Göttingen, Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Göttingen, Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Göttingen, Universiẗat, Diss., 2001.

Cellulose, the most abundant biopolymer on earth, is composed of β – 1,4 – linked glucose units, which in turn form a highly ordered crystalline structure that is insoluble and recalcitrant to degradation. It is the world’s most attractive, abundant and renewable energy resource, representing the bioconversion of carbon dioxide into green plants. Cellulosic biomass, such as agricultural and forestry residues, waste paper and industrial waste can therefore be used as an inexpensive and abundantly available source of sugar for fermentation into fuel ethanol. The combustion of biofuels releases carbon dioxide which is thus recycled and hence the use of these fuels in transportation provides an alternative to fossil fuels, solving many environmental problems. The ability to degrade crystalline cellulose seems to be restricted to a specialized group of microorganisms which includes for example Clostridium, Cellulomonas, Cytophaga, Trichoderma etc. Hence the aim of this project was to create BioBricks using different cellulases from cellulose degraders and express them in different expression hosts like Escherichia coli, Bacillus subtilis, Citrobacter freundii etc., using two different promoters, spac and lac. It was observed that the expression of Cytophaga hutchinsonii cellulases (CHU_2103 and CHU_2802) and dehydrogenases (CHU_1944 and CHU_2315) was toxic to the E. coli host for some unknown reason. Therefore it was decided to use cellulases from Cellulomonas fimi, which are well characterized. BioBricks of cellulases (cenA and cex) from C. fimi were introduced into different expression hosts. It was observed that under our experimental conditions Citrobacter freundii SBS197 gave the best results. Both Pspac and Plac were functional in this organism with expression being higher when Pspac was used. When E. coli JM109 was used as an expression host, activity was only detected when the lac promoter was used to control the expression. Although the expression was higher when E. coli JM109 (containing Plac) was used as an expression host, almost all of this activity was residing within the cells, whereas when C. freundii SBS197 was used as an expression host, considerable activity was detected in the surrounding medium, which is essential for cellulose degradation. Growth curve studies were done to see if heterologous cellulases enable the host to use cellulosic substrates as a source of carbon. It was observed that C. freundii SBS197 expressing cenA and cex was able to use filter paper and Avicel as a source of carbon with maximum growth of up to 8.8×108 cfu/ml and 1.2×109 cfu/ml respectively. This was about 2 – 5 fold higher when compared to the control (vector and/or negative) strains. Filter paper completely disappeared within 3 – 4 days when C. freundii SBS197 was used. Slight degradation was observed when E. coli JM109 was used but there was no physical degradation seen when B. subtilis 168 was used as an expression host. Hence it was concluded that heterologous cellulases impart to C. freundii SBS197 with the ability to use cellulosic substrates as a source of carbon. The maximum growth obtained using these cultures is to our knowledge higher than what has been reported so far for recombinant organisms expressing heterologous cellulases using cellulosic substrates as a source of carbon.

Citrobacter freundii sind Gram-negative, bewegliche, stäbchenförmige Bakterien aus der Familie der Enterobacteriaceae. Als opportunistischer Erreger kann C. freundii beispielsweise Harnwegsinfektionen und Neugeborenen-Meningitis verursachen. Die Internalisierung von C. freundii 3009 in das Endosom von Harnblasenepithelzellen (T24) erfolgt in vitro über einen ausschließlich Mikrotubuli-abhängigen Mechanismus. Als genetische Grundlage der Invasionskompetenz von C. freundii 3009 wurde in Vorarbeiten eine im Chromosom lokalisierte Invasionsdeterminante identifiziert, die eine hohe Identität zum Typ 1 Fimbrien Gencluster (fim) von Salmonella enterica serovar Typhimurium aufweist. Diese in Plasmid pTO3 klonierte Invasionsdeterminante vermittelt nicht-invasiven E. coli K12 Stämmen Invasivität. Im Gegensatz dazu sind rekombinante E. coli K12 Klone, die das fim Operon aus S. enterica serovar Typhimurium bzw. E. coli oder andere E. coli Adhäsindeterminanten tragen, nicht invasiv. Im Rahmen dieser Arbeit wurden die für die Invasionsfähigkeit von C. freundii 3009 essentiellen Gene der klonierten Invasionsdeterminante ermittelt. Dies geschah zum einen durch Subklonierung von Teilen des Plasmids pTO3. In anschließenden Invasionsassays wurden die entsprechenden E. coli K12 Klone hinsichtlich ihrer Fähigkeit zur Invasion untersucht. Die Internalisierung des Wildtyps C. freundii 3009 sowie der invasiven rekombinanten E. coli K12 Stämme konnte nicht nur, wie erwartet, mittels Mannose, sondern auch mittels Chitinhydrolysat [(GlcNAc)n] inhibiert werden. Zum anderen wurden C. freundii Wildtypmutanten konstruiert, in denen der zentrale Teil der Invasionsdeterminante deletiert ist. Diese chromosomalen Deletionsmutanten weisen im Vergleich zum Wildtyp 3009 eine deutlich reduzierte Invasionsrate in die humane Harnblasenepithelzellinie T24 auf. Für die Komplementante wurde die Wiederherstellung des invasiven Phänotyps demonstriert. Darüber hinaus ist bekannt, dass C. freundii 3009 in humane mikrovaskuläre Endothelzellen aus dem Gehirn (HBMEC) eindringen und dort replizieren kann. In der vorliegenden Arbeit wurde gezeigt, dass sowohl C. freundii 3009 als auch der, die fimCf Determinante tragende, rekombinante E. coli Stamm HB101pPH1 in der Lage sind, im Tiermodell mit neugeborenen Ratten die Blut-Hirn-Schranke zu überwinden. Neben der Analyse der Funktion der klonierten C. freundii 3009 fim Determinante in Invasionsassays und mittels Mannose-sensitiver Hefeagglutination, wurden die Genprodukte selbst ebenfalls nachgewiesen. Durch radioaktive Markierung der für die Invasivität essentiellen Proteine nach induzierter Transkription in einem T7 Phagenexpressionssystem konnten die Molekulargewichte von FimCfA, FimCfD, FimCfF und FimCfH ermittelt werden. Diese stimmen mit den von der DNA Sequenz abgeleiteten Molekulargewichten gut überein. Sowohl mit C. freundii 3009 als auch mit entsprechenden rekombinanten E. coli K12 Stämmen gelang es in Westernblots, das Adhäsin FimCfH an der Bakterienoberfläche nachzuweisen. Dies konnte auch für FimCfF in denselben rekombinanten E. coli K12 Stämmen gezeigt werden. Allerdings scheinen die FimCf Proteine nicht zu einem Pilus assembliert zu werden, da in elektronenmikroskopischen Untersuchungen von C. freundii 3009 oder die fimCf Determinante tragenden E. coli K12 Stämmen niemals Fimbrien beobachtet werden konnten. Da auch bestimmte Typ 1 Fimbrien Varianten aus E. coli über das Adhäsin FimEcH die Internalisierung der Bakterien in Blasenepithelzellen zu induzieren vermögen, wurden die fimEc Gencluster aus den beiden human pathogenen E. coli Stämmen 536 (uropathogenes Isolat) und IHE3034 (Neugeborenen-Meningitis Isolat) kloniert. Zudem wurde die Invasionsfähigkeit des E. coli K1 Stamms IHE3034 und der fim negativen Insertionsmutante IHE3034-2 charakterisiert. Typ 1 Fimbrien werden als wichtige Virulenzfaktoren von uropathogenen E. coli (UPEC) angesehen. Trotzdem konnte eine Reihe von klinischen UPEC Isolaten identifiziert werden, die diesen Adhäsintyp nicht mehr exprimieren. Genetische Untersuchungen im Rahmen dieser Arbeit ergaben, dass in 11 Isolaten das fimEc Operon vollständig aus dem Chromosom der Bakterien deletiert ist. In den übrigen 6 Isolaten ist noch ein Teil des 3´-Endes des Gens fimECH vorhanden. Außerdem ist in die Deletionsstelle dieser 6 Isolate ein IS1 Element von E. coli inseriert. In den übrigen uropathogenen Isolaten sind auch angrenzende DNA Bereiche von der Deletion betroffen. Abschließend kann festgestellt werden, dass eine fim ähnliche Determinante in C. freundii 3009 als Invasionssystem fungiert. Die Typ 1 Fimbrien der E. coli Stämme 536 und IHE3034 sind zwar notwendig, aber nicht hinreichend für die Invasivität. Obwohl Typ 1 Fimbrien als wichtige Virulenzfaktoren von uropathogenen E. coli gelten, ist dennoch in einer Reihe von klinischen UPEC Isolaten das fimEc Operon deletiert. / Citrobacter freundii, which belong to the family of Enterobacteriaceae, are Gram-negative, motile and rod shaped bacteria. As an opportunistic pathogen they are known to be involved in urinary tract infections as well as in newborn meningitis. In vitro, the internalization of C. freundii 3009 into the endosomes of the urinary bladder epithelial cell line T24 exclusively follows a microtubule dependent mechanism. Previously, a chromosomal invasion determinant of C. freundii 3009 has been identified and cloned in plasmid pTO3. This invasion determinant shows high identity to the type 1 fimbrial gene cluster (fim) of Salmonella enterica serovar Typhimurium. Interestingly, non-invasive E. coli K12 strains become invasive, when they are transformed with plasmid pTO3. In contrast, recombinant E. coli K12 strains harbouring the fim operon of either S. enterica serovar Typhimurium and E. coli, respectively, or other E. coli adhesin determinants were not invasive. In this work the genes of this chromosomal determinant, which are essential for invasiveness, were identified. This was achieved on one hand by subcloning parts of the invasion determinant of plasmid pTO3. Subsequently, the ability of the resulting plasmids to confer invasiveness to E. coli K12 strains was examined by gentamicin protection assays. Interestingly, the internalization of those recombinant E. coli K12 strains is not only inhibited by an analogue of the natural receptor of the adhesin FimCfH, namely mannose, but also by chitin hydrolysate [(GlcNAc)n]. On the other hand, mutants of wild type C. freundii 3009 were constructed by deleting the central part of this particular invasion determinant. These chromosomal deletion mutants showed a considerable lower invasion rate of the human epithelial cell line T24 in comparison to the wild type. Complementation of one of these mutants fully restored the wild type phenotype. Furthermore, it is known that C. freundii 3009 is capable to invade and replicate in human brain microvascular endothelial cells (HBMEC). The results presented here demonstrate that not only C. freundii 3009 is capable to breach the blood-brain-barrier in a newborn rat animal model, but also recombinant E. coli strain HB101pPH1 which carries the chromosomal fimCf determinant. In addition to the functional analysis of the C. freundii 3009 fim determinant by performing gentamicin protection assays and mannose sensitive yeast agglutination, the expression of the corresponding gene products was shown. Proteins essential for invasion, namely FimCfA, FimCfD, FimCfF and FimCfH, were specifically radio labelled using a T7 phage expression system and their molecular weight was determined. The observed molecular weights are in agreement with the calculated ones from deduced amino acid sequences. Furthermore, the presence of the fimbrial adhesin minor subunit FimCfH on the outer surface of C. freundii 3009 as well as appropriate recombinant E. coli K12 strains was shown using Westernblot technique. Expression of the minor subunit FimCfF on the outer surface was also shown for the recombinant E. coli K12 strains using the same technique. However, electron microscopic studies of C. freundii 3009 or E. coli K12 strains harbouring the fimCf determinant indicate that FimCf protein subunits are not assembled in hair like appendages called fimbriae. Since certain type 1 fimbrial variants of E. coli are able to induce bacterial internalization by bladder epithelial cells via the adhesin FimEcH, the fimEc gene cluster from human pathogenic E. coli strains 536 (uropathogenic isolate) and IHE3034 (newborn meningitis isolate) were cloned in order to examine the role of type 1 fimbriae of these strains in invasiveness. In addition, E. coli K1 strain IHE3034 and the isogenic fim negative insertion mutant IHE3034-2 were characterized for their invasion capability. Although type 1 fimbriae are known to be an indispensable virulence factor of uropathogenic E. coli (UPEC), several clinical UPEC strains, which are incapable of expressing this particular adhesin, were isolated and identified. By performing genetic analysis, it could be demonstrated that the fimEc operon is completely deleted from the chromosome in 11 of those isolates. In another 6 isolates the presence of only the 3´-end of the gene fimECH could be found. Furthermore, an IS1 element inserted in the fim deletion was identified in these 6 strains. In the remaining 11 isolates also the DNA region adjacent to the fimEc cluster was affected by this deletion process. In conclusion, it was ascertained that in C. freundii 3009 a fim like determinant functions as an invasion system. Type 1 fimbriae of E. coli strains 536 and IHE3034 are necessary but not sufficient to confer invasiveness. Although type 1 fimbriae of uropathogenic E. coli are known to be an important virulence factor, in several clinical uropathogenic E. coli strains the fimEc operon was deleted.

No description available.

No description available.

Os carbapenêmicos são os antimicrobianos mais amplamente utilizados no tratamento empírico de infecções graves por bacilos Gram-negativos. A pressão seletiva gerada pelo uso desses antimicrobianos ao longo das últimas três décadas contribuiu para a disseminação de enterobactérias e Gram-negativos não fermentadores produtores de carbapenemases, particularmente as do tipo KPC e NDM. Os genes que codificam essas enzimas usualmente estão localizados em plasmídeos e/ou transpósons. A hipótese atualmente mais aceita é que o gene blaNDM-1 seja uma quimera criada em Acinetobacter baumannii. A NDM-1 foi descrita em paciente proveniente da Índia e subsequentemente evidenciou-se sua ampla disseminação nesse país. A epidemiologia que tem sido observada nos casos detectados na Europa e Estados Unidos tem sido viagem à Índia, ou seja, sem casos autóctones. No Brasil, os primeiros casos foram identificados no Rio Grande do Sul, e a seguir no Rio de Janeiro e em São Paulo. Diferentemente dos casos da Europa e América do Norte, os casos do Brasil não tem relação epidemiológica com a Índia. O sequenciamento integral dos plasmídeos e cromossomos albergando o gene blaNDM permitirá entender como ocorre a disseminação desse mecanismo de resistência no Brasil. Para isso, foi avaliado o perfil de susceptibilidade dos isolados, bem como a capacidade conjugativa e clonalidade. Das vinte e oito amostras utilizadas neste trabalho, treze delas pertencem à espécie Enterobacter hormaechei, uma à espécie Citrobacter freundii, sete à espécie Escherichia coli, quatro à Klebsiella pneumoniae e três ao gênero Acinetobacter spp. Os primeiros isolados incluídos neste estudo (Escherichia coli e Enterobacter hormaechei produzindo NDM-1) foram isolados em agosto de 2013, de uma mesma amostra de swab retal de um paciente do Rio de Janeiro que nunca viajou para o exterior. O sequenciamento completo do DNA plasmidial utilizando a plataforma Illumina e a anotação de ambos os plasmídeos albergando o gene blaNDM-1 revelou que estes pertencem a grupos de incompatibilidade diferentes, IncFIIK (E. hormaechei) e IncX3 (E. coli), e abrigam um novo transpóson composto designado Tn3000. A comparação da sequência nucleotídica do Tn3000 com aquelas disponíveis no GenBank evidencia que a mesma estrutura está presente em plasmídeos de isolados da cidade de Porto Alegre e também em diferentes continentes. As espécies de Acinetobacter (A. radioresistens, A. ursingii e A. guillouiae) isoladas em São Paulo e Porto Alegre, possuem o gene blaNDM-1 albergados em um mesmo plasmídeo não tipável de 41.087 pb. A avaliação da clonalidade dos isolados de Enterobacter hormaechei \"subsp. oharae\" mostrou dois perfis diferentes através da técnica de PFGE, sendo que todos os microrganismos foram isolados de um surto no mesmo hospital no Rio de Janeiro. Isolados de Klebsiella pneumoniae de uma mesma paciente internada em hospital em Salvador, de sítios distintos - swab retal, hemocultura e urina, em ordem cronológica - obtiveram o mesmo perfil clonal pela técnica de PFGE. O mesmo ocorreu com três isolados de Escherichia coli, de um mesmo paciente do Rio de Janeiro, em amostras de swab retal. Os achados deste estudo evidenciam que no Brasil, Nepal, Marrocos e Índia há uma disseminação do gene blaNDM-1 mediada por um novo elemento móvel designado Tn3000 em enterobactérias. A detecção de um mesmo plasmídeo em diferentes espécies de Acinetobacter evidencia que neste gênero bacteriano, no Brasil, a disseminação do gene blaNDM-1 ocorre por conjugação. / Carbapenems are the antimicrobials most widely used in the empirical treatment of severe infections caused by Gram-negative bacilli. The selective pressure generated by the use of these antibiotics over the last three decades has contributed to the spread of enterobacteria and Gram-negative non-fermenting producing carbapenemases, mainly KPC and NDM. Genes encoding these enzymes are usually located in plasmids and/or transposons. Currently the most accepted hypothesis is that the blaNDM-1 gene is a chimera created in Acinetobacter baumannii. The NDM-1 was described in a patient from India and subsequently was reported to be broadly disseminate in this country. The epidemiology that has been observed in cases detected in Europe and United States is traveling to India, but no autochthonous cases. In Brazil, the first cases were identified in Rio Grande do Sul, and then in Rio de Janeiro and São Paulo. Differently from the cases described in Europe and North America, the cases from Brazil have no epidemiological link with India. The complete sequencing of plasmids and chromosomes harboring blaNDM gene will understanding how the dissemination of this resistance mechanism in Brazil occurs. In this work we will be evaluate the susceptibility profile of the isolates, and their conjugal capacity and clonality. Of the twenty-eight samples used in this study, thirteen of them belong to the species Enterobacter hormaechei, one to Citrobacter freundii, seven to Escherichia coli, four to Klebsiella pneumoniae and three to the genus Acinetobacter sp. The first two isolates included in this study (Escherichia coli and Enterobacter hormaechei) were isolated in August 2013, from the same rectal swab sample from a patient from Rio de Janeiro that never traveled abroad. Complete sequencing of plasmid DNA using Illumina platform and annotation of both plasmids harboring the blaNDM-1 gene revealed that they belong to different incompatibility groups, IncFIIK (E. hormaechei) and IncX3 (E. coli), and are harbor to a new transposon designated Tn3000. The comparison of the Tn3000 nucleotide sequence with those available at GenBank shows that the same structure is present in plasmids from other Porto Alegre and also in different continents. The Acinetobacter species (A. radioresistens, A. ursingii and A. guillouiae) isolated in São Paulo and Porto Alegre, have the blaNDM-1 gene harbored in a single non-typing plasmid of 41,087 bp. The evaluation of clonal relationship of Enterobacter hormaechei \"subsp. oharae\" showed two different profiles by PFGE technique; of note all microorganisms were isolated from an outbreak in the same hospital in Rio de Janeiro. Isolates of Klebsiella pneumoniae from a single patient hospitalized in Salvador, from different anatomical sites - rectal swab, blood culture and urine, in chronological order - obtained the same clonal profile by the PFGE technique. The same occurred with three Escherichia coli isolates, from the same patient from Rio de Janeiro, in swab rectal strains. Our findings suggest that in Brazil, Nepal, Morocco and India there is a spread of blaNDM-1 gene mediated by Tn3000 in enterobacteria. The detection of a same plasmid in different species of Acinetobacter shows that in this bacterial genus, in Brazil, the dissemination of the blaNDM-1 gene occurs by conjugation.

De nos jours plus que jamais, il est nécessaire d’anticiper et de comprendre les réponses évolutives des organismes vivants, face à des habitats instables et hétérogènes. Mais à quel point cela est-il possible ? Reproduire l’ensemble du déroulé d’une trajectoire évolutive nécessite de pouvoir décrire, d’une part, le « matériel » disponible pour s’adapter (c’est-à-dire les effets phénotypiques associés à la variabilité génétique produite), d’autre part, comment agissent les forces évolutives, associées à un contexte écologique, pour aboutir à un certain « assemblage » de ce matériel. Dans sa version la plus simple, ce processus évolutif peut-être décrit par plusieurs cycles d’évènements de mutations-sélection conduisant à l’adaptation d’une population à son environnement. Cette dynamique correspond assez bien à celle qui est décrite par les populations bactériennes dans les expériences d’évolution contrôlées en laboratoire. Parallèlement, les modèles de paysages adaptatifs (phénotypiques), et en particulier le modèle géométrique de Fisher, sont des outils très puissants pour formuler des prédictions générales et quantitativement testables sur ces trajectoires évolutives. Cependant, ils restent très théoriques et ont été largement pensés dans un contexte écologique simplifié. Au cours de cette thèse, nous avons identifié les déterminants (mutationnels et sélectifs) des trajectoires évolutives à long terme de populations bactériennes s’adaptant dans différents contextes environnementaux. Une première partie des résultats est mise en lumière par la validation expérimentale et la reconstruction de la topographie du paysage adaptatif généré par différentes doses d’un antibiotique, le long d’un gradient. Une deuxième partie expérimentale vise à intégrer une composante biotique (une autre bactérie) à ce même contexte environnemental. Les processus évolutifs intervenant au cours d’une coévolution à long terme maintenue par sélection fréquence-dépendante, y sont étudiés. / Today more than ever, it is crucial to anticipate and understand the evolutionary responses of living organisms faced with heterogeneous and unstable habitats. But to what extent is this possible? To reproduce an entire evolutionary trajectory, we must first describe the “material” available for adaptation (e.g. the phenotypic effects associated with the existing and novel genetic variability), and second describe the way evolutionary forces, shaped by the ecological context, result in specific “assemblies” of this material. At its simplest, this evolutionary process can be described by several cycles of mutation-selection events, leading to the adaptation of a population to an environment. This process is reflected in the evolutionary trajectories of bacterial lineages undergoing controlled experimental evolution in the lab. Concurrently, adaptive (phenotypic) landscape models, and especially Fisher’s geometrical model of adaptation, are powerful tools to formulate general predictions, which can then be tested on such evolutionary trajectories. However, they remain highly theoretical, and are widely conceived in a simple ecological context. In this thesis, we identified the (mutational and selective) determinants of the evolutionary trajectories of bacterial lines adapting to various environmental contexts. A first set of results regards evolution along a gradient of antibiotic doses, and their relevance is highlighted by experimental validation and by the reconstruction of the underlying adaptive landscape. A second experimental part integrates a biotic component (another bacteria) to the same environmental context. The evolutionary processes acting throughout the resulting long-term coevolution – maintained by frequency-dependent selection – are studied.

Os carbapenêmicos são os antimicrobianos mais amplamente utilizados no tratamento empírico de infecções graves por bacilos Gram-negativos. A pressão seletiva gerada pelo uso desses antimicrobianos ao longo das últimas três décadas contribuiu para a disseminação de enterobactérias e Gram-negativos não fermentadores produtores de carbapenemases, particularmente as do tipo KPC e NDM. Os genes que codificam essas enzimas usualmente estão localizados em plasmídeos e/ou transpósons. A hipótese atualmente mais aceita é que o gene blaNDM-1 seja uma quimera criada em Acinetobacter baumannii. A NDM-1 foi descrita em paciente proveniente da Índia e subsequentemente evidenciou-se sua ampla disseminação nesse país. A epidemiologia que tem sido observada nos casos detectados na Europa e Estados Unidos tem sido viagem à Índia, ou seja, sem casos autóctones. No Brasil, os primeiros casos foram identificados no Rio Grande do Sul, e a seguir no Rio de Janeiro e em São Paulo. Diferentemente dos casos da Europa e América do Norte, os casos do Brasil não tem relação epidemiológica com a Índia. O sequenciamento integral dos plasmídeos e cromossomos albergando o gene blaNDM permitirá entender como ocorre a disseminação desse mecanismo de resistência no Brasil. Para isso, foi avaliado o perfil de susceptibilidade dos isolados, bem como a capacidade conjugativa e clonalidade. Das vinte e oito amostras utilizadas neste trabalho, treze delas pertencem à espécie Enterobacter hormaechei, uma à espécie Citrobacter freundii, sete à espécie Escherichia coli, quatro à Klebsiella pneumoniae e três ao gênero Acinetobacter spp. Os primeiros isolados incluídos neste estudo (Escherichia coli e Enterobacter hormaechei produzindo NDM-1) foram isolados em agosto de 2013, de uma mesma amostra de swab retal de um paciente do Rio de Janeiro que nunca viajou para o exterior. O sequenciamento completo do DNA plasmidial utilizando a plataforma Illumina e a anotação de ambos os plasmídeos albergando o gene blaNDM-1 revelou que estes pertencem a grupos de incompatibilidade diferentes, IncFIIK (E. hormaechei) e IncX3 (E. coli), e abrigam um novo transpóson composto designado Tn3000. A comparação da sequência nucleotídica do Tn3000 com aquelas disponíveis no GenBank evidencia que a mesma estrutura está presente em plasmídeos de isolados da cidade de Porto Alegre e também em diferentes continentes. As espécies de Acinetobacter (A. radioresistens, A. ursingii e A. guillouiae) isoladas em São Paulo e Porto Alegre, possuem o gene blaNDM-1 albergados em um mesmo plasmídeo não tipável de 41.087 pb. A avaliação da clonalidade dos isolados de Enterobacter hormaechei \"subsp. oharae\" mostrou dois perfis diferentes através da técnica de PFGE, sendo que todos os microrganismos foram isolados de um surto no mesmo hospital no Rio de Janeiro. Isolados de Klebsiella pneumoniae de uma mesma paciente internada em hospital em Salvador, de sítios distintos - swab retal, hemocultura e urina, em ordem cronológica - obtiveram o mesmo perfil clonal pela técnica de PFGE. O mesmo ocorreu com três isolados de Escherichia coli, de um mesmo paciente do Rio de Janeiro, em amostras de swab retal. Os achados deste estudo evidenciam que no Brasil, Nepal, Marrocos e Índia há uma disseminação do gene blaNDM-1 mediada por um novo elemento móvel designado Tn3000 em enterobactérias. A detecção de um mesmo plasmídeo em diferentes espécies de Acinetobacter evidencia que neste gênero bacteriano, no Brasil, a disseminação do gene blaNDM-1 ocorre por conjugação. / Carbapenems are the antimicrobials most widely used in the empirical treatment of severe infections caused by Gram-negative bacilli. The selective pressure generated by the use of these antibiotics over the last three decades has contributed to the spread of enterobacteria and Gram-negative non-fermenting producing carbapenemases, mainly KPC and NDM. Genes encoding these enzymes are usually located in plasmids and/or transposons. Currently the most accepted hypothesis is that the blaNDM-1 gene is a chimera created in Acinetobacter baumannii. The NDM-1 was described in a patient from India and subsequently was reported to be broadly disseminate in this country. The epidemiology that has been observed in cases detected in Europe and United States is traveling to India, but no autochthonous cases. In Brazil, the first cases were identified in Rio Grande do Sul, and then in Rio de Janeiro and São Paulo. Differently from the cases described in Europe and North America, the cases from Brazil have no epidemiological link with India. The complete sequencing of plasmids and chromosomes harboring blaNDM gene will understanding how the dissemination of this resistance mechanism in Brazil occurs. In this work we will be evaluate the susceptibility profile of the isolates, and their conjugal capacity and clonality. Of the twenty-eight samples used in this study, thirteen of them belong to the species Enterobacter hormaechei, one to Citrobacter freundii, seven to Escherichia coli, four to Klebsiella pneumoniae and three to the genus Acinetobacter sp. The first two isolates included in this study (Escherichia coli and Enterobacter hormaechei) were isolated in August 2013, from the same rectal swab sample from a patient from Rio de Janeiro that never traveled abroad. Complete sequencing of plasmid DNA using Illumina platform and annotation of both plasmids harboring the blaNDM-1 gene revealed that they belong to different incompatibility groups, IncFIIK (E. hormaechei) and IncX3 (E. coli), and are harbor to a new transposon designated Tn3000. The comparison of the Tn3000 nucleotide sequence with those available at GenBank shows that the same structure is present in plasmids from other Porto Alegre and also in different continents. The Acinetobacter species (A. radioresistens, A. ursingii and A. guillouiae) isolated in São Paulo and Porto Alegre, have the blaNDM-1 gene harbored in a single non-typing plasmid of 41,087 bp. The evaluation of clonal relationship of Enterobacter hormaechei \"subsp. oharae\" showed two different profiles by PFGE technique; of note all microorganisms were isolated from an outbreak in the same hospital in Rio de Janeiro. Isolates of Klebsiella pneumoniae from a single patient hospitalized in Salvador, from different anatomical sites - rectal swab, blood culture and urine, in chronological order - obtained the same clonal profile by the PFGE technique. The same occurred with three Escherichia coli isolates, from the same patient from Rio de Janeiro, in swab rectal strains. Our findings suggest that in Brazil, Nepal, Morocco and India there is a spread of blaNDM-1 gene mediated by Tn3000 in enterobacteria. The detection of a same plasmid in different species of Acinetobacter shows that in this bacterial genus, in Brazil, the dissemination of the blaNDM-1 gene occurs by conjugation.