Journal of Medical Microbiology Papers in Press. Published February 13, 2015 as doi:10.1099/jmm.0.000041
Journal of Medical Microbiology Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low susceptibility to amphotericin B or itraconazole. --Manuscript Draft-Manuscript Number:
JMM-D-14-00341
Full Title:
Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low susceptibility to amphotericin B or itraconazole.
Short Title:
Miltefosine is active against Sporothrix brasiliensis isolates with low susceptibility standart antifungals.
Article Type:
Standard
Section/Category:
Antimicrobial agents and chemotherapy
Keywords:
Sporothrix brasiliensis, sporotrichosis, antifungal, miltefosine, yeasts alterations.
Corresponding Author:
Sonia Rozental Universidade Federal do Rio de Janeiro Rio de Janeiro, Rio de Janeiro BRAZIL
First Author:
Luana Pereira Borba-Santos, MSc
Order of Authors:
Luana Pereira Borba-Santos, MSc Thalita Gagini Kelly Ishida Wanderley de Souza, PhD Sonia Rozental
Manuscript Region of Origin:
BRAZIL
Abstract:
Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix schenckii complex. In recent years, sporotrichosis incidence rates have increased in the Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more frequently isolated from patients. The standard antifungals itraconazole and amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous and disseminated sporotrichosis, respectively, although decreased sensitivity to these drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the activity of the phospholipid analogue miltefosine - already in clinical use against leishmaniasis - towards the pathogenic yeast form of S. brasiliensis isolates with low sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal activity, with minimum inhibitory concentration (MIC) values between 1-2 µg.mL-1. Miltefosine exposure led to loss of plasma membrane integrity, and transmission electron microscopy (TEM) analysis revealed decrease in cytoplasmic electron density, alterations in the thickness of cell wall layers, and accumulation of an electron dense material in the cell wall. Flow cytometry analysis using an anti-melanin antibody revealed an increase in cell wall melanin in yeasts treated with miltefosine, when compared with control cells. The cytotoxicity of miltefosine were comparable to those of amphotericin B, but miltefosine showed higher selectivity index towards the fungus. Our results suggest that miltefosine could be an effective alternative for the treatment of S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo studies are required to confirm the antifungal potential of miltefosine for the treatment of sporotrichosis.
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Title: Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low
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susceptibility to amphotericin B or itraconazole.
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Running title: Miltefosine activity against Sporothrix brasiliensis
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Category: Antimicrobial Agents & Chemotherapy.
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Authors: Luana Pereira Borba-Santos1, Thalita Gagini1, Kelly Ishida2, Wanderley de
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Souza3,4, Sonia Rozental1#.
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Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brasil.
Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Duque de Caxias, Brasil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
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#Corresponding author:
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Mailing address: Laboratorio de Biologia Celular de Fungos, Instituto de Biofísica
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Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
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Avenida Carlos Chagas Filho 373, Centro de Ciências da Saúde, Bloco G, Sala C0-026,
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21.941-902, Rio de Janeiro/RJ, Brazil.Telefone number: +55-21-39386592
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Fax number: +55-21-3938-8193
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E-mail: [email protected]
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Abstract
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Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix
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schenckii complex. In recent years, sporotrichosis incidence rates have increased in the
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Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more
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frequently isolated from patients. The standard antifungals itraconazole and
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amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous
34
and disseminated sporotrichosis, respectively, although decreased sensitivity to these
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drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the
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activity of the phospholipid analogue miltefosine – already in clinical use against
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leishmaniasis - towards the pathogenic yeast form of S. brasiliensis isolates with low
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sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal
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activity, with minimum inhibitory concentration (MIC) values between 1-2 µg.mL-1.
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Miltefosine exposure led to loss of plasma membrane integrity, and transmission
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electron microscopy (TEM) analysis revealed decrease in cytoplasmic electron density,
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alterations in the thickness of cell wall layers, and accumulation of an electron dense
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material in the cell wall. Flow cytometry analysis using an anti-melanin antibody
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revealed an increase in cell wall melanin in yeasts treated with miltefosine, when
45
compared with control cells. The cytotoxicity of miltefosine were comparable to those
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of amphotericin B, but miltefosine showed higher selectivity index towards the fungus.
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Our results suggest that miltefosine could be an effective alternative for the treatment of
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S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo
49
studies are required to confirm the antifungal potential of miltefosine for the treatment
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of sporotrichosis.
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Key words: Sporothrix brasiliensis, sporotrichosis, antifungal, miltefosine, yeasts
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alterations.
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Introduction
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Sporotrichosis is the most frequent subcutaneous mycosis in Latin America and has
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high incidence rates in Brazil, especially in the Rio de Janeiro state, where zoonotic
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transmission prevails (Barros et al., 2010). This disease is caused by dimorphic fungal
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species from the Sporothrix schenckii complex (Marimon et al., 2007 and 2008),
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including Sporothrix brasiliensis, the most virulent species (Arrigala-Moncrieff et al.,
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2009; Fernandes et al., 2013) and the most prevalent in the clinical cases from the Rio
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de Janeiro state (Oliveira et al., 2011; Rodrigues et al., 2013; Borba-Santos et al.,
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2014). In zoonotic transmission, sporotrichosis is mainly acquired by scratches, bites or
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direct contact with lesion secretion from contaminated animals, where yeasts are the
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infective forms (Rodrigues et al., 2013b).
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The ‘gold standard’ treatment for cutaneous and lymphocutaneous forms of
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sporotrichosis is itraconazole, while amphotericin B is the first-line drug of choice for
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disseminated forms of the disease (Kauffman et al., 2007). However, recent reports
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demonstrated that these drugs are less potent against S. brasiliensis isolates, which have
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high minimum inhibitory concentration (MIC) values in vitro for these antifungals
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(Ottonelli et al., 2013; Rodrigues et al., 2014; Borba-Santos et al., 2014).
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Miltefosine is a phospholipid analogue originally developed as an antitumor drug, but
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later shown to have potent antiparasitic activity, which led to its licensing for the
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treatment of cutaneous and visceral leishmaniasis in India and Colombia (Dorlo et al.,
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2012). Miltefosine induces changes in the lipid composition of membranes (among
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other cellular effects), leading to a disruption of membrane structure that affects
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intracellular signaling processes essential for survival and cell growth (Jimenez-Lopes
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et al., 2010). Recently, Brilhante and co-workers (2014) showed that miltefosine is
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active against the filamentous (i.e., saprophytic) form of S. schenckii complex species,
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including S. brasiliensis. However, these authors did not determine whether miltefosine
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is active against the pathogenic (yeast) form of S. schenckii complex species, and did
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not examine the intracellular effects of miltefosine in these fungi.
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Given the clinical and epidemiological importance of S. brasiliensis, and the limited
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repertoire of therapeutic options available to treat sporotrichosis by this highly virulent
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species, the aim of our study was to evaluate the in vitro activity of miltefosine against
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the pathogenic yeast form of clinical isolates of S. brasiliensis that have low
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susceptibility to amphotericin B or itraconazole (Borba-Santos et al., 2014). In addition,
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we analyzed the cytotoxicity of miltefosine, and evaluated the alterations induced by
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exposure of yeast cells to this drug (by electron microscopy and flow cytometry), to
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clarify the mechanism of action of miltefosine in S. brasiliensis.
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Methods
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Microorganisms
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In this study, we used 13 clinical isolates of S. brasiliensis (Ss 34, Ss 37, Ss 56, Ss 68,
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B91, B182, B628, B735, B848, B972, B1008, B1086, HE06) that displayed low
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susceptibility to amphotericin B or itraconazole during in vitro testing (Borba-Santos et
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al., 2014). The genome strain ATCC MYA 4823 was used as a reference (Teixeira et
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al., 2014). The isolates were maintained in the filamentous (mycelial) form in potato
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dextrose agar (PDA; Difco, Detroit, USA) at 4°C until their use in experiments. The
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yeast phase was obtained from filamentous-form cultures by two successive 7-day
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passages in brain heart infusion (BHI, Difco, Detroit, USA) broth supplemented with
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2% glucose, at 36ºC, and with orbital agitation (Borba-Santos et al., 2014).
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Drugs
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Miltefosine (Cayman Chemical Company, MI, USA) was diluted in distilled water to
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obtain stock solutions of 2,000 µg mL-1 and maintained at -20°C. Amphotericin B and
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itraconazole (Sigma Chemical Co. St Louis, MO, USA) were used as reference
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antifungal and kept as 1,600 µg mL-1 in DMSO stock solutions, and stored at -20°C.
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Minimum inhibitory concentration (MIC)
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MIC values for the treatment of S. brasiliensis yeasts with miltefosine were determined
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using both microdilution methods adapted from the M27-A3 document from the
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Clinical and Laboratory Standards Institute (CLSI, 2008), as described in Borba-Santos
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et al. (2014). Briefly, yeast cells (0.5-2.5 x 103 colony forming units or CFU.mL-1) were
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treated with 0.01-16 µg.mL-1 miltefosine, in RPMI 1640 medium (Sigma Chemical Co.)
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supplemented with 2% glucose and buffered with 0.165 M 3-(N-morpholine) propane
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sulfonic acid (MOPS), pH 7.2. Samples were incubated at 35°C for 5 days and MIC was
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defined as the lowest concentration that prevents visible fungal growth in an inverted
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optical microscope (Axiovert 100, Zeiss, Germany).
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Minimal fungicidal concentration (MFC)
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To determine MFC values, 10-µL aliquots were collected from MIC samples at the end
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of the 5-day incubation period, and were plated in drug-free PDA. Plates were incubated
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at 35°C for 7 days, and the lowest concentration of miltefosine that failed to yield
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fungal growth was defined as the MFC.
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Drug interactions
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Interactions between miltefosine and itraconazole were assayed for the reference strain
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(ATCC MYA 4823) and also for two clinical isolates (Ss 37 and B737) with high MIC
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values (≥ 4 µg.mL-1) for itraconazole, using the checkerboard microdilution method
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(Eliopoulos and Moellering, 1991). Concentrations ranging from 0.03-16 µg.mL-1 for
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itraconazole and 0.25-16 µg.mL-1 for miltefosine were tested. MICs were determined
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for drugs alone and for their combinations, and the fractional inhibitory concentration
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index (FICI) was used to classify drug interactions. FICI values were determined using
134
the equation: FICI = (MICa in combination/MICa tested alone) + (MICb in
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combination/MICb tested alone), for drugs itraconazole (a) and miltefosine (b) tested
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against the same fungal strain (Eliopoulos and Moellering, 1991). Interactions were
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considered synergistic if FICI ≤ 0.5, absent if FICI > 0.5 and ≤ 4, and antagonistic if
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FICI > 4 (Odds, 2003).
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Time kill assays.
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For time kill assays, S. brasiliensis ATCC MYA 4823 yeasts (103 CFU.mL-1 starting
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inoculum) were exposed to different concentrations of miltefosine (0, MIC/2, MIC,
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MICx2, MICx4, MICx8) in RPMI 1640 medium supplemented with 2% glucose and
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buffered with 0.165 M MOPS (pH, 7.2), for 24, 48, 72, 96 and 168 hours, at 35ºC.
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Treated cultures were diluted and 50 µL of diluted cultures were plated on PDA
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medium and incubated at 35ºC for 7 days before CFU counting. Fungicidal activity was
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defined as a reduction of ≥ 99.9% in the number of CFU relative to that found in the
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starting inoculum; otherwise, the activity was considered fungistatic (Klepser et al.,
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1998).
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Membrane integrity assay
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S. brasiliensis yeast cells (ATCC MYA 4823) were treated with sub-inhibitory
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concentrations (1/2 MIC) of miltefosine, amphotericin B or itraconazole for 24 h at
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35ºC. Untreated and treated yeasts were washed in PBS and cells were incubated with
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10 µM of SYTOX® Blue dead cell stain (Molecular Probes®, Eugene, OR, USA) for
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20 min at room temperature, and in a dark chamber. After incubation, cells were washed
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in PBS and the fluorescence intensity was measured at 480 nm (excitation at 440 nm)
156
using a Spectra-MAX 340 tunable microplate reader (Molecular Devices Ltd, CA,
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USA).
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Transmission electron microscopy
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S. brasiliensis ATCC MYA 4823 yeast cells were treated with a sub-inhibitory
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concentration (1/2 MIC) of miltefosine for 24 h at 35ºC. Untreated and treated cells
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were washed in PBS, fixed in 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M
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cacodylate buffer, (for 24 h at 4°C), and post-fixed in 1% osmium tetroxide in 0.1 M
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cacodylate buffer containing 1.25 % potassium ferrocyanide and 5mM CaCl2, for 2 h at
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4°C. Then, cells were dehydrated in ethanol and embedded in Spurr resin. Ultrathin
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sections were stained in uranyl acetate and lead citrate, and observed in a JEOL 1200
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EX electron microscope. Cell wall thickness values – CW* (thickness of the innermost
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cell wall layer) and ML (thickness of the outer microfibrilar layer of cell wall) - were
168
measured in 20 cells for each sample, using the Image J software (NHI, USA).
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Flow cytometry analysis.
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Untreated and miltefosine-treated S. brasiliensis ATCC MYA 4823 yeast cells (treated
171
with sub-inhibitory concentration (1/2 MIC) for 24 h at 35ºC) were washed in PBS,
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fixed in 4% of formaldehyde in PBS, incubated with 1 % BSA for 30 min and then
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labeled with anti-melanin antibodies (0.1 mg/mL, for 30 min at room temperature)
174
purified from sera obtained from chromoblastomycosis patients (Alviano et al., 2004).
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Then, samples were incubated with FITC–labeled anti-human IgG (Molecular Probes®,
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Eugene, OR, USA) for 30 min at room temperature, and analyzed in a BD AccuriTM C6
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flow cytometer (10000 events/sample). Data were analyzed using the BD Accuri C6
178
Software. Negative control samples were not incubated with melanin antibodies.
179
Citotoxicity test
180
For cytotoxicity testing, aliquots of 100 µL of LLC-MK2 (Rhesus monkey kidney
181
epithelial cells) cell suspensions at 5 x 104 cells/mL were dispensed onto a 96-well
182
microtitre and incubated for 24 h. Then, monolayers were treated with different
183
concentrations of miltefosine or amphotericin B (1-100 µg.mL-1) for 48 h, at 37°C and
184
5% CO2. Cell viability was analyses by the tetrazolium (XTT) reduction assay and
185
selectivity indexes (SIs) were calculated using the formula: SI = CC50/MIC. CC50 values
186
correspond to the concentration that killed 50% of LLC-MK2 cells.
187
Statistical analysis
188
Statistical analyses were performed using the GraphPad Prism 5.0 software. Wilcoxon’s
189
tests were used to analyze differences between antifungal treatments and Mann
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Whitney’s tests were used to analyze differences in cell wall thickness values. Linear
191
regression was used to analyze correlations between MIC values. One-way ANOVA
192
was used (with Dunnett’s post hoc test) to compare membrane integrity evaluation
193
results. A 5% significance level was adopted (p < 0.05).
194 195
Results
196
The yeast form of S. brasiliensis is sensitive to low concentrations (≤ 2 g.mL-1) of
197
miltefosine.
198
The sensitivity of the infective yeast form of S. brasiliensis to miltefosine in vitro was
199
established by determining MIC and MFC values, using reference protocols, and these
200
values were compared to those reported previously for the standard antifungals
201
amphotericin B and itraconazole (Table 1). S. brasiliensis clinical isolates displaying
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high MIC values (MIC ≥ 4 µg.mL-1) for amphotericin B or itraconazole (Borba-Santos
203
et al., 2014) were sensitive to miltefosine (MIC ≤ 2 µg.mL-1), with MIC values between
204
1 and 2 µg.mL-1 for all isolates tested (Table 1). Miltefosine was more potent than
205
amphotericin B (p=0.005) and itraconazole (p=0.03) against all clinical S. brasiliensis
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isolates tested, and the reference strain ATCC MYA 4823 (from feline sporotrichosis)
207
was also susceptible to miltefosine treatment. No significant correlation was found
208
between MIC values for amphotericin B, itraconazole and miltefosine (r < 0.4).
209
According to MFC values, miltefosine was also more potent against S. brasiliensis than
210
both amphotericin B and itraconazole (Table 1). Additionally, time kill assays
211
performed using the reference strain (ATCC MYA 4823) showed that miltefosine has
212
fungicidal activity towards S. brasiliensis, with 2 µg.mL-1 (MICx2) of this drug
213
resulting in complete culture growth elimination after as little as 24 hours of treatment
214
(Fig. 1). After prolonged treatment (168 hours, or 7 days) with miltefosine, 1 µg.mL-1
215
(MIC) and 0.5 µg.mL-1 (MIC/2) of this drug inhibited 100% and ~80% (1Log10) of
216
starting inoculum growth, respectively (Fig. 1).
217
Combinations of itraconazole with miltefosine showed no synergistic effect against
218
yeast cells from the isolates ATCC MYA 4823, Ss 37 and B735 (0.5˂ FICI ≤ 4).
219
Exposure to miltefosine disrupts yeast membrane integrity, affects membrane-to-cell
220
wall interactions, and alters cell wall structure, increasing melanin content in the cell
221
wall.
222
To identify early changes in fungal morphology after treatment with miltefosine, S.
223
brasiliensis yeast cells were treated with sub-inhibitory concentration of this drug (0.5
224
µg.mL-1) for 24 hours, and plasma membrane integrity and ultrastructural characteristics
225
were analyzed.
226
Miltefosine promoted loss of plasma membrane integrity in S. brasiliensis yeast cells, as
227
demonstrated by the increase in Sytox blue fluorescence intensity of cell populations
228
(p16 µg mL-1 range) confirm that
288
miltefosine is potent against the tested isolates, and time kill experiments indicated that
289
miltefosine has fungicidal activity against the yeast form of S. brasiliensis, in agreement
290
with that previously described for other fungal species, including Candida sp.,
291
Cryptococcus sp. and Aspergillus sp. (Widmer et al., 2006; Ravu et al., 2013), and also
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for the mycelial form of S. schenckii complex species (Brilhante et al., 2014).
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Synergistic effects of miltefosine-azoles combinations have been described for some
294
fungal species (Biswas et al., 2013); however, we did not detected any interactions
295
between itraconazole and miltefosine when treating S. brasiliensis yeast cells from the
296
reference strain ATCC MYA 4823, or from two clinical isolates with high MIC values
297
for itraconazole (Ss 37 and B735).
298
Sytox blue staining of yeasts treated with miltefosine indicates that this phospholipid
299
analogue promotes cell death due to pronounced disturbances in the plasma membrane,
300
among other effects described previously. Miltefosine may have several cellular targets,
301
due to its ubiquitous effects on cell membranes (Barrat et al., 2009). In Trypanossama
302
cruzi and Leishmania species, the drug alters the phospholipid and sterol contents of
303
cells (Barrat et al., 2009), promoting a reduction in the amounts of phospholipids,
304
ergosterol and fatty acids (Dorlo et al., 2012). Despite the antifungal activity of
305
miltefosine and its potential as a therapeutic alternative to treat fungal infections, the
306
mode of action of this drug in fungi is poorly understood. Zuo et al. (2011)
307
demonstrated that miltefosine is rapidly incorporated into Saccharomyces cerevisiae
308
yeast cells, where it crosses the mitochondrial inner membrane, penetrating the
309
mitochondria and leading to an apoptosis-like cell death. In S. brasiliensis, miltefosine
310
may act on the synthesis of membrane components, which may promote instability and
311
loss of membrane integrity, ultimately leading to yeast cell death.
312
We also showed by TEM that the exposure to miltefosine promoted considerable
313
changes in S. brasiliensis yeast cells, including: loss in electron density (Fig. 3C),
314
accumulation of electron dense material in the cell wall (arrow in Fig.3D) and
315
alterations in cell wall thickness (Fig. 3E). Alterations in cell wall components after
316
miltefosine exposure were also described in C. albicans yeasts (Villa et al., 2013).
317
However, the aggregation of electron dense material in the cell wall following
318
miltefosine treatment had not been observed previously for other fungal species. This
319
electron dense material visualized by TEM is morphologically similar to the melanin
320
granules described by Teixeira et al (2010). In addition, flow cytometry analysis using
321
anti-melanin antibodies showed that miltefosine-treated cells had increased melanin
322
content compared to untreated cells. Thus, our results suggest that miltefosine treatment
323
could be modulates melanin turnover (breakdown and/or production) in S. brasiliensis
324
yeast cells, leading to the accumulation of aggregates of this pigment in the cell wall.
325
Similarly, Teixeira et al (2010) also showed that the amount of melanin in the cell wall
326
of S. schenckii could be modulated by varying culture medium composition (Teixeira et
327
al., 2010).
328
Although miltefosine displayed relatively high cytotoxicity towards kidney cells, this is
329
comparable to that observed for amphotericin B, the only available treatment option for
330
severe forms of sporotrichosis. Moreover, in vivo tests with BALB/c mice infected with
331
L. amazonensis showed that miltefosine was toxic only at doses higher than 50 mg kg-1
332
day-1 (Godinho et al., 2012). Given the lower MIC values for miltefosine against S.
333
brasiliensis in vitro, this drug shows higher selective indexes than amphotericin B for
334
the isolates tested (Table 2). Thus, miltefosine is likely to have a wider ‘therapeutic
335
window’ than amphotericin B against S. brasiliensis sporotrichosis, which should allow
336
the use of lower effective doses of this drug, to limit toxicity. Another advantage of
337
miltefosine treatment is that this drug can be administrated orally, whereas amphotericin
338
B treatment is intravenous.
339
Miltefosine is already used to treat some of the clinical manifestations of leishmaniasis
340
(Dorlo et al., 2012), which should facilitate its approval for use in the treatment of other
341
infectious diseases. Interestingly, miltefosine was approved for the topical treatment of
342
metastasized mammary carcinoma in Germany (Paparazafiri et al., 2005), in 1992; thus,
343
topical administration of miltefosine - which might be particularly useful for
344
sporotrichosis treatment – may be also effective in patients. Although some resistance to
345
miltefosine has been described in patients infected with Leishmania donovani, the main
346
resistance mechanisms seems to be associated with a defect in translocation of the drug
347
to the cytoplasm (Perez-Victoria et al., 2003).
348
The dose of miltefosine used for the treatment of leishmaniasis in humans is 2.5 mg kg-1
349
day-1, for 28 days (Dorlo et al., 2012). The reported IC50 value for the treatment of
350
intracellular amastigotes of Leishmania amazonensis with miltefosine in vitro was 9 µM
351
(3.6 µg.mL-1) (Santa-Rita et al., 2004), which is higher than the MIC values of 2.5-5
352
µM (1-2 µg.mL-1) reported here for the treatment of S. brasiliensis isolates. Therefore,
353
our results indicate that lower miltefosine doses than those used against human
354
leishmaniasis could be effective in the treatment of sporotrichosis.
355
In conclusion, ours results show that miltefosine might represent an interesting
356
alternative for the treatment of sporotrichosis caused by S. brasiliensis, especially when
357
standard treatment fails. Nevertheless, further in vivo studies in experimentally infected
358
animals are required to confirm the efficacy of miltefosine as an antifungal agent for the
359
treatment of sporotrichosis.
360
Declaration of interest: The authors report no conflict of interests.
361
Acknowledgements
362
The authors thank Dr. Zoilo Pires de Camargo and Dr. Anderson Messias Rodrigues
363
from the Universidade Estadual de São Paulo (São Paulo, SP, Brazil), Dra. Leila Maria
364
Lopes-Bezerra from Universidade Estadual do Rio de Janeiro (Rio de Janeiro, RJ,
365
Brazil) and Dr. Marcio Nucci from the Universidade Federal do Rio de Janeiro (Rio de
366
Janeiro, RJ, Brazil) for kindly providing the S. brasiliensis isolates used in this study.
367
This study was supported by the Brazilian funding agencies Fundação Carlos Chagas
368
Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de
369
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de
370
Desenvolvimento Científico e Tecnológico (CNPq).
371 372
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them. J Antimicrob Chemother 52, 1.
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Oliveira, M. M. E., Almeida-Paes, R., Muniz, M. M., Gutierrez-Galhardo, M. C. &
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Zancope-Oliveira, R. M. (2011). Phenotypic and molecular identification of
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Sporothrix isolates from an epidemic area of sporotrichosis in Brazil. Mycopathologia
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Heidrich, D., Valente, P. & Scroferneker, M. L. (2013). Antifungal susceptibilities
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Papazafiri, P., Avlonitis, N., Angelous, N., Calogeropoulou, T., Koufaki, M.,
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ring-substituted ether phopholipid derivatives. Cancer Chemother Pharmacol 56, 261-
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Ravu, R.R., Chen, Y.L., Jacob, M.R., Pan, X., Agarwal, A.K., Khan, S.I., Heitman,
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analogs. Bioorg Med Chem Lett 23, 4828-4831.
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Rodrigues, A.M., de Hoog, G.S., Pires, D., Brilhante, R.S., Sidrim, J.J., Gadelha,
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M.F., Colombo, A.L., de Camargo, Z.P. (2014). Genetic diversity and antifungal
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susceptibility profiles in causative agents of sporotrichosis. BMC Infect Dis 14, 219.
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Rodrigues, A.M., de Hoog, S., de Camargo, Z.P. (2013a). Emergence of
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pathogenicity in the Sporothrix schenckii complex. Med Mycol 51, 405-412.
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Rodrigues, A. M., Teixeira, M. M., de Hoog, G. S., Schubach, T. M. P., Pereira,
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S.A., Fernandes, G., F., Bezerra, L. M. L., Felipe, M. S. & de Camargo, Z. P.
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(2013b). Phylogenetic analysis reveals a high prevalence of Sporothrix brasiliensis in
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Leishmania amazonensis. J Antimicrob Chemother 54, 704-710.
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Teixeira, M.M., de Almeida, L.G.P., Kubitschek-Barreira, P., Alves, F.L.,
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Kioshima, E.S., Abadio, A.K.R., Fernandes, L., Derengowski, L.S., Ferreira, K.S.,
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Souza, R.C., Ruiz, J.C., de Andrade, N.C., Paes, H.C., Nicola, A.M., Albuquerque,
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P., Gerber, A.L., Martins, V.P., Peconick, L.D.F., Neto, A.V., Chaucanez, C.B.,
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Silva, P.A., Cunha, O.L., de Oliveira, F.F.M., dos Santos, T.C., Barros, A.L.N.,
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Soares, M.A., de Oliveira, L.M., Marini, M.M., Villalobos-Duno, H., Cunha,
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M.M.L., de Hoog, S., da Silveira, J.F., Henrissat, B., Niño-Vega, G.A., Cisalpino,
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P.S., Mora-Montes, H.M., Almeida, S.R., Stajich, J.E., Lopes-Bezerram L.M.,
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Vasconcelos, A.T.R., Felipe, M.S.S. (2014). Comparative genomics of the major
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fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix
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brasiliensis. BMC Genomics 15, 1-22.
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Teixeira, P.A., de Castro, R.A., Ferreira, F.R., Cunha, M.M., Torres, A.P., Penha,
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C.V., Rozental., S., Lopes-Bezerra, L.M. (2010). L-DOPA accessibility in culture
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medium increases melanin expression and virulence of Sporothrix schenckii yeast cells.
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Med Mycol 48, 687-695.
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Tong, Z., Widmer, F., Sorrel, T.C., Guse, Z., Jolliffe, K.A., Halliday, C., Lee, O.C.,
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Kong, F., Wright, L.C. & Chen, S.C. (2007). In vitro activities of miltefosine and two
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novel antifungal biscationic salts against a panel of 77 dermatophytes. Antimicrob
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Agents Chemother 51, 2219-2222.
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Urbina, J.A. (2006). Mechanisms of action of lysophospholipid analogues against
487
trypanossomatid parasites. Trans R Soc Trop Med Hyg 100, S9-S16.
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Villa, T.V., Ishida, K., de Souza, W., Prousis, K., Calogeropoulou, T., Rozental, S.
489
(2013). Effect of alkylphospholipids on Candida albicans biofilm formation and
490
maturation. J Antimicrob Chemother 68, 113-125.
491
Widmer, F., Wright, L.C., Obando, D., Handke, R., Ganendren, R., Ellis, D.H. &
492
Sorrel, T.C. (2006). Hexadecylphosphocoline (Miltefosine) has broad-spectrum
493
fungicidal activity and is efficacious in a mouse model of Cryptococcosis. Antimicrob
494
Agents Chemother 50, 414-441.
495
Zuo, X., Djordjevic, J.T., Bijosono Oei, J., Desmarini, D., Schibeci, S.D., Jolliffe,
496
K.A., Sorrell, T.C. (2011). Miltefosine induces apoptosis-like cell death in yeast via
497
Cox9p in cytochrome c oxidase. Mol Pharmacol 80,476-85.
498 499 500 501 502 503 504 505 506
507
Table 1. Minimum inhibitory concentration (MIC) and minimum fungicidal
508
concentration (MFC) for the treatment of Sporothrix brasiliensis yeasts with
509
miltefosine, compared to reported values for amphotericin B and itraconazole [Borba-
510
Santos et al., 2014]. All values are in µg mL-1.
S. brasiliensis isolates MYA 4823 B91 B182 B628 B735 B848 B972 B1008 B1036 HE06 Ss 68 Ss 34 Ss 37 Ss 56 511 512 513 514 515 516 517 518 519 520 521 522 523
a
a
Miltefosine MIC 1 2 2 1 2 2 2 2 2 2 1 1 1 1
MFC 2 4 2 1 2 >16 2 2 2 8 1 2 16 1
Amphotericin Bb MIC 0.5 2 1 4 2 4 4 4 4 1 4 4 4 4
MFC 2 16 16 4 2 4 4 4 4 1 4 4 4 4
Itraconazoleb MIC 0.5 8 4 1 16 2 0.5 1 2 8 2 2 4 2
MFC >16 >16 4 16 >16 16 1 1 >16 >16 8 16 8 16
Reference (genome) strain, from feline sporotrichosis. All other strains are from human sporotrichosis. b MIC and MFC values for amphotericin B and itraconazole reported in Borba-Santos et al., 2014.
524
Table 2. Analyses of miltefosine and amphothericin B cytotoxicity towards the
525
epithelial cell line LLC-MK2 (CC50) and selectivity index (SI) for the treatment of
526
Sporothrix brasiliensis yeast cells. LLC-MK2 Antifungal agents
mean MICa -1
CC50 -1
(µg mL )
(µg mL )
Miltefosine
2
5
2.5
Amphotericin B
4
6.25
1.56
527 528
a
Mean MIC values for treatment of the yeast form of S. brasiliensis.
529
b
SI = CC50/MIC.
530
SIb
531
Legends
532
Figure 1. Time-kill plot showing the activity of miltefosine against the yeast form of
533
Sporothrix brasiliensis ATCC MYA 4823. Yeast cell suspensions (103 colony forming
534
units or CFU ml-1) were treated with different concentrations of miltefosine at 35oC,
535
diluted and plated on potato dextrose agar (PDA). Plates were incubated for 7 days at
536
35oC, and CFU numbers were determined by manual counting. Experiments were made
537
in duplicate.
538
Figure 2. Membrane integrity evaluation after treatment of Sporothrix brasiliensis
539
(ATCC MYA 4823) yeast cells with miltefosine, amphotericin B and itraconazole. Cells
540
were left untreated (control) or were treated with 0.5 µg.mL-1 miltefosine, or with 0.25
541
µg.mL-1 of amphotericin B or itraconazole, for 24 hours, before staining with the
542
exclusion dye Sytox blue (Molecular Probes). Treatment with miltefosine only induced
543
a statistically significant loss in plasma membrane integrity compared to untreated
544
controls (**p
Journal of Medical Microbiology Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low susceptibility to amphotericin B or itraconazole. --Manuscript Draft-Manuscript Number:
JMM-D-14-00341
Full Title:
Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low susceptibility to amphotericin B or itraconazole.
Short Title:
Miltefosine is active against Sporothrix brasiliensis isolates with low susceptibility standart antifungals.
Article Type:
Standard
Section/Category:
Antimicrobial agents and chemotherapy
Keywords:
Sporothrix brasiliensis, sporotrichosis, antifungal, miltefosine, yeasts alterations.
Corresponding Author:
Sonia Rozental Universidade Federal do Rio de Janeiro Rio de Janeiro, Rio de Janeiro BRAZIL
First Author:
Luana Pereira Borba-Santos, MSc
Order of Authors:
Luana Pereira Borba-Santos, MSc Thalita Gagini Kelly Ishida Wanderley de Souza, PhD Sonia Rozental
Manuscript Region of Origin:
BRAZIL
Abstract:
Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix schenckii complex. In recent years, sporotrichosis incidence rates have increased in the Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more frequently isolated from patients. The standard antifungals itraconazole and amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous and disseminated sporotrichosis, respectively, although decreased sensitivity to these drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the activity of the phospholipid analogue miltefosine - already in clinical use against leishmaniasis - towards the pathogenic yeast form of S. brasiliensis isolates with low sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal activity, with minimum inhibitory concentration (MIC) values between 1-2 µg.mL-1. Miltefosine exposure led to loss of plasma membrane integrity, and transmission electron microscopy (TEM) analysis revealed decrease in cytoplasmic electron density, alterations in the thickness of cell wall layers, and accumulation of an electron dense material in the cell wall. Flow cytometry analysis using an anti-melanin antibody revealed an increase in cell wall melanin in yeasts treated with miltefosine, when compared with control cells. The cytotoxicity of miltefosine were comparable to those of amphotericin B, but miltefosine showed higher selectivity index towards the fungus. Our results suggest that miltefosine could be an effective alternative for the treatment of S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo studies are required to confirm the antifungal potential of miltefosine for the treatment of sporotrichosis.
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1
Title: Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low
2
susceptibility to amphotericin B or itraconazole.
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Running title: Miltefosine activity against Sporothrix brasiliensis
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Category: Antimicrobial Agents & Chemotherapy.
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Authors: Luana Pereira Borba-Santos1, Thalita Gagini1, Kelly Ishida2, Wanderley de
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Souza3,4, Sonia Rozental1#.
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1
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2
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3
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Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brasil.
Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Duque de Caxias, Brasil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
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#Corresponding author:
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Mailing address: Laboratorio de Biologia Celular de Fungos, Instituto de Biofísica
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Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
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Avenida Carlos Chagas Filho 373, Centro de Ciências da Saúde, Bloco G, Sala C0-026,
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21.941-902, Rio de Janeiro/RJ, Brazil.Telefone number: +55-21-39386592
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Fax number: +55-21-3938-8193
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E-mail: [email protected]
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Abstract
29
Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix
30
schenckii complex. In recent years, sporotrichosis incidence rates have increased in the
31
Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more
32
frequently isolated from patients. The standard antifungals itraconazole and
33
amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous
34
and disseminated sporotrichosis, respectively, although decreased sensitivity to these
35
drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the
36
activity of the phospholipid analogue miltefosine – already in clinical use against
37
leishmaniasis - towards the pathogenic yeast form of S. brasiliensis isolates with low
38
sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal
39
activity, with minimum inhibitory concentration (MIC) values between 1-2 µg.mL-1.
40
Miltefosine exposure led to loss of plasma membrane integrity, and transmission
41
electron microscopy (TEM) analysis revealed decrease in cytoplasmic electron density,
42
alterations in the thickness of cell wall layers, and accumulation of an electron dense
43
material in the cell wall. Flow cytometry analysis using an anti-melanin antibody
44
revealed an increase in cell wall melanin in yeasts treated with miltefosine, when
45
compared with control cells. The cytotoxicity of miltefosine were comparable to those
46
of amphotericin B, but miltefosine showed higher selectivity index towards the fungus.
47
Our results suggest that miltefosine could be an effective alternative for the treatment of
48
S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo
49
studies are required to confirm the antifungal potential of miltefosine for the treatment
50
of sporotrichosis.
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Key words: Sporothrix brasiliensis, sporotrichosis, antifungal, miltefosine, yeasts
53
alterations.
54 55 56 57 58
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Introduction
60
Sporotrichosis is the most frequent subcutaneous mycosis in Latin America and has
61
high incidence rates in Brazil, especially in the Rio de Janeiro state, where zoonotic
62
transmission prevails (Barros et al., 2010). This disease is caused by dimorphic fungal
63
species from the Sporothrix schenckii complex (Marimon et al., 2007 and 2008),
64
including Sporothrix brasiliensis, the most virulent species (Arrigala-Moncrieff et al.,
65
2009; Fernandes et al., 2013) and the most prevalent in the clinical cases from the Rio
66
de Janeiro state (Oliveira et al., 2011; Rodrigues et al., 2013; Borba-Santos et al.,
67
2014). In zoonotic transmission, sporotrichosis is mainly acquired by scratches, bites or
68
direct contact with lesion secretion from contaminated animals, where yeasts are the
69
infective forms (Rodrigues et al., 2013b).
70
The ‘gold standard’ treatment for cutaneous and lymphocutaneous forms of
71
sporotrichosis is itraconazole, while amphotericin B is the first-line drug of choice for
72
disseminated forms of the disease (Kauffman et al., 2007). However, recent reports
73
demonstrated that these drugs are less potent against S. brasiliensis isolates, which have
74
high minimum inhibitory concentration (MIC) values in vitro for these antifungals
75
(Ottonelli et al., 2013; Rodrigues et al., 2014; Borba-Santos et al., 2014).
76
Miltefosine is a phospholipid analogue originally developed as an antitumor drug, but
77
later shown to have potent antiparasitic activity, which led to its licensing for the
78
treatment of cutaneous and visceral leishmaniasis in India and Colombia (Dorlo et al.,
79
2012). Miltefosine induces changes in the lipid composition of membranes (among
80
other cellular effects), leading to a disruption of membrane structure that affects
81
intracellular signaling processes essential for survival and cell growth (Jimenez-Lopes
82
et al., 2010). Recently, Brilhante and co-workers (2014) showed that miltefosine is
83
active against the filamentous (i.e., saprophytic) form of S. schenckii complex species,
84
including S. brasiliensis. However, these authors did not determine whether miltefosine
85
is active against the pathogenic (yeast) form of S. schenckii complex species, and did
86
not examine the intracellular effects of miltefosine in these fungi.
87
Given the clinical and epidemiological importance of S. brasiliensis, and the limited
88
repertoire of therapeutic options available to treat sporotrichosis by this highly virulent
89
species, the aim of our study was to evaluate the in vitro activity of miltefosine against
90
the pathogenic yeast form of clinical isolates of S. brasiliensis that have low
91
susceptibility to amphotericin B or itraconazole (Borba-Santos et al., 2014). In addition,
92
we analyzed the cytotoxicity of miltefosine, and evaluated the alterations induced by
93
exposure of yeast cells to this drug (by electron microscopy and flow cytometry), to
94
clarify the mechanism of action of miltefosine in S. brasiliensis.
95
Methods
96
Microorganisms
97
In this study, we used 13 clinical isolates of S. brasiliensis (Ss 34, Ss 37, Ss 56, Ss 68,
98
B91, B182, B628, B735, B848, B972, B1008, B1086, HE06) that displayed low
99
susceptibility to amphotericin B or itraconazole during in vitro testing (Borba-Santos et
100
al., 2014). The genome strain ATCC MYA 4823 was used as a reference (Teixeira et
101
al., 2014). The isolates were maintained in the filamentous (mycelial) form in potato
102
dextrose agar (PDA; Difco, Detroit, USA) at 4°C until their use in experiments. The
103
yeast phase was obtained from filamentous-form cultures by two successive 7-day
104
passages in brain heart infusion (BHI, Difco, Detroit, USA) broth supplemented with
105
2% glucose, at 36ºC, and with orbital agitation (Borba-Santos et al., 2014).
106
Drugs
107
Miltefosine (Cayman Chemical Company, MI, USA) was diluted in distilled water to
108
obtain stock solutions of 2,000 µg mL-1 and maintained at -20°C. Amphotericin B and
109
itraconazole (Sigma Chemical Co. St Louis, MO, USA) were used as reference
110
antifungal and kept as 1,600 µg mL-1 in DMSO stock solutions, and stored at -20°C.
111
Minimum inhibitory concentration (MIC)
112
MIC values for the treatment of S. brasiliensis yeasts with miltefosine were determined
113
using both microdilution methods adapted from the M27-A3 document from the
114
Clinical and Laboratory Standards Institute (CLSI, 2008), as described in Borba-Santos
115
et al. (2014). Briefly, yeast cells (0.5-2.5 x 103 colony forming units or CFU.mL-1) were
116
treated with 0.01-16 µg.mL-1 miltefosine, in RPMI 1640 medium (Sigma Chemical Co.)
117
supplemented with 2% glucose and buffered with 0.165 M 3-(N-morpholine) propane
118
sulfonic acid (MOPS), pH 7.2. Samples were incubated at 35°C for 5 days and MIC was
119
defined as the lowest concentration that prevents visible fungal growth in an inverted
120
optical microscope (Axiovert 100, Zeiss, Germany).
121
Minimal fungicidal concentration (MFC)
122
To determine MFC values, 10-µL aliquots were collected from MIC samples at the end
123
of the 5-day incubation period, and were plated in drug-free PDA. Plates were incubated
124
at 35°C for 7 days, and the lowest concentration of miltefosine that failed to yield
125
fungal growth was defined as the MFC.
126
Drug interactions
127
Interactions between miltefosine and itraconazole were assayed for the reference strain
128
(ATCC MYA 4823) and also for two clinical isolates (Ss 37 and B737) with high MIC
129
values (≥ 4 µg.mL-1) for itraconazole, using the checkerboard microdilution method
130
(Eliopoulos and Moellering, 1991). Concentrations ranging from 0.03-16 µg.mL-1 for
131
itraconazole and 0.25-16 µg.mL-1 for miltefosine were tested. MICs were determined
132
for drugs alone and for their combinations, and the fractional inhibitory concentration
133
index (FICI) was used to classify drug interactions. FICI values were determined using
134
the equation: FICI = (MICa in combination/MICa tested alone) + (MICb in
135
combination/MICb tested alone), for drugs itraconazole (a) and miltefosine (b) tested
136
against the same fungal strain (Eliopoulos and Moellering, 1991). Interactions were
137
considered synergistic if FICI ≤ 0.5, absent if FICI > 0.5 and ≤ 4, and antagonistic if
138
FICI > 4 (Odds, 2003).
139
Time kill assays.
140
For time kill assays, S. brasiliensis ATCC MYA 4823 yeasts (103 CFU.mL-1 starting
141
inoculum) were exposed to different concentrations of miltefosine (0, MIC/2, MIC,
142
MICx2, MICx4, MICx8) in RPMI 1640 medium supplemented with 2% glucose and
143
buffered with 0.165 M MOPS (pH, 7.2), for 24, 48, 72, 96 and 168 hours, at 35ºC.
144
Treated cultures were diluted and 50 µL of diluted cultures were plated on PDA
145
medium and incubated at 35ºC for 7 days before CFU counting. Fungicidal activity was
146
defined as a reduction of ≥ 99.9% in the number of CFU relative to that found in the
147
starting inoculum; otherwise, the activity was considered fungistatic (Klepser et al.,
148
1998).
149
Membrane integrity assay
150
S. brasiliensis yeast cells (ATCC MYA 4823) were treated with sub-inhibitory
151
concentrations (1/2 MIC) of miltefosine, amphotericin B or itraconazole for 24 h at
152
35ºC. Untreated and treated yeasts were washed in PBS and cells were incubated with
153
10 µM of SYTOX® Blue dead cell stain (Molecular Probes®, Eugene, OR, USA) for
154
20 min at room temperature, and in a dark chamber. After incubation, cells were washed
155
in PBS and the fluorescence intensity was measured at 480 nm (excitation at 440 nm)
156
using a Spectra-MAX 340 tunable microplate reader (Molecular Devices Ltd, CA,
157
USA).
158
Transmission electron microscopy
159
S. brasiliensis ATCC MYA 4823 yeast cells were treated with a sub-inhibitory
160
concentration (1/2 MIC) of miltefosine for 24 h at 35ºC. Untreated and treated cells
161
were washed in PBS, fixed in 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M
162
cacodylate buffer, (for 24 h at 4°C), and post-fixed in 1% osmium tetroxide in 0.1 M
163
cacodylate buffer containing 1.25 % potassium ferrocyanide and 5mM CaCl2, for 2 h at
164
4°C. Then, cells were dehydrated in ethanol and embedded in Spurr resin. Ultrathin
165
sections were stained in uranyl acetate and lead citrate, and observed in a JEOL 1200
166
EX electron microscope. Cell wall thickness values – CW* (thickness of the innermost
167
cell wall layer) and ML (thickness of the outer microfibrilar layer of cell wall) - were
168
measured in 20 cells for each sample, using the Image J software (NHI, USA).
169
Flow cytometry analysis.
170
Untreated and miltefosine-treated S. brasiliensis ATCC MYA 4823 yeast cells (treated
171
with sub-inhibitory concentration (1/2 MIC) for 24 h at 35ºC) were washed in PBS,
172
fixed in 4% of formaldehyde in PBS, incubated with 1 % BSA for 30 min and then
173
labeled with anti-melanin antibodies (0.1 mg/mL, for 30 min at room temperature)
174
purified from sera obtained from chromoblastomycosis patients (Alviano et al., 2004).
175
Then, samples were incubated with FITC–labeled anti-human IgG (Molecular Probes®,
176
Eugene, OR, USA) for 30 min at room temperature, and analyzed in a BD AccuriTM C6
177
flow cytometer (10000 events/sample). Data were analyzed using the BD Accuri C6
178
Software. Negative control samples were not incubated with melanin antibodies.
179
Citotoxicity test
180
For cytotoxicity testing, aliquots of 100 µL of LLC-MK2 (Rhesus monkey kidney
181
epithelial cells) cell suspensions at 5 x 104 cells/mL were dispensed onto a 96-well
182
microtitre and incubated for 24 h. Then, monolayers were treated with different
183
concentrations of miltefosine or amphotericin B (1-100 µg.mL-1) for 48 h, at 37°C and
184
5% CO2. Cell viability was analyses by the tetrazolium (XTT) reduction assay and
185
selectivity indexes (SIs) were calculated using the formula: SI = CC50/MIC. CC50 values
186
correspond to the concentration that killed 50% of LLC-MK2 cells.
187
Statistical analysis
188
Statistical analyses were performed using the GraphPad Prism 5.0 software. Wilcoxon’s
189
tests were used to analyze differences between antifungal treatments and Mann
190
Whitney’s tests were used to analyze differences in cell wall thickness values. Linear
191
regression was used to analyze correlations between MIC values. One-way ANOVA
192
was used (with Dunnett’s post hoc test) to compare membrane integrity evaluation
193
results. A 5% significance level was adopted (p < 0.05).
194 195
Results
196
The yeast form of S. brasiliensis is sensitive to low concentrations (≤ 2 g.mL-1) of
197
miltefosine.
198
The sensitivity of the infective yeast form of S. brasiliensis to miltefosine in vitro was
199
established by determining MIC and MFC values, using reference protocols, and these
200
values were compared to those reported previously for the standard antifungals
201
amphotericin B and itraconazole (Table 1). S. brasiliensis clinical isolates displaying
202
high MIC values (MIC ≥ 4 µg.mL-1) for amphotericin B or itraconazole (Borba-Santos
203
et al., 2014) were sensitive to miltefosine (MIC ≤ 2 µg.mL-1), with MIC values between
204
1 and 2 µg.mL-1 for all isolates tested (Table 1). Miltefosine was more potent than
205
amphotericin B (p=0.005) and itraconazole (p=0.03) against all clinical S. brasiliensis
206
isolates tested, and the reference strain ATCC MYA 4823 (from feline sporotrichosis)
207
was also susceptible to miltefosine treatment. No significant correlation was found
208
between MIC values for amphotericin B, itraconazole and miltefosine (r < 0.4).
209
According to MFC values, miltefosine was also more potent against S. brasiliensis than
210
both amphotericin B and itraconazole (Table 1). Additionally, time kill assays
211
performed using the reference strain (ATCC MYA 4823) showed that miltefosine has
212
fungicidal activity towards S. brasiliensis, with 2 µg.mL-1 (MICx2) of this drug
213
resulting in complete culture growth elimination after as little as 24 hours of treatment
214
(Fig. 1). After prolonged treatment (168 hours, or 7 days) with miltefosine, 1 µg.mL-1
215
(MIC) and 0.5 µg.mL-1 (MIC/2) of this drug inhibited 100% and ~80% (1Log10) of
216
starting inoculum growth, respectively (Fig. 1).
217
Combinations of itraconazole with miltefosine showed no synergistic effect against
218
yeast cells from the isolates ATCC MYA 4823, Ss 37 and B735 (0.5˂ FICI ≤ 4).
219
Exposure to miltefosine disrupts yeast membrane integrity, affects membrane-to-cell
220
wall interactions, and alters cell wall structure, increasing melanin content in the cell
221
wall.
222
To identify early changes in fungal morphology after treatment with miltefosine, S.
223
brasiliensis yeast cells were treated with sub-inhibitory concentration of this drug (0.5
224
µg.mL-1) for 24 hours, and plasma membrane integrity and ultrastructural characteristics
225
were analyzed.
226
Miltefosine promoted loss of plasma membrane integrity in S. brasiliensis yeast cells, as
227
demonstrated by the increase in Sytox blue fluorescence intensity of cell populations
228
(p16 µg mL-1 range) confirm that
288
miltefosine is potent against the tested isolates, and time kill experiments indicated that
289
miltefosine has fungicidal activity against the yeast form of S. brasiliensis, in agreement
290
with that previously described for other fungal species, including Candida sp.,
291
Cryptococcus sp. and Aspergillus sp. (Widmer et al., 2006; Ravu et al., 2013), and also
292
for the mycelial form of S. schenckii complex species (Brilhante et al., 2014).
293
Synergistic effects of miltefosine-azoles combinations have been described for some
294
fungal species (Biswas et al., 2013); however, we did not detected any interactions
295
between itraconazole and miltefosine when treating S. brasiliensis yeast cells from the
296
reference strain ATCC MYA 4823, or from two clinical isolates with high MIC values
297
for itraconazole (Ss 37 and B735).
298
Sytox blue staining of yeasts treated with miltefosine indicates that this phospholipid
299
analogue promotes cell death due to pronounced disturbances in the plasma membrane,
300
among other effects described previously. Miltefosine may have several cellular targets,
301
due to its ubiquitous effects on cell membranes (Barrat et al., 2009). In Trypanossama
302
cruzi and Leishmania species, the drug alters the phospholipid and sterol contents of
303
cells (Barrat et al., 2009), promoting a reduction in the amounts of phospholipids,
304
ergosterol and fatty acids (Dorlo et al., 2012). Despite the antifungal activity of
305
miltefosine and its potential as a therapeutic alternative to treat fungal infections, the
306
mode of action of this drug in fungi is poorly understood. Zuo et al. (2011)
307
demonstrated that miltefosine is rapidly incorporated into Saccharomyces cerevisiae
308
yeast cells, where it crosses the mitochondrial inner membrane, penetrating the
309
mitochondria and leading to an apoptosis-like cell death. In S. brasiliensis, miltefosine
310
may act on the synthesis of membrane components, which may promote instability and
311
loss of membrane integrity, ultimately leading to yeast cell death.
312
We also showed by TEM that the exposure to miltefosine promoted considerable
313
changes in S. brasiliensis yeast cells, including: loss in electron density (Fig. 3C),
314
accumulation of electron dense material in the cell wall (arrow in Fig.3D) and
315
alterations in cell wall thickness (Fig. 3E). Alterations in cell wall components after
316
miltefosine exposure were also described in C. albicans yeasts (Villa et al., 2013).
317
However, the aggregation of electron dense material in the cell wall following
318
miltefosine treatment had not been observed previously for other fungal species. This
319
electron dense material visualized by TEM is morphologically similar to the melanin
320
granules described by Teixeira et al (2010). In addition, flow cytometry analysis using
321
anti-melanin antibodies showed that miltefosine-treated cells had increased melanin
322
content compared to untreated cells. Thus, our results suggest that miltefosine treatment
323
could be modulates melanin turnover (breakdown and/or production) in S. brasiliensis
324
yeast cells, leading to the accumulation of aggregates of this pigment in the cell wall.
325
Similarly, Teixeira et al (2010) also showed that the amount of melanin in the cell wall
326
of S. schenckii could be modulated by varying culture medium composition (Teixeira et
327
al., 2010).
328
Although miltefosine displayed relatively high cytotoxicity towards kidney cells, this is
329
comparable to that observed for amphotericin B, the only available treatment option for
330
severe forms of sporotrichosis. Moreover, in vivo tests with BALB/c mice infected with
331
L. amazonensis showed that miltefosine was toxic only at doses higher than 50 mg kg-1
332
day-1 (Godinho et al., 2012). Given the lower MIC values for miltefosine against S.
333
brasiliensis in vitro, this drug shows higher selective indexes than amphotericin B for
334
the isolates tested (Table 2). Thus, miltefosine is likely to have a wider ‘therapeutic
335
window’ than amphotericin B against S. brasiliensis sporotrichosis, which should allow
336
the use of lower effective doses of this drug, to limit toxicity. Another advantage of
337
miltefosine treatment is that this drug can be administrated orally, whereas amphotericin
338
B treatment is intravenous.
339
Miltefosine is already used to treat some of the clinical manifestations of leishmaniasis
340
(Dorlo et al., 2012), which should facilitate its approval for use in the treatment of other
341
infectious diseases. Interestingly, miltefosine was approved for the topical treatment of
342
metastasized mammary carcinoma in Germany (Paparazafiri et al., 2005), in 1992; thus,
343
topical administration of miltefosine - which might be particularly useful for
344
sporotrichosis treatment – may be also effective in patients. Although some resistance to
345
miltefosine has been described in patients infected with Leishmania donovani, the main
346
resistance mechanisms seems to be associated with a defect in translocation of the drug
347
to the cytoplasm (Perez-Victoria et al., 2003).
348
The dose of miltefosine used for the treatment of leishmaniasis in humans is 2.5 mg kg-1
349
day-1, for 28 days (Dorlo et al., 2012). The reported IC50 value for the treatment of
350
intracellular amastigotes of Leishmania amazonensis with miltefosine in vitro was 9 µM
351
(3.6 µg.mL-1) (Santa-Rita et al., 2004), which is higher than the MIC values of 2.5-5
352
µM (1-2 µg.mL-1) reported here for the treatment of S. brasiliensis isolates. Therefore,
353
our results indicate that lower miltefosine doses than those used against human
354
leishmaniasis could be effective in the treatment of sporotrichosis.
355
In conclusion, ours results show that miltefosine might represent an interesting
356
alternative for the treatment of sporotrichosis caused by S. brasiliensis, especially when
357
standard treatment fails. Nevertheless, further in vivo studies in experimentally infected
358
animals are required to confirm the efficacy of miltefosine as an antifungal agent for the
359
treatment of sporotrichosis.
360
Declaration of interest: The authors report no conflict of interests.
361
Acknowledgements
362
The authors thank Dr. Zoilo Pires de Camargo and Dr. Anderson Messias Rodrigues
363
from the Universidade Estadual de São Paulo (São Paulo, SP, Brazil), Dra. Leila Maria
364
Lopes-Bezerra from Universidade Estadual do Rio de Janeiro (Rio de Janeiro, RJ,
365
Brazil) and Dr. Marcio Nucci from the Universidade Federal do Rio de Janeiro (Rio de
366
Janeiro, RJ, Brazil) for kindly providing the S. brasiliensis isolates used in this study.
367
This study was supported by the Brazilian funding agencies Fundação Carlos Chagas
368
Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de
369
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de
370
Desenvolvimento Científico e Tecnológico (CNPq).
371 372
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498 499 500 501 502 503 504 505 506
507
Table 1. Minimum inhibitory concentration (MIC) and minimum fungicidal
508
concentration (MFC) for the treatment of Sporothrix brasiliensis yeasts with
509
miltefosine, compared to reported values for amphotericin B and itraconazole [Borba-
510
Santos et al., 2014]. All values are in µg mL-1.
S. brasiliensis isolates MYA 4823 B91 B182 B628 B735 B848 B972 B1008 B1036 HE06 Ss 68 Ss 34 Ss 37 Ss 56 511 512 513 514 515 516 517 518 519 520 521 522 523
a
a
Miltefosine MIC 1 2 2 1 2 2 2 2 2 2 1 1 1 1
MFC 2 4 2 1 2 >16 2 2 2 8 1 2 16 1
Amphotericin Bb MIC 0.5 2 1 4 2 4 4 4 4 1 4 4 4 4
MFC 2 16 16 4 2 4 4 4 4 1 4 4 4 4
Itraconazoleb MIC 0.5 8 4 1 16 2 0.5 1 2 8 2 2 4 2
MFC >16 >16 4 16 >16 16 1 1 >16 >16 8 16 8 16
Reference (genome) strain, from feline sporotrichosis. All other strains are from human sporotrichosis. b MIC and MFC values for amphotericin B and itraconazole reported in Borba-Santos et al., 2014.
524
Table 2. Analyses of miltefosine and amphothericin B cytotoxicity towards the
525
epithelial cell line LLC-MK2 (CC50) and selectivity index (SI) for the treatment of
526
Sporothrix brasiliensis yeast cells. LLC-MK2 Antifungal agents
mean MICa -1
CC50 -1
(µg mL )
(µg mL )
Miltefosine
2
5
2.5
Amphotericin B
4
6.25
1.56
527 528
a
Mean MIC values for treatment of the yeast form of S. brasiliensis.
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b
SI = CC50/MIC.
530
SIb
531
Legends
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Figure 1. Time-kill plot showing the activity of miltefosine against the yeast form of
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Sporothrix brasiliensis ATCC MYA 4823. Yeast cell suspensions (103 colony forming
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units or CFU ml-1) were treated with different concentrations of miltefosine at 35oC,
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diluted and plated on potato dextrose agar (PDA). Plates were incubated for 7 days at
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35oC, and CFU numbers were determined by manual counting. Experiments were made
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in duplicate.
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Figure 2. Membrane integrity evaluation after treatment of Sporothrix brasiliensis
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(ATCC MYA 4823) yeast cells with miltefosine, amphotericin B and itraconazole. Cells
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were left untreated (control) or were treated with 0.5 µg.mL-1 miltefosine, or with 0.25
541
µg.mL-1 of amphotericin B or itraconazole, for 24 hours, before staining with the
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exclusion dye Sytox blue (Molecular Probes). Treatment with miltefosine only induced
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a statistically significant loss in plasma membrane integrity compared to untreated
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controls (**p
