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In an attempt to determine the mating type of different Sporothrix schenckii sensu stricto isolates that remained viable after a long period of preservation in a culture collection and to correlate them with the degree of virulence/pathogenicity, a PCR technique using primers designed for the sequences of MAT1-1-1 and MAT1-2-1 genes and a murine experimental model were used. The results showed that there was no correlation between the mating type and virulence among the isolates. Furthermore, different degrees of virulence/pathogenicity, ranging from high to low, were found among them based on different virulence parameters. It was assumed that the long period of preservation favored the changes, yielding the isolation of variants. Thus, we believe that new technologies for studies on factors can improve our knowledge of the pathogenesis of sporotrichosis.
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Fusarium is a genus of ubiquitous fungi that comprises mycotoxigenic animal and plant pathogens. These fungi have the ability to exploit a wide range of substrates and hosts, indicating their great potential for enzyme production; however, this aspect is understudied. Therefore, the present study aimed for revaluating the identity of twenty-three Fusarium strains maintained in the University Recife Mycology (URM) culture collection, Brazil, and to evaluate their potential for proteases production and the milk-clotting activity of these proteases. According to phylogenetic analysis of translation elongation factor 1-alpha (TEF1) gene partial sequences, these strains belonged to 12 species representing four species complexes: Fusarium concolor, F. fujikuroi, F. incarnatum-equiseti, and F. oxysporum. Four of these species are putatively novel to science. Notably, novel associations of Fusarium spp. with certain hosts/substrates were documented. The proteolytic activity ranged from 1.67 U ml-1 to 22.03 U ml-1 among the evaluated fungal isolates, with specific proteolytic activity reaching 205.86 U mg-1. The values for coagulant activity and specific activity were up to 157.14 U ml-1 and 1,424.11 U mg-1, respectively. These results indicate the potential of URM Fusarium strains as a source for the production of enzymes of industrial interest. Additionally, they reinforce the importance of applying DNA-based methods for reviewing the identification of fungal strains preserved in biodiversity repositories.
Assuntos
Fusarium , Animais , Fusarium/genética , Filogenia , Brasil , Peptídeo Hidrolases/genética , LeiteRESUMO
Bacteria are an essential biotic component in freshwater environments. A group of 262 bacterial strains of freshwater environments from an altitudinal gradient in the Eastern Cordillera of Colombia was identified using the 16S rRNA gene sequence analysis. Hill numbers and related diversity indices were calculated to know the bacteria diversity in this collection and environments. In addition, the Bray-Curtis index was also calculated to know the differences in genera composition between sampled localities and their relationship with altitudinal gradient. The identified bacterial strains were grouped into 7 major phylogenetic groups (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Flavobacteriia, Actinomycetes, Clostridia, and Bacilli), 38 genera, and 84 distinctive species. Diversity analysis based on Hill numbers showed that the diversity concerning bacteria inhabiting freshwater environments was consistently high. Dominant genera were Klebsiella, Serratia, and Pseudomonas, although other genera such as Bacillus, Lelliottia, and Obesumbacterium were well represented per locality. The highest bacterial diversity came from localities Cimitarra and El Carmen del Chucurí, while those originating from Santa Bárbara and Páramo del Almorzadero were relatively lower diverse. Differences in diversity were found to be mainly due to the spatial replacement of one genus by another and, to a lesser extent, to the loss or gain of taxa.
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Bacillus , Bactérias , Colômbia , RNA Ribossômico 16S/genética , Filogenia , Bactérias/genética , Bacillus/genética , Enterobacteriaceae/genética , Água Doce , BiodiversidadeRESUMO
We developed a simple new selective LB-based medium, named CYP broth, suitable for recovering long-term stored Y. pestis subcultures and for isolation of Y. pestis strains from field-caught samples for the Plague surveillance. It aimed to inhibit the growth contaminating microorganisms and enrich Y. pestis growth through iron supplementation. The performance of CYP broth on microbial growth from different gram-negative and gram-positive strains from American Type Culture Collection (ATCC®) and other clinical isolates, field-caught rodent samples, and more importantly, on several vials of ancient Y. pestis subcultures was evaluated. Additionally, other pathogenic Yersinia species such as Y. pseudotuberculosis and Y. enterocolitica were also successfully isolated with CYP broth. Selectivity tests and bacterial growth performance on CYP broth (LB broth supplemented with Cefsulodine, Irgasan, Novobiocin, nystatin and ferrioxamine E) were evaluated in comparison with LB broth without additive; LB broth/CIN, LB broth/nystatin and with traditional agar media including LB agar without additive, and LB agar and Cefsulodin-Irgasan-Novobiocin Agar (CIN agar) supplemented with 50 µg/mL of nystatin. Of note, the CYP broth had a recovery twofold higher than those of the CIN supplemented media or other regular media. Additionally, selectivity tests and bacterial growth performance were also evaluated on CYP broth in the absence of ferrioxamine E. The cultures were incubated at 28 °C and visually inspected for microbiological growth analysis and O.D.625 nm measurement between 0 and 120 h. The presence and purity of Y. pestis growth were confirmed by bacteriophage and multiplex PCR tests. Altogether, CYP broth provides an enhanced growth of Y. pestis at 28 °C, while inhibiting contaminant microorganisms. The media is a simple, but powerful tool to improve the reactivation and decontamination of ancient Y. pestis culture collections and for the isolation of Y. pestis strains for the Plague surveillance from various backgrounds. KEY POINTS: ⢠The newly described CYP broth improves the recuperation of ancient/contaminated Yersinia pestis culture collections ⢠CYP broth was also efficient in reducing environmental contamination in field-capture samples, improving Y. pestis isolation ⢠CYP broth can also be used for the isolation of Y. enterocolitica and Y. pseudotuberculosis.
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Peste , Yersinia pestis , Humanos , Ágar , Peste/microbiologia , Novobiocina/farmacologia , Nistatina , Meios de Cultura/farmacologia , Cefsulodina/farmacologiaRESUMO
In recent years, ample research has focused on applying wild (especially non-Saccharomyces) yeasts in producing alcoholic beverages. Common characteristics of wild yeast strains include simultaneous high production of fruity and floral aroma compounds and low ethanol production. In this study, mead starter cultures were selected based on preliminary screening of wild yeast strains from a Brazilian culture collection (n = 63) for their ability to produce aroma-active compounds. The selected strains included one strain of Saccharomyces cerevisiae and three non-Saccharomyces strains (Pichia jadinii, Torulaspora delbrueckii, and Kluyveromyces lactis). These strains were used to ferment honey must prepared with Aroeira honey, adjusted to 24°Brix, which took 36 days to complete. Single culture fermentations and co-fermentations with S. cerevisiae and non-Saccharomyces strains were carried out. The quality of the produced beverages was evaluated by sugar consumption and production of alcohols and organic acids, analyzed with high-performance liquid chromatography. The volatile organic compound composition was analyzed with gas chromatography-mass spectrometry. Meads with various ethanol amounts (4.7-11.0% v/v) and residual sugar contents (70.81-160.25 g l-1) were produced. In addition, in both single-strain fermentation and co-fermentation with S. cerevisiae, meads produced with either Torulaspora delbrueckii or Kluyveromyces lactis had a roughly three-fold higher content of honey-aroma compound phenethyl acetate and a higher hedonic impression score than meads produced with only S. cerevisiae. These results demonstrated non-Saccharomyces yeasts' ability to increase aroma complexity and improve the sensory quality of low-alcoholic meads.
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Torulaspora , Vinho , Odorantes/análise , Saccharomyces cerevisiae , Leveduras , Fermentação , Etanol/análise , Vinho/análise , Vinho/microbiologiaRESUMO
Sporotrichosis is a subcutaneous mycosis worldwide distributed reaching hyperendemic proportions in Brazil. Many isolates from patients with sporotrichosis are preserved in culture collections by different methods around the world. The preservation methods are used to maintain the viability and the morphophysiological and genetic characteristics of isolates for long periods. In this study, we evaluated 34 isolates, previously, identified as S. schenckii by a classical identification method, initially preserved by periodical subcultures and then under mineral oil at culture collection of Oswaldo Cruz Institute/Fiocruz, to re-identify them by polyphasic identification. Our results showed that seven isolates remained viable for 34 to 64 years under oil, one isolate lost the ability to sporulate which was reverted by using a medium culture supplemented with rosebush branches and all of them were identified as Sporothrix schenckii sensu stricto by morphological, physiological, partial ß-tubulin gene sequencing and phylogenetic analysis.
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The International Culture Collection of (Vesicular-) Arbuscular Mycorrhizal Fungi-INVAM-the largest living culture collection of arbuscular mycorrhizal fungi (AMF) celebrated its 35th year in 2020. The authors record here the mission and goals of INVAM, its contribution as a living culture collection, some historical aspects of INVAM, and describe the advances in mycorrhizology and AMF systematics after INVAM moved to West Virginia University. This commentary emphasizes the importance of a living culture collection to preserve germplasm and to educate and assist researchers in mycorrhizal science.
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Glomeromycota , MicorrizasRESUMO
Reference fungal cultures (RFCs) are essential for the internal quality control of laboratories. The production of these cultures requires standardized procedures (IRAM 14950:2016 and ISO 17034:2016 standards) carried out by a recognized and accredited laboratory. The aim of this work was to produce RFC in paper disks of autochthonous strains, characterized by two, homogeneous and stable reference methods traceable at species level. RFC were produced using 14 regional species (7 yeasts and 7 filamentous fungi) from the fungal culture collection (DMic). Paper disks were impregnated with a culture suspension, dried and packed. Homogeneity, viability, identity and purity were verified. Short-and long-term stability at different temperatures and storage times were studied. Characterization of each strain allowed to confirm its identity and to ensure its traceability at international level. Produced batches were homogeneous and stable at -20 ±5 °C for 30 months. This method of production was adequate to produce homogeneous and stable RFC with phenotypic and genotypic characteristics correctly defined and internationally traceable. Standardized procedures were developed for the production of certified RFC that could be transferred to other microorganisms. Providing RFC that represent regional strains allows laboratories to produce more reliable results with a favorable impact on medical diagnosis, the environment or the food industry.
Los cultivos microbianos de referencia (CR) son imprescindibles para el control de calidad interno de los laboratorios. Asegurar su producción requiere de procedimientos estandarizados (IRAM 14950:2016 e ISO 17034:2016) realizados en un laboratorio reconocido y acreditado. El objetivo de este estudio fue producir cultivos fúngicos de referencia en discos de papel, a partir de un panel de cultivos autóctonos caracterizados por dos métodos de referencia, trazables a nivel taxonómico de especie, homogéneos y estables. Se produjeron CR de 14 especies circulantes en Argentina (7 de levaduras y 7 de hongos miceliales), depositadas en la colección de hongos de interés médico (DMic). Los discos de papel fueron embebidos con una suspensión del cultivo por producir, secados y envasados. Se verificó la homogeneidad, viabilidad, identidad y pureza de cada lote. Se evaluó la estabilidad a corto y largo plazo a distintas temperaturas y tiempos de almacenamiento. La caracterización de cada CR nos permitió confirmar su identidad y asegurar su trazabilidad a nivel internacional. Los lotes producidos fueron homogéneos y estables durante 30 meses conservados a -20 ±5 °C. Este método resultó adecuado para producir CR homogéneos y estables, con características fenotípicas y genotípicas correctamente definidas y trazables a nivel internacional. Los procedimientos estandarizados desarrollados en este trabajo pueden ser transferidos para producir CR certificados de otros microorganismos. La provisión de CR que represente cepas regionales permite a los laboratorios producir resultados más confiables con un impacto favorable en el diagnóstico médico, los estudios ambientales y la industria alimenticia.
Assuntos
Bancos de Espécimes Biológicos , Fungos , Micologia/normas , Preservação Biológica/instrumentação , Preservação Biológica/métodos , Controle de Qualidade , Padrões de Referência , Leveduras , Meios de Cultura , Micologia/métodosRESUMO
Reference fungal cultures (RFCs) are essential for the internal quality control of laboratories. The production of these cultures requires standardized procedures (IRAM 14950:2016 and ISO 17034:2016 standards) carried out by a recognized and accredited laboratory. The aim of this work was to produce RFC in paper disks of autochthonous strains, characterized by two, homogeneous and stable reference methods traceable at species level. RFC were produced using 14 regional species (7 yeasts and 7 filamentous fungi) from the fungal culture collection (DMic). Paper disks were impregnated with a culture suspension, dried and packed. Homogeneity, viability, identity and purity were verified. Short- and long-term stability at different temperatures and storage times were studied. Characterization of each strain allowed to confirm its identity and to ensure its traceability at international level. Produced batches were homogeneous and stable at -20±5°C for 30 months. This method of production was adequate to produce homogeneous and stable RFC with phenotypic and genotypic characteristics correctly defined and internationally traceable. Standardized procedures were developed for the production of certified RFC that could be transferred to other microorganisms. Providing RFC that represent regional strains allows laboratories to produce more reliable results with a favorable impact on medical diagnosis, the environment or the food industry.
Assuntos
Bancos de Espécimes Biológicos , Fungos , Micologia/normas , Meios de Cultura , Micologia/métodos , Preservação Biológica/instrumentação , Preservação Biológica/métodos , Controle de Qualidade , Padrões de Referência , LevedurasRESUMO
Recombinant simian IL-15 (siIL-15) was obtained for the preclinical assessment of an anti-human IL-15 vaccine. For this purpose, the cDNA from peripheral blood mononuclear cells of a Macaca fascicularis monkey was cloned into a pIL-2 vector. The siIL-15 was expressed in Escherichia coli strain W3110 as an insoluble protein which accounted for 13% of the total cellular proteins. Inclusion bodies were solubilized in an 8 M urea solution, which was purified by ion exchange and reverse phase chromatography up to 92% purity. The protein identity was validated by electrospray ionization-mass spectrometry, confirming the presence of the amino acids which distinguish the siIL-15 from human IL-15. The purified siIL-15 stimulates the proliferation of cytotoxic T-lymphocytes line (CTLL)-2 and Kit 225 cells with EC50 values of 3.1 and 32.5 ng/mL, respectively. Antisera from modified human IL-15-immunized macaques were reactive to human and simian IL-15 in enzyme-linked immunosorbent assays. Moreover, the anti-human IL-15 antibodies from immune sera inhibited siIL-15 activity in CTLL-2 and Kit 225 cells, supporting the activity and purity of recombinant siIL-15. These results indicate that the recombinant siIL-15 is biologically active in two IL-15-dependent cell lines, and it is also suitable for the preclinical evaluation of an IL-15-based therapeutic vaccine.