Caracterização e análise filogenética de ANAMMOX: uma bactéria chave no ciclo do nitrogênio

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-graduação em Ecologia, Florianópolis, 2009O presente estudo teve como objetivo caracterizar e analisar filogeneticamente bactérias anaeróbias oxidadoras de amônio (ANAMMOX) coletadas de bioreator de bancada de fluxo ascendente inoculado com lodo aclimatado proveniente de lagoa anaeróbia de um sistema de Lagoas de Tratamento de Dejetos de Suínos e padronizar técnica de Reação em Cadeia da Polimerase (PCR) objetivando amplificação de uma região de 436 pares de base (pb) correspondente a subunidade menor (16S rRNA) do ribossomo de bactérias ANAMMOX. Visando a identificação dos organismos presentes no bioreator, foram utilizadas as técnicas de PCR, clonagem e sequenciamento da região 16S rRNA e dos fragmentos de 436 pb amplificados. Os iniciadores desenvolvidos foram avaliados quanto sua especificidade e limite de detecção. Foram obtidos 29 clones, dos quais 17 carregavam o gene 16S rRNA e 12 carregavam o fragmento de 436 pb. Entre os 17 clones obtidos, três apresentaram 97% de identidade com ANAMMOX Candidatus Jettenia asiática e Planctomycete KSU-1, 12 tiveram identidade com Janthinobacterium (99%) e dois apresentaram similaridade com clones não cultivados. Dos clones carregando o fragmento de 436 pb, oito apresentaram 96-100% de semelhança com ANAMMOX Candidatus Anammoxoglobus propionicus, Planctomycete KSU-1 e Candidatus Jettenia asiatica. Um clone teve 99% de similaridade com Pseudomonas sp. e outros três clones apresentaram semelhança com clones não cultivados. Embora os iniciadores tenham amplificado fragmentos genômicos de organismos não-ANAMMOX, o teste de limite de detecção mostrou que com a PCR foi possível amplificar a região alvo usando concentrações extremamente baixas (0,3 ng) de material genético. A utilização de tais ferramentas (extração de material genômico e execução de PCR com os novos iniciadores aqui desenvolvidos) mostrou-se eficiente, econômica e de fácil execução para caracterização de organismos ANAMMOX, abrindo uma gama de oportunidades para ampliar nosso conhecimento sobre estas bactérias e consequentemente melhorar o tratamento de dejeto de suíno

Characterization of Planktochlorella nurekis Extracts and Virucidal Activity against a Coronavirus Model, the Murine Coronavirus 3

Certain members of the Coronaviridae family have emerged as zoonotic agents and have recently caused severe respiratory diseases in humans and animals, such as SARS, MERS, and, more recently, COVID-19. Antivirals (drugs and antiseptics) capable of controlling viruses at the site of infection are scarce. Microalgae from the Chlorellaceae family are sources of bioactive compounds with antioxidant, antiviral, and antitumor activity. In the present study, we aimed to evaluate various extracts from Planktochlorella nurekis in vitro against murine coronavirus-3 (MHV-3), which is an essential human coronavirus surrogate for laboratory assays. Methanol, hexane, and dichloromethane extracts of P. nurekis were tested in cells infected with MHV-3, and characterized by UV-vis spectrophotometry, nuclear magnetic resonance (NMR) spectroscopy, ultraperformance liquid chromatography-mass spectrometry (UPLC-MS), and the application of chemometrics through principal component analysis (PCA). All the extracts were highly efficient against MHV-3 (more than a 6 Log unit reduction), regardless of the solvent used or the concentration of the extract, but the dichloromethane extract was the most effective. Chemical characterization by spectrophotometry and NMR, with the aid of statistical analysis, showed that polyphenols, carbohydrates, and isoprene derivatives, such as terpenes and carotenoids have a more significant impact on the virucidal potential. Compounds identified by UPLC-MS were mainly lipids and only found in the dichloromethane extract. These results open new biotechnological possibilities to explore the biomass of P. nurekis; it is a natural extract and shows low cytotoxicity and an excellent antiviral effect, with low production costs, highlighting a promising potential for development and implementation of therapies against coronaviruses, such as SARS-CoV-2.This research was funded LVA-MIP-CCB-UFSC/Sigpex: 201917940, and CNPq, CAPES-DS

Desempenho de dois sistemas de tratamento de dejetos de suínos na redução de compostos químicos e de patógenos

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em BiotecnologiaA reutilização de águas residuárias geradas na suinocultura pode atender a várias finalidades merecendo destaque o reúso no próprio processo de produção animal ou em outras atividades agrícolas. Porém, para que se tenha segurança nesta prática, informações consistentes sobre a qualidade da água envolvendo aspectos físicos, químicos e microbiológicos precisam ser avaliadas. Atualmente, a inexistência de parâmetros para reúso de água na pecuária brasileira torna esta prática sem critérios para sua recomendação técnica. Diante disso, o presente estudo teve por objetivos padronizar e validar metodologias moleculares para o monitoramento de águas residuárias da suinocultura no que se refere à detecção viral; 2) Avaliar as águas residuárias da suinocultura por meio de estudo virológico de dois sistemas de tratamento de dejetos de suínos; 3) Estabelecer o perfil químico e microbiológico de ambos os sistemas de tratamento, apontando qual sistema é mais eficiente; 4) Apontar qual vírus seria o melhor candidato a marcador de contaminação ambiental por dejeto de suíno, tendo como base a incidência e persistência dos diferentes vírus nos sistemas de tratamento; 5) Apontar qual dos dois sistemas é mais eficiente na remoção dos compostos químicos e microbiológicos avaliados. Para isso, foram avaliados mensalmente (março de 2009 a dezembro de 2010), dois sistemas de tratamento de dejetos de suínos (sistema 1 e 2). Os resultados revelaram que o genoma de PAdV esteve presente em 66% das amostras coletadas o sistema 1 e em 78% das amostras do sistema 2. Já o PCV2 foi detectado em 96% das amostras coletadas no sistema 1 e em 86% das amostras do sistema 2. Partículas íntegras de PAdV e PCV2 estavam presentes em todos os pontos de amostragem, e partículas infecciosas de PCV2 (genótipos 2a e 2b) estavam presentes no efluente final. Foram detectados ainda PPV, TTSuV em ambos os sistemas . Dentre os vírus avaliados, recomenda-se o PAdV como marcador de contaminação ambiental por dejeto de suíno, por este ser um dos mais resistentes e frequentes. Em relação aos parâmetros químicos, a redução ao longo do sistema 1 atingiu níveis acima de 70% em todos os compostos e para o sistema 2, a redução atingiu níveis acima de 80% em todos os compostos avaliados. Para Salmonella sp., não houve redução no sistema 1, e a bactéria não foi detectada no efluente do sistema 2. Em resumo, o perfil químico e microbiológico do sistema 1 apresentou taxas de redução inferiores do que as encontradas no sistema 2, mostrando assim que o este último foi o mais indicado para o tratamento de dejetos de suíno visando o reúso de água. Porém, conclui-se que há a necessidade de se estudar um protocolo adicional de desinfecção do efluente final, visando a eliminação de micro-organismos patogênicos

IMPACT OF ANTHROPOGENIC ACTIVITIES IN THE SURFACE WATER QUALITY ABOVE GUARANI AQUIFER: A CASE STUDY IN SOUTHERN BRAZIL

This study evaluated the influence of anthropogenic activities in water quality from rural and urban areas in Southern Brazil, above the Guarani aquifer. Water samples were submitted to microbial and physical-chemical analyses and Trophic State Index calculation. Results showed high levels of total phosphorus, nitrite, and E. coli in the urban area, and consequently a high Trophic State Index, indicating severe stress. In rural areas, elevated nitrate concentrations were found to be related to soil fertilization practices. This study reinforces the alert on the impact of riparian forest recuperation and wastewater treatment on water quality, especially in areas above important aquifer systems such as the Guarani

Bacterial biodiversity from an anaerobic up flow bioreactor with ANAMMOX activity inoculated with swine sludge

The present study aimed to describe the bacterial community present at an anaerobic up flow bioreactor with ANAMMOX activity, inoculated with the sludge from the anaerobic pond of a swine slurry treatment system. The description was based on the molecular DNA techniques using primers for amplification of complete 16S rRNA gene and also new primers to amplify smaller fragments from 16S rRNA. During the bioreactor operation time, the bacterial community changed significantly, increasing the nitrogen removal efficiency, reaching after 500 days a removal rate of 94%. The complete PCR amplification of 16S rRNA gene generated 17 clones, where three presented similarity with Candidatus Jettenia asiatica (97%), twelve with Janthinobacterium (99%) and two with uncultured clones. The PCR amplification of 436 base pairs had generated 12 clones, of which eight presented 96-100% similarity with Candidatus Anammoxoglobus propionicus, Planctomycete KSU-1 and one with Pseudomonas sp. (99%) and three with uncultured clones

Salmonella enterica Serovar Enteritidis Control in Poultry Litter Mediated by Lytic Bacteriophage Isolated from Swine Manure

We report the use of bacteriophages for control of Salmonella Enteritidis in poultry production. Phage was isolated by the double-agar plate assay from agricultural waste samples, and one isolate, named SM1, was selected and propagated for application in poultry litter. Two experimental protocols were tested: single treatment and repeated treatment (re-application of phage SM1 after 6 h and 12 h). Each treatment cycle involved 25 g of poultry litter placed in plastic boxes and contaminated with 105 Colony Forming Units mL−1 (CFU mL−1) of S. Enteritidis, in independent duplicates. The contaminated litter was treated with 106 Plaque Forming Units mL−1 (PFU mL−1) of SM1 phage by dripping. Repeated application of phage SM1 reduced Salmonella counts by over 99.9%; the phage persisted in poultry litter for over 35 days. This study illustrates the application of SM1 treatment as a promising technology for bacterial control in production matrices that could allow safe and sustainable use of agricultural waste products as biofertilizers

Microbiological and physicochemical analysis of the coastal waters of southern Brazil

The aim of this study was to assess the impact of sewage discharge on coastal waters by evaluating the influence of physicochemical parameters on the presence of enteric microorganisms in seawater samples collected from 11 beaches in Florianopolis, Santa Catarina, Brazil, over a one-year period (August 2009 to July 2010). Samples were assessed for the presence of human adenoviruses (HAdV), polyomavirus (JCPyV), hepatitis A virus (HAV), and noroviruses (HuNoV GI and GII). Escherichia coli and physicochemical parameters (salinity, temperature, pH and dissolved oxygen) were also evaluated. From the 132 samples analyzed, 55% were positive for HAdV, 51.5% for HAV, 7.5% for HuNoV GI, 4.5% for HuNoV GII, and 3% for JCPyV. E. coli levels ranged from 8 to 1325 CFU/100 mL at all sites. The overall results highlight the problem of sewage discharge into coastal waters and confirm that there is no correlation between viral presence and bacterial contamination

Mineral waste containing high levels of iron from an environmental disaster (Bento Rodrigues, Mariana, Brazil) is associated with higher titers of enteric viruses.

Although the effects of heavy metals on the behavior, including infectivity, of bacteria have been studied, little information is available about their effects on enteric viruses. We report an investigation of effects on the biosynthesis of human adenoviruses (HAdV) and hepatitis A (HAV) of waters contaminated with mineral waste following an environmental disaster in Mariana City, Minas Gerais State, Brazil. The study area was affected on November 5, 2015, by 60 million m3 of mud (containing very high concentrations of iron salts) from a mining reservoir (Fund?o), reaching the Gualaxo do Norte River (sites evaluated in this study), the ?Rio Doce? River and finally the Atlantic Ocean. We found substantial counts of infectious HAdV and HAV (by qPCR) in all sampled sites from Gualaxo do Norte River, indicating poor basic sanitation in this area. The effects of iron on viral infection processes were evaluated using HAdV-2 and HAV-175, as DNA and RNA enteric virus models, respectively, propagated in the laboratory and exposed to this contaminated water. Experiments in field and laboratory scales found that the numbers of plaque forming units (PFU) of HAdV and HAV were significantly higher in contaminated water with high iron concentrations than in waters with low iron concentration (<?20 ?g/L of iron). These findings indicate that iron can potentiate enteric virus infectivity, posing a potential risk to human and animal health, particularly during pollution disasters such as that described here in Mariana, Brazil

Evaluation of the effective inactivation of enteric bacteria and viruses from swine effluent and sludge at tropical temperatures.

The safe recycling of organic wastes such as the nutrients obtained from agriculture activities is a relevant aspect of the ?One Health? strategy, a worldwide initiative including all aspects of health care for humans and animals, as well as aspects related to the conservation and development of the environment. It represents an important opportunity to mitigate the potential impact of microbial pathogens likely present in nutrient natural sources, as digestate, when applied to land as fertilizers. The objectives of the current study were to determine the inactivation of enteric microorganisms in swine digestate (effluent and sludge) during storage in anaerobic conditions and at different ambient temperatures of tropical zones (16, 22, and 37 ?C). Human adenovirus 2 (HAdV-2), bacteriophage PhiX-174, and Salmonella enterica serovar typhimurium (S. typhimurium) were used as enteric microbial models to determine the minimum storage time required to reduce at least 3log of enteric microorganisms load in both matrices at the mentioned temperatures. The minimum storage time required for S. typhimurium was shorter than those observed for viruses at 37 ?C, while reduction of at least 3log for S. typhimurium was observed after just 1 day; 20 and 90 days were needed for similar reductions for HAdV-2 and PhiX-174, respectively. Similar results were also observed at lower temperatures: 30 and 45 days were needed for S. typhimurium at 22 and 16 ?C, respectively, but 30 and 90 days were needed at 22 ?C for HAdV-2 PhiX-174, respectively, or 90 and 120 days at 16 ?C for HAdV-2 PhiX-174, respectively. This is a pioneering study on the evaluation of inactivation of enteric viruses in swine digestate in usual tropical ambient temperatures; it demonstrates that the time for a considerable inactivation is longer than that observed for a enteric microorganisms (S. typhimurium), and shows that the storage of swine digestate at the referred temperatures can be used as a low-cost post-treatment to inactivate enteric microorganisms, allowing a further use as safer fertilizer