Isolation of transcripts overexpressed in the human pathogen Trichophyton rubrum grown in lipid as carbon source

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts over-expressed in T rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-beta-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

Epidemiologic aspects and clinical outcome of fungal keratitis in southeastern Brazil

PURPOSE. Fungal keratitis (FK) is a sight-threatening disease, more prevalent in developing regions. The present retrospective study was conducted in order to evaluate the epidemiologic and clinical aspects and the progression of FK in patients treated at two ophthalmologic reference centers in Southeast Brazil. METHODS. The charts of patients with infectious keratitis treated between 2000 and 2004 were reviewed. For the 66 cases of FK confirmed by microbiological analysis, data related to patient, disease, and therapeutic approaches were obtained. RESULTS. Mean patient age was 40.7 +/- 16 years. Fifty-three were men and 13 were women. Ocular trauma occurred in 40% of cases (27). Previous medications taken by the patients were quinolone in 72.5% and antimycotics in 30%. Visual acuity (VA) at presentation was >0.3 in 16% and <0.1 in 74.5%. Penetrant keratoplasty was performed in 38% and evisceration in 15%. The causing agents were Fusarium sp in 67%, Aspergillus sp in 10.5%, and Candida sp in 10%. Medication alone resolved 39% of cases within a mean period of 24.5 +/- 12 days. Final VA was >0.3 in 28%, and <0.1 in 63%. CONCLUSIONS. Fungal keratitis presented as a disease with severe complications, predominantly among young males, and was mostly caused by filamentous fungi. The present information permits the establishment of preventive strategies. Reducing the time between onset and treatment and using more accessible specific medication would reverse the negative prognosis. (Eur J Ophthalmol 2009; 19: 355-61)

Transcriptional profiling reveals genes in the human pathogen Trichophyton rubrum that are expressed in response to pH signaling

Trichophyton rubrum is a dermatophyte that infects human skin and nails. Its growth on keratin as its carbon source shifts the ambient pH from acidic to alkaline, which may be an efficient strategy for its successful infection and maintenance in the host. In this study, we used suppression subtractive hybridization to identify genes preferentially expressed in T rubrum incubated at either pH 5.0 or pH 8.0. The functional grouping of the 341 overexpressed unigenes indicated proteins putatively involved in diverse cellular processes, such as membrane remodeling, cellular transport, metabolism, cellular protection, fungal pathogenesis, gene regulation, interaction with the environment, and iron uptake. Although the basic metabolic machinery identified under both growth conditions seems to be functionally similar, distinct genes are upregulated at acidic or alkaline pHs. We also isolated a large number of genes of unknown function, probably unique to T rubrum or dermatophytes. Interestingly, the transcriptional profiling of several genes in a pacC mutant suggests that, in T rubrum, the transcription factor PacC has a diversity of metabolic functions, in response to either acidic or alkaline ambient pH. (C) 2009 Elsevier Ltd. All rights reserved.

Analysis of the Nicotiana tabacum Stigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species

The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process.

Rinossinusite fúngica em pacientes com infecção nasossinusal crônica

OBJETIVO: Estudar a rinossinusite fúngica em pacientes com infecção nasossinusal crônica. Nas últimas décadas houve aumento das infecções fúngicas, e a rinossinusite fúngica (RSF) tem sido mais freqüentemente diagnosticada. O conhecimento da flora fúngica, da sua prevalência e da apresentação sintomática em pacientes portadores de rinossinusite crônica (RSC) permitirá um melhor entendimento da doença, fato importante para a realização do diagnóstico, estabelecimento do tratamento e formulação do prognóstico. FORMA DE ESTUDO: clínico retrospectivo com coorte transversal. MATERIAL E MÉTODO: 62 pacientes com diagnóstico de RSF foram selecionados entre 890 portadores de RSC submetidos à cirurgia endoscópica. Avaliou-se anamnese, exame otorrinolaringológico com videoendoscopia nasal, TC dos seios da face e exames microbiológicos e histopatológico. RESULTADOS: A prevalência de RSF foi de 6,7% em portadores de RSC submetidos à cirurgia endoscópica dos seios paranasais, e o tipo de fungo mais encontrado foi do gênero Aspergillus. Bola fúngica foi encontrada em mais da metade dos casos, e RSFA, em mais de um terço dos pacientes. CONCLUSÕES: A evolução sintomática após a cirurgia endoscópica foi mais favorável nos portadores de bola fúngica, que necessitaram menor número de reintervenções.

Solubilization of diabase and phonolite dust by filamentous fungus

The objective of this study was to evaluate the effect of the fungus Aspergillus niger strain CCT4355 in the release of nutrients contained in two types of rock powder (diabase and phonolite) by means of in vitro solubilization trials. The experimental design was completely randomized in a 5 x 4 factorial design with three replications. It was evaluated five treatments (phonolite dust + culture medium; phonolite dust + fungus + culture medium; diabase powder + culture medium; diabase powder + fungus + culture medium and fungus + culture medium) and four sampling dates (0, 10, 20 and 30 days). Rock dust (0.4% w/v) was added to 125 mL Erlenmeyer flasks containing 50 mL of liquid culture medium adapted to A. niger. The flasks were incubated at 30°C for 30 days, and analysis of pH (in water), titratable acidity, and concentrations of soluble potassium, calcium, magnesium, zinc, iron and manganese were made. The fungus A. niger was able to produce organic acids that solubilized ions. This result indicates its potential to alter minerals contained in rock dust, with the ability to interact in different ways with the nutrients. A significant increase in the amount of K was found in the treatment with phonolite dust in the presence of the fungus. The strain CCT4355 of A. niger can solubilize minerals contained in these rocks dust.

Assessment of fungal contamination in a group of Lisbon's gymnasiums with a swimming pool

The study’s main purpose was the assessment of the environmental fungal contamination, the exploration of possible associations between related environmental variables and the study of the relationship between fungal contamination of air and surfaces. A descriptive study was developed based upon air and surfaces monitoring for fungal contamination in ten indoor gymnasiums with a swimming pool located in Lisbon’s urban area. Fifty 200 litres air samples and 120 surface swabs were collected. Surfaces samples were collected before and after cleaning and disinfection and temperature and relative humidity values were registered during the collection period. Twenty five different species of fungi were identified in the air samples, being the three most commonly isolated genera the following: Cladosporium (36.6%), Penicillium (19.0%) and Aspergillus (10.2%). Thirty-seven different species of fungi were identified in the surface samples. Fusarium sp. was the most frequent genera before (19.1%) and after (17.2%) cleaning and disinfection. There was a significant association between the numbers of visitors and the fungal contamination determined in the surface samples (p<0.05). There was no significant association (p>0.05) between the contamination encountered in the air samples and the one registered in the surface samples and between the fungal contamination and the temperature or relative humidity measured on location. The data obtained enabled the assessment of the establishment’s fungal contamination and led the authors to conclude, consequently, that physical activity, which generally promotes health, can in fact be challenged by this factor.

Prevalence of fungi in indoor air with reference to gymnasiums with swimming pools

Fungal contamination of air in 10 gymnasiums with swimming pools was monitored. Fifty air samples of 200 L each were collected, using a Millipore air tester, from the area surrounding the pool, in training studios, in showers and changing rooms for both sexes, and also, outside premises, since these are the places regarded as reference. Simultaneously, environmental parameters – temperature and humidity – were also monitored. Some 25 different species of fungi were identified. The six most commonly isolated genera were the following: Cladosporium sp. (36.6%), Penicillium sp. (19.0%), Aspergillus sp. (10.2%), Mucor sp. (7%), Phoma sp. and Chrysonilia sp. (3.3%). For yeasts, three different genera were identified, namely, Rhodotorula sp. (70%), Trichosporon mucoides and Cryptococcus uniguttulattus (10%).

Air fungal contamination in two elementary schools in Lisbon, Portugal

A descriptive study was developed to monitor air fungal contamination in two elementary schools in Lisbon, Portugal. Eight air samples of 250 litres through impaction method were collected in canteen, library, classrooms and also, outside premises as reference place. Simultaneously, were also monitored environmental parameters, including temperature, and humidity through the equipment Babouc, LSI Sistems and according to the International Standard ISO 7726 - 1998. Considering both schools, sixteen different species of fungi in air were identified, being the 2 most commonly isolated Cladosporium sp. (51,1%) and Penicillium sp. (27,5%). Besides these genera Trichoderma, Aspergillus, Alternaria, Chrysonilia, Botritys, Ulocladium, Athrium, Aureobasidium, Phoma, Scedosporium e Geotrichum were also isolated. Regarding yeasts, Candida sp., Cryptococcus sp. and Rhodotorula sp. were isolated. The youngest school, as well canteens in each school, presented the worst results concerning the air fungal contamination, maybe due to the higher number of occupants. There was no significant relationship (p>0,05) between fungal contamination and temperature and humidity.

Possíveis implicações da contaminação fúngica num aviário

Introdução – Apesar de em Portugal se verificar o aumento da indústria da produção de aves para consumo humano, apenas alguns estudos incidem sobre a qualidade do ar interior e as implicações da sua degradação. Objectivos – Descrever a contaminação fúngica num aviário, analisar possíveis associações com a temperatura ambiente e a humidade relativa e o possível impacto na saúde dos consumidores e trabalhadores desta unidade. Métodos – Foi desenvolvido um estudo descritivo para avaliar a contaminação fúngica num aviário. Colheram‑se 5 amostras de ar de 100 litros através do método de compactação e 4 amostras de superfícies, utilizando a técnica da zaragatoa e um quadrado de 10 cm de lado de metal. Simultaneamente, os parâmetros ambientais – temperatura ambiente e humidade relativa – também foram medidos. Resultados – Foram identificadas vinte espécies de fungos no ar, sendo os seguintes os quatro géneros mais comummente isolados: Cladosporium (40,5%), Alternaria (10,8%), Chrysosporium e Aspergillus (6,8%). Nas superfícies, 21 espécies de fungos foram identificadas, sendo os 4 géneros mais identificados Penicillium (51,8%), Cladosporium (25,4%), Alternaria (6,1%) e Aspergillus (4,2%). Importa referir o facto de Aspergillus flavus, também isolado no ar, ser reconhecido como produtor de micotoxinas (aflatoxina) e Aspergillus fumigatus, uma das espécies isoladas no ar e superfícies, ser capaz de causar aspergilose grave ou fatal. Não se verificou relação significativa (p> 0,05) entre a contaminação fúngica e as variáveis ambientais. Conclusão – Caracterizou‑se a distribuição fúngica no ar e superfícies do aviário e analisou‑se a possível influência das variáveis ambientais. Foi reconhecido um potencial problema de Saúde Pública devido à contaminação fúngica e à possível produção de micotoxinas com a eventual contaminação dos produtos alimentares. A contaminação fúngica, particularmente causada pelo Aspergillus fumigatus, e a possível presença de micotoxinas no ar, devem ser encaradas também como fatores de risco neste contexto ocupacional. ABSTRACT - Background – Although there is an increasingly industry that produce whole chickens for domestic consumption in Portugal, only few investigations have reported on the indoor air of these plants and the consequences of their degradation. Objectives – Describe one poultry environmental fungal contamination analyse possible associations between temperature and relative humidity and its possible impact on the health of consumers and of the poultry workers. Methods – A descriptive study was developed to monitor one poultry fungal contamination. Five air samples of 100 litres through impaction method were collected and 4 swab samples from surfaces were also collected using a 10 cm square of metal. Simultaneously, environmental parameters – temperature and relative humidity – were also measured. Results – Twenty species of fungi in air were identified, being the 4 most commonly isolated the following genera: Cladosporium (40.5%), Alternaria (10.8%), Chrysosporium and Aspergillus (6.8%). In surfaces, 21 species of fungi were identified, being the 4 genera more identified Penicillium (51.8%), Cladosporium (25.4%), Alternaria (6.1%) and Aspergillus (4.2%). In addition, Aspergillus flavus also isolated in the poultry air is a well‑known producer of potent mycotoxins (aflatoxin), and Aspergillus fumigatus, one of the species isolated in air and surfaces, is capable of causing severe or fatal aspergillosis. There was no significant relationship (p>0,05) between fungal contamination and environmental variables. Conclusions – Was characterized fungal distribution in poultry air and surfaces and analyzed the association of environmental variables. It was recognized the Public Health problem because of fungal contamination and also due to probable mycotoxins production with the possible contamination of food products. Fungal contamination, particularly due to the presence of Aspergillus fumigatus and also the possible presence of mycotoxins in the air, should be seen as risk factor in this occupational setting.

Are air-borne mycotoxins a public health concern in Portugal?

Introduction - Microscopic filamentous fungi, under suitable environmental conditions, can lead to the production of highly toxic chemical substances, commonly known as mycotoxins. The most widespread and studied mycotoxins are metabolites of some genera of moulds such as Aspergillus, Penicillium and Fusarium. Quite peculiar conditions may influence mycotoxin biosynthesis, such as climate, geographical location, cultivation practices, storage and type of substrate. Toxicity has been extensively investigated for the most important mycotoxins, such as aflatoxins, ochratoxin A and Fusarium toxins, and much information derived from toxicokinetics in animal models has also been obtained. The adverse effects are mainly related to genotoxicity, carcinogenicity, mutagenicity, teratogenicity and immunotoxicity. Aim of the study - To identify fungal species able to produce important mycotoxins in different Portuguese settings.

Aflatoxinas: conceitos sobre mecanismos de toxicidade e seu envolvimento na etiologia do câncer hepático celular

Foram revistos os conceitos de maior relevância sobre mecanismos de toxicidade e evidências do envolvimento das aflatoxinas na etiologia do câncer hepático humano. A aflatoxina B1 (AFB1), principal metabólito produzido por fungos do gênero Aspergillus, manifesta seus efeitos tóxicos após conversão hepática em AFB1-epóxido, o qual reage com macromoléculas celulares, incluindo proteínas, RNA (ácido ribonucléico) e DNA (ácido desoxirribonucléico). A reação com o DNA ocorre através da ligação com guaninas, ao nível do códon 249, do gene supressor de tumores p53. Em seres humanos, estudos de biomonitoramento individual de derivados AFB1-N7-guanina tem demonstrado que as aflatoxinas constituem importantes fatores de risco, com uma provável interação sinergística com o vírus da hepatite B, para o desenvolvimento do carcinoma hepatocelular em populações expostas. Considerando-se a ocorrência freqüente das aflatoxinas em produtos alimentícios, no Brasil, ressalta-se a necessidade de estudos que avaliem criteriosamente o impacto dos níveis de exposição a estas toxinas sobre a saúde humana.

Air contaminants in animal production: the poultry case

A descriptive study was developed in order to assess air contamination caused by fungi and particles in seven poultry units. Twenty seven air samples of 25 litters were collected through impaction method. Air sampling and particle concentration measurement were performed in the pavilions’ interior and also outside premises, since this was the place regarded as reference. Simultaneously, temperature and relative humidity were also registered. Regarding fungal load in the air from the seven poultry farms, the highest value obtained was 24040 CFU/m3 and the lowest was 320 CFU/m3. Twenty eight species/genera of fungi were identified, being Scopulariopsis brevicaulis (39.0%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. From the Aspergillus genus, Aspergillus flavus (74.5%) was the most frequently detected species. There was a significant correlation (r=0.487; p=0.014) between temperature and the level of fungal contamination (CFU/m3). Considering contamination caused by particles, in this study, particles with larger dimensions (PM5.0 and PM10) have higher concentrations. There was also a significant correlation between relative humidity and concentration of smaller particles namely, PM0.5 (r=0.438; p=0.025) and PM1.0 (r=0.537; p=0.005). Characterizing typical exposure levels to these contaminants in this specific occupational setting is required to allow a more detailed risk assessment analysis and to set exposure limits to protect workers’ health.

Avaliação da qualidade química e microbiológica de tintas didácticas do ensino pré-escolar

As tintas utilizadas nas actividades didácticas possuem na sua composição ingredientes que dada a sua natureza, modo de fabrico e de utilização, podem representar um risco para a saúde das crianças. Neste âmbito, procurou-se com este estudo avaliar a qualidade química e microbiológica das tintas utilizadas pelas crianças no ensino pré-escolar. Vinte e nove amostras de tintas, incluindo guaches, tintas de águas, digitintas e tintas para pinturas faciais foram recolhidas em oito estabelecimentos de ensino, nomeadamente, Jardins de Infância, do conselho de Vila Nova de Gaia. A avaliação microbiológica envolveu não só a determinação da concentração microbiana presente nas amostras, como também, a avaliação da estabilidade microbiana nas tintas das espécies S.aureus e E.coli. Na avaliação química procedeu-se à determinação da concentração dos metais chumbo (Pb), cádmio (Cd), crómio (Cr), cobalto (Co), níquel (Nq), manganês (Mn), cobre (Cu) e zinco (Zn) quer em algumas das amostras recolhidas nos estabelecimentos de ensino, quer em tintas adquiridas em três estabelecimentos comerciais. Os resultados obtidos da avaliação microbiológica revelam uma contaminação estática na generalidade das tintas. Três amostras de tintas apresentaram ainda elevada contaminação por fungos, nomeadamente Aspergillus spp. e Trichophyton spp. Da avaliação da estabilidade microbiana das espécies S.aureus e E.coli observou-se uma sensibilidade das mesmas às tintas, sendo evidenciado, em alguns casos, um decrescimento da concentração ao longo do tempo de exposição, e noutros, uma sensibilidade imediata. A espécie S.aureus revelou, contudo, maior capacidade de resistência que a E.coli. Os resultados obtidos da avaliação química revelaram a presença de Cr em todas as amostras, registando as tintas adquiridas em estabelecimentos comerciais concentrações mais elevadas para este metal. Os metais Cu e Zn foram detectados, em algumas amostras de tintas artísticas, em concentrações acima dos valores limites. Nas tintas para a cara foram encontrados os metais Pb, Cd, Cr e Nq, cuja utilização é interdita nestes produtos. O conhecimento das características químicas e microbiológicas das tintas utilizadas por crianças do ensino pré-escolar revelou-se de grande importância, nomeadamente, para a determinação dos riscos a que este grupo de indivíduos pode estar exposto no seu dia-a-dia quando utilizam estes produtos.

Fungal and microbial volatile organic compounds exposure assessment in a waste sorting plant

In the management of solid waste, pollutants over a wide range are released with different routes of exposure for workers. The potential for synergism among the pollutants raises concerns about potential adverse health effects, and there are still many uncertainties involved in exposure assessment. In this study, conventional (culture-based) and molecular real-time polymerase chain reaction (RTPCR) methodologies were used to assess fungal air contamination in a waste-sorting plant which focused on the presence of three potential pathogenic/toxigenic fungal species: Aspergillus flavus, A. fumigatus, and Stachybotrys chartarum. In addition, microbial volatile organic compounds (MVOC) were measured by photoionization detection. For all analysis, samplings were performed at five different workstations inside the facilities and also outdoors as a reference. Penicillium sp. were the most common species found at all plant locations. Pathogenic/toxigenic species (A. fumigatus and S. chartarum) were detected at two different workstations by RTPCR but not by culture-based techniques. MVOC concentration indoors ranged between 0 and 8.9 ppm (average 5.3 ± 3.16 ppm). Our results illustrated the advantage of combining both conventional and molecular methodologies in fungal exposure assessment. Together with MVOC analyses in indoor air, data obtained allow for a more precise evaluation of potential health risks associated with bioaerosol exposure. Consequently, with this knowledge, strategies may be developed for effective protection of the workers.