Dissertations / Theses on the topic 'Escherichia coli Plasmid'

This study investigated the maintenance of several plasmid derivatives of pBR322 and ColEl present within Escherichia coli K-12, with a view to establishing a strategy that would ensure the stable maintenance of plasmid pBR322 and the identification of a postulated partitioning function on plasmid ColEl. Two strategies were adopted. The first of these used selective nitrogen-limited chemostat culture of a glutamate-dependent strain of E.coli, carrying a pBR322 derivative expressing glutamate dehydrogenase. Using this approach, it was found that the plasmid conferred a reproductive advantage and persisted, during nitrogen limitation, for several generations beyond that of pBR322 under glucose- or phosphate- limited conditions. The second strategy used nonselective chemostat culture of a strain of E.coli carrying derivatives of pBR322 encoding stability functions from either plasmid pSClOl, the partitioning region par, or plasmid R1, the cell division/plasmid inheritance coupling region parB. Although these plasmids persisted for many generations beyond that of pBR322 under similar chemostat culture conditions, no conclusions could be made with respect to the ability of these functions to ensure stable plasmid maintenance, since the copy numbers of the respective plasmids were several-fold greater than that of pBR322, a factor that in itself would contribute to the segregational stability of the plasmids. Plasmid- free segregants which did arise were found not to be isogenic with the host strain. These mutants exhibited an increased resistance to U.V.-light irradiation, a mucoid colony phenotype, an altered cell division cycle, giving rise to minicell, filament and Y-shaped cellular morphologies, an enhanced ability to form tandemly repeated plasmid multimers and an altered sensitivity to the DNA gyrase specific antibiotics novobiocin and nalidixic acid. A study of plasmid configuration in Ion and Ion sul strains of E.coli, which share similar phenotypic characteristics with the above mutants, revealed that strains which carry a sul or azi mutation express an enhanced capacity for plasmid multimerization. Finally, no conclusive evidence could be obtained to indicate the presence of a partitioning function on plasmid ColEl. This study concludes by postulating several factors which may affect the maintenance of plasmids in E.coli K-12.

Some strains of Escherichia coli possess an iron uptake system, first found on a ColV plasmid, which utilises the hydroxamate siderophore aerobactin and which significantly enhances the virulence of host bacteria in experimental infections of mice. The aerobactin system of plasmid ColV-K30, cloned as the multicopy recombinant plasmid pABN1, was localised to a 7.6 kb segment of DNA by transposon mapping. In the maxicell and minicell expression systems, the cluster of genes was found to specify five polypeptides, four of which are involved in aerobactin biosynthesis, the fifth being the outer membrane receptor for ferric-aerobactin. The linear order of the genes specifying these polypeptides was determined, the gene for the receptor being at the 3' end of the cluster. The 5' and 3' transcription initiation and termination sites were located by S1 nuclease transcriptional mapping, and further transcription studies revealed a probable internal promoter within the cluster of genes, a minor unregulated promoter for the receptor gene, in addition to the major regulated promoter(s) at the 5' end of the system. Transcription of the system on ColV-K30 and subcloned plasmids, and production of the siderophore by ColV-K30, as measured by b-galactosidase production by ColV: :Mu(Ap lac) aerobactin-deficient strains, were found to be under the control of the level of freely available iron in the growth medium. The aerobactin system on pABN1, like that on ColV-K30, was shown to enhance the virulence of host bacteria in experimental infections of mice, and was found to be widespread among E. coli strains isolated from a number of extraintestinal infections of man and animals. The system was found not to be exclusively associated with ColV plasmids among these strains, and was apparently located on the chromosome of some isolates.

Life is cellular. Cells grow and divide to give two new cells; this process is called the cell cycle. The chromosome in a bacterium is replicated into two identical copies before the cell divides. DNA replication is a fundamental process common to all forms of life. In my thesis, I have studied control of chromosome and plasmid replication in Escherichia coli, a rod-shaped bacterium. Plasmids are extrachromosomal autonomously replicating DNA molecules. I have combined the classical Meselson-Stahl density-shift and DNA hybridisation with theoretical analysis of DNA replication. The minimal time between two successive replications of the same molecule, the eclipse, was determined for both plasmid and chromosome. The aim was to investigate the processes ensuring the precise timing of chromosome replication in the cell cycle. In wild-type strains, the chromosomal eclipse was long. Mutations affecting the so-called sequestration process, the superhelicity of the DNA, and the initiation protein, DnaA, reduced the eclipse. Fast-growing E. coli has overlapping replicative phases with synchronous initiation from multiple initiation sites, oriC. I have investigated the complex interplay between different control processes by measuring the length of the eclipse and the degree of asynchronous initiation in various mutants. I have measured the eclipse period of plasmid R1 during up- and down-shifts in plasmid copy number. The length of the eclipse was found to be determined by structural events as well as by the properties of the copy-number-control system. During downshift from very high copy numbers, the rate of plasmid replication started very slowly and gradually increased until the normal copy number was achieved, in accordance with the +n model. The CopB system of plasmid R1 was shown to be a rescue system preventing cells with few plasmid copies from losing the plasmid in some of the daughter cells.

Els plasmidis són molècules d’ADN circular extracromosòmiques amb capacitat de replicació autònoma i, són descrits com els principals promotors de la disseminació de gens de resistència a antimicrobians. Escherichia coli i Klebsiella pneumoniae, són freqüents agents causals d’infeccions, importants pel seu caràcter multirresistent. En aquests bacteris, les betalactamases constitueixen un dels mecanismes de resistència més difosos mitjançant plasmidis. Mitjançant l’estudi i comparació del contingut plasmídic en bacteris d’individus sans i de pacients, l’objectiu va ser determinar les diferències o similituds d’aquests plasmidis entre les dues poblacions, per tal d’inferir en el seu origen i evolució en els soques causants d’infecció. Per a això, es van aïllar soques d’E. coli (n = 244) i K. pneumoniae (n = 115) de 150 mostres fecals d’individus sans i de 202 pacients amb bacterièmia. Es va realitzar un estudi de sensibilitat a antimicrobians mitjançant disc difusió, es van identificar les soques multirresistents i es van caracteritzar, per PCR i seqüenciació, els gens codificants de BLEE, AmpC i carbapenemases. L’estudi de plasmidis es va realitzar utilitzant la tècnica de PBRT i, es van subtipificar els plasmidis IncF, IncI1 i IncN mitjançant pMLST per a la seva diferenciació dins d’un mateix grup d’incompatibilitat. La localització dels gens de resistència i la definició de la fórmula FAB dels plasmidis IncF, es va realitzar mitjançant la tècnica de Southern-blot i hibridació. Un 29,2% de les soques de mostra clínica front d’un 10,8% en individus sans van resultar multirresistents. La prevalença de soques d’E. coli clíniques productores de BLEE va ser de 17,2% i 5% d’AmpC i, en K. pneumoniae, de 16,5% i 1%, respectivament, a més a més d’un 1,9% de carbapenemases . D’altra banda, es va observar una prevalença del 4,7% de portadors sans d’E. coli productores de BLEE i un 2,7% d’AmpC. Ambdues poblacions han sofert un augment en la detecció de soques productores d’aquests enzims sent també, en ambdós casos, la CTX-M-15 i la CMY-2 les betalactamases més freqüents. Els resultats del PBRT van mostrar com el contingut plasmídic de les dues poblacions seguia una mateixa tendència sense diferències significatives destacables. L’excepció es va observar en els replicons L, M, A/C i N, els quals només es van detectar en les soques de mostra clínica. A més, alguns d’aquests plasmidis amb replicó L o N van ser detectats en associació a gens blaBLEE. La subtipificació dels plasmidis, ens va permetre observar l’existència d’una gran diversitat de secuenciotips dins d’un mateix grup d’incompatibilitat. Dins dels plasmidis IncI1 es va observar una gran diferència entre els ST identificats en les soques d’individus sans i pacients. Només els ST12 i ST36 es van identificar en ambdues poblacions d’E. coli, i tots els ST12 en associació amb el gen blaCMY-2. Per contra, tot i que també es va detectar una gran diversitat de RST entre els plasmidis IncF, aquells detectats amb més freqüència, van estar presents en ambdues poblacions i, no es va identificar cap fórmula FAB destacable per la seva associació a gens bla. En conclusió, el contingut plasmídic en la població de soques clíniques és un reflex del contingut en soques d’individus sans però, certs plasmidis, només es detecten en soques causants d’infecció, suggerint una millor adaptació a ambients sotmesos a pressió selectiva. Secuenciotips IncI1 com el ST12, poden ser considerats plasmidis epidèmics per la seva alta prevalença i freqüència en associació amb gens blaCMY-2. En canvi, els plasmidis IncF semblen haver adquirit diferents gens de resistència a antimicrobians de forma aleatòria, suggerint que tota la família de plasmidis IncF és potencialment susceptible a adquirir i disseminar gens de resistència.<br>Los plásmidos son moléculas de ADN circular extracromosómicas con capacidad de replicación autónoma y, se describen como principales promotores de la diseminación de genes de resistencia a antimicrobianos. Escherichia coli y Klebsiella pneumoniae, son frecuentes agentes causales de infecciones, importantes por su carácter multirresistente. En estas bacterias, las betalactamasas constituyen uno de los mecanismos de resistencia más difundidos mediante plásmidos. Mediante el estudio y comparación del contenido plasmídico en bacterias de individuos sanos y de pacientes, el objetivo fue determinar las diferencias o similitudes de estos plásmidos entre ambas poblaciones, con el fin de inferir en su origen y evolución en las cepas causantes de infección. Para ello, se aislaron cepas de E. coli (n=244) y K. pneumoniae (n=115) de 150 muestras fecales de individuos sanos y de 202 pacientes con bacteriemia. Se realizó un estudio de sensibilidad a antimicrobianos mediante disco difusión, se identificaron las cepas multirresistentes y se caracterizó, por PCR y secuenciación, los genes codificantes de BLEE, AmpC y carbapenemasas. El estudio de plásmidos se realizó utilizando la técnica de PBRT y, se subtipificaron los plásmidos IncF, IncI1 e IncN mediante pMLST para su diferenciación dentro de un mismo grupo de incompatibilidad. La localización de los genes de resistencia y la definición de la fórmula FAB de los plásmidos IncF, se realizó mediante la técnica de Southern-blot e hibridación. Un 29,2% de las cepas de muestra clínica frente a un 10,8% en individuos sanos resultaron multirresistentes. La prevalencia de cepas de E. coli clínicas productoras de BLEE fue de 17,2% y 5% de AmpC y, en K. pneumoniae, de 16,5% y 1%, respectivamente, además de un 1,9% de carbapenemasas. Por otro lado, se observó una prevalencia del 4,7% de portadores sanos de E. coli productoras de BLEE y un 2,7% de AmpC. Ambas poblaciones han sufrido un aumento en la detección de cepas productoras de estas enzimas siendo también, en ambos casos, la CTX-M-15 y la CMY-2 las betalactamasas más frecuentes. Los resultados del PBRT mostraron como el contenido plasmídico de ambas poblaciones seguía una misma tendencia sin diferencias significativas destacables. La excepción se observó en los replicones L, M, A/C y N, los cuales solo se detectaron en las cepas de muestra clínica. Además, algunos de estos plásmidos con replicón L o N fueron detectados en asociación a genes blaBLEE. La subtipificación de los plásmidos, nos permitió observar cómo dentro de un mismo grupo de incompatibilidad existe una gran diversidad de secuenciotipos. Dentro de los plásmidos IncI1 se observó una gran diferencia entre los ST identificados en las cepas de individuos sanos y pacientes. Solo los ST12 y ST36 se identificaron en ambas poblaciones de E. coli, y todos los ST12 en asociación con el gen blaCMY-2. Por el contrario, a pesar de que también se detectó una gran diversidad de RST entre los plásmidos IncF, aquellos detectados con mayor frecuencia, estuvieron presentes en ambas poblaciones y, no se identificó ninguna fórmula FAB destacable por su asociación a genes bla. En conclusión, el contenido plasmídico en la población de cepas clínicas es un reflejo del contenido en cepas de individuos sanos, pero hay ciertos plásmidos, que solo se detectan en cepas causantes de infección, sugiriendo una mejor adaptación a ambientes sometidos a presión selectiva. Secuenciotipos IncI1 como el ST12 pueden ser considerados plásmidos epidémicos por su alta prevalencia y frecuencia en asociación con genes blaCMY-2. En cambio, los plásmidos IncF parecen haber adquirido distintos genes de resistencia a antimicrobianos de forma aleatoria, sin ningún RST destacable por ello; sugiriendo que toda la familia de plásmidos IncF es potencialmente susceptible a adquirir y diseminar genes de resistencia.<br>Plasmids are extracromosomal circular DNA molecules that can replicate autonomously. They are globally described as primary promoters for the dissemination of antimicrobial resistance genes. Escherichia coli and Klebsiella pneumoniae have been described as critical propriety microorganisms due to their increasing detection as multiresistant bacteria. In these bacteria, beta-lactamases constitute one of the most disseminated resistance-providing mechanisms through plasmids. Through the study and comparison of the plasmid content in the commensal microbiota from healthy individuals and patients with bacteriemia, the aim was to determine the differences and similarities of the plasmid content between these two populations, with the purpose to shed light on the origin and evolution of bacteria-causing infection. To achieve that goal, E. coli (n=244) and K. pneumoniae (n=115) strains from 150 faecal samples from healthy individuals and 202 patients with bacteraemia where isolated. An antimicrobial susceptibility testing was performed through the disc diffusion technique, the multiresistant strains were identified and, the blaESBL, blaAmpC and blacarbapenemase genes were characterised by PCR and sequencing approaches. The plasmid study was performed by using the PBRT technique. Plasmids IncF, Incl1 and IncN plasmids, were subtyped by pMLST, to differentiate each of them within the same incompatibility group. The location of the resistance genes was performed by using the Southern blot and hybridisation techniques. The same methodology was used to generate the FAB formula of the IncF plasmids. From the clinical samples, 29.2% of the strains turned out to be multiresistant against 10.8% from healthy individuals. The prevalence of clinical ESBL-producing and AmpC-producing E. coli strains was 17.2% and 5%, respectively. The prevalence of clinical ESBL-producing, AmpC-producing and carbapenemase-producing K. pneumoniae strains was 16.5%, 1% and 1.9%, respectively. Furthermore, 4.7% of the healthy individuals were carriers of ESBL-producing E. coli and 2.7% were carriers of AmpC-producing E. coli. Both populations have suffered an increased detection of strains that produce those enzymes and, in both cases, the beta-lactamases CTX-M-15 and CMY-2 were the most frequent. The PBRT results showed how the plasmid content in both populations kept the same trend without significant differences. The exception was observed in the replicons L, M, A/C and N, which were only detected in strains from clinical samples. What is more, some of these plasmids with L or N repliclon were detected in association with blaESBL genes. The plasmid subtyping, allowed us to observe a great diversity of sequence types (ST) within the same group of incompatibility. Within the Incl1-plasmids, a huge difference between the identified ST in healthy individuals and patient strains was observed. Only ST12 and ST36 were identified in both populations of E. coli. Noticeably, all the ST12 were found in association with the blaCMY-2 gene. In contrast, even though a great diversity of RSTs was detected within the IncF plasmids, those detected at higher rates were present in both populations. Furthermore, no formula was distinguishable by its association with bla genes. In conclusion, the replicon content of clinical strains mirrored that of comensal strains. Nevertheless, there are certain plasmids only detected in infection-causing strains, suggesting a better adaptation to an enviroment subjected to selective presure. IncI1 sequencetypes such as ST12 can be considered epidemic plasmids due to their high prevalence and frequency in association with blaCMY-2. On the other hand, the IncF plasmids have acquired multiple antimicrobial resistace genes randomly, with no evidence that the persitance of a single IncF is responsible for their dissemination. Therfore, the entire IncF family of plasmids is potentially capable of acquiring and propagating resitance genes.

Plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a member of the autotransporter subfamily termed SPATE (serine protease autotransporters of Enterobacteriaceae). Autotransporters, which are the most common Gram-negative secreted virulence factors, contain three functional domains: an amino terminal leader sequence, a mature protein or passenger domain, and a carboxy-terminal &#946; domain. The leader sequence targets the protein to the periplasmic space and the &#946; domain then forms a &#946;-barrel pore in the outer membrane of the bacterium which allows the passenger domain to enter the external milieu. In some cases the passenger domain is cleaved from the &#946;-barrel at the extracellular surface to release a soluble toxin. This is thought to be a self-contained process that does not require chaperones or ATP for folding and export of the passenger domain. Pet produces cytotoxic effects through cleavage of its target, the actin-binding protein &#945;- fodrin. Pet is secreted into the extracellular environment, but its target lies within the cytosol. To reach its target, Pet moves from the cell surface to the ER where it triggers ER-associated degradation (ERAD) to enter the cytosol. ERAD is a normal cellular process in which improperly folded proteins are exported from the ER to the cytosol for degradation. Other toxins that utilize this pathway are AB toxins such as cholera toxin (CT) and ricin. The A subunits of these toxins are thermally unstable, and this facilitates their ERAD-dependent translocation into the cytosol. Pet, however, is not an AB toxin. We predict that thermal unfolding is not the mechanism Pet employs to exploit ERAD. It was necessary to purify the toxin first in order to study the structural properties and ER export of Pet. Surprisingly, purified Pet eluted as two close peaks by size exclusion chromatography. Both peaks were Pet as demonstrated through immunoblotting. The folding efficiency of autotransporters has not been extensively elucidated, and based on our purification results, we hypothesized that there is inefficiency in the folding of autotransporters, specifically Pet. A toxicity assay showed that Pet peak one did not display cytopathic activity while Pet peak two did. CD and fluorescence spectroscopy measurements also detected structural differences between the two variants of Pet and demonstrated that Pet peak one was an unfolded variant of Pet peak two. Native gel electrophoresis and biophysical measurements indicated that Pet peak one did not exist as a dimer or aggregate. Our results indicate there are two forms of Pet, and thus the folding process of autotransporters appears to be inherently inefficient. Active Pet (peak two) was used for further biophysical measurements and biochemical assays. Circular dichroism and fluorescence spectroscopy showed that the secondary and tertiary structures of Pet are maintained at physiological temperature, 37&deg;C. Thermal unfolding of Pet occurred at temperatures above 50&deg;C. Fluorescence quenching of Pet was also performed and demonstrated that, at 37&deg;C, there are solvent-exposed aromatic amino acids. The slight structural alterations to Pet at physiological temperature as well as the exposed hydrophobic residues could trigger ERAD. In addition, a modeled structure of Pet revealed a hydrophobic loop which is surface-exposed and a likely target for toxin-ERAD interactions. The data suggests that translocation of Pet mediated by ERAD can occur by a mechanism different from certain AB toxins. An open, hydrophobic conformation likely triggers ERAD, but may also contribute to poor folding.<br>M.S.<br>Department of Biomolecular Science<br>Burnett College of Biomedical Sciences<br>Molecular and Microbiology MS

O pEO5, que codifica hemolisina, foi isolado de uma amostra de EPEC do sorotipo O26. Este plasmídio mostrou ser conjugativo e compatível com o pO157, e pelos testes de hibridização observou-se que estes plasmídios não são geneticamente relacionados. Para o estudo comparativo de similaridade foi seqüenciada uma região de 9227 pb de DNA do pEO5 que compreende todo o operon hlyCABD e suas regiões a montante e a jusante. A região do operon hemolitico (7225 pb) e a região promotora do operon foram similares às mesmas regiões do pHly152, que em uma amostra de E. coli isolada de roedor, codifica uma a hemolisina. No entanto, verificou-se a presença de elementos de inserção na região a montante do gene hlyC no pHly152. O pEO5 mostrou ser semelhante a outros plasmídios que também codificam a hemolisina em cepas de EPEC O26 de origem humana e de bovinos. A presença de estruturas semelhantes a transposons em ambas as extremidades do operon a hemolítico do pEO5 indica que esse fator de virulência provavelmente foi adquirido por transferência horizontal de genes.<br>The conjugative pEO5 encoding haemolysin in strains of EPEC O26 was investigated for its relationship with EHEC haemolysin-encoding of EHEC O26 and O157 strains. pEO5 was found to be compatible with EHEC virulence plasmids and did not hybridize in Southern blots with pO157, indicating that both plasmids were unrelated. A 9227 bp stretch pEO5 DNA encompassing the entire operon hlyCABD was sequenced and compared for similarity to plasmid and chromosomally inherited hly determinants. The a hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of pHly152, in particular, the structural a-hlyCABD and hlyR regions. pEO5 and hly of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural a-hlyCABD. The major difference found between the hly regions of pHly152 and pEO5 is caused by the IS2 upstream of the hlyC in pHly152. The presence of transposonlike structures at both ends of hly sequence indicates that pEO5 was probably acquired by horizontal gene transfer.

Three overlapping DNA fragments from the lower operon of Pseudomonas putida TOL plasmid pDK1, covering the xy1IH genes and downstream flanking region, were cloned into pUC19. They include a 2.8 kbp XhoI fragment, a 2.7 kbp PstI fragment and a 2.0 kbp EcoRI-HindIII fragment. They were subjected to DNA sequence analysis. The xy1I (4-oxalocrotonate decarboxylase) and xy1H (4-oxalocrotonate tautomerase) genes were found to possess coding regions of 792 and 189 nucleotides, respectively. A possible transcriptional terminator resembling E. coli rho-independent terminators was identified downstream of the translational stop of xy1H. An additional stem and loop structure was found in the intergenic region between xy1I and xy1H. The individual ORF's of the oxalocrotonate branch (xy1G, xy1I and xy1H) have been cloned into pUC18/19. The expression of the xy1G gene in E. coli was successfully assayed spectrophotometrically.

Les années 2000 sont marquées par l’émergence et la diffusion de souches résistantes aux antibiotiques en particulier chez les entérobactéries. Un des principaux mécanismes de résistance concerne les bêta-lactamines, ce sont les bêta-lactamases. Certains mécanismes de résistances ont connus ou connaissent actuellement un certain succès comme les bêta-lactamase à spectre étendu (BLSE) de type CTX-M puis récemment les carbapénèmases. La transmission de plasmides porteurs de ces enzymes joue un rôle prépondérant ainsi que certaines espèces comme Klebsiella pneumoniae et Escherichia coli qui est l’entérobactérie prépondérante dans le tube digestif de l’homme et des animaux et également la principale cause d’infection. Dans ce travail, nous avons étudié le transfert par transposition qui est le mécanisme clé de cette diffusion via différents plasmides parmi les plus prépondérants chez les entérobactéries dont essentiellement E. coli et K. pneumoniae. Nous avons également recherché une éventuelle relation avec le groupe phylogénétique, les gènes de résistance, type de plasmides et systèmes d’addiction plasmidique. Ce travail montre que les plasmides IncF ayant acquis TEM-1 ont de multiples systèmes d’addiction avec une fréquence plus élevée que les souches sensibles. Ce qui contribuerait à leur maintenance dans l’hôte. Les plasmides à large spectre d’hôte sont très rares chez ces souches de manière générale. Il ne semble pas y avoir de relation entre le groupe phylogénétique et les gènes de résistances dans cette étude. Quant au phénomène de transposition, il suscite la mise au point de conditions opératoires qui le régisse. Une détermination des conditions de stress a été réalisée et il reste à tracer le phénomène à proprement dit<br>The last years are characterized by the spread of antibiotics resistant strains particularly in Enterobacteriacae. Various mechanisms of resistances were acquired and transmitted by species like K. pneumoniae. Bacterial plasmids play an important role in the horizontal transfer of antimicrobial drug resistance genes like: CTX-M enzymes witch become the most prevalent family of ESBLs and recently carbapenemases. In this work, we studied the transfer by transposition, the key mechanism of resistance genes diffusion, between different plasmids. We also sought a possible relation between the genetic background and the frequency of transposition, by testing various types of plasmids among the most prevalent E. coli and K. pneumoniae. This work shows that IncF plasmids having acquired TEM-1 have multiple addiction systems with a frequency higher than the sensitive strains. It would contribute to their maintenance in the host. By the way, broad range plasmids are very rare at these strains. In addition, it does not seem to be a relation between the phylo-group and the genes of resistances in this study. For the transposition event, we start to settle the operating conditions which govern this phenomenon. A determination of the stress conditions was established and it remains to be completed

Den här rapporten presenterar ett antibiotikafritt, stabilt och kromosombaserat expressionssystem för läkemedelsproduktion i Escherichia coli på beställning av företaget Affibody AB. E. coli-stammen BL21(DE3) valdes som värdorganism för expressionssystemet. Systemet består av en genkassett som innehåller en T7-promotor, en 5′-UTR från genen ompA och en terminatorsekvens från RNA-operonet rrnB. Fyra kopior av genkassetten ska integreras i pseudogenerna caiB, yjjM, hsdS och yjiV. En datormodell som modellerar det egentliga kopietalet i cellerna har skapats i mjukvaran MATLAB, vilket visar att det uppskattas vara maximalt 32 kopior av genkassetten per cell på grund av replikation av kromosomen. Ett högt pH i fermentorn; att använda fed-batch och blandade kolhydratkällor; och att använda stammen BL21(DE3) minskar acetatproduktionen i cellen. En lägre acetatproduktion kan leda till en högre produkthalt. En proteinutbytesmodell för mjukvaran MATLAB har konstruerats för att uppskatta koncentrationen av Affibody®-molekylen i en E. coli cell.

Conselho Nacional de Desenvolvimento Científico e Tecnológico<br>Plasmídios são DNA extracromossômicos, com capacidade de se duplicarem de forma independente das células que os albergam, e são responsáveis pela expressão de uma variedade de características, como fatores de virulência. O material do presente estudo se constituiu da cepa receptora E. coli K12-R23, e de cepas de Klebsiella pneumoniae e de Escherichia coli, doadoras de plasmídios R e transconjugantes. O objetivo do presente estudo foi analisar os fenótipos conferidos em cepas transconjugantes de ambas bactérias pela transferência de plasmídios R de cepas doadoras para a receptora. Para a análise dos fenótipos, utilizaram-se, nas cepas do estudo, algumas variáveis: sensibilidade a antimicrobianos e a ERO, aderência a células HEp-2, e formação de slime e de biofilme. O marcador da presença de plasmídio, neste trabalho, foi a presença de resistênca à gentamicina nas cepas doadoras. Os resultados indicaram que houve transferência de plasmídio, pois as cepas transconjugantes de K. pneumoniae e de E. coli apesentaram este marcador (foram resistentes à gentamicina); além disso, as cepas transconjugantes mostraram perfis distintos da receptora em relação à sensibilidade aos antimicrobianos, às ERO, aos padrões de aderência às células HEp-2 e à formação de slime, apesar de a formação de biofilme nestas cepas não ter sofrido modificações. Observou-se, contudo, que várias características das cepas doadoras não foram encontradas nas cepas transconjugantes de E. coli e de K. pneumoniae.<br>Plasmids are extrachromosomal DNA that have the ability to duplicate independently of the host cells. Plasmids are responsible for the expression of a variety of characteristics of the cells, such as virulence factors. The present study utilized a receptor strain of E. Coli K12-R23 and strains of Klebsiella pneumoniae and E. coli which were R plasmids donor strains and transconjugant ones. The objective of this study was to analyze the attributed phenotypes of transconjugant strains in both bacteria caused by transference of R plasmids from the donor strains to the receptor ones. In order to analyze these phenotypes, the following variables were selected: sensitivity to antimicrobials and ROS, adherence to HEp-2 cells, and slime and biofilm structures. The marker of the presence of plasmids was gentamycin resistance in the donor strains. The observed results indicated that there was plasmid transfer; given that Klebsiella pneumoniae and E. coli strains presented the marker (e.g. they became resistant to gentamycin). Moreover, transconjugant strains have showed distinct profiles from the receptor strain concerning sensitivity to antimicrobials, ROS, adherence patterns to HEp-2 cells and slime structure. On the other hand, the biofilm structure of these strains did not present modifications. Yet, it was observed that several characteristics of the donor strains were not found in the transconjugant strains of E. coli and K. pneumoniae.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior<br>The marketing of fish has shown substantial increase; however, their place of origin and ways of handling and storage may pose health risk. The microorganisms in the food chain and in capture environments cause concern over the risk of disease transmission and the multiple resistance to several drugs. This study aimed at characterizing the antimicrobial resistance of 191 strains of Escherichia coli isolated from 20 fish samples, ten saltwater samples: (5) mackerel (S. cavalla) and (5) snapper (L. purpureus) and ten freshwater samples: (5) curimatà (P. cearenses) and (5) tilapia (O. niloticus), sold in street markets of the city of Fortaleza (Cearà State), Brazil. The strains were isolated, identified and then subjected to susceptibility testing (20 antibiotics) in order to provide the resistance profile. Moreover, it was also performed: Multiple Resistance Index (MRI), the Antimicrobial Resistance Index (ARI), Minimum Inhibitory Concentration (MIC), plasmid "cure" and analysis of plasmid profile through DNA extraction. Strains isolated from marine and freshwater fish species showed distinct multidrug resistance profiles, but with broad resistance to penicillins and tetracyclines. There was high frequency of saltwater isolates resistant to chloramphenicol. Overall, low resistance to betalactamase inhibitors (ampicillin/sulbactam, and piperacillin/tazobactam) and aminoglycosides was observed, excepting streptomycin. The MRI has shown that about 50% of the isolates were resistant to five of the twenty tested antimicrobials, highlighting the strains from mackerel (55.5%). As for MIC, the percentage of isolates resistant to NAL, CIP, MFX, SUT and AMO stood out in saltwater fish strains. For bacteria from freshwater, greater resistance to high concentrations of antimicrobials were most significant for AMO, SUL and NAL. Chromosomal profiles in freshwater isolates were mostly composed of amoxicillin, ampicillin, streptomycin, and sulfametazol/trimethoprim. For saltwater fish, there was predominance of quinolones. Analysis of the plasmid DNA showed the occurrence of a heterogeneous population of small plasmids distributed in various profiles. The greater diversity and lower molecular weight were observed in strains of marine fish samples. Thus, it is emphasized that the circulation of pathogenic E. coli with antimicrobial resistance characteristics poses a risk to the aquatic ecosystem and the marketing environment, there being need for continued vigilance to contaminant bacterial to fish so that the safety of these foods is guaranteed.<br>A comercializaÃÃo de pescado tem indicado aumento substancial, contudo, seu local de origem e formas de manipulaÃÃo e armazenamento, podem representar risco sanitÃrio. Os micro-organismos presentes na cadeia produtiva de alimentos e nos ambientes de captura ocasionam preocupaÃÃo pelo risco de transmissÃo de doenÃas e pela mÃltipla resistÃncia apresentada a diversos fÃrmacos. Este trabalho objetivou caracterizar a resistÃncia antimicrobiana de 191 cepas de Escherichia coli isoladas de 20 amostras de pescado, sendo dez amostras de pescado de Ãgua salgada: (5) cavala (S. cavalla) e (5) pargo (L. purpureus) e dez amostras de Ãgua doce: (5) curimatà (P. cearenses) e (5) tilÃpia (O.niloticus), comercializadas em feiras livres da cidade de Fortaleza, CE. As cepas foram isoladas, identificadas e posteriormente submetidas a testes de susceptibilidade (20 antimicrobianos) a fim de conferir o perfil de resistÃncia. AlÃm disso, foram realizados: Ãndice de MÃltipla ResistÃncia (IMR), Ãndice de ResistÃncia a Antimicrobianos (IRA), ConcentraÃÃo InibitÃria MÃnina (CIM), âcuraâ plasmidial e anÃlise de perfil plasmidial, atravÃs de extraÃÃo de DNA. As cepas isoladas das espÃcies de pescado marinho e de Ãgua doce apresentaram perfis de multirresistÃncia distintos, porÃm com ampla resistÃncia Ãs penicilinas e Ãs tetraciclinas. Houve elevada frequÃncia de isolados de pescado marinho resistentes ao cloranfenicol. Foi observada, de forma geral, baixa resistÃncia para inibidores de betalactamases (ampicilina/ sulbactam e piperacilina/ tazobactam) e aminoglicosÃdeos, excetuando, estreptomicina. O IMR demonstrou que cerca de 50% dos isolados foram resistentes a cinco dos vinte antimicrobianos testados, destacando as cepas provenientes de cavala (55,5%). Nos testes de CIM observou-se que, cepas isoladas de pescados de Ãgua salgada apresentaram maiores percentuais de isolados resistentes a NAL, CIP, MFX, SUT e AMO. Maiores resistÃncias a elevadas concentraÃÃes de antimicrobianos, quando testadas bactÃrias oriundas de Ãgua doce, foram observadas para AMO, SUL e NAL. Os perfis cromossomiais em isolados de Ãgua doce foram na maioria compostos por amoxicilina, ampicilina, estreptomicina e sulfametazol &#8725; trimetropim. Para o pescado de Ãgua salgada, foi verificado a predominÃncia de quinolonas. A anÃlise do DNA plasmidial mostrou a ocorrÃncia de uma populaÃÃo heterogÃnea de pequenos plasmÃdios distribuÃdos em vÃrios perfis. A maior diversidade e menor peso molecular foram observados em cepas de amostras de pescado de origem marinha. Dessa forma, destaca-se que a circulaÃÃo de E. coli patogÃnicas com caracterÃsticas de resistÃncia antimicrobiana representa um risco ao ecossistema aquÃtico e ao ambiente de comercializaÃÃo, havendo necessidade de vigilÃncia contÃnua a bactÃrias contaminantes do pescado, para que a seguranÃa desses alimentos seja garantida.

Dissertação de Mestrado em Segurança Alimentar<br>O conhecimento sobre a presença e frequência de genes de resistência às quinolonas mediada por plasmídeos (RQMP) em estirpes comensais de Escherichia coli de origem bovina é escasso a nível mundial. Foram estudadas um total de 237 amostras de E. coli de vitelos saudáveis previamente isoladas após pressão selectiva in vivo de enrofloxacina (ENR) e caracterizadas quanto à resistência aos antibióticos: 101, 79 e 57 isolados relativos a T0, T1 (6 semanas após administração de ENR) e T2 (10 semanas após administração de ENR), respectivamente. Os isolados foram caracterizados fenotipicamente por determinação da Concentração Inibitória Mínima (CIM) para os antimicrobianos ácido nalidíxico (AN), ciprofloxacina (CIP) e levofloxacina (LEV) e os resultados interpretados segundo os critérios epidemiológicos (ECOFF) estabelecidos pelo EUCAST. A frequência de genes de RQMP (qnr, aac(6’)-Ib-cr e qepA) foi determinada através de amplificação por PCR e sequenciação nucleotídica. A proporção de isolados de E. coli resistentes ao AN em T0, T1 e T2 foi de, respectivamente: 52,5% (n=53; CIM 64->256 μg/ml), 100% (n=79; CIM 128->256 μg/ml) e 82,5% (n=47; CIM 128->256 μg/ml). A resistência à CIP em T0, T1 e T2 foi de, respectivamente: 52,5% (n=53; CIM 0,125->256 μg/ml), 100% (n=79; CIM 0,25-128 μg/ml) e 89,5% (n=51; CIM 0,25-64 μg/ml). A resistência à LEV em T0, T1 e T2 foi de, respectivamente: 46,5% (n=47; CIM 0,5-64 μg/ml), 100% (n=79; CIM 0,5-64 μg/ml) e 87,7% (n=50; CIM 0,5-32 μg/ml). No que respeita aos determinantes de RQMP nos 237 isolados estudados, foram identificados: 11,8% (n=28) positivos para genes qnr (qnrB2, n=4; qnrD, n=11; qnrS1, n=13); e 0,8% (n=2) isolados positivos para o gene aac(6’)-Ib-cr. Da análise da frequência dos genes de RQMP nos isolados de E. coli observou-se: em T0, 3% de genes qnr (todos qnrS1) e 2% do gene aac(6’)-Ib-cr; em T1, 15,2% de genes qnr (10,1% qnrD e 5,1% qnrS1); em T2, 22,8% de genes qnr (7% qnrB2, 5,3% qnrD e 10,5% qnrS1). Os dados obtidos evidenciam um aumento significativo da prevalência de isolados resistentes ao longo do tempo de colheita, sugerindo que a pressão selectiva imposta pela exposição à ENR tem influência no aparecimento de resistência às quinolonas. Observou-se um aumento significativo da frequência de genes de RQMP ao longo do estudo longitudinal e mais de 80% dos isolados positivos para RQMP foram resistentes às quinolonas. Este é, para o nosso conhecimento, o primeiro estudo que descreve a identificação de resistência às quinolonas por qnrD em isolados de E. coli de bovinos.<br>ABSTRACT - The current knowledge about the presence and frequency of pasmid-mediated quinolone resistance (PMQR) genes in commensal Escherichia coli strains from cattle is scarce. Two hundred and thirty seven E. coli samples isolated from healthy calves were studied after in vivo enrofloxacin (ENR) selective pressure and previously characterized regarding antimicrobial susceptibility, including: 101, 79 and 57 isolates from T0, T1 (six weeks after ENR administration) and T2 (10 weeks after ENR administration), respectively. The phenotypic characterization was performed using Minimum Inhibitory Concentration (MIC) determinantion for nalidixic acid (NAL), ciprofloxacin (CIP) and levofloxacina (LEV) by the microdilution method and the results were interpreted according to EUCAST definitions of epidemiological cut-off values (ECOFF). PMQR genes (qnr, aac(6’)-Ib-cr and qepA) frequency was performed by PCR amplification and nucleotide sequencing. NAL resistant E. coli isolates in T0, T1 and T2 were, respectively: 52,5% (n=53; MICs 64 to >256 μg/ml), 100% (n=79; MICs 128 to >256 μg/ml) and 82,5% (n=47; MICs 128 to >256 μg/ml). CIP resistant isolates in T0, T1 and T2 were, respectively: 52,5% (n=53; MICs 0,125->256 μg/ml), 100% (n=79; MICs 0,25-128 μg/ml) and 89,5% (n=51; MICs 0,25-64 μg/ml). LEV resistant isolates in T0, T1 and T2 were, respectively: 46,5% (n=47; MICs 0,5-64 μg/ml), 100% (n=79; MICs 0,5-64 μg/ml) and 87,7% (n= 50; MICs 0,5-32 μg/ml). From the 237 E. coli isolates tested: 11,8% (n=28) harboured qnr genes (qnrB2, n=4; qnrD, n=11; and qnrS1, n=13) and 0,8% (n=2) were found positive for the aac(6’)-Ib-cr gene. The analysis of PMQR genes in E. coli at the different sampling times showed that: at T0, qnr genes were detected in 3% of the isolates (all found to be qnrS1) and 2% carried the aac(6’)-Ib-cr gene; at T1, 15,2% of the isolates carried qnr genes (10,1% qnrD and 5,1% qnrS1); and at T2, 22,8% of the isolates were found positive for qnr genes (7% qnrB2, 5,3% qnrD and 10,5% qnrS1). The results reveal an increased prevalence of resistant isolates along the time, suggesting that ENR selective pressure influences the emergence of quinolone resistance. A significant increased frequency of PMQR genes along the longitudinal study was observed and more than 80% of PMQR positive isolates were quinolone resistant. This is to our knowledge the first report on PMQR qnrD gene in E. coli isolates from cattle.

Escherichia coli patogênicas aviárias (APEC) causam infecções extraintestinais em aves, que podem ser localizadas ou sistêmicas, denominadas colibacilose. O patotipo de APEC não está definido, mas vários genes de virulência são associados a essas cepas, como os genes plasmidiais iroN, ompT, hlyF, iss e iutA, propostos, em 2008, como preditores da virulência de APEC. Além dos genes de virulência conhecidos, outros fatores podem estar associados à patogenicidade bacteriana, como as maquinarias de secreção protéica denominadas Sistemas de Secreção. O Sistema de Secreção do Tipo 6 (T6SS), descrito em 2006, tem sido associado à virulência de cepas APEC. Este trabalho divide-se em duas partes: a primeira teve como objetivo avaliar a frequência dos genes iroN, ompT, hlyF, iss e iutA em 401 cepas aviárias de E. coli e sua relação com a patogenicidade in vivo dessas cepas. A segunda parte teve como objetivos verificar a frequência de genes componentes do T6SS (clpV, vgrG, icmF e dotU), em uma coleção de 187 cepas APEC; e, em algumas cepas positivas para os genes, verificar a expressão de um fenótipo relacionado ao sistema, bem como a expressão do efetor vgrG e da ATPase do sistema clpV2, além da secreção de proteínas, no meio de cultura e durante contato com células eucarióticas. Os resultados da primeira parte indicam que cepas com dois ou mais dos genes analisados tem maior probabilidade de serem patogênicas do que cepas com apenas um ou nenhum dos genes. Já os da segunda parte mostram que várias cepas apresentaram duas cópias de pelo menos um dos genes testados, e algumas delas apresentaram resistência à predação por D. discoideum. Não foram encontradas diferenças entre a expressão dos genes vgrG (1 e 2) e clpV2 em cultura pura ou em contato com células eucarióticas. Este trabalho apresenta a triagem de genes plasmidiais em uma grande coleção de amostras de Escherichia coli, e a primeira triagem de genes do T6SS em uma coleção de isolados APEC.<br>Avian pathogenic Escherichia coli (APEC) causes extraintestinal infections in birds, which can be localized or systemic, known as colibacillosis. The APEC pathotype is still undefined, but several virulence genes are associated with these strains, as the plasmid-linked genes iroN, ompT, hlyF, iss and iutA, proposed in 2008 as APEC virulence predictors. Besides the known virulence genes, other factors may be associated with bacterial pathogenicity, such as the protein secretion machineries called Secretion Systems. Described in 2006, Type 6 Secretion System (T6SS) has been associated with virulence of APEC strains. This work is divided in two parts: the first aimed to evaluate the frequency of iroN, ompT, hlyF, iss and iutA genes in 401 avian strains of E. coli and its relationship with in vivo pathogenicity of these strains. The second part aimed to verify the frequency of T6SS genes (clpV, vgrG, icmF and dotU) in a collection of 187 APEC strains; and verify, in some positive strains, the expression of a phenotype related to the system, as well as the gene expression of effector vgrG and ATPase clpV2, besides the secretion of proteins into the culture medium and during contact with eukaryotic cells. The results of the first part of this study indicate that isolates harboring two or more of the genes analyzed were most likely to be pathogenic than strains harboring only one or none of the genes. The results of the second part show that several strains harbored two copies of at least one of the genes tested, and some of them were resistant to predation by D. discoideum. No differences were found between the expression of genes vgrG (1 and 2) and clpV2 in pure culture or in contact with eukaryotic cells. This work presents the screening of plasmidial genes in a large collection of Escherichia coli isolates, and the first screening of T6SS genes in a collection of APEC isolates.

L’émergence des déterminants de résistances plasmidiques aux quinolones (PMQR), auxquels appartient le gène qnrD, participent de manière significative à la sélection des résistances de haut-niveau aux permet les réparations de l’ADN lors des stress soumis aux bactéries, et d’autre part, que les aminosides, une autre classe d’antibiotiques que les fluoroquinolones, induisaient la réponse SOS chez Escherichia coli. En effet, nous avons montré que les petits plasmides-qnrD chez E. coli, induisent la formation de monoxyde de nitrogène et l’inhibition de la voie de détoxification Hmp-dépendante. Ces processus génèrent des lésions à l’ADN qui s’ajoutent à celles occasionnées par les aminosides concourant à activer la réponse SOS chez E. coli. L’ensemble de nos résultats montrent que l’émergence de la résistance aux fluoroquinolones peut être occasionnée par l’exposition d’E. coli à une autre classe d’antibiotiques, ici les aminosides<br>The emerging plasmid-mediated quinolones resistance (PMQR) determinants significantly participate in the selection of high-level of resistance to the major antibiotics fluoroquinolones, leading to numerous clinical failures. In this study, we reported for the first time that PMQR expression could be triggered by the fluoroquinolones but also by another major class of antibiotics, the aminoglycosides. We were able to show that this unique cross selection of antibiotic resistance was the consequence of the PMQR determinant qnrD being SOS-regulated in a RecA-LexA dependent manner. We demonstrated that sub inhibitory concentration of aminoglycoside induced nitric oxide formation associated with the repression of the Hmp-mediated detoxification pathway, resulting in the induction of the SOS response and thus up-regulation of the PMQR. Overall, our findings revealed an unexpected antibiotic resistance cross-selection with low aminoglycosides concentrations promoting emergence of fluoroquinolones resistance

The use of recombinant gene expression for industrial protein production has since the early 1970s become a multi-billion industry. Despite many years of intensive research, no universal expression system exists that can be used for high-level industrial production of any given recombinant gene. Because of this, much work must be done prior to production to identify the ideal production organism for the gene of interest, usually followed by optimization of an expression system useful in this organism. One of the most popular production organisms is the gram-negative bacterium Escherichia coli, which is regularly used for high-level production of both prokaryotic and eukaryotic proteins. Despite its popularity there are some well-known drawbacks of using E. coli for recombinant expression, including lack of some post-translational modifications of recombinant proteins, and few and inefficient systems for translocation and secretion. In the present work selected key processes in E. coli recombinant protein production has been studied with focus on exploring methods to improve protein production in this organism. The key processes include the use of signal peptides for efficient translocation of eukaryotic proteins to the periplasm, directed evolution of the transcription regulators for increased expression from their cognate promoters, and the use of codon-optimized synthetic genes. The use of signal peptides to achieve efficient translocation of recombinant genes is a well-known and common method. By testing the signal peptides sequences ompA, pelB and the novel, designed consensus signal peptide sequence CSP, in combination with the eukaryotic cDNA for the proteins hGM-CSF, scFv-phOx and hIFN-α2b, it was shown that the effect of signal peptides are gene-specific and, more importantly, that the presence of signal peptides not only directs the gene products to the periplasm but also increases the expression of the recombinant genes. Under high cell density culture (HCDC) expression production volumes of 2.3, 1.7 and 0.6 g/L of scFv-phOx, hGM-CSF and hIFN-α2b, respectively, were obtained.

This study was conducted to examine the rate of contamination and molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred and eighty raw food samples were tested and 60.8% of meat and 18.0% of shellfish samples were found to be contaminated with Salmonella spp. which belonged to variety of serogroups and serotypes. More than 90% of all food sources contained Escherichia coli and 32% of 50 shellfish samples were contaminated with Vibrio parahaemolyticus. PFGE was used to determine the degree of relatedness of Salmonella spp. There were 33 distinct PFGE patterns from 51 Salmonella spp. isolates tested, indicating that PFGE could be used as an alternative method for serotyping for use in epidemiology of Salmonella spp. Susceptibility of the isolates to 15 antimicrobial agents was investigated. Moderate to high frequencies of resistance to antibiotics were observed in Salmonella spp. and E. coli isolates and multi-resistance, defined as resistance to at least 4 antibiotics, was observed. All of the V. parahaemolyticus isolates were resistant to ampicillin/amoxicillin but not to other antibiotics. Betalactam TEM gene and tetracycline resistance tetA, tetB genes were widely distributed in both E. coli and Salmonella spp. isolates. Other resistance genes, including sulI, cmlA, aadA, aphA1, dhfrV, and aac(3)-IV were also present at high to moderate levels. Identification and characterisation of the mobile genetic elements, including identification of class 1 integrons and plasmids were carried out for multi-resistance isolates. The integrons harboured varying gene cassettes, including aadA1, aadA2, aadA5, aacA4, dhfrXII, drfA1 and dhfrA17, blaPSE1 and catB3. Thirty-five percent of Salmonella spp. isolates and 76% of E. coli isolates harboured plasmids of more than 95 kb. Transfer of resistance phenotypes between the isolates via conjugation and phage transduction was also demonstrated. Salmonella genomic island 1 (SGI1), a 43-kb genomic region contains a 13-kb antibiotic resistance gene cluster, has been identified in an isolate of S. Albany from chicken. The presence of Salmonella spp. virulence genes was investigated to examine the pathogenicity potential of the isolates. The invA gene was present in all Salmonella spp. isolates and the plasmid virulence gene spvC was detected in one S. Typhimurium isolate only, on a 95 kb virulence plasmid. Invasion assays performed in vitro demonstrated that all Salmonella isolates were capable of invading human intestine INT407 cells. In addition, the investigation for the presence of 58 selected virulence genes showed that all the tested isolates contained at least one virulence gene and there were 16 genes which are associated with different pathotypes detected. The data obtained in this study indicates that raw food in Vietnam is a potential reservoirs for many pathogenic organisms, and confirms the role of food animals as a reservoir of multidrug resistant E. coli and Salmonella spp.

Escherichia coli enteroagregativa (EAEC), destaca-se como um importante patógeno emergente causador de diarréia persistente em países em desenvolvimento e de diarréia aguda em países desenvolvidos. A grande heterogeneidade dos fatores de virulência caracteriza esta categoria, porém não foi estabelecido um marcador genético comum a todas as amostras de EAEC. O padrão de adesão agregativa (AA) em células HEp-2 e HeLa é a forma de caracterização e diagnóstico mais precisos desta categoria. Uma das toxinas envolvidas na patogênese é Plasmid-encoded toxin (Pet) pertencente à classe das proteínas autotransportadoras com características de uma serino protease denominada SPATEs. Iniciou-se este estudo com a determinação do padrão de adesão de 164 amostras EAEC, previamente caracterizadas como sonda pCVD432 ou onda AA positiva. Assim, 141 (86%) amostras, que apresentaram padrão de adesão agregativo, foram caracterizadas como EAEC. Face aos resultados obtidos, confirmou-se a baixa especificidade da sonda AA. A pesquisa do gene pet, por meio de ensaio de PCR, resultou na positividade de 12 (8,5%) amostras. Prosseguiu-se esse estudo com a padronização da reação de immuno-dot. Utilizando-se 300 &#181;L do sobrenadante bacteriano, soro policlonal anti-Pet e o conjugado nas diluições 1/50 e 1/2.500, respectivamente, resultados bastante reprodutíveis foram obtidos. O método foi mais sensível que a detecção do gene por PCR. Por esse ensaio, detectou-se a toxina Pet em 16 (11,3%) das 141 amostras EAEC. Nenhuma das amostras controle negativo foi reconhecida pelo soro anti-Pet, assim como as amostras de E. coli produtoras das mais diversas toxinas. Apesar da baixa prevalência de amostras de EAEC produtoras da toxina Pet, neste estudo padronizou-se um método rápido, sensível, específico e de baixo custo para pesquisa desta toxina mostrando o potencial diagnóstico deste ensaio para uso em inquéritos epidemiológicos, o que poderá permitir determinar o papel da Pet no desenvolvimento de diarréia aquosa.<br>Enteroaggregative Escherichia coli (EAggEC) is an emerging diarrheal pathogen, whose pathogenesis is thought to comprise colonization of the intestinal mucosa with the release of secretogenic toxins. One of the toxin involved is the plasmid-encoded toxin (Pet), which is secreted by the autotransporter mechanism and belongs to a growing class of Enterobacteriaceae autotransporter proteins. Since the characteristic aggregative adherence pattern of EAggEC is associated with the presence of a large plasmid called pCVD432, DNA probes and PCR primers derived from this plasmid have been recommended as a screening method for EAggEC in the clinicai laboratory. In this study 164 E. coli isolates positive for the pCVD432 probe were tested for adherence to HEp-2 cells in which 141 isolates showed aggregative pattern, 12 isolates from them amplify a 1037-bp DNA fragment corresponding to pet gene by PCR. Using this samples we standardized an immuno-dot assay for EAggEC detection through Pet toxin as target antigen. 300 &#181;l of bacterial supernatant were applied in a PVDF membrane, and using a rabbit polyclonal sera anti-Pet the expression of the toxin by immuno-dot was in the same isolates in which the gene was detected. Besides no negative controls reacted with Pet antisera, in which we included 40 isolates with no virulence markers for diarrheagenic E. coli and E. coli expressing toxins other than Pet. This method proves to be rapid, sensitive, specific and low cost, demonstrating this potential as diagnosis for Pet expression and its association with diarrhea.

Chez la bactérie Escherichia coli, la présence de lésions sur l'ADN induit l'expression d'un système de réparation de l'ADN, le système SOS. La protéine RecA activée, au contact de l'ADN simple brin créé par la réplication de l'ADN endommagé, clive la protéine LexA, represseur d'au moins 17 gènes qui participent à l'expression du système SOS. Le plasmide conjugatif R6-5 exprime une fonction appelée Psi ("Plasmid-mediated SOS Interference) qui perturbe l'expression constitutive du système SOS dans un mutant recA441. Ce travail est une contribution à la caractérisation de la fonction Psi. Nous montrons que la protéine PsiB de poids moléculaire 15 700 est l'inhibiteur de l'expression constitutive du système SOS. En effet, la protéine PsiB inhibe le clivage du répresseur LexA. Le plasmide F qui n'exprime pas la fonction Psi code une protéine PsiBF dont seulement cinq acides aminés sont différents de ceux de la protéine PsiB. L'inefficacité du plasmide F à exprimer la fonction Psi est due à une faible expression du gène psiBF plutôt qu'à une faible activité intrinsèque de la protéine PsiBF. Nous montrons aussi que la protéine PsiB inhibe le clivage du répresseur LexA après un rayonnement UV dans une bactérie recA+ seulement lorsque sa concentration cellulaire est augmentée grâce à une sur-expression du gène psiB cloné. La protéine PsiB inhibe donc l'induction du système SOS. Comme la protéine PsiB ne se fixe pas à de l'ADN simple brin, elle pourrait perturber indirectement l'activation de la protéine RecA en interférant avec le complexe de réplication bactérien.

La ségrégation du matériel génétique est une étape fondamentale du cycle cellulaire permettant la transmission du patrimoine génétique au cours des générations. Dans les cellules eucaryotes, la mitose est l'étape qui permet la répartition des chromosomes dupliqués dans chaque cellule fille. Des systèmes actifs, dédiés à la ségrégation de l'ADN sont retrouvés sur la majorité des plasmides et chromosomes bactériens. Ces systèmes ParABS, dits "de partition", sont constitués de deux protéines, ParA et ParB, et d'une séquence centromérique, parS. La protéine ParA est une ATPase capable de positionner les plasmides dans le cytoplasme tout au long du cycle cellulaire. Son comportement dynamique fait d'elle le moteur de la partition. La protéine ParB, l'autre acteur de la partition est une protéine adaptatrice entre la molécule d'ADN et la protéine motrice. ParB se fixe sur le centromère parS pour former une complexe nucléoprotéique appelée "complexe de partition". En utilisant différents modes de fixation à l'ADN et en établissant des interactions protéine-protéine multiples, ParB est aussi capable de s'organiser en complexe de partition dit "étendu" qui est le substrat de la réaction de ségrégation. La formation du complexe de partition "étendu" est la première étape du mécanisme de partition et est essentielle au processus de ségrégation. L'architecture de ce complexe n'est connue pour aucun des systèmes de partition parABS. L'un des systèmes modèles majeurs du mécanisme de ségrégation de l'ADN chez les bactéries est le système sopABC du plasmide F d'E. Coli. Ainsi, au cours de ma thèse, j'ai initié plusieurs projets en parallèle, visant à caractériser à la fois in vivo et in vitro, les différentes interactions impliquées dans l'organisation du complexe de partition et du complexe de partition "étendu" de ce plasmide. En collaboration avec l'équipe du Dr Véronique Le Berre au Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP-INSA) de Toulouse, j'ai contribué à la validation des déterminants nucléiques impliqués dans l'interaction de notre séquence centromérique modèle, parS, avec le motif hélice-tour-hélice (HTH) de ParB. Ensuite, nous avons identifié un nouveau motif en dehors du motif HTH et montré qu'une arginine de ce motif est essentielle à l'interaction spécifique avec le centromère. Ces résultats ont révélé une caractéristique conservée dans le règne bactérien : les protéines ParB contiennent un domaine de liaison au centromère, composé de deux motifs séparés et essentiels. Le cœur de mes travaux de thèse a été de comprendre l'organisation du complexe de partition "étendu" et son rôle dans la partition. Par une combinaison d'approche moléculaire in vivo et in vitro, j'ai montré que l'architecture de ce complexe étendu en dehors des sites parS, nécessitait deux types d'interaction, des interactions protéine/protéine mais également ADN/protéine. Afin d'étudier le mode d'interaction de ParB avec des séquences nucléiques non spécifiques avoisinant le complexe de partition, j'ai mis en œuvre une technique d'immunoprécipitation de chromatine, couplée à des techniques de hautes détections (qPCR ou ChIPseq). Cette technique nous a permis de montrer que ParB est capable de s'étendre sur une distance d'environ vingt kilobases de part et d'autre du centromère. Nos résultats semblent incompatibles avec le précédent modèle dans lequel ParB serait capable de polymériser sur l'ADN, à la manière d'un protéo-nucléofilament qui s'initierait au niveau du centromère. Ainsi ces travaux, nous ont permis de proposer un nouveau modèle dans lequel le complexe de partition "étendu" serait une structure concentrée, dynamique et résultant d'interactions stochastiques entre ParB et l'ADN avoisinant ParS<br>Segregation of genetic material over generations is an essential process ensuring that every daughter cell receives a copy of each DNA molecule. Similarly to Eukaryotes, Prokaryotes possess cytoskeletal machineries, named Par, responsible for DNA segregation. Bacterial Par systems, found on chromosomes as well as on various low copy number plasmids, are composed of three elements: a ParA protein, a ParB protein and a centromere site, parS. ParA ATPase is able to position plasmids in the cytoplasm during the cell cycle. Its dynamic pattern make it the motor of the partition. The centromere binding protein (CBP) ParB, binds the centromere to form a nucleoprotein assembly called the "partition complex". Using different mode of DNA binding and multiple protein-protein interactions, ParB is also capable of organizing into higher order complexes called the "extended" partition complex. This complex is the substrate for the partitioning process. Formation of the extended partition represents the first step in partition and is essential to segregation. The architecture of this complex is not known for any partition system parABS. Here, we focus on the assembly of the F partition complex. During my PhD, I initiated several projects in parallel to characterize the different interactions involved in the organization of the partition complex and the extended partition complex of this plasmid with in vivo and in vitro approaches. In collaboration with the laboratory of Dr. Veronique Le Berre in Toulouse (LISBP -INSA), we determined sopC basis involved in specific SopB-sopC interactions. Then, we identified a new ParB determinant, outside of the helix-turn-helix DNA binding motif, responsible for specific DNA binding to the centromere. These findings reveal that ParB have an extended DNA binding domain, composed of two separate DNA binding motifs. We extended our analysis to chromosomal ParB and show that this second centromere binding motif is highly conserved in a wide range of bacteria. Using in vivo and in vitro approaches, we show that the extended partition complex architecture requires both protein-protein and protein-DNA interactions. To investigate the overall organization of the SopB-sopC extended partition complex, we use chromatin immunoprecipitation (ChIP) coupled with high throughput sequencing. This technique allowed us to visualize that SopB is able to extend around sopC over ~20 Kb. Our results are thus inconsistent with previous models suggesting that SopB polymerize side by side in a proteo-nucleofilament emanating from the centromere. So, we propose a new model in which the extended partition complex of F plasmid assembles in a nucleoprotein complex from stochastic binding of SopB on neighboring sopC DNA

Le plasmide ColV pS88 joue un rôle majeur dans la virulence du clone de Escherichia coli O45:K1:H7 responsable de méningite néonatale. Ce plasmide est caractérisé par une région de virulence conservée CVP qui comprend des facteurs de virulence connus comme les systèmes de capture du fer ainsi que des gènes de fonction inconnue. Les objectifs de ce travail étaient d'identifier de nouveaux gènes de virulence et de mieux caractériser la distribution de la région CVP au sein des E. Coli extraintestinaux. Par analyse transcriptionnelle, nous avons identifié des gènes de fonction inconnue parmi lesquels le gène csbLp dont la transcription était fortement induite en situation de carence martiale. Ce gène apparaît toujours associé au sidérophore salmochéline. Par mutagénèse, spectrométrie de masse et modèle expérimental, nous avons montré qu'il était impliqué dans la virulence de pS88 en agissant comme booster de la synthèse de certains sidérophores via la voie métabolique du shikimate. Par ailleurs, nous avons montré une forte prévalence de la région CVP au sein du sous-groupe phylogénétique C, récemment isolé du groupe majeur B1, ce qui pourrait expliquer la virulence paradoxale de certaines souches B1 habituellement considérées comme commensales. Enfin, nous avons mis en évidence la présence d'une région CVP dans une souche productrice de shigatoxine responsable d'un syndrome hémolytique et urémique associé à une bactériémie. L'analyse fonctionnelle et épidémiologique de la région CVP a permis d'élaborer le concept de facteur de virulence auxiliaire des sidérophores et d'évaluer sa dissémination au sein des E. Coli responsables de pathogénicité intra et extraintestinale<br>The ColV plasmid pS88 plays a major role in the virulence of the neonatal meningitis Escherichia coli clone O45:K1:H7. This plasmid is characterized by a conserved virulence plasmidic (CVP) region including several known virulence factors as the iron uptake systems as well as genes of unknown function. The aims of this study were to identify new virulence genes and to better characterize the distribution of the CVP region within extraintestinal pathogenic E. Coli. Transcriptional analysis identified genes of unknown function including the gene csbLp whose transcription was strongly induced in iron-depleted conditions. This gene was present only in strains harboring the iro locus encoding salmochelins. By mutagenesis, mass spectrometry and experimental model, we showed that it was involved in the virulence of pS88 acting as a booster of catecholate/phenolate siderophores synthesis via the shikimate pathway. Furthermore, we have shown a high prevalence of the CVP region in the phylogenetic subgroup C, closely related to major group B1, which could explain the unexpected virulence of some B1 strains usually considered as commensal. Finally, we showed the presence of a CVP region in a Shiga toxin-producing E. Coli responsible for a hemolytic-uremic syndrome associated with bacteremia. The functional and epidemiological analysis of the CVP region pointed the concept of siderophore auxiliary virulence factors and showed its dissemination within extraintestinal and intestinal pathogenic E. Coli

La ségrégation, ou partition, des chromosomes et des plasmides bactériens est l'étape fondamentale du cycle cellulaire qui assure la transmission de l'ensemble du génome aux cellules filles. C'est l'équivalent procaryote de la mitose. Des systèmes de ségrégation, appelés les loci par, ont été identifiés sur les plasmides à bas nombre de copies, et des homologues de ces systèmes de partition sont présents sur la majorité des chromosomes bactériens. Le système code deux protéines, une ATPase et une protéine qui se fixe spécifiquement sur une région centromérique. Ces deux protéines interagissent entre elles, permettent la localisation subcellulaire des réplicons et assurent ainsi leur maintien dans les générations futures. Au laboratoire, nous étudions l'un des systèmes modèles majeurs, le système de partition du plasmide F d'Escherichia coli, afin de déterminer le mécanisme moléculaire assurant le processus de ségrégation et son contrôle pendant le cycle cellulaire. La stabilité du plasmide F est assurée par le système de partition sopABC. Après la réplication du plasmide, la protéine SopB s'assemble sur le centromère sopC pour former un complexe de partition qui permet aux copies du plasmide d'être positionnés au centre de la cellule. Avant la division cellulaire les plasmides migrent aux positions 1/4 et 3/4 de la cellule et assurent ainsi l'héritage des réplicons dans les futures cellules filles. L'ATPase SopA est essentielle dans le processus de partition, mais son rôle n'est pas bien défini. SopA pourrait être impliquée dans les étapes de positionnement et/ou de déplacement des plasmides de part et d'autre de la cellule. SopA possède plusieurs activités. In vivo, SopA agit comme autorépresseur de l'opéron sopAB en se fixant sur la région promotrice. De plus elle interagit avec le complexe de partition et forme des polymères en présence d'ATP. Nous avons montré que cette activité est régulée par SopB et par l'ADN. L'activité ATPase de SopA est essentielle pour la partition. Elle est légèrement stimulée par SopB et par l'ADN, mais lorsque ces deux facteurs sont présents, elle est fortement stimulée. Nous avons entrepris de caractériser les interactions existantes entre ces trois protagonistes. Ainsi, nous avons démontré que cette stimulation nécessite une interaction de SopA avec SopB d'une part et avec l'ADN d'autre part. Nous avons également montré que le site centromérique sopC potentialise la stimulation de l'activité ATPase par l'intermédiaire de SopB. Nous nous sommes intéressés ensuite à l'interaction SopA-SopB, et nous avons mis en évidence que SopB stimule l'activité ATPase de SopA via un motif arginine finger. Pour finir, nous avons montré que in vivo, la stimulation de l'activité ATPase de SopA joue un rôle dans la régulation de l'opéron sopAB mais aussi dans la partition du plasmide F<br>Mitotic segregation of chromosomes and plasmids, termed partition in bacteria, is a fundamental step of the cell cycle that ensures the transmission of the whole genome to daughter cells. It is governed by specific genetic loci named par, first identified in low copy number plasmids and later found to be present as homologues in most bacterial chromosomes. Par loci encode two proteins, an ATPase and a DNA binding protein, and include a cis-acting centromeric site. These components interact with each other to direct the subcellular localization that ensures stability of their replicons. To determine the molecular mechanisms of the partition process and its control during the cell cycle, we study the Sop partition system of the Escherichia coli plasmid, F. Sop is one of the best-known partition systems. After F plasmid replication, SopB protein binds to the sopC centromeric site to form a partition complex. The complex on each plasmid copy interacts with SopA, an ATPase, and activates it to move the plasmid molecules towards the two cell poles. SopA ATPase is essential to the segregation process but its role is not defined. SopA has many activities. In vivo it represses its own operon by binding to the sopAB promoter. Moreover, in addition to its interaction with the partition complex it polymerizes in the presence of ATP. We have shown that SopB and DNA regulate this activity. Although the ATP-binding site on SopA is essential for partition, ATP hydrolysis by SopA is very weak. It is stimulated modestly by DNA and by SopB and strongly in the presence of both. We have characterized the interactions necessary for stimulation of ATP hydrolysis. First we found that the SopB-sopC partition complex is required for maximal stimulation. Then we showed that SopB and DNA contact SopA by two distinct interactions to fully activate ATPase activity. We also found that SopB activates SopA ATPase through an arginine finger motif. Finally, we have shown that in vivo, stimulation of the ATPase activity is necessary for both regulation of the sopAB operon and partition of plasmid F to be efficient

Pour élucider certains facteurs ayant pu favoriser le succès des CTX-M-15, nous avons caractérisé les systèmes TA plasmidiques (pemKI, ccdAB, hok-sok, vagCD, pndAC, parDE, relBE et srn-B) de souches cliniques de 2 collections d’Escherichia coli producteurs de BLSE l’une tunisienne et l’autre française et nous avons montré que dans ces 2 collections, les plasmides IncF hébergeant les CTX-M-15 sont significativement plus riches en système TA (en moyenne plus de 2 systèmes TA) et particulièrement en vagCD, système TA identifié dans un plasmide de virulence de Salmonella. Ce système vagCD était significativement associé aux plasmides IncF hébergés par les souches appartenant au clone mondial O:25 H4 ST131. Nous avons montré aussi que les souches hébergeant les CTX-M-15 de la collection tunisienne sont assez riches en gènes de virulence particulièrement en gènes codant la cytotoxicité (hylA et cnfI) ce qui pourraient contribuer à leur diffusion massive dans nos hôpitaux. D'autre part, nous avons trouvé une forte prévalence des BLSE (30 %) chez les souches d’E. Coli commensales du tube digestif des volailles d’élevage intensif en Tunisie. La quasi-totalité de ces BLSE était plutôt de type CTX-M-1 que CTX-M-15 comme celles isolées chez l’homme et hébergées par des plasmides de groupe d’incompatibilité IncI1 tous renferment le système TA pndAC et rarement vagCD. Finalement, on a obtenu des simples mutants des systèmes TA (ccdAB, pemK et vagCD) du plasmide CTX-M-15 de la souche clonale O25, ST131 (TN03) en utilisant la technique de mutagenèse dirigée de Datsenko et al mais nous n’avons pas trouvé de perte significative des plasmides mutés au cours de la division cellulaire<br>To better understand the success of CTX-M, and particularly of CTX-M-15, we characterized the plasmid addiction systems (pemKI, ccdAB, hok-sok, vagCD, pndAC, parDE, relBE and srn-BC) in two collections of ESBL-producing Escherichia coli strains, one French and the other Tunisian. We have demonstrated that in both collections the addiction systems, mainly vagCD, were significantly more prevalent in IncF CTX-M-15-carrying plasmids. The vagCD system, which is associated to Salmonella virulence plasmids, was significantly associated to the pandemic ST131 clone. Moreover, the CTX-M-15 producing E. Coli isolates of the Tunisian collection had a high frequency of virulence factors particularly hylA and cnfI. On the other hand, we have detected a high prevalence of ESBLs in faecal samples of healthy poultry in Tunisia. The most common type of ESBLs identified was CTX-M-1, harboured by self conjugative IncI1 plasmids which all comprised the pndAC system and rarely vagCD. Finally, deletion mutants of the TA systems ccdAB, pemK and vagCD harboured by the CTX-M-15 plasmid of a French ST131 strain (TN03) were created using the method by Datsenko et al, but we didn’t observe a significant loss of mutant plasmids during cell division. Therefore, we plan to construct double or triple system TA mutants so that we could evaluate the role of the association of multiple addiction systems in plasmid stabilization

Toute l'information génétique nécessaire à la vie de la bactérie Escherichia coli est portée par son unique chromosome : une molécule d'ADN circulaire de 4,6 millions de paires de bases. En plus de son ADN chromosomique, une bactérie peut contenir des molécules d'ADN supplémentaires appelées plasmides. L'objet de cette thèse est la mise au point d'une expérience de mesure cellule par cellule du nombre de plasmides. Il s'agit de prendre, une par une, plusieurs milliers de bactéries et ensuite de compter le nombre de plasmides qu'elles contiennent. Pour cela nous avons utilisé des techniques de micro lithographie pour construire un dispositif constitué de réservoirs de 5 mm de diamètre reliés entre eux par un canal de 5 µm de large. Une goutte de liquide contenant les bactéries est déposée dans l'un des réservoirs. Une différence de hauteur de liquide et/ou une tension électrique appliquée entre les réservoirs permet de faire passer les bactéries d'un réservoir à l'autre, une par une, devant l'objectif d'un microscope inversé. Le nombre de plasmides dans la bactérie est mesuré de façon indirecte. Nous avons construit des plasmides qui portent tous un gène codant une protéine fluorescente verte, et lorsqu'une bactérie passe devant l'objectif du microscope nous mesurons la fluorescence de la cellule. Notre expérience repose sur l'hypothèse que le niveau d'expression est le même pour chaque copie du gène : la fluorescence de la cellule est alors proportionnelle au nombre de copies du plasmide. Cette thèse décrit l'expérience de microscopie de fluorescence ainsi que la construction des souches bactériennes. Les résultats préliminaires que nous avons obtenus montrent que le dispositif expérimental est au point : Lors d'une expérience, nous mesurons la fluorescence de plusieurs milliers de bactéries. Le signal est largement au dessus du bruit et les résultats sont reproductibles<br>All the genetic information necessary to the life of Escherichia coli is carried by its unique chromosome: a 4. 6 million base pair circular DNA molecule. Besides its chromosomal DNA this bacterium can also contain additionnal DNA molecules called plasmids. The aim of this thesis is to build up an experiment to measure cell by cell the number of plasmids. The point is to take, one by one, thousands of bacteria and then measure the number of plasmids they contain. To do this we used micro lithography techniques to built a ship made of 5 mm diameter reservoirs linked together by a 5 µm wide channel. A drop of liquid containing the bacteria is transfered in one of the reservoirs. A difference in the liquide height and/or some voltage applied between the reservoirs allows to make the bacteria pass from one reservoir to the other, one by one, in front of the objective of an inverted microscope. The number of plasmids is measured indirectly. We built plasmids which all carry a gene coding for a green fluorescent protein, and when a bacteriun passes by we measure the fluorescence of the cell. Our experiment is based on the assumption that the level of expression is the same for each copy of the gene: the fluorescence of the cell is proportionnal to the number of plasmids. This thesis describes the fluorescence microscopy experiment and the construction of the bacterial strains. The preliminary results that we obtained show that our experimental setup works. (In one experiment we measure the fluorescence of thousands of bacteria: The signal is far above the noise and the results are reproducible

La ségrégation de l'ADN, appelée partition chez les procaryotes, permet à tout organisme de transmettre son patrimoine génétique au cours des générations. Il existe des systèmes actifs de partition présents sur la majorité des plasmides et des chromosomes bactériens. Ces systèmes sont essentiels pour la ségrégation active des plasmides à bas nombre de copies tel que le plasmide F d'Escherichia coli, étudié au sein de notre équipe. Son système de partition, appelé sop, est composé de deux gènes, sopA et sopB et d'une séquence centromérique sopC. SopB se fixe à sopC pour former le complexe de partition, reconnu par l'ATPase SopA. SopA polymérise en présence d'ATP. Ce comportement pourrait être le " moteur " de la ségrégation des réplicons. Les travaux de notre équipe ont démontré que SopA se fixe de manière ATP-dépendante à l'ADN non spécifique. Cette fixation inhibe la polymérisation de SopA. SopB, de par sa capacité à se fixer à l'ADN non spécifique, contrebalance ainsi cette inhibition. Nous proposons un modèle dans lequel la polymérisation de SopA serait régulée dans la cellule par l'ADN du nucléoïde. En présence du plasmide, SopB, présent à forte concentration autour du complexe de partition, masquerait l'ADN, créant un environnement dans lequel SopA initierait sa polymérisation. Cette régulation de la dynamique de SopA serait nécessaire au processus de partition. Afin d'étayer notre modèle, nous avons recherché un domaine d'interaction à l'ADN dans SopA. Nous avons identifié un mutant ayant perdu sa fixation ATP-dépendante à l'ADN. Seules les activités de SopA dépendante de cette reconnaissance de l'ADN ont été affectées : l'inhibition de la polymérisation, la stimulation de l'activité ATPase basale et la localisation intracellulaire. Cette mutation entraîne aussi une perte majeure de stabilité du plasmide F correspondant. Ceci confirme l'implication de l'ADN du nucléoïde dans la régulation du comportement dynamique de SopA nécessaire à la partition du plasmide F<br>The segregation of the DNA, also called partition for procaryotes, is the process allowing any organisms to transmit its genetic heritage to next generation. In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems which comprise a centromere, a centromere-binding protein and an ATPase. We have taken as a model, the low-copy number plasmid F of Escherichia coli. Centromere-binding protein SopB binds to sopC centromere and forms the partition complex. This nucleoproteic complex is recognized by the SopA "Walker-box" ATPase. SopA shares with other partition ATPase the capacity of self assembly in presence of ATP. This dynamic self-assembly would allow active partition during bacterial division. Previous work in our team showed SopA is also able to bind to non specific DNA in an ATP-dependant manner whereby polymerization is inhibited. Indeed, DNA inhibited this polymerization and cause breakdown of pre-formed polymers. SopB counteracted this DNA effect by binding itself to and masking DNA. We had proposed a model in which the polymerization is spacially regulated. Nucleoid DNA prevent inappropriate SopA polymerization but when SopB is present in high concentration, it create a DNA-depleted zone within SopA can initiate polymerization. The regulation of the dynamic behaviour of the "driving" protein of the system would be necessary for the process of partition. To support our model, we looked for a DNA binding domain in SopA. We have found a SopA mutant, defective for ATP dependent DNA binding. Only the activities of SopA dependent on this binding were affected: the inhibition of the polymerisation is abolished, as the stimulation of the ATPase activity and the intracellular localization. Moreover, this mutant is defective for plasmid stabilization. This last result confirms the implication of the nucleoïd DNA in regulation of the dynamic behavior of SopA, which is necessary for the partition of the plasmide F

Introdução: As enzimas do grupo AmpC degradam cefalosporinas e cefamicinas e não são inibidas pelo ácido clavulânico, sulbactam ou tazobactam. Podem ser codificadas por genes de localização plasmidial ou cromossômica. A sua detecção tem importância epidemiológica, pois a expressão concomitante à perda de porinas em Enterobactérias pode levar à resistência aos carbapenêmicos, fármacos amplamente utilizados no tratamento de infecções graves. Os relatos de detecção de AmpC plasmidial no Brasil são escassos, mas a análise do perfil de sensibilidade de Escherichia coli isoladas de casos de infecções urinárias de pacientes atendidos em um laboratório privado da cidade de São Paulo, no período de 2006 à 2010, evidenciou um aumento considerável de resistência à cefoxitina, um marcador para expressão de AmpC plasmidial. Em 2006 a frequência era de 0,03% e em 2011 foi de 0,65%. Uma das hipóteses para esse aumento é a disseminação de clones ou de genes que codificam AmpC, transferidos em plasmídeos. Objetivo: Caracterizar os determinantes genéticos em E. coli com fenótipo compatível com produção de AmpC plasmidial. Material e métodos: Foram estudadas todas as E. coli isoladas de cultura de urina, não sensíveis à cefoxitina, detectadas no Fleury Medicina e Saúde em São Paulo, no período de 02 de janeiro a 01 de fevereiro de 2012. Os isolados foram avaliados fenotipicamente quanto à pureza, identificação da espécie e bloqueio enzimático com discos de cefoxitina e ceftazidima adicionados de ácido fenilborônico. A presença de genes que codificam AmpCs plasmidiais foi avaliada utilizando-se dois métodos distintos de PCR. Foi realizado o sequenciamento dos genes detectados. O desempenho do bloqueio enzimático com ácido fenilborônico foi determinado utilizando-se a PCR como padrão ouro. O perfil plasmidial e a clonalidade foram avaliados respectivamente por lise alcalina e ERIC-PCR. Resultados e conclusões: De um total de 2.494 E. coli 12 albergavam genes de AmpC plasmidial; a frequência de AmpC plasmidial foi portanto de 1,8% em pacientes hospitalizados 0,46% em pacientes ambulatoriais. O sequenciamento dos genes que codificam AmpCs plasmidiais evidenciou tratarem-se de 11 blaCMY-2 e uma blaCMY-4. O melhor desempenho do teste fenotípico com cefoxitina, ceftazidima e ácido fenilborônico foi obtido quando utilizados um mínimo de 5 mm de diferença entre o halo de inibição com o disco adicionado de ácido fenilborônico e puro para cefoxitina e ceftazidima. Com esses parâmetros a sensibilidade foi de 100,0%, a especificidade de 100%, o valor preditivo positivo 100 % e valor preditivo negativo 100%. Foi observada a presença e disseminação de três grupos clonais de E. coli produtoras de CMY entre hospitais na cidade de São Paulo.<br>Introduction: AmpC enzymes can degrade cephalosporins and cephamycins and are not inhibited by clavulanic acid, sulbactam or tazobactam. They can be encoded by genes of plasmid or chromosomal location. Detecting these enzymes is epidemiologically important because their expression and concomitant porins loss in Enterobacteriaceae can lead to resistance to carbapenems, antimicrobials widely used to treat serious infections. Reports of detection of plasmidial AmpCs in Brazil are scarce, but the analysis of the susceptibility profile of Escherichia coli isolated from cases of urinary tract infection in patients attended in a private laboratory, located at the city of São Paulo, from 2006 to 2010 indicated a considerable increase in cefoxitin resistance, a marker for plasmidial AmpC expression. In 2006 the rate was 0.03% and in 2011 it was 0.65%. One hypothesis for this increase was the spread of clones or genes encoding AmpC transferred into plasmids. Objective: To characterize the genetic determinants in E. coli presenting a phenotype consistent with plasmidial AmpC production. Material and Methods: We studied all E. coli not susceptible to cefoxitin isolated in urine cultures at Fleury Medicine and Health, in São Paulo, during the period from January 2nd to February 1st 2012. The isolates were phenotypically evaluated for purity, species identification and enzymatic blockage with cefoxitin and ceftazidime disks with added phenylboronic acid. The presence of genes encoding plasmidial AmpCs was evaluated using two different PCR methods. Sequencing of the genes detected was obtained. The performance of the enzymatic blockage with phenylboronic acid was determined using PCR as a gold standard. The plasmid profile and clonality were evaluated respectively by alkaline lysis and ERIC-PCR. Results and conclusions: Among 2,494 E. coli isolates, 12 had genes encoding for plasmidial AmpC; consequently the frequency of plasmidial AmpC in E. coli was 1.8% in inpatients and 0.46% in outpatients. Sequencing of the genes encoding plasmidial AmpCs showed them to be blaCMY-2 and just one blaCMY-4. The better performance of the phenotypic test was obtained when at least 5 mm inhibition zone difference was observed between the disc of cefoxitin and ceftazidime with added phenylboronic acid. With these parameters the sensitivity was 100%, specificity 100%, positive predictive value 100% and negative predictive value 100%. The presence and the dissemination of three clonal groups of CMY producing E. coli was observed among hospitals located at the city of São Paulo