Miltefosine inhibits Candida albicans and non-albicans Candida spp. biofilms and impairs the dispersion of infectious cells

ARTICLE IN PRESS International Journal of Antimicrobial Agents ■■ (2016) ■■–■■

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International Journal of Antimicrobial Agents j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j a n t i m i c a g

Miltefosine inhibits Candida albicans and non-albicans Candida spp. biofilms and impairs the dispersion of infectious cells

1

Q2

2 3 4

Q1 Taissa Vila a,*, Kelly Ishida b, Sergio Henrique Seabra c, Sonia Rozental a

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

a Laboratório de Biologia Celular de Fungos, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Av. Carlos Chagas Filho 373, 21941-902 Rio de Janeiro, RJ, Brazil b Laboratório de Quimioterapia Antifúngica, Departamento de Microbiologia, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-900 São Paulo, SP, Brazil c Laboratório de Tecnologia em Bioquímica e Microscopia (LTBM), Centro de Ciências Biológicas e da Saúde, Centro Universitário Estadual da Zona Oeste, Av. Manuel Caldeira de Alvarenga 1203, 23070-200 Rio de Janeiro, RJ, Brazil

A R T I C L E

I N F O

Article history: Received 24 March 2016 Accepted 30 July 2016 Keywords: Candida Miltefosine Biofilms Extracellular matrix Resistance Dispersion

A B S T R A C T

Candida spp. can adhere to and form biofilms over different surfaces, becoming less susceptible to antifungal treatment. Resistance of biofilms to antifungal agents is multifactorial and the extracellular matrix (ECM) appears to play an important role. Among the few available antifungals for treatment of candidaemia, only the lipid formulations of amphotericin B (AmB) and the echinocandins are effective against biofilms. Our group has previously demonstrated that miltefosine has an important effect against Candida albicans biofilms. Thus, the aim of this work was to expand the analyses of the in vitro antibiofilm activity of miltefosine to non-albicans Candida spp. Miltefosine had significant antifungal activity against planktonic cells and the development of biofilms of C. albicans, Candida parapsilosis, Candida tropicalis and Candida glabrata. The activity profile in biofilms was superior to fluconazole and was similar to that of AmB and caspofungin. Biofilm-derived cells with their ECM extracted became as susceptible to miltefosine as planktonic cells, confirming the importance of the ECM in the biofilm resistant behaviour. Miltefosine also inhibited biofilm dispersion of cells at the same concentration needed to inhibit planktonic cell growth. The data obtained in this work reinforce the potent inhibitory activity of miltefosine on biofilms of the four most pathogenic Candida spp. and encourage further studies for the utilisation of this drug and/or structural analogues on biofilm-related infections. © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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1. Introduction Invasive candidiasis has shown increasing importance in cases of nosocomial infection, and Candida spp. are the fourth most common pathogen isolated from hospital blood cultures [1]. Although Candida albicans remains the most frequently isolated species, the incidence of non-albicans Candida spp. has increased in recent decades [2]. Among non-albicans Candida spp., Candida glabrata is the most prevalent in North America and Europe, whilst Candida tropicalis and Candida parapsilosis are the most prevalent in South America and Asia [2]. Even with antifungal therapy, mortality due to candidaemia can be as high as 40% [1] and resistance to fluconazole and echinocandins has been shown to be more common in non-albicans Candida spp. than in C. albicans [3]. One of the major contributors to the pathogenicity of Candida spp. is their ability to form biofilms over different surfaces, including medical devices such as prostheses and catheters [4]. Biofilms

60 61 62 63 64

* Corresponding author. Department of Biology, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249, USA. Fax: +1 210 458 7023. E-mail address: [email protected] (T. Vila).

are microbial communities of cells that grow adhered to a surface, embedded in an extracellular matrix (ECM), and biofilm cells exhibit an altered phenotype compared with planktonic cells [5]. Biofilminfected catheters may function as reservoirs of infectious particles, releasing cells into the bloodstream and allowing metastatic infections of deep organs [6]. Moreover, cells that detach from the biofilm (‘dispersion cells’) are more virulent than their planktonic counterparts [6]. Increased resistance to drugs is the most clinically relevant phenotypic alteration displayed by cells that grow as biofilms [4,7] and several mechanisms have been proposed to explain this reduced susceptibility [8], including overexpression of drug efflux pumps [9,10] and the ability of the ECM to act as a drug sequestrant [11–13]. Some authors also argue that the increased cell density inside biofilms could explain resistance [14,15]. Among the few available antifungals for the treatment of candidaemia, only the lipid formulations of amphotericin B (AmB) and the echinocandins are effective against biofilms [16,17]. Miltefosine is an alkylphosphocholine that was initially developed as an antitumour agent, but its anticancer activity was shown to be limited. In parallel, this compound showed potent antiparasitic activity, particularly against Leishmania spp. and Trypanosoma

http://dx.doi.org/10.1016/j.ijantimicag.2016.07.022 0924-8579/© 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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cruzi [18,19]. Its antifungal activity on planktonic cells was first demonstrated by Widmer et al [20] and was further explored by others [20–23]. Recently, our group showed that miltefosine has an important inhibitory effect on in vitro biofilms formed by C. albicans and Fusarium oxysporum on catheter and fingernail surfaces [24,25] and in vivo using a candidiasis oropharyngeal model [26]. Thus, the aims of this work were (i) to extend the analysis of the in vitro antibiofilm activity of miltefosine against non-albicans Candida spp., (ii) to investigate whether miltefosine affects biofilm dispersion and (iii) to explore the role of the biofilm biomass in susceptibility to miltefosine. 2. Materials and methods 2.1. Strains Six Candida spp. strains were obtained from the American Type Culture Collection (ATCC®) and eleven clinical isolates were provided by Dr. Marcos Dornellas Ribeiro (Microbiology and Mycology Laboratory, HemoRio, Rio de Janeiro, Brazil) (Table 1). The clinical isolates with a heterogeneous susceptibility profile to standard drugs were selected based on their ability to display good biofilm formation. 2.2. Antifungal agents Miltefosine (Cayman Chemical, Ann Arbor, MI), fluconazole (Pfizer, São Paulo, Brazil) and caspofungin (Sigma-Aldrich, St Louis, MO) were diluted in distilled water, whilst amphotericin B deoxycholate (SigmaAldrich) was diluted in dimethyl sulphoxide (DMSO). Stock solutions were maintained at −20 °C. The final concentration of DMSO was not higher than 0.14%. 2.3. Minimum inhibitory concentration (MIC) determination MICs of the antifungal agents were determined for planktonic cells, dispersion cells and cells recovered from biofilms using the broth microdilution assay described by the Clinical and Laboratory Standards Institute (CLSI) [27]. Serial two-fold dilutions were performed to obtain concentration ranges from 0.03 to 16 μg/mL for AmB, miltefosine and caspofungin and from 0.12 to 128 μg/mL for fluconazole. The final concentration of yeasts in each well was

129 130 131 132 133

Table 1 Candida spp. strains obtained from the American Type Culture Collection (ATCC®) and clinical isolates from the Microbiology and Mycology Laboratory at the Institute of Hematology of Rio de Janeiro Estate (HemoRio), Rio de Janeiro, Brazil.

134

Strain

135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153

ATCC® C. albicans SC 5314 (MYA 2876™) C. albicans 10231™ C. parapsilosis 22019™ C. tropicalis 13803™ C. tropicalis 28707™ C. glabrata 2001™ Microbiology and Mycology Laboratory, HemoRioa C. albicans 44A C. albicans 58 C. albicans 119 C. parapsilosis 4 C. parapsilosis 13 C. parapsilosis 14 C. parapsilosis 81 C. tropicalis 5 C. tropicalis 6 C. tropicalis 75 C. glabrata 24

154

a

0.5 × 103 CFU/mL. The minimum concentrations that inhibited 50% and 90% of fungal growth (IC50 and IC90 values, respectively) were determined by visual analysis and by spectrophotometric readings at 492 nm (SpectraMAX® 340; Molecular Devices, Sunnyvale, CA). The susceptibility profile of standard drugs was analysed according to the CLSI [28]. 2.4. Drug susceptibility testing with higher-density planktonic (HDP) suspensions The IC was determined for HDP suspensions using the broth microdilution assay according to CLSI guidelines [27], except that the final concentration of yeasts in each well was 0.5 × 107 CFU/mL. In addition, read-outs were performed both by optical density (492 nm) and quantification of the metabolic activity of the cells using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulpho-phenyl)-2Htetrazolium-5-carboxanilide] assay. 2.5. Biofilm formation Biofilms were formed as described previously [29] with some modifications. Briefly, yeast suspensions (107 CFU/mL) were transferred into 96- or 24-well microplates (Techno Plastic Products, Trasadingen, Switzerland) and were incubated for 1.5 h (adhesion phase) at 36 °C under agitation. Non-adhered cells were then removed and freshly prepared RPMI 1640 with 2% glucose and 20% foetal bovine serum (Gibco®; Thermo Fisher Scientific, Waltham, MA) (‘supplemented RPMI’) was added to each well to allow biofilm formation. Microplates were incubated for 24 h at 36 °C under agitation. 2.6. Effects of antifungals on biofilms Antifungal susceptibility testing of biofilms during the developmental phase was performed based on a previously described protocol [29] with some modifications. To assess the effect of the drugs on biofilm formation, antifungal agents serially diluted in a separate microplate using supplemented RPMI were transferred to the wells of a microplate after the adhesion period (see Section 2.5) and biofilms were formed in the presence of the drug for 24 h at 36 °C under agitation. To assess the effect of antifungals on preformed biofilms, serially diluted antifungals were added to a microplate containing pre-formed biofilms (see Section 2.5) and were incubated for an additional 24 h at 36 °C under agitation. Biofilm was quantified using the XTT assay, and inhibition of 50% and 90% of the metabolic activity was defined as the biofilm IC (BIC50 and BIC90, respectively).

Isolation site

2.7. XTT reduction assay

Clinical (human) Male with bronchomycosis Case of sprue Not reported Human, pyelonephritis Faeces

Biofilms were quantified using the XTT reduction assay as described previously [29]. Colour change was measured in a SpectraMAX® 340 spectrophotometer.

Gastric lavage Blood Catheter tip Ulcer skin injury Ulcer skin injury Blood Blood Faeces Urine Blood Blood

Clinical isolates were kindly donated by Dr. Marcos Dornellas Ribeiro.

2.8. Confocal laser scanning microscopy (CLSM) Biofilms were formed on glass-bottomed Petri dishes (CELLview™; Greiner Bio-One GmbH, Frickenhausen, Germany) in the presence or absence (control biofilms) of the BIC50 of antifungals as described above. Biofilms were then incubated with fluorescent markers: Concanavalin A (ConA) conjugated to Alexa Fluor™ 488 (Invitrogen™; Thermo Fisher Scientific), which binds to glucose and mannose residues both on the cell wall and in the ECM; FilmTracer® SYPRO® Ruby (Invitrogen), which binds to glycoproteins in the ECM and inside the cells; and Calcofluor White M2R (Sigma-Aldrich), which dyes the fungal cell wall by binding

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to chitin residues. Biofilms were observed by CLSM [LSM 710 (Quasar Model); Zeiss, Oberkochen, Germany]. 2.9. Dispersion and recovery of biofilm cells Biofilms were formed on 24-well microplates in the presence or absence (control biofilms) of the BIC50 of antifungals as described above. The supernatant containing the dispersion cells was then gently aspirated, was washed in phosphate-buffered saline and was standardised to 103 CFU/mL in RPMI for the IC assay (see Section 2.3). Next, the biofilms were scrapped and transferred to microtubes for ECM extraction as previously described [11]. Briefly, the biomass was sonicated (10 min), vortexed (2 min) and centrifuged (3 cycles of 20 min, 4 °C). The supernatant was discarded and the cells were re-suspended in RPMI for the IC assay. 3. Results 3.1. Miltefosine inhibits C. albicans and non-albicans Candida spp. planktonic growth The planktonic susceptibility of 17 Candida spp. strains to miltefosine and standard antifungals was determined; the IC50 and IC90 values are shown in Table 2 and Supplementary Table S1. Miltefosine showed good inhibitory activity against Candida spp. planktonic cells, including non-albicans Candida spp. strains, with IC90 values ranging from 0.5 to >16 μg/mL (Table 2; Supplementary Table S1). C. albicans was the most susceptible species, showing a Q3 median IC90 of 1 μg/mL. The median IC90 values for C. parapsilosis and C. tropicalis strains were 4 μg/mL and 3 μg/mL, respectively, and C. glabrata presented variable susceptibility to miltefosine (Table 2; Supplementary Table S1). Susceptibility to fluconazole was highly variable among strains (IC50, 0.12 to >128 μg/mL) (Table 2). Ten of the strains showed reduced susceptibility to fluconazole, of which four showed a ‘dose-dependent susceptibility profile’, four presented a ‘trailing effect’ (i.e. the presence of residual growth at high drug concentrations) and six were resistant (Supplementary Table S1). C. albicans, C. parapsilosis and C. tropicalis had similar susceptibility profiles to fluconazole (median

259 260 261 262 263

Table 2 Susceptibility of Candida spp. planktonic cells to miltefosine and the standard antifungal drugs fluconazole, amphotericin B (AmB) and caspofungin. Species/antifungal

264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287

C. albicans (n = 5) Miltefosine Fluconazole AmB Caspofungin C. parapsilosis (n = 5) Miltefosine Fluconazole AmB Caspofungin C. tropicalis (n = 5) Miltefosine Fluconazole AmB Caspofungin C. glabrata (n = 2) Miltefosine Fluconazole AmB Caspofungin

IC50 (μg/mL)

IC90 (μg/mL)

Range

Median

Range

Median

*

ND 1 0.25 0.25

0.5–2 0.25 to >128 0.5–1 0.12–4

1 4 1 0.34

0.12–1 0.06–0.5 0.12–1 0.5–8 0.25–2 0.015–0.5 0.25–1

1.25 0.5 0.067 0.687

1–8 0.5 to >128 0.03–1 1–4

4 2 0.25 2

2 0.25 to >128 0.015–0.5 0.25–0.5

2 0.5 0.12 0.5

1–4 1 to >128 0.06–16 0.5–4

3 2 1 0.75

16 0.25–4 0.015–0.03 *

16 2.12 0.022 ND

2 to >16 0.5 to >128 0.06–0.12 0.5–2

9 ND 1.03 1.25

IC50/90, minimum concentrations that inhibited 50% and 90% of fungal growth, respectively; ND, not possible to determine. * Not determined because it was between dilutions in the microdilution assay used.

3

IC90 values of 4, 2 and 2 μg/mL, respectively), whilst C. glabrata showed the highest ICs (Table 2). Among all antifungals, AmB displayed the highest inhibitory activity against Candida spp. planktonic cells (IC50, 0.015–0.5 μg/mL; and IC90, 0.03–16 μg/mL) (Table 2). Only two strains (C. tropicalis 13803 and 28707) were resistant to AmB (Supplementary Table S1). The majority of strains were also susceptible to caspofungin (IC90, 0.12–4 μg/mL) (Table 2).

288 289 290 291 292 293 294 295 296

3.2. Candida spp. biofilms are susceptible to miltefosine both in initial and mature developmental phases The lowest concentrations of miltefosine and standard antifungals able to reduce the viability of biofilm cells (BIC50 and BIC90) are described in Table 3 and Supplementary Table S2. Candida spp. biofilm formation was inhibited by 50% at doses between 8 and 125 μg/mL miltefosine, whilst 90% inhibition was observed in the presence of 125 to >500 μg/mL (Table 3). Miltefosine inhibitory activity was more potent against C. parapsilosis biofilm formation, whilst C. glabrata formed biofilms with greater BICs (Table 3). Interestingly, the dose required to inhibit 90% of C. albicans, C. parapsilosis and C. tropicalis biofilm formation also reduced the viability of pre-formed biofilms by 50% (Table 3). Treatment with 62.5–250 μg/mL miltefosine reduced the viability of pre-formed Candida spp. biofilm cells by 50% in most strains (Table 3). C. tropicalis and C. glabrata pre-formed biofilms were less susceptible to miltefosine than the other species (Table 3). Comparison among standard antifungals revealed that fluconazole was the least active drug, showing no inhibitory effect either on initial or later biofilm development, whilst AmB and caspofungin had better antibiofilm activity (Table 3). AmB was particularly effective at inhibiting Candida spp. biofilm formation (BIC90, 0.125 to ≥16 μg/mL) (Table 3). Albeit, C. tropicalis ATCC 28707 was resistant to AmB in planktonic form and showed high BIC values (Supplementary Table S2). Concentrations of 0.12 to ≥16 μg/mL AmB reduced the viability of pre-formed biofilms by 50% (Table 3); however, the BIC90 was >16 μg/mL for most isolates (Supplementary Table S2). Similarly, 0.015–8 μg/mL caspofungin inhibited 50% of biofilm formation in all strains tested (Table 3), however concentrations >16 μg/mL were necessary to reduce 90% of biofilm formation and the viability of pre-formed biofilms in most strains tested (11/13 strains) (Table 3; Supplementary Table S2).

297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330

3.3. Miltefosine reduces the biomass and alters cell morphology inside biofilms To evaluate the inhibitory effect of miltefosine, biofilms were grown in the presence of their BIC50 (presented in Table 3) and were then analysed by CLSM. Reconstruction images of C. albicans control biofilms are ca. 40 μm thick and abundant ECM can be observed dispersed throughout the biofilm volume (Fig. 1A). Control biofilms revealed a robust network of interconnected hyphae (marked with Calcofluor staining) and budding yeasts (strongly marked by ConA) (Fig. 2A). In contrast, C. albicans biofilms formed in the presence of miltefosine had strongly reduced height (ca. 7 μm) and were possibly formed by a single layer of yeasts cells (Fig. 1B). Significant changes in the morphology of the cells were observed, with irregular ConA and Calcofluor distribution along hyphae and an absence of budding yeasts (Fig. 2B). Biofilm volume reduction was observed to a lesser extent in the presence of AmB or caspofungin (ca. 30 μm and 20 μm, respectively) (Fig. 1C,D). When AmB was present, C. albicans formed hyperfilamented biofilms and mannose/glucose accumulation areas were evident (Fig. 2C, white arrows indicate accumulation of carbohydrates stained by ConA). In the presence of caspofungin, biofilms grow exclusively as yeast (Fig. 2D) and ‘conical’

Please cite this article in press as: Taissa Vila, Kelly Ishida, Sergio Henrique Seabra, Sonia Rozental, Miltefosine inhibits Candida albicans and non-albicans Candida spp. biofilms and impairs the dispersion of infectious cells, International Journal of Antimicrobial Agents (2016), doi: 10.1016/j.ijantimicag.2016.07.022

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Table 3 Drug effect on Candida spp. biofilms: the minimum concentrations of miltefosine and the standard antifungals fluconazole, amphotericin B (AmB) and caspofungin that inhibit 50% (BIC50) and 90% (BIC90) of biofilm formation and that reduce 50% (BIC50) and 90% (BIC90) of pre-formed biofilm metabolic activity were determined using the XTT assay. Species/antifungal

Biofilm formation

Pre-formed biofilms

358

BIC50 (μg/mL)

359

Range

Median

Range

Median

Range

Median

Range

Median

62.5–125 4–1000 >0.06–1 >0.015–8

109.4 500 0.375 5

125–250 >1000 0.125–4 0.03 to >16

187.5 >1000 1.06 16

62.5–250 250 to >1000 2 to >16 >16

125 750 9 ND

250–500 >1000 16 to >16 0.06 to >16

312.5 >1000 16 16

8–16 4 to >1000 0.12–0.25 0.12–4

12 ND 0.185 ND

125 4 to >1000 0.125–1 >16

125 ND 0.5625 ND

125 125 to >1000 0.12–4 1 to >16

125 ND 2.53 ND

250–500 >1000 1 to >16 >16

312.5 ND ND ND

125 500 to >1000 0.5 to >16 >0.03–0.5

125 750 0.5 0.125

250 >1000 1 to >16 0.25 to >16

250 >1000 3 4

250 >1000 4 to >16 0.06 to >16

250 >1000 8 16

500 to >1000 >1000 16 to >16 16 to >16

750 >1000 16 16

125 1000 0.5 1

125 1000 0.5 1.0

250 to >500 >1000 1–2 0.5–16

ND >1000 1.25 8.25

125–1000 1000 to >1000 1–8 0.25 to >16

437.5 ND 4.5 ND

500 to >1000 >1000 8 to >16 >16

ND >1000 ND >16

360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380

C. albicans (n = 5) Miltefosine Fluconazole AmB Caspofungin C. parapsilosis (n = 2) Miltefosine Fluconazole AmB Caspofungin C. tropicalis (n = 3) Miltefosine Fluconazole AmB Caspofungin C. glabrata (n = 2) Miltefosine Fluconazole AmB Caspofungin

BIC90 (μg/mL)

BIC50 (μg/mL)

BIC90 (μg/mL)

ND, not possible to determine.

381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

cells intensely labelled by Calcofluor were observed (white arrowheads in Fig. 2D). Control C. parapsilosis biofilms were very compact (ca. 20 μm in height) and composed by elongated yeasts (Figs 1E, 2E). In the presence of miltefosine, biofilms showed slightly reduced biomass (Fig. 1F) and cells were distributed in small clumps (Fig. 2F). The presence of AmB or caspofungin culminated in thinner (ca. 12 μm and 8 μm, respectively) and less compact biofilms, with distinct colonised areas spread throughout the scanned area (Fig. 1G,H). Similar to C. albicans, the presence of AmB induced a hyperfilamented C. parapsilosis biofilm (Fig. 2G), whilst only elongated yeast cells composed the biofilm formed in the presence of caspofungin (Fig. 2H). C. tropicalis control biofilms were confluent and compact, composed mainly by elongated yeasts (Fig. 2I) that accounted for ca. 8 μm of the 20 μm total area reconstructed (Fig. 1I). The presence of miltefosine, AmB or caspofungin culminated in less extensive biofilms (Fig. 1J–L). Biofilms formed in the presence of miltefosine were looser, with cells distributed vertically in 20 μm (Fig. 1I,J), whilst compact biofilms, possibly formed by a single layer of cells, were formed in the presence of AmB (5–10 μm). Caspofungin induced a major reduction in biofilm formation, and a reduced number of cells grouped in clumps was observed (Fig. 1L). Morphologically altered yeasts were observed in biofilms formed in the presence of all three drugs (white arrowheads in Fig. 2J–L). 3.4. Dispersion cells from Candida spp. biofilms are susceptible to miltefosine Dispersion cells were collected from the supernatant of biofilms grown in RPMI medium (control) and from pre-treated biofilms grown in the presence of the BIC50 of each drug. The inhibitory activity of miltefosine and the standard antifungal drugs against dispersion cells from biofilms was evaluated using the broth microdilution assay [27]. Dispersion cells both from control and pretreated biofilms were susceptible to miltefosine (IC90, 1–4 μg/mL) (Table 4) and ICs were similar to those obtained for planktonic cells (Tables 2 and 4). Importantly, the presence of miltefosine during C. glabrata ATCC 2001 biofilm development inhibited the dispersion of cells, which impaired the microdilution assay. This behaviour

was also observed for C. albicans SC 5314 biofilms grown in the presence of caspofungin (Table 4). Considering the standard antifungal drugs, no major resistance increase was observed for dispersion cells (Table 4). Interestingly, dispersion cells from C. tropicalis 13803 and C. glabrata 2001 biofilms were more susceptible to fluconazole than planktonic cells (Table 4). Still, C. tropicalis and C. glabrata dispersion cells were less susceptible to AmB than planktonic cells (Table 4). 3.5. Recovered Candida spp. biofilm cells are susceptible to miltefosine To elucidate the role of the biofilm three-dimensional structure in the susceptibility to miltefosine and the standard antifungals, control and pre-treated biofilm cells were recovered and their susceptibility to antifungals was tested after the ECM was extracted. Cells recovered both from control and pre-treated Candida spp. biofilms were equally susceptible to miltefosine, with IC90 values of 1–4 μg/mL for all strains tested, similar to that obtained for planktonic cells (Table 5). Interestingly, cells recovered from C. albicans, C. tropicalis 13803 and C. glabrata biofilms were more susceptible to fluconazole than planktonic cells, whilst cells recovered from C. tropicalis 13803 and C. glabrata biofilms showed increased resistance to AmB (IC90, 8–16 μg/mL) (Table 5). C. tropicalis 28707 is resistant to fluconazole and AmB in planktonic form and remained resistant both in control and pre-treated biofilms after ECM extraction (IC90 ≥ 16 μg/mL) (Table 5). Candida spp. cells recovered from biofilms from all strains were as susceptible to caspofungin as planktonic cells (Table 5). 3.6. Candida spp. high-density planktonic (HDP) suspensions remain susceptible to miltefosine To further address whether the increased cell density inside the biofilm could be responsible for its reduced susceptibility, HDP suspensions (107 CFU/mL) were used to conduct the microdilution assay as described by the CLSI [27]. The results showed that even when the cell density is increased to correlate with the density of a biofilm, all Candida spp. remain susceptible to miltefosine (Table 6). A significant increase in the IC50 for HDP cells was observed (8–16 μg/mL)

Please cite this article in press as: Taissa Vila, Kelly Ishida, Sergio Henrique Seabra, Sonia Rozental, Miltefosine inhibits Candida albicans and non-albicans Candida spp. biofilms and impairs the dispersion of infectious cells, International Journal of Antimicrobial Agents (2016), doi: 10.1016/j.ijantimicag.2016.07.022

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Fig. 1. Confocal laser scanning microscopy images of Candida biofilms. Volume representation of spatial distributions of biofilm biomass stained with Concanavalin A–Alexa Fluor™ 488 (green), Calcofluor White M2R (blue) and FilmTracer® SYPRO® Ruby (red). (A–D) Candida albicans, (E–H) Candida parapsilosis and (I–L) Candida tropicalis biofilms were grown for 24 h in the presence of the respective BIC50 for biofilm formation of each drug: miltefosine (B,F,J), amphotericin B (C,G,K) and caspofungin (D,H,L). Control biofilms (A,E,I) received only RPMI supplemented with glucose (2%) and foetal bovine serum (20%). Magnification 63×. BIC50, concentration inhibiting 50% of the biofilm metabolic activity. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM02986. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Taissa Vila, Kelly Ishida, Sergio Henrique Seabra, Sonia Rozental, Miltefosine inhibits Candida albicans and non-albicans Candida spp. biofilms and impairs the dispersion of infectious cells, International Journal of Antimicrobial Agents (2016), doi: 10.1016/j.ijantimicag.2016.07.022

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Fig. 2. Confocal laser scanning microscopy images of Candida biofilms. Front view of the reconstructed Z-sections of the biofilms stained with Concanavalin A–Alexa Fluor™ 488 (green), Calcofluor White M2R (blue) and FilmTracer® SYPRO® Ruby (red). (A–D) Candida albicans, (E–H) Candida parapsilosis and (I–L) Candida tropicalis biofilms were grown for 24 h in the presence of the respective BIC50 for biofilm formation of each drug: miltefosine (B,F,J), amphotericin B (C,G,K) and caspofungin (D,H,L). Control biofilms (A,E,I) received only RPMI supplemented with glucose (2%) and foetal bovine serum (20%). White arrow in (C) indicates extracellular matrix (ECM) accumulation. White arrowheads in (J), (K), (D) and (L) indicate cell damage/surface alterations due to drug treatment. Magnification 63×. BIC50, concentration inhibiting 50% of the biofilm metabolic activity. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM02942. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 4 Susceptibility of Candida spp. planktonic and biofilm dispersion cells to miltefosine and the standard antifungals fluconazole, amphotericin B (AmB) and caspofungin. The minimum inhibitory concentrations of 50% and 90% of fungal growth (IC50 and IC90, respectively) were determined by the microdilution broth method [27]. Dispersion cells were recovered from drug-free formed biofilms (control) and from biofilms formed in the presence of its inhibitory concentration of 50% (BIC50) (pre-treated).a

477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510

7

Miltefosine Planktonic Biofilm dispersion cells Pre-treated biofilm dispersion cells Fluconazole Planktonic Biofilm dispersion cells Pre-treated biofilm dispersion cells AmB Planktonic Biofilm dispersion cells Pre-treated biofilm dispersion cells Caspofungin Planktonic Biofilm dispersion cells Pre-treated biofilm dispersion cells

C. albicans SC 5314

C. parapsilosis ATCC 22019

C. tropicalis ATCC 13803

C. tropicalis ATCC 28707

C. glabrata ATCC 2001

IC50 IC90 IC50 IC90 IC50 IC90

* 1 0.125 2 * 1–2

* 1–2 * 1 * 1

* 2 * 4 * 4

* 2–4 2 4 2 4

* 2–4 * 2 † †

IC50 IC90 IC50 IC90 IC50 IC90

0.125–0.25 4 0.12 1–4 0.12 1–2

2 4 1 2 0.5 1

0.25 2 <0.5 0.5–1 <0.5 0.5

>16R >16 >16R >16 >16R >16

4 >128R * 4 2 4

IC50 IC90 IC50 IC90 IC50 IC90

0.06 0.5 0.5 0.5–1 * 0.5

0.5 1 0.25 1 0.12–0.25 1

0.5 4R 1 8–16R 1 8–16R

0.125 >16R 8 16R * ≥16R

0.03 0.12 8 16R * 16R

IC50 IC90 IC50 IC90 IC50 IC90

0.125 0.125–0.5SDD 0.06 0.25 † †

* 1 * 1 * 1

* 0.5SDD * 0.12–0.25T 0.25 0.5T,SDD

* 0.5–1SDD–R 0.25 0.5SDD 0.25 0.5SDD

* 0.5 0.06–0.12 0.15–0.5R 0.12 0.5–1†

R, resistant; SDD, susceptible dose-dependent; T, trailing effect. Susceptibility analysis was based on the breakpoints defined by the Clinical and Laboratory Standards Institute (CLSI) [28]. a Values are expressed in μg/mL. * Not determined because it was between dilutions in the microdilution assay used. † Cell growth <103 CFU/mL (insufficient).

511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543

for miltefosine in comparison with that of lower-density suspensions (1–4 μg/mL), however, IC50 values of HDP cells were similar to IC90 values of lower-density suspensions (Table 6). On the other hand, HDP cells of all Candida spp. became resistant to fluconazole (IC50, 32 to ≥128 μg/mL) and showed reduced susceptibility to AmB (IC90, 2–16 μg/mL), and C. tropicalis HDP cells became resistant to caspofungin (IC50 > 16 μg/mL). 4. Discussion Biofilm formation on medical devices is a risk factor for invasive candidiasis and mortality [30,31]. Invasive Candida infections can be successfully treated with azoles, AmB and echinocandins [32], however biofilm-related infections are refractory to standard treatments [16,33]. Biofilms are up to 1000 times more resistant to azoles than planktonic cells [16], and echinocandin activity appears to be variable among Candida spp. [16,34]. Thus, an extremely limited drug arsenal is available to treat biofilm-related Candida infections. The activity of miltefosine against C. albicans biofilms was previously demonstrated by our group both using in vitro (central venous catheter) [24] and in vivo (oral candidiasis) [26] models. In the present study, this research was extended to include nonalbicans Candida spp. Miltefosine (115–500 μg/mL) inhibited biofilm formation by 90% in all Candida spp. tested. These concentrations, although higher than the BIC90 for AmB and caspofungin, are much lower than those found for fluconazole. These data are encouraging since fluconazole is the first choice for candidiasis treatment and, as previously showed by others [16] and confirmed here, is ineffective against Candida biofilms. Q4 Candida biofilm resistance is a multifactorial event still far from being completely elucidated [8]. Because several works have

confirmed the participation of the ECM in biofilm resistance, we tested whether the ECM was also impairing miltefosine activity. As expected, withdrawal of the ECM from the biofilm resulted in enhanced susceptibility of the cells of all Candida spp. to miltefosine, indicating a possible role for the ECM in biofilm response to this compound. Biofilm-recovered cells of C. albicans and C. parapsilosis also re-established their susceptibility to all other drugs, whilst C. tropicalis and C. glabrata recovered cells showed increased ICs for AmB. Seneviratne et al [15] also reported higher ICs for AmB in C. albicans biofilm-derived cells. This could indicate that other mechanisms may also be involved in the resistance of biofilm cells to AmB, such as changes in the ergosterol content of cell membranes, as previously reported [10,35]. After biofilm development, cell density is ca. 107–108 CFU/mL [15], which is much higher than the standardised inoculum used for IC determination (103 CFU/mL). Hence, it was postulated that the increased cell density could be the main reason responsible for the reduced activity of antifungal drugs against biofilms [14]. In agreement with that, we show here that planktonic suspensions of C. tropicalis adjusted to 107 CFU/mL were less susceptible to AmB and caspofungin, as also demonstrated by Perumal et al [14], and became completely resistant to fluconazole, corroborating two previous reports [14,15]. However, even though some reduction in susceptibility could be observed, the IC90 for HDP Candida spp. for miltefosine was still lower than the BIC90 both for biofilm formation and pre-formed biofilms, indicating that biofilm-related features other than (or together with) cell density may be responsible for its reduced susceptibility in comparison with planktonic cells. Reduced penetration of antifungals through the ECM of Candida biofilms has been previously attributed to a direct interaction of the drugs with polysaccharides of the ECM [11,13,36,37]. Direct

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Table 5 Antifungal susceptibility of planktonic cells compared with cells recovered from biofilms (after extracellular matrix extraction) grown as control (drug-free) or in the presence of the BIC50 of each drug. The minimum inhibitory concentrations of 50% (IC50) and 90% (IC90) of fungal growth.

579

IC value (μg/mL)

580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611

Miltefosine Planktonic cells Biofilm recovered cells Treated biofilm recovered cells Fluconazole Planktonic cells Biofilm recovered cells Treated biofilm recovered cells Amphotericin B Planktonic cells Biofilm recovered cells Treated biofilm recovered cells Caspofungin Planktonic cells Biofilm recovered cells Treated biofilm recovered cells

C. albicans SC 5314

C. parapsilosis ATCC 22019

C. tropicalis ATCC 13803

C. tropicalis ATCC 28707

C. glabrata ATCC 2001

IC50 IC90 IC50 IC90 IC50 IC90

* 1 0.06–0.12 2 0.06–0.12 2

* 1–2 * 1 * 1

* 2 * 4 * 4

* 2–4 2 4 * 4

* 2–4 * 2 * 2

IC50 IC90 IC50 IC90 IC50 IC90

0.12–0.25 4 0.12 0.5–1 0.12 0.25–0.5

2 4 * 2 1–2 2–4

0.25 2 <0.5 0.5–1 <0.5 1–2

>16R >16 >16R >16 >16R >16

4 >128R 2 4 2 4

IC50 IC90 IC50 IC90 IC50 IC90

0.06 0.5 * 0.5 * 1

0.5 1 * 1 0.12–0.25 0.5

0.5 4R 1–2 8R * 16R

0.12 >16R 8 16R 16 >16R

0.03 0.12 8 16R ND 8R

IC50 IC90 IC50 IC90 IC50 IC90

0.12 0.12–0.5 0.06 0.12 0.03 0.06

* 1 * 1 * 1

* 0.5 * 0.12T * 0.12–0.5T

* 0.5–1 * 0.25–0.5 * 0.12–0.5

* 0.5 0.25 I 0.5–1 * 1

BIC50, concentration inhibiting 50% of the biofilm metabolic activity; R, resistant; T, trailing effect; ND, not possible to determine. Susceptibility analysis was based on the breakpoints defined by the Clinical and Laboratory Standards Institute (CLSI) [28]. * Not determined because it was between dilutions in the microdilution assay used.

612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643

association of miltefosine and the ECM could explain the higher drug concentration necessary to affect Candida spp. biofilms compared with planktonic cells and also the recovered susceptibility achieved after ECM extraction. Based on these results in vitro, concentrations as low as 1 mg/mL miltefosine could effectively eliminate pre-formed biofilms of all Candida spp. tested. In vivo studies showed that topical administration of 2 mg/mL miltefosine is not toxic in a mouse model of oral candidiasis [26], and oral administration of doses as high as 18 mg/kg/day was not systemically toxic to mice [20]. Oral administration of miltefosine is currently used for the treatment of visceral leishmaniasis and has good oral bioavailability (82–95%) with distribution to all tissues and accumulation mainly in the liver, lungs, kidneys and spleen [19]. The maximum concentration reported in human serum ranges from 31 to 70 μg/mL (daily doses of 150 mg) [19]. This concentration is 7–16 times higher than the average IC90 for planktonic cells (4.4 μg/mL) but is still lower than the BIC50. Thus, oral administration of miltefosine alone might not successfully eliminate biofilms in vivo but it may be an important complementary drug for combined therapy. In addition, dispersion of cells from biofilms is directly correlated with dissemination and establishment of invasive disease [6]. Here we demonstrate that dispersion cells from Candida spp. biofilms are highly susceptible to miltefosine as well as to all standard antifungals tested, showing similar IC90 values to planktonic cells. Uppuluri et al reported similar results for AmB and caspofungin but not for fluconazole [38], which could be due to differences in biofilm growth models. It is important to reinforce that the presence of subinhibitory concentrations of miltefosine during C. glabrata biofilm growth inhibited the release of dispersion cells (Table 4). Inhibition of biofilm dispersion is of utmost importance during the

Table 6 Candida spp. planktonic susceptibility to miltefosine and the standard antifungal drugs fluconazole, amphotericin B (AmB) and caspofungin: comparison of low-density suspensions (103 CFU/mL), as defined by the Clinical and Laboratory Standards Institute (CLSI), and higher-density planktonic (HDP) suspensions (107 CFU/mL). The minimum inhibitory concentrations of 50% (IC50) and 90% (IC90) of fungal growth are expressed in μg/mL and were obtained by turbidity for all samples, as well as by measurement of the cell metabolic activity (XTT assay) for HDP cells. Strain/ antifungal

Low cell density (103 CFU/mL)

High cell density (107 CFU/mL)

Turbidity

Turbidity

IC50 C. albicans SC 5314 Miltefosine * Fluconazole 0.12–0.25 AmB 0.06 Caspofungin 0.12 C. parapsilosis ATCC 22019 Miltefosine * Fluconazole 2 AmB 0.5 Caspofungin * C. tropicalis ATCC 13803 Miltefosine * Fluconazole 0.25 AmB 0.5 Caspofungin * C. glabrata ATCC 2001 Miltefosine * Fluconazole 4 AmB 0.03 Caspofungin *

644 645 646 647 648 649 650 651 652 653 654

XTT assay

IC90

IC50

IC90

IC50

IC90

1 4 0.5 0.12–0.5

2–4 >128 0.5–0.25 <0.03T

16 >128 >16R >16

4 >128 2 >16R

16 >128 8–16R >16

1–2 4 1 1

2–4 32–128R 0.5 1–4T,SDD

8 >128 >16R >16

2–4 1 0.25–0.5 0.5–1

8 >128 2 16

2 2 4R 0.5

8 32–128R 0.125–0.25 >16R

16 >128 2R >16

8–16 >128R 0.03–0.06 <0.03T

16 >128 4R >16

2–4 >128 0.12 0.5

2–4 >128R 0.03–0.06 <0.03T

8–16 >128 1–2R 0.125

0.25 0.5–1 0.03–0.06 <0.03

>16 >128 1–2R 0.06–0.125

R, resistant; T, trailing effect; SDD, susceptible dose-dependent. Susceptibility analysis was based on the breakpoints defined by the CLSI [28]. * Not determined because it was between dilutions in the microdilution assay used.

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treatment of biofilm-related infections because it reduces dissemination and favours the elimination of the infection site. In this context, miltefosine is a promising drug to be used in lock therapy as a pre-treatment of medical devices to prevent biofilm formation and to help contain metastatic infections, as previously described for liposomal AmB [39]. 5. Conclusions In summary, this work shows that miltefosine is effective against fluconazole-resistant biofilms of the four most common pathogenic species of Candida (C. albicans, C. parapsilosis, C. tropicalis and C. glabrata). Miltefosine also inhibited the dispersion of C. glabrata biofilm cells, which in vivo could mean disrupting the re-infection cycle associated with biofilm-related infections. Together these data highlight the potential of miltefosine as an alternative drug in the treatment of biofilm-related Candida infections and point out that the synthesis of structural analogues could lead to an even better molecule, with increased antifungal activity and reduced toxicity. Acknowledgement The authors thank Dr. Christopher G. Pierce for the English revision. Q5 Funding: This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Brazil) and the Q6 Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (Brazil). Competing interests: None declared. Ethical approval: Not required. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijantimicag.2016.07.022. References [1] Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 2004;39:309– 17. [2] Nucci M, Queiroz-Telles F, Alvarado-Matute T, Tiraboschi IN, Cortes J, Zurita J, et al. Epidemiology of candidemia in Latin America: a laboratory-based survey. PLoS ONE 2013;8:e59373. [3] Cleveland AA, Farley MM, Harrison LH, Stein B, Hollick R, Lockhart SR, et al. Changes in incidence and antifungal drug resistance in candidemia: results from population-based laboratory surveillance in Atlanta and Baltimore, 2008–2011. Clin Infect Dis 2012;55:1352–61. [4] Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, McCormick T, Ghannoum MA. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol 2001;183:5385–94. [5] Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167–93. [6] Uppuluri P, Chaturvedi AK, Srinivasan A, Banerjee M, Ramasubramaniam AK, Köhler JR, et al. Dispersion as an important step in the Candida albicans biofilm developmental cycle. PLoS Pathog 2010;6:e1000828. [7] Ramage G, Rajendran R, Sherry L, Williams C. Fungal biofilm resistance. Int J Microbiol 2012;2012:1–14. [8] Taff HT, Mitchell KF, Edward JA, Andes DR. Mechanisms of Candida biofilm drug resistance. Future Microbiol 2013;8:1325–37. [9] Ramage G, Bachmann S, Patterson TF, Wickes BL, López-Ribot JL. Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J Antimicrob Chemother 2002;49:973–80. [10] Kuhn DM, Chandra J, Mukherjee PK, Ghannoum MA. Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols. Infect Immun 2002;71:878–88. [11] Nett J, Lincoln L, Marchillo K, Massey R, Holoyda K, Hoff B, et al. Putative role of β-1,3 glucans in Candida albicans biofilm resistance. Antimicrob Agents Chemother 2007;51:510–20. [12] Nett JE, Crawford K, Marchillo K, Andes DR. Role of Fks1p and matrix glucan in Candida albicans biofilm resistance to an echinocandin, pyrimidine, and polyene. Antimicrob Agents Chemother 2010;54:3505–8.

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[13] Mitchell KF, Taff HT, Cuevas MA, Reinicke EL, Sanchez H, Andes DR. Role of matrix β-1,3 glucan in antifungal resistance of non-albicans Candida biofilms. Antimicrob Agents Chemother 2013;57:1918–20. [14] Perumal P, Mekala S, Chaffin WL. Role for cell density in antifungal drug resistance in Candida albicans biofilms. Antimicrob Agents Chemother 2007;51:2454–63. [15] Seneviratne CJ, Jin LJ, Samaranayake YH, Samaranayake LP. Cell density and cell aging as factors modulating antifungal resistance of Candida albicans biofilms. Antimicrob Agents Chemother 2008;52:3259–66. [16] Kuhn DM, George T, Chandra J, Mukherjee PK, Ghannoum MA. Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother 2002;46:1773– 80. [17] Delattin N, Cammue BP, Thevissen K. Reactive oxygen species-inducing antifungal agents and their activity against fungal biofilms. Future Med Chem 2014;6:77–90. [18] Urbina JA. Mechanisms of action of lysophospholipid analogues against trypanosomatid parasites. Trans R Soc Trop Med Hyg 2006;100(Suppl. 1):S9–16. [19] Dorlo TPC, Balasegaram M, Beijnen JH, De Vries PJ. Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis. J Antimicrob Chemother 2012;67:2576–97. [20] Widmer F, Wright LC, Obando D, Handke R, Ganendren R, Ellis DH, et al. Hexadecylphosphocholine (miltefosine) has broad-spectrum fungicidal activity and is efficacious in a mouse model of cryptococcosis. Antimicrob Agents Chemother 2006;50:414–21. [21] Tong Z, Widmer F, Sorrell TC, Guse Z, Jolliffe KA, Halliday C, et al. In vitro activities of miltefosine and two novel antifungal biscationic salts against a panel of 77 dermatophytes. Antimicrob Agents Chemother 2007;51:2219–22. [22] Brilhante RS, Malaquias AD, Caetano EP, Castelo-Branco Dde S, Lima RA, Marques FJ, et al. In vitro inhibitory effect of miltefosine against strains of Histoplasma capsulatum var. capsulatum and Sporothrix spp. Med Mycol 2014;52:320–5. [23] Borba-Santos LP, Gagini T, Ishida K, de Souza W, Rozental S. Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low susceptibility to amphotericin B or itraconazole. J Med Microbiol 2015;64:415–22. [24] Vila TVM, Ishida K, de Souza W, Prousis K, Calogeropoulou T, Rozental S. Effect of alkylphospholipids on Candida albicans biofilm formation and maturation. J Antimicrob Chemother 2013;68:113–25. [25] Vila TVM, Quintanilha NS, Rozental S. Miltefosine is effective against Candida albicans and Fusarium oxysporum nail biofilms in vitro. J Med Microbiol 2015;64:1436–49. [26] Vila TV, Chaturvedi AK, Rozental S, Lopez-Ribot JL. In vitro activity of miltefosine against Candida albicans under planktonic and biofilm growth conditions and in vivo efficacy in a murine model of oral candidiasis. Antimicrob Agents Chemother 2015;59:7611–20. [27] Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard. 3rd ed. Wayne (PA): CLSI; 2008. Document M27-A3. [28] Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts; fourth informational supplement. Wayne (PA): CLSI; 2012. Document M27-S4. [29] Pierce CG, Uppuluri P, Tristan AR, Wormley FL, Mowat E, Ramage G, et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 2008;3:1494–500. [30] Kojic EM, Darouiche RO. Candida infections of medical devices. Clin Microbiol Rev 2004;17:255–67. [31] Rajendran R, Sherry L, Nile CJ, Sherriff A, Johnson EM, Hanson MF, et al. Biofilm formation is a risk factor for mortality in patients with Candida albicans bloodstream infection—Scotland, 2012–2013. Clin Microbiol Infect 2016;22:87– 93. [32] Pappas PG, Kauffman CA, Andes D, Benjamin DK Jr, Calandra TF, Edwards JE Jr, et al. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 2009;48:503–35. [33] Maubon D, Garnaud C, Calandra T, Sanglard D, Cornet M. Resistance of Candida spp. to antifungal drugs in the ICU: where are we now? Intensive Care Med 2014;40:1241–55. [34] Choi HW, Shin JH, Jung SI, Park KH, Cho D, Kee SJ, et al. Species-specific differences in the susceptibilities of biofilms formed by Candida bloodstream isolates to echinocandin antifungals. Antimicrob Agents Chemother 2007;51:1520–3. [35] Mukherjee PK, Chandra J. Candida biofilm resistance. Drug Resist Updat 2004;7:301–9. [36] Vediyappan G, Rossignol T, d’Enfert C. Interaction of Candida albicans biofilms with antifungals: transcriptional response and binding of antifungals to β-glucans. Antimicrob Agents Chemother 2010;54:2096–111. [37] Zarnowski R, Westler WM, Lacmbouh GA, Marita JM, Bothe JR, Bernhardt J, et al. Novel entries in a fungal biofilm matrix encyclopedia. MBio 2014;5:e01333-14. [38] Uppuluri P, Srinivasan A, Ramasubramanian A, Lopez-Ribot JL. Effects of fluconazole, amphotericin B, and caspofungin on Candida albicans biofilms under conditions of flow and on biofilm dispersion. Antimicrob Agents Chemother 2011;55:3591–3. [39] Schinabeck MK, Long LA, Mohammad AH, Chandra J, Mukherjee PK, Mohamed S, et al. Rabbit model of Candida albicans biofilm infection: liposomal amphotericin B antifungal lock therapy. Antimicrob Agents Chemother 2004;48:1727–32.

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