Aspects related to biofilm production and antifungal susceptibility of clinically relevant yeasts of the genus Trichosporon.

Trichosporonosis corresponds to a systemic fungal disease that leads to high mortality rates and is frequently associated with medical devices. It affects immunosuppressed patients in particular and is strongly linked to acquired human immunodeficiency, organ and tissue transplants, and malignant hematologic diseases such as leukemia and lymphomas. Trichosporon infections have been increasingly reported worldwide; however, little information is available either about their characteristics or the causative microorganism. Thus, the aims of the present study were: to investigate 59 yeasts of the genus Trichosporon by verifying the biofilm formation capacity of isolates; to analyze the susceptibility patterns of planktonic cells against the antifungals fluconazole, itraconazole, amphotericin-B, voriconazole, and caspofungin by comparing European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution technique with the commercial method Etest; and to assess the susceptibility patterns of biofilm cells (sessile) against the same antifungals through broth microdilution. The ability to form biofilm on the surface of polystyrene plates was noted for all isolates, and 54.3% of samples were considered strong producers. Comparison between the antifungal susceptibility techniques evidenced that Etest showed higher and discordant minimum inhibitory concentrations (MICs) from those obtained by the microdilution method, especially for fluconazole, itraconazole, and caspofungin. Considering the susceptibility of biofilms, most species had high MIC50 and MIC90 against the tested antifungals, showing 4-to-66-fold higher concentrations for amphotericin B and 2-to-33-fold greater concentrations for caspofungin. These results highlight the importance of further studies with Trichosporon spp. for comparison between laboratory findings and in vivo response, considering both the susceptibility tests and the behavior of biofilm cells against drugs.

This study investigated 59 isolates of the medically important yeast Trichosporon in relation to their ability to form biofilms and the susceptibility of biofilms to antifungal agents. All isolates were able to produce biofilms and biofilms showed lower antifungal susceptibility.

Inhibition of Klebsiella pneumoniae carbapenemases by mycocins produced by Wickerhamomyces anomalus.

New alternatives have been under study as treatment due to the problem of multidrug-resistant bacteria. Among them, Wickerhamomyces anomalus mycocins have shown a great potential against several microorganisms since they have high antimicrobial activity, as well as they can be used as fast available nutrients and stand several extreme conditions. In this way, Klebsiella pneumoniae carbapenemases inhibition by mycocins produced by W. anomalus is important. Microdilution assays were carried out to evaluate strains inhibition in liquid medium and the test in solid medium were carried out. Toxicity was evaluated by both hemolysis and Artemia salina Leach tests. W. anomalus supernatant showed 2.36 U/mg ß-glucanases activity, and antimicrobial activity was evidenced in 100% Klebsiella pneumoniae carbapenemase strains up to 0.12 U/mg concentration. Besides, there was low toxicity in hemolysis and Artemia salina Leach tests. It is suggested that W. anomalus mycocins may be an alternative to develop new antimicrobial substances.

Comparing the phenotypic, genotypic, and proteomic identification of Trichosporon species: A globally emerging yeast of medical importance.

Trichosporon spp. are widely distributed in the nature, comprising species that inhabit different ecological niches and can be found in the water, soil, and body surface of animals and humans. Such microorganisms have been classically associated with superficial infections; however, in the last decades, they have also been related to disseminated infections in immunocompromised patients, behaving as opportunistic agents, which demands rapid and accurate species identification for efficient therapy. Concordance level between the traditional phenotypic method and the molecular technique (gold standard) in the identification of all 59 Trichosporon samples was 59.3%. Identification concordance between MALDI-TOF spectrometry and the molecular technique was 71.2%. No isolate of environmental origin was identifiable by MALDI-TOF mass spectrometry (MS), and 100% of such environmental isolates were discordant for IGS region sequencing and phenotypic characterization. Both comparisons evidenced greatest concordance in the identification of T. asahii. The species T. debeurmannianum, T. dermatis, T. venhuisii and T. insectorum were not properly identified by both MALDI-TOF MS and the phenotypic technique. MALDI-TOF MS, in particular, seems to be appropriate to investigate yeasts of the genus Trichosporon; however, database updates are still necessary, especially for species that are not common in the clinical routine. With the aim of helping understand the aspects involved in early and accurate diagnosis of infections caused by this opportunistic agent, the present study compared the phenotypic, molecular (IGS region) and mass-spectrometry (MALDI-TOF) identification of 59 yeasts of the genus Trichosporon which had clinical and environmental origin and were kept in a mycology collection.

The present study compared the phenotypic, genotypic, and mass-spectrometry (MALDI-TOF) identification of 59 yeasts of the genus Trichosporon. MALDI-TOF MS, in particular, seems to be appropriate to investigate this yeasts when compared to a molecular technique (gold standard).

Infections Caused by Fusarium Species in Pediatric Cancer Patients and Review of Published Literature.

Fusarium species have emerged as responsible for a broad spectrum of infections, including superficial, locally invasive and disseminated ones, especially in the hospital environment. Since there are few reports of invasive and disseminated fusariosis in children, the aim of this study was to report four cases of nosocomial infection caused by this microorganism in children with cancer hospitalized in a public children's hospital located in Brazil. Two of these patients were female and two were male. All patients presented febrile neutropenia, while three patients had acute lymphocytic leukemia and one patient had Wilms' tumor as underlying disease. In two cases, fungi were isolated from blood and identified as Fusarium oxysporum species complex after phenotypic and genotypic studies, while in two other cases fungi were isolated from skin biopsies and identified as Fusarium solani species complex. One patient died 12 days after the onset of cutaneous lesions. All isolates, after susceptibility testing, presented high levels of minimum inhibitory concentration for itraconazole, voriconazole and amphotericin B. Considering the emergence of filamentous fungi as etiologic agents of nosocomial infections, health professionals should be aware of the problems these infections, especially fungal ones, may cause to debilitated patients.

Repression of Proteases and Hsp90 Chaperone Expression Induced by an Antiretroviral in Virulent Environmental Strains of Cryptococcus neoformans.

This study evaluated the effect of the antiretroviral ritonavir on protease secretion in different strains of Cryptococcus neoformans isolated from the environment and investigated the expression of heat shock protein (Hsp90), classically described virulence factors in other yeast in the presence of the same antiretroviral. The presence of the enzyme was detected by the formation of a degradation of the halo around the colonies. The results were classified as follows: level 1 (without proteases), level 2 (positive for proteases), and level 3 (strongly positive for proteases). Total protein extract isolated from the cell walls of the 12 strains incubated in the absence and presence of ritonavir (0.3125 mg mL-1) were resolved by SDS-PAGE and analyzed by Western blot assays using an antiserum against Hsp90 from Blastocladiella emersonii. All strains tested showed inhibition of proteinase activity in the presence of ritonavir at 0.3125 to 1.25 mg mL-1. High levels of Hsp90 were observed in the absence of ritonavir (0.3125 mg mL-1), except for the non-virulent control cells. In contrast, in the presence of the antiretroviral, a drastic reduction in the expression of the chaperone was observed. The data suggest that ritonavir, in addition to containing viral replication, could inhibit the expression of virulence factors in opportunistic yeast, as proteases and Hsp90. According to our current knowledge, this is the first time that the inhibition of Hsp90 by an antiretroviral was reported for environmental isolates of C. neoformans.

Dermatophytosis and immunovirological status of HIV-infected and AIDS patients from Sao Paulo city, Brazil.

Over the past decades, more people became infected with human immunodeficiency virus (HIV) and developed acquired immunodeficiency syndrome (AIDS). Because of that the incidence of fungal infections rose dramatically. It happened because this virus can modify the course of fungal diseases, leading to altered clinical pictures. The aim of this study was to evaluate epidemiological and biological aspects of dermatophytosis in HIV-positive and AIDS patients living in the city of São Paulo, Brazil. A total of 84 (44 HIV-positive and 40 AIDS) patients were enrolled in this study. The patients were tested for dermatophyte infections, as well as for the CD4(+) /CD8(+) and HIV viral load counts. Tinea unguium was most frequently observed in AIDS patients, whereas Tinea pedis was mostly observed in HIV-positive patients. The most frequent dermatophyte species was Trichophyton rubrum. CD4(+) counts and CD4(+) /CD8(+) ratios were not associated with a higher risk for dermatophytosis. On the other hand, viral load higher than 100 000 copies/ml was associated with a higher frequency of dermatophytosis. The results suggest to that although dermatophytosis is common in HIV-positive and AIDS patients, the degree of immunosuppression does not seems to correlate with increased risk of this fungal infection. In addition, high viral load as a predictive risk factor for dermatophyte infection should be subject of further evaluations.

Action of mycocins produced by Wickerhamomyces anomalus on Malassezia pachydermatis isolated from the ear canal of dogs.

This study aims to examine the effectiveness of mycocins produced by Wickerhamomyces anomalus in inhibiting Malassezia pachydermatis, a yeast commonly found in the ear canal of dogs. M. pachydermatis has a zoophilic origin and can be found in mammals, and frequently in dogs, where it mainly colonizes the ear canal region and the skin, leading to lesions that are difficult to treat. The antimicrobial mechanism was evaluated using dilutions of supernatant with enzymatic activity, which may include ß-glucanases, glycoproteins known to act on microorganism cell walls. However, it is important to note that this supernatant may contain other compounds as well. ß-glucanases in the mycocins supernatant were found at a concentration of 0.8 U/mg. The susceptibility of M. pachydermatis isolates was tested using the microdilution method. The isolates suffered 100% inhibition when tested with the culture supernatant containing mycocins. In the proteinases production test, 44% of the isolates tested were strong proteinases producers. Subsequently all these isolates suffered inhibition of their activity when tested in research medium containing mycocins supernatant at a subinhibitory concentration of ß-glucanases. This shows that mycocins can inhibit the production of proteinases, a virulence factor of M. pachydermatis. The viability test showed the antifungal action of mycocins in inhibiting the viability of M. pachydermatis cells after a period of 8  hours of contact. These results support the antimicrobial potential of mycocins and their promise as a therapeutic option.

Six-year trend analysis of nosocomial candidemia and risk factors in two intensive care hospitals in Mato Grosso, midwest region of Brazil.

We conducted this cross-sectional retrospective study using clinical and laboratory data from two tertiary hospitals in Cuiabá, Mato Grosso, Brazil, in order to explore the risk factors and estimate mortality, prevalence and lethality of candidemia between 2006 and 2011. A total of 130 episodes of candidemia were identified. The prevalence of candidemia was 1.8 per 1,000 admissions, the mortality rate was 0.9 per 1,000 admissions, and the lethality was 49.2 %. The main agent in this population was Candida parapsilosis (n = 50), followed by C. albicans (n = 45). Comparison between the numbers of episodes in the two triennia revealed that the non-albicans group grew by 48.2 %. The distribution of yeast species of Candida per hospital unit revealed that C. albicans was more prevalent than C. parapsilosis in the adult ICU and C. parapsilosis was more prevalent than C. albicans in the neonatal ICU. Patients remained hospitalized for an average of 53.5 days. Central venous catheters, parenteral nutrition and age were the variables that proved to be independent in the multivariate analysis and that maintained a statistically significant association with the incidence of death in patients with candidemia. The annual prevalence of candidemia showed a significant increase in the second triennium (2009-2011) compared with the first (2006-2008) probably due to increased exposure to risk factors: central venous catheter, H2 blockers, nutrition parenteral corticosteroids and mean hospital duration.

Candidemia by species of the Candida parapsilosis complex in children's hospital: prevalence, biofilm production and antifungal susceptibility.

Opportunistic infections are an increasingly common problem in hospitals, and the yeast Candida parapsilosis has emerged as an important nosocomial pathogen. The aims of this study were to determine and compare (i) the prevalence rate among C. parapsilosis complex organisms isolated from blood in a public children's hospital in São Paulo state, (ii) the ability of the complex C. parapsilosis species identified to produce biofilm and (iii) the antifungal susceptibility profiles. Forty-nine (49) specimens of isolated blood yeast were analyzed, previously identified as C. parapsilosis by conventional methods. After the molecular analysis, the isolates were characterized as C. parapsilosis sensu stricto (83.7 %), C. orthopsilosis (10.2 %) and C. metapsilosis (6.1 %). All species were able to form biofilm. The species with the highest biofilm production was C. parapsilosis sensu stricto, followed by C. orthopsilosis and further by C. metapsilosis. All of the strains have demonstrated similar susceptibility to fluconazole, caspofungin, voriconazole, cetoconazole and 5-flucytosine. Only one strain of C. parapsilosis was resistant to amphotericin B. Regarding itraconazole, 66.6 and 43.9 % isolates of C. metapsilosis and C. parapsilosis, respectively, have demonstrated to be susceptible dose-dependent, with one isolate of the latter species resistant to the drug. Candida parapsilosis sensu stricto has demonstrated to be the less susceptible, mainly to amphotericin B, caspofungin and "azoles" such as fluconazole. Therefore, C. metapsilosis and C. orthopsilosis are still involved in a restricted number of infections, but these data have become essential for there are very few studies of these species in Latin America.

Feline nasal granuloma due to Cryptoccocus gattii type VGII.

The aims of this study are to make a more precise identification of the etiologic agent of a nasal granuloma in a cat, to verify the susceptibility to the antifungal drugs: ketoconazole, itraconazole, fluconazole, posaconazole, voriconazole, amphotericin B and the proper treatment. Part of the granuloma's fragment was removed, added to a saline solution and sent to the Laboratory of Mycology. The solution was then seeded in Sabouraud dextrose agar, and the yeast was primarily identified by the traditional methods. The confirmation of the specie Cryptococcus gattii and its molecular type were performed using the PCR-RFLP molecular techniques. The antifungal susceptibility was verified by using the E-test method, and the cat was treated with itraconazole associated with 5-flucytosine. The isolated strain was identified as C. gattii type VGII and was susceptible to all antifungal drugs tested. The treatment with itraconazole associated with 5-flucytosine led to the cure of granulomatous lesions in the feline after 6 months. The characterization and molecular investigation of this microorganism are relevant because they could help us better understand the epidemiology of the infection and to guide us to treat properly the disease.

Voriconazole, combined with amphotericin B, in the treatment for pulmonary cryptococcosis caused by C. neoformans (serotype A) in mice with severe combined immunodeficiency (SCID).

Cryptococcosis is a subacute or chronic systemic mycosis with a cosmopolitan nature, caused by yeast of the genus Cryptococcus neoformans. The model of systemic cryptococcosis in mice with severe combined immunodeficiency (SCID) is useful for immunological and therapeutic study of the disease in immunodeficient hosts. Amphotericin B, fluconazole and flucytosine are the drugs most commonly used to treat cryptococcosis. Voriconazole is a triazole with high bioavailability, large distribution volume, and excellent penetration of the central nervous system. The objective of this study was to evaluate treatment with amphotericin B (AMB), voriconazole (VRC), and AMB, used in combination with VRC, of experimental pulmonary cryptococcosis in a murine model (SCID). The animals were inoculated intravenously (iv) with a solution containing 3.0 × 10(5) viable cells of C. neoformans ATCC 90112, (serotype A). Treatments were performed with amphotericin B (1.5 mg/kg/day), voriconazole (40.0 mg/kg/day) and AMB (1.5 mg/kg/day) combined with VRC (40.0 mg/kg/day); began 1 day after the initial infection; were daily; and lasted 15 days. Evaluations were performed using analysis of the survival curve and isolation of yeast in the lung tissue. There was a significant increase in survival in groups treated with AMB combined with VRC, compared with the untreated group and groups receiving other treatments (P < 0.05). In the group treated only with VRC and AMB combined with VRC, there was a significant reduction (P < 0.05) in the isolation of C. neoformans in lung tissue. Amphotericin B combined with voriconazole may be an effective alternative to increasing survival and may reduce yeast in the lung tissue of mice with pulmonary cryptococcosis and SCID.

Experimental model of arthritis induced by Paracoccidioides brasiliensis in rats.

Paracoccidioidomycosis (PCM), a disease caused by the fungus Paracoccidioides brasiliensis (Pb), is highly prevalent in Brazil, where it is the principal cause of death by systemic mycoses. The disease primarily affects men aged 30-50 year old and usually starts as a pulmonary focus and then may spread to other organs and systems, including the joints. The present study aimed to develop an experimental model of paracoccidioidomycotic arthritis. Two-month-old male Wistar rats (n = 48) were used, divided in 6 groups: test groups EG/15 and EG/45 (received one dose of 100 µl of saline containing 10(5) Pb viable yeasts in the knee); heat killed Pb-group HK/15 and HK/45 (received a suspension of 10(5) Pb nonviable yeasts in the knee) and control groups CG/15 and CG/45 (received only sterile saline in the knee). The rats were killed 15 and 45 days postinoculation. In contrast with the control rats, the histopathology of the joints of rats of the test groups (EG/15 and EG/45) revealed a picture of well-established PCM arthritis characterized by extensive sclerosing granulomatous inflammation with numerous multiple budding fungal cells. The X-ray examination revealed joint alterations in these groups. Only metabolic active fungi evoked inflammation. The experimental model was able to induce fungal arthritis in the knees of the rats infected with metabolic active P. brasiliensis. The disease tended to be regressive and restrained by the immune system. No evidence of fungal dissemination to the lungs was observed.

Diagnosis, treatment and maxillofacial rehabilitation in rhinocerebral mucormycosis patient: A case report and review of the literature.

Mucormycosis is an aggressive, rare and opportunistic infectious disease, with a high mortality rate. Etiologic agents are filamentous fungi, and infection among humans normally occurs through spore inhalation. A 61-year-old male individual, presenting left eye amaurosis, dark epistaxis, hyperalgesia and malodor underwent clinical examination, which detected ulcerative lesion and wide bone exposure in the hard palate and alveolar ridge. Direct microbiological examination, microbiological culture and lesion biopsy were performed. Non-septate smooth fungal hyphae forming right angles with each other were observed through the direct microbiological examination. Microbiological culture revealed fast-growing fungal colonies with cottony texture, identified as Rhizopus sp. Histopathological examination exhibited necrosis areas, intense mononuclear inflammatory infiltrate and bulky hyphae, thus concluding the mucormycosis diagnosis. Amphotericin B antifungal therapy and surgical intervention were adopted as treatment. The patient was then rehabilitated with maxillofacial prosthesis, subsequently to the healing of the surgical wound.

Combination efficacy of voriconazole and amphotericin B in the experimental disease in immunodeficient mice caused by fluconazole-resistant Cryptococcus neoformans.

The therapeutic efficacy of amphotericin B and voriconazole alone and in combination with one another were evaluated in immunodeficient mice (BALB/c-SCID) infected with a fluconazole-resistant strain of Cryptococcus neoformans var. grubii. The animals were infected intravenously with 3 × 10(5) cells and intraperitoneally treated with amphotericin B (1.5 mg/kg/day) in combination with voriconazole (40 mg/kg/days). Treatment began 1 day after inoculation and continued for 7 and 15 days post-inoculation. The treatments were evaluated by survival curves and yeast quantification (CFUs) in brain and lung tissues. Treatments for 15 days significantly promoted the survival of the animals compared to the control groups. Our results indicated that amphotericin B was effective in assuring longest-term survival of infected animals, but these animals still harbored the highest CFU of C. neoformans in lungs and brain at the end of the experiment. Voriconazole was not as effective alone, but in combination with amphotericin B, it prolonged survival for the second-longest time period and provided the lowest colonization of target organs by the fungus. None of the treatments were effective in complete eradication of the fungus in mice lungs and brain at the end of the experiment.

Preparation, Antimicrobial Properties, and Cytotoxicity of Acrylic Resins Containing Poly(diallyldimethylammonium chloride).

PURPOSE: To design and analyze the biologic properties (antibacterial and antifungal, as well as cytotoxicity) of a dental biomaterial based on incorporation of the biocide poly(diallyldimethylammonium chloride) (PDADMAC) into the masses of self- and thermopolymerizable acrylic resins. MATERIALS AND METHODS: PDADMAC was diluted into tetrahydrofuran (4 wt%) and incorporated into self- and thermopolymerizable acrylic resins. PDADMAC inclusion was verified by measuring the contact angle with water droplets. Plain resins were used as controls. The antibacterial activity was evaluated against Staphylococcus aureus (ATCC 6538P) and Escherichia coli (ATCC 8739), and the antifungal activity was tested against Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 16404). The cytotoxicity of substances leached from these materials was analyzed in human dental pulp stem cells using MTT reduction assay. RESULTS: Reduction of contact angle confirmed the incorporation of PDADMAC in the resins. Both resins containing PDADMAC were more effective against Gram-positive and Gram-negative bacteria than their controls. The modified resins were also significantly more effective against Candida albicans than controls, but no resin was effective against Aspergillus niger. The cell viability of cultures submitted to substances leached from the PDADMAC resins was similar to that of cells cultured under ideal conditions. CONCLUSION: The incorporation of PDADMAC into the acrylic resins achieved the desired antibacterial effect, with no changes in the biocompatibility properties of the resins. Moreover, the modified resins were effective against Candida albicans, the most common fungus in the oral cavity. Thus, the incorporation of PDADMAC in biomaterials seems to be promising in dentistry.

Correlation between Etest, disk diffusion, and microdilution methods for antifungal susceptibility testing of Candida species from infection and colonization.

The correlation between the microdilution (MD), Etest (ET), and disk diffusion (DD) methods was determined for amphotericin B, itraconazole and fluconazole. The minimal inhibitory concentration (MIC) of those antifungal agents was established for a total of 70 Candida spp. isolates from colonization and infection. The species distribution was: Candida albicans (n=27), C. tropicalis (n=17), C. glabrata (n=16), C. parapsilosis (n=8), and C. lusitaniae (n=2). Non-Candida albicans Candida species showed higher MICs for the three antifungal agents when compared with C. albicans isolates. The overall concordance (based on the MIC value obtained within two dilutions) between the ET and the MD method was 83% for amphotericin B, 63% for itraconazole, and 64% for fluconazole. Considering the breakpoint, the agreement between the DD and MD methods was 71% for itraconazole and 67% for fluconazole. The DD zone diameters are highly reproducible and correlate well with the MD method, making agar-based methods a viable alternative to MD for susceptibility testing. However, data on agar-based tests for itraconazole and amphotericin B are yet scarce. Thus, further research must still be carried out to ensure the standardization to other antifungal agents.

Antifungal efficiency of chemically and thermally-activated acrylic resins after surface treatment using poly (diallyldimethylammonium chloride).

INTRODUCTION: Acrylic resins are used in the preparation of facial prostheses and may be colonized by fungi. Here, we verified the antifungal efficacy of this material after surface treatment using poly (diallyldimethylammonium chloride). METHODS: Acrylic resin specimens with and without surface treatment were subjected to tests for fungistatic and fungicidal activities. Standard strains of Candida albicans and Aspergillus niger were used. RESULTS: After surface treatment, the fungistatic and fungicidal efficacies of the resins against C. albicans and fungistatic action against A. niger were verified. CONCLUSIONS: The surface treatment was a determinant of the antifungal activity of the material.

Apiotrichum veenhuisii isolated from a pediatric patient with acute myeloid leukemia: The first case in humans.

This is the first report of the yeast Apiotrichum veenhuisii (formerly Trichosporon veenhuisii) causing disease in humans; its virulence and in vitro behavior against antifungals were also studied. The sample was isolated from biopsy fragments of disseminated lesions on the skin of a pediatric patient with acute myeloid leukemia. The studied virulence factors evidenced that the strain tested negative for secretion of the enzymes proteinase, phospholipase, and hemolysin. The isolate was characterized as low biofilm producer. Except for amphotericin B and voriconazole, the sample presented high minimum inhibitory concentration values against azole and echinocandins.

Activity of Tri-N-Butyl Tin maleate in carpets against Staphylococcus aureus and Aspergillus niger, verified through two methodologies: Inhibition Halo (HZ) and Inhibition Surface (Print).

The aim of the present study was to verify the activity of the Tri-N-Butyl Tin maleate compound against Staphylococcus aureus and Aspergillus niger, after its industrial application in 40 samples of carpets of different materials (polypropylene, polyester, polyamide and wool). The qualitative assays were performed through two methodologies: Inhibition Halo (HZ) and Inhibition of Surface (Print). The carpet with the product inhibited 100% of bacterial (Staphylococcus aureus) and fungi (Aspergillus niger) growth, under the conditions of this study. The microbial inhibition was higher in upper portion of carpets. The methodologies employed appear to be adequate to test the bactericide and fungicide activities of the Tri-N-Butyl Tin maleate. The print methodology confirmed the results obtained by the inhibition zone assay. Further studies using the same methodologies are needed to confirm our results.