Fungicidal Activity of Fluconazole against Candida albicans in a Synthetic Vagina-Simulative Medium

Fluconazole (FLZ) has emerged as a highly successful agent inthe management of systemic infections of Candida. Cure ratesfor symptomatic candidiasis following single 150-mg FLZ dosetherapy exceed 90%. In vitro, however, FLZ is fungistatic onlyin a narrow pH range and is not effective at vaginal pH, 4.2.This study evaluated the effect of FLZ on Candida albicans underin vitro conditions resembling the vaginal microenvironment,using vagina-simulative medium (VS). We found that FLZ was fungicidalfor C. albicans in VS, but not in other media at the same pH,4.2. In VS, FLZ was fungicidal at concentrations of $>=$ 8µg/ml and reduced viability by greater than 99.9%. Analysisof the components of VS indicated that 17 mM acetic acid, aconcentration achieved in the vagina, was responsible for thesynergistic, fungicidal effect. This effect was not seen atneutral pH. Other substrates were not effective substitutesfor acetic acid; however, short-chained carboxylic acids, glyoxylateand malonate, were effective. Most strains of C. albicans thatwere resistant to FLZ under standard conditions were killedby FLZ plus acetate. Other species of Candida were also killed,except C. krusei and C. glabrata. This study shows that FLZhas fungicidal activity for Candida species under in vitro conditionsthat mimic the vaginal microenvironment. This raises the possibilitythat FLZ may also have fungicidal effects during treatment ofvaginal candidiasis. Elucidating the mechanism by which FLZand acetate interact may disclose vulnerable pathways that couldbe exploited in drug development.

INTRODUCTION

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Introduction

Over the last two decades, there has been a dramatic increasein the rate of superficial and invasive fungal infections (4,6, 12, 13, 34, 52, 53). Since its introduction in the early1990s, fluconazole (FLZ) has emerged as the primary treatmentoption for virtually all forms of susceptible Candida infectionsin both immunocompetent and immunocompromised hosts (27, 28,34, 35, 37, 38). FLZ inhibits 14 ${alpha}$ -lanosterol demethylase in theergosterol biosynthetic pathway, resulting in the accumulationof lanosterol and toxic 14 ${alpha}$ -methylated sterols in the fungalmembrane (5, 19-21).

Under NCCLS standardized conditions, FLZ consistently demonstratesin vitro inhibition of Candida microorganisms and is consideredfungistatic only (30). Fungistatic effects are narrowly defined,requiring a low initial cell density and heavily buffered mediaat neutral pH. NCCLS conditions attempt to reflect in vivo conditionsresembling systemic infections. Accordingly, MICs generatedwith these tests show reasonable correlation with patient outcomein FLZ-treated systemic infections (1, 2, 11, 22, 39, 48, 51).It is generally assumed that FLZ is fungistatic for Candidaspecies in vivo, inhibiting growth and allowing or aiding thebody's defenses that eliminate the infection (10, 14, 17, 29,49, 50). However, treatment of vaginal candidiasis with FLZis even more effective than for other sites of infection. Typically,a single dose suffices to rapidly eliminate infection (43, 44).Paradoxically, even at very high concentrations, at vaginalpH of 4.2 FLZ is ineffective in vitro.

Thus, the main objective of this paper was to determine whetherfactors present in the vaginal microenvironment are synergisticwith FLZ. In particular we wanted to determine if FLZ, basedon its clinical efficacy, was fungicidal under these conditions.To approach this, we used a synthetic medium with physical andchemical properties closely resembling those of vaginal secretionsfrom healthy nonpregnant premenopausal women (32). We show thatFLZ is fungicidal under these conditions and that acetic acid,present at concentrations found in the vagina, is responsiblefor this effect.

MATERIALS AND METHODS

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Materials and Methods

Organisms. Candida albicans strains used in these studies are listed inTable 1. Our Saccharomyces cerevisiae isolate was ATCC 22244.Organisms were stored in 25% glycerol in 0.5% yeast extract(Difco), 2% Proteose Peptone (Difco), and 1% glucose (YPD) at-20°C, or at -70°C for long-term storage. Cells werestreaked for single-colony isolation on YPD plates, and a freshcolony was inoculated into YPD broth and incubated at 37°Covernight with shaking at 200 rpm. These overnight cultureswere the source of cells used to inoculate various media toassess cell growth and death under various growth conditionsat 37°C.

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TABLE 1. MICs to FLZ and viabilities of C. albicans strains in YNB plus acetic acid plus FLZ

Drugs and reagents. FLZ (Pfizer-Roerig, Inc., New York, N.Y.) was prepared from100-mg tablets suspended in distilled water to a final stockconcentration of 2 mg/ml and filter sterilized.

Culture media, MICs, and viability assays. Vagina-simulative medium (VS) consists of 3.5 g of NaCl/liter,1.4 g of KOH/liter, 0.22 g of Ca(OH)₂/liter, 18 mg of bovineserum albumin/liter, 2.2 g of 90% lactic acid/liter, 1 g ofglacial acetic acid/liter, 0.32 g of 50% glycerol/liter, 0.4g of urea/liter, and 5 g of glucose/liter, adjusted to pH 4.2using concentrated HCl or 40 mM NaOH, as described elsewhere(32). Yeast nitrogen broth (YNB) was made up of 6.7 g of yeastnitrogen base/liter without amino acids (Difco, Detroit, Mich.),10 g of glucose/liter, and pH adjusted to 4.2 using concentratedHCl or 40 mM NaOH. Roswell Park Memorial Institute-1640 medium(RPMI 1640) with L-glutamine, without bicarbonate (Invitrogen,Carlsbad, Calif.) was buffered and adjusted to pH 7.0 with 165mM morpholinepropanesulfonic acid (MOPS).

MIC assays were performed according to the NCCLS M27A guidelines(30) in microtiter plates, using visual estimates of 80% reductionin turbidity of resuspended cultures.

Viability assays were performed using overnight cultures ofindicated Candida isolates grown in yeast extract (Difco)-Bactopeptone (Becton Dickinson and Co., Sparks, Md.)-glucose agar(YPD). Cultures were counted in a hemocytometer and dilutedin test media, typically to 2 x 10⁴ cells/ml. These cultureswere sampled at intervals and plated onto YPD plates, dilutingto achieve about 100 CFU. Cultures with low viability were platedwithout dilution (100 µl), so that the limit of detectionof viable cells was about 10 cells per 2 x 10⁴ initial cells,or <0.0005 viable cells per initial cell (viability ratio).Colony counts were verified to represent the number of livecells, despite clumping of cells in FLZ. Clumping altered colonycounts by at most twofold. Vital dye staining with methyleneblue followed a standard protocol (16).

RESULTS

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Results

FLZ is fungicidal in VS. C. albicans isolates responded to FLZ differently when grownin VS compared to YNB. Figure 1 shows an example of our standardlaboratory strain, with a low MIC for FLZ (0.5 µg/ml).In VS plus FLZ, viability decreased 4 orders of magnitude byday 4, after an initial 1-day lag. In contrast, C. albicanscells in YNB were not killed by FLZ but instead continued tomultiply, albeit at a lower rate and level of saturation thanwithout FLZ (Fig. 1).

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FIG. 1. Effect of medium composition on the fungicidal activity of FLZ in VS. An overnight colony of C. albicans ATCC 32354 (B311) was inoculated into duplicate cultures, in either 1 ml of YNB (squares) or 1 ml of VS (diamonds), to a density of 10⁴ cells/ml. One of each was supplemented with 64 µg of FLZ/ml (closed symbols). Cultures were incubated at 37°C without shaking. The number of viable organisms in each culture was determined as described in Materials and Methods.

Cells subjected to VS plus FLZ were evaluated with vital stainmethylene blue and were found to be intact (excluding dye),even though they were not viable by plating assay. In contrast,heat-killed cells (50°C for 1 h) were unable to excludethe dye. Even though intact, the cells were unable to replicate,since the plating assay diluted the cell suspension by 250-fold,well below effective concentrations for FLZ and acetate.

VS can be considered a growth medium for C. albicans, sincecell densities reached as much as 10⁷ cells/ml in 24 h fromstarting densities of 10⁴ cells/ml. Growth rates and final celldensities were lower in VS than in YNB. In YNB, cell densitiesof 1 x 10⁸ to 5 x 10⁸ cells/ml were typically achieved in thesame time interval.

Acetic acid is required for the fungicidal effect of FLZ. We attempted various manipulations of VS to determine whichcomponent(s) of VS might contribute to the fungicidal activityof FLZ. Data in Table 2 suggest that acetic acid is the criticalcomponent. In VS medium lacking acetic acid but containing 64µg of FLZ/ml, cell proliferation continued over 6 days,while in the presence of acetic acid viabilities decreased by94 to 99%. Components found to be unimportant included lacticacid and urea.

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TABLE 2. Effects of alterations in VS on the fungicidal effect of FLZ

The critical role of acetic acid in this fungicidal effect wasdetermined independently of VS by using YNB medium. Additionof 20 mM acetic acid to YNB, pH 4.2, resulted in marked FLZ-dependentfungicidal activity (Fig. 2A). Lactic acid had no effect. However,no fungicidal interaction between FLZ and acetic acid was seenin YNB buffered to maintain a pH above 7 (Fig. 2B). Similarly,no fungicidal interaction was seen in RPMI 1640, the NCCLS standardmedium, at pH 7 (Fig. 2C). FLZ (64 µg/ml) reduced therate and extent of growth compared to controls without FLZ,clearly demonstrating that FLZ inhibited but certainly did nothalt growth, which would be expected at this high initial celldensity. The effect of acetic acid was not mediated by pH alone,since control cultures without acetic acid were adjusted toa starting pH of 4.2. These data indicate that acetic acid issufficient to account for the fungicidal effects of FLZ in VSor YNB and that the interaction does not occur at neutral pH.

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FIG. 2. pH-dependent interaction of acetic acid with FLZ. (A) YNB was adjusted to pH 4.2, without supplement (diamonds), with acetic acid (20 mM; triangles), or with lactic acid (20 mM; squares). FLZ at 64 µg/ml was added to some cultures (closed symbols). (B) YNB was buffered with 165 mM MOPS, pH 7.4. Cultures are represented as for panel A. (C) RPMI 1640, pH 7, cultures are represented as described for panel A. An overnight culture of C. albicans ATCC 32354 was inoculated into each medium to a final cell density of $~$ 2 x 10⁵ cells/ml. Viability was determined as described in the legend for Fig. 1.

Consistent with the kill curves in Fig. 2, MIC tests also showeda dramatic synergy of FLZ with acetic acid. Strains of C. albicanswhich are susceptible under NCCLS methods had MICs of 32 to64 µg/ml in YNB at pH 4.2. However, these MICs were reducedto 1 to 2 µg/ml with 20 mM acetic acid. MICs were notaltered by acetic acid when using the highly buffered, neutralmedium under NCCLS conditions, consistent with the kill curvesin Fig. 2B and C.

The fungicidal effect of FLZ and acetate in YNB was seen inother Candida strains. Table 1 shows data for eight clinicalisolates of C. albicans, including six isolates highly resistantto FLZ under standard conditions. All but one were killed inFLZ-acetate-YNB. The single surviving isolate, Ca453, was moderatelyinhibited (two doublings) but was not killed. Thus, most butnot all FLZ-resistant strains were susceptible to FLZ-acetate.

FLZ-acetate killing in YNB was observed in some but not allnon-C. albicans Candida species. Clinical isolates of C. lusitaniae,C. dubliniensis, C. parapsilosis, and C. tropicalis were killedby the combination. However, neither of two independent clinicalisolates each of C. glabrata and C. krusei and only one of twoclinical isolates of C. dubliniensis were killed under the conditionsdescribed in Table 1. S. cerevisiae was also killed by FLZ-aceticacid in YNB at pH 4.2; at this pH, 20 mM acetic acid withoutFLZ was not inhibitory.

We tested a short list of other nonfermentable carbon sourcesto see if any mimicked acetic acid's interaction with FLZ, usingYNB adjusted to a final pH 4.2. No interactions were seen with20 mM citrate, succinate, fumarate, pyruvate, or ethanol. However,malonic acid and glyoxylic acid were effective and were notinhibitory in the absence of FLZ.

Minimal fungicidal concentration of FLZ in VS medium. Using an initial cell density of 2 x 10⁴ cells/ml, we testedthe minimum concentration of FLZ required for the fungicidaleffect. Concentrations of FLZ of $>=$ 8 µg/mlresulted in complete loss of viability by day 4 (Table 3). FLZat a concentration of 4 µg/ml inhibited growth by day4 but then over the next 2 days growth resumed, so that viabilitycounts reached those of the no-FLZ control. There was no differencein the growth of cells in VS containing less than 4 µgof FLZ/ml versus cultures without FLZ (data not shown).

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TABLE 3. Minimum fungicidal concentration of FLZ in VS for C. albicans^a

In vitro conditions influencing fungicidal activity of FLZ. It is possible that FLZ is fungicidal under any conditions whichdo not support growth (46). To test this possibility, we incubatedcells in three growth-limiting environments: YNB medium lackingglucose, distilled water, and complete YNB at 4°C. Figure3A shows that these conditions limited growth. However, noneof these conditions acted in synergy with FLZ to mimic the fungicidaleffects observed in VS or YNB supplemented with acetic acid.Thus, Fig. 2 and 3A together show that growth inhibition isneither sufficient nor necessary as a precondition allowingFLZ to mediate fungicidal effects.

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FIG. 3. Effect of growth-limiting conditions on activity of FLZ. (A) An overnight colony of C. albicans ATCC 32354 (B311) was suspended in sterile water and inoculated to an initial cell density of 10⁴ to 10⁵ cells/ml into 1 ml of the following media: YNB (triangles); YNB without glucose (diamonds); distilled water (dH₂O; circles with dashed lines). Open symbols, no FLZ; closed symbols, FLZ at 64 µg/ml. Curves for dH₂O with and without FLZ are nearly superimposed. (B) Similar 1-ml cultures were divided into three sets, all with FLZ at 64 µg/ml, as follows: static cultures in 1.5-ml tubes (diamonds), shaking cultures in 50-ml tubes (triangles), and shaking cultures contained in a candle jar to exclude oxygen (squares). Duplicates of each were supplemented with 20 mM acetic acid (closed symbols). Initial and subsequent cell viabilities were determined as described in the legend for Fig. 1.

Since the vaginal environment is microaerobic, assays performedto this point used static cultures to mimic this condition.To test whether this was important for the fungicidal effect,we compared static cultures with shaking cultures. Figure 3Bshows that the fungicidal effect of FLZ-acetic acid was greatlyreduced by shaking, although killing was observed after an extendedincubation time. The effect of shaking was negated in a shakingculture that was maintained in a microaerobic environment ofa candle jar. This shows that the effect was due to availableoxygen rather than to other possible differences between shakingand static cultures.

Effect of initial cell density on the time of killing by FLZ-acetic acid. The fungicidal effects seen to this point were slow, occurringover days, not hours. We tested whether this time was dependenton the initial cell density. Figure 4 shows that killing beganearlier at lower initial cell densities. This ranged from 1day for an inoculum of 10⁴ cells/ml up to about 10 days foran inoculum of 2 x 10⁵ cells/ml. Increasing densities imposedan increasing lag time for killing to begin; once begun, therate of decrease in viability was about the same for all cultures.Cultures started at >10⁶ cells/ml showed no decrease in viabilityover the time course of this experiment.

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FIG. 4. Effect of initial cell density on fungicidal activity of FLZ. An overnight colony of C. albicans ATCC 32354 (B311) was suspended in sterile water and inoculated into a series of VS cultures (1 ml) at 10⁴ to 10⁶ cells/ml, each indicated by a different symbol. FLZ was added to all cultures to a final concentration of 64 µg/ml. Cultures were incubated at 37°C without shaking. Viability was determined as described in the legend for Fig. 1.

DISCUSSION

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Discussion

This study evaluated the effect of FLZ on C. albicans in mediumclosely resembling the vaginal microenvironment. The effectof FLZ on C. albicans in this medium was found to be radicallydifferent from that seen in conventional yeast media (YNB).In VS, FLZ was fungicidal at or above 8 µg/ml and withcell densities up to 5 x 10⁵ cells/ml. In YNB at the same initialpH, FLZ alone was not even fungistatic and its only effect wasto reduce the saturation cell density. The component in VS responsiblefor fungicidal activity is acetate. This effect did not occurat neutral pH, suggesting that only the undissociated acid,predominant below pH 4.7, is effective. Adding other agentsthat could perturb the normal balance of metabolic pathwayswas generally not effective. The effectiveness of two agents,glyoxylate and malonate, in addition to acetic acid, suggeststhat the short-chain carboxylic acids are directly responsiblefor the fungicidal interaction with FLZ.

Most tested C. albicans isolates that were resistant to FLZ,by conventional NCCLS testing, were nevertheless killed by FLZ-acetate.Only one resistant strain was moderately inhibited but not killed.This suggests that vaginal therapy is potentially effectiveeven for many strains that have high MICs for FLZ. We speculatethat the single isolate that survived did so because its resistancemechanism differs from that of the other isolates.

The present study suggests that FLZ kills C. albicans underconditions easily created in vitro. This killing is dependenton a variety of factors including cell density, pH, and presenceof acetate. The vagina, a microaerobic, low pH microenvironment,contains adequate concentrations of acetate (10 to 20 mM) toproduce the fungicidal effect seen in our in vitro assay. Densitiesof C. albicans in symptomatic vulvovaginal candidiasis are typicallyless than 10⁶ cells/total cells in the vaginal fluid. Comparisonsof static to shaking cultures indicate that FLZ-acetate synergyis more effective in a microaerobic environment.

The similarities in the in vitro conditions needed for acetate-FLZsynergy in vitro and the vaginal microenvironment are consistentwith the clinical and mycological success observed with FLZin clinical practice. Vaginal yeast cultures predictably becomenegative within 72 to 96 h of a single dose of FLZ in more than90% of women with Candida vaginitis (42, 43). However, 40 to60% of women with Candida vaginitis who experience mycologicalcure with FLZ become vaginal culture positive with the identicalstrain within 30 days of treatment (44). This implies that killingin vivo in some women is inefficient, allowing for persistenceof a small number of FLZ-susceptible organisms during treatmentand regrowth in the days following drug withdrawal. Based onour observations, it makes sense now to ask whether these womenhave reduced levels of acetate compared to women who achievelong-term cures after FLZ treatment.

Our data indicate that acetic acid promotes a fungicidal effectwith FLZ under conditions in which FLZ is minimally effectivealone: acidic pH and high initial cell densities. The relationshipbetween pH and FLZ efficacy is controversial. Most studies showthat FLZ or other azoles are not effective at acidic pH (7,26, 33), similar to our experience. In contrast, one group foundthat MICs only increased marginally at pH 5.5 compared to 7.4(15). This study, however, used modifications of the NCCLS MICmethod that compromised the effect of the initial pH: they inoculatedwith 100-fold-higher cell numbers and under-buffered the mediumwith 1/15 the buffer concentration used in RPMI 1640. In ourexperience, these two changes would result in a rapid declinein pH during incubation with or without FLZ, so that C. albicanswould respond in pH 7.4 medium as it would in the pH 5.5 medium.The most contradictory study showed that pH 4.5 medium reduces"trailing" effects, residual growth above the MIC that compromisesendpoint determination, so that FLZ appeared more effectivethan at neutral pH (25). It will be interesting to see if thesedifferences are due to strain variations or to subtle differencesin the media, e.g., 165 mM MOPS adjusted to pH 4.5. However,we have shown that under conditions in which FLZ alone is onlymarginally inhibitory, FLZ and acetic acid are fungicidal.

Other work has addressed whether FLZ can be fungicidal underspecial in vitro circumstances. Sohnle et al. have shown thatFLZ increases the rate at which C. albicans loses viabilityduring prolonged incubations (more than 7 days) in distilledwater (46, 47). Our results extend this observation, since bothVS and YNB with acetate were capable of sustaining numerouscell divisions. This shows that FLZ can be fungicidal underconditions that permit growth. Others have shown that FLZ canbecome fungicidal in combination with calcineurin inhibitorssuch as cyclosporine A (31); whether the mechanism behind thisinteraction is related to FLZ-acetate is yet to be determined.

The antifungal effects and interactions of acetate and FLZ onC. albicans has been previously studied (40, 41). Those investigatorsdemonstrated that acetate is inhibitory at 240 mM for cellsthat are deficient in 14 ${alpha}$ -lanosterol demethylase, due eitherto mutation or to the presence of FLZ. They cited, but did notshow, data suggesting the inhibition was not fungicidal (40).In contrast, we report that acetate converts a fungistatic responseinto a fungicidal response. Further, our observation documentsan effect with acetate concentrations about 10-fold lower thanthey used (40, 41). These contrasting observations are mostlikely due to key differences in the methods. They used shakingcultures while we used static cultures. We observed that theacetic acid-FLZ fungicidal effect in VS fluid was greatly reducedif the cultures were incubated with shaking. Another difference,which may account for the disparity in acetate concentrations,is that our medium was synthetic and adjusted to vaginal pH4.2, whereas they used complex YPD medium.

Shimokawa and Nakayama suggested that, since wild-type cellswere inhibited by 240 mM acetate alone below pH 5 whereas demethylase-deficientcells were inhibited even above pH 7, 14 ${alpha}$ -demethylase deficiencypermeabilizes the cell membrane to dissociated acetate abovepH 5 (40). Below this pH, the predominant, undissociated acidis permeable even to intact membranes and thus inhibits untreatedwild-type cells. The premise of this suggestion is that acetatealone is directly inhibitory, assisted by FLZ or 14 ${alpha}$ -demethylasedeficiency when above pH 5.

Our data do not determine whether FLZ potentiates killing byacetate or acetate potentiates killing by FLZ. Fungicidal effectsof acetate are not a new topic to food microbiologists, whouse acetate and other monocarboxylic acids as preservatives(9). Its effects on S. cerevisiae have been studied increasinglyof late. Acetate enters S. cerevisiae in the anionic form viaproton symporters that are glucose repressed. In the presenceof glucose, acetate enters via simple diffusion of the undissociatedacid, prevalent only below pH 4.75 (8, 9). Once inside, theacid dissociates, but enzymes for its metabolism are subjectto negative regulation by glucose. Under these conditions, acetateaccumulates and becomes toxic. One mechanism for its toxicitymay be that the increase in protons from the dissociating acidmust be pumped out by the H⁺-ATPase, effectively uncouplingthe proton motive force at the plasma membrane. Another is theinhibition of metabolic pathways by the reduced intracellularpH. Perfusion studies in S. cerevisiae show that exposure to24 mM undissociated acetic acid in an acidic medium causes adramatic drop in intracellular pH (1 to 2 units) that persistsfor several minutes and activates the H⁺-ATPase (3, 18). A flowcytometry study documented a rapid loss in cell membrane integrityfollowing exposure to higher concentrations of acetic acid (36).

These observations suggest that acetate may increase intracellularconcentrations of FLZ by increasing membrane permeability orby decreasing the ability of the cell to efflux FLZ. Conversely,energy required to efflux FLZ may impair the cell's abilityto restore intracellular pH via the H⁺-ATPase. In C. albicansacetate is ineffective under aerobic conditions, presumablythe result of depletion of acetate by respiration.

A specific mechanism for acetate toxicity in S. cerevisiae isemerging. Recent data show that acetate as low as 20 mM killsS. cerevisiae at pH 3 in a manner that suggests apoptosis: cycloheximide-sensitivechromosome condensation, DNA breaks, and exposure of phosphtidylserineon the outer layer of the plasma membrane (24). This processis accompanied by a release of cytochrome c from the mitochondria,loss of cytochrome c oxidase, and an increase in reactive oxygenspecies. Most importantly, several mitochondrial mutants didnot show this response, suggesting that, as in mammalian cells,the death response requires mitochondrial involvement (23).C. albicans cells killed by FLZ plus acetate are intriguinglyintact, not necrotic. It remains to be seen whether apoptotic-likeprocesses are induced by the combined effects of FLZ and acetatein C. albicans.

We have used the term fungicidal to indicate a significant declinein viability over the time course of several days, clearly differentthan the result seen with FLZ alone. Purists may demand thatthe term should be restricted to conditions in which 99.9% reductionin viability is achieved within 24 h. Without disputing this,recognizing and acknowledging that killing is occurring bothin vivo and in vitro is crucial in evaluating the therapeuticeffects of any antimicrobial drug. Clinical responses to single-doseoral FLZ in Candida vaginitis, achieving a negative 10% KOHexamination within 24 h and negative cultures within 48 h, stronglysupport the concept of in vivo, vaginal fungicidal activity,especially in the absence of a host polymorphonuclear leukocyteresponse.

The synergistic in vitro fungicidal activity of FLZ plus acetateagainst isolates defined as resistant by NCCLS M27A criteriais significant. Recently, Sobel et al. demonstrated that so-calledhighly FLZ-resistant strains of C. albicans were susceptiblein patients with Candida vaginitis, indicating a unique in vivoactivity of FLZ (45). It is also noteworthy that vaginal activityof FLZ is considerably enhanced compared to therapeutic activityin oral candidiasis. The latter site is composed of differentbacterial flora and organic acids and a higher pH.

In conclusion, although FLZ is considered a prototypic fungistaticagent, it has a potent fungicidal activity in the presence ofacetate under in vitro conditions that closely simulate thehuman vaginal environment. This fungicidal activity againstC. albicans is concentration dependent, pH dependent, and celldensity dependent. Of interest and reflecting clinical experience,no fungicidal activity is evident for C. krusei and C. glabrata.Understanding the synergistic interaction may be valuable inidentifying new antifungal drug targets.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine Detroit, MI 48201. Phone: (313) 577-0419. Fax: (313) 577-0419. E-mail: rakins{at}med.wayne.edu.

REFERENCES

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