Survey of the gram negaive bacterial pollution in some areas of Anzali Lagoon

For this survey, eight stations were selected in the Anzali Lagoon based on vicinity to the urban area, agricultural fields, in lets and out lets of the rivers. Sampling was carried out seasonally and in each season triplicate water samples were taken from surface layer of each station. Totally 96 water samples were collected during one year period in sterile condition.Samples were transferred to the laboratoray at 4ºC temperature using ice box. The pollution by Coliform was examind using standard technigue of MPN and for count of bacteria differential, specific test and pourplate method isolate was used. In this survey different genus of bacteria were isolated and identified as: E.Coli (%19.65), Shigella (%18.21), Klebsiella (%17.86), Proteus (%13.21), Enterobacter (%11.07), Morganella (%9.65), Salmonella (%3.92), Serratia (%2.5), Providencia (%2.14) and Citrobacter (%1.79) which belonged to family Enterobacteriacea. Also the most important isolated genus of Vibrionacea were Vibrio (%47.55), Aeromonas (%28.67) & Plesiomonas (%4.9) and nonfermented-bacilli containg Suedomonace (%18.88). There was significant differences in the bacterial pollution between stations (P<0.05). The highest Coliform count was recorded during summer amounting to (147.71±171.36) ind. 100 cm3 and also the highest Fecal Coliform count was observed in summer (135.125±173.19) ind. 100 cm3 in the water. Generally the pollution in summer was higher in comparison to rest of the year due to increasing temperature and decreasing water flow of rivers.Higher bacterial pollution during autum in comparison to winter was attributed to heavay rain, erosion of the soil, flooding of the rivers and run off of the waste waters. Pirbazar River, Psikhan and Shambe Bazaar Roga drained high volume of urban agricultural and animal waste waters.The western area of the lagoon which is the bigest part of the Anzali lagoon had the least pollution

A new theta-type thermosensitive replicon from Lactoccocus lactis as an integration vector for Enterococcus faecalis

We isolated a replication thermosensitive mutant of the theta-type lactococcal pUCL22 replicon. An improved version of this thermosensitive replicon was obtained by fusioning the replication repA gene with the downstream repB gene. The resulting plasmid was named pUCB3522Ts. It is highly instable at 42°C in Enterococcus faecalis. Integration into the chromosome via homologous recombination was monitored using the npr gene of E. faecalis JH2-2 as a target. A 513 bp PCR amplification product from an internal region of this npr gene was cloned into pUCB3522Ts. Integration of this construction into the JH2-2 npr gene was selected by shift temperature, from 30°C to 42°C. 85% of the analysed clones showed integration into the npr gene, demonstrating the practicality of this thermosensitive replicon as a genetic integrative tool for E. faecali

The Extracytoplasmic Function Sigma Factor SigV Plays a Key Role in the Original Model of Lysozyme Resistance and Virulence of Enterococcus faecalis

Background: Enterococcus faecalis is one of the leading agents of nosocomial infections. To cause diseases, pathogens or opportunistic bacteria have to adapt and survive to the defense systems encountered in the host. One of the most important compounds of the host innate defense response against invading microorganisms is lysozyme. It is found in a wide variety of body fluids, as well as in cells of the innate immune system. Lysozyme could act either as a muramidase and/ or as a cationic antimicrobial peptide. Like Staphylococcus aureus, E. faecalis is one of the few bacteria that are completely lysozyme resistant. Results: This study revealed that oatA (O-acetyl transferase) and dlt (D-Alanylation of lipoteicoic acids) genes contribute only partly to the lysozyme resistance of E. faecalis and that a specific transcriptional regulator, the extracytoplasmic function SigV sigma factor plays a key role in this event. Indeed, the sigV single mutant is as sensitive as the oatA/dltA double mutant, and the sigV/oatA/dltA triple mutant displays the highest level of lysozyme sensitivity suggesting synergistic effects of these genes. In S. aureus, mutation of both oatA and dlt genes abolishes completely the lysozyme resistance, whereas this is not the case in E. faecalis. Interestingly SigV does not control neither oatA nor dlt genes. Moreover, the sigV mutants clearly showed a reduced capacity to colonize host tissues, as they are significantly less recovered than the parental JH2-2 strain from organs of mice subjected to intravenous or urinary tract infections

Automodification de l'énolase par le 2-phosphoglycérate et son rôle potentiel

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

INCIDENCE DE STRESS PHYSICO-CHIMIQUES LIES AU PROCESSUS DE FABRICATION DU SAUMON FUME SUR UNE BACTERIE D'ALTERATION SHEWANELLA PUTREFACIENS

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Caractérisation de la réponse au stress acide chez Lactococcus lactis

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Identification et caractérisation des gènes induits in vivo chez Enterococcus faecalis

Enterocoocus faecalis fait partie du microbiote commensal humain, son habitat principal étant le tractus gastro-intestinal. S il est inoffensif pour des individus en bonne santé, E. faecalis a récemment émergé en tant que cause majeure d infections nosocomiales. Dans le but de mieux comprendre le passage de la bactérie d un état commensal à celui de pathogène, nous avons développé une approche de type R-IVET (Recombination-based in vivo expression technology) pour ce microorganisme. Deux systèmes R-IVET avec différents niveaux de sensibilité ont été construits dans une souche dérivée d E. faecalis V583 et testés chez l insecte Galleria mellonella, dans des modèles murins de septicémie ou de péritonite ainsi que lors d une croissance en urine. L ensemble des résultats a conduit à l identification de 79 gènes dont l expression est activée in vivo. Parmi ceux-ci, l opéron ef_3197/6 a été montré comme étant fortement induit chez l insecte. La délétion de cette structure opéronique a démontré que le système à deux composants codé par ces gènes est essentiel au potentiel pathogène d E. faecalis chez G. mellonella. Le gène ef_0377, induit à la fois dans les modèles insecte et murins a également été étudié plus en détail, ce qui a permis de montrer que ce gène codant une protéine ankyrine était également impliqué dans la virulence. Il en est de même pour le gène ef_3282, codant la sous-unité de liaison à l ATP ClpC du complexe protéolytique Clp. Ainsi, les différents criblages R-IVET ont permis la mise en évidence de nouveaux facteurs d E. faecalis impliqués dans la persistance in vivo et le potentiel de virulence de ce pathogène opportuniste.Enterococcus faecalis is part of the commensal microbiota of humans and its main habitat is the gastrointestinal tract. Although harmless in healthy individuals, E. faecalis has recently emerged as a major cause of nosocomial infections. In order to better understand the transformation of a harmless commensal into a life-threatening pathogen, we developed a R-IVET approach (Recombination-based in vivo expression technology) for this microorganism. Two R-IVET systems with different levels of sensitivity have been constructed in a E. faecalis V583 derivative strain and tested in the insect model Galleria mellonella, in mouse bacteremia and peritonitis models and during growth in urine. Our combined results led to the identification of 79 in vivo activated genes. Among them, the ef_3197/6 operon was shown to be strongly induced in the insect host model. Deletion of this operonic structure demonstrated that this two-component system encoded by these genes was essential to the E. faecalis pathogenic potential in G. mellonella. Gene ef_0377, induced in both insect and mammalian models, has also been further analyzed and it has been demonstrated that this ankyrin-encoding gene was also involved in E. faecalis virulence. Gene ef_3282, encoding the ATP-binding subunit ClpC from the Clp proteolytic complexe, has also been demonstrated to be involved in the pathogenic potential of this organism. Thus these different R-IVET screenings led to the identification of new E. faecalis factors implied in in vivo persistence and pathogenic potential of this opportunistic pathogen.CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Étude de la résistance au lysozyme chez Enterococcus faecalis

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Construction of Plasmid Vectors for Screening Replicons from Gram-Positive Bacteria and Their Use as Shuttle Cloning Vectors

International audiencePlasmids play a central role in engineering recombinant bacteria because they are the primary vehicles used to manipulate targeted sequences. In some cases, bacteria of interest are poorly provided with suitable tools for these molecular or genetic manipulations. In this context, we constructed from two shuttle cloning vectors, pUCB2871 and pUCB2872, the basic vectors pUCB30 and pUCB31, which could represent suitable tools to isolate replicons from Gram-positive bacteria. These plasmid vectors are characterized by the following after-features: (a) the pUC origin of replication is unable to replicate in Gram-positive bacteria; (b) an erythromycin-resistance encoding gene that is functional in both Gram-negative and -positive bacteria; (c) the pUC19 multiple cloning site (MCS) within the lacZα reporter gene; and (4) an additional multiple cloning site (MCS). Cloning replicons from Gram-positive bacteria in this additional MCS would allow the derivative vectors to function directly as shuttle cloning vectors

Analyse moléculaire de l'adaptation à son environnement d'Enterococcus faecalis (caractérisation du système à deux composants Err05-Ehk05 impliqué dans la régulation du gène sagA codant d'une protéine générale de stress extracellulaire)

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF