Effect of growth time on the surface and adhesion properties of Lactobacillus rhamnosus GG

Aims: To investigate the changes in the surface properties of Lactobacillus rhamnosus GG during growth, and relate them with the ability of the Lactobacillus cells to adhere to Caco-2 cells. Methods and Results: Lactobacillus rhamnosus GG was grown in complex medium, and cell samples taken at four time points and freeze dried. Untreated and trypsin treated freeze dried samples were analysed for their composition using SDS-PAGE analysis and Fourier transform infrared spectroscopy (FTIR), hydrophobicity and zeta potential, and for their ability to adhere to Caco-2 cells. The results suggested that in the case of early exponential phase samples (4 and 8 h), the net surface properties, i.e. hydrophobicity and charge, were determined to a large extent by anionic hydrophilic components, whereas in the case of stationary phase samples (13 and 26 h), hydrophobic proteins seemed to play the biggest role. Considerable differences were also observed between the ability of the different samples to adhere to Caco-2 cells; maximum adhesion was observed for the early stationary phase sample (13 h). The results suggested that the adhesion to Caco-2 cells was influenced by both proteins and non-proteinaceous compounds present on the surface of the Lactobacillus cells. Conclusion: The surface properties of Lact. rhamnosus GG changed during growth, which in return affected the ability of the Lactobacillus cells to adhere to Caco-2 cells. Significance and Impact of the Study: The levels of adhesion of Lactobacillus cells to Caco-2 cells were influenced by the growth time and reflected changes on the bacterial surface. This study provides critical information on the physicochemical factors that influence bacterial adhesion to intestinal cells.

Effect of food models and low-temperature storage on the adhesion of Lactobacillus rhamnosus GG to Caco-2 cells

This study evaluated the effects of fat and sugar levels on the surface properties of Lactobacillus rhamnosus GG during storage in food model systems, simulating yogurt and ice cream, and related them with the ability of the bacterial cells to adhere to Caco-2 cells. Freeze-dried L. rhamnosus GG cells were added to the model food systems and stored for 7 days. The bacterial cells were analyzed for cell viability, hydrophobicity, ζ potential, and their ability to adhere to Caco-2 cells. The results indicated that the food type and its composition affected the surface and adhesion properties of the bacterial cells during storage, with yogurt being a better delivery vehicle than ice cream in terms of bacterial adhesion to Caco-2 cells. The most important factor influencing bacterial adhesion was the storage time rather than the levels of fats and sugars, indicating that conformational changes were taking place on the surface of the bacterial cells during storage.

Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro

Intimin and EspA proteins are virulence factors expressed by attaching and effacing Escherichia coli (AEEC) such as enteropathogenic and enterohaemorrhagic E. coli. The EspA protein makes up a filament structure forming part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Bacterial surface displayed intimin interacts with translocated intimin receptor in the host cell membrane leading to intimate attachment of the bacterium and subsequent attaching and effacing lesions. Here, we have assessed the use of recombinant monoclonal antibodies against E. coli O157:147 EspA and intimin for the disruption of AEEC interaction with the host cell. Anti-gamma intimin antibodies did not reduce either adhesion of E. coli O157:H7 to host cell mono-layers or subsequent host cell actin rearrangement. Anti-EspA antibodies similarly had no effect on bacterial adhesion however they had a marked effect upon E. coli O157:H7-induced host cell actin rearrangement, where both monoclonal and polyclonal antibodies completely blocked cytoskeletal changes within the host cell. Furthermore, these anti-EspA antibodies were shown to reduce actin rearrangement induced by some but not all other AEEC serotypes tested. Both polyclonal and monoclonal antibodies could be used to label E. coli O157 EspA filaments and these immunoreagents did not inhibit the formation of such filaments. This is the first report of monoclonal antibodies to EspA capable of disrupting the TTSS function of E. coli O157:H7. (c) 2005 Elsevier SAS. All rights reserved.

Development of a fermentation system to model sessile bacterial populations in the human colon

A fermentation system was designed to model the human colonic microflora in vitro. The system provided a framework of mucin beads to encourage the adhesion of bacteria, which was encased within a dialysis membrane. The void between the beads was inoculated with faeces from human donors. Water and metabolites were removed from the fermentation by osmosis using a solution of polyethylene glycol (PEG). The system was concomitantly inoculated alongside a conventional single-stage chemostat. Three fermentations were carried out using inocula from three healthy human donors. Bacterial populations from the chemostat and biofilm system were enumerated using fluorescence in situ hybridization. The culture fluid was also analysed for its short-chain fatty acid (SCFA) content. A higher cell density was achieved in the biofilm fermentation system (taking into account the contribution made by the bead-associated bacteria) as compared with the chemostat, owing to the removal of water and metabolites. Evaluation of the bacterial populations revealed that the biofilm system was able to support two distinct groups of bacteria: bacteria growing in association with the mucin beads and planktonic bacteria in the culture fluid. Furthermore, distinct differences were observed between populations in the biofilm fermenter system and the chemostat, with the former supporting higher populations of clostridia and Escherichia coli. SCFA levels were lower in the biofilm system than in the chemostat, as in the former they were removed via the osmotic effect of the PEG. These experiments demonstrated the potential usefulness of the biofilm system for investigating the complexity of the human colonic microflora and the contribution made by sessile bacterial populations.

In vitro study on the cell adhesion ability of immobilized lactobacilli on natural supports

The aim of the present study was to investigate the effect of probiotic immobilization onto wheat grains, both wet and freeze dried, on the adhesion properties of the probiotic cells and make comparisons with wet and freeze dried free cells. Lactobacillus casei ATCC 393 and Lactobacillus plantarum NCIMB 8826 were used as model probiotic strains. The results showed satisfactory adhesion ability of free cells to a monolayer of Caco-2 cells (> 1000 CFU/100 Caco-2 cells for wet cells). Cell immobilization resulted in a significant decrease in adhesion, for both wet and freeze dried formulations, most likely because immobilized cells did not have direct access to the Caco-2 cells, but it still remained in adequate levels (> 100 CFU/100 Caco-2 cells for wet cells). No clear correlation could be observed between cell adhesion and the hydrophobicity of the bacterial cells, measured by the hexadecane adhesion assay. Most notably, immobilization enhanced the monolayer integrity of Caco-2 cells, demonstrated by a more than 2-fold increase in transepithelial electrical resistance (TEER) compared to free cells. SEM micrographs ascertained the adhesion of both immobilized and free cells to the brush border microvilli. Finally, the impact of the food matrix on the adhesion properties of probiotic bacteria and on the design of novel functional products is discussed.

Determination of the impact of continuous defoliation of Lolium perenne and Trifolium repens on bacterial and fungal community structure in rhizosphere soil

To determine the effects of defoliation on microbial community structure, rhizosphere soil samples were taken pre-, and post-defoliation from the root tip and mature root regions of Trifolium repens L. and Lolium perenne L. Microbial DNA isolated from samples was used to generate polymerase chain reaction-denaturing gradient gel electrophoresis molecular profiles of bacterial and fungal communities. Bacterial plate counts were also obtained. Neither plant species nor defoliation affected the bacterial and fungal community structures in both the root tip and mature root regions, but there were significant differences in the bacterial and fungal community profiles between the two root regions for each plant. Prior to defoliation, there was no difference between plants for bacterial plate counts of soils from the root tip regions; however, counts were greater in the mature root region of L. perenne than T. repens. Bacterial plate counts for T. repens were higher in the root tip than the mature root region. After defoliation, there was no effect of plant type, position along the root or defoliation status on bacterial plate counts, although there were significant increases in bacterial plate counts with time. The results indicate that a general effect existed during maturation in the root regions of each plant, which had a greater impact on microbial community structure than either plant type or the effect of defoliation. In addition there were no generic consequences with regard to microbial populations in the rhizosphere as a response to plant defoliation.

Use of an artificial root to examine the influence of 8-hydroxyquinoline on soil microbial activity and bacterial community structure

Invasive plant species have been shown to alter the microbial community composition of the soils they invade and it is suggested that this below-ground perturbation of potential pathogens, decomposers or symbionts may feedback positively to allow invasive success. Whether these perturbations are mediated through specific components of root exudation are not understood. We focussed on 8-hydroxyquinoline, a putative allelochemical of Centaurea diffusa (diffuse knapweed) and used an artificial root system to differentiate the effects of 8-hydroxyquinoline against a background of total rhizodeposition as mimicked through supply of a synthetic exudate solution. In soil proximal (0-10 cm) to the artificial root, synthetic exudates had a highly significant (P < 0.001) influence on dehydrogenase, fluorescein diacetate hydrolysis and urease activity. in addition, 8-hydroxyquinoline was significant (p = 0.003) as a main effect on dehydrogenase activity and interacted with synthetic exudates to affect urease activity (p = 0.09). Hierarchical cluster analysis of 16S rDNA-based DGGE band patterns also identified a primary affect of synthetic exudates and a secondary affect of 8-hydroxyquinoline on bacterial community structure. Thus, we show that the artificial rhizosphere produced by the synthetic exudates was the predominant effect, but, that the influence of the 8-hydroxyquinoline signal on the activity and structure of soil microbial communities could also be detected. (C) 2009 Elsevier Ltd. All rights reserved.

Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

Formulation and delivery of the bacterial antagonist Bacillus subtilis for management of lentil vascular wilt caused by Fusarium oxysporum f. sp lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log(10) CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log(10) CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc-based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.

Factors affecting biological control effectiveness of Pasteuria penetrans in Meloidogyne javanica and the bacterial development in the nematode body

The invasion and infectivity of Meloidogyne javanica juveniles (J2) encumbered with spore of Pasteuria Penetrans were influenced by the temperature and the time J2 were in the soil before exposure to roots. The percentage of infected females decreased as the time juveniles spent in soil increased. When spore encumbered J2 were maintained at 30 degrees C the decrease in infection was greater than that at 18 degrees C. The thermal time requirements and the base temperature for P. penetrans development were estimated. The rate of development followed an exponential curve between 21 and 36 degrees C and the base temperature for development was estimated by extrapolation to be 18.5 degrees C. The effect of integrating a nematode resistant tomato cultivar with the biocontrol agent P. penetrans also was investigated. The ability of the biocontrol agent to reduce numbers of root-knot nematodes was dependent on the densities of the nematode and P. penetrans spores in the soil.

Antagonistic potential of bacterial isolates associated with entomopathogenic nematodes against tomato wilt caused by Fusarium oxysporum f.sp., Lycopersici under greenhouse conditions

Three concentrations of Xenorhabdus nematophila and Xenorhabdus spp., (4x10(5,) 4x10(6,) 4x10(7) cells/ml) were evaluated in the laboratory and in pot experiments to test their antagonistic effects on Fusarium oxysporum f.sp., lycopersici. All concentrations effectively inhibited its growth on agar plates. In soil under greenhouse conditions treatments with each bacterium at 4x10(7) cells/ml reduced the disease incidence of tomato by up to 40.38 and 47.54% respectively and there were significant increases of plant biomass by 198 and 211% respectively. The rhizosphere population of Fusarium oxysporum f.sp., lycopersici was reduced by 97%. The Xenorhabdus spp., was comparatively more effective than X. nematophila.

Susceptibility of different insect pupae to the bacterial symbiont, Xenorhabdus nematophila, isolated from the entomopathogenic nematode Steinernema carpocapsae

Cells of the bacterial symbiont Xenorhabdus nematophila from the entomopathogenic nematode, Steinernema carpocapsae entered the pupae of Plutella xylostella after 15 minutes treatment with suspensions containing the bacterial cells. Secretions of Xenorhabdus nematophila, in either broth or water, were found lethal to the pupae of P. xylostella when applied in moist sand. The bacterial symbiont Xenorhabdus nematophila was found lethal to the pupae of greater wax moth (Galleria mellonella), beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella) and black vine weevil (Otiorhynchus sulcatus) in the absence of the nematode vector and the cells of X. nematophila entered the haemocoele of the pupae.

Iron-regulated surface determinant protein a mediates adhesion of staphylococcus aureus to human corneocyte envelope proteins

The ability of Staphylococcus aureus to colonize the human nares is a crucial prerequisite for disease. IsdA is a major S. aureus surface protein that is expressed during human infection and required for nasal colonization and survival on human skin. In this work, we show that IsdA binds to involucrin, loricrin, and cytokeratin K10, proteins that are present in the cornified envelope of human desquamated epithelial cells. To measure the forces and dynamics of the interaction between IsdA and loricrin (the most abundant protein of the cornified envelope), single-molecule force spectroscopy was used, demonstrating high-specificity binding. IsdA acts as a cellular adhesin to the human ligands, promoting whole-cell binding to immobilized proteins, even in the absence of other S. aureus components (as shown by heterologous expression in Lactococcus lactis). Inhibition experiments revealed the binding of the human ligands to the same IsdA region. This region was mapped to the NEAT domain of IsdA. The NEAT domain also was found to be required for S. aureus whole-cell binding to the ligands as well as to human nasal cells. Thus, IsdA is an important adhesin to human ligands, which predominate in its primary ecological niche.