Pablo Perez

Bacterial imprinting of the neonatal immune system: lessons from maternal cells?

OBJECTIVE. We examined the presence of a natural bacterial inoculum in breast milk and its intracellular transport from the maternal intestine to the breast through the circulation. METHODS. Breast milk and peripheral blood were collected aseptically from healthy donors at various times after delivery, and the presence of viable bacteria was determined through plating. Temporal temperature gradient gel electrophoresis was used to examine the bacterial ribosomal DNA content in milk cells, maternal peripheral blood mononuclear cells, and feces and in corresponding infant feces. Blood from nongravid nonlactating women served as control samples. Bacterial translocation to extraintestinal tissues was also evaluated in virgin, pregnant, and lactating mice. RESULTS. Breast milk contained a low total concentration of microbes of <103 colony-forming units per mL. Temporal temperature gradient gel electrophoresis revealed that maternal blood and milk cells contained the genetic material of a greater biodiversity of enteric bacteria. Some bacterial signatures were common to infant feces and to samples of maternal origin. Bacterial translocation from the gut to mesenteric lymph nodes and mammary gland occurred during late pregnancy and lactation in mice. CONCLUSIONS. Bacterial translocation is a unique physiologic event, which is increased during pregnancy and lactation in rodents. Human breast milk cells contain a limited number of viable bacteria but a range of bacterial DNA signatures, as also found in maternal peripheral blood mononuclear cells. Those peripheral blood mononuclear cells showed greater biodiversity than did peripheral blood mononuclear cells from control women. Taken together, our results suggest that intestinally derived bacterial components are transported to the lactating breast within mononuclear cells. We speculate that this programs the neonatal immune system to recognize specific bacterial molecular patterns and to respond appropriately to pathogens and commensal organisms.

Inhibition of Giardia intestinalis by Extracellular Factors from Lactobacilli: an In Vitro Study

ABSTRACT The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent.Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G1 phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation ofGiardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.

Lactobacillus johnsonii La1 Antagonizes Giardia intestinalis In Vivo

ABSTRACT This study describes the in vivo activity of Lactobacillus johnsonii La1 (NCC533) in Giardia intestinalis-infected gerbils (Meriones unguiculatus). Daily administration of lactobacilli in the drinking water from 7 days before inoculation with Giardia trophozoites efficiently prevented G. intestinalis strain WB clone C6 from infecting gerbils. More specifically, shedding of fecal Giardia antigens (GSA65 protein) was diminished in the La1-treated group, and resolution of infection was observed by 21 days postinoculation. Histology and analysis of enzymatic markers of microvillus membrane integrity revealed that probiotic administration also protected against parasite-induced mucosal damage. In addition, a cellular response to Giardia antigens was stimulated in spleen cells from La1-treated gerbils. Results show for the first time the antigiardial effect of probiotic lactobacilli in vivo and provide further insight into the antagonistic properties of lactic acid bacteria against protozoa involved in intestinal infections.

Disassembly of F-Actin Cytoskeleton after Interaction of Bacillus cereus with Fully Differentiated Human Intestinal Caco-2 Cells

ABSTRACT In the present study, the role of direct procaryote-eucaryote interactions in the virulence of Bacillus cereus was investigated. As a model of human enterocytes, differentiated Caco-2 cells were used. Infection of fully differentiated Caco-2 cells with B. cereus in the exponential phase of growth, in order to minimize the concentration of spores or sporulating microorganisms, shows that a strain-dependent cytopathic effect develops. Interestingly, addition of 3-h-old cultures of some strains resulted in complete detachment of the cultured cells after a 3-h infection whereas no such effect was found after a 3-h infection with 16-h-old cultures. Infection of enterocyte-like cells with B. cereus leads to disruption of the F-actin network and necrosis. Even though the effect of secreted factors cannot be ruled out, direct eucaryote-procaryote interaction seems to be necessary. In addition, we observed that some B. cereus strains were able to be internalized in Caco-2 cells. Our findings add a new insight into the mechanisms of virulence of B. cereus in the context of intestinal infection.

Oral Administration of Kefiran Induces Changes in the Balance of Immune Cells in a Murine Model

The aim of the present study was to evaluate the effect of the oral administration of kefiran on the balance of immune cells in a murine model. Six week old BALB/c mice were treated with kefiran (300 mg/L) for 0, 2 and 7 days. Kefiran treatment increased the number of IgA+ cells in lamina propria after 2 and 7 days. Percentage of B220+/MHCII(high) cells in mesenteric lymph nodes (2 days) and Peyers patches (7 days) was higher compared to untreated control mice. An increase of macrophages (F4/80+ cells) was observed in lamina propria and peritoneal cavity (2 and 7 days). In contrast, at day 7, macrophage population decreased in Peyers patches. These results show the ability of kefiran to modify the balance of immune cells in intestinal mucosa. This property could be highly relevant for the comprehension of the probiotic effect attributed to kefir.

Intestinal microflora and the interaction with immunocompetent cells

The intestinal mucosal surface is colonised by the comensal microflora that attains very high numbers of bacterial cells in the distal intestine, more specifically in the colon. At the same time these extensive areas are the interface with the external environment, through which most pathogens initiate infectious processes in mammals. Intestinal mechanisms of defense need to discriminate accurately between comensal, symbiotic microflora, and exogenous pathogens. Today we do not fully understand the essence of the mechanism of discrimination but, probably, innate as well as adaptive immune responses participate in this process. We have explored , in in vitro models, the capacity of mucosal immunocompetent cells to discriminate amongst signals delivered by different types of bacteria. We have found at least two different patterns of innate response to gram-negative and gram-positive bacteria, and within this last group big differences are observed between species. We have only wo rked with non-pathogenic bacteria in what may represent the modulation of the physiological host status. The understanding of these modulatory functions could render a unique possibility for the use of food-borne bacteria to prevent or correct intestinal problems associated with food allergy, inflammatory bowel disease, and autoimmunity.

Surface characterization and adhesive properties of bifidobacteria.

Publisher Summary This chapter discusses the surface characterization and adhesive properties of Bifidobacteria. The colonization of different portions of the intestinal tract by beneficial microorganisms constitutes the first defensive barrier against the invasion of pathogenic microorganisms or toxic substances. The ability to adhere to the intestinal epithelia and to compete with other microorganisms seems to be crucial for a probiotic strain to colonize the gastrointestinal tract. Bacteria of the genus Bifidobacterium are normal inhabitants of the gut of humans and animals, where they play an important role in the prevention of gastrointestinal disorders. Bifidobacteria have been successfully employed to prevent antibioticrelated diarrhea and acute infant diarrhea. One approach to obtaining information regarding adherent behavior in the intestinal ecosystem is to study the surface properties of the selected strains. The adhesion of microorganisms to natural environments can be inferred by studying the surface properties of bacteria, employing a combination of methodologies. This procedure includes several determinations such as partitioning of bacteria in hydrocarbon-aqueous interfaces, ζ potential, binding to solid surfaces, capacity to aggregate particles, and adhesion to monolayers of cell cultures. These methodologies are applied to a collection of bifidobacteria isolated at CIDCA (Centro de Investigacion y Desarrollo en Criotecnologia de Alimentos, La Plata, Argentina) and to reference strains.

Inhibition of Bacillus cereus in milk fermented with kefir grains.

The effects of kefir-fermented milk were tested against a toxigenic strain of Bacillus cereus. The incubation of milk with B. cereus spores plus 5% kefir grains prevented spore germination and growth of vegetative forms. In contrast, when 1% kefir grains was used, no effects were observed. The presence of metabolically active kefir grains diminished titers of nonhemolytic enterotoxin A, as assessed by enzyme-linked immunosorbent assay. During fermentation, kefir microorganisms produce extracellular metabolites such as organic acids, which could play a role in the inhibition of spore germination and growth of B. cereus, although the effect of other factors cannot be ruled out. Results of the present study show that kefir-fermented milk is able to antagonize key mechanisms involved in the growth of B. cereus as well as interfere with the biological activity of this microorganism.

In vitro interaction between Bacillus megaterium strains and Caco-2 cells

To further our understanding of the virulence potential of Bacillus megaterium strains, cell association and invasion assays were conducted in vitro by infecting human enterocytes (Caco-2 cells) with 53 strains of this bacterium isolated from honey. Two series of experiments were performed: (i) necrosis and cell detachment assays with the supernatants of bacterial culture filtrates from 16-h cultures and (ii) adhesion/invasion assays in which cultured enterocytes incubated with bacteria from 3-h cultures were resuspended in Dulbeccos modified Eagles medium and chloramphenicol. The detachment of Caco-2 cells was evaluated by staining the cells with crystal violet. Necrosis was assessed by fluorescence microscopy of cells labeled with propidium iodide. Association (adhesion plus invasion) was determined by plate counts and invasion in an aminoglycoside protection assay. The results showed that spent culture supernatants detached and necrotized Caco-2 cells in a strain-dependent manner. Seven out of 53 B. megaterium filtered culture supernatants caused complete cell detachment. Suspensions of these same bacterial strains adhered and invaded enterocytes in 2-h infection experiments. To our knowledge, this is the first report on the interaction between B. megaterium and intestinal epithelial Caco-2 cells.

Interaction between Bacillus cereus and cultured human enterocytes: effect of calcium, cell differentiation, and bacterial extracellular factors.

Bacillus cereus interaction with cultured human enterocytes and the signaling pathways responsible for the biological effects of the infection were investigated. Results demonstrate that calcium depletion increases the ability of strains T1 and 2 to invade cells. Bacteria associated in greater extent to undifferentiated enterocytes and extracellular factors from strain 2 increased its own association and invasion. Inhibitors of signaling pathways related to phosphorylated lipids (U73122 and wortmannin) were able to significantly reduce cytoskeleton disruption induced by B. cereus infection. Adhesion of strain T1 decreased in the presence of U73122 and of wortmannin, as well as when those inhibitors were used together. In contrast, invasion values were diminished only by U73122. Results show that different factors are involved in the interaction between B. cereus and cultured human enterocytes. Following infection, disruption of the cytoskeleton could facilitate invasion of the eukaryotic cells.