INTRODUCTION

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LY303366, a semisynthetic echinocandin B derivative, is an investigational antifungal agent possessing activity against a broad range of Candida species and filamentous fungi (7, 9, 10). LY303366 disrupts glucan formation in the cell wall of fungi via inhibition of (1, 3)--D-glucan synthase, resulting in cell damage and lysis (8). According to the manufacturer, LY303366 exhibits fungicidal activity, and therefore, it has been recommended that 100% inhibition of visual growth be used as the MIC endpoint for in vitro susceptibility determinations. This endpoint is the same as that currently utilized for amphotericin B, another fungicidal agent.

We have previously described the antifungal characteristics of LY303366 against various Candida species as determined by time-kill curve methods (1, 2). Initially, the antifungal activity of LY303366 was studied with RPMI 1640 medium buffered to a pH of 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) against two isolates each of Candida albicans, Candida glabrata, and Candida tropicalis (1). The activity of LY303366 was assessed according to the rate and extent of fungicidal activity observed over a series of concentrations ranging from 0.125 to 16×MIC for each isolate. MICs were determined with 100% inhibition of visual growth as the endpoint. Surprisingly, LY303366 exhibited fungicidal activity, defined as a reduction in the CFU per milliliter of 99.9% compared with the starting inoculum, against only three of the test isolates. Additionally, there did not appear to be a discernible correlation between the MIC for test isolates and the degree of fungicidal activity observed, a marked departure from our experience with fluconazole and amphotericin B (4). Lastly, when MICs were determined for LY303366 in RPMI 1640 medium, appreciable trailing was observed, a phenomenon that was essentially absent when antibiotic medium no. 3 (AM 3) served as growth medium. As a result, we decided to evaluate the effect of growth medium on the correlation between LY303366 MICs and time-kill studies (2). In this series of studies, the same isolates that had been previously tested in RPMI 1640 medium were now evaluated with AM 3 as growth medium. In AM 3, the fungicidal activity of LY303366 exhibited better correlation with MICs, i.e., subinhibitory concentrations produced little or no antifungal activity whereas suprainhibitory concentrations resulted in marked decreases in colony counts. Lastly, in this medium, LY303366 exhibited fungicidal activity against all six isolates.

In light of our findings, we concluded that the fungicidal activity of LY303366 is significantly influenced by the growth medium selected for testing. Furthermore, since LY303366 does not consistently exhibit fungicidal activity in RPMI 1640 medium and MICs demonstrate poor correlation with time-kill results in this medium, use of 100% inhibition of visual growth as the endpoint for susceptibility determinations may inaccurately reflect the activity of LY303366 in this medium. Therefore, the current study was devised to evaluate use of an alternative endpoint in the determination of MICs of LY303366.

(This work was previously presented at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy [1a].)

	MATERIALS AND METHODS

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Antifungal agents. LY303366 (Eli Lilly and Co., Indianapolis, Ind.) was utilized for susceptibility determinations and time-kill studies. A stock solution of LY303366 (1,000 µg/ml) was prepared in dimethyl sulfoxide (DMSO) and subsequently diluted with RPMI 1640 medium (Sigma Chemical Co., St. Louis, Mo.) buffered to a pH of 7.0 with 0.165 M MOPS buffer (Sigma). The final concentration of DMSO in the time-kill test solutions was 1% (vol/vol) of the solution composition. Growth curves were determined in the presence of 1% (vol/vol) DMSO and compared with growth curves naive for DMSO exposure to verify that the solvent did not affect the viability of the test isolates. LY303366 stock solution was separated into unit-of-use aliquots and stored at 70°C until used.

Test isolates. Two clinical isolates each of C. glabrata (582 and 350) and C. tropicalis (2697 and 3829) were selected for testing. Additionally, one American Type Culture Collection strain, ATCC 90028, and one clinical isolate, OY31.5, of C. albicans, were utilized. Since we previously examined the activity of LY303366 against these six isolates in earlier studies, they were again selected for use in the current series to facilitate comparisons among studies (1, 2). Isolates were obtained from the Department of Pathology, The University of Iowa College of Medicine.

Antifungal susceptibility testing. The MIC of LY303366 against each of the test isolates was determined by broth microdilution techniques as described by the National Committee for Clinical Laboratory Standards (5). MICs were determined in RPMI 1640 medium buffered with MOPS utilizing an initial inoculum of approximately 1 × 10³ to 5 × 10³ CFU/ml. Microtiter trays were incubated at 35°C in a moist, dark chamber, and MICs were recorded after 48 h of incubation. Two susceptibility endpoints were recorded for each isolate. The MIC₁₀₀ was defined as the lowest concentration of drug which resulted in total inhibition of visible growth. This is the endpoint currently recommended by the manufacturer. The MIC₈₀ was defined as the lowest concentration of drug which produced an 80% reduction in visible growth compared with control. Eighty percent reduction in visible growth is an endpoint similar to that used for the susceptibility determination for fungistatic agents such as fluconazole.

Scanning electron microscopy. Samples corresponding to control, 0.25 to 0.5× MIC₈₀, and 2 to 4× MIC₈₀, were collected from microtiter trays following MIC determination and transferred to a 0.4-µm-pore-size polycarbonate Nuclepore filter (Nuclepore Corp., Pleasanton, Calif.), and suction was applied to remove excess liquid. The filter was immersed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Samples were fixed in osmium tetroxide and dehydrated with a graded series of ethanol washes followed by immersion in hexamethyldisilizane. Following removal from hexamethyldisilizane, samples were air dried, mounted onto aluminum stubs, and sputter coated with Pd-Au. A Hitachi S4000 scanning electron microscope (Hitachi Scientific Instruments, Mountain View, Calif.) was used for visualization of samples.

Time-kill curve methodology. Time-kill procedures were conducted as previously described (1, 2, 4). Fungi were obtained from banked samples and subcultured twice on potato dextrose agar plates (Remel) prior to testing. Fungal suspensions were prepared in sterile water by touching three to five colonies from a 24- to 48-h-old culture plate and adjusting the resulting suspension to a 0.5 McFarland turbidity standard (approximately 1 × 10⁶ to 5 × 10⁶ CFU/ml) by spectrophotometric methods. A 1:10 dilution of this suspension was made by adding 1 ml of fungal suspension to 9 ml of RPMI 1640 medium with or without the desired amount of LY303366. This dilution yielded a starting inoculum of approximately 1 × 10⁵ to 5 × 10⁵ CFU/ml. The antifungal activity of LY303366 was studied over a range of multiples of the MIC₈₀ encompassing 0.25 to 16× MIC₈₀. Test solutions were placed on an orbital shaker and incubated at 35°C. At predetermined time points (0, 2, 4, 6, 8, 12, and 24 h), a 0.1-ml sample was removed from each test solution and serially diluted 10-fold in sterile water and a 10-µl aliquot was plated onto a potato dextrose agar plate for colony count determination. Following inoculation, plates were incubated at 35°C for 24 to 48 h before being read. When colony counts were expected to be less than 1,000 CFU/ml, a 30-µl sample was taken directly from the test solutions and plated onto a potato dextrose agar plate without dilution. Following these procedures, the lower limit of fungal quantitation was 50 CFU/ml. Additionally, according to these sampling procedures antifungal carryover was not observed (3). All time-kill curve experiments were conducted in duplicate. For these studies, fungicidal activity was defined as greater than or equal to a 3-log₁₀ (99.9%) reduction in CFU per milliliter compared with the starting inoculum (6).

Analysis. Mean colony count data (log₁₀ CFU per milliliter) were plotted as a function of time for each isolate and compared with respective MIC₈₀, MIC₁₀₀, and MFC data.

	RESULTS

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Antifungal susceptibility. The results of susceptibility tests with LY303366 (i.e., MIC₈₀, MIC₁₀₀, and MFC) are presented in Table 1. For a given isolate, median MIC₁₀₀s were noted to be two to five wells greater than corresponding MIC₈₀s. The greatest difference between MIC₈₀ and MIC₁₀₀ was noted for C. albicans 90028, a difference of five doubling dilutions or wells. MFCs were consistently greater than MIC₈₀ determinations for each of the isolates. In contrast, MFCs were found to be greater than or equal to the MIC₁₀₀ for only two of the six isolates (C. albicans 90028 and C. albicans OY31.5).

Time-kill curves. (i) C. albicans. Strain OY31.5 demonstrated good correlation between the MIC₈₀ and time-kill results (Fig. 1A). Exposure of OY31.5 to LY303366 at multiples of MIC₈₀ resulted in almost no reduction in CFU compared to the starting inoculum. In contrast, concentrations of LY303366 of >2× MIC₈₀ exhibited fungicidal activity. For this isolate, the MIC₁₀₀ was equal to 32× MIC₈₀.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Representative time-kill curve plots for C. albicans OY31.5 (A), C. glabrata 350 (B), and C. tropicalis 3829 (C) at the following concentrations: control (), 0.25× MIC80 (), 0.5× MIC80 (), MIC80 (), 2× MIC80 (), 4× MIC80 (), 8× MIC80 (), and 16× MIC80 ().

(ii) C. glabrata. Both strains of C. glabrata, 350 (Fig. 1B) and 582 (data not shown), demonstrated excellent correlation between the MIC₈₀ and time-kill results. Minimal inhibition of fungal growth was exhibited by these isolates when exposed to a concentration of LY303366 of 0.25× their respective MIC₈₀s. Furthermore, increasing the concentration of drug in solution, up to the maximal concentration tested, resulted in improved antifungal activity against both strains. Fungicidal activity was noted with concentrations of 0.5× MIC₈₀ and 2× MIC₈₀ for strains 350 (MIC₁₀₀ = 4× MIC₈₀) and 582 (MIC₁₀₀ = 8× MIC₈₀), respectively.

(iii) C. tropicalis. LY303366 produced fungistatic activity against both isolates, C. tropicalis 2697 (data not shown) and 3829 (Fig. 1C). Partial inhibition of growth was noted for both strains at concentrations of <MIC₈₀; however, the maximum inhibitory effect was produced by concentrations greater than or equal to the MIC₈₀. The MIC₁₀₀ of each isolate would have correlated with a time-kill sample containing a 32× MIC of LY303366.

Scanning electron microscopy. Samples taken from control wells displayed well-formed cells with smooth unadulterated surfaces (Fig. 2A). Additionally, these cells appear to be viable and actively growing, as evident from budding cells. Specimens taken from fungal samples exposed to sub-MIC₈₀s of LY303366 generally resembled control populations (Fig. 2B). The majority of cells appeared normal, and budding was evident; however, rare cells that exhibited minor abnormalities, dimples or folds, in cell wall structure were observed. In general, micrographs of fungi exposed to LY303366 concentrations of >MIC₈₀ exhibited substantial ultrastructure abnormalities such as cell deformation, dimpling, and clumping (Fig. 2C). Cellular fragments were also commonly observed. Lastly, when intact cells were detected, signs of viability (i.e., budding) were severely diminished or absent.

View larger version (144K): [in this window] [in a new window]   View larger version (154K): [in this window] [in a new window]   FIG. 2.   Electron micrographs of C. glabrata 582 exposed to concentrations of LY303366 equal to control (A), MIC80 (B), and 16× MIC (C). Bars, 1 µm.

	DISCUSSION

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Using 80% reduction in growth compared to control as the endpoint for susceptibility testing for LY303366, we were able to demonstrate improved correlation between MICs and fungicidal activity as assessed by MFC determination and time-kill curves. The rationale behind our investigation of this alternative susceptibility endpoint stems from our previous work with LY303366 (1, 2). LY303366 does not always exhibit fungicidal activity when tested in RPMI medium; in fact, the fungicidal threshold was achieved by only half of our test isolates (1). This finding, coupled with observations of microcolony trailing in MIC trays and the need for test concentrations as low as 0.02× MIC₁₀₀ before the antifungal effect of LY303366 subsided, led us to question the validity of the recommended susceptibility endpoint. In this first series of experiments, although RPMI 1640 was used as the primary test medium, MICs were also determined in AM 3. Interestingly, when AM 3 served as the growth medium, trailing in the MIC trays was virtually absent. Subsequently, we repeated time-kill experiments with AM 3 as growth medium and found a much stronger correlation between MIC₁₀₀ and MFC and time-kill results (2). We also noted that, in AM 3, LY303366 was fungicidal against all six test isolates. Selection of a susceptibility endpoint of 100% inhibition of visual growth is appropriate for an agent that consistently demonstrates fungicidal activity, such as amphotericin B or LY303366 in AM 3. However, if fungicidal activity is not routinely observed, as is the case with the azole antifungals and LY303366 in RPMI, then an endpoint reflecting fungistatic activity should be utilized for susceptibility determinations (MIC₈₀).

In the present study, we evaluated the relation between MICs of LY303366, determined with 80% reduction in growth as an endpoint, and two additional measures of antifungal activity (MFCs and time-kill curves). We were able to demonstrate a stronger relationship between MIC₈₀ and MFCs than that in our previous report (1). The minimum concentration of an antimicrobial necessary to kill an organism, MFC, should be equal to or greater than the MIC for that microbe. In this study, however, four of the six isolates had MFCs which were one to three wells less than the corresponding MIC₁₀₀. Similar discrepancies were not noted among MIC₈₀ and MFC data. Furthermore, it has been our experience with time-kill data generated with other antifungal agents that the time-kill sample which corresponds to the MIC, plus or minus one dilution, represents a transition from minimal activity (subinhibitory concentrations) to maximal antifungal effect (suprainhibitory concentrations) and encompasses the concentration which produces 50% of the maximal effect. Such a relationship between the LY303366 MIC₁₀₀s and time-kill results in RPMI medium was nonexistent. In contrast, a correlation was evident between MIC₈₀ of LY303366 and time-kill results for all isolates except C. albicans 90028.

Inspection of electron micrographs revealed the existence of a recognizable subinhibitory effect produced by LY303366. Visible dimpling or folding was noted for each of the isolates at concentrations less than the MIC₈₀. It was further noted that the frequency of cell wall alterations and the presence of cell fragments increased whereas signs of viability such as budding declined as the concentration of drug in solution increased. Although we were not able to identify the MIC for an isolate by inspection of micrographs, we were able to conclude that the maximal cell wall effect was generally obtained following exposure of the isolates to concentrations of LY303366 less than the MIC₁₀₀. These findings strengthen our argument that the MIC₁₀₀ tends to underestimate the activity of LY303366.

LY303366 is not a uniformly fungicidal agent; therefore, a susceptibility testing endpoint which assumes fungicidal activity should not be advocated. Rather, an endpoint of 80% reduction in visible growth, similar to the endpoint currently utilized for determination of azole MICs, should be used. The MIC₈₀ provides better correlation with other measurements of antifungal activity such as MFCs and time-kill curves. Therefore, we recommend that an 80% reduction in fungal growth be accepted as the proper endpoint for susceptibility determinations with this agent and that these recommendations be incorporated into future National Committee for Clinical Laboratory Standards documentation concerning LY303366. Failure to promptly recognize the MIC₈₀ as the appropriate endpoint for susceptibility testing may result in serious underestimation of the activity of LY303366.