Effect of Melanin Biosynthesis Inhibition on the Antifungal Susceptibility of Chromoblastomycosis Agents

ABSTRACT

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by genera of melanized fungi: Fonsecaea, Cladophialophora, Phialophora, Exophiala, and Rhinocladiella. Melanin is a virulence factor known to influence antifungal susceptibility. A specific inhibitor of melanin biosynthesis is tricyclazole. The aim of this study was to evaluate the effect of melanin inhibition on antifungal susceptibility of chromoblastomycosis agents and describe the susceptibility profiles of some unusual CBM agents. Seventy-six clinical isolates, representing 13 species of the five main genera of CBM agents, were studied. The antifungal susceptibility testing was performed according to the M38-A2 protocol of CLSI (Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi, 3rd ed., CLSI Standard M38, 2017). In the melanin inhibition test, 16 mg/liter of tricyclazole was added to the medium used in the inoculum preparation and the susceptibility assay. CBM agents were less susceptible to amphotericin B than azoles and terbinafine. The unusual species showed similar susceptibility profiles to those of other species of the same genera. With tricyclazole exposure, MICs of terbinafine, posaconazole, and itraconazole for Fonsecaea spp. significantly decreased (P < 0.05). For Phialophora spp., this reduction was significant for posaconazole and itraconazole. For the other genera, there was a reduction in MICs of terbinafine and itraconazole; however, the statistical tests were not significant. Melanin inhibition can increase the antifungal susceptibility of most CBM agents to itraconazole and terbinafine, the main drugs used in the disease treatment. This increased susceptibility may open up new possibilities for therapy in refractory cases of CBM and/or cases caused by resistant fungal strains. Further studies are needed to confirm the same results in vivo.

INTRODUCTION

Chromoblastomycosis (CBM) is a chronic, disabling, and recalcitrant subcutaneous mycosis that is endemic in tropical and subtropical regions of the planet, affecting mainly the most impoverished populations in Latin America, Africa, and Asia (1). Its etiological agents are melanized fungi of the family Herpotrichiellaceaea: the main genera involved are Fonsecaea, Cladophialophora, Phialophora, Exophiala, and Rhinocladiella (2).

Treatment is based on antifungal therapy, which can be combined with physical and surgical methods in severe cases due to the recalcitrant nature of CBM (3). The first-line drug is itraconazole (ITC), followed by terbinafine (TRB) (1). In refractory cases, the disease can be treated with posaconazole (POS) or voriconazole (VRC). Ketoconazole (KTC) and amphotericin B (AMB) are currently not being used due to the severe side effects of these drugs and frequent failure in treatment (2, 4). It is important to emphasize that there are no randomized clinical trials for choosing the best therapy (1). In this context, in vitro antifungal susceptibility trials may be useful.

An antifungal resistance factor of CBM agents is the melanin present in its vegetative and parasitic cells (5). Specifically for these fungi, the type of melanin produced is 1,8-dihydroxynaphthalene melanin (DHN-melanin) (6, 7), which protects the fungi against aggressive environmental factors (8, 9). Moreover, DHN-melanin is considered a virulence factor and an immunologically active compound. It helps to escape from phagocytosis, promotes resistance to oxidative stress, and activates the host’s complement system (10). It is known that melanin can reduce the susceptibility of melanized cells to antifungals (11), as already demonstrated for amphotericin B and caspofungin against Cryptococcus spp. and Histoplasma capsulatum (12, 13) and itraconazole and ketoconazole against Madurella mycetomatis (14). Therefore, the use of drugs capable of inhibiting melanin biosynthesis in vivo may be a possibility to improve the treatment of diseases caused by black fungi (15).

A specific inhibitor of DHN-melanin biosynthesis is the fungicidal agrochemical 5-methyl-1,2,4-triazol[3,4]benzothiazole (tricyclazole). Tricyclazole is used in in vitro studies at concentrations of 16 to 32 μg/ml, and this range does not affect the viability of the fungal cells (16, 17). The presence of tricyclazole in the culture medium changes the colonies’ color from dark green, gray, or black (due to melanin) to reddish brown; the medium also has this color due to the accumulation of intermediary metabolites of the DHN-melanin biosynthesis pathway (18).

Therefore, this study’s main objective was to evaluate the influence of DHN-melanin on the antifungal susceptibility of CBM agents, since it is a well-known factor of virulence and resistance of black fungi. Additionally, we aimed to describe the susceptibility profile of CBM agents, including some unusual etiological agents of this disease.

RESULTS AND DISCUSSION

The geometric mean and range of MIC values for each antifungal for treatment of CBM agents by genus and species are shown in Tables 1 and 2. Most MICs of the antifungals (without tricyclazole) in the present study are similar to those found in other studies, as described in the systematic review by Hellwig et al. (19) and other research, like studies by Andrade et al. (20), Coelho et al. (21), and Silva et al. (22), which also evaluated strains from Brazil. These studies showed that CBM agents are less susceptible to amphotericin B than azoles and terbinafine, which was corroborated by our results. However, in another study that evaluated strains of the Fonsecaea species F. pedrosoi, F. monophora, and F. nubica from China, South and Central America, and Europe, it was found that these strains were more susceptible to itraconazole, posaconazole, and even amphotericin B compared with our results (23). This may be due to differences in the geographical origins of the strains.

TABLE 1.

MICs of antifungals for each genus of CBM agent exposed or not exposed to the melanin inhibitor tricyclazole

Genus (n isolates)	Antifungal MIC (mg/liter) without or with 16 μg/ml tricyclazolea
	TRB		VRC		POS		KTC		ITC		AMB
	Without	With	Without	With	Without	With	Without	With	Without	With	Without	With
Fonsecaea (57)	0.06 (0.03–0.5)*	0.03 (0.03–0.25)	0.125 (0.06–1)*	0.25 (0.06–0.5)	0.25 (0.03–2)*	0.125 (0.03–1)	0.25 (0.03–2)	0.25 (0.03–2)	0.5 (0.06–>16)*	0.5 (0.03–2)	4 (1–16)	4 (2–16)
Phialophora (6)	0.06 (0.03–0.125)	0.03 (0.03–0.125)	0.5 (0.25–4)	1(0.125–2)	0.5 (0.25–1)*	0.125 (0.06–0.5)	1 (0.125–2)	0.5 (0.06–2)	0.5 (0.25–1)*	0.125 (0.06–1)	8 (4–8)	4 (0.25–16)
Exophiala (5)	1 (0.25–8)	0.25 (0.03–4)	1 (1–4)	0.5 (0.125–8)	0.25 (0.125–0.5)	0.25 (0.03–1)	0.5 (0.25–4)	1 (0.125–4)	1 (0.25–4)	0.5 (0.06–1)	4 (0.5–16)	8 (0.5–16)
Cladophialo-phora (4)	0.5 (0.25–4)	0.045 (0.03–1)	0.187 (0.06–1)	0.25 (0.125–4)	0.25 (0.06–0.5)	0.187 (0.125–0.5)	0.25 (0.125–1)	0.187 (0.125–2)	0.75 (0.5–1)	0.375 (0.125–1)	5 (2–8)	3 (2–8)
Rhinocladiella (4)	0.5 (0.5–0.5)	0.25 (0.125–1)	1.125 (0.125–2)	1 (0.5–2)	0.312 (0.125–1)	0.25 (0.125–1)	2 (2–2)	2 (2–4)	0.75 (0.5–2)	0.5 (0.25–0.5)	6 (1–8)	12 (2–16)

^aThe values shown are geometric means with ranges in parentheses. TRB, terbinafine; VRC, voriconazole; POS, posaconazole; KTC, ketoconazole; ITC, itraconazole; AMB, amphotericin B. Asterisks indicate MIC values with a statistically significant difference (P < 0.05) without tricyclazole versus the same antifungal with tricyclazole by Wilcoxon statistical test using SPSS v.18.

TABLE 2.

MICs of antifungals for each CBM agent species exposed or not exposed to the melanin inhibitor tricyclazole

Species (n isolates)a	Antifungal MIC (mg/liter) without or with 16 μg/ml tricyclazoleb
	TRB		VRC		POS		KTC		ITC		AMB
	Without	With	Without	With	Without	With	Without	With	Without	With	Without	With
F. pedrosoi (39)	0.06 (0.03–0.5)	0.03 (0.03–0.125)	0.25 (0.06–1)	0.25 (0.125–0.5)	0.25 (0.06–1)	0.125 (0.03–1)	0.25 (0.06–2)	0.25 (0.125–1)	1 (0.125–>16)	0.5 (0.06–2)	4 (1–16)	4 (2–16)
F. monophora (14)	0.092 (0.03–0.5)	0.045 (0.03–0.125)	0.125 (0.125–0.25)	0.25 (0.125–0.5)	0.25 (0.03–2)	0.06 (0.03–0.25)	0.25 (0.03–1)	0.125 (0.03–2)	0.5 (0.06–1)	0.25 (0.03–1)	8 (1–8)	4 (2–>16)
F. nubica (2)	0.092 (0.06–0.125)	0.045 (0.03–0.06)	0.125 (0.125–0.125)	0.187 (0.125–0.25)	0.375 (0.25–0.5)	0.375 (0.25–0.5)	0.562 (0.125–1)	0.25 (0.25–0.25)	0.75 (0.5–1)	0.75 (0.5–1)	5 (2–8)	5 (2–8)
F. pugnacius (2)	0.28 (0.06–0.5)	0.140 (0.03–0.25)	0.092 (0.06–0.125)	0.155 (0.06–0.25)	0.925 (0.06–0.125)	0.03 (0.03–0.03)	0.562 (0.125–1)	0.077 (0.03–0.125)	0.312 (0.125–0.5)	0.14 (0.03–0.25)	3 (2–4)	8 (8–8)
P. americana (5)	0.125 (0.03–0.125)	0.03 (0.03–0.125)	0.5 (0.25–4)	1 (1–2)	0.5 (0.25–1)	0.125 (0.06–0.5)	1 (0.25–2)	0.5 (0.5–2)	1 (0.5–1)	0.5 (0.06–1)	8 (8–8)	4 (2–16)
P. macrospora (1)	0.06	0.03	0.25	0.125	0.25	0.06	0.125	0.06	0.25	0.125	4	0.25
C. carrionii (3)	0.5 (0.25–0.5)	0.03 (0.03–0.06)	0.125 (0.06–0.25)	0.25 (0.125–0.25)	0.25 (0.06–0.5)	0.25 (0.125–0.5)	0.25 (0.125–0.25)	0.125 (0.125–0.25)	1 (0.5–1)	0.25 (0.125–1)	8 (2–8)	4 (2–8)
C. bantiana (1)	4	1	1	4	0.25	0.125	1	2	0.5	0.5	2	2
E. spinifera (3)	1 (1–8)	1 (0.25–4)	1 (1–4)	1 (0.5–8)	0.5 (0.25–0.5)	0.25 (0.03–1)	0.5 (0.5–4)	1 (1–4)	1 (0.25–4)	0.25 (0.06–1)	2 (0.5–4)	2 (0.5–8)
E. xenobiotica (2)	2.125 (0.25–4)	0.077 (0.03–0.125)	2.5 (1–4)	0.187 (0.125–0.25)	0.187 (0.125–0.25)	0.25 (0.25–0.25)	0.25 (0.25–0.25)	0.312 (0.125–0.5)	0.75 (0.5–1)	0.5 (0.5–0.5)	12 (8–16)	12 (8–16)
R. aquaspersa (1)	0.5	0.125	2	1	1	1	2	4	2	0.5	1	16
R. tropicalis (2)	0.5 (0.5–0.5)	0.625 (0.25–1)	0.188 (0.125–0.25)	0.75 (0.5–1)	0.125 (0.125–0.125)	0.187 (0.125–0.25)	2 (2–2)	2 (2–4)	0.5 (0.5–0.5)	0.375 (0.25–0.5)	6 (4–8)	9 (2–16)
R. similis (1)	0.5	0.25	2	2	0.5	0.25	2	2	1	0.5	8	8

^aShown are species of the genera Fonsecaea, Cladophialophora, Phialophora, Exophiala, and Rhinocladiella.

^bThe values shown are geometric means with ranges in parentheses. TRB, terbinafine; VRC, voriconazole; POS, posaconazole; KTC, ketoconazole; ITC, itraconazole; AMB, amphotericin B.

To the best of our knowledge, this is the first study to determine the in vitro antifungal susceptibility of Fonsecaea pugnacius, Phialophora americana, Phialophora macrospora, and Rhinocladiella tropicalis, which are unusual species as etiologic agents of CBM. The two strains of F. pugnacius had results similar to those of the other species of Fonsecaea. P. americana and Phialophora macrospora showed similar susceptibility profiles between them and profiles similar to that observed for Phialophora verrucosa in other studies (19, 24). R. tropicalis was 8- to 16-fold more susceptible to caspofungin than the Rhinocladiella species R. aquaspersa and R. similis. For R. aquaspersa, which is another rare etiological agent of CBM, our tested strain presented higher MIC values for itraconazole and posaconazole, while for voriconazole and amphotericin B, the MIC values were similar to those of previous studies (25).

Regarding melanin inhibition, according to the mean and range values in Table 1, MICs of terbinafine, posaconazole, and itraconazole significantly (P < 0.05) decreased when isolates of the genus Fonsecaea were exposed to tricyclazole. Similarly, for Phialophora isolates, the MIC values for posaconazole and itraconazole were also significantly reduced with tricyclazole exposure. For all other genera, itraconazole and terbinafine MICs decreased in the presence of tricyclazole. However, due to the low number of isolates, the results of statistical tests comparing MIC values were not significant. For several species, only a few isolates were studied because these species are rarely found to cause chromoblastomycosis (2), and 84.4% of the isolates used are from Brazil, where F. pedrosoi is the most prevalent etiologic agent of CBM (1, 2).

Considering the genus Fonsecaea, more than 60% of the isolates had relevant reductions in MIC values for itraconazole, posaconazole, and terbinafine, ranging from 2-fold to 64-fold (Fig. 1 and Table 2). The four Fonsecaea strains with high MIC values for itraconazole (>16 mg/liter) were F. pedrosoi and had a decrease of 32- or 64-fold in the presence of tricyclazole (MIC values reduced to 1 or 0.5 mg/liter, respectively). There were no Fonsecaea strains with high posaconazole and terbinafine MICs (Table 2). However, some isolates had relevant MIC value reductions (Fig. 1).

FIG 1.

Percentages of isolates of the genus Fonsecaea that presented relevant reductions (2-fold to 64-fold) in MIC values with DHN-melanin inhibition.

Analyzing the isolates of the other genera (Table 2), inhibition of melanin led to a reduction in MIC values of itraconazole from 4-fold to 16-fold for Phialophora (2/6), Cladophialophora (2/4), Exophiala (3/5), and Rhinocladiella (1/4). For terbinafine, the reduction was also from 4-fold to 16-fold for Cladophialophora (all isolates), Phialophora (2/6), Exophiala (1/4), and Rhinocladiella (2/4).

There are no established breakpoints (BPs) in CLSI for chromoblastomycosis agents. BPs are used to define which isolates are sensitive and which are resistant to a determined antifungal agent and to predict clinical response (26). Therefore, to be able to compare isolates, we used the interpretative values cited by Hellwig et al. (19), who considered the isolates resistant at a MIC of ≥4 mg/liter. It is important to note that this breakpoint was used in our study for comparative purposes only and not to predict clinical responses.

Considering this value, nearly 80% of the isolates were resistant to amphotericin B. With the melanin inhibition, only one F. pedrosoi strain and one P. macrospora strain had an 8-fold reduction in MIC values. For the other antifungals, all the isolates considered resistant presented MICs of <4 mg/liter with melanin inhibition: four isolates of F. pedrosoi and one of Exophiala spinifera for itraconazole, one isolate of P. americana and one of Exophiala xenobiotica for voriconazole, and one isolate of E. xenobiotica for terbinafine. These results raise the possibility of using a drug to inhibit the fungal melanin biosynthesis in the host tissues to improve itraconazole and terbinafine action. Further studies are needed to prove this possibility and demonstrate if these results are reproducible in vivo.

To the best of our knowledge, this is the first study to evaluate the influence of DHN-melanin on the antifungal susceptibility of a considerable number of isolates of CBM agents and two or more species for each of the five main genera. As far as we are aware, to date, the only other study that evaluated this effect of melanin in CBM agents was conducted by Sun et al. in 2011 (9). In this study, they found no difference between the responses to eight antifungals of two pairs of Fonsecaea monophora isolates. We hypothesized that this might be due to the small number of tested isolates, because in our study, with a large number, we found relevant differences between the MIC values. Moreover, in this study, melanin mutant strains from the cultivation of a meristematic mutant strain were analyzed, not wild-type isolates.

The effect of melanin on antifungal profiles may be related to the binding of antifungals to DHN-melanin, as found for DOPA (3,4-dihydroxyphenylalanine)-melanin in other studies, suggesting that fungal melanin binds to antifungals and prevents them from reaching their target sites (11–13, 27). In agreement with this hypothesis, Fernandes et al. (28) found that Alternaria infectoria activates DHN-melanin synthesis in response to certain antifungal drugs (including itraconazole)—possibly as a protective mechanism against these drugs. In another study, coumaric acid analogs showed antimelanogenic activity, potentializing at 16× the amphotericin B fungicidal activity in Cryptococcus neoformans (29). Moreover, in Madurella mycetomatis, when melanin was added in the culture medium, the MICs obtained were 16-fold higher for itraconazole and 32-fold higher for ketoconazole (14), and the inhibition of DOPA-melanin in Paracoccidiodes brasiliensis decreased the MICs of itraconazole (30), indicating protection of DHN- and DOPA-melanin against antifungals. However, in another study, no alteration in the voriconazole MIC of Cryptococcus neoformans was observed in the presence or absence of DOPA-melanin (27).

The binding of DHN-melanin and some antifungals has already been confirmed in the study by van de Sande et al. (14) with Madurella mycetomatis. Van de Sande et al. found that this melanin binds with itraconazole and ketoconazole (with similar molecular structures), but not with the other azoles fluconazole and voriconazole. The interaction of DHN-melanin with these antifungals is probably mediated by the dichlorobenzene ring, the long polyaromatic side chain, or a combination of both. The binding of DHN-melanin with amphotericin B was also verified (14). Even with this binding, and similarly to our study, these authors found for some isolates an increase in the MIC values of amphotericin B when melanin was not present. The results are the opposite of what was expected, and even though these differences in our study were not statistically significant, this fact deserves further studies and clarifications.

This binding of DHN-melanin and some antifungals may partially explain the increased susceptibility for many isolates that were found in our study. Another possible factor is the difference in the amount of melanin produced by each species of each genus. Indeed, when DHN-melanin biosynthesis was inhibited, the differences between species of the same genus were especially pronounced in Cladophialophora, where C. bantiana had higher ketoconazole and voriconazole MIC values than C. carrionii, and Exophiala, where E. spinifera had higher voriconazole MIC values than E. xenobiotica. For Phialophora, P. macrospora had a lower amphotericin B MIC value than the other two Phialophora species tested, presenting a 16-fold decrease in the presence of tricyclazole (Table 2). However, the results for melanin quantification in the same strains, published previously by Heidrich et al. (31), indicated no correlation between the amount of extracted melanin and the changes in the MICs of these strains in the present study (data not shown). Despite these somewhat contradictory facts, it is important to highlight that the present study showed that, for most CBM agents and the most used antifungals, melanin has a relevant action in reducing susceptibility. Considering that, of the 39 isolates of F. pedrosoi studied, 10.2% had a resistance profile to itraconazole (the first-line treatment) (1). It is important to verify which factors contribute to this resistance and how it can be reduced.

In conclusion, this study shows that inhibition of DHN-melanin could increase most CBM agents’ susceptibility in vitro to itraconazole, terbinafine, and posaconazole. However, there were variations in the reduction of MIC values in species of the same genus and between the same species’ isolates. The present study corroborates with other studies that evaluated the antifungal susceptibility of CBM agents, which found less effectiveness of amphotericin B compared to azoles and terbinafine. For the first time, this study also has described the susceptibility profiles of some unusual etiological agents of CBM (F. pugnacius, P. americana, P. macrospora, and R. tropicalis) and has shown that some CBM agents have in vitro resistance to itraconazole. These findings reinforce the importance of conducting antifungal susceptibility assays in clinical practice. Finally, our study shows the possibility of developing drugs that inhibit the melanization of CBM agents to improve treatment efficacy. Further studies are needed to prove that the results found here can be reproduced in vivo.

MATERIALS AND METHODS

Microorganisms.

Seventy-six clinical isolates of CBM agents from the fungal culture collection of the Laboratory of Pathogenic Fungi, Department of Microbiology, ICBS, Universidade Federal do Rio Grande do Sul were evaluated. Previously (32), the strains had been molecularly identified by the sequencing of the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 DNA region (Table 3), and they were representative of 13 species distributed among five genera: Fonsecaea pedrosoi (n = 39), F. monophora (n = 14), F. nubica (n = 2), F. pugnacius (n = 2), Phialophora americana (n = 5), P. macrospora (n = 1), Exophiala spinifera (n = 3), E. xenobiotica (n = 2), Cladophialophora carrionii (n = 3), C. bantiana (n = 1), Rhinocladiella tropicalis (n = 2), R. aquaspersa (n = 1), and R. similis (n = 1). The strain Candida krusei ATCC 6258 was used as a quality control in the antifungal susceptibility tests.

TABLE 3.

GenBank accession numbers of the fungal isolates used in the study

Species	No. of isolates	Genbank accession no.
Fonsecaea pedrosoi	39	MH382046, MH382050, MH382054, MH382051, MH382089, MH382031, MH382081, MH382028, MH382033, MH382030, MH382038, MH382086, MH382079, MH382041, MH382085, MH444807, MH382052, MH382045, MH368488, MH382037, MH382083, MH382029, MH382036, MH382047, MH382080, MH382044, MH382087, MH382082, MH444810, MH382034, MH382049, MH382053, MH382048, MH382042, MH382043, MH382035, MH382039, MH382040, MH382032
Fonsecaea monophora	14	MH382090, MH382065, MH382069, MH368490, MH382061, MH382066, MH382084, MH382068, MH382063, MH382064, MH382070, MH382062, MH382067, MH497043
Fonsecaea nubica	2	MH382071, MH444806
Fonsecaea pugnacius	2	MH444808, MH444809
Phialophora americana	5	MH382077, MH444811, MH382088, MH382078, MH382076
Phialophora macrospora	1	MH382075
Exophiala spinifera	3	MH382060, MH382073, MH382072
Exophiala xenobiotica	2	MH444805, MH382059
Cladophialophora carrionii	3	MH382058, MH382056, MH382057
Cladophialophora bantiana	1	MH382055
Rhinocladiella tropicalis	2	KU854928/KX356663, MH444804
Rhinocladiella aquaspersa	1	MH444803
Rhinocladiella similis	1	KY657562

Inoculum preparation.

Each strain was inoculated on potato dextrose agar (Himedia, Mumbai, India) in the presence and absence of 16 μg/ml tricyclazole (Sigma-Aldrich, MO, USA), previously dissolved with ethyl alcohol (96%). The inhibition of melanin was verified by the color change of the colonies and the culture medium. After 14 days at 30°C, the conidial suspensions were prepared by scraping the fungal colonies’ surface with sterile plastic loops and sterile saline solution (0.85%), and then the suspensions were filtered with Whatman no. 1 filter paper (Sigma-Aldrich, USA) to separate hyphae and conidia. The presence of conidia in the suspensions was verified by optical microscopy, and the conidial counting was performed with a Neubauer chamber. The determination of conidial viability was confirmed by colony counting. The standardized final concentration was (1.5 to 4) × 10⁴ conidia/ml in each well in the susceptibility assay.

Antifungal susceptibility testing.

The microdilution technique was used to determine the MICs according to the M38-A2 protocol of CLSI (26). For the DHN-melanin inhibition assay, the same protocol was followed; however, 16 μg/ml of tricyclazole was added to RPMI 1640 and 3-(N-morpholino) propane sulfonic acid (MOPS) medium.

The evaluated antifungals were terbinafine (TRB), itraconazole (ITC), posaconazole (POS), voriconazole (VRC), ketoconazole (KTC), and amphotericin B (AMB), all acquired from Sigma-Aldrich (MO, USA) and tested in the final concentration range of 0.03 to 16 μg/ml. The MICs were determined after 5 days of incubation at 35°C, considering 100% of visual inhibition compared to the growth in the drug-free wells (growth control). The susceptibility test was performed in triplicate on two different days for each strain.

Statistical analysis.

A paired Wilcoxon statistical test was performed using SPSS version 18, considering α = 0.05.