Sporothrix schenckii COMPLEX:SUSCEPTIBILITIES TO COMBINED ANTIFUNGAL AGENTS AND CHARACTERIZATION OF ENZYMATIC PROFILES.

Sporothrix schenckii was reclassified as a complex encompassing six cryptic species, which calls for the reassessment of clinical and epidemiological data of these new species. We evaluated the susceptibility of Sporothrix albicans(n = 1) , S. brasiliensis(n = 6) , S. globosa(n = 1), S. mexicana(n = 1) and S. schenckii(n = 36) to terbinafine (TRB) alone and in combination with itraconazole (ITZ), ketoconazole (KTZ), and voriconazole (VRZ) by a checkerboard microdilution method and determined the enzymatic profile of these species with the API-ZYM kit. Most interactions were additive (27.5%, 32.5% and 5%) or indifferent (70%, 50% and 52.5%) for TRB+KTZ, TRB+ITZ and TRB+VRZ, respectively. Antagonisms were observed in 42.5% of isolates for the TRB+VRZ combination. Based on enzymatic profiling, the Sporothrix schenckii strains were categorized into 14 biotypes. Leucine arylamidase (LA) activity was observed only for S. albicans and S. mexicana. The species S. globosa and S. Mexicana were the only species without β-glucosidase (GS) activity. Our results may contribute to a better understanding of virulence and resistance among species of the genus Sporothrix in further studies.

INTRODUCTION

Sporotrichosis is caused by the dimorphic fungus Sporothrix schenckii, which is one of the predominant microorganisms responsible for cutaneous and lymphocutaneous mycosis, affecting both humans and animals. Sporotrichosis has a worldwide distribution, especially in tropical and subtropical regions of Latin America where endemic areas have been recognized2 , 5. Furthermore, an increase in the frequency of sporotrichosis as an opportunistic disease has been observed in the past few years, and this increase is associated with significant morbidity and mortality rates in immunocompromised patients5 , 10.

For many years, local hyperthermia and oral potassium iodide solution have been therapeutic options for the treatment of sporotrichosis21 , 22. Additionally, with over 50 years of clinical use, amphotericin B (AMB) is still considered the "gold standard" for the treatment of serious fungal infections. However, the use of AMB is associated with significant adverse effects related to toxicity13. According to the Clinical Practice Guidelines for the Management of Sporotrichosis 13, monotherapy with itraconazole is the first choice for subcutaneous sporotrichosis treatment. Although treatment with itraconazole has been shown to elicit an excellent therapeutic response in humans4, reports of sporotrichosis refractory to oral itraconazole treatment have been described, particularly in animals11.

In vitro susceptibility studies for terbinafine have been encouraging2 , 18, and studies using terbinafine with human patients have shown good efficacy9. Additionally, cases of refractory and disseminated sporotrichosis have been treated with a combination of antifungal drugs, presenting satisfactory results6 , 11.

Recently, it was proposed that Sporothrix schenckii is a species complex encompassing six cryptic species15, which calls for the reassessment of clinical and epidemiological data, in particular with regard to antifungal susceptibility and phenotypic characteristics which may be related to virulence3 , 16 , 17. In this context, the aim of this study was to evaluate the in vitro susceptibility of Sporothrix albicans, S. brasiliensis, S. globosa, S. mexicana and S. schenckii to terbinafine in combination with itraconazole, ketoconazole or voriconazole, as well as the enzymatic profile of these species.

MATERIAL AND METHODS

Microorganisms: Thirty-eight isolates obtained from human (n = 30) and feline (n = 8) cases of sporotrichosis diagnosed in the hinterland of Rio Grande do Sul State (Brazil). These strains were maintained in the Department of Microbiology of the Federal University of Santa Maria and were previously identified by phenotypic and molecular tests17 as S. schenckii (n = 36), S. albicans (n = 1), and S. brasiliensis (n = 6). The species S. globosa (CBS 132922) and S. mexicana (CBS 120341) were also included.

Antifungal drugs, in vitro susceptibility and drug interactions test: The antifungal agents itraconazole (ITZ), ketoconazole (KTZ) (ITZ and KTZ: Janssen Pharmaceutica, Beerse, Belgium), terbinafine (TRB) (Novartis, Basel, Switzerland) and voriconazole (VRZ) (Pfizer, Rome, Italy) were obtained commercially. Susceptibility tests were performed according to the CLSI protocol M38-A2 microdilution technique7, with filamentous phase of Sporothrix strains. Aspergillus flavus (ATCC 204304) was included as a control strain. Drug interactions were evaluated by the checkerboard method20, and the fractional inhibitory concentration (FIC) was calculated for each agent by dividing the minimal inhibitory concentration (MIC) of each drug in combination by the MIC of the drug alone. When computing the FIC index, off-scale MICs were converted to the next highest concentration.

FIC values were then summed up to determine the fractional inhibitory concentration index (FICI) resulting from the combination. Synergism was defined as FICI ≤ 0.5 and additionally as 0.5 < FICI ≤ 1.0. Indifference was defined as 1.0 < FICI ≤ 4, whereas antagonism was defined as FICI > 412.

Enzymatic profile: Enzymatic activity was determined using the APIZYM(r) commercial kit system (BioMérieux, Marcy-l'Étoile, France) in accordance with the manufacturer's instructions. The kit consists of 19 enzymatic substrates: alkaline phosphatase (KP), esterase (ES), esterase lipase (EL), lipase (LP), leucine arylamidase (LA), valine arylamidase (VA), cystine arylamidase (CA), trypsin (TR), α-chymotrypsin (CH), acid phosphatase (AP), naphthol-AS-BI-phosphohydrolase (NP), α-galactosidase (GL), β-galactosidase (GA), β-glucuronidase (GU), α-glucosidase (GC), β-glucosidase (GS), N-acetyl-β-glucosaminidase (NG), α-mannosidase (MN) and α-fucosidase (FU).

To prepare a conidia suspension with a turbidity of 5-6 McFarland in API suspension medium (2 mL), 65 μL of liquid phase from incubated cultures was added to each cupule of the APY-ZYM(r) tray and maintained at 37 ºC for four h. Tests were carried out in triplicate, and subsequently the enzymatic activities of all strains were analyzed, and results of the reactions were recorded. Interpretation of the results was based on the production and intensity of color development: 0 corresponded to a negative reaction, 5 to a reaction of maximum intensity and values 1, 2, 3, and 4 were intermediate reactions with varying levels of intensity; however, according to the kit, reactions are only considered positiveintermediate when intensities are 3 and 4.

RESULTS

The results of the in vitro susceptibility test, FIC index, and the resulting drug interactions against the 40 Sporothrix sp. isolates are described in Table 1. MICs ranged from 0.03 to 0.5 μg/mL for TRB, 0.06 to 4.0 μg/mL for KTZ, 0.03 to >128 μg/mL for ITZ and 1.0 to 16 μg/mL for VRZ. The MIC of the control strain (A. flavus ATCC 204304) was within the range provided by CLSI7.

Table 1

In vitro susceptibility and interactions between terbinafine (TRB), itraconazole (ITZ), ketoconazole (KTZ), and voriconazole (VRZ) against clinical isolates of Sporothrix schenckiicomplex

Isolate	MIC (µg/mL)					MIC combination				MIC combination				MIC combination
	TRB	KTZ	ITZ	VRZ		TRB/KTZ	FICI	Int1		TRB/ITZ	FICI	Int1		TRB/VRZ	FICI	Int1
SB01	0.25	0.5	0.25	8		0.125/0.25	1.00	Ad		0.125/0.25	1.50	I		0.5/0.5	2.06	I
SB251	0.5	0.5	0.5	2		0.06/0.06	0.25	S		0.125/0.5	1.25	I		1/0.06	2.03	I
SB245	0.5	0.25	0.25	1		0.06/0.06	0.37	S		0.06/0.06	0.37	S		0.5/0.125	0.75	Ad
FMR8314	0.06	0.125	0.125	4		0.06/0.06	1.54	I		0.06/0.125	2.04	I		0.015/2	0.76	Ad
FMR8319	0.06	0.06	0.25	1		0.06/0.125	3.12	I		0.03/0.5	2.04	I		0.06/1	2.04	I
FMR8326	0.125	0.25	0.125	4		0.06/0.125	1.00	Ad		0.06/0.06	0.96	Ad		0.06/0.25	0.56	Ad
SS02	0.25	0.25	0.125	8		0.125/0.125	1.00	Ad		0.03/0.125	1.12	I		0.25/0.25	1.03	I
SS03	0.125	0.25	0.125	2		0.125/0.25	2.00	I		0.016/0.125	1.12	I		0.25/0.25	3.0	I
SS04	0.06	0.125	0.125	4		0.125/0.03	2.33	I		0.125/0.25	2.0	I		0.25/0.25	4.23	A
SS05	0.25	0.25	0.125	8		0.125/0.25	1.50	I		0.125/0.25	2.0	I		0.5/0.5	2.06	I
SS06	0.03	0.5	0.5	4		0.016/0.5	1.52	I		0.016/0.125	0.77	Ad		0.25/0.25	8.36	A
SS07	0.125	0.25	0.25	4		0.125/0.25	2.00	I		0.016/0.125	0.62	Ad		0.5/0.5	4.12	A
SS08	0.25	0.5	0.25	8		0.125/0.25	1.00	Ad		0.125/0.25	2.0	I		0.25/0.125	1.02	I
SS09	0.06	0.25	0.125	8		0.125/0.25	3.08	I		0.125/0.125	3.08	I		0.25/0.125	4.18	A
SS10	0.125	0.125	0.03	4		0.125/0.25	2.00	I		0.125/0.25	2.0	I		0.25/0.25	2.06	I
SS11	0.03	0.125	0.125	8		0.016/0.5	4.52	A		0.016/0.125	1.52	I		0.25/0.25	8.36	A
SS12	0.25	0.5	0.5	8		0.125/0.125	0.75	Ad		0.016/0.125	0.31	S		0.25/0.25	1.03	I
SS13	0.25	0.06	0.03	1		0.25/0.016	1.26	I		0.5/0.016	2.52	I		1.0/0.016	4.01	A
SS14	0.06	0.25	0.25	8		0.125/0.25	3.00	I		0.016/0.125	0.76	Ad		1.0/0.25	16.7	A
SS15	0.125	0.125	0.125	4		0.125/0.25	3.00	I		0.25/1.0	10.0	A		1.0/0.5	8.13	A
SS16	0.25	0.5	0.25	8		0.125/0.125	0.75	Ad		0.016/0.125	0.56	Ad		0.25/0.125	1.02	I
SS17	0.125	0.125	0.125	16		0.125/0.125	2.00	I		0.016/0.125	1.13	I		0.25/0.125	2.00	I
SS18	0.03	0.5	0.25	4		0.016/1.0	2.52	I		0.016/0.125	1.02	I		0.25/0.125	8.36	A
SS19	0.125	2	0.5	16		0.016/1.0	0.62	Ad		0.06/0.125	0.75	Ad		0.25/0.25	2.01	I
SS20	0.25	0.25	0.125	8		0.125/0.25	1.50	I		0.125/0.25	2.5	I		1.0/0.5	4.06	A
SS21	0.125	0.5	0.25	16		0.125/0.25	1.50	I		0.031/0.25	1.25	I		0.25/0.125	2.0	I
SS22	0.06	0.25	0.125	4		0.125/0.25	3.08	I		0.016/0.5	4.26	A		2.0/0.016	33.3	A
SS23	0.125	0.25	0.5	8		0.125/0.25	2.00	I		0.016/0.25	0.62	Ad		0.25/0.25	2.03	I
SS24	0.25	0.125	0.25	4		0.5/0.016	2.12	I		0.125/0.016	0.56	Ad		0.25/0.125	1.03	I
SS25	0.06	0.5	0.25	4		0.125/0.25	2.58	I		0.125/0.25	3.08	I		0.25/0.125	4.20	A
SS26	0.06	1	0.25	4		0.016/0.5	0.76	Ad		0.016/0.125	0.76	Ad		0.25/0.125	4.20	A
SS27	0.125	0.5	0.25	8		0.125/0.25	1.50	I		0.125/0.25	2.00	I		0.25/0.125	2.02	I
SS28	0.125	0.25	0.25	4		0.125/0.25	2.00	I		0.016/0.125	0.62	Ad		0.5/1.0	4.25	A
SS29	0.06	0.5	0.125	4		0.016/1.0	2.26	I		0.125/0.25	4.08	A		0.25/0.25	4.23	A
SS33	0.25	2	1	16		0.125/0.25	0.63	Ad		0.125/0.25	0.75	Ad		0.25/0.125	1.01	I
SS34	0.125	0.5	1	4		0.125/0.25	1.50	I		0.016/1.0	1.12	I		0.5/0.03	4.01	A
SS35	0.125	0.5	0.5	8		0.125/0.25	1.50	I		0.125/0.25	1.50	I		0.25/0.125	2.02	I
SS36	0.125	0.5	0.5	8		0.1250.25	1.50	I		0.016/0.25	0.62	Ad		0.25/0.125	2.02	I
SS37	0.125	0.25	0.5	16		0.125/0.25	2.00	I		0.016/0.25	1.13	I		0.25/0.125	2.02	I
SS38	0.125	2	1	8		0.125/0.25	1.13	I		0.5/0.5	1.0	Ad		0.25/0.125	2.02	I
SS39	0.25	0.5	0.5	8		0.125/0.25	1.00	Ad		0.125/0.25	1.00	Ad		1/0.016	4.01	A
SS40	0.125	0.25	0.03	8		0.125/0.25	2.00	I		1.0/0.016	8.52	A		0.25/0.125	2.02	I
SA32	0.25	4	>128	8		0.125/0.25	0.56	Ad		0.5/2.0	2.0	I		0.125/1.0	0.63	Ad
SG00	0.25	2	8	16		0.25 / 0.5	1.25	I		0.125/1.0	0.625	A		0.25/8.0	1.5	I
SM00	0.25	4	32	16		0.03/4	1.0	Ad		0.125/4.0	0.625	A		0.125/8.0	1.0	Ad

Interaction; A: antagonism; Ad: additive; S: synergism; I: indifference. SA (S. albicans), SB (S. brasiliensis), SG (S. globosa), SM (S. mexicana), SS (S. schenckii).

When TRB was combined with KTZ, ITZ or VRZ, synergism was observed in one isolate (2.2%) for TRB+ITZ and two for TRB+KTZ (2.25%). Additive interactions were observed for TRB+KTZ (26.7% of isolates), TRB+ITZ (33.3% of isolates) and TRB+VRZ (11.1% of isolates). Most interactions were indifferent, as was the case for TRB+KTZ (66.7% of isolates), TRB+ITZ (51.1% of isolates) and TRB+VRZ (51.1% of isolates). Antagonism was observed only with one isolate when TRB was combined with KTZ, with four (8.89%) isolates for the TRB+ITZ combination and with 17 (37.78%) isolates for the TRB+VRZ combination.

The enzymatic profiles of the Sporothrix spp., analyzed using the API ZYM(r), system are listed in Table 2. Based on the 11 substrates, the 36 isolates of Sporothrix schenckii tested could be categorized into 14 separate biotypes, from which 100% showed leucine arylamidase (LA) activity negative for S. schenckii isolates and 20% showed positive reactions for esterase (ES), esterase lipase (EL), alkaline phosphatase (KP), and naphtol-AS-BI-phosphohydrolase (NP). The non-schenckii species presented unique and very unusual features, as shown in Table 2. S. albicans and S. mexicana were the only strains with a positive reaction for leucine arylamidase (LA). The enzymatic activities of acid phosphatase (AP) and naphtol-AS-BI-phosphohydrolase (NP) were observed for all isolates studied and β-glucosidase (GS activity) was absent only for S. globosa and S. mexicana (Table 2).

Table 2

Enzymatic profile of clinical isolates of Sporothrix schenckii (SS), Sporothrix brasiliensis (SB), Sporothrix albicans (SL), Sporothrix globosa (SG) and Sporothrix mexicana (SM)

Species	Strains showing activity for:											Number of isolates (%)
	KP	ES	EL	LP	LA	AP	NP	GC	GS	NG	MN
SB	+	+	+	-	-	+	+	-	+	+	-	1 (2.2)
SB	+	+	-	-	-	+	+	+	+	-	-	1 (2.2)
SB	+	+	+	-	-	+	+	+	+	+	+	1 (2.2)
SB	+	-	-	-	-	+	+	-	+	-	-	2 (4.4)
SB	+	-	-	-	-	+	+	-	+	-	+	1 (2.2)
SS	+	+	+	-	-	+	+	-	+	+	-	2 (4.4)
SS	-	+	+	-	-	+	+	+	+	-	-	3 (6.6)
SS	-	+	+	-	-	+	+	-	+	-	-	14 (31.1)
SS	+	+	+	-	-	+	+	-	+	+	+	1 (2.2)
SS	+	+	+	-	-	+	+	+	+	+	+	1 (2.2)
SS	+	+	-	-	-	+	+	-	+	-	-	1 (2.2)
SS	-	-	+	-	-	+	+	-	+	-	-	2 (4.4)
SS	+	+	+	-	-	+	+	-	+	-	-	3 (6.6)
SS	+	+	+	-	-	+	+	+	+	-	-	1 (2.2)
SS	-	-	-	-	-	+	+	-	+	-	-	1 (2.2)
SS	-	+	-	-	-	+	+	-	+	-	-	2 (4.4)
SS	-	+	+	-	-	+	+	-	+	+	-	3 (6.6)
SS	-	-	-	-	-	+	+	-	+	+	-	1 (2.2)
SS	-	+	-	-	-	+	+	+	+	-	-	1 (2.2)
SA	-	+	+	-	+	+	+	-	+	-	-	1 (2.2
SG	-	+	+	-	-	+	+	-	-	-	-	1 (2.2
SM	+	+	+	-	+	+	+	-	-	-	-	1 (2.2)
TOTAL	16	38	35	0	3	45	45	8	43	10	4

a Substrates for enzymes: alkaline phosphatase (KP); esterase (ES); esterase lipase (EL); lipase (LP); leucine arylamidase (LA); acid phosphatase (AP); naphthol-AS-BI-phosphohydrolase (NP); a-glucosidase (GC); b-glucosidase (GS); N-acetyl-b-glucosaminidase (NG); a-mannosidase (MN).

DISCUSSION

To overcome antifungal resistance and toxicity from monotherapy with AMB, antifungal combinations represent an alternative approach to enhance the efficacy of individual drugs and lower dosages; these combined drug treatments may result in reduced toxicity and therapeutic success in cases that do not respond to treatment with single antifungal agents12. High success rates for the treatment of sporotrichosis with ITZ alone4 have not encouraged the use of antifungal combinations against Sporothrix spp. in the past few years. However, the recent description of Sporothrix complex species which are refractory to ITZ treatment has stimulated research for combined antifungal therapeutic alternatives to treat sporotrichosis6 , 8 , 11 , 24.

The susceptibility tests for antifungal agents have not established break points for S. schenckii complex, albeit document M38-A2 (CLSI, 2008) suggests that for analytical purposes, a MIC ≥ 4.0 μg/mL for itraconazole may be considered resistant to some filamentous fungi17. Here all the S. schenckii and S. brasiliensis isolates were susceptible to ITZ (MIC range 0.03-0.5 μg/mL). On the other hand, S. albicans, S. globosa and S. mexicana have shown high MICs to itraconazole, which have been demonstrated by some authors8 , 16 , 17 , 19.

Taking this fact into account, we evaluated the activity of TRB, the second choice for subcutaneous sporotrichosis treatment13, in association with ITZ, KTZ and VRZ. The TRB+KTZ and TRB+ITZ combinations produced indifferent and additive interactions for 97.5% and 87.5% of isolates, respectively. These data are in accordance with ZHANG et al. 24, who found predominantly additive and indifferent interactions with TRB+ITZ, highlighting the use of TRB alone or in combination with other antifungal agents in sporotrichosis cases that are not responsive to ITZ treatment.

We have also observed that VRZ demonstrated the highest MICs against Sporothrix spp. when compared to KTZ and ITZ. This reduced susceptibility has been previously described1 , 16 , 17, and the TRB+VRZ combination employed here predominantly showed indifferent (55%) and antagonistic (42%) interactions. Thus, the use of VRZ alone or in combination with TRB in sporotrichosis treatment does not appear to be advantageous.

Importantly, there are no protocols describing the use (or not) of the classification of additive in drug synergism testing for fungi, which is often subject of discussion and misunderstanding. Mathematically, additive is logical for the classical Loewe additivity model, as discussed by JOHNSON et al.12, and it was used in this study didactically. The terms additive and indifferent are both related to the neutral nature of drugs interaction, i.e. neither synergistic nor antagonistic interactions. However, biological differences that can be observed between these interpretations are multifactorial.

Several studies have demonstrated the ability of fungi to produce extracellular enzymes such as proteases, lipases, keratinases and others, which represents a useful tool to identify microorganisms and thus providing an understanding of the relationships between enzyme production and pathogenicity. The use of the API ZYM(r) system to study the enzymatic profiles of different fungal species was previously reported14 , 23. The Api Zym(r)detection system was considered as a suitable option for the characterization of S. schenckii species complex as a result of presenting well-defined profiles.

Our results have demonstrated high intraspecific variability for the enzymatic activity of S. schenckii strains and revealed the presence of 14 different biotypes. The main biotype (35% of S. schenckii) demonstrated ES, EL, AP, NP activities, and these enzymatic activities were also observed for all the species studied. We believe these enzymes could be associated with virulence factors required for the development of sporotrichosis and so, deserve further investigation.

Differential identification of the Sporothrix complex by phenotype is based on the size of radial colony growth, the morphology of conidia, the assimilation of sucrose and raffinose, and the ability to grow at 37 °C15 , 16. Our results bring new contributions to the phenotypic identification of species enrolled in the S. schenckii complex based on the pattern of enzymatic activities. Thus, as expected, we must emphasize the following profiles: i) S. albicans and S. mexicanawere the only ones to show LA activity and ii) S. globosaand S. mexicana were the only species showing absent GS activity. Further studies with a larger number of non-schenckii species are required in order to detect intraspecific variability. Even so, our results will be helpful when associated with the phenotypic tests recommended by MARIMON et al. 15, rendering the phenotypic tests more effective in order to identify cryptic species of the Sporothrix complex.

In conclusion, the in vitro combinations of TRB with KTZ and TRB with ITZ were shown to be advantageous compared to the use of these drugs individually, but antagonism was common with the TRB+VRZ combination. The enzymatic screening data represent an addition to the available methods for phenotypic characterization of new Sporothrix schenckiicomplex species, as well as an interesting area for further research to identify and explore the virulence of the Sporothrix complex.