Microbiologia Agrícola
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/190
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Item Development and evaluation of chimeric recombinant antigen, nano vaccine and probiotics for fish to Aeromonas hydrophila and Pseudomonas fluorescens control(Universidade Federal de Viçosa, 2025-02-26) Rodrigues, Jojo; Mendes, Tiago Antonio de Oliveira; http://lattes.cnpq.br/9161106617289234Aquaculture is an aquatic food production system whose main products are the cultivation of fish, bivalves and algae. It contributes approximately US$230 billion to the world economy annually. Growth estimates project trends until 2050 and, therefore, aquaculture is considered one of the most promising global markets. The high consumption of fish in the human diet has intensified in recent years due to the benefits it brings to human health and nutrition, since fish are a source of proteins, vitamins, minerals and “good” fats, which help prevent cardiovascular, bone, ophthalmological and degenerative diseases. However, fish production has been threatened by the establishment of pathogenic bacteria that cause a wide range of diseases and comorbidities in fish and are associated with high mortality rates and production losses in aquaculture production. The species Aeromonas hydrophila and Pseudomonas fluorescens are representatives of a group of Gram-negative bacteria that threaten fish health and have an alarming zoonotic potential. Vaccination of fish, as well as alternative methods such as the use of probiotics in supplementation, are considered the gold standard in the fight against these infectious agents. Therefore, this study aimed to develop and evaluate a chimeric, nanoencapsulated, probiotic antigen for fish against pathogenic strains of Aeromonas hydrophila and Pseudomonas fluorescens. The antigen was constructed using immunoinformatics tools using the sequence of the protein UDP-N-acetylmuramate-L-alanine ligase, which is important for the biogenesis of the precursor of the cell wall of bacteria, as a skeleton. A prediction of B and T cell epitopes was performed on the BepiPred 2.0 and NetCLT web servers, and the most immunogenic protein sequences of both bacteria were used to construct the chimeric antigen. The evaluation of the immunogenic property was evaluated in the animal model, zebrafish (Danio rerio), and the gene expression of genes related to the fish immune system was evaluated. Protection after immunization was evaluated by challenging the LD50 or lethal doses of A. hydrophila in vivo and survival was recorded over a period of seven days after challenge. The nano antigen was also nanoencapsulated by nanostructured lipid nanocarriers and the physicochemical characteristics of the formulations as well as the immunogenic and protective properties in vivo were evaluated. In addition, the antimicrobial and probiotic potential of two strains, Lactococcos lactis and Lactobacillus acidophilus, was performed and characterized in vivo. The results were promising and demonstrated an ease of production of the antigen on a large scale through expression in bacterial systems, as well as an immunogenic and protective potential culminating in the upregulation of genes of the fish immune system and in the high survival percentage of immunized fish that were challenged with the LD50 and lethal doses of A. hydrophila. The results were even more promising when the antigen was nanoencapsulated, demonstrating that after nanoencapsulation, the protection and survival of the fish were significant at low concentrations of the antigen and challenge of the fish with a highly lethal dose of A. hydrophila. However, the probiotic and antimicrobial evaluation of the strains of L. lactis and L. acidophilus was positive only in vitro, culminating in a direct antimicrobial activity against pathogenic strains of A. hydrophila and P. fluorescens, but the same was not observed in vivo, and the protection and survival of the fish that received the probiotic treatments were not significant in relation to the fish that were not treated. The data presented in this work demonstrate the power of using immunoinformatics and nanotechnology for the construction and production of products with antimicrobial and vaccine potential against pathogenic agents. Keywords: Aquaculture; Fish; Pathogenic bacteria; Aeromonas hydrophila; Vaccination; Immunoinformatics.Item Isolation and characterization of foodborne pathogen-infecting phages and genetic determinants of phage susceptibility in Staphylococcus aureus(Universidade Federal de Viçosa, 2025-05-21) Cunha, Paloma Cavalcante; Paula, Sergio Oliveira de; http://lattes.cnpq.br/1842375517176650The growing prevalence of antibiotic-resistant bacterial pathogens in clinical, veterinary, and food production settings demands alternative control strategies. Bacteriophages (phages), due to their host specificity and safety, have emerged as promising antimicrobial agents for both therapeutic and biocontrol applications. This thesis explores the isolation, genomic and functional characterization, and application of lytic phages targeting Salmonella enterica and Staphylococcus aureus, two major foodborne and zoonotic pathogens. The study also investigates bacterial genetic determinants that modulate susceptibility to phage infection. In Chapter 1, the polyvalent Tequintavirus phage UFVCit2 was isolated and shown to infect Citrobacter freundii, Shigella flexneri, and different S. enterica serovars. Genomic analysis confirmed its strictly lytic nature and genetic safety. UFVCit2 significantly reduced S. enterica counts on chicken meat and lettuce in food model experiments, highlighting its potential for biocontrol applications in complex matrices. Chapter 2 presents the characterization of phage CapO46, a newly identified Rosenblumvirus with broad activity against S. aureus strains from clinical and environmental origins. CapO46 exhibited rapid adsorption kinetics, a short latent period, and stable lytic activity in ultra-high-temperature (UHT) milk, achieving up to 7.2 log10 CFU/mL reduction after 12 hours. Genomic analyses confirmed the absence of lysogeny, virulence, or resistance genes, further supporting its suitability as a safe and effective biocontrol agent for dairy applications. In Chapter 3, a genome-wide CRISPR interference (CRISPRi-seq) screen was conducted to identify host factors in S. aureus that influence susceptibility to CapO46. The screen revealed that repression of mgrA, sarZ, and SAOUHSC_00695 conferred resistance to infection, while silencing rpiRc increased susceptibility. Functional validation via bacterial growth, phage replication, adhesion, and biofilm formation assays confirmed the involvement of these genes in regulating both phage sensitivity and surface-associated virulence traits. Altogether, this work demonstrates the potential of phages as targeted, safe, and sustainable alternatives to antibiotics, and underscores the importance of genetic context in modulating bacterial susceptibility. The integration of phage genomics, food model applications, and functional bacterial genetics provides a comprehensive framework for the rational development ofphage-based biocontrol and therapeutic strategies. Keywords: Salmonella enterica; Staphylococcus aureus; Biocontrol; CRISPR interference; Host factors; Phage resistance.Item Efeito do etanol, cloreto de sódio e peróxido de hidrogênio sobre o crescimento de linhagens de Spathaspora passalidarum isoladas do bioma amazônico(Universidade Federal de Viçosa, 2025-02-26) Andrade, Julia Martins de; Fietto, Luciano GomesO Brasil é o segundo maior produtor de etanol do mundo. O uso de combustíveis sustentáveis tem sido cada vez mais desejável visando menor impacto ambiental. Como forma de suprir a demanda energética sem utilizar hidrocarbonetos e derivados agrícolas, o Etanol de Segunda Geração (E2G) surge como uma possibilidade. No caso deste biocombustível, os microrganismos e as biomassas lignocelulósicas exercem um papel crucial. Por isso, este trabalho teve como objetivo caracterizar fisiologicamente 12 linhagens da levedura Spathaspora passalidarum isoladas do bioma brasileiro amazônico, visando futuras aplicações na indústria sucroalcooleira. Esta espécie possui potencial para a produção de E2G por ser fermentadora de xilose. Os experimentos avaliaram o perfil de crescimento das 12 linhagens em estresses provocados por etanol (5%, 6% e 7%), cloreto de sódio (0,5 M, 0,75 M e 1 M), peróxido de hidrogênio (7 mM e 10 mM) e diferentes concentrações de glicose (2%, 18%, 20%,25% e 30%). Os ensaios de estresse foram realizados em meio YPD e meio YPX utilizando o equipamento Growth Profiler 960 (EnzyScreen). Ademais, foram realizados dois experimentos de co fermentação glicose/xilose a fim de identificar fontes de carbono preferenciais e rendimento de produção de etanol (g/L). Os resultados apontaram que o perfil de crescimento das linhagens apresentou variações de acordo com o estresse e o tipo de fonte de carbono (glicose ou xilose). Em meio YPX (xilose), as linhagens apresentaram maior resiliência ao estresse por etanol. Em condições de estresse por NaCl, a levedura de referência obteve o maior crescimento em 1 M, com OD (600 nm) de aproximadamente 1,2. Na primeira cofermentação, observou-se consumo preferencial de glicose (esgotada em até 12 horas por todas as linhagens). A linhagem 6 demonstrou consumo eficiente de xilose e produziu 10,5 g/L de etanol em 24 horas. A segunda cofermentação foi realizada com a levedura NRRL Y-27907 e o a levedura isolada número 6, sendo o isolado mais bem sucedido na produção de etanol. Portanto, conclui-se que a linhagem 6 de S. passalidarum apresentou melhor desempenho em relação aos outros isolados nos ensaios realizados. Palavras-chave: Leveduras; Spathaspora passalidarum; Resposta ao estresse; Produção de etanol; Biocombustíveis; Etanol 2G.Item Bioprospection and adaptive mechanisms of halotolerant bacteria: applications in biotechnology and astrobiological studies(Universidade Federal de Viçosa, 2025-02-25) Vieira, Camila de Souza; Totola, Marcos Rogerio; http://lattes.cnpq.br/9880193425166992Halophilic and halotolerant bacteria can be applied in environments where conventional organisms cannot thrive, such as in the bioremediation of salinized soils or the treatment of saline effluents. These organisms are also frequently used in astrobiology studies due to their well-recognized ability to tolerate or adapt to extreme conditions. Brazil, with its diverse biomes and high biodiversity, harbors saline environments that remain largely unexplored in terms of microbial biodiversity. This study aimed to investigate the biotechnological potential and the feasibility of using halotolerant bacteria isolated from extreme Brazilian environments (arid and saline) as model organisms in astrobiological studies, as well as to expand the understanding of the adaptive capacity of halotolerant microorganisms to NaCl. The bacteria were collected from three areas in Camocim, Ceará, Brazil. Their identification was carried out through fatty acid methyl ester (FAME) profiling and the analysis of the DNA sequence encoding the 16S rRNA. The requirement for or tolerance to NaCl and sodium perchlorate (NaClO4) were evaluated by determining the specific growth rates (µ) in culture media containing different salt concentrations. The ability to use NaClO4 as a final electron acceptor was also assessed. The biotechnological potential of the isolates was evaluated by analyzing their production of biosurfactants, exopolysaccharides, and the enzymes lipase, protease, and amylase. To study the adaptive capacity of microorganisms to sodium chloride concentrations above the maximum originally tolerated by the wild-type strain, the technique of adaptive laboratory evolution (ALE) was applied. The study used one of the isolates collected in Camocim, selected based on its µ value at 100 g L-1 of NaCl, and a bacterium previously isolated from Trindade Island, Brazil, which had demonstrated high adaptability to NaCl. The evolved and parental/wild-type strains were compared using growth curves and specific growth rate values at different NaCl concentrations. Additionally, it was assessed whether the mutations generated by ALE altered the growth rate in the presence of NaClO4 as a stressor and whether they affected the fatty acid profile in response to NaCl concentration in the growth medium. Among the isolates, those belonging to the genus Bacillus were predominant (18/20). All isolates were able to grow at 100 g L-1 NaCl and at least 30 g L-1 NaClO4, in addition to being able to reduce NaClO4 in the absence of other electron acceptors. The isolates could produce biosurfactants, exopolysaccharides, and enzymes of industrial/biotechnological interest. ALE resulted in variants with a higher specific growth rate and greater tolerance to higher NaCl concentrations compared to the parental strains. Furthermore, an increase in µ at 70 g L-1 NaClO4 was observed, along with changes in the fatty acid profile under both saline and non-saline stress conditions. Keywords: extremophiles; astrobiology; salt adaptation; adaptive laboratory evolution.Item Promotion of microbial phosphate solubilization by clay minerals(Universidade Federal de Viçosa, 2024-08-23) Iglesias Barrera, Gustavo Andrés ; Costa, Maurício Dutra; http://lattes.cnpq.br/8820351730014631A number of physical and chemical interactions between clays and microorganisms have been reported with strong influence on microbial physiology and ecology. However, clay influences on phosphate-solubilizing microorganisms (PSM) remains unknown. PSM are capable of converting low solubility phosphates into soluble orthophosphate that can be taken up by plants. PSM are commonly present in the soil, particularly in the rhizosphere where they maintain associative symbiosis with the host plant. This study aimed at investigating the influence of soil clays (kaolinite, gibbsite, goethite, and hematite) on phosphate solubilization by the fungus Aspergillus niger. In the first chapter, a literature review is presented addressing the potential use of PSM in agriculture. The interaction between clays and microorganisms is reported and the strategies to enhance microbial phosphate solubilization are described. In the second chapter, an investigation on the influence of kaolinite, gibbsite, goethite, and hematite, on organic acid production and phosphate solubilization by the fungal isolate A. niger FS1. The experiments were conducted in modified NBRIP medium containing 3 g L! of Araxá rock phosphate (RP) supplemented with clays at 250, 500, 1000, 1500, 2000, 2500, and 3000 g L'!. The media were inoculated with 10º fungal conidia and incubated for 7 days at 28ºC. AII clays promoted increases in Araxá RP solubilization. The addition of gibbsite and kaolinite to the culture media led to the highest values of soluble P recorded (453.68 and 407.61 mg L', respectively). Gibbsite increased oxalic acid production by the fungus 8.36 times that of the control treatment. Gibbsite and kaolinite also promoted higher yields of citric acid, with 15.69 and 8.93 mmol L' ', respectively. In the third chapter, the hypothetical mechanisms of promotion of phosphate solubilization by clays were verified. Clays were added directly to the culture media as described above or placed in plastic capsules to prevent direct contact of the particles with the fungus. The effects of increasing concentrations of Fe-EDTA, AlCls, and silicic acid on Araxá RP solubilization and organic acid production were also evaluated. The encapsulation of clay particles did not decrease RP solubilization, indicating that the direct contact of the clays with the fungal mycelium is not required to induce RP solubilization. Significant increases in soluble P (3.89 times that of the control treatment) and oxalic acid were observed with increasing concentrations of AlCl;. Fe-EDTA and silicic acid increased phosphate solubilization by only 29 and 11%, respectively, with no significant increases in oxalic and citric acid production. In the fourth chapter, we investigated the effect of kaolinite, gibbsite, goethite, and hematite on the solubilization of Araxá, Argélia, Bayovar, Catalão, Marrocos, and Patos de Minas RPs and aluminum, calcium, and iron phosphate. For this, modified NBRIP medium was supplemented with 3 g L! of each P source above. The media were inoculated with 10º fungal conidia and incubated for 7 days at 28ºC. Gibbsite was the most efficient clay at promoting the solubilization of all RPs tested, reaching 100% cof solubilization for the majority. Kaolinite was particularly effective at the solubilization of iron phosphate, reaching 80.9% of solubilization. Only goethite was capable of enhancing aluminum phosphate solubilization (22./2%). In the fifth chapter, a brief review is presented discussing the question: Are there really microorganisms specialized in phosphate solubilization or is this process merely a side-effect of other microbial metabolic activities? All the findings presented here offer new possibilities for developing innovative strategies to enhance RP solubilization using soil clays. Our results shed light on the interactions between soil clays and fungal hyphae that may operate in the soil to increase P availability to plants. They also improve our understanding on P dynamics in the soil involving PSM and clay minerals. Keywords: Soil clays, phosphate solubilization, organic acids, kaolinite, gibbsite, goethite, hematite, phosphate solubilizing microorganisms.Item Actinobactérias como agentes de biocontrole e promoção de crescimento de soja(Universidade Federal de Viçosa, 2024-08-26) Fernandes, Fábulo Junior Nogueira; Queiroz, Marisa Vieira de; http://lattes.cnpq.br/2541389172357294Item Penicillium sp. 658F5R-AM endofítico de Hevea brasiliensis: anotação do genoma, controle de fitopatógenos, promoção de crescimento vegetal e produção de metabólitos secundários(Universidade Federal de Viçosa, 2024-08-24) Silva, Mirele Lopes da; Queiroz, Marisa Vieira de; http://lattes.cnpq.br/9323296572880336Item Application of eco-friendly alternative methods for controlling Pseudomonas spp. in dairy products(Universidade Federal de Viçosa, 2024-09-27) Rossi, Letícia Elisa; Machado, Solimar Goncalves; http://lattes.cnpq.br/5622267108419962Item Análise de resposta imune de planta induzida por vesículas extracelulares de Ralstonia pseudosolanacearum(Universidade Federal de Viçosa, 2024-08-30) Oliveira, Phedra Gusmão da Silva de; Zerbini, Poliane Alfenas; http://lattes.cnpq.br/0559671336115754O Complexo de Espécies Ralstonia solanacearum (CERS) é composto por bactérias Gram-negativas fitopatogênicas, causadoras da murcha bacteriana, de grande importância no setor agrícola. Esse complexo é formado por três espécies (R. solanacearum, R. pseudosolanacearum e R. syzygii), classificadas em quatro filotipos distribuídos em diferentes regiões geográficas no mundo. A alta diversidade genética, combinada com ampla gama de hospedeiros e alta taxa de sobrevivência da bactéria na ausência de uma planta hospedeira torna a doença de difícil controle. Neste contexto, a utilização de bacteriófagos tem se mostrado uma estratégia promissora para o controle da murcha bacteriana causada pelas bactérias do CERS. Os vírus que infectam bactérias podem se multiplicar de várias maneiras, resultando em diferentes consequências para o hospedeiro, desde a lise celular até infecções crônicas. As infecções crônicas, por sua vez, podem induzir alterações fenotípicas significativas no hospedeiro, afetando características como patogenicidade, crescimento, formação de biofilme entre outras. Vírus classificados na família Inoviridae são vírus que infectam bactérias de forma crônica, e, como consequência desta estratégia de multiplicação, causam diversas modificações fenotípicas nas células infectadas, incluindo alteração da agressividade em bactérias patogênicas. Uma das alterações já descritas é afetar a produção e composição das vesículas extracelulares bacterianas (VEBs). VEBs são nanopartículas complexas de composição e estrutura variável de acordo com o momento fisiológico e interação com o ambiente em que a bactéria está inserida. Com diversas funções, as VEBs podem ser carreadoras de fatores de virulência bastante eficientes, e induzir a resposta imune em plantas ativando mecanismos de resposta de defesa e necrose. Entretanto a resposta imune da planta em resposta à presença de vesículas extracelulares produzidas por bactérias fitopatogências tem sido pouco explorada. O objetivo deste trabalho foi caracterizar VEBs produzidas por Ralstonia pseudosolanacearum, infectadas pelo Ralstonia solanacearum inovirus brazil 1 (RSIBR1), analisar a resposta imune de plantas e possibilidade de imunização contra a murcha bacteriana por VEBs no processo de infecção por R. pseudosolanacearum. O tamanho das vesículas de bactérias não infectadas foi homogêneo, em contraste com bactérias infectadas, onde o tamanho das vesículas produzidas foi bastante heterogêneo. A produção de VEBs também foi afetada em bactérias infectadas pelo inovírus, com dez vezes menos vesículas produzidas quando comparadas com bactérias não infectadas. As vesículas purificadas a partir de culturas de bactérias infectadas e não infectadas causaram reação de hipersensibilidade e necrose tecidual em folhas de tomate, sendo possível observar um perfil fenotípico de resposta mais intensa em plantas infiltradas com VEBs purificadas, a partir da bactéria infectada. A expressão relativa dos genes das proteínas de patogênese classe 1a (pr1A), e semelhante a osmotina (olP) do sistema imune de plantas, obteve um aumento significativo para VEBs purificadas a partir de bactérias infectadas em relação as VEBs de bactérias não infectadas, nos dois períodos de avaliação. A infiltração de plantas com vesículas causou proteção parcial em plantas de tomate contra a murcha bacteriana, com sintomas brandos e reduzida taxa de mortalidade. Esse foi o primeiro relato de VEBs de R. pseudosolanacearum causarem proteção e contra efeitos graves da murcha bacteriana, sendo necessário maior investigação do conteúdo presente nas vesículas. Palavras-chave: vesículas extracelulares; ralstonia spp.; sistema imune de planta.Item Exploring lynronne-1 andcombinational therapy as alternatives to antibiotics for controlling bovinemastitis(Universidade Federal de Viçosa, 2024-05-16) Moreira, Ana Júlia Silva; Mantovani, Hilário Cuquetto; http://lattes.cnpq.br/8282028148510029