In vitro antibacterial activity of Lactobacillus helveticus strain KS300 against diarrhoeagenic, uropathogenic and vaginosis-associated bacteria.

AIMS: The purpose of this study was to investigate in vitro the antibacterial activity of the Lactobacillus helveticus strain KS300 against vaginosis-associated bacteria including Gardnerella vaginalis and Prevotella bivia, uropathogenic Escherichia coli, and diarrhoeagenic Salmonella enterica serovar Typhimurium. METHODS AND RESULTS: The KS300 strain inhibited the growth of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. After direct co-culture, data show that the Lactobacillus strain decreased the viability of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. The adhering KS300 strain inhibited the adhesion of G. vaginalis DSM 4944 and uropathogenic Dr-positive E. coli IH11128 onto HeLa cells. Moreover, the KS300 strain inhibited the internalization of uropathogenic Dr-positive E. coli IH11128 within HeLa cells and S. typhimurium SL1344 within Caco-2/TC7 cells. CONCLUSIONS: The findings demonstrate that L. helveticus strain KS300 is adhesive onto cultured human cells and has antagonistic activities against vaginosis-associated, uropathogenic and diarrhoeagenic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhering L. helveticus strain KS300 is a potential probiotic strain displaying a strain-specific array of in vitro antibacterial activities.

Inhibition of Giardia intestinalis by extracellular factors from Lactobacilli: an in vitro study.

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 G(1) 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 of Giardia 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.

Favourable effect of an acidified milk (LC-1) on Helicobacter pylori gastritis in man.

OBJECTIVE: The supernatant of Lactobacillus johnsonii La1 culture was shown to be bactericidal and to have a partial, acid-independent suppressive effect on Helicobacter pylori in humans. The aim of the present study was to investigate the effect of L. johnsonii La1-acidified milk (LC-1) on H. pylori infection. DESIGN AND METHODS: Fifty-three volunteers infected with H. pylori as determined by positive 13C-urea breath test and positive serology were randomized to receive either LC-1 or a placebo 180 ml twice a day for 3 weeks. All subjects also received clarithromycin 500 mg bid during the last two weeks of acidified milk therapy. Oesophagogastroduodenoscopy and biopsies were performed at inclusion and repeated 4-8 weeks after the end of the treatment. H. pylori infection was confirmed by urease test and histology. H. pylori density and inflammation were scored using a modified Sydney classification. RESULTS: LC-1 ingestion induced a decrease in H. pylori density in the antrum (P= 0.02) and the corpus (P= 0.04). LC-1 also reduced inflammation and gastritis activity in the antrum (P= 0.02 and P= 0.01, respectively) and of activity in the corpus (P= 0.02). Clarithromycin eradicated H. pylori in 26% of the subjects; LC-1 did not improve the antibiotic effect. CONCLUSION: These results suggest that H. pylori infection and gastritis can be down-regulated by LC-1.

Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity.

BACKGROUND AND AIMS: The gastrointestinal microflora exerts a barrier effect against enteropathogens. The aim of this study was to examine if bifidobacteria, a major species of the human colonic microflora, participates in the barrier effect by developing antimicrobial activity against enterovirulent bacteria. METHODS: Antibacterial activity was examined in vitro against a wide range of Gram negative and Gram positive pathogens. Inhibition of Salmonella typhimurium SL1334 cell association and cell invasion was investigated in vitro using Caco-2 cells. Colonisation of the gastrointestinal tract in vivo by bifidobacteria was examined in axenic C3/He/Oujco mice. Antimicrobial activity was examined in vivo in axenic C3/He/Oujco mice infected by the lethal S typhimurium C5 strain. RESULTS: Fourteen human bifidobacterium strains isolated from infant stools were examined for antimicrobial activity. Two strains (CA1 and F9) expressed antagonistic activity against pathogens in vitro, inhibited cell entry, and killed intracellular S typhimurium SL1344 in Caco-2 cells. An antibacterial component(s) produced by CA1 and F9 was found to be a lipophilic molecule(s) with a molecular weight of less than 3500. In the axenic C3/He/Oujco mice, CA1 and F9 strains colonised the intestinal tract and protected mice against S typhimurium C5 lethal infection. CONCLUSION: Several bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity, suggesting that they could participate in the "barrier effect" produced by the indigenous microflora.

Effect of whey-based culture supernatant of Lactobacillus acidophilus (johnsonii) La1 on Helicobacter pylori infection in humans.

BACKGROUND: Specific strains of Lactobacillus acidophilus are known to inhibit intestinal cell adhesion and invasion by enterovirulent bacteria. As L. acidophilus can survive transiently in the human stomach, it may downregulate Helicobacter pylori infection. METHODS: The ability of L. acidophilus (johnsonii) La1 supernatant to interfere with H. pylori bacterial growth, urease activity, and adhesion to epithelial cells was tested in vitro. Its effect on H. pylori infection in volunteers was monitored in a randomized, double-blind, controlled clinical trial, using a drinkable, whey-based, La1 culture supernatant. H. pylori infected volunteers were treated 14 days with 50 ml of La1 supernatant four times a day combined with either omeprazole 20 mg four times a day or with placebo. Infection was assessed by breath test, endoscopy, and biopsy sampling, performed at inclusion, immediately at the end of the treatment (breath test only), and 4 weeks after the end of the treatment. RESULTS: La1 supernatant inhibited H. pylori growth in vitro, regardless of previous binding of H. pylori to epithelial cells. In 20 subjects (8 females, 12 males, mean age 33.1 years) a marked decrease in breath test values was observed immediately after treatment with La1 supernatant, both in the omeprazole and in the placebo group (median 12.3 vs. 28.8 and 9.4 vs. 20.4, respectively; p < 0.03). In both treatment groups, breath test values remained low 6 weeks after treatment (omeprazole treated 19.2, placebo treated 8. 3; p < 0.03 vs. pretreatment), but the persistence of H. pylori infection was confirmed in gastric biopsies. CONCLUSION: La1 culture supernatant shown to be effective in vitro has a partial, acid-independent long-term suppressive effect on H. pylori in humans.

Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells.

The influence of pH on the adhesion of two Lactobacillus strains to Caco-2 human intestinal cells was investigated. One strain, Lactobacillus johnsonii La1, was adherent at any pH between 4 and 7. The other one, L. acidophilus La10, did not attach to this cell line under the same experimental conditions. On the basis of these results, we used the monoclonal antibody technique as a tool to determine differences on the surface of these bacteria and to identify a factor for adhesion. Mice were immunized with live La1, and the hybridomas produced by fusion of spleen cells with ONS1 cells were screened for the production of antibodies specific for L. johnsonii La1. A set of these monoclonal antibodies was directed against a nonproteinaceous component of the L. johnsonii La1 surface. It was identified as lipoteichoic acid (LTA). This molecule was isolated, chemically characterized, and tested in adhesion experiments in the same system. The adhesion of L. johnsonii La1 to Caco-2 cells was inhibited in a concentration-dependent way by purified LTA as well as by L. johnsonii La1 culture supernatant that contained LTA. These results showed that the mechanism of adhesion of L. johnsonii La1 to human Caco-2 cells involves LTA.

Demonstration of safety of probiotics -- a review.

Probiotics are commonly defined as viable microorganisms (bacteria or yeasts) that exhibit a beneficial effect on the health of the host when they are ingested. They are used in foods, especially in fermented dairy products, but also in pharmaceutical preparations. The development of new probiotic strains aims at more active beneficial organisms. In the case of novel microorganisms and modified organisms the question of their safety and the risk to benefit ratio have to be assessed. Lactic acid bacteria (LAB) in foods have a long history of safe use. Members of the genera Lactococcus and Lactobacillus are most commonly given generally-recognised-as-safe (GRAS) status whilst members of the genera Streptococcus and Enterococcus and some other genera of LAB contain some opportunistic pathogens. Lactic acid bacteria are intrinsically resistant to many antibiotics. In many cases resistances are not, however, transmissible, and the species are also sensitive to many clinically used antibiotics even in the case of a lactic acid bacteria- associated opportunistic infection. Therefore no particular safety concern is associated with intrinsic type of resistance. Plasmid-associated antibiotic resistance, which occasionally occurs, is another matter because of the possibility of the resistance spreading to other, more harmful species and genera. The transmissible enterococcal resistance against glycopeptide antibiotics (vancomycin and teicoplanin) is particularly noteworthy, as vancomycin is one of the last effective antibiotics left in the treatment of certain multidrug-resistant pathogens. New species and more specific strains of probiotic bacteria are constantly identified. Prior to incorporating new strains into products their efficacy should be carefully assessed, and a case by case evaluation as to whether they share the safety status of traditional food-grade organisms should be made. The current documentation of adverse effects in the literature is reviewed. Future recommendations for the safety of already existing and new probiotics will be given.

Immune modulation of blood leukocytes in humans by lactic acid bacteria: criteria for strain selection.

Lactic acid bacteria in food can transiently colonize the intestine and exert beneficial effects (probiotic). Survival during intestinal transit or adhesion to epithelium or both seem to be important for modifying the host's immune reactivity. Because Lactobacillus acidophilus strain La1 is adherent to enterocytes in vitro, we hypothesize that contact with immune cells may occur in vivo. However, Bifidobacterium bifidum strain Bb12, which shows high fecal colonization, is another potential immunomodulator. Twenty-eight volunteers were divided into two groups and given a fermented product containing one of the two strains. Lymphocyte subsets and leukocyte phagocytic activity were studied in blood. No modifications were detected in lymphocyte subsets. In contrast, phagocytosis of Escherichia coli ssp. was enhanced in both groups (P < 0.001 for both). Bacterial adhesion to enterocytes, fecal colonization, or both seem to be valuable selection criteria for immunomodulation. Antiinfective mechanisms of defense can be enhanced after ingestion of specific lactic acid bacteria strains.

The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo.

The adhering human Lactobacillus acidophilus strain LA1 inhibits the cell association and cell invasion of enteropathogens in cultured human intestinal Caco-2 cells (M. F. Bernet, D. Brassard, J. R. Neeser, and A. L. Servin, Gut 35:483-489, 1994). Here, we demonstrate that strain LA1 developed its antibacterial activity in conventional or germ-free mouse models orally infected by Salmonella typhimurium. We present evidence that the spent culture supernatant of strain LA1 (LA1-SCS) contained antibacterial components active against S. typhimurium infecting the cultured human intestinal Caco-2 cells. The LA1-SCS antibacterial activity was observed in vitro against a wide range of gram-negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, S. typhimurium, Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter cloacae. By contrast, no activity was observed against species of the normal gut flora, such as lactobacilli and bifidobacteria. The LA1-SCS antibacterial activity was insensitive to proteases and independent of lactic acid production.

Effects of intrajejunal perfusion and chronic ingestion of Lactobacillus johnsonii strain La1 on serum concentrations and jejunal secretions of immunoglobulins and serum proteins in healthy humans.

OBJECTIVES: Link-Amster reported an increase in serum IgA when healthy subjects ingested a fermented dairy product containing Lactobacillus johnsonii La1. We aimed to assess the effects of La1 on the jejunal secretions and serum concentrations of total and specific immunoglobulins and proteins. METHODS: Twelve healthy volunteers ingested a fermented milk containing La1 or a control from day 1 till day 28, following a randomised double blind protocol. At days 0 and 28, the jejunum was successively perfused with a control solution and with a La1 suspension. The serum concentrations and jejunal secretions of albumin, orosomucoid, transferrin, alpha 2-macroglobulin, m-IgA, p-IgA, IgG, IgM, secretory component, and specific antibodies against La1 were assessed. RESULTS: Serum concentrations of IgA slightly increased between d0 and d28 in the group receiving La1 (1.85 +/- 0.64 g/L vs 1.76 +/- 0.76; P = 0.02). The other parameters were not altered. CONCLUSION: This study shows that the immunomodulating effects of La1 ingestion in man are not due to modification of jejunal protein permeability.

A 23 kDa membrane glycoprotein bearing NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked carbohydrate chains acts as a receptor for Streptococcus sanguis OMZ 9 on human buccal epithelial cells.

Streptococcus sanguis colonizes several human oral surfaces, including both hard and soft tissues. Large salivary mucin-like glycoproteins bearing sialic acid residues are known to bind various S.sanguis strains. However, the molecular basis for the adhesion of S.sanguis to human buccal epithelial cells (HBEC) has not been established. The present study shows that S.sanguis OMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner. The desialylation of such cells invariably abolishes adhesion of S.sanguis OMZ 9 to the cell surface. A soluble glycopeptide bearing short sialylated O-linked carbohydrate chains behaves as a potent inhibitor of the attachment of S.sanguis OMZ 9 to exfoliated HBEC. The resialylation of desialylated HBEC with CMP-sialic acid and Gal beta 1,3GalNAc alpha 2,3-sialyltransferase specific for O-glycans restores the receptor function for S.sanguis OMZ 9, whereas a similar cell resialylation with the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase specific for N-glycans is without effect. Finally, the same resialylation reaction carried out with CMP-9-fluoresceinyl-sialic acid as a substrate yields exfoliated HBEC bearing fluorescence on a single 23 kDa protein, when using the alpha 2,3-sialyltransferase as the catalyst. The latter finding demonstrates that this 23 kDa cell surface glycoprotein bears NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked sugar chains, a carbohydrate sequence which is recognized by S.sanguis OMZ 9 on exfoliated HBEC. In similar experiments carried out with a buccal carcinoma cell line termed SqCC/Y1, S.sanguis OMZ 9 did not attach in great numbers to such cultured cells, and these cells were shown to not express membrane glycoprotein bearing alpha 2,3-sialylated O-linked carbohydrate chains.

Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria.

Four human Lactobacillus acidophilus strains were tested for their ability to adhere onto human enterocyte like Caco-2 cells in culture. The LA 1 strain exhibited a high calcium independent adhesive property. This adhesion onto Caco-2 cells required a proteinaceous adhesion promoting factor, which was present in the spent bacterial broth culture supernatant. LA 1 strain also strongly bound to the mucus secreted by the homogeneous cultured human goblet cell line HT29-MTX. The inhibitory effect of LA 1 organisms against Caco-2 cell adhesion and cell invasion by a large variety of diarrhoeagenic bacteria was investigated. As a result, the following dose dependent inhibitions were obtained: (a) against the cell association of enterotoxigenic, diffusely adhering and enteropathogenic Escherichia coli, and Salmonella typhimurium; (b) against the cell invasion by enteropathogenic Escherichia coli, Yersinia pseudotuberculosis, and Salmonella typhimurium. Incubations of L acidophilus LA 1 before and together with enterovirulent E coli were more effective than incubation after infection by E coli.

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.

An intestinal galactose-specific lectin mediates the binding of murine IgE to mouse intestinal epithelial cells.

A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy.

In vitro inhibition of adhesion of Candida albicans clinical isolates to human buccal epithelial cells by Fuc alpha 1----2Gal beta-bearing complex carbohydrates.

The role of cell surface glycoconjugates as possible adhesion receptors for Candida albicans yeasts on human buccal epithelial cells was investigated by using a quantitative radiometric assay involving 14C-metabolically labeled microorganisms. Various structurally defined soluble glycopeptides and oligosaccharides were tested at a low concentration (1 mg/ml) for their ability to competitively inhibit yeast adhesion to such exfoliated cells. Comparisons were also made with various molecular species previously proposed to act as adhesion molecules. A preparation of glycopeptides derived from pooled human newborn meconiums inhibited the attachment (up to 55%) of all three clinical isolates examined. The mild hydrolysis of fucosyl residues from the above mixture totally abolished its inhibitory potency. By using human milk oligosaccharide probes, the minimal structural requirement for activity was found to be the Fuc alpha 1----2Gal beta determinant (the H sugar sequence found on all blood group substances of the ABO [H] system). By contrast, the fucosylated determinants of the Lewis blood group system were found to be totally inactive. Total adhesion inhibitions were never obtained in the present experiments, suggesting that H disaccharide-bearing cell surface glycoconjugates could act as host receptors for C. albicans on human buccal epithelial cells as a part of a mechanism involving multireceptor specificities.

Catabolism of N-glycosylprotein glycans: evidence for a degradation pathway of sialylglyco-asparagines resulting from the combined action of the lysosomal aspartylglucosaminidase and endo-N-acetyl-beta-D-glucosaminidase. A 400-MHz 1H-NMR study.

A mixture of sialylglycoasparagines and sialylglycopeptides was successively incubated with lysosomal extracts, at two pH values, first at pH 7.5 and then at pH 4. The 1H-NMR analysis of the sialyloligosaccharides released during the enzymatic digestion demonstrates the sequential action of aspartylglucosaminidase and an endo-N-acetyl-beta-D-glucosaminidase which release sialyloligosaccharides identical to the reference sugars isolated from the urine of patients suffering from sialidosis. This process represents a new catabolic pathway for N-glycosyl-proteins which may account for the appearance of the oligosaccharides stored in tissues and urine of patients suffering from lysosomal diseases.