INTRODUCTION

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The past decade has witnessed a significant increase in the prevalence of resistance to antibacterial and antifungal agents. Resistance to antimicrobial agents has important implications for morbidity, mortality and health care costs in U.S. hospitals, as well as in the community. Hence, substantial attention has been focused on developing a more detailed understanding of the mechanisms of antimicrobial resistance, improved methods to detect resistance when it occurs, new antimicrobial options for the treatment of infections caused by resistant organisms, and methods to prevent the emergence and spread of resistance in the first place. Most of this attention has been devoted to the study of antibiotic resistance in bacteria for several reasons: (i) bacterial infections are responsible for the bulk of community-acquired and nosocomial infections; (ii) the large and expanding number of antibacterial classes offers a more diverse range of resistance mechanisms to study; and (iii) the ability to move bacterial resistance determinants into standard well-characterized bacterial strains facilitates the detailed study of molecular mechanisms of resistance in bacterial species.

The study of resistance to antifungal agents has lagged behind that of antibacterial resistance for several reasons. Perhaps most importantly, fungal diseases were not recognized as important pathogens until relatively recently (2, 148). For example, the annual death rate due to candidiasis was steady between 1950 and about 1970. Since 1970, this rate increased significantly in association with several changes in medical practice, including more widespread use of therapies that depress the immune system, the frequent and often indiscriminate use of broad-spectrum antibacterial agents, the common use of indwelling intravenous devices, and the advent of chronic immunosuppressive viral infections such as AIDS. These developments and the associated increase in fungal infections (5) intensified the search for new, safer, and more efficacious agents to combat serious fungal infections.

For nearly 30 years, amphotericin B (Fig. 1), which is known to cause significant nephrotoxicity, was the sole drug available to control serious fungal infections. The approval of the imidazoles and the triazoles in late 1980s and early 1990s were major advances in our ability to safely and effectively treat local and systemic fungal infections. The high safety profile of triazoles, in particular fluconazole (Fig. 1), has led to their extensive use. Fluconazole has been used to treat in excess of 16 million patients, including over 300,000 AIDS patients, in the United States alone since the launch of this drug (124a). Concomitant with this widespread use, there have been increasing reports of antifungal resistance (115). The clinical impact of antifungal resistance has been recently reviewed (115). Also, three excellent reviews concentrating on various aspects of antifungal resistance including clinical implications have been published recently (27, 86, 153). Therefore, the clinical impact of resistance is not covered in this review. Instead, our goal is to focus on the molecular mechanisms of antifungal resistance. Since mechanisms of antibacterial resistance are characterized in considerably more detail than those of antifungal resistance, we have chosen to use well-described mechanisms of bacterial resistance as a framework for understanding fungal mechanisms of resistance, insofar as such comparisons can be logically applied. In so doing, we hope to make an understanding of antifungal resistance mechanisms accessible to those who use these agents clinically, as well as those who may wish to study them in the future.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Structures of representative antifungal agents.

Although it is our premise that a comparison between mechanisms of resistance to antifungals and antibacterials is a useful way of developing a perspective on antimicrobial resistance in the two kingdoms, the comparison is necessarily limited by several factors. First, the structures of fungi and bacteria differ in very significant ways (such as the diploid nature of most fungi and the longer generation time of fungi compared to bacteria), and the available antibacterial and antifungal agents target structures and functions most relevant to the organisms to be inhibited. For example, many antibacterial agents inhibit steps important for the formation of peptidoglycan, the essential component of the bacterial cell wall. In contrast, most antifungal compounds target either the formation or the function of ergosterol, an important component of the fungal cell membrane. Nevertheless, there are important parallels between the mechanisms by which fungi develop resistance to ergosterol biosynthesis inhibitors and bacteria develop resistance to anti-cell wall agents. Regarding other types of bacterial resistance, comparisons are limited by the fact that antifungal analogues of many classes of antibacterial agents (protein synthesis inhibitors such as aminoglycosides, macrolides, and tetracyclines; topoisomerase inhibitors such as fluoroquinolones; and metabolic pathway inhibitors such as trimethoprim-sulfamethoxazole) do not exist. Conversely, antifungal nucleoside analogues such as 5-fluorocytosine (5FC) have no counterparts among clinically available antibacterial agents (although they are represented among the antiviral compounds). As such, the capacity for fungi to develop ribosomal resistance or topoisomerase mutations is unknown, as is the capacity for bacteria to develop resistance to nucleoside analogues. Interestingly, the antibacterial RNA polymerase inhibitor rifampin, which demonstrates no intrinsic activity against fungi, appears quite active against several fungal species when used in combination with amphotericin B (8). This synergistic activity has been attributed to increased uptake of the rifampin into the fungal cell resulting from the action of amphotericin B on the fungal membrane. Similar synergism has been demonstrated between amphotericin B and 5FC by Polak et al. (109), using murine models of candidiasis. The mechanism for this synergism has been postulated by some investigators to be improved uptake of the 5FC as a result of membrane disorganization due to amphotericin B-ergosterol interaction (87). This synergistic effect resembles the postulated mechanism of bactericidal synergism between cell wall-active agents and aminoglycosides against enterococci, in which increased intracellular concentrations of streptomycin are detectable when streptomycin is combined with penicillin in vitro against Enterococcus faecalis (91). In contrast to the notion that amphotericin B improves the uptake of 5FC, data obtained by Beggs et al. (7) suggest that these two agents act sequentially and not in combination against Candida albicans to affect synergy.

The second limitation to the comparison between antifungal and antibacterial resistance mechanisms is that some general classes of resistance mechanisms have not yet been identified in fungi. Resistance to antibacterial agents results from modification of the antibiotic, modification of the antimicrobial target, reduced access to the target, or some combination of these mechanisms. Antibiotic modification is arguably the most important mechanism of resistance to the -lactam (-lactamases) and aminoglycoside (aminoglycoside-modifying enzymes) classes of antibacterials. In contrast, although there has been a single, unconfirmed report of degradation of nystatin by dermatophytic fungi (13), there are no data to suggest that antibiotic modification is an important mechanism of antifungal resistance. On the other hand, accumulating evidence suggests that both target alterations and reduced access to targets (sometimes in combination) are important mechanisms of resistance to antifungal agents. These mechanisms have important parallels in antibacterial resistance.

The third limitation to the comparison is that our knowledge of genetic exchange mechanisms in bacteria is far more advanced than our knowledge of exchange mechanisms, if they exist, in fungi. Bacteria employ an extensive repertoire of plasmids, transposons, and bacteriophages to facilitate the exchange of resistance and virulence determinants among and between species. As a result, the opportunity for rapid emergence of high-level resistance and the potential for emergence and dissemination of resistance even in the absence of direct selection by specific antimicrobial pressure abound. Conversely, antifungal resistance described to date generally involves the emergence of naturally resistant species (as in the increasing importance of Candida krusei in areas of extensive use in certain medical centers) or the progressive, stepwise alterations of cellular structures or functions to avoid the activity of an antifungal agent to which there has been extensive exposure.

The final important limitation in comparing mechanisms of resistance to antifungals and antibacterials lies in the availability of standardized bacterial strains and plasmids for use in the study of antimicrobial resistance in bacteria. This availability allows the isolation of resistance determinants in well-characterized backgrounds, so that the specific contribution of different resistance mechanisms can be assessed. The availability of well-characterized strains and systems for DNA delivery allows a much more rigorous approach to the study of the genetics and physiology of bacterial resistance mechanisms, in comparison to fungal mechanisms of resistance. For example, the first step in analyzing plasmid-mediated -lactamases in bacteria is the transfer of the plasmid to a well-characterized strain, generally Escherichia coli. In this way, complicating mechanisms such as membrane alteration can be controlled and reasonable comparisons of the level of resistance conferred by different -lactamases can be made. The fact that similar standardized systems are not available in fungi means that the study of resistance almost always occurs in the clinical strains themselves, making an assessment of the precise contribution of individual resistance mechanisms to the phenotypic expression of resistance difficult and often impossible.

Since clinicians tend to depend, predominantly, on MIC breakpoints to determine the susceptibility of an isolate, it is important to briefly update the status of antifungal susceptibility testing. Unlike antibacterial agents, for which standardized susceptibility testing methods with interpretive breakpoints are well established, acceptable testing methods and tentative breakpoints for antifungal agents have only recently been suggested (116) (Table 1). Even these breakpoints are limited to yeast, particularly Candida, and fluconazole and itraconazole. Suggested breakpoints were based on the analysis of in vitro MIC (involving 883 isolates) and clinical data from 729 patients treated with these two antifungal agents. Based on currently available data, it appears that appropriately measured resistance in vitro (MICs of fluconazole and itraconazole of 64 and 1 µg/ml, respectively) often correlates with failure in the treatment of clinical infections (116).

It is important to emphasize that the predictability of clinical success or failure in response to the administration of a specific antifungal (or antibacterial) agent depends on more than just its MIC for the infecting organism. Other factors (clinical status of the patient, presence of foreign material, location of infection) have a significant impact on the likelihood of therapeutic success. The role of these other factors in determining the clinical outcome of fungal infections is best illustrated by a study investigating the correlation of MIC and clinical outcome from patients with AIDS-associated cryptococcal meningitis (155). In this study, our group determined the fluconazole MIC against Cryptococcus neoformans isolates from 76 patients enrolled in two collaborative clinical trials (75, 89). When the MIC was correlated with the clinical response, several interesting observations were made. First, a statistically significant correlation between MIC data and clinical success or failure was apparent (P = 0.012). Further, upon multivariate analysis, it became clear that use of 5FC and the presence of a positive blood culture at the time of study entry also had a significant impact on response (155). When the patients were subdivided into these four possible groups (did/did not receive 5FC, did/did not have a positive blood culture), a distinct and strong correlation of MIC with outcome became apparent. For example, the predicted probability of treatment failure for a patient who did not receive 5FC, had a negative blood culture, and an isolate with a fluconazole MIC of 16 µg/ml was over 40%. This probability decreased to roughly 10% if the patient received 5FC. The need to consider other factors to predict clinical outcome, in addition to MICs, was also emphasized by the findings of others (115), who showed that host factors, such as the presence of an indwelling catheter, influenced clinical outcome.

Understanding the mechanism(s) of action of different antimicrobial agents is an important prerequisite to understanding mechanisms of resistance. In fact, in many cases an elucidation of resistance mechanisms has allowed or enhanced our understanding of specific mechanisms of action. We therefore combine our discussions of mechanisms of action and resistance to individual antimicrobial classes, although the bulk of our attention is focused on mechanisms of resistance. Readers interested in a more in-depth discussion of the mechanisms of action of different antifungal agents are referred to thorough reviews (35, 36, 48, 52, 118, 124).

The three major groups of antifungal agents in clinical use, azoles, polyenes, and allylamine/thiocarbamates, all owe their antifungal activities to inhibition of synthesis of or direct interaction with ergosterol. Ergosterol is the predominant component of the fungal cell membrane (104).

(i) Mechanism of action. The first reports of the antifungal properties of N-substituted imidazoles were published in the late 1960s (55, 125). These original compounds, such as miconazole and econazole, and those that followed, such as ketoconazole, fluconazole, and itraconazole, proved to be important drugs for combating human fungal infections. The clinical efficacy and safety of fluconazole in particular has resulted in widespread use. The resultant emergence of resistance to azoles has intensified the search for new compounds that are active against resistant organisms (29, 47, 58, 76, 93, 94, 106, 129, 133, 136, 156). A review of the abstracts presented at the 1995 and 1996 Interscience Conference on Antimicrobial Agents and Chemotherapy revealed that 10 azole-related agents are currently under development for the treatment of fungal infections.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Ergosterol biosynthetic pathway. Steps at which various antifungal agents exert their inhibitory activities are shown. TERB, terbinafine; FLU, fluconazole; ITRA, itraconazole; VOR, voriconazole.

(ii) Mechanisms of resistance to azoles. As noted above, there are as yet no reports of modification of azole antimicrobials as a mechanism of resistance. Resistant strains therefore either exhibit a modification in the quality or quantity of target enzyme, reduced access to the target, or some combination of these mechanisms. These mechanisms are discussed in detail below and are summarized in Fig. 3 and Table 2.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Mechanisms by which microbial cells might develop resistance. 1, The target enzyme is overproduced, so that the drug does not inhibit the biochemical reaction completely. 2, The drug target is altered so that the drug cannot bind to the target. 3, The drug is pumped out by an efflux pump. 4, The entry of the drug is prevented at the cell membrane/cell wall level. 5, The cell has a bypass pathway that compensates for the loss-of-function inhibition due to the drug activity. 6, Some fungal "enzymes" that convert an inactive drug to its active form are inhibited. 7, The cell secretes some enzymes to the extracellular medium, which degrade the drug.

View larger version (28K): [in this window] [in a new window]   FIG. 4.   Relationship between MIC, fluconazole dose, and emergence of resistance in oropharyngeal candidiasis. , MIC of fluconazole for the clinical isolate; , effective daily dose of fluconazole. MICs are represented on the secondary y axis, in logarithmic scale. Boxes above the graph represent genetic changes identified at each stage. Based on data from references 114 and 151.

(i) Mechanism of action. From the 1950s until the discovery of the azoles, polyene antifungal agents such as amphotericin B represented the standard of therapy for systemic fungal infections (132). There is an association between polyene susceptibility and the presence of sterols in the plasma membrane of the cells. All organisms susceptible to polyenes, e.g., yeasts, algae, and protozoa, contain sterols in their outer membrane, while resistant organisms do not (97). The importance of membrane sterols for polyene activity is also supported by earlier studies, where it was shown that fungi can be protected from the inhibitory action of certain polyenes by the addition of sterol to the growth medium (41, 73, 159). It was suggested that this effect is due to a physicochemical interaction between added sterols and the polyenes, which prevents the drug from binding with the cellular sterols. The interaction between the sterols and polyenes is further supported by direct spectrophotometric evidence that adding sterols to aqueous solutions of the polyene filipin or nystatin decreases the UV absorbance significantly (73), suggesting a direct interaction between the added sterol and the antifungal agent (69, 98).

View larger version (69K): [in this window] [in a new window]   FIG. 5.   Schematic representation of the interaction between amphotericin B and cholesterol in a phospholipid bilayer. (A) The conducting pore is formed by the end-to-end union of two wells or half pores. Adapted from reference 21 with permission of the publisher. (B) Molecular orientation in an amphotericin B-cholesterol pore. The dotted lines between the hydrocarbon chains of phospholipids represent short-range London-van der Waals forces. The dashed lines represent hydrogen bonds formed between amphotericin B and cholesterol molecules.

(ii) Mechanism of resistance to polyenes. Despite more than 30 years of clinical use, resistance to polyene antibiotics, such as amphotericin B and nystatin, is rare, with resistant isolates being confined mostly to the less common species of Candida, such as C. lusitaniae, C. glabrata, and C. guilliermondii (84). Fryberg (31) suggested that development of resistance occurs by selection of naturally occurring resistant cells, present in small numbers in the population. These naturally resistant cells produce modified sterols that bind nystatin with lower affinity. The growth rate in the presence of nystatin is therefore dependent upon the normal growth rate (in the absence of nystatin) and the rate at which nystatin causes cell membrane damage. This latter rate is presumed to be a function of the affinity of nystatin for the membrane sterols: the greater the nystatin-sterol affinity, the greater the rate of membrane damage. As a corollary, one would expect each resistant strain to exhibit slower growth than its more susceptible parent. A difference between resistant and susceptible strains was in fact observed (31). The fact that resistance to polyenes is gradually lost after serial passage on media devoid of nystatin presumably represents repopulation of the culture by cells producing sterols with a higher affinity for nystatin. The molecular genetics underlying these shifts in sterol content are not well worked out. Athar and Winner (4), however, have suggested that resistance results from mutation rather than selection.

(i) Mechanism of action. Allylamines, such as terbinafine and naftifine, have been developed as a new class of ergosterol biosynthetic inhibitors that are functionally as well as chemically distinct from the other major classes of ergosterol-inhibiting antifungal agents (118, 119). Terbinafine (Fig. 1) is highly effective against dermatophytes in vivo and in vitro. A recent study of terbinafine by the National Committee for Clinical Laboratory Standards M27 method showed that its geometric mean MIC against 179 clinical isolates of C. albicans was 1.2 µg/ml (61, 118). Furthermore, preliminary evidence from our group and from Ryder and coworkers indicates that terbinafine has good activity against at least some azole-resistant C. albicans strains (61, 118). By using the same assay system, terbinafine appears highly active against Cryptococcus neoformans (118). Studies investigating the efficacy of this agent against disseminated candidiasis in an animal model are under way.

(ii) Mechanism of resistance to allylamines. Although clinical failure has been observed in patients treated with terbinafine, allylamine resistance in association with clinical use of terbinafine and naftifine has not been found in human pathogenic fungi. However, with the increased use of this agent, resistance may be expected, since Vanden Bossche et al. (141) have reported a C. glabrata strain that became resistant to fluconazole and expressed cross-resistance to terbinafine. Other investigators report that CDR1 can use terbinafine as a substrate (122). The machinery to develop resistance to allylamines is therefore already present in some fungal species.

The fungal cell wall contains compounds, such as mannan, chitin, and - and -glucans, that are unique to the fungal kingdom. Since these components are not found elsewhere in nature, they have been identified as possible targets that provide selective toxicity advantages (48). Our knowledge of the cell wall composition of medically important fungi comes mainly from studies conducted with C. albicans. The cell wall of this yeast is a multilayered structure composed of chitin, -glucan and mannoprotein, with the last two constituents making up to 80% of the wall mass (16, 110, 134). The outer layers are composed of mannan, mannoprotein, and -(1,6)-glucan, while the inner layers are predominantly -(1,3)-glucan and chitin with some mannoprotein (135).

A number of compounds that have the ability to affect the cell walls of fungi have been discovered and described over the past 30 years (48). We will concentrate in this review on glucan synthesis inhibitors only, since at least one antifungal agent that belongs to this class of compounds is being evaluated in clinical trials (MK-0991, being developed by Merck & Co.). Chitin synthesis inhibitors, such as nikkomycins, have been extensively investigated, but no product has been commercially developed.

Inhibitors of glucan synthesis. Of the three groups of compounds (aculeacins, echinocandins, and papulacandins) that are specific inhibitors of fungal 3-glucan synthase, only echinocandins (Fig. 1) are being actively pursued in clinical trials to evaluate their safety, tolerability, and efficacy against candidiasis. Echinocandins, which are lipopeptides, have fungicidal activity both in vitro and in vivo against Candida and Aspergillus species (15, 138, 147).

(i) Mechanism of action. -Glucan inhibitors act as specific noncompetitive inhibitors of -(1,3)-glucan synthetase, a large (210-kDa) integral membrane heterodimeric protein (48). Treatment of fungi with these compounds inhibits the synthesis of the structural glucan component without affecting nucleic acid or mannan synthesis (90, 137). Inhibitors of glucan synthesis also have secondary effects on other components of intact cells including a reduction in the ergosterol and lanosterol content and an increase in the chitin content of the cell wall (107). Inhibition of -(1,3)-glucan synthetase results in cytological and ultrastructural changes in fungi characterized by growth as pseudohyphae, thickened cell wall, and buds failing to separate from mother cells. Cells also become osmotically sensitive (15, 139), with lysis being restricted largely to the growing tips of budding cells (12).

(ii) Mechanism of resistance to glucan synthesis inhibitors. Since clinical use of glucan synthesis inhibitors has not occurred, resistant mutants resulting from clinical therapy are not available. Therefore, knowledge of mechanisms of glucan synthesis inhibitors resistance is based entirely on analysis of laboratory-derived mutants. The following discussion is based on the results of laboratory mutation experiments reported by Kurtz and coworkers (70, 71), who analyzed resistant mutants of S. cerevisiae. The target of lipopeptides, including echinocandins, is glucan synthase (a heterodimeric enzyme), which in S. cerevisiae is encoded by FKS1 and RHO1. S. cerevisiae also contains another gene, FKS2, which is highly homologous to FKS1. Mutations in the FKS1 gene confer high-level in vitro resistance to lipopeptides. Low-level resistance (<10-fold) is associated with mutations in another cell wall synthesis gene, GNS1, that encodes an enzyme involved in fatty acid elongation. Mutations in FKS2 gene do not confer resistance. Additionally, activation of MDR-like genes or selection of pathway bypass mutations does not seem to be important as resistance mechanisms to the lipopeptides. Finally, since lipopeptides do not traverse the cell membrane, entry mechanisms may not play a role in their action and thus probably cannot play a role in the response of the fungal cell to them. These findings, taken together with the low rate of mutation (10⁸) per generation of fungal cells, suggest that at least in vitro, S. cerevisiae develops resistance to lipopeptide antimycotic agents via mutations that alter the protein encoded by FKS1, which is the main target of the inhibitor and is presumed to be the catalytic component of the fungal cell wall glucan synthase.

(i) Mechanism of action. 5FC is a fluorinated pyrimidine with inhibitory activity against many yeasts, including Candida and Cryptococcus neoformans. The initial promise of this agent has been diminished by the high prevalence of primary resistance in many fungal species. Two surveys of C. albicans conducted by Stiller et al. (130) and Defever et al. (21) provided estimates of resistance frequencies. The majority of the candidal isolates studied were susceptible (60 and 57%), but significant percentages were partially resistant (36 and 37%) or highly resistant (4 and 6%). Today, 5FC is used in combination with other antifungals, such as amphotericin B and fluconazole, but only rarely as a single agent.

(ii) Mechanism of resistance to 5-fluorocytosine. 5FC resistance mechanisms have been fully investigated and reviewed in depth (59, 149). In principle, resistance to 5FC may result from decreased uptake (loss of permease activity) or loss of enzymatic activity responsible for conversion to FUMP. Although resistance due to decreased 5FC uptake has been found in S. cerevisiae and C. glabrata, this mechanism does not seem to be important in C. albicans or Cryptococcus neoformans (63, 149).

(a) Correlation with antibacterial resistance. Although there are no nucleoside analogues among antibacterial compounds, there are agents that require chemical modification for activity inside the bacterial cell. Among these is metronidazole, a 5-nitroimidazole molecule whose activation depends on reduction of the nitro group in the absence of oxygen. Resistance to metronidazole is rare (perhaps because of its relatively infrequent use in comparison to other agents) and is believed to be due to decreased uptake or reduced rate of reduction (26). A more relevant comparison involves resistance of herpes simplex virus to the antiviral compound acyclovir. This agent is phosphorylated intracellularly by virus-encoded thymidine kinase. Acyclovir monophosphate then becomes converted to acyclovir triphosphate by cellular enzymes, at which point the molecule becomes integrated into replicating viral DNA, where it acts as a chain terminator. The most common mechanism of resistance to acyclovir is either a deficiency or altered substrate specificity of the viral thymidine kinase (50). These alterations result in the failure to convert acyclovir to its active form inside the cell.

It is generally accepted that a difference in the pathogenicity and resistance pattern of various candidal species exists. For example, C. albicans is both more pathogenic and more susceptible to antifungals than C. krusei (115). This raises the question whether there is a correlation between the ability of an organism to cause infection and its resistance to antifungals.

A number of investigators have attempted to answer this question. Anaissie et al. (1) evaluated the pathogenicity of C. krusei in normal and immunocompromised mice and compared its virulence to C. albicans. Unlike C. albicans, a combination of a large inoculum and immunosuppression was needed to establish severe infection. A high inoculum was also required to establish hematogenously disseminated candidal infection in a neutropenic guinea pig model (38). These data underline the low pathogenicity of C. krusei. In another study, Graybill et al. (43) demonstrated decreased virulence of serial C. albicans isolates with increasing fluconazole MICs in a mouse model of systemic candidiasis. In another study by the same group (42), a murine model of systemic candidiasis was used to assess the virulence of serial C. albicans strains (obtained from five patients with 17 episodes of oropharyngeal candidiasis) for which the fluconazole MICs were increasing. The fluconazole MICs for these isolates exhibited at least an eightfold progressive increase from susceptible (MIC < 8 µg/ml) to resistant (MIC > 16 µg/ml). When the virulence of these isolates was tested in the animal model, a fivefold progressive decrease in the dose, accounting for a 50% mortality rate, was noted. Consistent with a reduction in virulence of the serial isolates was the finding that a decreased fungal burden in the kidneys occurred in mice challenged with two of three resistant strains. Therefore, there is a suggestion from animal studies with species that are innately resistant, such as C. krusei strains and C. albicans isolates that have attained resistance, that the presence of resistance is correlated with diminished virulence. However, establishing a direct cause-and-effect relationship requires more investigation.

There is evidence that virulence is an intrinsic trait related to species-specific genetic determinants. Studies show that unlike C. albicans, C. krusei lacks virulence determinants. Therefore, virulence is not associated with resistance or susceptibility of an organism per se. Data from our group and others show that C. krusei adheres poorly to host cells (epithelial and endothelial) as well as to nonbiological surfaces (30, 120). Moreover, C. krusei-mediated endothelial-cell damage requires a longer incubation period and higher initial inoculum compared to C. albicans (1 × 10⁶ and 2 × 10⁵ cells, respectively) (30). Additionally, C. krusei showed less invasiveness of dorsal tongue mucosal cultured cells than did C. albicans or C. tropicalis (120). These characteristics seem to be maintained across different C. krusei strains, suggesting that virulence attenuation is a general characteristic of C. krusei and is not related to whether the strain is susceptible or resistant to antifungal agents. Studies comparing the virulence of a number of C. krusei strains that differ in their susceptibility to antifungals are necessary to prove this hypothesis.

Examples abound of bacterial species in which resistant variants are no less virulent than their more susceptible counterparts. Methicillin-resistant staphylococci are just as virulent as their susceptible counterparts, as are penicillin-resistant pneumococci and ampicillin-resistant E. coli strains. It is conceivable that some resistance traits, such as those mediated by the decreased expression of outer membrane proteins, may confer a selective disadvantage in the absence of antibiotic selective pressure, since these channels presumably perform other functions important for the survival of the cell. Conversely, in the setting of widespread antimicrobial use, resistance determinants may indeed behave as virulence determinants by favoring colonization, which predisposes to infection. Much more work is required to define mechanisms of bacterial virulence before a precise correlation between virulence and resistance can be made.

Strategies to avoid and suppress the emergence of antifungal resistance have not been defined. However, approaches analogous to those recommended for antibacterials (19, 78, 126) could be suggested. These measures include (i) prudent use of antifungals, (ii) appropriate dosing with special emphasis on avoiding treatment with low antifungal dosage, (iii) therapy with combinations of existing agents, (iv) treatment with the appropriate antifungal (in cases where the etiological agent is known), and (v) use of surveillance studies to determine the true frequency of antifungal resistance. It should be emphasized that data supporting the use of the suggested measures is largely lacking, and ongoing studies may provide some specific guidelines in the near future. Additionally, advances in rapid diagnosis of fungi may be helpful in reducing the use of inappropriate antifungals to treat organisms that are resistant to a particular agent. Unfortunately, progress in developing diagnostic methods specific to fungi has been slow. The recent approval of a reference method for the antifungal susceptibility testing of yeast (95) is encouraging and provides a means for performing surveillance studies.