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L’insorgenza e la diffusione dell’antibiotico resistenza sta diventando un problema a livello mondiale. Molti sono gli ambienti in cui può avvenire tale diffusione, ma una delle principali vie di trasmissione passa attraverso la catena alimentare. Infatti, l’utilizzo di sostanze antimicrobiche è largamente diffuso negli allevamenti di animali ad uso alimentare e in agricoltura. In particolare, negli allevamenti gli antibiotici non solo vengono usati per trattare eventuali patologie, ma anche come profilassi e come promotori di crescita. Di conseguenza, questo uso a volte sconsiderato ha portato all’insorgenza di batteri resistenti a tali sostanze. Un ruolo fondamentale nella trasmissione e diffusione di tali resistenze a livello alimentare è svolto da batteri non patogeni che sono parte del naturale microbiota degli alimenti. Questi microorganismi infatti, pur non essendo essi stessi nocivi per l’uomo, possono fungere da reservoir di antibiotico resistenze per eventuali batteri patogeni. I batteri che generalmente svolgono questo ruolo sono i batteri lattici. Per questo motivo molto importante è stato identificare e studiare l’antibiotico resistenza anche di tali microorganismi. Negli ultimi anni, tuttavia, c’è stato un crescente interesse per un’altra classe di microorganismi, chiamata Haloarchaea o alobatteri o archaea alofili, poiché la loro presenza è stata rilevata in alimenti particolarmente salati. Dal momento che in letteratura ci sono pochi lavori che studiano i profili di antibiotico resistenza di tali microorganismi e, comunque, tali profili non sono stati studiati su un numero significativo di microorganismi appartenenti alla stessa specie, il presente lavoro di tesi è volto a definire il profilo di antibiotico resistenza del capostipite degli archaea alofili, che è l’Halobacterium salinarum, verificare se ci sono ceppi che presentano antibiotico resistenze e controllare se tali resistenze possono essere trasferite a batteri patogeni. / Antimicrobial resistance is now widely acknowledged as a major global public health challenge. There are many environments through which the transmission and diffusion of antibiotic resistance could happen, but one of the main routes of transmission is the food chain. As a matter of fact, antibiotic use is widely spread in animal husbandry and in agriculture. In particular, in animal husbandry antimicrobials have been used both for therapeutic reasons and as growth promoters. As a consequence, a selective pressure on pathogenic and commensal bacteria of animal origin has been exerted during the time, leading to the onset of microorganisms resistant to such compounds. A pivotal role in the spread in the food chain of antibiotic resistance has been played by non-pathogenic bacteria present in food. These microorganisms are not harmful for humans, but they could represent a reservoir of antibiotic resistance for foodborne pathogenic bacteria. Usually lactic acid bacteria play this role, since they are present in all fermented food. For this reason, the antibiotic resistance profile of lactic acid bacteria has been assessed. In recent years, another class of microorganisms called halophilic archaea have raised an increasing scientific interest, since they have been found in the human intestinal mucosa as well as in foods such as salted codfish and fermented Asiatic seafood. As a few papers have studied the antibiotic resistance profiles of halophilic archaea, and the only present do not consider a statistically significant number of microorganisms belonging to the same species, the aim of the present work is to define the antibiotic resistance profile of the major exponent of halophilic archaea, named Halobacterium salinarum, and consequently to verify if some strains present antibiotic resistances and if they can transfer these resistances to bacteria present in the food chain.

Made available in DSpace on 2014-12-17T14:05:23Z (GMT). No. of bitstreams: 1 UaskaBS_TESE.pdf: 2951660 bytes, checksum: 93152a3c6ea30c7f4eaa465f3f5d2969 (MD5) Previous issue date: 2013-04-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundia?-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundia? river / A busca por genes baseada na constru??o e an?lise de bibliotecas metagen?micas a partir de solo gera oportunidades para explorar uma enorme diversidade gen?tica e metab?lica de microrganismos. Os rios s?o ecossistemas com alta diversidade biol?gica, mas ainda pouco explorados por meio de metagen?mica. Com o objetivo de explorar a diversidade microbiana, uma biblioteca metagen?mica foi constru?da a partir de DNA extra?do de substrato de rio em tr?s pontos ao longo do rio Jundia? (Rio Grande do Norte-Brasil). Os pontos de amostragem s?o derivados de ?rea aberta, terreno acidentado e com a incid?ncia direta da luz solar. Esta biblioteca foi analisada funcionalmente e tamb?m com base em sequ?ncias. Para a an?lise funcional foi utilizado meio de cultura s?lido LB com concentra??o de NaCl variando de 0,17M a 0,85M. Foram obtidos 15 clones positivos com caracter?sticas halotolerantes. Os DNAs recombinantes foram extra?dos e retransformados em cepa de Escherichia coli DH10B e curvas de sobreviv?ncia foram obtidas para confirma??o e quantifica??o da resist?ncia ao estresse abi?tico. As sequ?ncias dos clones foram obtidas e submetidas a ferramenta BLASTX e assim foi comprovado que alguns clones codificavam prote?nas hipot?ticas. Um dos clones apresentou uma ORF completa com elevada similaridade de glucose-6-fosfato-isomerase que participa na s?ntese do precursor de glicerol, sendo um soluto compat?vel para equilibrar a press?o osm?tica no interior e no exterior das c?lulas. Posteriormente, para identifica??o de genes que codificam osm?litos relacionados com halotoler?ncia e identifica??o da diversidade microbiol?gica, amostras de DNA ambiental do substrato do rio e da coluna d??gua do estu?rio e oceano foram coletadas e pirosequenciadas. As sequ?ncias de osm?litos de diferentes microrganismos foram obtidas a partir do UniProt e utilizadas como RefSeqs para a identifica??o por homologia (TBLASTN) nos bancos de dados metagen?micos. As sequ?ncias identificadas nos bancos de dados ambientais foram submetidas ao programa HMMER com o fim de identificar dom?nios funcionais. Foram identificadas as enzimas: alfa-trealose-fosfato sintase, L-ectoina sintase (ectC), transaminase do ?cido L-2,4-diaminobut?rico (EctB), ?cido L-2 ,4-diaminobut?rico acetiltransferase (EctA), L-treonina 3-desidrogenase (via de s?ntese do sorbitol), Glicerol-3-fosfato desidrogenase, inositol-3-fosfato desidrogenase, chaperonas, L-prolina glicina beta?na liga??o transportador ABC, mio-inositol-1-fosfato sintase, a prote?na simportadora de prolina/s?dio -PutP e trealose-6-fosfato fosfatase. Estas s?o enzimas que participam da s?ntese de osm?litos comumente relacionados a ambientes salinos, no entanto a identifica??o desses solutos em ambiente de rio ? justificada pela elevada concentra??o salina no solo durante prolongadas esta??es de seca neste rio. Quanto ? riqueza da microbiota foi identificado que o substrato do rio possui uma abund?ncia de halobact?rias semelhante a do mar e superior a do estu?rio. Esses dados confirmam a exist?ncia de uma resposta especializada contra o estresse salino por microrganismos no ambiente do rio Jundia?

Exploité depuis toujours, le sel a connu, au temps de la Gabelle notamment, des heures de gloire certaines, faisant de lui un métal blanc précieux, recherché et coûteux. Cependant, de nos jours, il est considéré comme un minéral essentiel, certes, mais aussi dangereux pour la santé si surconsommé. Ainsi, les paludiers d'aujourd’hui doivent mettre en avant le caractère authentique de leur produit et leur savoir-faire ancestral, symboles de qualité dans l'esprit des contemporains, pour réussir à maintenir leur activité et leur part de marché. L'objectif de cette étude était ainsi de définir des pistes qui garantiraient aux paludiers la protection de leur travail, par exemple dans le cadre d'une démarche d'appellation d'origine contrôlée ou protégée. Pour ce faire, un partenariat avec des sauniers de la Côte Atlantique Française (Ile de Ré, Ile de Noirmoutier, presqu'Ile de Guérande, salines de Saint-Armel) a permis de collecter divers échantillons (eaux des marais, sels). Dans un premier temps, une recherche de microorganismes et une étude sur les traces d'ADNr-16S présent sur les cristaux de sel, ont permis de caractériser une partie du microbiote halophile se développent au niveau des bassins de production du sel de mer, alors que leur teneur en sel peut aller jusqu'à 25 %. Dans un second temps, une recherche de composés volatils a été conduite afin de déterminer si l'environnement pouvait influencer l'empreinte olfactive des eaux des marais et du sel lors de sa formation et / ou de sa récolte. Un protocole d'extraction et d'analyses a été développé et a permis la mise en évidence d'un profil en composés volatils propre à chaque bassin. Parmi les composés volatils détectés, de nombreux norisoprénoïdes, provenant probablement de la dégradation de caroténoïdes produits par les microorganismes halophiles, ont ainsi été identifiés : pour le « bassin » Ile de Ré, 21 composés volatils ont été identifiés dont 8 composés dérivés des caroténoïdes (CDC) ; pour le « bassin » de Noirmoutier, 13 composés dont 7 CDC ; pour le « bassin » de Saint-Armel, 54 composés dont 25 CDC ; pour le « bassin » Guérandais, 19 composés dont 10 CDC.D'une façon générale, les résultats obtenus aussi bien d'un point de vue microbiologique que chimique, ont révélé une forte corrélation entre les marais salants et le sel qu'ils produisent : les microorganismes spécifiques d'un environnement laissent des empreintes sur les eaux et le sel, ce qui permettrait notamment aux paludiers de caractériser leur produit en vue d'une protection basée sur de véritables marqueurs propres à chaque marais (pour des origines distantes de quelques kilomètres, des différences sont déjà notables entre salines tant au niveau « odeur » qu'au niveau microbiote). / Salt has always been exploited in living memory. It has known its heyday at several occasions throughout history, particularly during the period of the French Gabelle, and therefore came to be seen as a precious white metal both sought after and expensive. Yet, nowadays, although it has been considered as an essential mineral, it has also turned up to be unhealthy when taken in excessive amounts. Consequently, salt workers of our day and age have to highlight the genuine nature of their product as well as their ancestral skills, for they both stand out as tokens of quality for our contemporaries. Meeting the expectations of the consumer is the only way for them to keep up with their work and maintain their share on the market. Thus, this study aimed to define ways for salt workers to have their work preserved, for instance throughout a Protected Designation of Origin. Therefore, a partnership with Atlantic French salt workers (from Ré Island, Noirmoutier, Guérande and Saint-Armel) has been established, allowing us to collect samples of salt marsh water and salts.First, an overview of microorganism population and 16S-rDNA in each water or salt sample permitted to define what kinds of microorganism populations were to be found in salt marshes. Secondly, a search for volatile components was led so as to determine whether the environment might affect the olfactory footprint of salt marshes and of salt itself during its formation and its harvest. A process of extraction and analysis has been developed, shedding light on a link between the origin and the olfactory footprint of salt. As an example, the halophilous microorganisms which are extremely rich in carotenoid (hence the red-orange colour of some marshes) are partly responsible for the presence of norisoprenoids in the volatile components which have been identified: 21 compounds were identified in Ré Island (including 8 norisoprenoids), 13 in Noirmoutier (including 7 norisoprenoids), 54 in Saint-Armel (including 25 norisoprenoids),19 in Guérande (including 10 norisoprenoids).For each area, DNA traces and volatile profiles were identified. Therefore, a strong link can be established between salt marshes and the salt they produce. It appears that the differences between salt flats regarding either their smell or their microbiota is always noteworthy, even when marshes are only a few miles apart. Thus, the specific pool of the identified microorganisms which leave prints on the salt would allow saltworkers to define their product so as to ensure a form of protection based on specific markers which are proper to each marsh.