Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was con-ducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhe-sion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27kDa proteins. Indeed, adhesion to Caco-2 cell monoiayers of C. difficiie strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficiie may utilize blood components as adhesins to adhere to human intestinal cultured cells.     

The electrophoretic polymorphism of bacterial esterases

Abstract: The specific activities of esterases and certain other molecular properties including immunospecificity indicate that the electrophoretic variations of these enzymes in bacterial populations are the result of allelic variations at specific gene loci. The esterase polymorphism of Enterobacteriaceae and some other species isolated from man or animals demonstrates that esterases can distinguish between bacteria at the species or subspecies level, both by their biochemical properties and by their electrophoretic differences. The esterase data complement DNA hybridization studies and agree with ribosomal DNA polymorphism, especially for delineating a phylogenetically distinct group of highly pathogenic strains in Escherichia coli . A two-dimensional electrophoretic profile obtained by establishing a direct correspondence between homologous esterase bands resolved by independent runs of isoelectric focusing and standard electrophoresis considerably improves the detection of allelic variations, whereas protein titration curves (electrophoresis in pH gradient) can be used to demonstrate the real electrophoretic homogeneity of allozymes or evalue their molecular relationship in terms of apparent amino acid substitutions. This overview establishes that esterases, by their significant electrophoretic polymorphism, are reliable molecular markers for systematics and epidemiology, and are suitable enzyme systems for studying population genetics and phylogeny.     

Seasonal incidence and ecology of the tick Ixodes ricinus (Acari: Ixodidae) on grazing pastures in Western France

A longitudinal survey was carried out during a 2 year period in Western France to assess the infestation level of grazing pastures byIxodes ricinus ticks. Four farms were visited once a month and each of the grazing pastures was sampled in the centre and at the border using the blanket dragging method. A total of 3562I. ricinus (34 adults, 900 nymphs and 2628 larvae) were collected and the infestation was significantly higher during the first year (p<0.0001). The infestation level byI. ricinus varied between grazing pastures and farms. Grazing pastures in the vicinity of forest were more infested than the others, all through the study. The seasonal distribution of ticks showed peaks, with low fluctuations between farms, years and stages. Tick abundance could not be related to vegetation, but only to the vicinity of woods.     

The effect of temperature on eicosane substrate uptake modes by a marine bacterium Pseudomonas nautica strain 617: relationship with the biochemical content of cells and supernatants

Three hydrocarbon uptake modes (adherence, emulsification and solubilization) were identified and quantified in cells and supernatants of a mesophilic marine bacterium Pseudomonas nautica strain 617 grown on eicosane. The adherence capacity was related to the enrichment of cells with wax esters and glycolipids. The emulsifying activity was related to the presence of extracellular biosurfactants composed of proteins, carbohydrates and lipids (35:63:2). The intensity of substrate uptake modes was sensitive to temperatures currently found in the original environment of P. nautica (16°C, 20°C and 32°C). When temperature decreased, a significant increase in adherence and emulsifying activity was observed in relation to biochemical changes, whereas solubilizing activity decreased. The marine bacterium was able to degrade 53–59% eicosane at the end of exponential growth after 13, 5 and 3 days incubation at 16°C, 20°C and 32°C respectively.     

Sexual tetramorphism in Thymelaea hirsuta (Thymelaeaceae): morph ratios in open-pollinated progeny

Natural populations of Thymelaea hirsuta have previously been shown to comprise four distinct sexual morphs: males, females, protogynous individuals, i.e., first female then male, and protandrous individuals, i.e., first male then female. The objective of the present study has been to confirm the genetic basis of this sexual tetramorphism by quantifying morph ratios in the open-pollinated progeny of the four sexual phenotypes growing in a natural population. All four phenotypes were recovered in the progeny of each morph. All observed plants displayed a single sexual phenotype, thus confirming the genetic basis of the tetramorphism. The progeny sex ratios indicate that the genetic determination of sex in this species may be influenced by cytoplasmic factors, while the observed levels of functional female fertility suggest a near-dioecious system. The evolutionary significance of this tetramorphism as a transitional stage in the evolution of dioecy is discussed.     

Patchiness and disturbance: plant community responses to porcupine diggings in the central Negev

We tested the hypothesis that diversity and productivity of herbaceous plant communities in disturbed soil are related to the physical and biological heterogeneity of the landscape Our study was earned out on vegetation responses in porcupine diggings on a rocky slope in the central Negev desert in Israel We measured aboveground bio-mass and plant density per species in 150 porcupine diggings (15 cm deep and 15 to 20 cm wide) and in equally sized adjacent control samples in the undisturbed soil matrix We calculated mean annual biomass production, plant density and species richness for 10 sample areas along the slope In addition, we divided the plants into groups according to propagule size and dispersal mode We denoted two types of landscape heterogeneity, which we called physical and biological patchiness Physical patchiness was measured as the ratio of bare rock to soil surface Biological patchiness was the area of the soil covered by shrubs with associated soil mound and under-story relative to the total soil surface We also measured disturbance density, as the long term (17 yr) average density of newly made porcupine diggings We found that 1) the physical patchiness explained 30% of the variation of biological patchiness along the slope, while 2) the patterns of disturbance intensity and biological patchiness were similar (R-=0 386) 3) Biomass, density and species richness were significantly higher in diggings than m the soil matrix 4) Plant density in the matrix, but not m the diggings, was significantly correlated with physical patchiness, 5) species richness in diggings was significantly correlated with biological patchiness, but 6) biomass production in diggings and matrix was not affected by either physical or biological patchiness of the landscape 7) Disturbance density did not affect vegetation responses in diggings and matrix 8) A shift in the plant communities in the matrix towards plants with smaller seeds was associated with increasing physical patchiness, while m diggings there was an opposite shift 9) The proportion of wind dispersers was higher in diggings than outside, while the proportion of runoff dispersers was lower, 10) the densities of runoff dispersers in diggings and matrix were positively correlated with physical and biological patchiness 11) Physical and biological patchiness formed the two major gradients of species composition, explaining 30 and 25% respectively We conclude that the network of physical and biological patchiness and soil disturbance are important in the redistribution of resources and seeds, which control plant biomass, density, species richness and diversity The bare rock surface is the main source for runoff flow with associated soil, organic matter and nutrients The understory vegetation of shrubs provides seeds for creating and maintaining diversity The soil matrix absorbs runoff flow, and disturbances absorb runoff and trap seeds Thus, differences in landscape heterogeneity and their effects on resource and seed movement interact in controlling plant community productivity and diversity in the landscape     

Osteodentine and vascular osteodentine of Anarhichas lupus (L.)

Summary TEM, SEM and X-ray diffraction analysis demonstrate the heterogeneity of the dentinal tissue of Anarhichas lupus, a vascular osteodentine. The disordered aspect of collagen fibres, incompletely mineralized (the periodical striation being still visible), explains the scattered distribution of crystallites since they are responsible for their arrangement. The low degree of mineralization revealed by the visible collagen striation is confirmed by X-ray diffraction analysis (the crystallinity of vascular osteodentine being much lower than that of the peripheral dental tissue) as well as by high resolution TEM, since no lattice planes could be observed. Osteodentine, supporting bone and proper bone have in common a mineral phase, more or less organized, different from the apatite system.The authors thank Mireille Cottrel-Gengoux for her technical assistance