Identification and characterization of Aspergillus flavus and aflatoxins

Aspergillus flavus is the main producer of the well known carcinogenic aflatoxins. The presence of this fungus and aflatoxins is of huge concern in terms of food safety. The identification of A. flavus is not straightforward due to similarities with closely related species (e.g. A. parasiticus and A. nomius). Also, from the biochemical point of view the closely-related species are able to produce different mycotoxins. In order to clarify the differentiation between species the identification schemes is revisited. Selective media, data from mycotoxins production and molecular biology tools are discussed in order to clarify the concept of A. flavus species.Fundação para a Ciência e a Tecnologia (FCT

Modern polyphasic methods that include MALDI-TOF analyses for fungal identifications and authentications

[Excerpt] The identification of species is an important goal in taxonomic mycology. Information about each fungus (e.g. morphological description, physiological and biochemical properties, ecological roles, and societal risks or benefits) is the key element in this process. Identifications can be a long and seemingly never-ending process with frequent revisions of the taxonomic schemes. These changes make identifications even more complicated for the non specialised researchers as each taxonomic group has specialized literature, terminology and characters. This occurs to the extend that identifications can only be undertaken by a narrow group of scientists especially skilled in the "art", which can make the procedures appear to be subjective. [...

A new species of Penicillium section Ramosa from Tunisian Apples

One of the limiting factors that influence the fruits economic chain value is the relatively short shelf-life period caused by fungal infections. The symptoms of fungal infection may be observed at different times but many fungi may remain dormant for varying periods until post-harvest favorable conditions become available for their development. In a mycotoxin contamination survey of apples from markets in Tunisia, 54 Penicillium strains were isolated. However, two isolates could not be assigned to any described species based on morphological and molecular phylogenetic analyses. The aim of this study was the characterisation and description of this new putative species. For morphological analyses, MUM 17.62 and MUM 17.80 were inoculated in triplicate in CYA, YES, G25N, CSN and MEA media and incubated in the dark at 25 ºC for 7 days. CYA plates were also incubated at 30ºC and 37ºC. Colony size was measured and for microscopy analysis fungi grown in MEA was used. Multilocus sequence analysis (MLSA) was performed through comparison of partial β-tubulin (benA), calmodulin (cmd) and nuclear ribosomal internal transcribed spacer (ITS) region with sequences available in GenBank derived from type strains of Penicillium species. All the sequences were aligned and phylogenetic trees were assembled using MEGA. For MUM 17.62 and MUM 17.80 morphologically, the colonies growth was very restricted in the different media. No growth was observed on CYA at 30 ºC and 37 ºC. The strains show slight differences in green colour. Both present velutionous, sulcate and irregular colonies in MEA. Microscopically, the conidiophores are biverticillate and conidia ellipsoidal. MLSA revealed that the two strains belong to Penicillium section Ramosa. Fingerprinting using the M13 microsatellite showed that the two strains are not clones and analysis of the isoepoxydon dehydrogenase (idh) gene revealed that they lack the ability to produce patulin. In summary, in terms of multigene phylogeny the two strains are closely related to P. lenticrescens, P. chroogomphum and P. soppii of the section Ramosa however they well-circumscribe a sp. nov. coined as Penicillium tunisinus.info:eu-repo/semantics/publishedVersio

Biotipagem, sorotipagem e ribotipagem na avaliação epidemiológica de A. baumannii em unidades hospitalares, Sorocaba, São Paulo, Brasil

Dissemination of Acinetobacter baumannii strains in different units of a hospital in Sorocaba, São Paulo, Brazil was evaluated over a period of two years. By using biotyping, serotyping and ribotyping, 27 distinct clones were differentiated among 76 strains isolated between 1993-94, from clinical specimens of hospitalized patients. Two clones, 2:O4:A (biotype:serotype:ribotype) and 2:O29:A accounted for the majority of strains widely disseminated in the units during 1993. The introduction in the hospital setting, of a new clone, 6:O13:B, at the end of 1993 and its predominance through 1994 is discussed. Among 15 strains isolated from neonates, 6 (40%) belonged to the same clone, 2:O4:A. Interestingly, this clone was almost all recovered in neonatal intensive care unit, nursery and in pediatric unit. All strains were susceptible to imipenem and polymyxcin B. Multiresistant strains (up to 12 antimicrobial agents) accounted for 66.7% and 84.8% of the strains isolated in 1993 and in 1994, respectively.Foi avaliada a disseminação, durante um período de dois anos, de cepas de Acinetobacter baumannii em diferentes unidades de um hospital de Sorocaba, São Paulo.Usando as técnicas de biotipagem, sorotipagem e ribotipagem, as 76 cepas isoladas no período 1993-94, foram diferenciadas em 27 distintos clones. Dois clones, 2:O4:A (biotipo:sorotipo:ribotipo) e 2:O29:A predominaram, em 1993, entre as cepas disseminadas nas várias unidades. Observou-se no final de 1993, a introdução de um novo clone, 6:O13:B e a sua predominância em 1994. Entre as 15 cepas isoladas de recém nascidos, 6 (40%) pertenciam ao mesmo clone, 2:O4:A . A maioria das cepas desse clone foram isoladas da unidade de terapia intensiva neonatal, de berçário e de unidades pediátricas. Quanto à resistência aos agentes antimicrobianos, todas as cepas foram sensíveis à polimixina B e ao imipenen. Em 1993, 66,7% das cepas eram multi-resistentes (resistentes de 4 a 12 dos 15 antimicrobianos testados), enquanto que, em 1994, 84,8% das 46 cepas isoladas foram multi-resistentes a mais de 4 e até a 12 drogas

Micotoxinas: Contributos da Micoteca da Universidade do Minho (MUM) para a segurança alimentar

Micotoxinas são metabolites produzidos por fungos que quando ingeridos, inalados ou absorvidos através da pele causam no homem, ou em animais, problemas de saúde ou mesmo, em situações extremas, a morte. Nos últimos anos, no seio da Micoteca da Universidade do Minho, tem-se desenvolvido projectos que procuram dar resposta aos riscos alimentares derivados da contaminação fúngica. Assim, usando a técnica de cromatografia em camada fina (TLC), a patulina e a citrinina foram estudadas para avaliar o potencial micotoxigénico de Penicillium expansum isolados de uvas utilizadas na produção de vinho. Citrinina foi produzida por todas as estirpes estudadas quando estas forma crescidas em meio de sacarose e extracto de levedura (YES) mas somente uma estirpe das 51 foi capaz de produzir esta micotoxina quando foram crescidas em meio de sumo de uva (GJ). Patulina foi produzida por 20 e 31 estirpes quando as 51 estirpes cresceram respectivamente em YES ou GJ. Pelos resultados obtidos as uvas contaminadas com patulina parecem não contribuir para a contaminação do sumo de uva com esta micotoxina. Por sua vez a micoflora das uvas saudáveis utilizadas para a produção de vinho em Portugal foram analisadas quanto ao potencial de produzirem ocratoxina A (OTA). Num total de 386 estirpes foram isoladas pelo método de plaqueamento. Os géneros mais frequentes não-ocratoxigénicos foram Cladosporium (28%), Penicillium (24%), Botrytis (13%) e Aspergillus (9%). Estirpes produtoras de OTA pertenceram às espécies de Aspergillus carbonarius e A. ochraceus. Neste estudo houve ainda a possibilidade de descriminar do grupo dos aspergilli negros a nova espécie Aspergillus ibericus não produtora de OTA. Esta espécie foi totalmente caracterizada fenotipicamente, pela biologia molecular e, mais recentemente, pela análise espectral de MALDI-TOF MS. Mais recentemente, na Micoteca da Universidade do Minho, a contaminação dos frutos secos, nomeadamente, na amêndoa, tem sido um tópico de estudo. As espécies aflatoxigénicas Aspergillus flavus e A. parasiticus têm sido isoladas e identificadas seguindo a abordagem polifásica. A biologia molecular e a análise espectral por MALDI-TOF MS têm-se revelado técnicas robustas em paralelo com a análise por HPLC das aflatoxinas B1/B 2 e G1/G2 e o ácido ciclopiazónico para a compreensão dos contributos destes contaminantes neste alimento

Contributions of micoteca da Universidade do Minho to food mycology

Micoteca da Universidade do Minho (MUM) is a collection of fungi established in 1996 where research into secondary metabolites in the form of mycotoxins is paramount. An objective is to maintain and provide authentic strains for research in biotechnology, life sciences and challenge testing. Knowledge, information and training in mycology are promulgated by the biological resource centre. MUM is involved in projects which address the health and quality risks from fungal contamination of grape products (e.g. wine), apples, cheese, chillies, nuts and corn: Fungi in drinking water are of particular concern. Aspergillus ochraceous, Aspergillus flavus, Penicillium expansum and Fusarium graminearum especially are relevant to our work: Associated mycotoxins are ochratoxin A, aflatoxins, patulin, citrinin and zearalenone. We study the earthy off-flavours in wine from Botrytis cinearum and P. expansum. The analytical techniques employed are PCR, HPLC, GC-MS and MALDITOF MS for strain characterization. MUM participates in several European and national projects. Post-graduate studies on mycotoxigenic fungi and food security particularly are important. A key objective is international collaboration. MUM strains are well characterized for toxigenicity, making the collection relevant to its mission and industry. This presentation will describe the progress made in this innovative Portuguese laboratory over 10 years

Penicillium crustosum as a potential OTA producer - new insights from whole - genome sequencing of strain MUM 16.125

Ochratoxin A (OTA) is a well-studied mycotoxin that poses severe health risks. OTA is mainly produced by Aspergillus and Penicillium species associated with food spoilage and it is present in a wide diversity of food and feed products. Recent studies have reported the presence of OTA in food matrices where known OTA producers are not present1,2. For that reason, other species such as P. crustosum are now being considered. A recent study using comparative genomic analysis3 clarified the OTA biosynthetic gene cluster composition. In order to gain insight into the secondary metabolism of P. crustosum, this study aimed to sequence and explore the complete genome of strain MUM 16.125. This strain was isolated from cheese rind sample contaminated with OTA in which no known OTA producers were present1. The genome assembly comprises 199 contigs with a total length of 30.95 Mb and contains 10975 predicted protein-coding genes. In total, 109 gene clusters potentially related with secondary metabolism were identified, including putative gene clusters for penitrem, clavaric acid or naphthopyrones biosynthesis. Nevertheless, no evidence of an OTA biosynthetic gene cluster was found. A total of 83 complete and 49 partial protein sequences from published OTA biosynthetic genes from 11 Aspergillus and 3 Penicillium species were queried against the predicted P. crustosum proteins. Only 3 strong matches were found (to a short partial P. verrucosum PKS and 2 P. thymicola chloroperoxidases) but matches to complete key genes were absent. Considering these findings, it appears that strain MUM 16.125 lacks the most common genetic pathway to produce OTA, providing important information relevant to understand the role of P. crustosum as putative OTA producer. Nevertheless, the additional secondary metabolism gene clusters found (such as penitrem, clavaric acid or naphthopyrones) highlight the potential of this strain for metabolite production, including other mycotoxins or compounds with antioxidant, anticancer or antibiotic properties.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of CEB (UID/BIO/04469/2019) and iBiMED (UIDB/04501/2020) units; and by CANCYL (POCI-01-0145-FEDER-031849) and GenomePT (POCI-01-0145-FEDER-022184) projectsinfo:eu-repo/semantics/publishedVersio