generation
330 was in 128 µg/ml of fluconazole). Third, in population D12, the
decrease in MIC from 64 µg/ml at generation 260 to 4 µg/ml at
generation 330 was accompanied by no significant change in fitness
compared to the progenitor measured in 128 µg of fluconazole per ml.
The drop in MIC that occurred repeatedly in the experimental
populations without an accompanying reduction in fitness in the
presence of a high drug concentration may reflect differences between
conditions of the MIC test and the experimental liquid culture.
Alternatively, the drop in MIC may be a function of interactions
between mixed genotypes (evolved versus progenitor), which occur in
competitions but not in MIC tests.
The relationship between cell growth parameters and fitness. In addition to the discordance between MIC and fitness, there was no consistent relationship between cell doubling time or stationary-phase cell density and fitness relative to the progenitor for populations reared in the presence of drug. For example, population samples that did not differ from each other in either of these parameters, such as D7-260 and D7-330 or D10-200 and D10-330, differed significantly from each other in fitness (Fig. 1 and 2). These parameters do not account for the fitness advantage of D8-330 over the progenitor in the absence of drug (Fig. 2). In D9-330, fitness increased significantly in the presence of drug (Fig. 1A) despite a longer doubling time (Fig. 1B) and a stationary-phase cell density that did not differ significantly from that of the progenitor (Fig. 1C). A more consistent relationship was detected in some cases, such as with D12-260, with a decrease in fitness in the absence of drug (Fig. 2A) coupled with a significantly longer doubling time than that of the progenitor in the absence of drug (Fig. 2B) and no difference in stationary-phase cell density (Fig. 2C).
In contrast to the populations evolved with drug, the relationship between growth parameters and fitness was more predictable for the populations evolved in the absence of drug. At generation 330, among populations N1, N2, N3, N4, N5, and N6 in competitions without drug, four of the six had a fitness advantage (Fig. 4A) and all had shorter doubling times (Fig. 4B) as well as stationary-phase cell densities that did not differ significantly from that of the progenitor (Fig. 4C). In the presence of drug, the larger variances in fitness may have obscured very small increases in fitness over the progenitor (Fig. 3A), but all had shorter doubling times (Fig. 3B) and stationary-phase cell densities that were not significantly different from that of the progenitor (Fig. 3C). Despite the trend toward a relationship between growth parameters and fitness of the populations reared without drug, the correlation between doubling time and fitness or stationary-phase cell density and fitness for populations evolved with drug or evolved without drug was not significant when measured in either environment (P > 0.25 in all cases). The lack of correlation between growth parameters and fitness provides further support for the importance of interactions between genotypes in determining the outcome of competitions.Implications for the emergence and spread of drug resistance. The divergence in fitness associated with the evolution of drug resistance in experimental populations of C. albicans has important implications for the emergence and spread of drug resistance. Some resistant isolates may incur a fitness cost and consequently may decline in frequency with suspension of the use of an antimicrobial agent. Other isolates may evolve resistance, with a decrease in the frequency of the resistant type precluded by a fitness advantage maintained both in the presence and in the absence of drug. Our previous results (12) confirmed that drug resistance was remarkably stable relative to the progenitor after 50 generations of further evolution in the absence of drug. Another study (3) suggests that the frequency of drug-resistant types increases faster under constant selection than it decreases in the absence of selection. While a cost of resistance will ensure that the frequency of resistant bacteria will decline following cessation of use of a given antibiotic, they will rapidly increase in frequency upon reintroduction of the drug if even a low frequency of resistant bacteria remains (24). The paucity of fitness cost associated with the evolution of drug resistance in the experimental populations of C. albicans raises the possibility that once resistant genotypes emerge, it will be very difficult to control their spread, even with a severe reduction in drug use.
Identifying the attributes that make one genotype or species a better competitor than another in a given environment remains an important challenge in microbial ecology. The difficulty is at least partly attributable to the integration of component genes and pathways in the composite phenotype of competitive fitness (21). Our results suggest that fitness is the result of complex interactions between mixed genotypes in competition assays. Fitness in competition assays is not predicted when the competitors are evaluated separately for MIC or growth parameters such as doubling time and stationary-phase cell density. While evolution of microbial populations in an infected host may be different from that occurring in a laboratory environment (7, 10), our results are consistent with evidence from natural populations. The most common mechanisms of azole resistance identified among resistant isolates of C. albicans recovered from patients (42) were also detected in the experimental populations (12). In addition, the clonal spread of fluconazole-resistant isolates of C. albicans to patients who had never received treatment with the drug (45) might be expected given the negligible cost of resistance in the experimental populations. Even in this simple laboratory system, however, we found complex interactions between mixed genotypes in competition. In the host, the potential for interactions among genotypes of C. albicans is far greater due to the additional complexities of the host immune system, other microbes, and heterogeneity among different anatomical sites.| |
ACKNOWLEDGMENTS |
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We thank the reviewers V. Perrot and P. T. Magee for helpful suggestions.
This work was supported by a grant-in-aid from Pfizer Canada Inc. and research grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to L.M.K. and J.B.A. L.E.C. was supported by an Ontario graduate scholarship and an NSERC postgraduate scholarship.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Botany, University of Toronto at Mississauga, 3359 Mississauga Rd. North, Mississauga, Ontario, Canada L5L 1C6. Phone: (905) 828-5338. Fax: (905) 828-3792. E-mail: lcowen{at}credit.erin.utoronto.ca.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, May 2001, p. 2971-2978, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.2971-2978.2001
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