Pablo F. Pérez

Isolation and characterization of Bifidobacterium strains for probiotic formulation

Twenty-five Bifidobacterium strains isolated from infant feces were identified by sugar fermentation patterns and whole-cell protein analysis. Using gradient SDS-PAGE, six characteristic protein bands of the genus were detected in 40 strains of bifidobacteria but not in lactobacilli. Computerized numerical analysis enabled strains to be grouped in two main clusters. Strains of Bifidobacterium bifidum belong to a well-differentiated cluster that joins the cluster of the remaining species at 0.582 similarity. The predominant species among isolated strains from infant feces were B. bifidum, B. longum, and B. breve. Probiotic and technological indicators such as surface properties, inhibitory capacity, resistance to bile and low pH, and ability to grow under aerobic conditions were studied. Not all desirable characteristics were present in a single strain. In general, adherent and inhibitory strains were not resistant to bile, low pH, and aerobic conditions. Only 10 of 40 strains were resistant to 0.5% bile.

Effect of bile on the lipid composition and surface properties of bifidobacteria

Aim: The changes produced on the bacterial surface of Bifidobacteria cells when they are grown in bile were compared with those provoked by bile added to bacteria grown in the absence of bile.

Lactobacilli isolated from kefir grains: evidence of the presence of S-layer proteins

In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains. Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66-71 kDa as measured by SDS-PAGE. The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl. The above mentioned proteins were recovered in the LiCl extracts. After dialysis and concentration, the proteins extracted were able to reassemble in a regular array. Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen. Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment. The production of this surface structure under different culture conditions was also evaluated. Finally, the relationship between the presence of S-layer proteins and surface properties (e.g. adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated.

Lactobacilli antagonize biological effects of enterohaemorrhagic Escherichia coli in vitro.

Aims:  To assess the effect of two lactobacilli on the biological activity of enterohaemorrhagic Escherichia coli (EHEC) in vitro.

Kefiran antagonizes cytopathic effects of Bacillus cereus extracellular factors

Kefiran, the polysaccharide produced by microorganisms present in kefir grains, is a water-soluble branched glucogalactan containing equal amounts of D-glucose and D-galactose. In this study, the effect of kefiran on the biological activity of Bacillus cereus strain B10502 extracellular factors was assessed by using cultured human enterocytes (Caco-2 cells) and human erythrocytes. In the presence of kefiran concentrations ranging from 300 to 1000 mg/L, the ability of B. cereus B10502 spent culture supernatants to detach and damage cultured human enterocytes was significantly abrogated. In addition, mitochondrial dehydrogenase activity was higher when kefiran was present during the cell toxicity assays. Protection was also demonstrated in hemolysis and apoptosis/necrosis assays. Scanning electron microscopy showed the protective effect of kefiran against structural cell damages produced by factors synthesized by B. cereus strain B10502. Protective effect of kefiran depended on strain of B. cereus. Our findings demonstrate the ability of kefiran to antagonize key events of B. cereus B10502 virulence. This property, although strain-specific, gives new perspectives for the role of bacterial exopolysaccharides in functional foods.

Surface proteins from Lactobacillus kefir antagonize in vitro cytotoxic effect of Clostridium difficile toxins

In this work, the ability of S-layer proteins from kefir-isolated Lactobacillus kefir strains to antagonize the cytophatic effects of toxins from Clostridium difficile (TcdA and TcdB) on eukaryotic cells in vitro was tested by cell detachment assay. S-layer proteins from eight different L. kefir strains were able to inhibit the damage induced by C. difficile spent culture supernatant to Vero cells. Besides, same protective effect was observed by F-actin network staining. S-layer proteins from aggregating L. kefir strains (CIDCA 83115, 8321, 8345 and 8348) showed a higher inhibitory ability than those belonging to non-aggregating ones (CIDCA 83111, 83113, JCM 5818 and ATCC 8007), suggesting that differences in the structure could be related to the ability to antagonize the effect of clostridial toxins. Similar results were obtained using purified TcdA and TcdB. Protective effect was not affected by proteases inhibitors or heat treatment, thus indicating that proteolytic activity is not involved. Only preincubation with specific anti-S-layer antibodies significantly reduced the inhibitory effect of S-layer proteins, suggesting that this could be attributed to a direct interaction between clostridial toxins and L. kefir S-layer protein. Interestingly, the interaction of toxins with S-layer carrying bacteria was observed by dot blot and fluorescence microscopy with specific anti-TcdA or anti-TcdB antibodies, although L. kefir cells did not show protective effects. We hypothesize that the interaction between clostridial toxins and soluble S-layer molecules is different from the interaction with S-layer on the surface of the bacteria thus leading a different ability to antagonize cytotoxic effect. This is the first report showing the ability of S-layer proteins from kefir lactobacilli to antagonize biological effects of bacterial toxins. These results encourage further research on the role of bacterial surface molecules to the probiotic properties of L. kefir and could contribute to strain selection with potential therapeutic or prophylactic benefits towards CDAD.

Screening of bile resistance and bile precipitation in lactic acid bacteria and bifidobacteria

A modification of the ecometric method was developed for a rapid screening of bile resistance in lactic acid bacteria and bifidobacteria. Validation of the MEM bile assay (modified ecometric method) was performed comparing the bile resistance index (RIbile) and the bile resistance ratio (R%). Most Bifidobacterium strains assayed were bile sensitive (83.3%), while only 62.1% of lactobacilli showed that behavior. Some bifidobacterial strains (55.6%) showed a crystalline precipitate when grown on solid medium supplemented with 0.5% ox bile. The crystalline structures produced by B. pseudolongum CIDCA 531 were isolated and analyzed by optical and scanning electron microscopy, thin-layer chromatography, melting point, and specific cholesterol reactions. Those studies confirmed the presence of cholesterol in these crystalline structures. On the other hand, none of the lactobacilli and streptococci studied had the ability to produce crystalline precipitates.

Trehalose, a cryoprotectant for Lactobacillus bulgaricus

Abstract The cryoprotective effect of trehalose in the freezing-thawing process was assayed in two strains of Lactobacillus bulgaricus. The viability and acidification properties before and after the process were compared. At a concentration of 0.3 M, trehalose was able to preserve the viability and the acidification power. The degree of protection is dependent on the strain and those compounds present in the medium. This effect is more noticeable in water an culture medium than in milk. However, the milk supplied with trehalose has the highest effectiveness.

Effect of Bacillus cereus Exocellular Factors on Human Intestinal Epithelial Cells

To gain insight on the biological effects of the exocellular factors produced by Bacillus cereus, culture filtrate supernatants of different strains were coincubated with differentiated Caco-2 cells. Exocellular factors were able to detach enterocyte-like cells from the substratum after 1 h of incubation. In addition, microvilli effacing and dramatic changes on the cellular surface of enterocytes were found after incubation periods as short as 20 min. Since cell detachment was not inhibited by fetal calf serum, thiol activated cholesterol-binding cytolysin, cereolysin O, does not seem to be involved. Also, translocation of phosphatidylserine from the inner to the outer leaflets of the plasma membrane was demonstrated by using fluorescein isothiocyanate (FITC)-Annexin V. In contrast to the high capability of detaching Caco-2 cells shown by all the strains under study, the mitochondrial dehydrogenase activity was lowered by culture filtrate supernatants in a strain-dependent manner. For strain M2, the decrease in dehydrogenase activity was already evident after 30 min of incubation. Production of biologically active factors depends on the growth phase, and maximal activity was found in late exponential-early stationary phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of concentrated exocellular factors showed a very complex scenery supporting the multifactorial character of the biological activity of B. cereus.

Lipid raft‐dependent adhesion of Giardia intestinalis trophozoites to a cultured human enterocyte‐like Caco‐2/TC7 cell monolayer leads to cytoskeleton‐dependent functional injuries

Gardia intestinalis, the aetiological agent of giardiasis, one of the most common intestinal diseases in both developing and developed countries, induces a loss of epithelial barrier function and functional injuries of the enterocyte by mechanisms that remain unknown. Three possible mechanisms have been proposed: (i) Giardia may directly alter the epithelial barrier after a close interaction between the trophozoite and polarized intestinal cells, (ii) intestinal functions may be altered by factors secreted by Giardia including an ‘enterotoxin’, proteinases and lectins, and (iii) based on mouse studies, a mechanism involving the intervention of activated T lymphocytes. We used fully differentiated cultured human intestinal Caco‐2/TC7 cells forming a monolayer and expressing several polarized functions of enterocytes of small intestine to investigate the mechanisms by which G. intestinalis induces structural and functional alterations in the host intestinal epithelium. We first report that adhesion of G. intestinalis at the brush border of enterocyte‐like cells involves the lipid raft membrane microdomains of the trophozoite. We report an adhesion‐dependent disorganization of the apical F‐actin cytoskeleton that, in turn, results in a dramatic loss of distribution of functional brush border‐associated proteins, including sucrase‐isomaltase (SI), dipeptidylpeptidase IV (DPP IV) and fructose transporter, GLUT5, and a decrease in sucrose enzyme activity in G. intestinalis‐infected enterocyte‐like cells. We observed that the G. intestinalis trophozoite promotes an adhesion‐dependent decrease in transepithelial electrical resistance (TER) accompanied by a rearrangement of functional tight junction (TJ)‐associated occludin, and delocalization of claudin‐1. Finally, we found that whereas the occludin rearrangement induced by G. intestinalis was related to apical F‐actin disorganization, the delocalization of claudin‐1 was not.