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potential of IMS against multidrug-resistant dermatophytes, without presenting toxicity to human leucocyte cells at MIC. Significance and Impact of the Study: The ...
Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Imidazolium salts with antifungal potential against multidrug-resistant dermatophytes € ndchen3, C.M. Guez4, V.Z. Bergamo1, L.F.S. de Oliveira4, D.F. Dalla Lana1, R.K. Donato2, C. Bu 4 2 M.M. Machado , H.S. Schrekker and A.M. Fuentefria1 1 Laboratory of Applied Mycology, Department of Analysis, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brazil 2 Laboratory of Technological Processes and Catalysis, Institute of Chemistry, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brazil 3 Laboratory for Product and Process Optimization, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brazil 4 Center for Studies in Biochemistry, Immunology and Toxicology, Universidade Federal do Pampa – UNIPAMPA, Uruguaiana, RS, Brazil

Keywords antidermatophytic activity, imidazolium salts, ionic drugs, multidrug-resistant dermatophytes, toxicity. Correspondence Alexandre M. Fuentefria, Laboratory of Applied Mycology, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga 2752, Porto Alegre, Rio Grande do Sul, Brazil. E-mail: [email protected] 2015/0255: received 11 February 2015, revised 6 May 2015 and accepted 20 May 2015 doi:10.1111/jam.12862

Abstract Aims: To investigate the antidermatophytic action of a complementary set imidazolium salts (IMS), determining structure-activity relationships and characterizing the IMS toxicological profiles. Methods and Results: The susceptibility evaluation of 45 dermatophytic clinical isolates, treated in vitro with eleven different IMS (ionic compounds) and commercial antifungals (nonionic compounds), was performed by broth microdilution, following the standard norm of CLSI M38-A2. All dermatophytes were inhibited by IMS, where the lowest minimum inhibitory concentration (MIC) values were observed for salts with n-hexadecyl segment in the cation side chain, containing either the chloride or methanesulfonate anion. 1-n-Hexadecyl-3-methylimidazolium chloride (C16MImCl) and 1-nhexadecyl-3-methylimidazolium methanesulfonate (C16MImMeS) acted as fungicides, even in extremely low concentrations, wherein C16MImMeS exerted this effect on 100% of the tested dermatophytes. Some of these IMS provoked evident alterations on the fungi cell morphology, causing a total cell damage of ≥70%. Importantly, none of the screened IMS were cytotoxic, mutagenic or genotoxic to human leucocyte cells. Conclusions: This report demonstrates for the first time the strong antifungal potential of IMS against multidrug-resistant dermatophytes, without presenting toxicity to human leucocyte cells at MIC. Significance and Impact of the Study: The expressive antifungal activity of IMS, combined with the in vitro nontoxicity, makes them promising compounds for the safe and effective treatment of dermatophytoses, mainly when this skin mycosis is unresponsive to conventional drugs.

Introduction Dermatophytoses, also known as tinea, are one of the most common cutaneous mycoses worldwide. They belong to the earliest fungal infections reported, caused by dermatophytes of three anamorphic genera; Microsporum, Trichophyton and Epidermophyton (Indira et al. 2014; Koch and English 2014). Epidemiological studies indicate that the

dermatophytoses are considered the third most frequent dermatological disorder in children and the second in adults (Cortez et al. 2012). They are of extreme relevance to public and veterinary health, as they invade and propagate in keratinized tissues, such as skin, hair and nails, of humans and animals. Hence, they often cause very contagious, sometimes chronic and uncontrolled infections (Straten et al. 2003; Chermette et al. 2008).

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The treatment of dermatophytoses is carried out, generally, with the use of topical and/or systemic antifungal agents associated or not with keratolytic substances (Lakshmipathy and Kannabiran 2010; Bikowski 2014). The most effective and commercially available antifungals are mostly expensive and cause side effects, discouraging the patients to fully perform prolonged therapeutic treatments (Choudhary et al. 2014). Withal, the dermatophytic fungi are associated with an even more serious problem, which is their frequent multidrug-resistance to conventional antifungal treatments. This causes a large variation in susceptibility among species, decreasing the favourable response necessary for a progressive improvement and subsequent cure (Mukherjee et al. 2003; Choudhary et al. 2014). Imidazolium salts (IMS) are ionic compounds, generally composed of a(n) (a)symmetrically substituted organic cation and an (in)organic anion (Riduan and Zhang 2013; Biczak et al. 2014). Some of these IMS are denominated imidazolium ionic liquids (IL), for being in the liquid state at temperatures of 100°C or below. The favourable physicochemical properties—including negligible vapour pressure, high thermal and chemical stability, negligible flammability and high ionic conductivity— make IMS a promising class of compounds for the development of new drugs (Riduan and Zhang 2013; Biczak et al. 2014). Additionally, various important biological activities have been reported; such as anti-inflammatory (Gao et al. 2013), antiarrhythmic (Lis et al. 1987), anticancer (Malhotra and Kumar 2010; Gao et al. 2013), antifibrotic (Zhang et al. 2009), antibacterial (Coleman et al. 2012), anti-yeasts (Schrekker et al. 2013) and antimicrobial in general (Anderson and Long 2010). Herein, we report for the first time on the in vitro antifungal activity of a complementary set of IMS against multidrug-resistant dermatophytes, together with the human leucocyte toxicity profile of the most promising

+ N N (CH2)n

n=3 N

Materials and methods Imidazolium salts Eleven IMS (Fig. 1) were synthesized through methods described in the literature and the spectral data were in agreement with those reported previously (final purity ≥98%) (Cassol et al. 2006; Schrekker et al. 2008; Wasserscheid and Welton 2008): 1-n-butyl-3-methylimidazolium chloride (C4MImCl), 1-n-butyl-3-methylimidazolium methanesulfonate (C4MImMeS), 1-n-butyl-3-methylimidazolium octanesulfonate (C4MImOcS), 1-cyclohexylpropyl-3-methylimidazolium methanesulfonate (C9MImMeS), 1-n-decyl-3-methylimidazolium chloride (C10MImCl), 1,3-didecyl-2-methylimidazolium chloride ((C10)2MimCl), 1-n-hexadecyl-3-methylimidazolium chloride (C16MImCl), 1-n-hexadecyl-3-methylimidazolium methanesulfonate (C16MImMeS), 1-n-hexadecyl-3-methylimidazolium bis (trifluoromethylsulfonyl)imide (C16MImNTf2), 1-n-hexadecyl-3-methylimidazolium hydroxybenzoate (salicylate) (C16 MImSaC) and 1-n-octadecyl-3-methylimidazolium chloride (C18MImCl). The molecular weights (g mol 1) of C4MImCl, C4MImMeS, C4MImOcS, C9MImMeS, C10MImCl, (C10)2MImCl, C16MImCl, C16MImMeS, C16MImNTf2, C16MImSaC and C18MImCl are 17467, 23431, 33250, 30246, 25883, 39909, 34299, 40232, 58771, 44467 and 37104 respectively. The IMS solutions were prepared in sterile ultrapure water. Reference commercial antifungal agents Terbinafine (TBF), purity ≥97%, was supplied by Cristalia (Sao Paulo, Brazil), griseofulvin (GSF), purity ≥97%, was

Anions

Cations n=3 n=9 n = 15 n = 17

substances. This allowed establishing structure-activity relationships and identifying compounds for the safe and effective treatment of dermatophytoses.

C4MIm C10MIm C16MIm C18MIm

C9MIm

+ N (CH2)n

– O CI (CI)

O – O O S O (MeS)

OO

CF3 S N O O O – (NTf2)

F3C

S

O – O O S O (OcS)

HO N (CH2)n

378

+ N (CH2)n

n=9

(C10)2MIm

(SaC) – O O

O

Figure 1 Imidazolium salts evaluated in this study: MIm: methylimidazolium cation; (Cl): chloride anion; (MeS): methanesulfonate anion; (NTf2): bis(trifluoromethylsulfonyl)imide anion; (OcS): octanesulfonate anion; (SaC): salicylate anion.

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acquired from Wallace Pharmaceuticals (Mumbai, India) and ketoconazole (KTZ), purity ≥96%, was obtained from All Chemistry (Sao Paulo, Brazil). TBF, GSF and KTZ stock solutions of 1600 lg ml 1 were prepared using sterile ultrapure water and DMSO, and stored at 4°C. Serial micro-dilutions of each stock solution were performed to obtain a final concentration of 6400 lg ml 1 and 05% (v/v) for water and DMSO, respectively (CLSI, M38-A2 2008). Dermatophytic clinical isolates A total of 45 clinical isolates of four dermatophytic species, of human origin, were tested for the antifungal susceptibility: Nine Microsporum canis (MCA 01, MCA 29, MCA 32, MCA 33, MCA 36*, MCA W3, MCA 38, MCA 39, MCA 40*), 12 Microsporum gypseum (MGY 42, MGY 45, MGY 46, MGY 48, MGY 49, MGY 50, MGY 51, MGY 52, MGY 53, MGY 54, MGY 57, MGY 58*), 12 Trichophyton mentagrophytes (TME 16*, TME 18, TME 31, TME 32, TME 33, TME 34*, TME 35, TME 36, TME 38, TME 40, TME 44, TME 46) and 12 Trichophyton rubrum (TRU 20, TRU 23, TRU 25*, TRU 40, TRU 42, TRU 43*, TRU 46, TRU 48, TRU 49, TRU 50, TRU 52, TRU 53). A total of 38 isolates were sensitive to the evaluated commercial antifungal agents, and seven were identified as in vitro resistant to at least two of the antimycotics, thus being characterized as multidrug-resistant isolates (marked with an asterisk (*) for differentiation). This resistance was established according to the increase in minimum inhibitory concentration (MIC) values for these isolates, considering the following resistance threshold concentrations: TBF MIC ≥ 100 lg ml 1, GSF MIC ≥ 400 lg ml 1 and KTZ MIC ≥ 800 lg ml 1 (considerably higher concentrations than observed for the most isolates). All the clinical isolates used in this study are from a variety of dermatophytic infections (tinea capitis, tinea corporis, tinea cruris, tinea unguium, tinea barbae, tinea manuum and tinea pedis) and are deposited in the Mycology Collection of Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. Susceptibility tests Susceptibility tests were performed for determining the MIC and the minimum fungicidal concentration (MFC). The MIC was defined by the broth microdilution method, according to the protocol established for filamentous fungi (CLSI, M38-A2 2008). The conidial inocula (10 9 103 to 30 9 103 CFU ml 1) were prepared from cultures grown on potato dextrose agar (PDA) at 32°C. The assays were conducted with RPMI medium, containing L-glutamine (without sodium bicarbonate),

Dermatophytic imidazolium salt antifungals

buffered to pH 70 with 0165 mol l 1 MOPS. The concentrations of the antifungal agents and IMS ranged from 003 to 3200 lg ml 1 and 002 to 5000 lg ml 1 respectively. MIC was defined as the lowest concentration of the substance capable of inhibiting the visible fungal growth. Controls were used in parallel: Sterility control (negative control as a drug-free medium) and positive control for fungal cells viability. Commercial antifungals were used as antifungal activity reference in all assays. The experiments were carried out in duplicate, incubating the microplates at 32°C for 4 days (96 h) and reading the MIC visually. To determine the MFC, aliquots of each serial microdilution (corresponding to the MIC, 2xMIC and 4xMIC) were sown on PDA agar plates (Pronadisa, Madrid-Spain), which were incubated at 32°C for 96 h and analysed. The MFC was defined as the lowest drug concentration that yielded three or fewer colonies (i.e. ≥99% of the inoculum was killed) (Espinel-Ingroff et al. 2002). This assay was performed in triplicate. Time kill assay The fungicidal activity of C16MImMeS against four multidrug-resistant isolates (M. canis - MCA 36*, M. gypseum MGY 58*, T. mentagrophytes - TME 34* and T. rubrum TRU 43*) was studied by kill curve experiments (Natesan et al. 2008). The dermatophytic conidial inocula (10 9 103 to 30 9 103 CFU ml 1) were incubated in the presence of concentrations corresponding to the MIC, 2xMIC and 4xMIC of C16MImMeS, for each dermatophyte, and compared to an untreated control. Aliquots of the conidial suspension were collected after 1, 6, 12, 24 and 48 h and diluted to obtain 10–10 3 dilutions. After, 010 ml aliquots were spread on PDA plates and incubated at 32°C for 96 h. The number of CFU ml 1 was determined and kill curves were constructed by plotting mean log10 CFU ml 1 against the exposure time of dermatophytic conidia to various concentrations of the salt, including the standard deviations. The assay was performed in triplicate. Scanning electron microscopy analysis Aliquots of microplate susceptibility testing fungal inoculum (TME 16* treated with C16MImCl, C16MImMeS and C16MImNTf2 in subinhibitory concentrations of 078, 039 and 156 lg ml 1 respectively), as well as untreated control, were incubated at 32°C for 24 h. Subsequently, these samples were fixed for 24–48 h at 4°C with 25% glutaraldehyde in 005 mol l 1 cacodylate buffer (pH 72). The samples were dehydrated in acetone and dried by a CO2 critical-point dryer (Baltec CPD 030). The dried samples were finally coated with osmium in an osmium plasma coater (Nippon laser & Electronics

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Laboratory, Nagoya City, Japan), and examined using a Carl Zeiss MEV Evo 50 (Inouye et al. 2007). Cell damage evaluation by the MTT-based assay In order to assess the cell damage of the dermatophytes, a colorimetric assay using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) was performed for an isolate multidrug-resistant of T. rubrum after treatment in vitro with the IMS, at the MIC. The absorbance (A) was measured at 570 and 690 nm on a multiscan-enzyme-linked immunosorbent assay reader (2104 EnVisionâ Multilabel Reader; PerkinElmer, Turku, Finland). The percentage cell damage (CD %) was calculated by the following equation: CD %=1 [(A570 nm - A690 nm with drug)/(A570 nm A690 nm without drug)] 9 100. As such, the absorbance was adjusted for nonspecific absorption by subtracting absorbance at 690 nm from absorbance at 570 nm. The yellow tetrazolium salt undergoes a dehydrogenase promoted chemical transformation, inside mitochondria or in other cell locations possessing dehydrogenase activity, to form the purple formazan derivative, which can be measured spectrophotometrically at 570 nm (Chiou et al. 2001). Toxicity evaluation The three most effective (with lower MIC values against all dermatophytes) IMS were selected for toxicological analysis: C16MImCl, C16MImMeS and C16MImNTf2. Cell culture The Committee of Ethics in Research approved the following protocol (authorization no 23081005770/2009-38):

Human leucocyte cell cultures were prepared according to Dos Santos et al. (2010). The tested salt concentrations ranged from the geometric mean of the minimum concentrations that inhibit 50% of the fungal isolates analysed (MIC50), up to 100 times higher (Table 1). All the compounds were diluted in phosphate buffer saline (PBS). Hydrogen peroxide solution (H2O2, 100 lmol l 1), which is reported to be toxic for cells, was used as a positive control (Nakamura et al. 2003). Subsequently, all the cell cultures were maintained in a CO2 incubator (Model MCO19AIC, Sanyo) for 72 h at 37°C. After this period, cytotoxic, genotoxic and mutagenic parameters were established. Effects on cell proliferation and viability (cytotoxicity analysis) In order to perform the cell proliferation and cell viability analysis, the cell suspension was mixed with Turk’s solution and 02% trypan blue, respectively, with subsequent microscopic observation (Olympusâ model CH-30, Tokyo, Japan; 4009 magnification). The tests were performed in triplicate, as described by Burow et al. (1998). Mutagenicity, genotoxicity and chromosomal stability analysis Micronucleus (MN) frequency evaluation (mutagenicity analysis), comet assay (genotoxicity analysis) and analysis of the effects on chromosomal stability were performed. For MN frequency evaluation, the treated cell cultures were fixed on slides by the smear slide method and allowed to dry at room temperature. Slides, in duplicate, were stained by Pan otico Rapidoâ (Labor Clean) and then analysed by optical microscopy (Olympusâ model

Table 1 MIC50 of the IMS, terbinafine (TBF), griseofulvin (GSF) and ketoconazole (KTZ) against dermatophytes strains Isolates (n = 45)

C4MIm Cl

C4MIm MeS

C4MIm OcS

C9MIm MeS

C10MIm Cl

(C10)2Mim Cl

MCA (n = 9) MGY (n = 12) TME (n = 12) TRU (n = 12) Geometric mean

1250/7156 1250/7156 1250/7156 1250/7156 1250/7156

1250/5335 1250/5335 1250/5335 1250/5335 1250/5335

1250/3759 1250/3759 2500/7519 1250/3759 1487/4470

1250/4133 625/2066 313/1033 313/1033 526/1737

156/604 039/151 1250/4829 625/2415 263/1016

313/784 313/784 313/784 156/392 263/659

Isolates (n = 45)

C16MIm Cl

C16MIm MeS

C16MIm NTf2

C16MIm SaC

C18MIm Cl

TBF

GSF

KTZ

MCA (n = 9) MGY (n = 12) TME (n = 12) TRU (n = 12) Geometric mean

005/014 039/114 002/007 005/014 007/020

005/012 005/012 005/012 005/012 005/012

156/266 156/266 313/532 313/532 221/376

313/703 1250/2811 625/1406 156/351 442/994

625/1684 625/1684 625/1684 1250/3369 743/2003

003 003 003 003 003

050 100 050 100 071

050 100 200 100 100

MIC50 and geometric mean values of IMS expressed in lg ml 1/nmol ml 1 and MIC50 and geometric mean of TBF, GSF and CTZ in lg ml 1; n = the number of fungal isolates of Microsporum canis (MCA), Microsporum gypseum (MGY), Trichophyton mentagrophytes (TME) and Trichophyton rubrum (TRU).

380

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CH-30) with immersion lens, as described by Thomas et al. (2008). For the comet assay in single cell gel electrophoresis test, the samples were electrophoresed (20 min at 300 mA and 25 V) in a horizontal chamber (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently stained with silver. Each cell was evaluated as for their level of damage (from 1 to 4). The sum of the damage values provided a damage index (DI). This index can vary between 0 (100 cells 9 0) to 400 (100 cells 9 4). The assay was performed in triplicate, as described by Singh et al. (1988). For the analysis of the effects on chromosomal stability, colcemid solution (10 lg ml 1) was added to the leucocytes suspension. The cells were examined microscopically (Olympusâ model CH-30) to analyse the chromosomes in the metaphase stage, as described by Yunis (1976). Statistical analyses Statistical analyses, comparing IMS and commercial antifungals, were performed to determine statistically significant differences (P < 005), using the Kruskal–Wallis nonparametric test, followed by the Dunn post-test. For the cell damage statistical analysis, only the Kruskal–Wallis test was applied, considering a significance level P < 005. These statistical tests were conducted using STATISTICA v10.0 statistical software (Statsoft, Tulsa, OK). For the assessment of toxicity tests, analyses of variance (ANOVA) were employed, followed by the post hoc Bonferroni test. Results with P < 005 were considered significant. The data were analysed by using GRAPHPAD PRISM software (GraphPad Software, San Diego, CA—ver. 5.02 for Windows). Results Susceptibility and structure-activity relationships All of the 11 IMS evaluated (C4MImCl, C4MImMeS, C4MImOcS, C9MImMeS, C10MImCl, (C10)2MImCl, C16MImCl, C16MImMeS, C16MImNTf2, C16MImSaC and C18MImCl) were able to inhibit the in vitro growth of the tested dermatophytes. A broad range of MIC values were determined, where the IMS caused fungal growth inhibition from eminently low (approx 005 lg ml 1) to higher concentrations (approx. 2500 lg ml 1) (Table 1). Comparing the MIC values in mass and molar unities (lg ml 1 vs nmol ml 1) allowed evaluating the performance of each compound, considering the molar mass differences. The obtained MIC values followed the same trend for both unities, indicating that the chemical com-

Dermatophytic imidazolium salt antifungals

position contribution surpassed the IMS molar mass differences. Regarding the IMS-cation side chain length, the most effective molecules were those with C10 and C16 alkyl segments. The highest MIC50 values (Table 1) were ascertained for the IMS with the shortest (C4MImCl, C4MImMeS and C4MImOcS), as well as the longest side chains (C18MImCl), showing a trend for an optimum chain length of C16 for efficacy. This corroborates with the statistical analysis result, in which C4MImCl, C4MImMeS and C4MImOcS were statistically nonequivalent to any of the three commercial antifungals analysed, independent of the species tested. Specifically for T. rubrum, C18MImCl also did not present statistical similarity with any of the three antifungals investigated. Also the IMS-anion plays an important role (Table 1). Although the observed differences could be due to multiple anion-effects, i.e. volume, coordination strength and water solubility, the volume appears being prevalent: increasing anion volume causes higher MIC values. As a consequence, the salts with methanesulfonate and chloride anions were identified as the most promising antifungals. Within the IMS tested, the best cation and anion combinations were determined for C16MImCl and C16MImMeS. Statistically, C16MImCl and C16MImMeS showed being similar to TBF (Fig. 2), one of the most employed antifungal and reference fungicidal drug for dermatophytosis treatment. This fungicidal activity was also confirmed for both C16MImCl and C16MImMeS. The MFC values are presented in Table 2 for the Microsporum and Trichophyton genus. All remnant IMS, as well as griseofulvin and ketoconazole, presented only fungistatic activity in all tested concentrations (MIC, 2xMIC e 4xMIC). Importantly, TBF did not present the fungicidal effect against some of the tested multidrug-resistant isolates. In these cases, the MFC values corresponded to the actual MIC. Similar TBF MFC values were determined for the two most promising IMS. The IMS C16MImCl was fungicide at 4xMIC for the majority of the dermatophytic isolates. Interestingly, C16MImMeS was fungicide for all of the 45 dermatophytic species, at very low concentrations, which were always four times higher than the MIC (Table 2). Regarding the fungicidal kinetics of C16MImMeS, the curves relating the number of colonies of multidrug-resistant dermatophytic isolates, in function of time, with the IMS at different concentrations are presented in Fig. 3. These plots express the logarithmic number of viable dermatophyte colonies after the in vitro treatment with the broad spectrum fungicidal IMS. This allows determining the effective fungicidal concentration and the duration of this fungitoxic action. At concentrations corresponding to the MIC and 2xMIC, a gradual and periodic decrease of CFU ml 1 in function of time was identified. At the

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Trichophyton mentagrophytes 50

Trichophyton rubrum 50

40

40

Concentration (µg ml–1)

Concentration (µg ml–1)

Dermatophytic imidazolium salt antifungals

30 20 10 0 –10 C4MImC1 C16MImNTf2 C16MImC1 Terbinafine C18MImC1 C10MImC1 C16MImMeS

30 20 10 0 –10 C16MImC1 C4MImC1 C16MImNTf2 Terbinafine C18MImC1 C16MImMeS C10MImC1

Figure 2 Graphical analysis of the statistical differentiation of some imidazolium salts compared with terbinafine in boxplot of concentration.

Table 2 Minimum fungicidal concentration (lg ml 1) of C16MImCl, C16MImMeS and terbinafine against dermatophytes of the Microsporum and Trichophyton genera Isolates

C16MImCl

Microsporum canis MCA 01 156‡ MCA 29 020‡ MCA 32 Fungistatic MCA 33 020‡ MCA 36* Fungistatic MCA W3 156‡ MCA 38 020‡ MCA 39 080‡ MCA 40* Fungistatic Microsporum gypseum MGY 42 156‡ MGY 45 156‡ MGY 46 156‡ MGY 48 1250‡ MGY 49 Fungistatic MGY 50 1250‡ MGY 51 156‡ MGY 52 156‡ MGY 53 156‡ MGY 54 156‡ MGY 57 Fungistatic MGY 58* Fungistatic

C16MImMeS

Terbinafine

156‡ 020‡ 020‡ 625‡ 625‡ 020‡ 080‡ 020‡ 020‡

003† 013† 013† – Fungistatic 006† 003† 003† Fungistatic

020‡ 156‡ 313‡ 625‡ 020‡ 020‡ 020‡ 625‡ 020‡ 020‡ 020‡ 1250‡

003† 003† 003† 013† 003† 013† 025† 013† 006† 006† 003† Fungistatic

Isolates

C16MImCl

Trichophyton mentagrophytes TME 16* Fungistatic TME 18 008‡ TME 31 1250‡ TME 32 008‡ TME 33 008‡ TME 34* Fungistatic TME 35 1250‡ TME 36 020‡ TME 38 625‡ TME 40 008‡ TME 44 008‡ TME 46 Fungistatic Trichophyton rubrum TRU 20 020‡ TRU 23 020‡ TRU 25* Fungistatic TRU 40 020‡ TRU 42 020‡ TRU 43* Fungistatic TRU 46 Fungistatic TRU 48 020‡ TRU 49 Fungistatic TRU 50 020‡ TRU 52 Fungistatic TRU 53 020‡

C16MImMeS

Terbinafine

313‡ 020‡ 1250‡ 040‡ 008‡ 156‡ 020‡ 040‡ 020‡ 020‡ 020‡ 156‡

Fungistatic 006† 003† 006† 003† Fungistatic – – 003† 003† 003† 013†

020‡ 020‡ 625‡ 020‡ 008‡ 625‡ 020‡ 020‡ 040‡ 020‡ 020‡ 020‡

006† 003† Fungistatic 006† 003† 003† 003† 003† 003† 006† 003† –

*Multidrug-resistant dermatophytic isolates. †Fungicide at MIC values. ‡Fungicide at 4xMIC values.

concentration of 4xMIC, a total absence of fungal growth was observed, where this IMS had its action onset since the first hour of contact with the fungal species and maintained its strong fungicidal effect during the whole 48 h of analysis. 382

Additionally, the IMS C16MImCl, C16MImMeS and C16MImNTf2 caused clear alterations in the morphology of the multidrug-resistant clinical isolate of TME, as shown in Fig. 4. In twice-lower concentrations than the MIC, these compounds were already able to cause severe

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Log10 CFU mL–1

4

1·56 µg ml–1

3 2

MCA 36*

MGY 58*

1 12·50 µg ml–1

6·25 µg ml–1 Control

4 Log10 CFU mL–1

6·25 µg ml–1

3·13 µg ml–1

0

Figure 3 Time Kill curves of 4 multidrugresistant dermatophytes; Microsporum canis (MCA 36*), Microsporum gypseum (MGY 58*), Trichophyton mentagrophytes (TME 34*) and Trichophyton rubrum (TRU 43*); based on the fungicidal action of C16MImMeS compared to untreated control.

Control 3·13 µg ml–1

Control

Control

0·39 µg ml–1

3

1·56 µg ml–1

0·78 µg ml–1

2

3·13 µg ml–1

TME 34*

TRU 43*

1 1·56 µg ml–1

6·25 µg ml–1

0 0

10

30 20 Time (h)

40

0

10

20 30 Time (h)

40

50

structural damage, as evidenced by the scanning electron microscopy images. The untreated control (Fig. 4a,b) presents a set of hyphae involved in a dense, firm and compact way, representing a micromorphologically intact fungal cell. After in vitro treatment with C16MImCl, hyphal system disaggregation occurred (Fig. 4c) and cell agglomerate dehydration (Fig. 4d). With the application of C16MImMeS, even stronger structural fragmentation (Fig. 4e) and more severe osmotic effect on the TME hyphae aggregate (Fig. 4f) was observed. Differently, the structure of the dermatophytic hyphae treated with C16MImNTf2 appeared more conserved, including the presence of intact hypha (Fig. 4g,h), suggesting that this IMS inhibits the growth of T. mentagrophytes through a different mechanism. The total cell damage also played an important role. Ketoconazole induced damage percentages higher than 80%, which is in agreement with the parameters established by CLSI (M38-A2, 2008), indicating a reduction in dermatophyte growth of at least 80% for this imidazolic antifungal. C16MImCl and C16MImMeS were statistically equivalent to KTZ, causing a minimum cell damage of 80% for T. rubrum. On the other hand, C16MImNTf2 showed weaker cell damage ability, with a CD% of approx. 70% for T. rubrum. Statistically, only the IMS C16MImNTf2 cell damage values were inferior to those of KTZ.

20% cell viability reduction. Differently, none of the IMS interfered significantly in the cell proliferation process and did not harm the assessed leucocyte’s viability. The three IMS differed from the hydrogen peroxide, in all treatments, considering the appraised concentrations equivalent to the geometric mean of the MIC50, 10xMIC50 and 100xMIC50, for the 45 dermatophytes (Fig. 5). Thus, showing that C16MImCl, C16MImMeS and C16MImNTf2 were not cytotoxic to human leucocytes at these concentrations. The mutagenesis, genotoxicity and chromosomal instability caused to human cells after exposure to C16MImCl, C16MImMeS and C16MImNTf2, expressed, as the MN frequency, DNA index and numeric chromosomal abnormalities, are observed in Fig. 5. Once more, none of the IMS analysed influenced the MN emergence or caused significant DNA damage. All the salts were statistically different to the H2O2 solution and similar to the PBS buffer (negative control), i.e. no mutations and DNA damage, as well as no cellular numeric chromosomal changes could be detected for these IMS. The IMS and PBS were statistically different from hydrogen peroxide, which caused approx. 15% of chromosomal defects. The whole set of toxicity tests, using human leucocytes, suggest that C16MImCl, C16MImMeS and C16MImNTf2, are safe for bio-applications at concentrations at which these are highly effective in vitro antifungals.

Toxicity evaluation

Discussion

The toxicity analysis data of IMS are shown in Fig. 5. Hydrogen peroxide, a positive control for cytotoxicity, decreased the number of leucocytes and caused an approx.

The IMS evaluated in this study have shown potent antidermatophytic activity, including against multidrug-resistant dermatophytes. Important IMS structure- antifungal

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Figure 4 Scanning electron microscopy images of the multidrug-resistant Trichophyton mentagrophytes isolate (TME 16*): untreated control (a and b) and after in vitro treatment with C16MImCl (c and d), C16MImMeS (e and f) and C16MImNTf2 (g and h); in subinhibitory concentrations.

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activity relationships were established, showing that the cation alkyl chain length performs an important role in the inhibition of these pathogenic species. The optimum chain length for inhibiting all fungi has been defined as n-hexadecyl, independent of the associated anion. IMS with either shorter (n-butyl) or longer (n-octadecyl) side chains were less effective. These observations corroborate with previously published results of IMS action against different fungal genera, suggesting their broad antifungal and antibiofilm effectiveness (Pernak et al. 2003; Carson et al. 2009; Bergamo et al. 2014). Furthermore, C16MImCl and C16MImMeS were able to inhibit the fungal 384

growth with MIC values comparable to that of TBF and lower than those determined for GSF and KTZ. Interestingly, IMS showed such strong activity also against multidrug-resistant dermatophytes, when TBF exerted only a small or no effect at all (Tables 1 and 2). The MIC50 of these two IMS varied between 002 and 039 lg ml 1 (Table 1), values up to 100 times smaller than the ones recently presented in the literature as effective antidermatophytic compounds, such as; peptides (200– 6400 lg ml 1, Simonetti et al. 2009), synthetic thiazoles (200 to >6400 lg ml 1, Ouf et al. 2013), and essential oils (025–400 mg ml 1, Khosravi et al. 2013).

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2·21 µg ml–1

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Figure 5 Graphical representation of the toxicological analysis; effects of different concentrations of C16MImCl, C16MImMeS and C16MImNTf2 on (1) cell proliferation and (2) cell viability, (3) micronucleus frequency (4) DNA damage and (5) numerical chromosomal abnormalities, for evaluating the cytotoxicity, mutagenicity, genotoxicity and capacity to cause chromosomal instability to human leucocytes. Bars with the same letters indicate statistical similarity.

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Synergistically with the predominant influence of the IMS-cation side chain length, the anion also presented an extremely important contribution. IMS with smaller anions showed the best in vitro antifungal performances against dermatophytes, identifying C16MImCl and C16MImMeS as the most promising antifungal candidates. Only these two IMS presented fungicidal activity, wherein the C16MImMeS was the only compound, con-

C16MImC1

C16MImMeS

C16MImNTf2

sidering also the commercial antifungals tested, able to exterminate 100% of the studied clinical isolates, including the multidrug-resistant ones. Concentrations four times higher than the MIC values appear to be the threshold for this fungicidal action. This was confirmed by both the MFC determination and the time kill assay. At these concentrations, especially C16MImMeS promoted a quick (complete fungal death in 1 h) and long-lasting

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fungal death (48 h) for all studied dermatophytes. The performance presented by this IMS is far superior to those presented in recent studies in the literature, using Melaleuca alternifolia oil, naftifine hydrochloride and a synthetic analogue of protegrin peptide, as fungicides against dermatophytes (Hammer et al. 2002; Ghannoum et al. 2013; Simonetti et al. 2014). The IMS C16MImCl, C16MImMeS and C16MImNTf2 cause micromorphological damage on the dermatophytes cells, which seems to be originated from a type of osmotic stress, followed by de-structuring of the TME hyphae. Inouye et al. (2007) reported similar micromorphological disruptions on TME, using a treatment with a suspension of essential oils (thymol 02% and tea tree oil 16%) and salt (sodium chloride 10%). Cell surface modifications were reported, including the formation of vesicles and wrinkled hyphas, and cell wall dissolution, where the authors noted that salt addition potentiated the fungicidal activity of the association, used as footbath for tinea pedis cases (Inouye et al. 2007). Yamaguchi et al. (2009) reported about the hyphae and cell wall detrimental effect of a thiosemicarbazide camphene derivative at a concentration of 55 nmol ml 1. Comparatively, C16MImCl and C16MImMeS caused even more severe hyphae alterations on a TME multidrug-resistant isolate at concentrations approx. 50 times lower. The relevant dermatophyte morphological damage corroborated with the total cell damC16MImMeS and age caused by C16MImCl, C16MImNTf2. At MIC values these IMS triggered high levels of cell injury, even higher than KTZ. Therewithal, the evaluation of IMS toxicological profiles over human leucocyte cells provided important input about their safety in bioapplications. The MN test is one of the most used methods to evaluate possible mutagenic damage caused by drugs or new biomolecules. Currently, the MN frequency investigation follows the mutagenicity test pattern OECD (Guidelines for the testing of chemicals/ section 4: Health Effects, Mammalian Erythrocytes Micronucleos (Test: n° 474) and recommended by GeneTox Program, Environmental Protection Agency – EPA/ US), enabling the accurate identification of possible clastogenic (chromosome breakage) and aneugenic (abnormal chromosome segregation) agents (Fenech 2000). Equally important, the comet test is an extremely sensitive method for studying DNA damage in individual cells (Mukhopadhyay et al. 2004; Collins 2009). While the comet assay detects reversible lesions, the MN test detects most persistent DNA damage or unrepairable aneugenic effects (Hartmann et al. 2003). Applying these tests, IMS presented no evidence of mutagenicity and genotoxicity, at concentrations up to 100 times higher than their MIC. This validated the IMS use for in vitro bioaplications, especially for antidermatophytosis formulations. Never386

theless, information about the in vivo cytotoxicity, genotoxicity and mutagenicity will be necessary for a definitive conclusion about the safe use of IMS. Altogether, the IMS showed broad antidermatophytic activity at very low concentrations, presenting rapid and prolonged fungicidal effect, causing evident morphological changes and pronounced cell damage to pathogenic fungal species. Concurrently, the thriving discovery that these salts are not cytotoxic, mutagenic or genotoxic to human leucocytes, strongly corroborates to a prominent position as a new class of potent and safe antifungal agents.

Acknowledgements The authors are grateful to the Brazilian agencies CAPES, CNPq (Public Notice Universal MCTI/CNPq No 14/ 2013) and FAPERGS for the financial support. R. K. Donato is thankful to FAPERGS-CAPES for the DOCFIX post-doctoral fellowship. Conflict of Interest No conflict of interest declared.

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