Multi-well fungal co-culture for de novo metabolite-induction in time-series studies based on untargeted metabolomics.

The induction of fungal metabolites by fungal co-cultures grown on solid media was explored using multi-well co-cultures in 2 cm diameter Petri dishes. Fungi were grown in 12-well plates to easily and rapidly obtain the large number of replicates necessary for employing metabolomic approaches. Fungal culture using such a format accelerated the production of metabolites by several weeks compared with using the large-format 9 cm Petri dishes. This strategy was applied to a co-culture of a Fusarium and an Aspergillus strain. The metabolite composition of the cultures was assessed using ultra-high pressure liquid chromatography coupled to electrospray ionisation and time-of-flight mass spectrometry, followed by automated data mining. The de novo production of metabolites was dramatically increased by nutriment reduction. A time-series study of the induction of the fungal metabolites of interest over nine days revealed that they exhibited various induction patterns. The concentrations of most of the de novo induced metabolites increased over time. However, interesting patterns were observed, such as with the presence of some compounds only at certain time points. This result indicates the complexity and dynamic nature of fungal metabolism. The large-scale production of the compounds of interest was verified by co-culture in 15 cm Petri dishes; most of the induced metabolites of interest (16/18) were found to be produced as effectively as on a small scale, although not in the same time frames. Large-scale production is a practical solution for the future production, identification and biological evaluation of these metabolites.

Comparison of three antigenic extracts of Eurotium amstelodami in serological diagnosis of farmer's lung disease.

In France and Finland, farmer's lung disease (FLD), a hypersensitivity pneumonitis common in agricultural areas, is mainly caused by Eurotium species. The presence of antibodies in patients' serum is an important criterion for diagnosis. Our study aimed to improve the serological diagnosis of FLD by using common fungal particles that pollute the farm environment as antigens. Fungal particles of the Eurotium species were observed in handled hay. A strain of Eurotium amstelodami was grown in vitro using selected culture media; and antigen extracts from sexual (ascospores), asexual (conidia), and vegetative (hyphae) forms were made. Antigens were tested by enzyme-linked immunosorbent assay (ELISA), which was used to test for immunoglobulin G antibodies from the sera of 17 FLD patients, 40 healthy exposed farmers, and 20 nonexposed controls. The antigens were compared by receiver operating characteristic analysis, and a threshold was then established. The ascospores contained in asci enclosed within cleistothecia were present in 38% of the hay blades observed; conidial heads of aspergillus were less prevalent. The same protocol was followed to make the three antigen extracts. A comparison of the results for FLD patients and exposed controls showed the area under the curve to be 0.850 for the ascospore antigen, 0.731 for the conidia, and 0.690 for the hyphae. The cutoffs that we determined, with the standard deviation for measures being taken into account, showed 67% for sensitivity and 92% for specificity with the ascospore antigen. In conclusion, the serological diagnosis of FLD by ELISA was improved by the adjunction of ascospore antigen.

Impact of targeted antifungal prophylaxis in heart transplant recipients at high risk for early invasive fungal infection.

BACKGROUND: Invasive fungal infection (IFI) is associated with high mortality after heart transplantation (HTx). After two undiagnosed fatal cases of early disseminated fungal infections in our heart transplant program, a retrospective analysis was conducted to identify risk factors for the development of IFI and implement a new antifungal prophylaxis policy. METHODS: Clinical characteristics of HTx recipients hospitalized in our center (2004-2010) were recorded (Period 1), and risk factors associated with IFI were investigated using Cox regression analysis. From October 2010 to October 2012 (Period 2), targeted caspofungin prophylaxis was administered to all recipients at high risk for IFI, based on the results of the Period 1 analysis. RESULTS: During Period 1, 10% (6/59) of the patients developed IFI at a median onset of 9 days after transplantation. By multivariate analysis, the use of posttransplant extracorporeal membrane oxygenation (ECMO) was the strongest predictor for fungal infection (OR, 29.93; 95% CI, 1.51-592.57, P=0.03), whereas renal replacement therapy (RRT) and Aspergillus colonization were significant predictors only by univariate analysis. During Period 2, only 4% (1/26) of the patients developed IFI. In patients at high risk for IFI, antifungal prophylaxis was administered to 17% (4/23) in Period 1 versus 100% (13/13) in Period 2 (P<0.01). By survival analysis, antifungal prophylaxis was associated with a reduction in 90-day IFI incidence (HR, 0.14; 95% CI, 0.03-0.84, P=0.03) and 30-day mortality (HR, 0.25; 95% CI, 0.09-0.8, P=0.02). CONCLUSION: Extracorporeal membrane oxygenation was identified an important risk factor for IFI after HTx, and its use may require targeted administration of antifungal prophylaxis in the immediate posttransplant period.

Multiplex blood PCR in combination with blood cultures for improvement of microbiological documentation of infection in febrile neutropenia.

The frequent lack of microbiological documentation of infection by blood cultures (BC) has a major impact on clinical management of febrile neutropenic patients, especially in cases of unexplained persistent fever. We assessed the diagnostic utility of the LightCycler SeptiFast test (SF), a multiplex blood PCR, in febrile neutropenia. Blood for BC and SF was drawn at the onset of fever and every 3 days of persistent fever. SF results were compared with those of BC, clinical documentation of infection, and standard clinical, radiological, and microbiological criteria for invasive fungal infections (IFI). A total of 141 febrile neutropenic episodes in 86 hematological patients were studied: 44 (31%) microbiologically and 49 (35%) clinically documented infections and 48 (34%) unexplained fevers. At the onset of fever, BC detected 44 microorganisms in 35/141 (25%) episodes. Together, BC and SF identified 78 microorganisms in 61/141 (43%) episodes (P = 0.002 versus BC or SF alone): 12 were detected by BC and SF, 32 by BC only, and 34 by SF only. In 19/52 (37%) episodes of persistent fever, SF detected 28 new microorganisms (7 Gram-positive bacterial species, 15 Gram-negative bacterial species, and 6 fungal species [89% with a clinically documented site of infection]) whereas BC detected only 4 pathogens (8%) (P = 0.001). While BC did not detect fungi, SF identified 5 Candida spp. and 1 Aspergillus sp. in 5/7 probable or possible cases of IFI. Using SeptiFast PCR combined with blood cultures improves microbiological documentation in febrile neutropenia, especially when fever persists and invasive fungal infection is suspected. Technical adjustments may enhance the efficiency of this new molecular tool in this specific setting.

Sensing of mammalian IL-17A regulates fungal adaptation and virulence.

Infections by opportunistic fungi have traditionally been viewed as the gross result of a pathogenic automatism, which makes a weakened host more vulnerable to microbial insults. However, fungal sensing of a host's immune environment might render this process more elaborate than previously appreciated. Here we show that interleukin (IL)-17A binds fungal cells, thus tackling both sides of the host-pathogen interaction in experimental settings of host colonization and/or chronic infection. Global transcriptional profiling reveals that IL-17A induces artificial nutrient starvation conditions in Candida albicans, resulting in a downregulation of the target of rapamycin signalling pathway and in an increase in autophagic responses and intracellular cAMP. The augmented adhesion and filamentous growth, also observed with Aspergillus fumigatus, eventually translates into enhanced biofilm formation and resistance to local antifungal defenses. This might exemplify a mechanism whereby fungi have evolved a means of sensing host immunity to ensure their own persistence in an immunologically dynamic environment.

Antifungal drug resistance mechanisms in fungal pathogens from the perspective of transcriptional gene regulation.

Fungi are primitive eukaryotes and have adapted to a variety of niches during evolution. Some fungal species may interact with other life forms (plants, insects, mammals), but are considered as pathogens when they cause mild to severe diseases. Chemical control strategies have emerged with the development of several drugs with antifungal activity against pathogenic fungi. Antifungal agents have demonstrated their efficacy by improving patient health in medicine. However, fungi have counteracted antifungal agents in several cases by developing resistance mechanisms. These mechanisms rely on drug resistance genes including multidrug transporters and drug targets. Their regulation is crucial for the development of antifungal drug resistance and therefore transcriptional factors critical for their regulation are being characterized. Recent genome-wide studies have revealed complex regulatory circuits involving these genetic and transcriptional regulators. Here, we review the current understanding of the transcriptional regulation of drug resistance genes from several fungal pathogens including Candida and Aspergillus species.

Comportamento de fungos e de sementes de feijoeiro durante o teste de envelhecimento artificial

O teste de envelhecimento artificial, recomendado para avaliar o vigor de lotes de sementes, apresenta variabilidade em seus resultados; a ação dos fungos é considerada uma das causas dessa variabilidade. Este trabalho objetivou verificar os efeitos de diferentes períodos de envelhecimento artificial, no comportamento fisiológico de sementes do feijoeiro e dos fungos Aspergillus spp., Penicillium spp., Fusarium oxysporum e Colletotrichum lindemuthianum, inoculados artificialmente. Foram conduzidos testes de sanidade, germinação, tetrazólio, emergência, condutividade elétrica e lixiviação de potássio. As respostas obtidas, dependentes da duração do período de envelhecimento, indicaram efeitos da espécie fúngica presente. Concluiu-se que o teste de envelhecimento artificial associa a expressão de causas fisiológicas e sanitárias, o que prejudica a interpretação dos dados obtidos; a presença de fungos, principalmente de Aspergillus spp., pode ser considerada como capaz de interferir de modo negativo no desempenho das sementes envelhecidas artificialmente.

Alterações dos padrões de isoenzimas em sementes de milho infectadas por fungos

Este trabalho teve como objetivo estudar a interferência dos fungos Aspergillus flavus, Fusarium moniliforme e Penicillium spp. sobre padrões eletroforéticos das sementes de milho. Tais padrões são, normalmente, utilizados na identificação de cultivares e na certificação da pureza genética da espécie em estudo. Sementes da cultivar C-805 foram infectadas artificialmente com os referidos fungos; outra parte delas foi tratada com Benomil e Thiabendazol, e ainda outra parte (controle) não foi tratada. As amostras foram acondicionadas em câmara de crescimento (25°C, 95% de umidade relativa) por um período de 30 dias. Na análise eletroforética foi avaliada também uma amostra de sementes que não permaneceu em câmara de crescimento, visando detectar possíveis interferências das condições do ambiente de crescimento sobre os padrões eletroforéticos. Os resultados obtidos permitiram concluir que a infecção das sementes com os fungos Aspergillus flavus, Fusarium moniliforme e Penicillium spp. promove alterações nos padrões eletroforéticos das isoenzimas malato-desidrogenase, esterase, fosfatase ácida, peroxidase e glutamato-oxalacetato-transaminase. A infecção das sementes com Aspergillus flavus promove alterações tanto na intensidade como no número de bandas dos padrões isoenzimáticos da álcool-desidrogenase e malato-desidrogenase.

Histone deacetylase inhibition as an alternative strategy against invasive aspergillosis.

Invasive aspergillosis (IA) is a life-threatening infection due to Aspergillus fumigatus and other Aspergillus spp. Drugs targeting the fungal cell membrane (triazoles, amphotericin B) or cell wall (echinocandins) are currently the sole therapeutic options against IA. Their limited efficacy and the emergence of resistance warrant the identification of new antifungal targets. Histone deacetylases (HDACs) are enzymes responsible of the deacetylation of lysine residues of core histones, thus controlling chromatin remodeling and transcriptional activation. HDACs also control the acetylation and activation status of multiple non-histone proteins, including the heat shock protein 90 (Hsp90), an essential molecular chaperone for fungal virulence and antifungal resistance. This review provides an overview of the different HDACs in Aspergillus spp. as well as their respective contribution to total HDAC activity, fungal growth, stress responses, and virulence. The potential of HDAC inhibitors, currently under development for cancer therapy, as novel alternative antifungal agents against IA is discussed.

Diagnostic des onychomycose a moisissures et importance pour le traitement [Diagnosis of non dermatophyte onychomycosis ant its relevance for treatment].

PCR methods are reliable and suitable to in situ identify dermatophytes, yeasts and non dermatophyte moulds (NDM) in onychomycosis. Onychomycosis insensitive to standard treatment with topical agents as well as with oral terbinafine or itraconazole revealed Fusarium spp., Acremonium spp. and Aspergillus spp. as infectious agents. However, NDM onychomycosis could be efficiently cured using topical amphotericin B. In conclusion, correct fungal species identification is important in onychomycoses in order to prescribe adequate treatments since dermatophytes and moulds have different sensitivities to antifungal drugs.

Atividade de microrganismos solubilizadores de fosfatos na presença de nitrogênio, ferro, cálcio e potássio

A capacidade e o potencial de solubilização de 21 isolados de microrganismos solubilizadores de fosfatos (Bacillus, Pseudomonas, Enterobacteriaceae, Penicillium, Aspergillus e Paecilomyces) foram avaliados em cultivos em meio de cultura Glicose-Extrato de Levedura contendo diferentes fosfatos (Ca, Al ou Fe), na presença de fontes de N (peptona, amônio e nitrato) e teores de Fe, Ca e K. O crescimento e a atividade solubilizadora variaram em função do tipo de microrganismo e dos fatores nutricionais. Em relação às fontes de N, a presença de amônio favoreceu a solubilização em seis isolados; destes, três solubilizaram somente nesta fonte. O nitrato diminuiu a atividade solubilizadora, reduzindo ou inibindo a solubilização. Para a maioria dos microrganismos, a atividade solubilizadora não foi afetada pelas variações nos teores de ferro. Baixos teores de Ca e K limitaram o crescimento de cinco isolados que apresentam características de amplo crescimento (Aspergillus). Em dois desses isolados, a solubilização de fosfato de Ca foi favorecida. Variações na capacidade e no potencial de solubilização dos microrganismos, em resposta às condições do meio de cultura, indicam que o processo ocorre com eficiência variável ou sugerem a presença de diferentes mecanismos de solubilização.

Effect of two microbial phytase preparations on phosphorus utilisation in broilers fed maize-soybean meal based diets

Selostus: Fytaasientsyymilisäyksen vaikutus fosforin hyväksikäyttöön maissi-soijarouhepohjaisessa broilerrehussa

Respostas da cultura do rabanete à inoculação de fungos solubilizadores de fosfatos

O objetivo deste trabalho foi estudar o efeito dos fungos solubilizadores de fosfatos MSF-044, MSF-062 (Penicillium sp.) e MSF-087 (Aspergillus sp.) e da aplicação de fosfato solúvel [Ca(H2PO4)2.2H 2O], nas doses de 0,0 , 4,5 , 9,0 , 17,5 , 35,0 , 70,0 e 140,0 mg kg-1 de P, na produção de matéria seca e na absorção de fósforo pela cultura do rabanete. O experimento foi realizado em solo não-estéril, em casa de vegetação. Os isolados diferiram quanto à capacidade de promover a produção de matéria seca, dependendo da dose de P aplicada. Os isolados MSF-044 e MSF-062 foram mais eficientes sob baixas doses (4,5 a 17,5 mg kg-1 de P), enquanto o isolado MSF-087 proporcionou maior produção de matéria seca somente na dose mais alta (140,0 mg kg-1 de P). Plantas submetidas à inoculação do isolado MSF-062 (Penicillium sp.) e com 17,5 mg kg-1 de P apresentaram produção de matéria seca equivalente às obtidas por plantas com até 70,0 mg de P por kg de solo, sem inoculação. A quantidade total de P absorvida pelas plantas não foi influenciada pela inoculação, no solo, dos isolados testados.

Efeito da calagem, da colheita e da secagem na qualidade sanitária de amendoim na seca

O objetivo deste trabalho foi avaliar o efeito da calagem, da época de colheita e do método de secagem na qualidade sanitária do amendoim (Arachis hypogaea L.), cultivar Botutatu, cultivado no campo na época da seca. O delineamento experimental foi o de blocos ao acaso em parcela subsubdividida, com quatro repetições. As parcelas foram constituídas por ausência e presença de calcário dolomítico (1,8 t/ha), as subparcelas, por quatro épocas de colheita, a partir de 104 dias após a semeadura, e as subsubparcelas, por duas condições de secagem (estufa a 30ºC e ambiente a 24ºC e 60% de umidade relativa do ar). Em cada colheita, foi realizada a avaliação da população de fungos no solo, nas sementes e nas vagens, assim como do potencial de produção de aflatoxina destes isolados. A calagem não interfere na população de Aspergillus spp. no solo e não previne sua contaminação nas vagens e nas sementes do amendoim; o atraso na época de colheita proporciona aumento da contaminação de Aspergillus flavus nas vagens e da produção de aflatoxina G1 e G2; as condições de secagem em ambiente propiciam maior incidência por Rhizopus spp. nas vagens e menor incidência por Aspergillus flavus nas sementes.

Diagnostic and identification in situ of fungal pathogens in superficial mycosis

Fungi are divided in 3 groups in the field of medical mycology. The dermatophytes are filamentous fungi able to grow on keratinized tissues from human or animals. They are the main cause of superficial and cutaneous mycoses of the skin and its appendix (hair and nail). The yeasts, or dimorphic fungi, can be responsible of diverse types of infections (superficial to deep mycoses). The moulds include all Non-dermatophyte Filamentous Fungi (NDF). In medical mycology, the most representative moulds are Aspergillus spp., Fusarium spp. and Mucor spp. Diagnosis of mycosis is currently based on direct mycological examination of biological samples, as well as macroscopic and microscopic identification of the infectious fungus in culture assay. However, culture assays were found to remain sterile in roughly 40% of cases otherwise positive by direct mycological examinations. Additionally, results from culture assays are often difficult to interpret as various NDF are sometimes isolated. This thesis work is composed of three projects focusing on the development of new assays for direct in situ identification of fungi from dermatological samples. Part 1. A Polymerase Chain Reaction - Terminal Restriction Fragment Length Polymorphism assay (PCR-TRFLP) targeting the 28S rDNA was developed to identify dermatophytes and NDF in nails with suspected onychomycosis. This method is faster and more efficient than culture. It further enables the distinction of more than one agent in case of mixed infection. A fast and reliable assay for the identification of dermatophytes and NDF in onychomycosis was found to be highly relevant since onychomycosis with Fusarium spp. or other NDF are weakly responsive or unresponsive to standard onychomycosis treatments with oral terbinafine and itraconazole. Part 2. A nested PCR-sequencing assay targeting the 28S rDNA was developed to identify dermatophyte species in skin and hair samples. This method is especially suitable for tinea capitis where dermatophytes identification is critical for subsequently prescribing the adequate treatment. The challenge presented when performing direct PCR fungi identification in skin and hair differs from that seen in onychomycosis as small amount of material is generally collected, few fungal elements are present in the clinical sample and one dermatophyte among a dozen species must be identified. Part 3. Fusarium spp. is currently isolated from nails with a frequency of 15% of that of dermatophytes in the laboratory of Mycology of the CHUV (2005-2012). The aim of this work was to examine if the intensive use of terbinafine and itraconazole could be a cause of the high incidence of Fusarium nail infections. For that purpose, two different methods, specific PCR and TRFLP, were used to detect both Fusarium spp. and Trichophyton spp. in nails of previously treated or untreated patients. TRFLP assay was found to be less sensitive than classical PCR assays specifically detecting Fusarium spp. or Trichophyton spp. Independently of the detection method used, the prevalence of Fusarium spp. appears not to be higher in patients previously treated by oral standard treatment with terbinafine and azoles which are highly effective to fight Trichophyton spp. in nails. In many cases Fusarium sp. was detected in samples of patients not previously subjected to antifungal therapy. Therefore, these treatments do not appear to favor the establishment of Fusarium spp. after elimination of a dermatophyte in nail infection. - En mycologie médicale, les champignons sont classés en 3 groupes. Les dermatophytes sont des champignons filamenteux capables de se développer dans les tissus kératinisés des hommes et des animaux, ils représentent la principale cause des mycoses superficielles et cutanées de la peau et de ses appendices (ongles et cheveux). Les levures, ou champignons dimorphiques, peuvent être responsables de divers types d'infections (superficielles à profondes). Les moisissures incluent tous les champignons filamenteux non-dermatophytes (NDF), les Aspergillus spp., les Fusarium spp. et les Mucor spp. sont les principales espèces rencontrées. Le diagnostic d'une mycose est basé sur un examen mycologique direct des prélèvements biologiques ainsi que sur l'identification macroscopique et microscopique du champignon infectieux isolé en culture. Cependant, dans environ 40% des cas, l'identification de l'agent pathogène est impossible par cette méthode car la culture reste stérile, bien que l'examen direct soit positif. De plus, la croissance de moisissures et/ou autres contaminants peut rendre l'interprétation de l'examen difficile. Ce travail de thèse est composé de trois projets focalisés sur le développement de nouvelles méthodes d'identification des champignons directement à partir d'échantillons dermatologiques. Projet 1. Une méthode de Réaction en chaîne de polymérase couplée à du polymorphisme de longueur des fragments de restriction terminaux (PCR-TRFLP), en ciblant l'ADN ribosomal 28S, a été développée pour l'identification des dermatophytes et moisissures dans les ongles avec suspicion d'onychomycoses. Cette technique s'est avérée plus rapide et plus efficace que la culture, permettant l'identification de plusieurs champignons en même temps. Posséder une méthode d'identification rapide et fiable des dermatophytes et des NDF dans les onychomycoses a été jugée nécessaire du fait que les Fusarium et d'autres NDF sont peu ou pas sensibles aux traitements oraux standards à la terbinafine et à Γ itraconazole. Projet 2. Une PCR nichée couplée au séquençage d'un fragment de l'ADN ribosomal 28S a été développée afin de différencier les dermatophytes dans la peau et les cheveux. Cette méthode est particulièrement adaptée au cas de tinea capitis, où l'identification du dermatophyte est essentielle afin de prescrire le traitement adéquat. Le problème de l'identification du pathogène fongique dans les cheveux et la peau diffère des onychomycoses car de petites quantités sont prélevées chez les patients, peu d'éléments fongiques sont présents et il faut discriminer un dermatophyte parmi une douzaine d'espèces potentielles. Projet 3. Au laboratoire de Mycologie du CHUV, les Fusarium ont été isolé dans les ongles à une fréquence de 15% pour la période 2005-2012. Le but de ce travail était d'examiner si l'utilisation intensive de terbinafine et d'itraconazole pouvait être une des causes de la forte incidence des infections des ongles par Fusarium. A cet effet, deux méthodes ont été utilisées pour détecter à la fois Fusarium spp. et Trichophyton spp., la PCR spécifique et le TRFLP. Indépendamment de la méthode choisie, il en résulte que la prévalence des Fusarium η'apparaît pas liée à un traitement au préalable des patients avec de la terbinafine ou des azoles, thérapies très efficaces contre les Trichophyton spp. dans les ongles. De plus, il existe de nombreux cas où Fusarium était détecté chez des patients non traités.