Abstract Yeasts are unicellular fungi widespread in the environment, and studies suggest that there is a positive correlation between yeast and polluted aquatic environments. The aim of this study was to analyze the diversity and resistance of yeasts isolated from water samples collected along the Arroio Dilúvio in Porto Alegre to antifungals. Yeast isolates from the Arroio Dilúvio were subjected to susceptibility assays against antifungals using the minimum inhibitory concentration (MIC) test, and amplification of the ITS1-5.8S-IT2 region; in addition, the ITS-5.8S region was sequenced for some of the isolates. The amplification product was subjected to PCR-RFLP, and the restriction profile allowed the construction of a dendrogram. Susceptibility tests showed a high prevalence of resistance to azole antifungals, where 16.8% of the isolates had a resistance profile to amphotericin B. The sequence analysis allowed the identification of Candida species, including potentially pathogenic species, and species of the Debaryomyces spp. The resistance to antifungals in yeasts isolated from Arroio Dilúvio reinforces the importance of studies of environmental microbiota, and indicates that environmental degradation influences the phenotype displayed.

ABSTRACT During composting processes, the degradation of organic waste is accomplished and driven by a succession of microbial populations exhibiting a broad range of functional competencies. A total of 183 bacteria, isolated from a composting process, were evaluated for cellulase activity at different temperatures (37, 50, 60, and 70°C) and pH values. Out of the 22 isolates that showed activity, isolate 380 showed the highest cellulase activity. Its ability to produce cellulase was evaluated in culture medium supplemented with carboxymethyl cellulose, microcrystalline cellulose, wheat straw, and rice husk. The culture medium supplemented with carboxymethyl cellulose induced higher enzyme activity after 6 hours of incubation (0.12 UEA mL-1 min-1). For wheat straw and rice husk, the results were 0.08 UEA mL-1 min-1 for both, while for microcrystalline cellulose, 0.04 UEA mL-1 min-1 were observed. The highest carboxymethyl cellulase activity was observed at 60°C (0.14 UEA mL-1 min-1) for both crude and partially purified enzyme after 30 and 120 min of incubation, respectively. Alkalinization of the medium was observed during cultivation in all substrates. The cellulase had a molecular mass of 20 kDa determined by SDS-Page. Isolate 380 was identified as Bacillus licheniformis. This work provides a basis for further studies on composting optimization.

Os actinomicetos endofíticos são promissoras fontes de compostos antimicrobianos. Este estudo avaliou a atividade dos metabólitos produzidos pelo actinomiceto endofítico R18(6) contra bactérias Gram-negativas multirresistentes a antimicrobianos. O isolado R18(6) foi cultivado em culturas submersas sob diferentes condições: fonte de carbono, temperatura, pH e tempo de incubação para otimização da produção do composto. O actinomiceto cultivado em meio base suplementado com 1% de glicose, pH 6.5 e incubação a 30 ºC sob agitação constante de 100 rpm durante 96 h, mostrou a melhor atividade contra as bactérias Gram-negativas testadas. A concentração inibitória mínima produzida pelo microorganismo variou entre 1/32 e 1/256. Revelou atividade bactericida ou bacteriostática de acordo com o isolado Gram-negativo. O extrato ativo mostrou-se estável em altas temperaturas e instável em meio contendo enzimas proteolíticas. A micromorfologia R18(6) foi avaliada, sob microscopia óptica e de varredura revelando que era morfologicamente similar ao gêneroStreptomyces.

Endophytic actinomycetes are promising sources of antimicrobial substances. This study evaluates the activity of metabolites produced by the endophytic actinomycete R18(6) against Gram-negative bacteria multiresistant to antimicrobials. R18(6) isolate was grown in submerged cultures under different conditions: carbon source, temperature, pH and incubation time to optimize antimicrobials production. The actinomycete grown in base medium supplemented with 1% glucose, pH 6.5 and incubation at 30 ºC for 96 h with shaking at 100 rpm, exhibited the highest activity against the used Gram-negative bacteria. Minimum inhibitory concentration (MIC) of the crude extract produced by the microorganism varied between 1/32 and 1/256. It had bactericide or bacteriostatic activity, depending on the Gram-negative organism. The active extract was stable at high temperatures, and unstable in medium containing proteolytic enzymes. Micromorphology of R18(6) was investigated by optical and scan microscopy, revealing that it was morphologically similar to the genusStreptomyces.

A técnica da compostagem é um recurso empregado com o objetivo de reciclar resíduos orgânicos domésticos e sanitizar o lodo produzido nas estações de tratamento de efluentes de esgoto (ETE) podendo-se obter um composto com propriedades de fertilizante agrícola e/ou corretor de solos degradados. A alta temperatura atingida pelo sistema deve ser responsável pela redução de micro-organismos patogênicos presentes no início do processo assegurando, desta forma, a qualidade microbiológica do composto sem oferecer riscos de contaminação, conforme preconiza a Resolução 354/2006 do CONAMA. Objetivou-se, com o trabalho, avaliar a influência da temperatura sobre a redução de Escherichia coli, Salmonella sp., ovos de helmintos e vírus entéricos durante o processo de compostagem, além da contagem de bactérias heterotróficas. Foram realizados testes colimétricos, semeaduras em meios de cultivo, testes de visualização de ovos de helmintos e detecção molecular de vírus entéricos. Os resultados indicaram oscilação nas contagens de E. coli e de bactérias heterotróficas, mesmo após a fase termofílica. Por outro lado, não foi detectada a presença de Salmonella sp., vírus entéricos nem de ovos viáveis de helmintos, ao final do processo.

The composting technique is a promising alternative for the treatment of solid organic residues, along with the sludge produced in sewage treatment plants. The final compost might show fertilizer properties, which can be used to recover poor soils. The high temperatures that the composting process can achieve are responsible for the reduction of the pathogenic microbial population, helminth eggs and virus present in the beginning of the process. This behavior assures the microbial quality of the compost according to the 375/2006 Resolution of the CONAMA. The aim of this study was to evaluate the influence of temperature on Escherichia coli, Salmonella sp., helminth egg and enteric virus presence in the final product from a composting process. For this matter colimetric assays were performed together with the seedling of the samples in different culture media, the assay for helminth eggs visualization under the microscope and the identification of enteric virus. The results showed an oscillation on E. coli and heterotrophic bacteria counts, even after the thermophilic phase. On the other hand, Salmonella sp., helminth eggs and enteric virus were not found in the final compost.

INTRODUÇÃO: Estafilococos são patógenos responsáveis por surtos de doenças transmitidas por alimentos. O estudo investigou a prevalência de genes de enterotoxinas e o perfil de resistência aos antimicrobianos em estafilococos coagulase-negativo (CoNS) e estafilococos coagulase-positivo (CoPS) isolados de morcilhas no sul do Brasil. MÉTODOS: Duzentas colônias típicas e atípicas do ágar Baird-Parker foram inoculadas em ágar sal-manitol. Oitenta e dois estafilococos manitol-positivos foram submetidos a testes bioquímicos e perfil de susceptibilidade antimicrobiana. A presença dos genes da coagulase (coa) e enterotoxinas (se) foi investigada por reação em cadeia da polimerase (PCR). RESULTADOS: Os isolados foram divididos em dois grupos: 75,6% (62/82) CoNS e 24,4% (20/82) CoPS. Através dos testes bioquímicos, 9 espécies foram determinadas, Staphylococcus saprophyticus (37,8%) e Staphylococcus carnosus (15,9%) foram as mais prevalentes. Testes de susceptibilidade demostraram fenótipos de resistência aos antibióticos administrados em humanos, como gentamicina, tetraciclina, cloranfenicol e eritromicina. O gene coa foi detectado em 19,5% (16/82) das cepas e quatro fragmentos de DNA polimórficos foram observados. Cinco CoNS contendo o gene coa foram submetidos ao sequenciamento do 16S rRNA e três mostraram similaridade com CoNS. Quarenta amostras foram positivas para pelo menos um gene se, os mais frequentes foram sea (28,6%) e seb (27,5%). CONCLUSÕES: A presença de resistência aos antimicrobianos e de genes se nos isolados de morcilha indicou que este alimento pode representar um risco potencial à saúde, já que a presença nos alimentos pode causar doenças de origem alimentar ou ser uma possível rota de transferência de estafilococos resistentes aos humanos.

INTRODUCTION: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. METHODS: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. RESULTS: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). CONCLUSIONS: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

INTRODUÇÃO: O objetivo deste estudo foi confirmar a identificação de amostras clínicas e alimentos de Enterococcus gallinarum e Enterococcus casseliflavus por PCR-RFLP. MÉTODOS: Cinquenta e duas cepas identificadas por exames bioquímicos convencionais foram submetidos a amplificação por PCR e digestão com HinfI. Apenas 20 (38,5%) das 52 amostras apresentaram um padrão de DNA esperado E. gallinarum e E. casseliflavus. RESULTADOS: Analise dos resultados deste estudo demonstraram que, algumas vezes E. gallinarum e E. casseliflavus são erroneamente identificados e confirmaram a potencial aplicação da análise do 16S rDNA para identificação exata destas espécies. CONCLUSÕES: A correta identificação é importante a fim de distinguir entre resistência intrínseca e adquirida à vancomicina.

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

The phytopathogenic fungus Drechslera tritici-repentis causes tan spot, an important disease of wheat in the southern Brazilian state of Rio Grande do Sul. Twelve D. tritici-repentis isolates were obtained from wheat seeds from different locations in the state. Their colony morphology on potato dextrose agar and polymorphisms in genomic DNA by the random amplified polymorphic DNA (RAPD) method were investigated. For the RAPD method, 23 primers were tested of which nine were selected for use in the study of D. tritici-repentis polymorphisms. The degree of similarity between isolates was calculated using a simple matching coefficient and dendrograms constructed by the unweighted pair-group method with arithmetical averages (UPGMA). The morphological and RAPD analyses showed intraspecific polymorphisms within the isolates, but it was not possible to establish a relationship between these polymorphisms and the geographical regions from where the host seeds were collected.

A presença das biovares I e/ou II de Ralstonia solanacearum em uma lavoura de batatas (Solanum tuberosum) tem influência direta no sucesso das medidas adotadas para controlar a murcha bacteriana. As biovares diferem entre si em relação à agressividade, latência e sobrevivência. Assim, um experimento de campo foi conduzido em uma área naturalmente infestada em duas épocas de cultivo com os objetivos de verificar (1) a incidência de biovares I e/ou II, (2) a relação entre biovar e época de plantio e (3) a relação entre biovar e cultivar de batata. Os isolados obtidos de plantas das cultivares Achat, Baronesa, Elvira, Macaca, Monte Bonito e Trapeira foram identificados como biovar I ou II através da PCR, utilizando os oligonucleotídeos iniciadores T3A e T5A. Ambas as biovares foram encontradas na área naturalmente infestada. De 73 isolados de R. solanacearum, 94,5% foram identificados como biovar II e 5,5% como biovar I. A biovar II foi isolada dos cultivos de primavera e de outono, independente da cultivar, mas a I apenas do cultivo de primavera e de plantas assintomáticas das cultivares Achat e Macaca. A maior população da biovar I nestas duas cultivares pode ser uma evidência da possível relação entre biovar e cultivar.

The occurrence of biovars I and/or II of Ralstonia solanacearum in a potato (Solanum tuberosum) field has direct consequences for the success of the measures adopted to control bacterial wilt. Biovars differ regarding aggressiveness, latency and survival. An experiment was conducted in a naturally infested potato field in two seasons to find (1) the incidence of biovars I and II, (2) the relationship between biovar and planting season, and (3) the relationship between biovar and potato cultivar. Bacterial isolates from potato cultivars Achat, Baronesa, Elvira, Macaca, Monte Bonito, and Trapeira were identified as biovar I or II through PCR, using T3A and T5A primers. Both biovars I and II were found in the naturally infested field. Among 73 strains, 5.5 and 94.5 % were identified as biovar I and II, respectively. Biovar II was found in the spring and in the fall, regardless of the cultivar, but biovar I only in the spring season and on symptomless plants of Achat and Macaca cultivars. The highest population of biovar I on these two cultivars may be evidence for a possible relationship between biovar and cultivar.