Recherche d'un système d'efflux multidrogue chez Aeromonas hydrophila (impact sur la résistance aux antibiotiques)

Au cours de la dernière décennie, Aeromonas hydrophila s'est révélée être un pathogène opportuniste chez l'homme. Chez les bactéries à Gram négatif, les systèmes d'efflux contribuent de manière importante aux résistances multidrogues. Ce travail rapporte pour la première fois une analyse fonctionnelle d'un système d'efflux multidrug resistant (MDR) de type resistance-nodulation-cell division (RND) chez le genre Aeromonas. L'implication du système d'efflux AheABC dans la résistance intrinsèque MDR à 13 substrats antibiotiques et autres a été montrée par des expériences d'inactivation/complémentation chez la souche ATCC 7966T d'Aeromonas hydrophila. Possédant un spectre plus étroit que son homologue AcrAB d'E. coli, ce système n'efflue pas les quinolones et fluoroquinolones. La protéine AheR interagit avec la séquence promotrice d'aheABC et serait impliquée dans la régulation spécifique. De plus, un motif "marbox" présent dans cette région promotrice et un homologue de l'activateur global Rob d'E. coli ont été identifiés suggérant aussi un niveau de régulation global. Par ailleurs, la présence d'autre(s) pompe(s) d'efflux chez cette espèce a été confirmée par l'utilisation du PAbN (Phe-Arg-b-naphtilamide), inhibiteur de pompes RND. L'analyse de l'activité du PAbN sur 10 souches d'Aeromonas provenant de l'environnement ou d'origine clinique montre un rôle du système AheABC et d'autres pompes d'efflux dans la résistance de ces souches. La résistance aux quinolones et fluoroquinolones pour certaines de ces souches associe l'efflux actif à d'autres mécanismes de résistances comme des mutations dans la Quinolone Resistance Determining Region des gènes gyrA et parC.During the last decade, Aeromonas hydrophila has been shown to be an opportunistic pathogen. In Gram-negative bacteria, the efflux pumps play a major role in the development of the multi-drug resistance phenotype (MDR). This work demonstrated for the first time the funtional analysis of an efflux system of the resistance-nodulation-cell division (RND) type in the genus Aeromonas Inactivation and complementation experiments have shown that AheABC efflux mechanism is responsible for the intrinsic resistance to 13 antimicrobials in the 7966T reference strain. It is noteworthy that the system has nevertheless a narrower spectrum than its homologous in E. coli as it doesn't include the quinolones antibiotics. The AheR protein interacts with the promoter sequence of the AheABC operon and would be involved in the specific regulation. Furthermore, the finding of a "marbox" motif in this region and an E. coli Rb-like global activator together implies a global regulation. In addition, the presence of other various pumps in this species has been confirmed by the use of PAbN (Phe-Arg-bnaphthilamide) which is an inhibitor of efflux pumps. Analysis of the activity of PABN on 10 Aeromonas strains from environmental and clinical sources has shown that the AheABC syqtem and other pumps play an important role in the resistance phenotype exhibited by these strains. Regarding the genetic basis of the mechanisms of resistance to quinolones in these strains we have shown that in addition to efflux pumps activity, mutations in the quinolone resistance determining region (QRDR) are also involved in the overall resistant phenotype.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Role of the AheABC Efflux Pump in Aeromonas hydrophila Intrinsic Multidrug Resistance▿

Gene inactivation and complementation experiments showed that the tripartite AheABC efflux pump of Aeromonas hydrophila extruded at least 13 substrates, including nine antibiotics. The use of phenylalanine-arginine-β-naphthylamide (PAβN) revealed an additional system(s) contributing to intrinsic resistance. This is the first analysis of the role of multidrug efflux systems in Aeromonas spp

Profiling of Campylobacter jejuni Proteome in Exponential and Stationary Phase of Growth

Campylobacter jejuni has been reported as a major cause of bacterial food-borne enteritides in developed countries during the last decade. Despite its fastidious growth requirements, including low level of oxygen and high level of CO2, this pathogen is able to persist in the environment without permanent loss of its viability and virulence. As C. jejuni is not able to multiply outside a host, the cells spend significant amount of time in stationary phase of growth. The entry into the stationary phase is often correlated to resistance to various stresses in bacteria. The switching between exponential and stationary phases is frequently mediated by the regulator sigma S (RpoS). However, this factor is absent in C. jejuni and molecular mechanisms responsible for transition of cells to the stationary phase remain elusive. In this work, proteomic profiles of cells from exponential and stationary phases were compared using 2-D electrophoresis (2DE) fingerprinting combined with mass spectrometry analysis and qRT-PCR. The identified proteins, whose expression differed between the two phases, are mostly involved in protein biosynthesis, carbon metabolism, stress response and motility. Altered expression was observed also in the pleiotropic regulator CosR that was over-expressed during stationary phase. A shift between transcript and protein level evolution of CosR throughout the growth of C. jejuni was observed using qRT-PCR and (2DE). From these data, we hypothesized that CosR could undergo a negative autoregulation in stationary phase. A consensus sequence resulting from promoter sequence alignment of genes potentially regulated by CosR, including its own upstream region, among C. jejuni strains is proposed. To verify experimentally the potential autoregulation of CosR at the DNA level, electrophoretic mobility shift assay was performed with DNA fragments of CosR promoter region and rCosR. Different migration pattern of the promoter fragments indicates the binding capacity of CosR, suggesting its auto-regulation potential

Influence of food processing environments on structure initiation of static biofilm of Listeria monocytogenes

International audienceDeciphering the initial steps and the initial structure of biofilm formation is important to understand their effects on the subsequent formation of mature listerial biofilms on food-processing equipment as well as on remedial options to control their proliferation. In this research the achievement of an initial 2D biofilm structure, observed using fluorescence microscopy, could be divided into four steps: (i) cell cluster formation with attached cells side-by-side; (ii) branch elongation by cell division; (iii) open geometric forms connected through branches and (iv) honeycomb-like structure. Among the four test strains able to reach a honeycomb-like structure, there was one (EGD-e) which could not enter to the second step and subsequently could not develop complex biofilms. Only time-shift differences according to growth medium and surface were observed except for EGD-e when incubated on plastic surface. Interestingly, the first step of the primary structure of Listeria monocytogenes was impaired for all strains in acidic conditions. The biofilm primary structure is a critical stage for the development of listerial biofilms with elaborated structure. Some environmental conditions such as acidic pH could impair the structure of the biofilm and prevent complex biofilm formation

Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions

Campylobacter jejuni is responsible for the major foodborne bacterial enteritis in humans. In contradiction with its fastidious growth requirements, this microaerobic pathogen can survive in aerobic food environments, suggesting that it must employ a variety of protection mechanisms to resist oxidative stress. For the first time, C. jejuni 81-176 inner and outer membrane subproteomes were analyzed separately using two-dimensional protein electrophoresis (2-DE) of oxygen-acclimated cells and microaerobically grown cells. LC-MS/MS analyses successfully identified 42 and 25 spots which exhibited a significantly altered abundance in the IMP-enriched fraction and in the OMP-enriched fraction, respectively, in response to oxidative conditions. These spots corresponded to 38 membrane proteins that could be grouped into different functional classes: (i) transporters, (ii) chaperones, (iii) fatty acid metabolism, (iv) adhesion/virulence and (v) other metabolisms. Some of these proteins were up-regulated at the transcriptional level in oxygen-acclimated cells as confirmed by qRT-PCR. Downstream analyses revealed that adhesion of C. jejuni to inert surfaces and swarming motility were enhanced in oxygen-acclimated cells or paraquat-stressed cells, which could be explained by the higher abundance of membrane proteins involved in adhesion and biofilm formation. The virulence factor CadF, over-expressed in the outer membrane of oxygen-acclimated cells, contributes to the complex process of C. jejuni adhesion to inert surfaces as revealed by a reduction in the capability of C. jejuni 81-176 DCadF cells compared to the isogenic strain. Taken together, these data demonstrate that oxygen-enriched conditions promote the over-expression of membrane proteins involved in both the biofilm initiation and virulence of C. jejuni

Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain

Background: Campylobacter jejuni is a leading cause of bacterial enteritis worldwide. This microaerophilic bacterium can survive in aerobic environments, suggesting it has protective mechanisms against oxidative stress. The clinical C. jejuni Bf strain is characterized by an increased resistance to oxygen. This study aimed to characterize the behavior of the clinical C. jejuni Bf strain under an aerobic atmosphere and in response to ROS promoter agents. Methods: Growth was studied in both aerobic and microaerobic conditions using classic cultivable methods. Electronic microscopy and mreB gene expression were used to evaluate the morphology of this strain under aerobic conditions. The survival under oxidative stress was tested in the presence of different concentrations of hydrogen peroxide (H2O2) and paraquat (PQ). Results: The results showed that C. jejuni Bf strain can grow aerobically, unlike other strains of C. jejuni tested. Cells of C. jejuni Bf exposed to oxidative stress presented changes in morphology and the gene mreB, responsible for maintaining the bacillary cell morphology, was down‑expressed. In aerobically acclimated conditions, C. jejuni Bf exhibited a higher survival rate of 52% in the presence of H2O2 (1mM) compared to the reference strain NCTC 11168. Concentrations above 1mM PQ were lethal for the reference strain but not for C. jejuni Bf. Conclusions: Taken together, these data highlight the resistance to oxidative stress conditions of C. jejuni Bf, indicating that this microorganism seems more adapted to survival in hostile environmental conditions

PFRU, a single dominant locus regulates the balance between sexual and asexual plant reproduction in cultivated strawberry.

Strawberry (Fragaria sp.) stands as an interesting model for studying flowering behaviour and its relationship with asexual plant reproduction in polycarpic perennial plants. Strawberry produces both inflorescences and stolons (also called runners), which are lateral stems growing at the soil surface and producing new clone plants. In this study, the flowering and runnering behaviour of two cultivated octoploid strawberry (Fragaria×ananassa Duch., 2n=8×=56) genotypes, a seasonal flowering genotype CF1116 and a perpetual flowering genotype Capitola, were studied along the growing season. The genetic bases of the perpetual flowering and runnering traits were investigated further using a pseudo full-sibling F1 population issued from a cross between these two genotypes. The results showed that a single major quantitative trait locus (QTL) named FaPFRU controlled both traits in the cultivated octoploid strawberry. This locus was not orthologous to the loci affecting perpetual flowering (SFL) and runnering (R) in Fragaria vesca, therefore suggesting different genetic control of perpetual flowering and runnering in the diploid and octoploid Fragaria spp. Furthermore, the FaPFRU QTL displayed opposite effects on flowering (positive effect) and on runnering (negative effect), indicating that both traits share common physiological control. These results suggest that this locus plays a major role in strawberry plant fitness by controlling the balance between sexual and asexual plant reproduction

Elucidating the functional role of endoreduplication in tomato fruit development

International audienceBackgroundEndoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague,although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination.ScopeHere recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described.ConclusionsThe cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits