The Chilean Network of Microbial Culture Collections: Establishment and Operation

According to the data available at the World Data Center for Microorganism-WDCM from the Word Federation for Culture Collection-WFCC, Chile has four registered culture collections that preserve 2777 microbial strains. At the global point of view, the culture collections in Chile are in different level of operation regarding its own infrastructure and compliancy with quality standards for preservation of strains and for services provide. The absence of funding to support the preservation of the Chilean microbial assets is a key issue for the development of the Chilean bioeconomy. Considering this, the Chilean culture collections started working together to establish the Chilean Network of Microbial Culture Collections (RCCCM, acronym in Spanish). In this note, the establishment and operation of the RCCCM is presented and discussed

MALDI-TOF ICMS: avanços na identificação de fungos

O sistema mais antigo pam a classificação das espécies de fungos, que incluem fungos filamentosos e levedums, são baseados em dados morfulógicos, principalmente naqueles ligados às estrutums reprodutivas. No entanto, este método de classificação apresenta limitações criticas, tais como as cultums de fungos que não desenvolvem estrutums reprodutivas, ou a semelbança morfológica entre membros de espécies diferentes. A incorpomção de testes bioquímicos e moleculares em taxonomia de fungos tem ajudado a resolver esses problemas. Apesar destes avanços as principais limitações situam""e por (I) os testes fisiológicos rápidos e fiáveis e os dados das sequências estão disponíveis ainda apenas pam um número limitado de taxa; (2) a aplicação de métodos moleculares à rotina é relativamente caro e exige mão-de-obm altamente especializada; (3) apresentam atmsos na identificação (que em alguns casos pode ser de semanas) bem como limites na discriminação de espécies relacionadas. A espectrometria de massa, pela técnica de MALDI-TOF ICMS, tem sido usada como uma abordagem fenotípica pam a identificação rápida de fungos. Quando aplicada à identificação de fungos, esta técnica está fundamentada na análise das proteínas constituintes das células microbianas intactas, onde o espectro de massa das proteínas é gemdo e interpretado como um fingerprint celular. Pam tal, uma pequena quantidade da amostm do material biológico - cerca de 50 J!g de biomassa - é tmnsferida directamente da placa de cu! tum pam a placa de MALDI-TOF e recoberta por uma matriz orgãnica em solução aquosa e acidificada. A acidez desta solução é fundamental pam uma extmcção proteica óptima. Depois de evapomda a tàse líquida, obtém-se um material cristalizado, necessário à ionização das moléculas. As amostms são, então, submetidas a um sistema de vácuo e irmdiadas por um laser pulsado de nitrogénio a 337 um. Esta irmdiação conduz à ionização suave das moléculas, onde a matriz orgãnica previne a fragmentação molecular. A nuvem de iões gemda dumnte a ionização é acelemda pam dentro do tubo "TOF', onde esses iões são sepamdos de acordo com os seus tempos de voos individuais. O tempo de voo de cada ião ocorre em função da mzão massa/carga (m/z) e os espectros finais são obtidos numa escala de 2 a 20 kDa. Finalmente, esses espectros são tmtados numa base de dados contendo espectros teóricos e experimentais pam as diferentes espécies de fungos. A presente técnica é bastante robusta na identificação de fungos até ao nível de espécie. Contudo, em alguns casos, é possível a diferenciação desses microrganismos até ao nível de estirpe. No presente tmbalbo serão apresentados os últimos avanços sobre a técnica de MALDI-TOF aplicada à identificação de fungos, desenvolvidos na Micoteca da Universidade do Minho (www.micoteca.deb.uminbo.pt)

Public service culture collections of microorganisms: are they important for biotechnology?

Microbiology as a scientific discipline recognised the need to preserve microorganisms for scientific studies establishing from its very beginning research culture collections (CC). Later on, to better serve different scientific fields and bioindustries with the increasing number of strains of scientific, medical, ecological and biotechnological importance public service CC were established with the specific aims to support their user communities. Currently, the more developed public service CC are recognised as microBiological Resources Centres (mBRC). mBRC are considered to be one of the key elements for sustainable international scientific infrastructure, which is necessary to underpin successful delivery of the benefits of biotechnology, whether within the health sector, the industrial sector or other sectors, and in turn ensure that these advances help drive economic growth. In more detail, mBRCs are defined by Organisation for Economic Co-operation and Development (OECD) as service providers and repositories of the living cells, genomes of organisms, and information relating to heredity and functions of biological systems. mBRCs contain collections of culturable organisms (e.g., microorganisms, plant, animal cells), replicable parts of these (e.g. genomes, plasmids, virus, cDNAs), viable but not yet culturable organisms, cells and tissues, as well as database containing molecular, physiological and structural information relevant to these collections and related bioinformatics. Thus mBRCs are fundamental to harnessing and preserving the world’s microbial biodiversity and genetic resources and serve as an essential element of the infrastructure for research and development. mBRCs serve a multitude of functions and assume a range of shapes and forms. Some are large national centres performing a comprehensive role providing access to diverse organisms. Other centres play much narrower, yet important, roles supplying limited but crucial specialised resources. In the era of the knowledge-based bio-economy mBRCs are recognised as vital element to underpinning the biotechnology

MALDI-TOF MS: uma década de experiência na aplicação em microbiologia, sempre olhando o futuro

Matrix Assisted Laser Desorption/Ionisation – Time Of Flight Mass Spectrometry (MALDI-TOF MS) é uma técnica físico-química recentemente difundida internacionalmente no campo da microbiologia. A técnica tem dado um grande contributo para o conhecimento científico acerca da identificação de microrganismos ao nível de espécie e, em alguns casos, ao nível de linhagem. Trata-se de uma ferramenta que já tem sido eficazmente utilizada em testes de identificação rápida em microbiologia clínica, alimentar e ambiental. Ao longo da última década, foram desenvolvidos e implementados na Micoteca da Universidade do Minho (MUM), um conjunto de metodologias para a análise, caracterização e identificação de microrganimos em culturas puras, bem como para a detecção de microrganimos e de seus metabólitos, em amostras complexas. Neste período, diferentes grupos taxonômicos de fungos filaentosos, leveduras, bactérias, fagos e diatomáceas foram estudados. Neste contexto, o presente trabalho tem como objetivo principal fazer uma retrospectiva detalhada sobre os trabalhos desenvolvidos ao longo da última década na MUM em colaborações inter-institucionais, bem como apontar o que se espera para o futuro da aplicação da técnica de MALDI-TOF MS para um maior desenvolvimento da microbiologia

Limitations of MALDI-TOF MS in the fungal identification: could LC-MS/MS be a solution?

Fungal polyphasic identification aims the integration of different taxonomic characters. By using numerous techniques, it is assumed the level of variation in the technique can be reduced, although variation in the fungal specimen remains. In the ground-breaking paper by Cain et al. (1994), a new methodology for the identification of bacteria by Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) was presented, where sample preparation involved minimal purification of cells. Holland et al. (1996) described for the first time an improved method for the rapid identification of whole bacterial cells by MALDI-TOF MS, establishing the basis of the current methodology. This inspired the use of MALDI-TOF MS in fungal identifications (Kallow et al. 2006, Santos et al. 2010). MALDI-TOF MS has now been applied routinely to analyse the chemical cellular composition of microorganisms, providing rapid and discriminatory proteomic profiles for identification and subtyping. The application of this technique for the identification of clinical fungal samples is currently well-established based on the remarkable reproducibility for the measurement of constantly expressed and highly abundant proteins, such as ribosomal proteins, that are used as biomarkers to generate a fingerprint profile that range between 2 and 20 kDa. However, the fungal identification by MALDI-TOF MS can be limited in some fungal taxonomic group, especially when genetically closely related species are under evaluation. In order to overcome this limitation, new methodologies based on liquid chromatography coupled to mass spectrometry (LC-MS/MS system) have been evaluated. Although it means a methodology which involves consumables more expansive, the preliminary results obtained for fungal species have been satisfactory and have cut-edge the limitation faced in the use of MALDI-TOF MS technique. In this poster, a detailed comparison MALDI-TOF MS vs. LC-MS/MS for fungal identification will be presented and discussed.Universidad de La Frontera (Temuco, Chile) - Projects DIUFRO DI16-0135, DIUFRO PIA16-0009, Doctoral Programme in Science of Natural Resource

Intramolecular cyclization: a promising strategy in the prodrug conception

Many drugs used for the treatment of common diseases are associated with various adverse effects and limited bioavailability. The suppression of such problems continues to be a very important target for scientists. The development of prodrugs for controlled release in vivo is an attractive way to overcome these problems. Classical in prodrug is an inactive molecule which the parent drug is covalently bonded to a carrier unit, and which can liberate the drug through chemical or enzymatic pathways. A new and interesting prodrug system for amine, alcohol, and thiol drugs takes advantage of several easy intramolecular cyclization reactions. So, the cyclization process can control the release rate of the parent drug. In this paper is a review about the prodrug strategies based on intramolecular cyclization reactions is presented.Muitos fármacos estão associados a vários efeitos adversos e limitada biodisponibilidade. A resolução destes problemas continua sendo um alvo importante para a comunidade científica. O desenvolvimento de pró-fármacos que conduzam a uma liberação controlada in vivo é um caminho atrativo para resolver estes problemas. Um pró-fármaco clássico é molécula inativa, em que o princípio ativo é ligado covalentemente a uma unidade transportadora, de modo que o fármaco pode ser liberado através de uma reação química ou enzimática. Um sistema novo e interessante de pró-fármaco, que tem sido apresentado para fármacos possuidores de grupos amino, álcool e tiol, é aquele no qual o princípio ativo é liberado através de uma ciclização intramolecular. Neste caso, o processo de ciclização, dependendo da cadeia da unidade transportadora e do próprio fármaco, pode controlar a velocidade de liberação do princípio ativo. Desta forma, este trabalho apresenta uma revisão do desenvolvimento de pró-fármacos baseados na liberação do princípio ativo através de uma ciclização intramolecular

Characterization and identification of aspergillus section flavi isolates from portuguese almonds using a polyphasic approach including MALDI-TOF ICMS

Aspergillus is a large genus, with a complex and ever evolving taxonomy. Section Flavi is one of the most significant sections in the genus, and is one of the best studied among fungi, for the numerous industrial applications as well as for food safety issues. Section Flavi is composed of a large number of very closely related species. While these species are difficult to differentiate morphologically and even genetically, they differ in a characteristic that is of paramount importance for food safety, as some are responsible for the production of the highly toxigenic aflatoxins. Taxonomy and species identification are therefore subject of great interest for scientists aiming to clarify the species concept and limits within the section. In this sense, the establishment of schemes for species and for aflatoxigenic strains identification that are simultaneously accurate, sensitive, robust and expedite is mandatory. At present, reliable identification schemes typically imply the analysis of a wide variety of morphological, biochemical and molecular traits. Recently, Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) has been used to generate spectra of protein masses in a range of 2,000 to 20,000 Da, which result in a taxa specific fingerprint. This technique has already shown high potentialities to discriminate very closely related taxa, but has rarely been used in fungal species identification, either on its one or as part of a polyphasic scheme of identification. This work aimed to: i) characterize the population of Aspergillus section Flavi collected from Portuguese almonds in relation to their aflatoxigenic potential; ii) identify the isolates by applying a set of morphological, biochemical, molecular and spectral analyses (polyphasic approach); iii) compare the data obtained from the various approaches in terms of sensitivity, reliability and user-friendliness; and iii) determine the validity of MALDI-TOF technique for the identification of closely related field isolates of section Flavi

Utilization of white rot fungi for textile dye decolourisation under alkaline condition and high salt concentration in solid medium

A large amount of azo dyes are used for dyeing textiles. However, the dyes contaminate wastewaters and need to be treated. This is important because of the aesthetic, toxic and carcinogenic effects of the affected waters. Recently there has been an increase in interest in using white rot fungi (wrf) which degrade xenobiotic compounds including azo dyes. Wrf degrade lignin and others recalcitrant molecules using nonspecific extracellular enzymes. Four white rot fungi obtained from the Micoteca da Universidade do Minho (MUM) culture collection were used to screen for degradability capabilities. Reactice Black 5 (RB5) was selected in the present work because these dyes are most commonly used in the textile dyeing. Screening for RB5 decolourisation was carried out on solid medium in plates. Two wrf showed good growth and decolourisation abilities. These are now under study to determine which ligninolytic enzymes are produced