Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.     

Human ileostomy glycoproteins as a model for small intestinal mucus to investigate adhesion of probiotics

Human ileostomy glycoproteins were used as a model for small intestinal mucus to investigate the adhesion of 12 Lactobacillus strains, one Lactococcus strain and one Propionibacterium strain, Both probiotic and dairy strains were tested. Adhesive and non-adhesive Escherichia coli strains were used as controls. All the strains were also tested for their adhesion to polystyrene. Adhesion to ileostomy glycoproteins and to polystyrene varied significantly among the strains tested. Lactobacillus rhamnosus (human isolate), Lactobacillus GG, Lact. acidophilus 1 and P. freudenreichii adhered to ileostomy glycoproteins. Adhesion was concentration-dependent and the most adhesive strains were able to saturate the substratum. These results indicate that human ileostomy glycoproteins can be used as a model system to select potential probiotic strains to complement the adhesion test with intestinal cell lines.     

Lactobacillus casei DN-114 001 inhibits the ability of adherent-invasive Escherichia coli isolated from Crohn's disease patients to adhere to and to invade intestinal epithelial cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.     

Porcine E. coli: Virulence-Associated Genes,Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars.Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation.In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.     

Influence of manufacturing processes on in vitro properties of the probiotic strain Lactobacillus rhamnosus Lcr35®

Probiotics are administered as complex manufactured products and yet most studies on probiotic bacterial strains have been performed with native culture strains. Little is known about the influence of industrial processes on the properties of the microorganisms. In this study, we comparatively assessed the characteristics of the probiotic bacterial strain Lactobacillus rhamnosus (Lcr35(?)) together with four of its commercial formulations, including three intestinal formulas (BACILOR with Lcr Restituo(?) packet and capsule and FLOREA Lcr Lenio(?)) and one vaginal formula (GYNOPHILUS Lcr Regenerans(?)). Lcr35(?) grown from the intestinal formulas displayed increased resistance to acidic pH and bile stress, especially FLOREA (Lcr Lenio(?)), which showed a 4.5log higher number of viable bacteria compared to the results obtained with the control native Lcr35(?) strain. Adhesion to intestinal cells was significantly higher with Lcr Restituo(?) packet and Lcr Restituo(?) capsule vs Lcr35(?). Bacteria from the vaginal formulation GYNOPHILUS had increased ability to metabolize glycogen thereby increasing lactic acid production. In vitro growth inhibition of the pathogen Candida albicans was significantly higher with bacteria from the vaginal formulation (4.5 log difference) and in the presence of vaginal epithelial cells than with the native strain. Our results show that the manufacturing process influences strain properties and should therefore be adapted according to the strain and the therapeutic indication.     

OmpD but not OmpC is involved in adherence of Salmonella enterica serovar typhimurium to human cells

Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 degrees C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.     

Adherence and colonization properties of Lactobacillus rhamnosus TB1, a broiler chicken isolate

AIMS: Selected lactic acid bacteria (LAB) isolated from intestinal tract of chicken have been studied in order to investigate their ability to adhere in vitro to Basement Membrane Matrigel (BMM). A selected strain showing a good adherence in BMM test was used for in vivo colonization assays. METHODS AND RESULTS: In vitro assessment of adhesion of broiler chicken isolates was performed using BMM assay. Among LAB strains tested, Lactobacillus rhamnosus TB1 showed a good adherence that was comparable to the one of an Escherichia coli EPEC strain used as positive control. For in vivo colonization assays this strain was fluorescently stained with the carboxyfluorescein diacetate succinimidyl ester (cFDA-SE) thus allowing its detection in different layers of intestinal tract after inoculation in broiler chicken. Further, stained L. rhamnosus were found with a highest value in rectum, jejunum and ileum both 3 and 24 h after administration. CONCLUSIONS: BMM assay is a quick method to test in vitro adhesion properties of bacterial strains and cFDA-SE-stained bacteria may be considered as an alternative method to test in vivo adhesion and colonization properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus rhamnosus TB1 was therefore showed to be able to adhere strongly in vitro to BMM and in vivo to intestinal epithelial cells of chicken and may be considered as a potential probiotic for chicken.     

Genome sequence of Lactobacillus amylovorus GRL1118, isolated from pig ileum

Lactobacillus amylovorus is a common member of the beneficial microbiota present in the pig gastrointestinal tract. Here, we report the genome sequence of the surface layer (S-layer) protein-carrying and potentially probiotic strain L. amylovorus GRL1118, which was isolated from porcine ileum and which shows strong adherence to pig intestinal epithelial cells.     

Factors involved in adherence of lactobacilli to human Caco-2 cells.

A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kernéis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany

AIMS: To verify the presence of Escherichia coli Nissle 1917 as a natural isolate in swine and to characterize in vitro probiotic properties as well as in vivo persistence in a feeding experiment. METHODS AND RESULTS: During studies on the intestinal microflora of pigs, we isolated E. coli Nissle 1917 sporadically from a pig population over a period of 1 year. The identity of the isolates as E. coli Nissle 1917 was verified by serotyping, Nissle-specific PCR, macrorestriction analysis (pulsed field gel electrophoresis) and the determination of in vitro probiotic properties in invasion and adhesion assays using a porcine intestinal epithelial cell line. Both the E. coli isolates and the E. coli Nissle 1917 strain showed strong reductions in adhesion of porcine enteropathogenic E. coli and invasion of Salmonella typhimurium with epithelial cells in vitro, with a probiotic effect. Screening of five epidemiologically unlinked swine farms and two wild boar groups showed one farm positive for E. coli Nissle 1917. A feeding experiment with four piglets showed viable E. coli Nissle 1917 in the intestine of three animals. CONCLUSIONS: The results of this study suggest that the E. coli Nissle 1917 strain is already partially established in swine herds, but the colonization of individual animals is variable. SIGNIFICANCE AND IMPACT OF THE STUDY: We report natural, long-term colonization and transmission of the probiotic E. coli Nissle 1917 strain in a swine herd, characterized individual persistence and colonization properties in swine and established an in vitro porcine intestinal epithelial cell model of probiotic action. The results of this study would have implications in the use of this strain as a probiotic in swine and contribute to a better understanding of the individual nature of intestinal bacterial persistence and establishment.     

Inhibitory effects of bovine lactoferrin on the adherence of enterotoxigenic Escherichia coli to host cells

Adherence is an essential and prerequisite step for the colonization of mucosal surfaces by enterotoxigenic Escherichia coli (ETEC). We studied the effect of bovine lactoferrin (BLF) on the adherence of ETEC to human epithelial cells in vitro, and to intestinal mucosa of ICR germfree mice in vivo. In the in vitro study, BLF was found to inhibit the adherence of ETEC. This adhesion-inhibiting activity of BLF was found to lessen with decreasing BLF concentration, but the data obtained suggest a positive inhibitory effect of BLF against the adhesion of ETEC cells. In the in vivo study, the counts of adherent bacteria in various sections of the intestinal tract (duodenum, jejunoileum, and large intestine) were lower in the BLF group than in the control group, suggesting the possible action of BLF as an intestinal tract adherence-blocking agent with regards to ETEC.     

Screening for probiotic properties of strains isolated from feces of various human groups

The present study searched for potential probiotic strains from various human fecal samples. A total of 67 aerobic and 38 anaerobic strains were isolated from 5 different categories of human feces. Systematic procedures were used to evaluate the probiotic properties of the isolated strains. These showed about 75-97% survivability in acidic and bile salt environments. Adhesion to intestinal cell line Caco-2 was also high. The isolates exhibited hydrophobic properties in hexadecane. The culture supernatants of these strains showed antagonistic effects against pathogens. The isolates were resistant to a simulated gastrointestinal environment in vitro. Of the 4 best isolates, MAbB4 (Staphylococcus succinus) and FIdM3 (Enterococcus fecium), were promising candidates for a potential probiotic. S. succinus was found to be a probiotic strain, which is the second such species reported to date in this particular genus. A substantial zone of inhibition was found against Salmonella spp., which adds further support to the suggestion that the probiotic strain could help prevent intestinal infection. This study suggested that the human flora itself is a potential source of probiotics.     

Lactobacillus casei DN-114 001 Inhibits the Ability of Adherent-Invasive Escherichia coli Isolated from Crohn's Disease Patients To Adhere to and To Invade Intestinal Epithelial Cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.     

Adhesion of Lactobacillus reuteri strain Lr1 to equine epithelial cells and competitive exclusion of Clostridium difficile from the gastro-intestinal tract of horses

Lactobacillus reuteri Lr1, isolated from healthy horses, remained viable after 2 h at pH 2.0 and in the presence of 1.5 % (w/v) bile. Strain Lr1 survived passage through the equine gastro-intestinal tract (GIT). However, no viable cells of L. reuteri Lr1 were detected on the third day after administration, suggesting that the strain did not colonise the GIT for longer than two days. Strain Lr1 adhered to non-viable, but not to viable, buccal epithelial cells in vitro. Adherence of strain Lr1 to buccal epithelial cells increased 25 % after treatment of the bacterial cells with pepsin. Treatment with pronase prevented the adhesion to epithelial cells. This suggested that specific proteins on the cell surface of L. reuteri Lr1 are involved in adhesion to epithelial cells. Strain Lr1 aggregated with Clostridium difficile C6, isolated from the GIT of a horse that died from severe colic. Adherence of C. difficile C6 to epithelial cells declined from 60 % to 3 % when challenged with L. reuteri Lr1 and the number of viable clostridia decreased tenfold during dosage. Red blood cell, haemoglobin and haemocrit levels were significantly (P ≤ 0.05) lower after dosage with L. reuteri Lr1. Cholesterol and glucose levels were mildly elevated for one day during dosage, but decreased significantly thereafter to levels similar than before dosage. Genes encoding adhesion to collagen, production of aggregation substances, cytolysin and β hemolysin III, resistance to vancomycin A, B and C, and gelatinase activity were not detected, suggesting that L. reuteri Lr1 is a potential probiotic that may be used to control C. difficile cell numbers in the GIT.     

Decrease in adherence of bacteria and yeasts to human mucosal epithelial cells by noxythiolin in vitro

Adherence of buccal and vaginal isolates of Candida albicans to buccal epithelial cells and the adherence of urine isolates of Escherichia coli and Staphylococcus saprophyticus to uroepithelial cells was quantified by light microscopy. The antimicrobial agent noxythiolin reduced the adherence of these micro-organisms in both exponential and stationary growth phases. Adherence of both the blastospore and pseudohyphal forms of C. albicans was reduced. Treatment of epithelial cells and/or micro-organisms with noxythiolin resulted in decreased adherence. No anti-adherence effect was observed with formaldehyde and N-methylthiourea, the degradative products of noxythiolin.     

Impact of spray-drying on the pili of Lactobacillus rhamnosus GG

The preservation of the viability of microorganisms in probiotic formulations is the most important parameter ensuring the adequate concentration of live microorganisms at the time of administration. The formulation and processing techniques used to produce these probiotic formulations can influence the preservation of the microbial viability. However, it is also required that the bacteria maintain their key probiotic capacities during processing, formulation and shelf life. In this study, we investigated the impact of spray-drying on different cell wall properties of the model probiotic strain Lactobacillus rhamnosus GG, including its adherence to intestinal epithelial cells. The dltD gene knock-out mutant, L. rhamnosus GG CMPG5540, displaying modified cell wall lipoteichoic acids, showed significantly increased colony-forming units after spray-drying and subsequent storage under standard conditions compared to wild-type L. rhamnosus GG. In contrast, disruption of the biosynthesis of exopolysaccharides or pili expression did not impact survival. However, spray-drying did significantly affect the adherence capacity of L. rhamnosus GG. Scanning electron microscopy confirmed that the pili, key surface factors for adherence to intestinal cells and mucus, were sheared off during the spray-drying process. These data thus highlight that both the functionality and viability of probiotics should be assessed during the spray-drying process and subsequent storage.     

Genome sequence of Lactobacillus amylovorus GRL1112

Lactobacillus amylovorus is a common member of the normal gastrointestinal tract (GIT) microbiota in pigs. Here, we report the genome sequence of L. amylovorus GRL1112, a porcine feces isolate displaying strong adherence to the pig intestinal epithelial cells. The strain is of interest, as it is a potential probiotic bacterium.     

Dairy bacteria remove in vitro dietary lectins with toxic effects on colonic cells

Aims:  To assess in vitro the ability of some dairy bacteria to bind concanavalin A (Con A), peanut agglutinin (PNA) and jacalin (AIL), preventing their toxicity on mouse intestinal epithelial cells (IEC).
Methods and Results:  Con A and AIL reduced significantly IEC viability in vitro , as determined by Trypan Blue dye exclusion or by propidium iodide/fluorescein diacetate/Hoescht staining. Different strains of dairy bacteria were able to remove lectins from the media. Two strains were subjected to treatments used to remove S-layer, cell wall proteins, polysaccharides and lectin-like adhesins. They were then assayed for the ability to bind dietary lectins and reduce toxicity against IEC and to adhere to IEC after interaction with lectins. Con A and AIL were removed by Propionibacterium acidipropionici and Propionibacterium freudenreichii by binding with specific sugar moieties on the bacterial surface. Removal of lectins by bacteria impaired IEC protection. Adhesion of P. acidipropionici to IEC was reduced but not abolished after binding Con A or AIL.
Conclusions:  Removal of Con A or AIL by dairy propionibacteria was effective to avoid the toxic effect against colonic cells in vitro.
Significance and Impact of the Study:  Consumption of foods containing these bacteria would be a tool to protect the intestinal epithelia.     

Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM

Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.     

A Potent Probiotic Strain from Cheddar Cheese

A lactic acid bacteria Leuconostoc paramesenteroides was isolated and characterized from cheddar cheese and was adapted to grow at low pH (2.0) and high bile salt concentration (2%) by sequential sub-culturing so that it can survive the extreme environmental condition of gut. Cell hydrophobicity assay shows the maximum adherence of the culture to toluene (46.11%). Adhesion ability was confirmed by in vitro assay using rat intestinal epithelial layer. The culture has an antimicrobial activity against food borne pathogens and is vancomycin sensitive. The culture shows a β-galactosidase activity of 3.42 μM/mg protein, which indicates the ability of the culture to hydrolyze lactose for easy absorption. All these properties determine the ability of the culture to be used as a probiotic.