Orientador: Oswaldo Durival Rossi Junior / Banca: Angela Cleusa de Fatima Banzatto de Carvalho / Banca: Francisca Neide Costa / Resumo: Tem sido evidente o aumento no consumo de pescado, especialmente do salmão (Salmo salar) sob a forma "in natura", em pratos da cozinha oriental. Como conseqüência, tem havido maior preocupação quanto às suas características higiênico-sanitárias, tendo em vista a facilidade que microrganismos encontram para se desenvolverem em sua carne, o que pode expor os consumidores a agentes que causam desde uma simples gastrenterite até o óbito. Diante desta preocupação, desenvolveu-se este estudo com objetivos de avaliar características microbiológicas do salmão por meio da quantificação de microrganismos heterotróficos mesófilos, coliformes totais e termotolerantes, o perigo de veiculação de Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella sp., Escherichia coli e Aeromonas sp. através da carne e contribuir com subsídios técnicos para criar uma legislação brasileira com padrões microbiológicos específicos para o pescado consumido cru. Foram colhidas 31 amostras de salmão, 16 refrigeradas e 15 congeladas, no comércio varejista de cidades da região nordeste do estado de São Paulo. Os resultados obtidos mostram populações de microrganismos heterotróficos mesófilos variando entre 1,0 x 10 e 3,9 x 106 UFC/g, coliformes totais e termotolerantes em, respectivamente, 32,24% e 19,33% das amostras e Aeromonas sp. em 35,48% das amostras com variação populacional de 2,0 x 102 a 8,0 x 103 UFC/g. Ainda houve a presença de Staphylococcus aureus em uma amostra e ausência de Vibrio parahaemolyticus, Salmonella sp. e Escherichia coli. Os resultados obtidos podem servir de parâmetro para a criação de um padrão microbiológico específico para o pescado consumido cru e servem também de alerta para os consumidores do produto tendo em vista a veiculação de microrganismos potencialmente patogênicos. / Abstract: The increasing of seafood consumption has become evident especially in the use of salmon (Salmo salar) consumed raw in oriental dishes. Consequently, it has risen up the concern related to their hygienic-sanitary characteristics due to the facility that microorganisms multiply in the meat which can expose consumers to the causative agents of a mild gastroenteritis until the death. Regarding such informations, this study was aimed to evaluate microbiological characteristics of salmon by quantifying microrganisms heterotrophic mesophiles, total coliforms and thermotolerant. It was also evaluated the danger of transmission of Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella sp., Escherichia coli and Aeromonas sp. on the fish muscle and contributed to technical informations to create a Brazilian regulations about specific microbiological standards for consumption of raw seafood. Thirty-one samples of salmon were collected, 16 chilled and 15 frozen, from the retail market in cities of the northeast region of São Paulo State. The results show populations of mesophilic heterotrophic microorganisms ranging from 1.0 x 10 and 3.9 x 106 CFU/g, in total and fecal coliforms, respectively, 32.24% and 19.33% of samples and Aeromonas sp. in 35.48% of samples ranging population of 2.0 x 102 to 8.0 x 103 CFU/g. Staphylococcus aureus was present in one sample and were not found Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli. The results may serve as a parameter for the establishment of a microbiological standard for the consumption of raw seafood and also as a warning to consumers of the product for the presence of potentially pathogenic microorganisms. / Mestre

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-10-09T20:37:27Z No. of bitstreams: 1 tamiryssilvabarino.pdf: 1193174 bytes, checksum: 4ad4ca2cfdc016bc7f65fda94a5fb98d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-10-10T12:20:20Z (GMT) No. of bitstreams: 1 tamiryssilvabarino.pdf: 1193174 bytes, checksum: 4ad4ca2cfdc016bc7f65fda94a5fb98d (MD5) / Made available in DSpace on 2017-10-10T12:20:20Z (GMT). No. of bitstreams: 1 tamiryssilvabarino.pdf: 1193174 bytes, checksum: 4ad4ca2cfdc016bc7f65fda94a5fb98d (MD5) Previous issue date: 2017-02-13 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Microrganismos patogênicos são responsáveis pelo desenvolvimento de diversas infecções, e em ambientes hospitalares, levam muitos pacientes ao óbito. Modificar esse quadro é necessário, todavia as bactérias apresentam distintos mecanismos de resistência após mutações em seu DNA. Além disso, o uso indiscriminado de antibióticos gera continuamente a seleção natural e propagação de cepas mais resistentes fazendo com que os antibióticos percam sua eficácia com o passar dos anos. Diante desse panorama, a elaboração de um nanocarreador contendo peptídeos antimicrobianos (PAMs ou AMPs do inglês Antimicrobial Peptides) tornase uma interessante alternativa contra bactérias patogênicas. O objetivo deste trabalho foi sintetizar nanoesferas de alginato de sódio pelo método de Gelificação Ionotrópica para a entrega de AMPs Lrot3 às células bacterianas de Escherichia Coli 25922 e Staphylococcus epidermidis 12228. A atividade antimicrobiana dos peptídeos livres e nanoestruturados (128, 64, 32, 16, 8 e 4µg/mL), foi avaliada por meio da concentração mínima inibitória (MIC), e o potencial citotóxico investigado em células HEK 293 humanas utilizando o ensaio de MTT (Tetrazólio de azul de tiazolilo). Os dados foram avaliados por ANOVA e as médias comparada pelo teste de Tukey. Para a bactéria E. coli 25922, o peptídeo livre na concentração de 64 µg/mL destacou-se por ser a única concentração com atividade antimicrobiana significativa (P˂0,01), apresentando ação similar ao do cloranfenicol (128 µg/mL e 64 µg/mL). As nanopartículas contendo AMPs, por sua vez, mostraram-se eficazes em todas as concentrações (P˂0,01). Diferentemente se deu no S. epidermidis 12228, onde observou-se maior resistência da bactéria a todos tratamentos. Os resultados do MTT sugerem que as nanopartículas de alginato não apresentam citotoxicidade in vitro, motivando a continuidade deste estudo. Concluindo, os AMPs nanoestruturados tem potencial para serem utilizados no combate a infecções ocasionadas por E. coli. / Pathogenic microorganisms are responsible for the development of various infections, particularly in hospital environment, leading to death in many patients. It is therefore necessary to change this scenario, it is difficult, however, because the bacteria have very distinct resistance mechanisms after mutations in their DNA. Furthermore, the indiscriminate use of antibiotics continuously generates the selection and propagation of more resistant strains, causing loss of effectiveness of antibiotics over the years. In this case, the development of a nanocarrier containing Antimicrobial Peptides (AMPs) becomes an interesting alternative against pathogenic bacteria. This paper aims to synthesize the delivery of AMPs to bacterial cells of Escherichia coli 25922 and Staphylococcus epidermidis 12228 through the use of nanospheres of sodium alginate by ionotropic gelation method. Alginate stands out for being a polyanionic, biodegradable and biocompatible polymer, which show interaction with Lrot3 cationic AMP. The antimicrobial activity of free and nanostructured peptides (128, 64, 32, 16, 8 e 4 µg/mL) was evaluated by the minimum inhibitory concentration (MIC) and the cytotoxic potential investigated in human HEK 293 cells using the MTT Assay (Tetrazolium thiazolyl blue). The data were evaluated by ANOVA and the means compared by Tukey test. For Gramnegative E. coli 25922 bacteria, the free peptide at concentration of 64 µg/mL stood out for being the only concentration with significant antimicrobial activity (P˂0,01), showing similar action to the chloramphenicol, at concentrations of 128 µg/mL and 64 µg/mL, respectively. NPs, in turn, showed inhibitory activity at all concentrations (P˂0,01). Unlike occurred in S. epidermidis 12228, which was observed more bacterial resistance to all treatments. The MTT data suggest that alginate nanoparticles do not exhibit in vitro cytotoxicity, encouraging the continuation of this study. In conclusion, the nanostructured AMPs has the potential to be used to fight infections caused by E. coli.

M.Tech. (Biomedical Technology) / Drinking water in the greater Johannesburg area is produced by Rand Water and is transported to local Johannesburg Water where it is stored in reservoirs for distribution. At any point during the production, distribution and storage of the water, contamination with free-living amoebae, potentially containing amoeba resistant bacteria, may occur. Free-living amoebae are often resistant to the biocides used by water treatment industries and may thus be transmitted to public facilities, consumers’ homes and informal settlements through water distribution systems and during storage in small containers. The aim of our study was to analyse the water quality around Johannesburg with regard to free-living amoebae and amoeba resistant bacteria. A total of 182 tap and 5 storage tank water samples, collected from Hillbrow, Bertrams, Riverlea, Braamfischerville and Hospital Hill, were analysed for amoebae, indicator organisms, Legionellae, environmental mycobacteria, Shigella, Salmonella and Vibrio species using amoebal enrichment method. Direct microscopy indicated the presence of amoebae in 96.1% of samples. Acanthamoeba cysts were present in 69.0% of the samples. In 55.0% of these samples visibly active intracellular bacteria were observed within the sample suspensions. In the 46 samples analysed by polymerase chain reaction, the presence of Acanthamoeba species was confirmed in 65.2%, and the intracellular bacteria such as Legionella pneumophila and Mycobacterium avium was confirmed in 23.9% and 73.9% respectively. All samples indicated the presence of Shigella species while one sample contained Salmonella species on xylose lysine desoxycholate agar after amoebal enrichment processing. Vibrio species was not confirmed in the samples. Our results indicated a high risk of transmission of amoeba resistant bacteria through drinking water to people living in these areas.

La pathogénicité des bactéries a toujours été attribuée à des facteurs de virulence et les bactéries pathogènes sont considérées comme étant mieux armées, comparé à des bactéries ne provoquant pas de maladies. Selon les premières études génomiques, le fait de supprimer un certain nombre de gènes des bactéries pathogènes, limiterait leur capacité à infecter leurs hôtes. Au contraire, des études de génomique comparatives récentes, démontrent que la spécialisation des bactéries dans les cellules eucaryotes est associée à une perte de gènes massive, en particulier pour les endosymbiontes allopatriques qui sont isolés depuis longtemps dans une niche intracellulaire. En effet, les bactéries sympatriques, extracellulaires, ont souvent des génomes plus grands et présentent une résistance et une plasticité plus importante. Ces bactéries constituent, de fait, plutôt des complexes d’espèces que de vraies espèces. Certaines bactéries spécialistes, comme les bactéries pathogènes, arrivent à s’échapper de ces complexes et à coloniser une niche, bénéficiant alors d’un nom d’espèce. Leur spécialisation leur permet de devenir allopatriques et leurs pertes de gènes favorisent une évolution réductive. Ces observations nous ont conduits à réaliser une étude afin de quantifier le taux de perte de gènes lors de l’évolution de ces bactéries extracellulaires vers celle de bactéries spécialistes intracellulaires. Notre objectif était de vérifier que ce qui caractérise l’évolution des bactéries intracellulaires est bien la réduction génomique, en prenant en compte tous les événements possibles de gains de gènes. Par ailleurs, dans une étude neutre comparant les 12 espèces pandémiques les plus dangereuses pour l’homme avec les espèces non-épidémiques les plus proches, nous avons voulu identifier des spécificités génomiques associées à la capacité virulente de bactéries pathogènes et démontrer que, à part les toxines et les modules toxine-antitoxine, ce qui caractérise ces espèces ce ne sont pas les facteurs de virulence, mais la perte des gènes de régulation. Au final, les bactéries pathogènes ont un répertoire virulent dans lequel les gènes absents sont aussi importants que les gènes présents. / The virulence of pathogenic bacteria has been attributed to virulence factors and pathogenic bacteria are considered to have more genes compared to bacteria that do not cause disease. According to the first genomic studies, removing a certain number of genes from pathogenic bacteria impairs their capacity to infect hosts. However, more recent studies have demonstrated that the specialization of bacteria in eukaryotic cells is associated with massive gene loss, especially for allopatric endosymbionts that have been isolated for a long time in an intracellular niche. Indeed, bacteria living in sympatry often have bigger genomes and exhibit greater resistance and plasticity and constitute species complexes rather than true species. Specialists, including specific pathogenic bacteria, escape these bacterial complexes and colonize a niche; thereby gaining a species name. Their specialization allows them to adopt allopatric lifestyle and experience reductive genome evolution. These observations led us to design a study to quantify the rate of gene losses during the evolution of free-living bacteria to intracellular specialists. Our objective was to verify that what characterizes the evolution of intracellular bacteria is genomic reduction, taking under consideration all possible gene gain events. Furthermore, in another neutral study comparing the 12 most dangerous pandemic bacteria to Humans to their closest non-epidemic species, we wished to identify any genomic specificities associated to the virulent capacity of pathogenic bacteria and demonstrate that, besides toxins and surprisingly, toxin-antitoxin modules, pathogenic bacteria are not characterized by more virulence factors, but rather by a loss of regulatory genes. Finally, virulent bacteria exhibit a genomic repertoire in which absent genes are as important as present ones.

La qualité de l'eau est une préoccupation majeure pour la santé publique et la préservation de l'environnement. Nous proposons d’utiliser les phages comme des biosenseurs pour détecter les pathogènes humains ou animaux présents dans les eaux de surface, un outil nommé Phagosensor.La mise au point d’un phagosensor prototype a permis d’optimiser la détection des bactéries par cytométrie en flux. Les résultats montrent que cet outil est extrêmement rapide, sensible, et très spécifique. L’outil phagosensor permet de détecter des bactéries cibles présentes dans un environnement complexe tel que l’eau de mer.Le prototype fonctionnel a servi de base à la construction de phagosensors spécifiques de bactéries pathogènes. Les résultats montrent que la stratégie développée à partir du prototype peut être rapidement transposée à la détection de pathogènes tel que Salmonella. Le séquençage de novo et l’annotation des génomes de trois phages isolés de l’environnement permettront la construction de nouveaux phagosensors spécifiques de souches d’E. coli pathogènes.Cette stratégie a été adaptée à la détection d’un signal luminescent post-infection en utilisant les phages recombinants portant l’opéron luxCDABE. Les bactéries infectées sont rapidement détectables.L’ensemble de ces résultats démontre que la stratégie développée est applicable à la construction de phagosensor sur demande pour une détection rapide, sensible et spécifique des bactéries d’intérêt que ce soit en fluorescence ou en luminescence. La détection dans l’eau de mer suggère, qu’à terme, des outils pourront être conçus pour la détection de bactéries pathogènes dans d’autres matrices telles que le sang ou les aliments. / Water quality is a major concern for public health and natural environment preservation. We propose to use phages to develop biosensor tools able to detect human and animal pathogens present in water. The construction of a phagosensor prototype using an optimized genetic engineering strategy, infection and detection conditions, allowed the specific detection of bacteria. The results show that detection is fast, specific and highly sensitive. Moreover, the phagosensor tool detects target bacteria in a complex environment such as seawater. Phagosensors specific of pathogenic bacteria were constructed following the strategy developed for the prototype. Results show that the strategy we designed can be successfully transposed to detect pathogens such as Salmonella. De novo sequencing and genomes annotation of three phages isolated from the environment were carried out to develop phagosensors that are specific of pathogenic E. coli. This technology was then adapted to detect a luminescent signal arising post-infection using genetically modified phages carrying the entire luxCDABE operon. The bacteria infected with the lux recombinant phages were rapidly detected by luminescence emission. Together, these results demonstrate that our technology can be applied to construct various phagosensors adapted to the detection of different bacterial species of interest and using at least two output signals. These tools allow a rapid, specific and highly sensitive detection that are close to the European guideline. Efficient bacterial detection in seawater suggests that phagosensors could be developed to detect pathogenic bacteria in other matrices such as blood or food.

A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. Apesar de sua importância, a patogênese e virulência permanecem não elucidadas. As leptospiras não apresentam proteases conhecidas de degradação de matriz extracelular, atividade crucial para a penetração e disseminação nos hospedeiros. Assim, foi proposta a investigação da interação de leptospiras com plasminogênio/plasmina e as implicações para a infecção. As leptospiras capturam plasminogênio na superfície, e este é convertido à plasmina por ativadores do hospedeiro. A plasmina associada propicia degradação de componentes de matriz extracelular, habilidade de penetração e evasão imune. Adicionalmente, as leptospiras estimulam a expressão de ativadores de plasminogênio e metaloproteases de matriz. Os resultados contribuem para o conhecimento do processo infeccioso das leptospiras, descrevendo um novo mecanismo de patogenicidade. / Leptospirosis is a zoonosis caused by pathogenic bacteria from genus Leptospira. Despite its importance, the pathogenicity and virulence remain to be elucidated. The leptospires do not present known proteases able to degrade extracellular matrix, an activity essential for the penetration and dissemination within the hosts. Therefore, we proposed the investigation of the leptospiral interaction with plasminogen/plasmin and its implications for infection. Leptospires capture plasminogen on the surface, which is converted to plasmin by hosts activators. Surface-bound plasmin confers extracellular matrix components degradation, penetration ability and immune evasion. Additionally, leptospires stimulate plasminogen activators and matrix metaloproteases expressions. The results constitute one possible mechanism that contributes to the invasion process and the rapid dissemination of Leptospira.

Thesis (M.Sc.(Microbiology)) -- University of Limpopo, 2019 / Salmonella enterica serovar Gallinarum biovars Gallinarum, and Pullorum, Pasteurella multocida and Escherichia coli are among the most important pathogens in poultry and are the causal agents of fowl typhoid, pullorum disease, fowl cholera and collibacillosis in poultry. The present study was designed to identify and determine the distribution of these pathogens in household-raised chickens and their antibiotic and virulence profiles. For this purpose, 40 chickens were bought from household families at Ga-Dikgale, GaMolepo and Ga-Mphahlele in the Capricorn district of Limpopo Province and sacrificed for sampling. Tissues including breast meat, lungs, small and large intestines were harvested from each chicken. Bacteria associated with these samples were cultured in selective bacteriological media followed by biotyping using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) for identification. Out of a total of 160 tissue samples evaluated, E. coli and Salmonella were detected in these tissues. Furthermore, determination of the pathogenic E. coli and Salmonella strains at species level using primer sets that target selected genes of interest in the polymerase chain reaction (PCR) assay was employed. The invA gene, a confirmatory gene for Salmonella species was detected in all the Salmonella isolates using PCR. For the pathogenic E. coli, astA, eae, hlyA, fIiCH7, stxI and the fimbrial genes (F6 and F41) were detected in some of the E. coli isolates recovered from the samples. Disk diffusion test was also performed to determine the antibiotic susceptibility of the bacteria. The results from the current samples reveals that there is a high distribution of Salmonella and pathogenic E. coli in these areas and therefore further epidemiological and identification studies are needed to determine these organisms at species level and investigate their pathogenicity. The antimicrobial susceptibly data generated from this study can be a valuable reference to veterinarians for treating bacterial diseases in poultry.

Yeast probiotics and paraprobiotics, live and inactivated yeast cells, respectively, improve health and performance of livestock by stabilizing the intestinal microbial community. They have also been used for infection prevention and treatment. Despite much research already conducted, the mechanism of direct antagonism, or adhesion of bacteria to the probiotic/paraprobiotic, is under characterized. Additionally, it is unknown which probiotic/paraprobiotic is optimal to use for specific infections. The interactions between the yeast and certain pathogens were analyzed qualitatively with scanning electron microscopy (SEM) and quantitatively with membrane filtration assays. Gram-positive bacteria were found to exhibit specificity under SEM. Through membrane filtration, Listeria monocytogenes exhibited binding to all samples (P<0.05), while Salmonella Typhimurium exhibited binding (P<0.001) with all samples except with 2338. Escherichia coli O157:H7 only bound to the probiotics (P<0.001). With a better understanding of how specific yeast probiotics and paraprobiotics interact with bacteria, specific therapies can be administered to combat infections.

This study is entitled, “determination of the presence of antibiotics and bacterial pathogens with their susceptibility in fresh milk from the San Nicolas community and the cities of Penipe, Guano, and Chambo in the Chimborazo province”. It was funded by the Benson Institute of Brigham Young University and Nestle. The objectives were: 1) to determine the presence of antibiotics in fresh milk from the San Nicolas community and the cities of Penipe, Guano, and Chambo, 2) to determine the presence of bacterial pathogens in milk from the places previously mentioned, and 3) to detect the susceptibility of the isolated bacteria to the most commonly used antibiotics on the market. With regard to the presence of antibiotics, they were only found once in Chambo (beta-lactams) and once in San Nicolas (tetracycline). Three types of pathogenic bacteria were found: Escherichia coli, Streptococcus agalactiae, and Staphylococcus aureus. Only one bacterium was found that has not been studied: Pseudomonas aureoqinosa. This bacterium was found in milk from San Nicolas. With regard to the susceptibility of the bacteria to antibiotics, it was found that Escherichia coli was sensitive to the following antibiotics: nitrofurans, erimethoprina, trimethoprim and sulfonamides, and tetracyclines and spectomicina. Escherichia coli also showed medium sensitivity to chloramphenicol as well as resistance to ampicillin and sulfonamides. Streptococcus agalactiae was sensitive to lincomycin, neomycin, enrofloxacin, and clindamycin. It also showed medium sensitivity to erythromycin and low tetracycline and penicillin as well as resistance to gentamicin. Lastly, Staphylococcus aureus was sensitive to neomycin and enrofloxacin. In addition, it showed medium sensitivity to lincomycin, gentamicin, clindamycin, and tetracycline as well as resistance to low erythromycin and penicillin. This study was done over a four month period with at least 12 samples being collected from each area. A total of 60 samples were analyzed. Based on the results obtained, our recommendation is that hygienic measures are taken in obtaining and transporting milk to eliminate the growth of bacterial pathogens. Also, we recommend the use of the effective antibiotics listed above in order to completely eradicate these microorganisms.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24898号 / 工博第5178号 / 新制||工||1988(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 伊藤 禎彦, 教授 松田 知成, 教授 越後 信哉 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM