LY Exhibits Rapid and Potent Fungicidal Activity in Flow Cytometric Assays of Yeast Viability

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1999, p Vol. 43, No /99/$ Copyright 1999, American Society for Microbiology. All Rights Reserved. LY Exhibits Rapid and Potent Fungicidal Activity in Flow Cytometric Assays of Yeast Viability LISA J. GREEN,* PHILIP MARDER, LARRY L. MANN, LI-CHUN CHIO, AND WILLIAM L. CURRENT Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana Received 21 October 1998/Returned for modification 14 December 1998/Accepted 12 January 1999 LY is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)- -Dglucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY may be useful for antifungal therapy. New antifungal agents are urgently needed to combat an increasing number of life-threatening, systemic fungal infections. A fungicidal mode of action is highly desirable since many patients requiring antifungal therapy are immunocompromised (28). Enzymes required for fungal cell wall synthesis are excellent targets for development of nontoxic fungicidal drugs. Lipopeptide antifungals of the echinocandin class of compounds are noncompetitive inhibitors of (1,3)- -D-glucan synthase of Candida spp. (24, 25) and Aspergillus spp. (2) and are also effective against other pathogenic fungi, such as Pneumocystis carinii (1, 12). Previous studies demonstrated the utility of flow cytometric technology in detecting the rapid (3 h) fungicidal activity of cilofungin, a narrow-spectrum semisynthetic echinocandin B (ECB) analog. Those studies provided a positive correlation between flow cytometric detection of fluorescent-dye uptake and classical microbiological techniques (10) for measurement of fungicidal activity. In the present study, we used the same methods and a single-cell-deposition technique to compare the fungicidal activities of amphotericin B (AMB) and cilofungin to that of a new, more potent ECB analog, LY We also varied the in vitro drug treatment conditions to examine relationships between compound exposure time and subsequent killing of target yeasts. * Corresponding author. Mailing address: DC0444, Lilly Corporate Center, Indianapolis, IN Phone: (317) Fax: (317) Green_Lisa_J@lilly.com. MATERIALS AND METHODS Yeast cultures. Candida albicans A26 (ATCC 90234) and Saccharomyces cerevisiae YPH499 were obtained from Eli Lilly & Co. (Indianapolis, Ind.) Infectious Disease Research stock cultures and maintained on yeast-peptone-dextrose (YPD; Difco, Detroit, Mich.) agar at 4 C. For experiments, organisms were grown overnight in YPD broth in 32 C shaker bath and diluted to cells/ ml in fresh YPD broth just before use. Antifungal agents. Antifungal agents were diluted in their appropriate solvents (listed below) to prepare 1.0-mg/ml stock solutions. Cilofungin (Eli Lilly & Co.) was diluted in ethanol, LY (Eli Lilly & Co.) was dissolved in reagentgrade methanol, and crystalline AMB (Sigma, St. Louis, Mo.) was dissolved in dimethyl formamide. Fresh dilutions of compound in YPD broth were prepared for each experiment. Compound treatments. Active cultures and experimental compounds were both diluted in YPD broth (1 ml each), combined in sterile 12- by 75-mm polypropylene test tubes, and placed in a 32 C shaker bath for defined time intervals. For pulsed-compound-exposure experiments, 2 ml of phosphate-buffered saline (PBS) was added to tubes after pulsed exposure, the tubes were centrifuged for 5 min at 400 g, and the supernatant was aspirated from these pulsed cultures. The pulsed-exposure cells were then resuspended in 2.0 ml of fresh YPD broth containing a drug vehicle, whereas the continuous-exposure cells were resuspended in the existing broth containing the drug and vehicle. Tubes were returned to the 32 C shaker bath to complete a 3- or 5-h incubation period. In experiments where cultures were exposed to drug for the last 5 min of the experiment, 20 lofa100 drug solution was added to the 2-ml culture, and further processing for propidium iodide (PI) staining, sorting, or CFU assay began after the 5-min exposure. PI staining and flow cytometric analysis. For PI staining of cultures, cells were pelleted by centrifugation for 5 min at 400 g, the supernatant was discarded, and the cells were resuspended in PI (Sigma) solution (25 g/ml in PBS) for 20 min at room temperature before analysis on an EPICS Elite flow cytometer (Coulter Corp., Hialeah, Fla.). During flow cytometric analysis, individual cells were detected and categorized by their forward-angle and right-angle scatter of incident 488-nm laser light as displayed in Fig. 1A. This light scatter data plot was used to establish a gated region that excluded cell clusters ( 2 cells) from the fluorescence analysis. The light scatter gated PI fluorescence of individual cells was acquired by using a 630-nm band-pass filter (FL3) and displayed in singleparameter histograms (Fig. 1B through E). By using the cytometer s onboard software, the fluorescence histograms were integrated to determine the percentage of PI (living) and PI (dead) yeast cells. Flow cytometric cell sorting. Vehicle control or compound-treated yeast cells were single-cell sorted with a Coulter EPICS Elite ESP cell sorter as described below. For each test sample, the light scatter data were acquired and used to establish a sorting region that included only single cells and budding cells. By using the cell sorter s capabilities, single cells or budding cells were electrostatically deflected into individual microculture wells containing 200 l ofypd broth in 96-well tissue culture plates (Becton Dickinson Labware, Franklin Lakes, N.J., item 3072). These plates were incubated at 30 C without shaking for 48 h and then visually inspected for yeast growth. The percentage of single cells that formed visible colonies in wells (percent single-cell growth [SCG%]) was determined as follows: (no. of wells with a visible yeast colony after 48 h/total no. of wells inoculated by single-cell deposition) 100. CFU assay. Aliquots of cultures were removed just prior to PI staining or sorting and serially diluted in PBS. Selected dilutions were plated in duplicate on YPD agar. The plates were incubated for a minimum of 48 h at 30 C, colonies were counted, and results were expressed as the percent reduction in CFU per milliliter compared to the growth in nontreated control cultures. 830

2 VOL. 43, 1999 LY FUNGICIDAL ACTIVITY 831 a representative experiment are displayed in Fig. 1. Figure 1A shows the light scatter gating region used for analysis of red fluorescence. In Fig. 1B, the PI fluorescence for untreated C. albicans cultures illustrates the very dim autofluorescence profile attained. Figures 1C and D display the PI fluorescence for other treatments of C. albicans. All cells that developed a PI fluorescence intensity greater than that noted for the untreated control cells (Fig. 1B) were defined as PI. Treatment of cells with 70% ethanol (Fig. 1C) gave a 100-fold increase in PI fluorescence intensity, resulting in virtually all of the cells becoming PI. Continuous treatment of cultures with the minimal fungicidal concentration (MFC), the lowest concentration of drug resulting in more than 99% killing of cells, of LY (for C. albicans, 0.01 g/ml) for 3 h (Fig. 1D) resulted in 87.7% of the C. albicans cells becoming PI. Finally, cultures that were pulse treated with LY at the MFC for only 5 min (Fig. 1E), followed by one wash and further incubation in compound-free broth for the remainder of the 3-h incubation, displayed a similar percentage of PI cells (85.7%) as did those that were continuously treated during the 3-h incubation. Virtually identical results were obtained when S. cerevisiae cells were similarly tested (data not shown). Determination of SCG% of PI and PI cultures. S. cerevisiae cultures were treated with a moderately potent concentration of LY (0.02 g/ml) for 3 h, stained with PI, and analyzed on the cytometer. Data from a representative experiment are summarized in Fig. 2. From this PI fluorescence profile, cell-sorting regions were established around PI and PI regions (Fig. 2). Single cells from these two regions were deposited into microculture wells of 96-well plates and incubated at 30 C for 48 h. The number of wells (out of 48 from each condition per plate) that displayed positive yeast growth are shown in Fig. 2. These data demonstrate that after treatment with LY303366, virtually all the Saccharomyces cells that Downloaded from on October 22, 2018 by guest FIG. 1. Flow cytometric evaluation of treated C. albicans cells that were stained with PI. Actively growing yeast cultures were treated with fungicidal agents as described in the text, mixed with PI, and analyzed on the flow cytometer. (A) Light scatter gating region used for the analysis of red fluorescence of each sample. FS, forward scatter; SS, side scatter. (B through E) Red fluorescence histograms displaying the integrated percentage of PI cells. The treatments for the panels were medium only (control) (B), 70% ethanol (C), LY for 3 h (D), and LY for a 5-min pulse (E). RESULTS Effect of LY on PI staining of yeast. Antifungal effects can be detected by flow cytometric analysis of PI-treated yeast cells (10). Certain antifungal agents increase the PI staining of yeast. The effects of two fungicidal agents on C. albicans from FIG. 2. Single-cell sorting of cells treated with LY and stained with PI. Active cultures of S. cerevisiae were treated with LY (0.02 g/ml) for 3 h and mixed with PI. The cells were analyzed on the cytometer, and individual cells were sorted into microwell cultures based on their red fluorescence intensity as defined by regions M2 (PI ) and M1 (PI ). After 2 days of static incubation at 30 C, the microwells were visually inspected for determination of cell growth. The number of wells (out of 48 used) with S. cerevisiae growth is displayed for each of three plates in this representative experiment.

3 832 GREEN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Determination of PI staining and CFU reduction for C. albicans treated with LY or AMB continuously or for an initial 5-min pulse Compound (concn) Treatment a %PI cells (SD) 10 4 CFU/ml (SD) FIG. 3. PI staining and SCG% for S. cerevisiae treated for 3 h with LY (A) Active yeast cultures were treated with twofold serial dilutions (0.005 to 0.16 g/ml) of LY and either stained with PI and flow cytometrically analyzed (F) or single-cell sorted for SCG% determinations (E). Each data point represents the mean 1 standard error of the mean (SEM) for four experiments. (B) Mean SCG% and mean percentage of PI cells for each LY concentration tested. The associated linear-regression line and correlation coefficient are shown. stained with PI were nonviable (SCG% 4.2), while the PI cells exhibited a SCG% (95.8) equal to that of the untreated controls (96.0). These data also indicate that PI itself (in the absence of fungicidal agents) does not alter yeast SCG%. Similar results were obtained with C. albicans (data not shown). Concentration response of LY on PI staining and single-cell sorting. We studied the effect of LY concentration on S. cerevisiae viability. In addition to studying the effect of LY on PI uptake, we also utilized a cell sorter to confirm the fungicidal effect. This instrument facilitates automated, selective single-yeast-cell deposition into microculture wells of a 96-well plate. In the first series of cell-sorting experiments, we examined the effect of treatments of various concentrations of LY on S. cerevisiae viability by using both the PI-staining technique and single-cell sorting during the same experiments. Results of these studies are displayed in Fig. 3. After a 3-h incubation with LY303366, the percentage of PI cells (Fig. 3A) increased with increasing compound concentration. There was a concomitant decrease in the SCG% in the wells, as determined by cell sorting (Fig. 3A), as the concentration of LY increased. Linear-regression analysis of this data (Fig. 3B) demonstrated a very high correlation (r Vehicle None (control) 7.6 (1.8) 83 (23) LY (0.01 g/ml) Continuous 87.0 (6.1) 0.39 (0.29) Pulse 88.3 (4.6) 0.24 (0.31) Vehicle None (control) 5.0 (0.5) 154 (66) AMB (2.5 g/ml) Continuous 13.0 (5.9) (0.01) Pulse 5.0 (3.2) 43 (24) a C. albicans cells were treated with compound continuously or pulsed for the first 5 min, as described in Materials and Methods. Following a total treatment period of 3 h (LY303366) or 5 h (AMB), cultures were further processed for CFU and PI assays as described in Materials and Methods. Data are means for four experiments ) of effect for these two measurements of cell viability. The same correlation was observed in experiments using C. albicans (data not shown). Time course of LY fungicidal activity. We compared PI staining and SCG% for C. albicans cultures that were incubated with LY throughout a 3-h period. At 30-min intervals, aliquots of LY (0.01 g/ml)-treated cultures were either analyzed for PI fluorescence or sorted as single cells into the wells of 96-well microculture plates. As shown in Fig. 4, percentages of PI cells increased throughout the 3-h period with an attendant decrease in SCG%. After 180 min of treatment, 78.1% of the cells were PI and had an SCG% of Fungicidal effects of an initial 5-min exposure to antifungals. Results from a previous study with ECB analogs (10) demonstrated that ECB analogs kill C. albicans rapidly. In that study, treatment with cilofungin caused 90% of the C. albicans cells to become PI within 3 h. In the present study, we modified that method to include compound removal (by washing with PBS) in order to investigate the effects of even shorter exposure times on PI fluorescence and reduction in CFU. The data in Table 1 demonstrate that an initial 5-min exposure to LY was FIG. 4. Time course of the fungicidal activity of LY against C. albicans. An actively growing C. albicans culture was incubated with LY (0.01 g/ml) for a total of3hat32 C. At 30-min intervals, an aliquot of treated culture was removed and either cell sorted for determination of SCG% (E) or stained with PI (F) and flow cytometrically analyzed. Each data point represents the mean percentage of each activity 1 standard error of the mean (SEM) for three separate experiments. The mean SCG% and the mean percentage of PI cells for 3-h control cultures were 95 and 2, respectively.

4 VOL. 43, 1999 LY FUNGICIDAL ACTIVITY 833 Compound TABLE 2. Minimum pulsed-exposure time for inducing maximum fungicidal effects Pulsed-exposure time (min) a Pulsed fungicidal concn ( g/ml) Dose relationship to MFC LY Cilofungin AMB a C. albicans cells were incubated for the indicated intervals, washed, and then incubated in fresh media to complete either a 3-h (LY and cilofungin) or 5-h (AMB) total treatment. equally as potent as a full 3-h exposure period. A 5-min pulse with LY at the MFC (0.01 g/ml) followed by a 175-min incubation in drug-free broth resulted in a mean of 88.3% PI cells and an average reduction in CFU per milliliter of 99.7%, compared with a mean of 87.0% PI cells and an average reduction in CFU per milliliter of 99.6% for the 3-h continuous drug treatment. While the 3-h PI assay correlates well with the CFU and SCG% results for LY (Fig. 2), in this study a 5-h PI assay underestimated the killing activity of AMB as determined by CFU assays. Continuous or initial 5-min pulsed exposure to AMB at 2.5 g/ml (four times the MFC) produced lower percentages of PI cells than with the LY treatments. An initial 5-min pulsed exposure to AMB was less effective than a continuous 5-h incubation in AMB, as measured by the PI and CFU reduction assays (Table 1). Determination of minimum fungicidal exposure time. We used the pulsed-exposure technique and PI fluorescence analysis in conjunction with CFU assays to compare the potencies and activities of selected antifungals for C. albicans. Cultures were incubated with antifungals for defined periods, washed with PBS, further incubated in compound-free YPD broth (for 180 or 300 min total), and tested by the PI fluorescence and CFU assays. The concentrations tested were multiples of the MFCs of the antifungal compounds for each strain as previously established by a conventional broth microdilution technique (9). After multiple drug concentrations were tested over various exposure periods, the minimum compound exposure times that induced the maximal fungicidal activity of each compound were determined; they are summarized in Table 2. The minimum exposure time required for LY at the MFC (0.01 g/ml) to exert its maximal fungicidal effect was only 5 min. Cilofungin at a concentration of 1.6 g/ml (four times the MFC) was not effective in pulsed exposures below the full 180-min period. For AMB, the minimal exposure time determination shown in Table 2 was based on CFU results, since the PI assay underestimates C. albicans killing by this compound (Table 1). A 30-min exposure (2.5 g/ml, or four times the MFC) was as effective in the CFU assay as a full 5-h exposure. Effect of timing of pulsed exposure to LY on fungicidal activity. C. albicans cultures were exposed to LY (0.01 g/ml) by three different treatment regimens in order to help elucidate the importance of an active-growth period following compound exposure for manifestation of fungicidal effects. Four cultures were started, with the first being an untreated control. The second was incubated with LY continuously for 3 h. The third culture was pulse incubated with LY for 5 min, washed in PBS, and returned to a 32 C shaker bath for 175 min. The fourth culture was incubated without compound for 175 min, followed by the addition of LY during the last 5 min. At 180 min, aliquots from each test culture were assayed by the PI fluorescence, CFU reduction, and SCG% assays. Data from three such experiments are summarized in Table 3. Results obtained by all three methods indicate that an initial 5-min pulsed exposure to LY303366, followed by 175 min of incubation without compound, was as effective in killing C. albicans cells as a continuous 3-h exposure. In contrast, a 5-min exposure, which occurred at the end of the 180-min experiment and was analyzed immediately for antifungal activity, displayed a significantly decreased fungicidal effect. DISCUSSION Treatment options for serious fungal infections are currently limited. New antifungal drugs are urgently needed, especially compounds with a fungicidal mode of action. In this study, we used flow cytometric analysis, single-cell-sorting techniques, and standard microbiological methods to investigate the activity of LY against C. albicans and S. cerevisiae. Our results indicate that LY is a highly potent and rapidly acting fungicidal compound. While our methods are novel, our results are consistent with an accumulating body of knowledge regarding the potency and broad-spectrum fungicidal activity of this promising antifungal compound (11, 20, 26). Advancements in flow cytometry instrumentation have pushed the technology into microbiology laboratories worldwide. A thorough review of flow cytometric applications for microbiology was published recently (4). Flow cytometry offers unique advantages for studying the fungicidal activities of selected compounds against yeasts, providing the ability to examine the effects of compounds on thousands of individual cells within seconds. This type of analysis facilitates the understanding of the heterogeneous nature of yeast populations with respect to drug sensitivity. In a previous study (10), members of our group showed how flow cytometric measurement of PI fluorescence could distinguish the activity of fungicidal agents, such as cilofungin, from that of a fungistatic compound such as fluconazole. The PI method was particularly well suited for determining the extent of fungal cell killing by cilofungin; more than 90% of the cells treated with cilofungin became PI within 3 h of treatment at the MFC. In the present study, we applied this method to investigate the antifungal effects of a more potent ECB analog, LY Using this approach, we demonstrated an approximately 40-fold increase in potency of LY over cilofungin. Other investigators, using standard in vitro and in vivo microbiology assays, have demonstrated that LY has potent, broad-spectrum fungicidal activity (11, 16, 23, 29 31). The additional cell-sorting experiments described in this paper supported our hypothesis that LY treated, PI yeast cells were indeed nonviable (Fig. 2). This confirmation is important because others have shown that certain bacteria may TABLE 3. Effect of timing on 5-min pulsed exposure of LY for fungicidal effects on C. albicans Time period of LY treatment a %PI cells (SEM) % CFU reduction (SEM) SCG% (SEM) None 1.1 (0.3) 91.9 (2.7) Continuous 75.6 (5.6) 90.5 (1.3) 27.6 (6.5) First 5 min 86.2 (2.7) 86.9 (2.7) 21.8 (3.5) Last 5 min 27.2 (6.5) 44.2 (14.1) 46.0 (4.5) a C. albicans cells were treated with compound for the time period indicated, as described in Materials and Methods. When a total incubation of 3 h was reached for all cultures, culture aliquots were further processed for PI staining, CFU assay, and SCG% assay as described in Materials and Methods. Data are means for three experiments.

5 834 GREEN ET AL. ANTIMICROB. AGENTS CHEMOTHER. exhibit staining with selected viability dyes and yet be able to recover and grow (4, 7, 13). In addition, other studies have indicated that treatment of yeast with the fungistatic agent fluconazole can increase PI fluorescence slightly, yet these cells resume their growth and exhibit normal PI-staining patterns after removal from compound (19). Results from the concentration response and time course experiments (Fig. 3 and 4) demonstrate that the PI assay and single-cell sorting for determination of SCG% complement each other. In the LY concentration response experiments, PI staining correlated (r 0.99) with decreased SCG%, further validating the PI method for measuring fungicidal activity of LY against C. albicans and S. cerevisiae. After confirming the lethal effects of a 3-h treatment with LY303366, we then focused on determining the minimal exposure period sufficient to kill C. albicans by comparing pulsedcompound exposures to continuous 3- or 5-h incubations. Using this approach, we determined (Table 2) that binding of LY to C. albicans is rapid; exposure to the MFC (0.01 g/ml) for 5 min killed 99% of the cells. These data demonstrate that chemical modifications of the fatty acid side chains of the ECB nucleus (6) incorporated in the synthesis of LY resulted in an agent with much greater potency than cilofungin. The rapid killing of yeast cells by LY after a short exposure period may contribute to its potent efficacy in vivo (3). The rapid action and enhanced potency of LY compare favorably with those of AMB, a polyene antifungal that has been used clinically since the 1950s (8). AMB has been reported to bind ergosterol in fungal cell membranes within 30 min (27). Cell membrane permeability is increased, first for potassium ions and then for other cell constituents, resulting in cell death within hours after exposure. Binding of AMB was recently shown to decrease yeast plasma membrane potential within 30 min of AMB exposure (18). Flow cytometric analysis of PI staining has been used to study AMB activity against yeasts (22) and to investigate serum effects (15) or synergy (21) or antagonism (14) between antifungals. Our studies also demonstrated that a 30-min incubation with AMB has an effect on C. albicans, an effect measured by increased PI fluorescence (Table 2). Although AMB binds rapidly, our data indicate that AMB does not increase yeast cell membrane permeability to PI as early as LY does. C. albicans cultures treated with AMB for 5 h display only one-fifth of the PI cells displayed by cultures treated with LY for 3 h (Table 1). Some investigators have used deoxycholate to enhance PI entry into AMB-treated cells (14). AMB is highly effective against most clinical fungal isolates (17); however, nephrotoxicity limits its clinical utility. Among its effects, LY targets a fungusspecific enzyme, glucan synthase (5), which may contribute to an improved toxicity profile. Because LY binds rapidly to yeast cells and has an inhibitory effect on cell wall synthesis, its antifungal effects would be expected to occur over time in a growing culture. One paradox emerged from the pulsed-exposure experiments: while a 5-min pulsed exposure to LY was shown to be sufficient to eventually kill target cells (Table 1), deposition of single cells into microwells following 30 min of incubation with LY resulted in a smaller-than-expected decrease in SCG% (Fig. 4). We hypothesized that active-growth conditions are important for maximal killing activity of LY To test this idea, we conducted experiments in which cells were treated with LY for 5 min, with or without an active-growth period (incubation in a shaker water bath) for 175 min in compound-free medium (Table 3). The PI staining, CFU, and SCG% assays all indicated that a 5-min pulse with compound without the active-growth period resulted in a significantly reduced fungicidal effect compared to the effect on cultures that were actively grown in a shaking water bath for an additional 175 min. To reconcile these results, we considered the fact that for each of the three fungicidal activity assays, cells were suspended in drug-free medium following the drug pulse. It is possible that when LY treated cells are suspended in drug-free broth, some of the drug will come off of the cells. We hypothesize that if yeast cells remain in log-phase growth, as promoted by the shaker bath, most will begin cell division and, therefore, die before much drug comes off. With static culture conditions following the drug pulse, such as the microtiter well or agar plate, drug release could become a more significant factor because fewer cells will divide while a lethalthreshold level of drug remains bound to the cell. Our results imply that yeasts growing at lower rates may require somewhat longer exposure periods in order to be killed by this agent. In summary, we have utilized flow cytometric analysis, cell sorting, and standard microbiological plating methods to study the fungicidal activities of selected antifungals against S. cerevisiae and C. albicans. Our data indicate that cell sorting may be a valuable tool for studying and differentiating various antifungal activities. The PI fluorescence and cell-sorting methods provide tools for evaluating certain modes of action that are not easily accomplished by standard microbiological techniques. In addition, our data indicate that LY is a potent, rapidly acting antifungal agent. In our experiments, we confirmed that LY treated PI yeast cells are nonviable. Our data also suggest that LY works best against actively growing yeasts. The potency and rapid action of LY suggest that it may be useful for antifungal therapy of human disease. REFERENCES 1. Bartlett, M. S., W. L. Current, M. P. Goheen, C. J. Boylan, C. H. Lee, M. M. Shaw, S. F. Queener, and J. W. Smith Semisynthetic echinocandins affect cell wall deposition of Pneumocystis carinii in vitro and in vivo. Antimicrob. Agents Chemother. 40: Beaulieu, D., J. Tang, S. B. Yan, J. M. Vessels, J. A. Radding, and T. R. Parr, Jr Characterization and cilofungin inhibition of solubilized Aspergillus fumigatus (1,3)- -D-glucan synthase. Antimicrob. Agents Chemother. 38: Current, W., and W. Turner Echinocandin antifungal agents, p In W. Strohl (ed.), Biotechnology of antibiotics. Marcel Dekker, Inc., New York, N.Y. 4. Davey, H. M., and D. B. Kell Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol. Rev. 60: Debono, M., and R. S. Gordee Antibiotics that inhibit fungal cell wall development. Annu. Rev. Microbiol. 48: Debono, M., W. W. Turner, L. LaGrandeur, F. J. Burkhardt, J. S. Nissen, K. K. Nichols, M. J. Rodriguez, M. J. Zweifel, D. J. Zeckner, R. S. Gordee, et al Semisynthetic chemical modification of the antifungal lipopeptide echinocandin B (ECB): structure-activity studies of the lipophilic and geometric parameters of polyarylated acyl analogs of ECB. J. Med. Chem. 38: Diaper, J. P., K. Tither, and C. Edwards Rapid assessment of bacterial viability by flow cytometry. Appl. Microbiol. Biotechnol. 38: Gallis, H. A., R. H. Drew, and W. W. Pickard Amphotericin B: 30 years of clinical experience. Rev. Infect. Dis. 12: Gordee, R. S., D. J. Zeckner, L. C. Howard, W. E. Alborn, Jr., and M. Debono Anti-Candida activity and toxicology of LY121019, a novel semisynthetic polypeptide antifungal antibiotic. Ann. N. Y. Acad. Sci. 544: Green, L., B. Petersen, L. Steimel, P. Haeber, and W. Current Rapid determination of antifungal activity by flow cytometry. J. Clin. Microbiol. 32: Krishnarao, T. V., and J. N. Galgiani Comparison of the in vitro activities of the echinocandin LY303366, the pneumocandin MK-0991, and fluconazole against Candida species and Cryptococcus neoformans. Antimicrob. Agents Chemother. 41: Kurtz, M. B., I. B. Heath, J. Marrinan, S. Dreikorn, J. Onishi, and C. Douglas Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)- -D-glucan synthase. Antimicrob.

6 VOL. 43, 1999 LY FUNGICIDAL ACTIVITY 835 Agents Chemother. 38: (Erratum, 38:2516.) 13. Lloyd, D., and A. J. Hayes Vigour, vitality and viability of microorganisms. FEMS Microbiol. Lett. 133: Martin, E., F. Maier, and S. Bhakdi Antagonistic effects of fluconazole and 5-fluorocytosine on candidacidal action of amphotericin B in human serum. Antimicrob. Agents Chemother. 38: Martin, E., U. Schlasius, and S. Bhakdi Flow cytometric assay for estimating fungicidal activity of amphotericin B in human serum. Med. Microbiol. Immunol. 181: Moore, C. B., K. L. Oakley, and D. W. Denning In vitro activity of LY compared with fluconazole, flucytosine and amphotericin B against Candida spp., abstr. F-67, p In Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 17. Odds, F. C Resistance of clinically important yeasts to antifungal agents. Int. J. Antimicrob. Agents 6: Ordonez, J. V., and N. M. Wehman Amphotericin B susceptibility of Candida species assessed by rapid flow cytometric membrane potential assay. Cytometry 22: Petersen, B. H., L. Green, L. Steimel, D. Hyslop, and W. L. Current Use of flow cytometry for determination of minimal exposure time for effective antifungal activity of LY and other antifungal compounds, abstr. 362, p In Program and abstracts of the 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 20. Pfaller, M. A., S. A. Messer, and S. Coffman In vitro susceptibilities of clinical yeast isolates to a new echinocandin derivative, LY303366, and other antifungal agents. Antimicrob. Agents Chemother. 41: Pore, R. S Amphotericin B synergy testing by the FCST. Curr. Microbiol. 24: Pore, R. S Antibiotic susceptibility testing of Candida albicans by flow cytometry. Curr. Microbiol. 20: Rennie, R., C. Sand, and S. Smith In vitro activity of antifungal agent LY against Candida species, other yeasts and Aspergillus species, abstr. F45, p In Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 24. Taft, C. S., T. Stark, and C. P. Selitrennikoff Cilofungin (LY121019) inhibits Candida albicans (1-3)- -D-glucan synthase activity. Antimicrob. Agents Chemother. 32: Tang, J., and T. R. Parr, Jr W-1 solubilization and kinetics of inhibition by cilofungin of Candida albicans (1,3)- -D-glucan synthase. Antimicrob. Agents Chemother. 35: Uzun, Ö., S. Kocagöz, Y. Çetinkaya, S. Arikan, and S. Ünal In vitro activity of a new echinocandin, LY303366, compared with those of amphotericin B and fluconazole against clinical yeast isolates. Antimicrob. Agents Chemother. 41: Vanden Bossche, H., G. Willemsens, and P. Marichal Anti-Candida drugs the biochemical basis for their activity. Crit. Rev. Microbiol. 15: Wade, J. C Treatment of fungal and other opportunistic infections in immunocompromised patients. Leukemia 11(Suppl. 4):S38 S Zeckner, D., T. Butler, C. Boylan, B. Boyll, Y. Lin, P. Raab, J. Schmidtke, and W. Current LY303366, activity in a murine systemic candidiasis model, abstr. 365, p In Program and abstracts of the 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 30. Zeckner, D., T. Butler, C. Boylan, B. Boyll, P. Watson, and W. Current In vitro evaluation of LY against Candida spp. clinical isolates and evaluation of efficacy in a murine model of systemic candidiasis, abstr. F98, p In Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 31. Zelenitsky, S., J. Karlowsky, D. Hoban, N. Laing, T. Balko, A. Kabani, M. Turik, and G. Zhanel Activity of an investigational echinocandin, LY against fluconazole susceptible and resistant Candida albicans in a multiple dose in vitro pharmacodynamic model, abstr. F-72, p In Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. Downloaded from on October 22, 2018 by guest