Schenckii Strains Research Articles

Tenebrio molitor as an Alternative Model to Analyze the Sporothrix Species Virulence.

BackgroundSporotrichosis is an increasing threat for humans, affecting mainly skin and subcutaneous tissues but that can cause disseminated infection in immunocompromised patients. Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main etiological agents of this mycosis, and each species show different virulence levels. The gold standard to assess fungal virulence is the mouse model that is expensive and time-consuming. Thus, invertebrate models have been reported as an alternative for the evaluation of fungal virulence. Here, we assessed whether Tenebrio molitor larvae could be a new alternative to study Sporothrix spp. virulence.MethodsT. molitor larvae were inoculated with different doses of S. schenckii, S. brasiliensis, and S. globosa, and animal mortality, cytotoxicity, and immunological parameters were analyzed, including the ability to stimulate immunological priming.ResultsMortality curves demonstrated that yeast-like cells were the best fungal morphology to kill larvae and showed a similar ranking in virulence than that reported in other animal models, ie, being S. brasiliensis and S. globosa the species with the highest and lowest virulence, respectively. The usefulness of this model was validated with the analysis of several S. schenckii strains with different virulence degrees, and changes in cytotoxicity, humoral and cellular immunological parameters. Low-virulence strains stimulated low levels of cytotoxicity, phenoloxidase activity, and hemocyte countings, and these immunological cells poorly uptake fungi. Moreover, using recombinant Gp70 from S. schenckii immunological priming was stimulated in larvae and this protected against a lethal dose of fungal cells from any of the three species under study.ConclusionThe study demonstrated that T. molitor larvae are an appropriate alternative invertebrate model to analyze the virulence of S. schenckii, S. brasiliensis, and S. globosa. Additionally, hemocyte levels, phenoloxidase activity, cytotoxicity, uptake by hemocytes, and immunological priming are biological parameters that can be used to study the Sporothrix–T. molitor interaction.

Sporotrichosis: Review of Innate and Acquired Immune Mechanisms

Context: Different factors such as the site of infection, the etiological agent, and the immune system can modify the antifungal response of the host. Differences in Sporothrix schenckii strains’ virulence and the host’s immune competency may be involved in the development of sporotrichosis. Nevertheless, the mechanisms related to the disease’s development and progression remain not fully elucidated. Nowadays, no model outweighs the usefulness of mice in biological studies. In these models, transient controlled immunity is created by depressed inflammatory cells during the acute phase of the disease. This is also related to nitric oxide-induced T-cell apoptosis and the lack of a mitogen response. Evidence Acquisition: The recognition of the lipid components of S. schenckii can induce and prolong inflammation. This recognition occurs mainly through the Toll-like receptor (TLR)-4 or the inflammasome. At the same time, TLR-2-mediated identification of fungal exoantigen can serve as an immune evasion process, continuing and worsening the infection. Cell-mediated immune mechanisms have a predominant influence on modulating the clinical expression of sporotrichosis, which is mainly related to Th1/Th17 immunity. Conclusions: In this study, we aimed to explore the innate and acquired immune mechanisms involved in sporotrichosis, as well as the most commonly used animal models for experimental studies.

Fungicidal efficacy of photodynamic therapy using methylene blue against Sporothrix globosa in vitro and in vivo.

Sporothrix schenckii strains are the aetiological agents of sporotrichosis, which is endemic in China. The most common clinical manifestation of sporotrichosis is cutaneous and subcutaneous nodular lesions with lymphangitis involvement. Currently, antifungal therapy is commonly used to treat sporotrichosis, however, drug resistance and complications are the major concerns, especially in patients who have asymptomatic liver injury or existing liver disorders, children, and pregnant women. To assess the in vitro and in vivo antifungal efficacy of photodynamic therapy (PDT) using photosensitizer methylene blue against Sporothrix strains isolated from patients. A light-emitting diode (LED) lamp was used as the light source with a wavelength of 640 ± 10nm. For the in vitro study, the presence of five Sporothrix strains was assayed following LED irradiation with and without photosensitizer (L + M + , L + M-), with photosensitizer alone (L-M + ), or no exposure to LED light or photosensitizer (L-M-). For the in vivo study, mice were infected with the fungi and then treated with sodium chloride (control group), antifungal itraconazole alone (itraconazole group), and a combination of antifungal itraconazole and PDT (itraconazole +PDT group), or PDT alone (PDT group). The results showed that, in vitro, PDT (L + M + ) effectively inhibited fungal growth at an energy density of 40J/cm2. In vivo, itraconazole + PDT effectively inhibited growth of the fungus with the highest level of efficacy. PDT with methylene blue is an effective adjuvant therapy for resistant infections of Sporothrix.

Prevalence and antifungal susceptibility of Sporothrix species in Jiangxi, central China

Sporotrichosis is a subcutaneous mycosis caused by traumatic inoculation of pathogenic Sporothrix species. Until recently, Sporothrix globosa was considered as the unique Chinese species causing this disorder. In the present study, 33 clinical Sporothrix strains isolated from Jiangxi, China, were classified and antifungal susceptibility for each strain was determined. Thirteen S. globosa strains and 20 S. schenckii strains were identified by morphology and by multilocus analysis using rDNA ITS, CAL, and EF1α (i.e., internal transcribed spacer, calmodulin and elongation factor-1α). In vitro antifungal susceptibility testing of yeast phases indicated that itraconazole, terbinafine, and posaconazole were most effective against both species, followed by amphotericin B and voriconazole, while fluconazole, 5-fluorocytosine had low efficacy with high MICs. Co-occurrence of S. schenckii and S. globosa in central China may indicate different routes of transmission in this area.

Melanins Protect Sporothrix brasiliensis and Sporothrix schenckii from the Antifungal Effects of Terbinafine.

Terbinafine is a recommended therapeutic alternative for patients with sporotrichosis who cannot use itraconazole due to drug interactions or side effects. Melanins are involved in resistance to antifungal drugs and Sporothrix species produce three different types of melanin. Therefore, in this study we evaluated whether Sporothrix melanins impact the efficacy of antifungal drugs. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of two Sporothrix brasiliensis and four Sporothrix schenckii strains grown in the presence of the melanin precursors L-DOPA and L-tyrosine were similar to the MIC determined by the CLSI standard protocol for S. schenckii susceptibility to amphotericin B, ketoconazole, itraconazole or terbinafine. When MICs were determined in the presence of inhibitors to three pathways of melanin synthesis, we observed, in four strains, an increase in terbinafine susceptibility in the presence of tricyclazole, a DHN-melanin inhibitor. In addition, one S. schenckii strain grown in the presence of L-DOPA had a higher MFC value when compared to the control. Growth curves in presence of 2×MIC concentrations of terbinafine showed that pyomelanin and, to a lesser extent, eumelanin were able to protect the fungi against the fungicidal effect of this antifungal drug. Our results suggest that melanin protects the major pathogenic species of the Sporothrix complex from the effects of terbinafine and that the development of new antifungal drugs targeting melanin synthesis may improve sporotrichosis therapies.

Cutaneous Sporotrichosis Presenting as Clinical Feature of Facial Cellulitis in an Adult.

Dear Editor: Sporotrichosis is subcutaneous mycosis due to the dimorphic fungus Sporothrix schenckii complex1,2,3. Fixed cutaneous sporotrichosis may present as acneiform, nodular, ulcerated, or verrucous. Less often the patients may present with facial cellulitis. Clinically initial cutaneous lesion is most frequently on an exposed area, commonly the face in children and the arms in adults4. We report the case of cutaneous sporotrichosis of the upper eyelid in adult. The patient was a 57-year-old female who showed with localized erythematous plaques with swelling on the right upper eyelid for 1 month (Fig. 1A). She has done persimmon tree farming without any recongnizable injury and took blepharoplasty 3 months before the lesion appears. Both of them were suspicious as a route of the infection. Cultures from biopsy specimen were performed in Sabouraud's dextrose agar slants. As a result, black, moist and wrinkled colonies of S. schenckii were observed in two slants (Fig. 2A). Septate branched mycelia and clustered conidia were observed in slide culture (Fig. 2B). Histopathologically, pseudoepitheliomatous hyperplasia and chronic granulomatous inflammation were observed on H&E stain. Many round spores were found around dermis in periodic acid Schiff stain (Fig. 2C). The nucleotide sequence of internal transcribed spacer for clinical isolate was identical to that of S. schenckii strain KMU 3360 (GenBank accession number {type:entrez-nucleotide,attrs:{text:AB122043.1,term_id:40714495,term_text:AB122043.1}}AB122043.1). Fig. 1 (A) Localized erythematous plaques with swelling on the right upper eyelid. (B) Skin lesions showing completely cured after six months of treatment. Fig. 2 (A) Dark brown to black, moist and wrinkled colonies on Sabouraud's dextrose agar slants at 25℃ for 2 weeks. (B) Septate branched mycelia and clustered conidia were observed in slide culture (lactophenol cotton blue stain, ×1,000), (C) ... Itraconazole is the drug of choice for treatment of sporotrichosis5. The patient was treated with oral itraconazole 200 mg/day for six months. The skin lesions were completely cured and no recurrence is observed during 1 year of follow up (Fig. 1B). Although cutaneous sporotrichosis is not unusual, the clinical presentation and the location of the lesion might have led dermatologist into misdiagnosis as facial cellulitis. Therefore, we report this case to emphasize the fungal culture of the skin lesions presenting as clinical feature of faical cellulitis.

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

SUMMARY Sporothrix schenckiiwas 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. globosaand S. mexicanawere 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 Sporothrixin further studies.

Sporothrix schenckii(sensu strictS. globosa) mating type 1-2 (MAT1-2) gene

Sporotix schenckii is a pathogenic fungus that causes human and animal sporotrichosis, and based on morphology of the sessile conidia and molecular analysis, it was recently recognized as a species complex comprising at least the following six sibling species: S.albicans, S.brasiliensis, S.globosa, S.luriei, S.mexicana and S.schenckii. However, apart from S.schenckii sensu strict, only S.brasiliensis, S.globosa and S.luriei are associated with human and animal infection. S.globosa has been most commonly isolated in Asia, Europe and the USA; therefore, molecular epidemiological study for S.globosa is important in relation to human sporotrichosis in Japan. To the best of our knowledge, this is the first study to determine the mating type 1-2 (MAT1-2) gene of Sporothrix schenckii with the aim of understanding the taxonomy of the genus Sporothrix. The MAT1-2 gene (1618bp) encodes a protein sequence of 198 amino acids. Reverse transcription polymerase chain reaction analysis also detected MAT1-2 gene mRNA expression in all of the S.schenckii strains examined, indicating that this gene is expressed in S.schenckii cells. Phylogenetic analysis of the MAT1-2 gene fragments of Ophiostoma himal-ulmi, O.novo-ulmi, O.ulmi and S.schenckii indicated that these isolates could be classified into four clusters. MAT1-1 gene-specific polymerase chain reaction was positive in 15 isolates, but negative in four human isolates and one feline isolate.

Different virulence levels of the species of Sporothrix in a murine model

A comparative study on the experimental pathogenicity of five species of Sporothrix of clinical interest, Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix mexicana, and Sporothrix schenckii sensu stricto, was performed using an immunocompetent murine model. Two strains of each species and two levels of inoculum for each strain (2x10(7) and 2x10(4) conidia/animal) were tested by intravenous inoculation of mice (ten per group). Mortality was caused by the low inoculum of one strain of S. brasiliensis only, and the high inocula of S. brasiliensis and S. schenckii strains. Other inocula and other species tested did not kill any of the experimental animals. Tissue burden studies showed fungal spread to kidneys, lungs, spleen, brain, and testicles. S. brasiliensis was recovered extensively from all of the studied organs, and S. schenckii and S. globosa were recovered in lower amounts. Histopathological studies revealed differences in the lesions, which ranged from local inflammation with a low number of fungal cells at the injection site in mice infected with S. globosa, to massive infiltration of fungal cells in organs of those infected with S. brasiliensis. Our findings showed that S. brasiliensis and S. schenckii were the most virulent species, and suggest that lesional mechanisms could be species-specific.

Growth conditions influence melanization of Brazilian clinical Sporothrix schenckii isolates

Sporothrix schenckii is known to produce DHN melanin on both conidial and yeast cells, however little information is available regarding the factors inducing fungal melanization. We evaluated whether culture conditions influenced melanization of 25 Brazilian S. schenckii strains and one control strain (ATCC 10212). Tested conditions included different media, pH, temperature, incubation time, glucose concentrations, and presence or absence of tricyclazole or L-DOPA. Melanization was reduced on Sabouraud compared to defined chemical medium. The majority of strains produced small amounts of melanin at 37 °C and none melanized at basic pH. Increased glucose concentrations did not inhibit melanization, rather increasing glucose enhanced pigment production in 27% of strains. Melanin synthesis was also enhanced by the addition of L-DOPA and its addition to medium with tricyclazole, an inhibitor of melanin synthesis, resulted in fungal melanization, including hyphal melanin production. Our results suggest that different S. schenckii strains have distinct control of melanization and that this fungus can use phenolic compounds to enhance melanization in vitro.

A comparative serological study of theSsCBF antigenic fraction isolated from threeSporothrix schenckiistrains

A previous work showed that an antigenic fraction of Sporothrix schenckii, SsCBF, was specifically recognized by IgG antibodies present in patients' clinical specimens. The objective of this investigation was to compare the reactivity of this antigen recovered from three S. schenckii strains. ROC curve analysis revealed a variation in the sensitivity and specificity of the antigen derived from each of the strains, with a higher AUC for strain 1099-18. Moreover, the presence of the main O-glycosidically-linked epitopes described in the SsCBF fraction was ascertained. A significant reduction in SsCBF reactivity of all the strains was observed after beta-elimination confirming the presence of O-glycan epitopes. The antigen isolated from strain 1099-18 proved to be a more accurate diagnostic tool for serodiagnosis of sporotrichosis.

Mitochondrial DNA Analysis of Sporothrix schenckii in India, Thailand, Brazil, Colombia, Guatemala and Mexico

Up to now, 30 mitochondrial DNA(mtDNA)and 4 rDNA types of Sporothrix schenckii strains have been identified. Here, seventy-six isolates of S. schenckii from Mexico, Guatemala, Brazil, Thailand and India were genotyped and studied epidemiologically by mtDNA restriction fragment length polymorphisms(RFLP)and internal transcribed spacer region(ITS)-RFLP analysis and two new mtDNA types, Type 31 and Type 32, were found. Type 30, previously reported by Mora-Cabrera et al. was confirmed to be Type 3 and designated as blank. Of 48 isolates from Mexico, 41 belonged to Group A wherein Type 2(13 isolates), Type 3(10)and Type 28(7)were dominant. All ten isolates from India and Thailand belonged to Group B. The 52 Group A and 24 Group B isolates corresponded to rDNA Type I and Type IV , respectively, reported by Watanabe et al.(Nippon Ishinkin Gakkai Zasshi 45: 165-175, 2004).

A comparative serological study of the SsCBF antigenic fraction isolated from three Sporothrix schenckii strains

A comparative serological study of the SsCBF antigenic fraction isolated from three Sporothrix schenckii strains

Comparison of virulence of differentSporothrix schenckiiclinical isolates using experimental murine model

The virulence of two strains of Sporothrix schenckii isolated from patients with lymphocutaneous or disseminated sporotrichosis were examined in BALB/c mice (Group 1 and 2, respectively). The mice were inoculated subcutaneously into the left hind footpad with 4 x 10(6) S. schenckii yeast cells in order to evaluate (i) the development of cutaneous lesions, (ii) signs of inactivity, (iii) weight loss, (iv) survival rates, (v) number of viable yeast cells in the lungs and spleen, (vi) splenic index, (vii) extent of organ lesions, and (viii) immunological responses. Comparison of the two groups showed more severe disease in Group 2 mice that developed significant weight and hair loss associated with inactivity and left hind footpad lesions that extended close to the testicular area. The histopathology and large number of viable microorganisms isolated from the spleen confirmed the higher invasive ability of this strain. Moreover, a decrease of an in vitro specific lymphoproliferative response and IFN-gamma production were observed over time in Group 2 mice. As a result, at the end of the experiment, the S. schenckii-antigen (Ss-Ag) response was considered negative with a stimulation index (SI) = 2. In contrast, Group 1 mice presented a positive response to Ss-Ag (SI = 14.1). These results confirm the existence of different virulence profiles in S. schenckii strains. In addition, the use of subcutaneous inoculation as a suitable route for verification of the pathogenicity of this fungus in the murine model was confirmed.

Differential induction of Th1-prone immunity by human dendritic cells activated with Sporothrix schenckii of cutaneous and visceral origins to determine their different virulence

Sporotrichosis is caused by a thermo-dependent dimorphic fungus, Sporothrix schenckii. The major clinical manifestations occur in the skin; however, cases of visceral manifestations have also been increasingly reported with some being observed in immune compromised patients. Different virulence of individual S. schenckii strain as well as immune status of the host could contribute to form such different clinical manifestations. Thus, the purpose of the study was to investigate whether different virulence of individual S. schenckii could be a factor for such clinical difference. We investigated the interactions between human monocyte-derived dendritic cells (MoDCs) and S. schenckii, assessed by (i) morphological features, (ii) surface marker expressions, cytokine productions, (iii) signaling pathways and (iv) allostimulatory activity of the activated MoDCs. Immature MoDCs, obtained from peripheral blood monocytes supplemented with granulocyte macrophage colony-stimulating factor and IL-4, were stimulated with S. schenckii strains of both yeasts and conidia forms of different origins (cutaneous isolates: KMU4649, IFM5906 and IFM46010; visceral isolates: KMU4648, IFM41598 and ATCC26331) to be used for various assays. Through the analysis, we found that the cutaneous S. shenckii of cutaneous origins were more potent to activate MoDCs to induce strong T(h)1 response, as evidenced by abundant IFN-gamma production, while the S. shenckii of visceral origins induced only minimal dendritic cell activation and T(h)1 induction. The p38 mitogen-activated protein kinase and c-Jun N-terminal kinase signaling pathways appeared to be associated with the differential activation of the MoDCs by S. schenckii of cutaneous and the visceral origins. Overall, we concluded that the differential activation of MoDCs by S. schenckii of cutaneous and visceral origins to induce T(h)1 response, other than immune status or the host, may be a factor for their different clinical manifestations.

Genotyping of Sporothrix schenckii by analysis of ribosomal DNA regions

An attempt was made to identify the genotypes of 31 Sporothrix schenckii strains and the relationship between the genotypes, the geographic distributions and clinical manifestations using restriction fragment length polymorphic analysis. The total DNA was extracted by cetyltrimethyl ammonium bromide. The polymorphisms were detected by hybridisation of ApaI-digested Sporothrix genomic DNA with a probe amplified from the small subunit ribosomal DNA and adjacent internal transcribed spacer regions. The band patterns manifested by Southern blotting were explored to investigate the genotypes of the 31 strains of S. schenckii collected from five different areas in China. Of the data from the 31 strains of S. schenckii studied, 15 individual patterns (DNA type A-O) were recognised. Type A-C accounted for 51.61% of all the strains. The DNA typing of S. schenckii by Southern blotting was highly sensitive and highly distinguishable. We also found a significant correlation between DNA patterns and different geographic areas and clinical manifestations.

Glutathione-dependent extracellular ferric reductase activities in dimorphic zoopathogenic fungi

In this study, extracellular glutathione-dependent ferric reductase (GSH-FeR) activities in different dimorphic zoopathogenic fungal species were characterized. Supernatants from Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis and Sporothrix schenckii strains grown in their yeast form were able to reduce iron enzymically with glutathione as a cofactor. Some variations in the level of reduction were noted amongst the strains. This activity was stable in acidic, neutral and slightly alkaline environments and was inhibited when trivalent aluminium and gallium ions were present. Using zymography, single bands of GSH-FeRs with apparent molecular masses varying from 430 to 460 kDa were identified in all strains. The same molecular mass range was determined by size exclusion chromatography. These data demonstrate that dimorphic zoopathogenic fungi produce and secrete a family of similar GSH-FeRs that may be involved in the acquisition and utilization of iron. Siderophore production by these and other fungi has sometimes been considered to provide a full explanation of iron acquisition in these organisms. Our work reveals an additional common mechanism that may be biologically and pathogenically important. Furthermore, while some characteristics of these enzymes such as extracellular location, cofactor utilization and large size are not individually unique, when considered together and shared across a range of fungi, they represent an important novel physiological feature.

Evaluation of the in vitro and in vivo dimorphism of Sporothrix schenckii, Blastomyces dermatitidis, and Paracoccidioides brasiliensis isolates after preservation in mineral oil.

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 degrees C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 degrees C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 degrees C and their micromorphology showed thin filaments with single hyaline conidia. At 36 degrees C the colonies did not differ from those grown at 25 degrees C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 degrees C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.

Variabilidad genética en cepas de Sporothrix schenckii aisladas en Abancay, Perú

Objective: To identify S. schenckii genotypes circulating in 2 districts from the Abancay province in Peru. Material and Methods: 17 strains from patients with lympho-cutaneous and fixed cutaneous lesions using RAPD-PCR techniques (Randomized Amplified Polymorphic DNA-Polymerase Chain Reaction) with A GTG5 primer (GTG GTG GTG GTG GTG). Results: We identified 6 genotypes, being genotype I the most frequent in the studied geographical areas. There was not any association with clinical and geographical characteristics. Conclusions: Our results prove that there is genetic biodiversity between circulating S. schenckii strains in the two districts assessed.

Variants of Sporothrix schenckii with attenuated virulence for mice.

Strains of Sporothrix schenckii preserved under mineral oil were examined for virulence in BALB/c mice. The mice were inoculated with S. schenckii conidia and development of cutaneous lesions, signs of inactivity, weight loss, survival rates, number of viable yeast cells in lung and spleen, splenomegaly and organ lesions were evaluated. After intravenous injection of 7.5 x 10(6) conidia, two of five S. schenckii strains were unable to induce systemic disease and to kill the mice, only producing cord-like lesions on the tail that regressed with mouse maturation. Very small numbers of viable cells isolated from the spleen confirmed the lower invasive ability of these strains when compared with other strains studied here. These results suggest a relationship between the attenuation of virulence and the storage method under mineral oil after long periods of time.