1 Anidulafungin is fungicidal and exerts a variety of post-antifungal effects against 1 Candida ...

30 Pages·2009·4.1 MB·English

AAC Accepts, published online ahead of print on 13 April 2009
Antimicrob. Agents Chemother. doi:10.1128/AAC.01480-08
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Anidulafungin is fungicidal and exerts a variety of post-antifungal effects against
2 Candida albicans, C. glabrata, C. parapsilosis and C. krusei isolates.
3
4 Katherine T. Nguyen1
5 Philip Ta1 D
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6 Bich Thu Hoang1 n
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7 Shaoji Cheng1,4 ad
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8 Binghua Hao1,4 f
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9 M. Hong Nguyen1,2,3,4

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10 *Cornelius J. Clancy1,3,4,5 p
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13 Departments of 1Medicine and 2Molecular Genetics and Microbiology, University of sm
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14 Florida College of Medicine, the 3North Florida/South Georgia Veterans Health System, r
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15 Gainesville, FL, the 4Department of Medicine, University of Pittsburgh, and the 5V.A. n

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16 Pittsburgh Healthcare System, Pittsburgh, PA n
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18 * Corresponding author (current address): Cornelius J. Clancy, M.D. 5
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19 University of Pittsburgh 2
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20 867 Scaife Hall 1
21 3550 Terrace Street, 9

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22 Pittsburgh, PA 15261 y
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24 Phone: [phone] e
25 Fax: [phone] s
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26 e-mail: [email protected]
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29 Key words: Anidulafungin/time-kill/post-antifungal effect/Candida
30
31 Running title: Anidulafungin post-antifungal effects against Candida
1
1 Abstract.
2
3 Anidulafungin targets the cell walls of Candida species by inhibiting β-1,3-glucan
4 synthase, thereby killing isolates and exerting prolonged post-antifungal effects (PAFEs).
5 We performed time-kill and PAFE experiments on C. albicans (n=4), C. glabrata (3), C.
6 parapsilosis (3) and C. krusei (2) isolates, and characterized PAFEs in greater detail. D
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7 Minimum inhibitory concentrations (MICs) were 0.008-0.125 µg/mL against C. albicans, n
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8 C. glabrata, and C. krusei, and 1.0-2.0 µg/mL against C. parapsilosis. During time-kill d
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9 experiments, anidulafungin caused significant kills at 16x (range: log 2.68-3.89) and 4x f
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10 MIC (log 1.87-3.19), achieving fungicidal levels (≥log 3) against nine isolates. One hour

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11 drug exposure during PAFE experiments resulted in kills ranging from log 1.55-3.47 and p
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12 log 1.18-2.89 (16x and 4x MIC, respectively), achieving fungicidal levels against four a
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13 isolates. Regrowth of all 12 isolates was inhibited for ≥ 12 hours after drug wash-out. m
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14 Isolates of each species collected 8 hours after a one hour exposure to anidulafungin (16x r
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15 and 4x MIC) were hypersusceptible to SDS (0.01-0.04%) and calcofluor white (40 n

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16 µg/mL). Moreover, PAFEs were associated with major cell wall disturbances, as evident n
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17 in electron micrographs of viable cells, and significant reductions in adherence to buccal ry

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18 epithelial cells (p≤0.01). Finally, three of four PAFE isolates tested were 2
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19 hypersusceptible to killing by J774 macrophages (p≤0.007). Our data suggest that the 9

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20 efficacy of anidulafungin in the treatment of candidiasis might stem from both direct g
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21 fungicidal activity and indirect PAFEs that lessen the ability of Candida cells to establish s
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22 invasive disease and persist within infected hosts.
2
1 Introduction.
2
3 Anidulafungin is an echinocandin agent that disrupts the cell wall of Candida
4 species by inhibiting β-1,3-D-glucan synthase. In recent studies of treatment for invasive
5 candidiasis, the agent was shown to be at least as effective as the frontline azole agent
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6 fluconazole (12,22) . Additional clinical trial data demonstrating the efficacy of o
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7 caspofungin and micafungin in the treatment of diverse types of candidiasis make clear lo
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8 that the echinocandins are significant additions to the antifungal armamentarium (13,17). e
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10 In general, MIC90 values of anidulafungin are low against the common pathogens tt
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11 C. albicans, C. glabrata, C. tropicalis and C. krusei (0.06 – 0.12 µg/mL), including /a
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12 isolates that are resistant to azole agents (20,21). MIC90 values against C. parapsilosis .a
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13 and C. guilliermondii isolates are higher (2 µg/mL), as also noted for other echinocandins .
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14 (20,21). Diminished susceptibility to anidulafungin might reflect changes in the glucan /
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15 synthase subunit Fks1p (2,18). Regardless of the mechanism, the clinical significance of J
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16 elevated anidulafungin MICs remains unclear (22). To date, only a few studies have a
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17 assessed the anti-candidal activity of anidulafungin by time-kill or post-antifungal effect 5
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18 (PAFE) methods. Similar to other echinocandins, anidulafungin exhibited concentration- 1
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19 dependent fungicidal activity against C. albicans, C. glabrata, C. tropicalis and C. krusei y

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20 isolates during time-kill experiments at concentrations of 4x and 16x MIC (10,23). In e
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21 PAFE experiments, one hour exposure to anidulafungin at 4x MIC resulted in prolonged
22 growth inhibition of C. albicans (9). To our knowledge, time-kill or PAFE data have not
23 been published for anidulafungin against C. parapsilosis. Caspofungin, however, is
3
1 fungicidal and causes prolonged PAFE growth inhibition of C. parapsilosis at
2 concentrations ≥ 4x MIC (7).
3
4 We hypothesized that anidulafungin would demonstrate significant PAFEs against
5 Candida isolates of diverse species, as measured by growth inhibition following brief D
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6 drug exposure in vitro. In addition, we hypothesized that anidulafungin’s PAFEs would n
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7 cause changes to the candidal cell wall that result in decreased cell integrity and d
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8 adherence to host cells, and increased susceptibility to killing by phagocytes. In this f
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9 study, we assessed the fungicidal activity of anidulafungin against twelve Candida

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10 isolates (4 C. albicans, 3 C. glabrata, 3 C. parapsilosis and 2 C. krusei) by time-kill and p
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11 PAFE methods. We then tested PAFE-inhibited cells for susceptibility to cell wall-active a
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12 drugs and visualized cell walls by electron microscopy. Finally, we assessed adherence m
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13 to human epithelial cells and killing by macrophages. r
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16 Materials and methods. n
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18 Anidulafungin, Candida isolates and measurement of growth rates. Anidulafungin ry

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19 was provided by Pfizer, Inc (Groton, CT). Ten Candida isolates (3 C. albicans, 3 C. 2
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20 glabrata, 3 C. parapsilosis and 1 C. krusei) were recovered from the bloodstreams of 9

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21 patients with candidemia. C. parapsilosis isolates were confirmed as belonging to group g
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22 I (C. parapsilosis sensu stricto) by ITS1 sequences, using previously described methods s
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23 (15). C. albicans ATCC 90028 and C. krusei ATCC 6258 were included as quality
24 controls. The in vitro growth rates were determined in yeast peptone dextrose (YPD) and
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1 RPMI 1640 liquid media at 30 and 37°C in microtitre plates as described in our previous
2 publication (5).
3
4 Antifungal susceptibility testing. MICs of anidulafungin were determined using a broth
5 microdilution technique (M27-A3) in RPMI 1640 medium buffered to pH of 7.0 with D
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6 MOPS, as recommended by the Clinical Laboratory Standards Institute Antifungal n
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7 Subcommittee (8). The starting inoculum was 0.5 x 103 to 2.5 x 103 cells/mL, and ad
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8 incubation was at 35˚ C. The MICs were read as the lowest concentration of drug that f
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9 caused a significant diminution ( 50%) of growth below control levels (19). The range of

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10 anidulafungin concentrations was 0.008 to 32 µg/µL. Each isolate was tested on least p
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11 three occasions. a
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12 m
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13 Antifungal carryover. Antifungal carryover was excluded using standard methods (11). r
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14 A fungal suspension of approximately 5 x 103 CFU/mL was prepared, and 100 µL added n

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15 to 900 µL of sterile water or sterile water plus anidulafungin. Immediately after the n
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16 addition of the fungal suspension to the aqueous solution, the test tube was vortexed and ry

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17 a 30 µL aliquot plated on Sabouraud dextrose agar (SDA) plates. Following 48 hours of 2
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18 incubation at 35°C, the numbers of colony forming units (CFUs) were determined. The 9

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19 mean colony count at each multiple of MIC tested was compared with the data for the g
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20 control. Significant antifungal carryover was defined as a reduction of CFU > 25% of the s
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21 control.
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1 Time-kill and PAFEs. We measured time-kills and PAFEs simultaneously for each
2 isolate using methods previously described by our lab (7,14). Prior to testing, isolates
3 were subcultured twice on SDA plates. Colonies from a 24- to 48-hour culture were
4 suspended in 9 mL of sterile water and adjusted to 0.5 McFarland standard. Then, 1 mL
5 of the suspension was added to 9 mL of either RPMI 1640 buffered to pH of 7.0 with D
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6 MOPS or a solution of medium plus anidulafungin. These methods resulted in a starting n
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7 inoculum of approximately 1 x 105 to 5 x 105 CFU/mL. Test solutions were placed on an ad
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8 orbital shaker and incubated at 35°C with agitation. At the desired time points, 100 µL f
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9 were obtained from each solution, serially diluted ten-fold in sterile water, and 10 µL

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10 plated on SDA plates. Following incubation at 35°C for 48 hours, the number of CFU p
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11 was determined. a
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12 m
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13 Using this protocol, we set up time-kill and PAFE experiments for control (no drug), r
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14 0.25x, 1x, 4x and 16x MIC tubes in duplicate tubes (labeled as “time-kill” and “PAFE”). n

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15 We plated all tubes at time 0. After an incubation period of one hour, we removed the n
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16 respective “PAFE” tubes of the set, washed and centrifuged cells at 1400 x g for 10 ry

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17 minutes (3 cycles), and resuspended the fungal pellet in warm RPMI medium (9 mL) 2
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18 prior to re-incubation with the “time-kill” tubes. Both sets of tubes were plated on SDA 9

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19 plates after 2, 4, 8, 12, 24 and 48 hours, and CFUs enumerated after incubation at 35°C g
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20 for 48 hours. Time-kill and PAFE experiments were conducted twice for each isolate, and s
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21 data presented as mean values.
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1 “Persister” Candida isolates were not killed by anidulafungin after 12 hours of time-
2 kill and PAFE experiments. For each isolate, three colonies growing on SDA plates at 48
3 hours were selected, and their in vitro growth rates and susceptibility to anidulafungin
4 were tested using the methods above.
5 D
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6 Sensitivities to cell wall agents SDS and calcofluor white (6). Each of the following n
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7 experiments was performed in triplicate. Candida cells were recovered 8 hours after a ad
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8 one hour exposure to anidulafungin at 0, 1x and 4x MIC. Cells were subcultured in YPD f
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9 liquid medium with 1% glucose until exponential growth, and diluted to an OD of 0.1.
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10 Four microlitres of undiluted and serial 10-fold dilutions of each culture were spotted p
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11 onto YPD plates containing calcofluor white (40 µg/mL) or SDS (0.01-0.04 %). The a
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12 plates were incubated at 30 °C for 72 h. In preliminary experiments, Candida isolates not sm
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13 exposed to anidulafungin were only slightly susceptibile to these concentrations of r
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14 calcofluor white and SDS. For this reason, we hypothesized that the concentrations n

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15 would be useful for showing increased susceptibility among Candida isolates exposed to n
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16 anidulafungin. ry

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17 2
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18 Electron microscopy. Transmission electron microscopy was performed by the 9

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19 Electron Microscopy Laboratory at the North Florida/South Georgia Veterans Health g
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20 System, in accordance with their established protocol. Briefly, Candida cells recovered 8 s
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21 hours after a one hour exposure to anidulafungin at 0, 1x and 4x MIC were cultured on
22 SDA plates for 24 hours at 35˚ C. Cells selected from colonies on the plates were fixed
23 at 4˚ C in 0.1 M Na cacodylate buffer (pH 7.2) containing 2% glutaraldehyde and 2%
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1 paraformaldehyde. The samples were then dehydrated through a graded series of ethanol
2 and embedded in Lowicryl K4M (Electron Microscopy Sciences, Hatfield, PA). Thin
3 sections were imaged with a Zeiss EM902 electron microscope.
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5 Adherence of C. albicans to human buccal epithelial cells (BECs) (6). Buccal D
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6 epithelial cells were collected from three investigators by gently scraping the cheek n
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7 mucosa with a cotton swab and dispensed into 10 mL PBS. The pooled BECs were then d
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8 washed four times with PBS, and counted using a haemocytometer. A final concentration f
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9 of 1 × 105 epithelial cells/mL was adjusted in PBS. Candida cells recovered 8 hours after

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10 a one hour exposure to anidulafungin at 4x MIC were cultured on SDA for 24 hours at p
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11 35˚ C. To perform the adherence assay, 0.5 mL of the washed epithelial cells were a
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12 incubated in a glass tube with 0.5 mL of washed Candida cells in PBS at a concentration m
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13 of 1 × 106 per mL in a shaking incubator at 37°C for one hour; for control, 0.5 mL of r
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14 BECs were mixed with 0.5 mL of PBS. Following incubation, the cells were vacuum n

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15 filtered through prewet 20-mm diameter polycarbonate filters with 12 mm pore size n
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16 (Costar, MA, USA) mounted on a filter manifold (Millipore, Bedford, MA). Each filter ry

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17 was washed 10 times with PBS to remove unattached candidal cells. The washed filters 2
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18 were then removed and pressed gently onto glass slides. The slides were air-dried, heat 9

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19 fixed for 1 min, and Gram stained. The slides were examined under light microscopy in g
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20 1-mm intervals, and the number of candidal cells attached to 100 BECs was counted. s
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21 Each experiment was performed at least two separate occasions, using BECs harvested
22 from the same individuals at the same time on successive days; on each occasion, the
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1 experiments were performed in duplicate. Differences between isolates exposed to
2 anidulafungin and the corresponding controls were determined using Student's t-test.
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4 Macrophage phagocytosis assay (4).
5 Candida cells were recovered as described for adherence assays, opsonized with 50% D
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6 human serum for 30 minutes at 37 °C, washed with PBS, counted in a haemacytomer n
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7 chamber and transferred to liquid RPMI medium. The murine macrophage-like tumour d
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8 cell line J774A.1 (ECACC 85011428, from a female BALB/c mouse) was obtained from f
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9 American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured at 37°C

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10 and 5% CO in complete medium (RPMI supplemented with heat-inactivated fetal calf
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11 serum 10% v/v, 5 mM L-glutamine, streptomycin 100 mg/L and penicillin 50 mg/L). a
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12 Viability was assessed by trypan blue exclusion (Gibco) and was >95%. For m
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13 quantification experiments, J774A.1 monolayer cells were challenged with Candida cells r
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14 at a ratio of 1:10, and incubated for 2 hours at 37 °C. The medium was then removed and n

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15 the monolayers were washed thoroughly 10 times with cold PBS, after which the n
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16 mixtures were resuspended in 1 mL of sterile distilled water at 37°C for 5 minutes and ry

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17 agitated vigorously with a micropipette until the macrophages were completely lysed. 2
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18 Serial 10-fold dilutions were made, and a colony count was performed following 9

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19 incubation at 30°C for 48 hours. The percentage of surviving Candida was calculated in g
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20 comparison with the CFUs of Candida grown in the same conditions without s
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21 macrophages. The assays were performed in triplicate and repeated at least twice.
22 Differences between the null mutant and CAI12 were determined using Student's t-test.
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1 Results.
2
3 Anidulafungin is fungicidal and exerts prolonged post-antifungal effects (PAFEs)
4 against diverse Candida spp. MICs of anidulafungin are shown in Table 1. The ranges
5 of MICs are consistent with values for the given species reported in large surveillance
6 studies of anidulafungin activity in vitro (20). As anticipated, MICs against C. D
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7 parapsilosis isolates were higher than other species. n
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8 The results of time-kill and PAFE experiments are summarized in Table 2. e
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9 Representative kill- and PAFE curves are shown in Figure 1 for three Candida isolates. o
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10 In the time-kill experiments at 16x MIC, the range of log time-kills was 2.68 to 3.89. t
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11 Anidulafungin was fungicidal against nine isolates (defined as ≥ log 3 kill), including all /a
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12 C. parapsilosis, C. glabrata and C. krusei. At 4x MIC, the range of log time-kills was .a
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13 1.87 to 3.19, and anidulafungin was fungicidal against two of the C. parapsilosis isolates. .
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14 One hour exposure to anidulafungin during PAFE experiments also resulted in significant /
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15 time-kills, ranging from log 1.55 - 3.47 at 16x MIC and log 1.18 - 2.89 at 4x MIC. J
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16 Indeed, one hour exposure to 16x MIC was fungicidal against four isolates, including all a
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17 three C. parapsilosis isolates. Moreover, regrowth of all twelve isolates was inhibited for 5
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18 at least 12 hours after anidulafungin was washed out, consistent with sustained PAFEs. 1
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19 It is notable that persister Candida isolates that were not killed by anidulafungin g
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20 in our study did not exhibit elevated MICs upon re-testing. Moreover, growth of the s
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21 persisters in liquid media after recovery from time-kill and PAFE experiments was not
22 reduced.
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