Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-03-27T12:00:11Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-03-27T13:49:39Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2018-03-27T13:49:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-03-02 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O queijo Minas Frescal (QMF) representa um dos queijos mais consumidos no Brasil. Diversos fatores do seu processamento influenciam suas características microbiológicas, e consequentemente, sua qualidade e propriedades organolépticas. Seu alto teor de umidade e os riscos de contaminação durante a cadeia produtiva favorecem a ocorrência de microrganismos contaminantes, muitas vezes apresentando resistência aos antimicrobianos. Dessa forma, do ponto de vista da segurança alimentar e frente ao crescente fenômeno da resistência bacteriana às drogas, torna-se importante a investigação sobre a estrutura da comunidade bacteriana em QMF, bem como a avaliação da ocorrência de marcadores genéticos microbianos relacionados à resistência a drogas e seu potencial de mobilização. Neste estudo foram obtidas 5 amostras de um mesmo lote de 7 marcas de QMF identificadas de A a G, totalizando 35 amostras. Após a extração de DNA total microbiano das amostras, foram utilizadas abordagens de DNA fingerprint, pela amplificação de sequências palindrômicas extragênicas repetitivas (rep-PCR) para avaliação comparativa da similaridade da estrutura global da comunidade bacteriana. Posteriormente, PCR-DGGE foi utilizada para avaliar o perfil e a riqueza das amostras com relação a grupos de bactérias láticas. Matrizes de similaridade foram obtidas utilizando o método de agrupamento UPGMA. Os resultados obtidos pela técnica de rep-PCR revelaram que as amostras de queijos foram claramente agrupadas de acordo com as suas respectivas marcas. Além disso, perfis semelhantes entre amostras de marcas diferentes foram observados, indicando a presença de um núcleo microbiano comum. As amostras avaliadas também foram agrupadas de acordo com suas respectivas marcas de fabricação de acordo com os padrões de DGGE obtidos para bactérias láticas. A elevada similaridade entre a maioria das amostras do mesmo lote obtida nas técnicas de fingerprint sugere a reprodutibilidade e aplicabilidade das técnicas, e controle no processamento dos queijos ao longo da cadeia produtiva. Para a avaliação do resistoma clínico, a presença de 40 marcadores de resistência a diferentes classes de antibióticos foi avaliada por reação de PCR. Um núcleo comum de marcadores genéticos em todas as marcas foi detectado, associado à resistência aos beta-lactâmicos, tetraciclinas, quinolonas e sulfonamidas. Outros marcadores, incluindo aqueles relacionados a bombas de efluxo e resistência aos aminoglicosídeos, também foram observados. Integrons de classes 1 e 2 foram detectados, respectivamente, em 77% e 97% das amostras. As diferentes amostras de QMF puderam ser agrupadas de acordo com seu perfil de marcadores genéticos de resistência aos antimicrobianos, o que sugere epidemiologia peculiar que pode estar relacionada a qualidade e aos níveis de contaminação dos queijos ao longo da cadeia produtiva. Em conjunto, os dados sugerem que embora a cadeia produtiva do QMF seja controlada na indústria, riscos sanitários são inerentes pela contaminação dos queijos por bactérias putativas resistentes a antimicrobianos. Como um todo, os dados apontam para a necessidade de discussão dos parâmetros de qualidade microbiológica na produção, armazenamento e distribuição de QMF. Além disso, a detecção de integrons de classe 1 e 2 levanta questões a respeito do potencial de transferência horizontal de genes de resistência para a microbiota humana através do consumo destes alimentos. / Minas Frescal cheese (QMF) represents one of the most consumed cheeses in the country. Several factors of its processing influence its microbiological characteristics, and, consequently, its quality and properties. Its high moisture content and the risks of contamination during the production chain favor the occurrence of contaminating microorganisms, often presenting antimicrobial resistance. Thus, from the point of view of food safety and the growing phenomenon of bacterial resistance to drugs, it is important to investigate the structure of the bacterial community in Minas Frescal cheese, as well as the evaluation of the occurrence of microbial genetic markers related to drug resistance and its potential for mobilization. In this study 5 samples from the same batch of 7 brands of Minas Frescal cheeses were identified from A to G, totaling 35 samples. After the extraction of total DNA from the samples, DNA fingerprint approaches were used, by the amplification of repetitive extragenic palindromic sequences (rep-PCR) to evaluate the similarity of the global structure of the bacterial community. Afterwards, PCR-DGGE was used to evaluate the profile and richness of the samples in relation to groups of lactic bacteria. Similarity matrices were obtained using the UPGMA clustering method. The results obtained by the rep-PCR technique revealed that the cheese samples were clearly brand-clustered. In addition, similar profiles among samples of different brands were observed, indicating the presence of a common microbial nucleus. The evaluated samples were also separated according to their respective manufacturing brands by the DGGE for lactic acid bacteria. The high similarity among the majority of the samples from the same batch obtained in the fingeprint techniques suggests the reproducibility and applicability of the techniques, and control in the cheese processing along the production chain. For the evaluation of clinical resistance, the presence of resistance markers to different classes of antibiotics was evaluated by PCR reaction. A common core of genetic markers was detected, associated with resistance to beta-lactams, tetracyclines, quinolones and sulfonamides. Other markers, including those related to efflux pumps and aminoglycoside resistance, have also been observed, but not in all brands. Integrons of classes 1 and 2 were detected, respectively, in 77% and 97% of the samples. The different QMF samples could be grouped according to their profile of genetic markers of antimicrobial resistance, which suggests peculiar epidemiology that may be related to the quality and levels of contamination of the cheeses along the production chain. Taken together, the data suggest that although the productive chain of QMF is controlled in the industry, health risks are inherent in the contamination of cheeses by putative antimicrobial resistant bacteria. As a whole, the data point to the need to discuss the parameters of microbiological quality in the production, storage and distribution of QMF. In addition, the detection of class 1 and 2 integrons raises questions about the potential for horizontal transfer of resistance genes to the human microbiota through the consumption of these foods.

WGS permet d'analyser et de déchiffrer l'étude de résistances de bactéries multirésistantes(MDR), en comprenant les différents mécanismes de résistance, les annuaires génétiques. Au cours de ma thèse de doctorat, j'ai réalisé: 1 revue de la littérature sur l'utilisation de nouveaux outils de diagnostic contemporains et les capacités dans la détection des foyers dans les maladies infectieuses causées par MDR. L'identification et l'analyse de résistances de bactéries multirésistantes Comme étant des Shewanellalgae, normalement de l'environnement marin, dans notre cas une souche clinique isolée du lavage bronchoalvéolaire d'un patient hospitalis avec pneumonie et Chryseobacteriumin dologenes, isolé d'une fibrose kystique du patient. Dans cette analyse, nous pouvons montrer que les bactéries environnementales telles que les S.algae peuvent être un réservoir de gènes de résistance aux antibiotiques. L'analyse exhaustive de ces bactéries a montré leur capacité à s'adapter à leurs écosystèmes, y compris l'acquisition de nouveaux éléments génétiques par transfert latéral de gènes. La détection des gènes impliqués dans la synthèse de peptides synthetasenon ribosomale et de polycétide synthétase peut avoir un rôle dans leur capacité à survivre dans des environnements hostiles tels que le tractus respiratoire des patients atteints de fibrose kystique ou leur présence chez des patients ayant subi plusieurs antibiotiques. Nous avons réalisé une analyse standardisée «insilico» afin de déterminer la résistance de ces bactéries et la présence de métabolites secondaires associés aux bactériocines et aux NRPS/PKS. L'application du NTS pour le séquençage du génome bactérien de nouvelles espèces bactériennes isolées dans le microbiome humain nous a permis de développer une plateforme capable d'analyser ces nouvelles espèces dans les 48heures. Ce travail permet de mieux comprendre la biodiversité des bactéries isolées dans le microbiome humain. / Theuse of WG Sallows to analyze and to decipherthe study of resistome of Multi Drug Resistant bacteria (MDR), understanding the different resistance mechanisms, genetic directories and their dissemination mechanisms at global level. During them y thesis I have achieved: 1. A literature review on the use of new contemporary diagnostic tools and capabilities in detecting out breaksin infectious diseases caused by MDR. 2: The identification and the analysis of resistome of multidrug resistant bacteria from clinical isolates suchasShewanellaalgae, normally marine environmental, in our case clinical strain isolated from the broncho alveolar lavage of a hospitalized patient with pneumonia and Chryseobacteriumin dologenes, isolated from a patient cysticfibrosis. In this analysis, we can show that environment albacteria suchas S.algae can be a reservoir of antibiotic resistance genes. The exhaustive analysis of these bacteria showed their ability to a dapttotheirecosystemsincludingtheacquisitionofnewgeneticelementsbylateralgenetransfer. The detection of genes in volved in the synthesis of nonribosomal peptide synthetase and polyketide synthases may have a role in their ability to survive in hostile environments suchas therespiratorytractofCFpatients or their presence inpatients having suffered multipleofantibiotic. 3:In this work,through theuse of the NTS onnew bacterial species isolated from human microbiome,we have a chieveda standardized analysis"insilico"to determine there sistome of these bacteria and the presence of secondary metabolites associated bacteriocins and the NRPS/PKS. The application of the NTS for sequenc in go bacterial genome of new bacterial species isolated in the human microbiome, allowe dus to develop a platform capable of analyzing the senew species within 48

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Antibiotics are one of the greatest discoveries in medicine, and emerged resistances have become a global threat. It is theorized that a big part of the antibiotic resistance genes come from the environment, and wastewater treatment plants and hospitals are considered a great breeding ground for the spread of these. The aim of this project is to analyse the microbiome and resistome of the wastewater of Uppsala and to evaluate the efficiency in the elimination of antibiotic resistance genes and bacteria. Samples from the University Hospital and the influents, sand filter and effluent of the Wastewater Treatment Plant were collected, DNA was extracted and sequenced to be analysed through metagenomics to explore them taxonomically and looking for resistance genes. Bacteria were also isolated, and their resistances were analysed. Taxonomical differences became noticeable in Order, Family, Genus and Species, with an increase of diversity in the Effluent samples. A total of 233 resistance genes were found in all the samples. There was a clear reduction in the number of resistance genes in the Effluent samples. However, there was an important number of genes carried in these and some prevail through all the path. Within all the isolates collected, from a total of 11, three E. coli isolates, one C. freundii and one E. cloacae presented resistances. Our study shows that the effluent of the wastewater treatment plant of Uppsala is potentially causing a negative impact on the environment, flushing out water not completely free of antibiotic resistance genes and resistant bacteria.

Widespread use of antibiotics in livestock production can result in the dissemination of bacteria carrying antibiotic resistance genes (ARGs) to the broader environment. Within agroecosystems, ARGs can pose a risk to livestock handlers, farmers, and ultimately consumers. The overall goals of this dissertation are to examine the presence of resistance (antibiotic, metal) in agricultural soils and evaluate the most critical potential points of best management control of antibiotic resistance spread along the agricultural production chain. The relative impacts of agricultural practices, manure management, native soil microbiota, and type of crop grown and harvested on the agricultural resistome are multi-dimensional and cannot be captured via a single analytical technique or by focusing on one specific point in the agricultural process. Culture-, molecular "indicator"-, and next-generation sequencing- based methods were employed to characterize antibiotic resistance via taxonomic and functional profiles on the broader manure, soil, and vegetable surface microbial communities through 16S rRNA amplicon sequencing and shotgun metagenomics. Although antibiotic concentrations dissipated in the soil after 28 days after amendment application, antibiotic resistance presence was recoverable throughout the entire 120d growing season in the compost and manure amendments, the amended soil, and on vegetable surfaces. The addition of organic fertilizers increased antibiotic resistance presence compared to background levels. Further, metals and metal resistance were also measured in the amended soils and were found to be at greater levels in the inorganically fertilized soils compared to the manures and compost amended soils. Analysis of the widespread agroecosystem microbial community composition and broader metagenome has characterized varying genera profiles in the soil and on the vegetable surfaces and specific ARG and mobile genetic element (plasmid) co-occurrences. These co-occurrences highlight which ARGs may be most critical for future antibiotic resistance dissemination research. It is imperative to employ multiple methods when measuring agricultural resistance, as one method alone may miss significant patterns and lead to different best management recommendations. Linking the livestock manure, soil, and vegetable surface-associated ARBs, ARGs, resistomes, and microbiomes will help identify critical control points for mitigation of agricultural dissemination of antibiotic resistance to the environment and food production. / Doctor of Philosophy / By 2050, it is estimated that antibiotic resistant infections will be the leading cause of death worldwide. It is important to consider human, animal, and environmental health when researching antibiotic resistance, which is known as a "One Health" approach. In this dissertation work, I focus on the environmental side of antibiotic resistance in our agricultural systems. Agriculture is a known source of antibiotic resistance due to its use of antibiotics in livestock as a treatment for illness, and in some instances, as a growth promoter. Over one growing season, I measured antibiotic resistance in an agricultural setting using many techniques. First, I analyzed the effects of inorganic (chemical) versus organic (manure and compost) fertilization on antibiotic resistance in the soil. I measured antibiotic resistance by growing antibiotic resistant bacteria, quantifying specific antibiotic resistant genes (ARGs) using DNA amplification, and quantifying all the ARGs in the soil using a next-generation sequencing (NGS) technique called shotgun metagenomics. I found that adding manure to the soil increases ARGs compared to background soil levels, and that composting in an effective management strategy in decreasing ARGs in the soil over time. Second, I analyzed the same effects of fertilization on metal resistance in the soil. I was able to use the same NGS dataset to measure metal resistance genes (MRGs). I found that adding inorganic chemical fertilizer increases MRGs in the agricultural soils compared to the organic (manure or compost) fertilizer. Additionally, I studied the microbes that live in the agricultural soils using another kind of NGS data specific for microbial identification. I found that although there were small differences between the microbial populations in the soil when fertilizers were added, they returned to similar composition over the growing season. Lastly, I measured antibiotic resistance and microbes throughout the entire agricultural system. I picked the point of fertilization (manure management), soil, and the lettuce surface to evaluate if antibiotic resistance spreads from the farm to the vegetable that ends up on a consumer's plate. I found that at each point antibiotic resistance is present, but at different levels. Composting reduces ARGs compared to raw manure. Agricultural soils may act as a natural buffer to antibiotic resistance. Lettuce plants grown in compost fertilized soils have less ARGs than lettuce plants grown in manure. There are many agricultural management practices that effectively reduce antibiotic resistance and using all of them plus many measurement methods will ultimately help farmers and consumers reduce antibiotic resistance in our agricultural systems.