Adhesion of inactivated probiotic strains to intestinal mucus

It has been suggested that probiotics should be viable in order to elicit beneficial health effects. Inactivation of probiotics has been suggested to interfere with the binding to the mucosa and thereby with the immune modulating activity of probiotics. The effect of different inactivation methods on the mucus adhesion of nine probiotic strains was studied. Inactivation by heat or gamma-irradiation generally decreased the adhesive abilities. However, heat treatment increased the adhesion of Propionibacterium freudenreichii and gamma-irradiation enhanced the adhesion of Lactobacillus casei Shirota. Inactivation by u.v. was not observed to modulate the adhesion of the tested strains and it was concluded to be the most appropriate method for studying non-viable probiotics and preparing control products.     

The adhesion of putative probiotic lactobacilli to cultured epithelial cells and porcine intestinal mucus

Aims: To investigate the adhesion of lactobacilli and their subsequent competitive exclusion ability against pathogens. Methods and Results: Four species of putative probiotic lactobacilli were studied for their adhesion abilities. First, the adhesion to Caco‐2 cells was examined by light and electron microscopy. The four species were then labelled by [methyl‐³H] thymidine and their adhesion to porcine intestinal mucus was determined by radioactivity. The tested lactobacilli showed best adhesion on ileal mucus compared with duodenal and jujenal mucus. Oxidative compound pre‐treatment (NaIO₃ and NaIO₄) dramatically decreased the adhesion of the lactobacilli to mucus. Pre‐treating mucus with proteolytic enzymes (proteinase K and trypsin) resulted in the increase of adhesion in Lactobacillus serotype Reuteri I2021, but the results in the other species were variable. Lactobacillus serotype Fermentum I5007 showed greatest adhesion potential and exerted the best competitive exclusion against Salmonella and Escherichia. Conclusions: Adhesion ability in lactobacilli is species‐specific. Lactobacilli with higher adhesion index have better competitive exclusion ability. Significance and Impact of the Study: This study suggests that there is a positive correlation between adhesion and competitive exclusion ability of lactobacilli. Additionally, the in vitro adhesion assay is a feasible way to screen unknown lactobacilli, potentially for future industrial applications.     

Colonization by lactobacilli of piglet small intestinal mucus

The colonization potential of lactobacilli was investigated using small intestinal mucus extracts from 35-d-old pigs. Mucus-secreting tissue from the small intestine of piglets was gently rinsed to remove contents and then shaken in buffer to release mucus from the surface. Numbers of lactobacilli in different portions of the small intestine of 35-d-old pigs were enumerated. Also, mucus isolated from the small intestine of pigs was investigated for its capacity to support the growth of lactobacilli. Results indicated that Lactobacillus spp. inhabit the mucus layer of the small intestine and can grow and adhere to ileal mucus. From adhesion studies of Lactobacillus fermentum 104R to mucus analysed by Scatchard plot, it is suggested that an associating system showing positive cooperativity is involved. Proteinaceous compound(s) involved in the adhesion to mucus were detected in the spent culture fluid from the growth of strain 104R. Studies are continuing in order to identify and characterize the adhesion-promoting protein(s). From the data, it is proposed that lactobacilli colonize the mucus layer of the small intestine of pigs.     

Characterization of intestinal lactobacilli as putative probiotic candidates

AIMS: To use antioxidative activity and antagonistic properties of lactobacilli against selected pathogens and members of the normal microflora as a basis for screening probiotic candidates. METHODS AND RESULTS: Antagonistic activity of lactobacilli against target bacteria in both microaerobic and anaerobic environments was tested. Production of antagonistic metabolites (ethanol, hydrogen peroxide (H2O2), acetic, lactic and succinic acid) by lactobacilli as well as their total antioxidative activity were assessed. In general, the lactobacilli tested were most effective against Gram-negative bacteria and their antagonistic activity was strain-specific. However, obligately heterofermentative lactobacilli had the strongest activity when tested in a microaerobic environment. Additionally, facultatively heterofermentative lactobacilli were equally effective in either milieu and produced significant levels of acetic and lactic acid. Moreover, obligately homofermentative lactobacilli had high H2O2 production and total antioxidative activity but weak antagonistic activity. CONCLUSIONS: Antioxidative and antagonistic activity of intestinal lactobacilli is strain-specific but typically can be related to their fermentation type which may be used for rapidly screening large numbers of lactobacilli for probiotic candidates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first report on the utilization of group characteristics to screen lactobacilli intended for specific probiotic use. Such uses include the targeting of particular gut niches and pathogens as well as allowing for long-term benefits to the host.     

Adhesion of Bifidobacterium spp. to human intestinal mucus

Twenty-four Bifidobacterium strains were examined for their ability to bind to immobilized human and bovine intestinal mucus glycoproteins. Each of the tested bacteria exhibited its characteristic adhesion to human and bovine fecal mucus. No significant differences were found among the taxonomic species. Among the tested bacteria, B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum and B. pseudocatenulatum adhered to human fecal mucus better than bovine fecal mucus, while the binding of B. animalis and B. lactis was not preferential. These results suggest that the mucosal adhesive properties of bifidobacteria may be a strain dependent feature, and the mucosal binding of the human bifidobacteria may be more host specific.     

In vitro appraisal of the probiotic value of intestinal lactobacilli

Lactobacilli play a distinctive role in the microbial balance of the chicken gut. In experiments simulating the chicken crop, the antagonism of lactobacilli against Enterobacteriaceae and Salmonella typhimurium was demonstrated and was attributed to lactic acid production. Moreover, adhesion to the crop epithelium was a common characteristic of intestinal lactobacilli. As opposed to salmonellas, lactobacilli were sensitive to deconjugated bile salts at 2.5mm. This sensitivity could lower their chance of proliferation in the small bowel of the chicken tract.The authors are at the Laboratory of Microbial Ecology, Faculty of Agricultural Sciences, State University of Gent, Coupure L., 653, B-9000 Gent, Belgium.     

Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus

AIMS: The aims of this study present were to assess and to evaluate in vitro the abilities of commercial probiotic strains derived from fermented milk products and related sources currently marketed in European countries, to inhibit, compete and displace the adhesion of selected potential pathogens to immobilized human mucus. METHODS AND RESULTS: The adhesion was assessed by measuring the radioactivity of bacteria adhered to the human mucus. We tested 12 probiotic strains against eight selected pathogens. All strains tested were able to adhere to mucus. All probiotic strains tested were able to inhibit and displace (P<0.05) the adhesion of Bacteroides, Clostridium, Staphylococcus and Enterobacter. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting that several complementary mechanisms are implied in the processes. CONCLUSIONS: Our results indicate the need for a case-by-case assessment in order to select strains with the ability to inhibit or displace a specific pathogen. Probiotics could be useful to correct deviations observed in intestinal microbiota associated with specific diseases and also, to prevent pathogen infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The competitive exclusion properties of probiotics as well as their ability to displace and inhibit pathogens are the most importance for therapeutic manipulation of the enteric microbiota. The application of such strategies could contribute to expand the beneficial properties on human health against pathogen infection.     

The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.     

Specific probiotic strains and their combinations counteract adhesion of Enterobacter sakazakii to intestinal mucus

Enterobacter sakazakii is an opportunistic pathogen and an occasional contaminant in powdered infant formula. Interaction between specific probiotics and E. sakazakii may reduce the risk of infection. The aim of this study was to characterize in vitro the ability of probiotics (alone and in combinations) to inhibit, compete with and displace the adhesion of E. sakazakii to immobilized human mucus and to assess their capacity to aggregate with pathogen. Specific probiotic strains have proved to aggregate E. sakazakii cells and, through competitive exclusion, inhibition and displacement of the adhered pathogen, were able to inhibit E. sakazakii action on intestinal mucus. The ability to inhibit and to displace adhered pathogen depended on both the probiotic and the pathogen, suggesting that several complementary mechanisms are involved in the processes. We suggest that the selection of specific probiotic strains and their combinations may be a useful means of counteracting E. sakazakii contamination in infant formula and thus to reduce the risk of emerging infection. This approach may also allow the development of new probiotic combinations to counteract the risks associated with other pathogens by improving the intestinal barrier against pathogens.     

Structural basis for adaptation of lactobacilli to gastrointestinal mucus

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the underpinning mechanisms of these interactions are not understood. Here, we provide structural and functional insights into the canonical mucus‐binding protein (MUB), a multi‐repeat cell‐surface adhesin found in Lactobacillus inhabitants of the GI tract. X‐ray crystallography together with small‐angle X‐ray scattering demonstrated a ‘beads on a string’ arrangement of repeats, generating 174 nm long protein fibrils, as shown by atomic force microscopy. Each repeat consists of tandemly arranged Ig‐ and mucin‐binding protein (MucBP) modules. The binding of full‐length MUB was confined to mucus via multiple interactions involving terminal sialylated mucin glycans. While individual MUB domains showed structural similarity to fimbrial proteins from Gram‐positive pathogens, the particular organization of MUB provides a structural explanation for the mechanisms in which lactobacilli have adapted to their host niche by maximizing interactions with the mucus receptors, potentiating the retention of bacteria within the mucus layer. Together, this study reveals functional and structural features which may affect tropism of microbes across mucus and along the GI tract, providing unique insights into the mechanisms adopted by commensals and probiotics to adapt to the mucosal environment.     

Some probiotic properties of chicken lactobacilli

The beneficial effect of lactobacilli has been attributed to their ability to colonize human and animal gastrointestinal tracts. In this work, adhesion assays with three lactobacillus strains and intestinal fragments obtained from chickens were assessed. Lactobacillus animalis and L. fermentum were able to adhere to three kinds of epithelial cells (crop, small and large intestines) with predominance to small intestine. Among the strains considered, L. fermentum subsp. cellobiosus showed the lowest and L. animalis the highest adhesion ability. Scanning electron microphotographs showing L. animalis and L. fermentum adhering to intestinal cells were obtained. The characterization of L. animalis adhesion indicated that lectin-like structure of this strain has glucose/mannose as specific sugars of binding. However, a calcium requirement was not observed. The adhesion of L. fermentum was reduced by addition of sialic acid or mannose (P < 0.01). These carbohydrates can be involved in the interaction between adhesin and epithelial surface. In this case, the dependence on bivalent cations was demonstrated. Lactobacillus fermentum was effective in reducing the attachment of Salmonella pullorum by 77%, while L. animalis was able to inhibit (90%, 88%, and 78%) the adhesion of S. pullorum, S. enteritidis, and S. gallinarum to host-specific epithelial fragments respectively. Our results from this in vitro model suggest that these lactobacilli are able to block the binding sites for Salmonella adhesion.     

Adhesion of four Bifidobacterium strains to human intestinal mucus from subjects in different age groups

The number of bifidobacteria in faeces and intestinal contents has been shown to be reduced with increasing age of the subject. The adhesion of four Bifidobacterium strains was tested to mucus isolated from subjects of different age. All strains bound significantly less to mucus isolated from elderly subjects, compared to mucus from the other age groups. Two of the tested strains also showed decreased adhesion to mucus isolated from 6-month-old and adult subjects compared to the adhesion to mucus from 2-month-old subjects. The results suggest that reduced adhesion may be a factor involved in the decreasing colonisation of elderly subjects by bifidobacteria.     

Selection of lactobacilli for chicken probiotic adjuncts

During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains (Salmonella enteritidis and Escherichia coli). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3.0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo. It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.     

Selection of lactobacilli for chicken probiotic adjuncts

During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains ( Salmonella enteritidis and Escherichia coli ). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3·0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo . It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.     

Application of fluorescent techniques to evaluate the survival of probiotic lactobacilli to bile acid

Purpose of work  

To apply a fluorescent dye as an alternative technique to evaluate the survival of potentially probiotic lactobacilli to bile acids (BA) as first step in the design of probiotic functional foods.     

Adhesion of lactobacilli to polymer surfaces in vivo and in vitro

The ability of bacteria to attach to surfaces has been recognized as an important phenomenon, particularly for pathogenic organisms that utilize this capacity to initiate disease. The present investigation was undertaken to determine whether indigenous urogenital bacteria, lactobacilli, colonized prosthetic devices in vivo and in vitro and attached to specific polymer surfaces in vitro. Polyethylene intrauterine devices (IUDs) in place for 2 years were removed from six women who were asymptomatic and free of signs of cervical or uterine infection. Lactobacilli were found attached to the IUDs, as determined by culture, and fluorescent antibody and acridine orange staining techniques. This demonstrated that bacterial biofilms consisting of indigenous bacteria can occur on prosthetic devices without inducing a symptomatic infection. In vitro studies were then undertaken with well-documented lactobacilli strainsL. acidophilus T-13,L. casei GR-1, GR-2, and RC-17, andL. fermentum A-60. These organisms were found to adhere to IUDs and urinary catheters within 24 hours. A quantitative assay was designed to examine the mechanisms of adhesion ofL. acidophilus T-13 to specific polymer surfaces that are commonly used as prosthetic devices. The lactobacilli adhered optimally to fluorinated ethylene propylene when 10⁸ bacteria were incubated for 9 hours at 37°C in phosphate buffered saline, pH 7.1. Additional experiments verified that the lactobacilli adhered to polyethyleneterephthalate, polystyrene, and sulfonated polystyrene and to silkolatex catheter material. There was a linear relationship found between polymer hydrophobicity and bacterial adherence. These results demonstrate that lactobacilli bind to various surfaces in vivo and in vitro, and that the nature of the substratum can affect the colonization.     

Adherence capacities of oral lactobacilli for potential probiotic purposes

The most abundantly used probiotic strains come from the genus Lactobacillus and only a few studies have investigated their role in oral health. Even if a positive correlation has been established between the saliva Lactobacillus count and dental caries, this genus is generally recognized as safe (GRAS). Moreover, lactobacilli could in some cases play a beneficial role by inhibiting the growth of some oral pathogenic bacteria. This activity could justify their use as probiotic. To establish the potential health benefit of probiotic candidates, appropriate in?vitro tests are required, particularly on their adhesive capacity. The aim of this work was to investigate the adhesive properties and surface characteristics of 70 oral lactobacilli that could be used as probiotics for oral health. For this, three methods were used: biofilm formation on a glass surface and on saliva-coated hydroxyapatite discs and the microbial adhesion to solvent method. The results of the biofilm formed on glass surface showed 13 strains with an adhesion score equal to or higher than 3. 57/70 (81%) of the tested lactobacilli did not form any biofilm on glass surfaces. All of the 13 strains formed biofilms on HA discs. Among these 13 strains, 10/13 (77%) showed low surface hydrophobicity (0-35%) and 3/13 (23%) showed medium hydrophobicity (36-70%). Some of the selected strains showed potentially useful adhesive capacity. This work paves the way for the selection of probiotics that could be used for oral health purposes with the aim to reduce carious risk.     

Isolation of lactobacilli with probiotic properties from the human stomach

Aims: Recent evidence suggests that the human gastric microbiota is much more diverse than previously thought. The aim of this study was to assess the potential for isolating lactobacilli from the human stomach. Methods and Results: Lactobacilli were selectively cultured from gastric biopsies from 12 patients undergoing routine endoscopy. Lactobacilli were present in four of 12 biopsies. We isolated, in total 10 different strains representing five species (Lactobacillus gasseri, L. fermentum, L. vaginalis, L. reuteri and L. salivarius). The 10 isolates varied greatly in their ability to inhibit the growth of two Gram‐positive bacteria and two Gram‐negative bacteria. Furthermore, the acid and bile resistance profiles of the 10 isolates spanned a wide range. Conclusions: Five different Lactobacillus species were cultured from human gastric biopsies for the first time. Significance and Impact of the Study: Diverse Lactobacillus species are more prevalent in the human stomach than previously recognized, representing an untapped source of bacteria with beneficial probiotic and/or biotechnological properties.