We have documented that C. albicans can become resistant to azole antifungals through a mechanism that involves selection of a resistant isolate from a heterogeneous population of cells. These findings highlight the complexity of antimicrobial susceptibility testing and clinical interpretation for both bacteria and yeasts.
Recently, a heterogeneous phenotype in clinical isolates of Cryptococcus neoformans
isolated from AIDS patients with persistent meningitis was described (11
). The two series of C. albicans
isolates described here also acquired clinically significant resistance, as they became resistant to fluconazole in vivo and caused disseminated infection despite prophylactic administration of large doses of the drug in BMT recipients. The prevalence of these organisms and whether they account for clinical failures of fluconazole therapy in other situations are unknown.
Inducible drug resistance associated with a heterogeneous phenotype in C. albicans
is consistent with previous reports of heterogeneity in susceptibility phenotypes (22
) and resistance mechanisms (4
) within clonal populations of Candida
spp. Cultures of C. glabrata
, C. krusei
, and C. albicans
contain heterogeneous populations of colonies that vary in susceptibility to fluconazole and itraconazole (22
). Also, the rapid development of reversible fluconazole resistance in C. albicans
after serial growth in the presence of drug was reported (2
), although the mechanisms associated with resistance were not determined. Other investigators found that populations of C. albicans
that are experimentally induced to become resistant to fluconazole acquire resistance through multiple mechanisms, despite clonal derivation by single-cell progenitors (4
). More recently, in vitro induction of resistance in C. tropicalis
was documented, with associated increased expression of CDR1
). Our findings complement these observations by documenting that the variability in the susceptibility phenotype within cellular populations is associated with the clinical development of resistance and an inducible phenotype in vitro.
This phenomenon might be similar to the heterogeneous resistance that occurs in staphylococci and enterococci that become resistant to β-lactam and glycopeptide antibiotics (3
). In staphylococci, heterogeneous resistance appears to involve several genes and control is complex (3
). Although the mechanisms remain elusive, factors in the cellular environment (temperature and medium pH, etc.) affect the phenotypic expression of resistance (3
). One mechanism by which these organisms become resistant to β-lactam and glycopeptide antibiotics appears to be mediated by a change in cell wall components, such as penicillin-binding proteins and peptidoglycan, resulting in a decrease in susceptibility due to altered binding (and possibly cellular entry) of the drug (12
). Whether similar mechanisms are associated with heterogeneous azole drug resistance in C. albicans
is currently unknown, but it is of interest that all of the antimicrobials that have been associated with heterogeneous resistance (β-lactams, glycopeptides, and azoles) are static drugs that directly or indirectly target the cell wall or membrane.
As noted previously (22
), another possible cellular mechanism associated with heterogeneity in C. albicans
might involve phenotypic switching between susceptibility phenotypes (26
). Isolates of Candida lusitaniae
undergo a reversible phenotypic switch that is associated with the development of resistance to amphotericin B (30
). Since different strains of C. albicans
undergo reversible phase switching, this is a potential mechanism for heterogeneity between susceptibility phenotypes (26
The high frequency at which resistant cellular subpopulations are detected suggests that genetic mutations are not responsible for the generation of resistance, because mutations occur at frequencies approximating 10−6
per gene. Although we utilized a DNA fingerprinting method that has the best resolution, our conclusions are limited by the fact that no methods to fully ascertain the genetic distance between strains are available (25
). Also, our molecular studies suggest that the two series of isolates both became resistant to the drug in vivo and in vitro along with associated increased mRNA levels for CDR1
and this study). Although we do not know how the cellular and molecular makeups of these isolates otherwise compare, it is possible that the heterogeneous phenotype is specifically associated with CDR
expression, as increased expression of this efflux pump might cause a metabolic growth disadvantage that could explain the transient nature of resistance. Studies investigating the cellular phenotypes and molecular mechanisms of heterogeneous isolates are ongoing.
The development of resistance in these strains should be differentiated from the trailing phenotype that is observed in serial dilution susceptibility testing. Isolates that trail exhibit a low azole MIC after 24 h of growth and a high MIC after 48 h (18
). The isolates described here do not trail. This is an important distinction, as trailing isolates behave as susceptible strains in vivo (18
), while these heterogeneous isolates were clearly the cause of resistant disease in patients on prophylactic fluconazole. Preliminary population analyses on trailing isolates do not show a heterogeneous pattern as described in this study, but instead these isolates have a high degree of variability in colony morphology in the absence of drug (data not shown). Also, the trailing isolates do not become resistant with serial passage in the presence of drug in vitro (data not shown). These findings suggest that the heterogeneous and trailing phenotypes differ in both in vitro and in vivo behavior. While isolates that have the heterogeneous phenotype are a potential cause of clinically resistant infection, the significance of the trailing phenotype appears to be limited to being a cause of falsely elevated MICs in susceptibility testing.
The finding that isolates of C. albicans
can become resistant to fluconazole rapidly, despite MICs being within the susceptible range, has important implications for the clinical microbiology laboratory. Previous studies have noted that serial dilution susceptibility testing is less successful in predicting therapeutic success than in predicting failure (19
). Although it is likely that host factors are the major explanation, it is possible that isolate heterogeneity contributes to these limitations. Given the use of fluconazole as a single agent for candidemia in immunocompetent hosts (17
), the laboratory should be able to differentiate homogeneous, susceptible isolates from heterogeneous, susceptible isolates that can become resistant after drug exposure. Our preliminary results suggest that E tests might be useful, but further studies are necessary to characterize large numbers of heterogeneous isolates and to document the correlation between heterogeneity and clinical failure.
In summary, we have described an important mechanism by which C. albicans can become resistant to azole antifungals in immunosuppressed patients. Clinical microbiologists and clinicians should be aware of this as a potential cause of azole treatment failure. Further studies are required to determine the prevalence of this phenotype and the cellular and molecular factors involved in conferring resistance.