Adhesion and biofilm formation by two clinical isolates of Trichosporon Cutaneum in various environmental conditions.

Trichosporon spp. is an emerging opportunistic pathogen and a common cause of both superficial and invasive infections. Although Trichosporon asahii is the most frequently isolated species, Trichosporon cutaneum is also widely observed, as it is the predominant agent in cases of white Piedra and onychomycosis. Trichosporon spp. is a known to produce biofilms, which serve as one of its virulence mechanisms, however, there is limited data available on biofilms formed by T. cutaneum. Thus, the aim of this study was to assess the adhesion and biofilm formation of two clinical isolates of T. cutaneum under various environmental conditions (including temperature, nutrient availability, and carbon source), as well as their tolerance to fluconazole. Adhesion was tested on common abiotic substrates (such as silicone, glass, and stainless steel), revealing that T. cutaneum readily adhered to all surfaces tested. CV staining was applied for the evaluation of the environment influence on biofilm efficiency and it was proved that the nutrient availability has a major impact. Additionaly, fluorescent staining was employed to visualize the morphology of T. cutaneum biofilm and its survival in the presence of fluconazole. Hyphae production was shown to play a role in elevated biofilm production in minimal medium and increased tolerance to fluconazole.

MALDI-TOF MS characterisation, genetic diversity and antifungal susceptibility of Trichosporon species from Iranian clinical samples.

BACKGROUND: Trichosporonosis is an emerging fungal infection caused by Trichosporon species, a genus of yeast-like fungi, which are frequently encountered in human infections ranging from mild cutaneous lesions to fungemia in immunocompromised patients. The incidence of trichosporonosis has increased in recent years, owing to higher numbers of individuals at risk for this infection. Although amphotericin B, posaconazole and isavuconazole are generally effective against Trichosporon species, some isolates may have variable susceptibility to these antifungals. OBJECTIVES: Herein, we evaluated the species distribution, genetic diversity and antifungal susceptibility profiles of Trichosporon isolates in Iran. METHODS: The yeasts were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Phylogenetic analysis was performed based on amplified fragment length polymorphism (AFLP). The in vitro susceptibilities of eight antifungal agents were analysed using the Clinical and Laboratory Standards Institute broth microdilution methods. RESULTS: The isolates belonged to the species T asahii (n = 20), T japonicum (n = 4) and T faecale (n = 3). A dendrogram of the AFLP analysis demonstrated that T asahii and non-asahii Trichosporon strains (T japonicum and T faecale) are phylogenetically distinct. While voriconazole was the most active agent (GM MIC = 0.075 µg/ml), high fluconazole MICs (8 µg/ml) were observed for a quarter of Trichosporon isolates. The GM MIC value of amphotericin B for T asahii and non-asahii Trichosporon species was 0.9 µg/ml. CONCLUSIONS: The distribution and antifungal susceptibility patterns of the identified Trichosporon species could inform therapeutic choices for treating these emerging life-threatening fungi.

Vancomycin enhances growth and virulence of Trichosporon spp. planktonic cells and biofilms.

Invasive fungal infections (IFIs) are important worldwide health problem, affecting the growing population of immunocompromised patients. Although the majority of IFIs are caused by Candida spp., other fungal species have been increasingly recognized as relevant opportunistic pathogens. Trichosporon spp. are members of skin and gut human microbiota. Since 1980's, invasive trichosporonosis has been considered a significant cause of fungemia in patients with hematological malignancies. As prolonged antibiotic therapy is an important risk factor for IFIs, the present study investigated if vancomycin enhances growth and virulence of Trichosporon. Vancomycin was tested against T. inkin (n = 6) and T. asahii (n = 6) clinical strains. Planktonic cells were evaluated for their metabolic activity and virulence against Caenorhabditis elegans. Biofilms were evaluated for metabolic activity, biomass production, amphotericin B tolerance, induction of persister cells, and ultrastructure. Vancomycin stimulated planktonic growth of Trichosporon spp., increased tolerance to AMB, and potentiates virulence against C. elegans. Vancomycin stimulated growth (metabolic activity and biomass) of Trichosporon spp. biofilms during all stages of development. The antibiotic increased the number of persister cells inside Trichosporon biofilms. These cells showed higher tolerance to AMB than persister cells from VAN-free biofilms. Microscopic analysis showed that VAN increased production of extracellular matrix and cells in T. inkin and T. asahii biofilms. These results suggest that antibiotic exposure may have a direct impact on the pathophysiology of opportunistic trichosporonosis in patients at risk. LAY ABSTRACT: This study showed that the vancomycin stimulated Trichosporon growth, induced morphological and physiological changes on their biofilms, and also enhanced their in vivo virulence. Although speculative, the stimulatory effect of vancomycin on fungal cells should be considered in a clinical scenario.

Study on the bacteriostatic action of Chinese herbal medicine on avian Trichosporon.

In this study, a strain of Trichosporon was isolated from white pseudomembranes and ulcers formed on mucous membranes of pigeon bursas and was identified through gene sequencing. Bacteriostatic actions of Acorus gramineus, Sophora flavescens, Polygonum hydropiper, and Chinese herbal mixture on this species were explored in vitro, and the minimum inhibitory concentration of herbal medicines against Trichosporon was determined through microdilution method. Therapeutic effects of herbal medicines on chickens infected by Trichosporon were studied, whose results showed that minimum inhibitory concentration of A. gramineus was 32 µg/µL, that of S. flavescens was 2 µg/µL, that of P. hydropiper was 120 µg/µL, and that of Chinese herbal mixture was 36 µg/µL. Antibacterial effects of S. flavescens were the best. In accordance with animal experiments, therapeutic effects of Chinese herbal medicines on infected chickens were better than those of fluconazole. The mortality rate of the Chinese herbal medicine treatment group was 33.33%, that of the fluconazole treatment group was 46.67%, and that of the Chinese medicine protection group was 23.33%. The longer the time of Chinese medicine treatments was, the better the treatment effects would be. Glutamic oxaloacetylase values of the serum and liver in the Chinese herbal medicine treatment group were both significantly lower than those of the nontreatment group. From the results, it can be seen that A. gramineus, S. flavescens, P. hydropiper, and Chinese herbal mixture have certain inhibitory effects on Trichosporon spp. Chinese herbal medicine protections in advance could reduce Trichosporon infections.

A novel silkworm infection model with fluorescence imaging using transgenic Trichosporon asahii expressing eGFP.

Trichosporon asahii is a pathogenic fungus that causes deep mycosis in patients with neutropenia. Establishing an experimental animal model for quantitatively evaluating pathogenicity and developing a genetic recombination technology will help to elucidate the infection mechanism of T. asahii and promote the development of antifungal drugs. Here we established a silkworm infection model with a transgenic T. asahii strain expressing eGFP. Injecting T. asahii into silkworms eventually killed the silkworms. Moreover, the administration of antifungal agents, such as amphotericin B, fluconazole, and voriconazole, prolonged the survival time of silkworms infected with T. asahii. A transgenic T. asahii strain expressing eGFP was obtained using a gene recombination method with Agrobacterium tumefaciens. The T. asahii strain expressing eGFP showed hyphal formation in the silkworm hemolymph. Both hyphal growth and the inhibition of hyphal growth by the administration of antifungal agents were quantitatively estimated by monitoring fluorescence. Our findings suggest that a silkworm infection model using T. asahii expressing eGFP is useful for evaluating both the pathogenicity of T. asahii and the efficacy of antifungal drugs.

N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, a zinc chelator, inhibits biofilm and hyphal formation in Trichosporon asahii.

OBJECTIVE: Trichosporon asahii is the major causative fungus of disseminated or deep-seated trichosporonosis and forms a biofilm on medical devices. Biofilm formation leads to antifungal drug resistance, so biofilm-related infections are relatively difficult to treat and infected devices often require surgical removal. Therefore, prevention of biofilm formation is important in clinical settings. In this study, to identify metal cations that affect biofilm formation, we evaluated the effects of cation chelators on biofilm formation in T. asahii. RESULTS: We evaluated the effect of cation chelators on biofilm formation, since microorganisms must assimilate essential nutrients from their hosts to form and maintain biofilms. The inhibition by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) was greater than those by other cation chelators, such as deferoxamine, triethylenetetramine, and ethylenediaminetetraacetic acid. The inhibitory effect of TPEN was suppressed by the addition of zinc. TPEN also inhibited T. asahii hyphal formation, which is related to biofilm formation, and the inhibition was suppressed by the addition of zinc. These results suggest that zinc is essential for biofilm formation and hyphal formation. Thus, zinc chelators have the potential to be developed into a new treatment for biofilm-related infection caused by T. asahii.

Genomic and transcriptome identification of fluconazole-resistant genes for Trichosporon asahii.

Trichosporon asahii infection is difficult to control clinically. This study identified a case with over 15 years of T. asahii infection-related systemic dissemination disease and conducted genome and transcriptome sequencing to identify fluconazole-resistant genes in fluconazole-resistant versus susceptible strains isolated from this patient's facial skin lesions. The data revealed mutations of the ergosterol biosynthetic pathway-related genes in the T. asahii genome of the fluconazole-resistant strain, that is, there were 36 novel mutations of the ERG11 gene, three point mutations (V458L, D457V, and D334S) in the ERG3, and a missense mutation (E349D) in ERG5 in the fluconazole-resistant strain of the T. asahii genome. To ensure that ERG11 is responsible for the fluconazole resistance, we thus simultaneously cultured the strains in vitro and cloned the ERG11 CDS sequences of both fluconazole-susceptible and -resistant strains into the Saccharomyces cerevisiae. These experiments confirmed that these mutations of ERG11 gene affected fluconazole resistance (> 64 µg/ml vs. <8 µg/ml of the MIC value between fluconazole-resistant and -susceptible strains) in Saccharomyces cerevisiae. In addition, expression of ergosterol biosynthesis pathway genes and drug transporter was upregulated in the fluconazole-resistant strain of T. asahii. Collectively, the fluconazole resistance in this female patient was associated with mutations of ERG11, ERG3, and ERG5 and the differential expression of drug transporter and fatty acid metabolic genes.

Mixed Infection of Toe Nail Caused by Trichosporon asahii and Rhodotorula mucilaginosa.

Trichosporon asahii and Rhodotorula mucilaginosa are important fungal species causing disseminated disease in immunocompromised patients. Onychomycosis prevalence rate ranges from 2 to 30%, which were 50% of nail diseases and 30% of superficial mycosis, respectively. Although important, little is known about the co-habitation of T. asahii and R. mucilaginosa in the causation of onychomycosis. Here, we present the co-habitation of T. asahii and R. mucilaginosa as causative agents of onychomycosis in a healthy 41-year-old male in China. Direct microscopic examination, fungal culture and MALDI-TOF MS were employed in isolated pathogens; hence, antifungal susceptibility test was evaluated. T. asahii was sensitive to posaconazole, voriconazole and itraconazole, whereas R. mucilaginosa was sensitive to both 5-flucytosine and amphotericin B. This report highlights the co-habitation of T. asahii and R. mucilaginosa in the causation of onychomycosis and to raise the awareness of this infection among dermatologists.

Molecular Identification, Genotyping, Phenotyping, and Antifungal Susceptibilities of Medically Important Trichosporon, Apiotrichum, and Cutaneotrichosporon Species.

Recently, Trichosporon taxonomy has been reevaluated and new genera of the Trichosporonaceae family have been described. Here, 26 clinical isolates were submitted for identification via sequencing of the intergenic space 1 (IGS1) region, genotyping, and investigation of virulence factors. Antifungal susceptibility was determined using the CLSI broth microdilution method for fluconazole (FLC), itraconazole (ITC), and amphotericin B (AMB). Of these, 24 isolates were identified, including 12 T. asahii, 4 T. inkin, 3 T. faecale, 1 T. coremiiforme, 1 T. japonicum, 2 Cutaneotrichosporon dermatis (formerly T. dermatis), and 1 Apiotrichum mycotoxinivorans (formerly T. mycotoxinivorans). Species-level identification of 2 isolates was not successful; they were described as Trichosporon sp. We observed optimal colonial development at 35-40 °C. Lipase was the major extracellular enzyme produced (100%); caseinase was not produced (0%). Biofilms were produced by all isolates (classified as low). High AMB minimum inhibitory concentration (MIC) was observed, with all strains resistant. Fluconazole was the most active drug among the antifungals tested. However, high MICs for FLC were observed in C. dermatis and A. mycotoxinivorans species, which also showed resistance to ITC and AMB. This study, conducted in the Northern region of Brazil, identified 5 Trichosporon species along with C. dermatis and A. mycotoxinivorans and demonstrated their pathogenic potential through their ability to produce important virulence factors. This may contribute to our understanding of the epidemiology and factors related to the pathogeneses of species in the Trichosporonaceae family.

Exploring the resistance mechanisms in Trichosporon asahii: Triazoles as the last defense for invasive trichosporonosis.

Trichosporon asahii has recently been recognized as an emergent fungal pathogen able to cause invasive infections in neutropenic cancer patients as well as in critically ill patients submitted to invasive medical procedures and broad-spectrum antibiotic therapy. T. asahii is the main pathogen associated with invasive trichosporonosis worldwide. Treatment of patients with invasive trichosporonosis remains a controversial issue, but triazoles are mentioned by most authors as the best first-line antifungal therapy. There is mounting evidence supporting the claim that fluconazole (FLC) resistance in T. asahii is emerging worldwide. Since 2000, 15 publications involving large collections of T. asahii isolates described non-wild type isolates for FLC and/or voriconazole. However, very few papers have addressed the epidemiology and molecular mechanism of antifungal resistance in Trichosporon spp. Data available suggest that continuous exposure to azoles can induce mutations in the ERG11 gene, resulting in resistance to this class of antifungal drugs. A recent report characterizing T. asahii azole-resistant strains found several genes differentially expressed and highly mutated, including genes related to the Target of Rapamycin (TOR) pathway, indicating that evolutionary modifications on this pathway induced by FLC stress may be involved in developing azole resistance. Finally, we provided new data suggesting that hyperactive efflux pumps may play a role as drug transporters in FLC resistant T. asahii strains.

Species distribution and antifungal susceptibility of 358 Trichosporon clinical isolates collected in 24 medical centres.

OBJECTIVES: To provide species distribution and antifungal susceptibility profiles of 358 Trichosporon clinical isolates collected from 24 tertiary-care hospitals. METHODS: Species identification was performed by sequencing the IGS1 region of rDNA. Antifungal susceptibility testing for amphotericin B, fluconazole, voriconazole and posaconazole followed the Clinical and Laboratory Standards Institute reference method. Tentative epidemiologic cutoff values (97.5% ECVs) of antifungals for Trichosporon asahii were also calculated. RESULTS: Isolates were cultured mostly from urine (155/358, 43.3%) and blood (82/358, 23%) samples. Trichosporon asahii was the most common species (273/358, 76.3%), followed by T. inkin (35/358, 9.7%). Isolation of non-T. asahii species increased substantially over the last 11 years [11/77 (14.2%) from 1997 to 2007 vs. 74/281, (26.3%) from 2008 to 2018, p0.03]. Antifungal susceptibility testing showed high amphotericin B minimum inhibitory concentrations against Trichosporon isolates, with higher values for T. faecale. The ECV for amphotericin B and T. asahii was set at 4 µg/mL. Among the triazole derivatives, fluconazole was the least active drug. The ECVs for fluconazole and posaconazole against T. asahii were set at 8 and 0.5 µg/mL, respectively. Voriconazole showed the strongest in vitro activity against the Trichosporon isolates; its ECV for T. asahii was set at 0.25 µg/mL after 48 hours' incubation. CONCLUSIONS: Trichosporon species diversity has increased over the years in human samples, and antifungal susceptibility profiles were species specific. Trichosporon asahii antifungal ECVs were proposed, which may be helpful to guide antifungal therapy.

Disseminated Trichosporon asahii infection in a combined liver-kidney transplant recipient successfully treated with voriconazole.

INTRODUCTION: Trichosporon asahii is an emerging cause of systemic fungal infection in an immunocompromised host. Several life threatening disseminated T. asahii infection in single solid organ (liver or kidney) transplant recipients, in neutropenic and hematological malignancy patients have been reported. CASE PRESENTATION (METHODS AND RESULTS): A 49-year old gentleman who underwent simultaneous living-donor liver transplantation (donor sister) and kidney transplant (donor wife) developed fever and subsegmental patchy consolidation with right sided pleural effusion on fourth postoperative day. Central line blood stream infection was suspected. Blood culture grew creamy white colonies of T. asahii on blood agar with characteristic dirty-green colonies on CHROMagar. Laboratory analysis of pleural fluid also revealed budding yeast cells identified as T. asahii. Microscopy of the isolates showed hyphae, arthroconidia, and blastospores. The isolates were identified as T. asahii by VITEK MS which uses matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology. Initially liposomal amphotericin B and micafungin was initiated, but due to lack of clinical and microbiological response, patient was switched to voriconazole. Simultaneously, tacrolimus doses were reduced to one-third in view of interaction with voriconazole. Subsequently, patient improved with resolution of fever and microbiological cure. CONCLUSION: This is the first case report of disseminated T. asahii infection in a combined liver-kidney transplant recipient successfully treated with voriconazole. Azole antifungal are the promising drug of choice for systemic T. asahii infection. Drug interactions should be considered while using these antifungal agents.

Molecular identification and biological characteristic analysis of an Apiotrichum mycotoxinivorans (formerly Trichosporon mycotoxinivorans) strain isolated from sputum specimens of a pediatric patient with pneumonia.

Apiotrichum mycotoxinivorans (formerly Trichosporon mycotoxinivorans) has long been used to degrade fungal toxins in livestock feed. However, clinic reports about this type of fungus are rare. In this study, we report the morphology, biochemistry, and molecular characteristics of an A. mycotoxinivorans strain isolated from a pediatric patient with congenital ventricular septal defect and pneumonia. A female patient, 26 months old, presented with congenital ventricular septal defect. Pulmonary infection symptoms were observed after the patient received cardiac repair surgery. Sputum bacterial and fungal cultures were positive for Elizabethkingia anophelis and a fungus, which was not readily identifiable using biochemical identification, or MALDI-TOF MS analysis. The strain was finally identified as A. mycotoxinivorans using amplification and sequencing of the D1/D2 region of 26S rDNA, ITS, and IGS1. Antifungal susceptibility test results suggested that fluconazole or voriconazole may be an appropriate choice for antifungal therapy. A biodegradability of ochratoxin A was considered as a characteristic of the fungal strain. Our results support the existing evidence that A. mycotoxinivorans is an opportunistic pathogen for human beings. Nucleic acid analysis allows for the accurate identification of the species in instances where conventional identification methods such as biochemical testing and MALDI-TOF MS may be unsuccessful.

Sodium butyrate inhibits planktonic cells and biofilms of Trichosporon spp.

Trichosporon spp. have been increasingly recognized as an important pathogen of invasive and disseminated infections in immunocompromised patients. These species are prone to form biofilms in medical devices such as catheters and prosthesis, which are associated with antifungal resistance and therapeutic failure. Therefore, new antifungals with a broader anti-biofilm activity need to be discovered. In the present study we evaluate the inhibitory potential of sodium butyrate (NaBut) - a histone deacetylase inhibitor that can alter chromatin conformation - against planktonic and sessile cells of T. asahii and T. inkin. Minimum inhibitory concentration (MIC) of NaBut against planktonic cells was evaluated by microdilution and morphological changes were analyzed by optical microscopy on malt agar supplemented with NaBut. Biofilms were evaluated during adhesion, development and after maturation for metabolic activity and biomass, as well as regarding ultrastructure by scanning electron microscopy and confocal laser scanning microscopy. NaBut inhibited the growth of planktonic cells by 50% at 60 mM or 120 mM (p < 0.05) and also reduced filamentation of Trichosporon spp. NaBut reduced adhesion of Trichosporon cells by 45% (10xMIC) on average (p < 0.05). During biofilm development, NatBut (10xMIC) reduced metabolic activity and biomass up to 63% and 81%, respectively (p < 0.05). Mature biofilms were affected by NaBut (10xMIC), showing reduction of metabolic activity and biomass of approximately 48% and 77%, respectively (p < 0.05). Ultrastructure analysis showed that NaBut (MIC and 10xMIC) was able to disassemble mature biofilms. The present study describes the antifungal and anti-biofilm potential of NaBut against these opportunist emerging fungi.

In vitro activity of diphenyl diselenide and ebselen alone and in combination with antifungal agents against Trichosporon asahii.

This study evaluated the in vitro susceptibility of Trichosporon asahii strains to diphenyl diselenide (DPDS) and ebselen (EBS) alone and in combination with amphotericin B (AMB), fluconazole (FCZ), itraconazole (ITZ) and caspofungin (CAS) using the microdilution method. EBS showed in vitro activity against T asahii strains with minimal inhibitory concentration (MIC) ranged from 0.25 to 8.0 µg/mL. For DPDS, the MIC ranged from 8.0 to 64 µg/mL. The combinations demonstrating the greatest synergism rate against fluconazole-resistant T asahii strains were the following: CAS + DPDS (96.67%), AMB + DPDS (93.33%), FCZ + DPDS (86.67%) and ITZ + DPDS (83.33%). The combinations AMB + DPDS and AMB + EBS exhibited the highest synergism rate against the fluconazole-susceptible (FS) T asahii strains (90%). Antagonism was observed in the following combinations: FCZ + EBS (80%) and FCZ + DPDS (13.33%) against the FS strains, and ITZ + EBS (20%) against the FR strains. Our findings suggest that the antimicrobial activity of DPDS and EBS against T. asahii and its use as an adjuvant therapy with antifungal agents warrant in vivo experimental investigation.

Farnesol inhibits planktonic cells and antifungal-tolerant biofilms of Trichosporon asahii and Trichosporon inkin.

Trichosporon species have been considered important agents of opportunistic systemic infections, mainly among immunocompromised patients. Infections by Trichosporon spp. are generally associated with biofilm formation in invasive medical devices. These communities are resistant to therapeutic antifungals, and therefore the search for anti-biofilm molecules is necessary. This study evaluated the inhibitory effect of farnesol against planktonic and sessile cells of clinical Trichosporon asahii (n = 3) andTrichosporon inkin (n = 7) strains. Biofilms were evaluated during adhesion, development stages and after maturation for metabolic activity, biomass and protease activity, as well as regarding morphology and ultrastructure by optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy. Farnesol inhibited Trichosporon planktonic growth by 80% at concentrations ranging from 600 to 1200 µM for T. asahii and from 75 to 600 µM for T. inkin. Farnesol was able to reduce cell adhesion by 80% at 300 µM for T. asahii and T. inkin at 600 µM, while biofilm development of both species was inhibited by 80% at concentration of 150 µM, altering their structure. After biofilm maturation, farnesol decreased T. asahii biofilm formation by 50% at 600 µM concentration and T. inkin formation at 300 µM. Farnesol inhibited gradual filamentation in a concentration range between 600 and 1200 µM. Farnesol caused reduction of filament structures of Trichosporon spp. at every stage of biofilm development analyzed. These data show the potential of farnesol as an anti-biofilm molecule.

Invasive Infections Due to Trichosporon: Species Distribution, Genotyping, and Antifungal Susceptibilities from a Multicenter Study in China.

A total of 133 clinical Trichosporon isolates were collected in the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program in 2009 to 2016. Accurate identification was performed by sequencing of the intergenic spacer 1 (IGS1) region. Among these isolates, Trichosporon asahii (108 isolates [81.2%]) was the leading species, followed by Trichosporon dermatis (7 isolates [5.3%]), Trichosporon asteroides (5 isolates [3.8%]), Trichosporon inkin (5 isolates [3.8%]), Trichosporon dohaense (3 isolates [2.3%]), and 1 isolate (0.7%) each of Trichosporon faecale, Trichosporon jirovecii, Trichosporon mucoides, Trichosporon coremiiforme, and Trichosporon montevideense Both the Vitek mass spectrometry (MS) (bioMérieux, Marcy l'Etoile, France) and Bruker Biotyper MS (Bruker Daltonics GmbH, Germany) platforms gave high levels (>97.5%) of correct identification when the species were present in the database. The geometric mean (GM) of amphotericin B MICs for T. asahii was 2-fold higher than that for non-asahii Trichosporon High fluconazole MICs (≥8 µg/ml) were observed for 25% of T. asahii isolates (27/108 isolates) and 16% of non-asahii Trichosporon (4/25 isolates) isolates. Itraconazole MICs were ≤0.5 µg/ml for 89.5% of the isolates. Voriconazole was the most potent antifungal agent in vitro, with a GM of 0.09 µg/ml. Genotyping of the isolates using IGS1 sequence alignment revealed that genotype 1 was most common (41.7%), followed by genotype 4 (31.5%), genotype 3 (23.1%), genotype 5 (0.9%), genotype 6 (0.9%), and genotype 7 (1.8%). Our data on species distribution, genotypes, and antifungal susceptibilities may contribute to a better understanding of the epidemiology of invasive Trichosporon infections throughout China.

Effect of resveratrol and Regrapex-R-forte on Trichosporon cutaneum biofilm.

Microorganisms that cause chronic infections exist predominantly as surface-attached stable communities known as biofilms. Microbial cells in biofilms are highly resistant to conventional antibiotics and other forms of antimicrobial treatment; therefore, modern medicine tries to develop new drugs that exhibit anti-biofilm activity. We investigated the influence of a plant polyphenolic compound resveratrol (representative of the stilbene family) on the opportunistic pathogen Trichosporon cutaneum. Besides the influence on the planktonic cells of T. cutaneum, the ability to inhibit biofilm formation and to eradicate mature biofilm was studied. We have tested resveratrol as pure compound, as well as resveratrol in complex plant extract-the commercially available dietary supplement Regrapex-R-forte, which contains the extract of Vitis vinifera grape and extract of Polygonum cuspidatum root. Regrapex-R-forte is rich in stilbenes and other biologically active substances. Light microscopy imaging, confocal microscopy, and crystal violet staining were used to quantify and visualize the biofilm. The metabolic activity of biofilm-forming cells was studied by the tetrazolium salt assay. Amphotericin B had higher activity against planktonic cells; however, resveratrol and Regrapex-R-forte showed anti-biofilm effects, both in inhibition of biofilm formation and in the eradication of mature biofilm. The minimum biofilm eradicating concentration (MBEC80) for Regrapex-R-forte was found to be 2222 mg/L (in which resveratrol concentration is 200 mg/L). These methods demonstrated that Regrapex-R-forte can be employed as an anti-biofilm agent, as it has similar effect as amphotericin B (MBEC80 = 700 mg/L), which is routinely used in clinical practice.

Trichosporon inkin meningitis in Northeast Brazil: first case report and review of the literature.

BACKGROUND: Trichosporon species may colonize the skin, respiratory tract and gastrointestinal tract of human beings. The yeast is recognized as etiological agent of white piedra, a superficial mycosis. Nevertheless, immunocompromised hosts may develop invasive Trichosporonosis. Central nervous system trichosporonosis is a very rare clinical manifestation. In fact, only a few cases have been published in the literature and none of them was caused by Trichosporon inkin. CASE PRESENTATION: Here we report the first clinical case of meningoencephalitis due to this species in a female previously healthy patient under corticosteroids and antibiotics therapy for several months. She was submitted to an invasive procedure to remove a left sided acoustic neuroma and further developed a cerebrospinal fistula. After some days of the procedure, she presented a predominantly and intensive occipital holocranial headache, followed by vomiting, hyporexia, weight loss, asthenia, irritability, difficulty to concentrate and rotator vertigo. The patient further developed a cerebrospinal fistula in the occipital region and was submitted to a surgical correction. After several months of clinical interventions, she was diagnosed with CNS Trichosporonosis, after Magnetic Resonance Imaging and positive microbiological cultures obtained within two different occasions (2 weeks apart). Despite the antifungal therapy with Amphotericin B and Voriconazole, the patient did not survive. CONCLUSIONS: Despite CNS Fungal infections are mostly due to Cryptococcus spp., other emergent yeasts, such as T. inkin may be considered as a likely etiological agent. This is the first case report of CNS Trichosporonosis, where species identification was performed with rDNA sequencing.

Inhibitory effect of a lipopeptide biosurfactant produced by Bacillus subtilis on planktonic and sessile cells of Trichosporon spp.

The present study aimed to investigate the inhibitory effect of a bacterial biosurfactant (TIM96) on clinical strains of Trichosporon. Additionally, the effect of TIM96 on the ergosterol content, cell membrane integrity, and the hydrophobicity of planktonic cells was assessed. The inhibitory activity of TIM96 against Trichosporon biofilms was evaluated by analyzing metabolic activity, biomass and morphology. MIC values ranged from 78.125 to 312.5 µg ml-1 for TIM96; time-kill curves revealed that the decline in the number of fungal cells started after incubation for 6 h with TIM96 at both MIC and 2×MIC. The biosurfactant reduced the cellular ergosterol content and altered the membrane permeability and the surface hydrophobicity of planktonic cells. Incubation at 10×MIC TIM96 reduced cell adhesion by up to 96.89%, thus interfering with biofilm formation. This concentration also caused up to a 99.2% reduction in the metabolic activity of mature biofilms. The results indicate potential perspectives for the development of new antifungal strategies.