To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an ...adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins ( spaCBA ) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.
AIM： To evaluate the effects of three potentially anti-inflammatory probiotic bacteria from three different genera on immune variables in setting based on previous in cytokine responses. healthy ...adults in a clinical vitro characterization of METHODS： A total of 62 volunteers participated in this randomized, double-blind and placebo-controlled parallel group intervention study. The volunteers were randomized to receive a milk-based drink containing either Lactobacillus rhamnosus GG （LGG）, Bifidobacterium animalis ssp. lactis Bb12 （Bb12）, or Propionibacterium freudenreichii ssp. shermanii JS （PJS） or a placebo drink for 3 wk. Venous blood and saliva samples were taken at baseline and on d 1, 7 and 21. Fecal samples were collected at baseline and at the end of intervention. RESULTS： The serum hsCRP expressed as the median AUC0-21 （minus baseline） was 0.018 mg/L in the placebo group, -0.240 mg/L in the LGG group, 0.090 mg/L in the Bb12 group and -0.085 mg/L in the PJS group （P = 0.014）. In vitro production of TNF-α from in vitro cultured peripheral blood mononuclear cells （PBMC） was significantly lower in subjects receiving LGG vs placebo. IL-2 production from PBMC in the Bb12 group was significantly lower compared with the other groups. CONCLUSION： In conclusion, probiotic bacteria have strain-specific anti-inflammatory effects in healthy adults.
Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are ...likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.
Lactobacillus rhamnosus GG (GG) is a widely used and intensively studied probiotic bacterium. Although the health benefits of strain GG are well documented, the systematic exploration of mechanisms ...by which this strain exerts probiotic effects in the host has only recently been initiated. The ability to survive the harsh conditions of the gastrointestinal tract, including gastric juice containing bile salts, is one of the vital characteristics that enables a probiotic bacterium to transiently colonize the host. Here we used gene expression profiling at the transcriptome and proteome levels to investigate the cellular response of strain GG toward bile under defined bioreactor conditions. The analyses revealed that in response to growth of strain GG in the presence of 0.2% ox gall the transcript levels of 316 genes changed significantly (p < 0.01, t test), and 42 proteins, including both intracellular and surface-exposed proteins (i.e. surfome), were differentially abundant (p < 0.01, t test in total proteome analysis; p < 0.05, t test in surfome analysis). Protein abundance changes correlated with transcriptome level changes for 14 of these proteins. The identified proteins suggest diverse and specific changes in general stress responses as well as in cell envelope-related functions, including in pathways affecting fatty acid composition, cell surface charge, and thickness of the exopolysaccharide layer. These changes are likely to strengthen the cell envelope against bile-induced stress and signal the GG cells of gut entrance. Notably, the surfome analyses demonstrated significant reduction in the abundance of a protein catalyzing the synthesis of exopolysaccharides, whereas a protein dedicated for active removal of bile compounds from the cells was up-regulated. These findings suggest a role for these proteins in facilitating the well founded interaction of strain GG with the host mucus in the presence of sublethal doses of bile. The significance of these findings in terms of the functionality of a probiotic bacterium is discussed.
Insight into the diversity and function of the human intestinal microbiota has been stimulated by clinical studies with bacteria that exhibit specific functions and which are marketed as probiotics ...to positively affect our health. Initial efforts concentrated on establishing sound scientific support for the efficacy of these probiotic bacteria, which mainly include
Lactobacillus and
Bifidobacterium species. Following these evidence-based functional approaches, considerable research is now focused on the mechanisms of action of probiotic bacteria. The mechanisms identified to date mainly relate to the stimulation of host defence systems, immune modulation and the competitive exclusion of pathogens. Recent efficacy, molecular and genomics-based studies have also been reported for some probiotic strains that have found their position in the market place.
Lactobacillus bacteremia is a rare entity, and its clinical significance is poorly defined. We have reviewed the risk factors and outcome for 89 case patients with Lactobacillus bacteremia. Species ...characterization was done in 53% of the cases, revealing 25 L. rhamnosus strains and 22 other Lactobacillus species. In 11 cases, the strain was identical with the probiotic L. rhamnosus GG. In 82% of the cases, the patients had severe or fatal comorbidities. Predisposing factors to bacteremia were immunosuppression, prior prolonged hospitalization, and prior surgical interventions. No significant differences were observed in these predisposing factors or clinical features between patients with cases associated with the various Lactobacillus species, other than higher C-reactive protein values in patients with L. rhamnosus bacteremia. Mortality was 26% at 1 month and was 48% at 1 year. In multivariate analysis, severe underlying diseases were a significant predictor for mortality (odds ratio OR, 15.8), whereas treatment with antimicrobials effective in vitro was associated with lower mortality (OR, 0.22). We conclude that lactobacilli in blood cultures are of clinical significance and that their susceptibility should guide decisions about antimicrobial treatment.
AIM： To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyte-derived dendritic cells （moDCs）.
METHODS： Cytokine ...production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay （ELISA） and flow cytometric analysis （FACS）, respectively. The kinetics of mRNA expression of cytokine genes was determined by Northern blotting. The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors.
RESULTS： All studied bacteria induced the maturation of moDCs in a dose-dependent manner. More detailed analysis with S. thermophilus THS, B. breve Bb99, and L. lactis subsp, cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class Ⅱ and CD86 as efficiently as pathogenic bacteria. However, these bacteria differed in their ability to induce moDC cytokine gene expression. S. thermophilus induced the expression of pro-inflammatory （TNF-α, IL-12, IL-6, and CCL20） and Thl type （IL-12 and IFN-γ） cytokines, while B. breve and L. lactis were also potent inducers of antiinflammatory IL-10. Mitogen-activated protein kinase （MAPK） p38, phosphatidylinositol 3 （PI3） kinase, and nuclear factor-kappa B （NF-κB） signaling pathways were shown to be involved in bacteria-induced cytokine production. CONCLUSION： Our results indicate that potentially probiotic bacteria are able to induce moDC maturation, but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another,
Lactobacilli supposedly have low pathogenicity; they are seldom detected in blood culture. Lactobacillus rhamnosus GG, which originates indigenously in the human intestine, became available for use ...as a probiotic in 1990 in Finland. We evaluated the possible effects of the increased probiotic use of L. rhamnosus GG on the occurrence of bacteremia due to lactobacilli. Lactobacilli were isolated in 0.02% of all blood cultures and 0.2% of all blood cultures with positive results in Helsinki University Central Hospital and in Finland as a whole, and no trends were seen that suggested an increase in Lactobacillus bacteremia. The average incidence was 0.3 cases/100,000 inhabitants/year in 1995-2000 in Finland. Identification to the species level was done for 66 cases of Lactobacillus bacteremia, and 48 isolates were confirmed to be Lactobacillus strains. Twenty-six of these strains were L. rhamnosus, and 11 isolates were identical to L. rhamnosus GG. The results indicate that increased probiotic use of L. rhamnosus GG has not led to an increase in Lactobacillus bacteremia.
Although generally innocuous, Lactobacillus species have been isolated infrequently in connection to serious infections such as endocarditis and sepsis. We report, to our knowledge, the first case of ...severe infection due to a Lactobacillus rhamnosus strain indistinguishable from L. rhamnosus strain GG.
The effects of a probiotic mixture (PRO), supplemented with either galacto-oligosaccharide (GOS) or polydextrose (PDX), on cell numbers of lactic acid bacteria (LAB) and bifidobacteria (BIF) were ...studied in conventional rats and healthy human subjects. In rats the baseline BIF cell numbers were below the detection limit and were increased by the 2-week GOSPRO intervention. In contrast baseline LAB numbers in rats were high and not affected by the treatments. The human study consisted of two independent but concurrent trials; both started with PRO followed by GOSPRO or PDXPRO periods. In the human subjects variation in numbers of BIF and LAB were high. The GOSPRO group exhibited high counts of faecal LAB and BIF at the start and showed little or no effects of the interventions. In contrast, the PDX group had low faecal LAB and BIF numbers at the start and clearly increased cell numbers of BIF after the PDXPRO period, and LAB after the PRO and PDXPRO period, compared with the run-in period. We propose here that responses to pro- and prebiotics are dependent on baseline numbers of LAB and/or BIF, and that the conventional rat model does not predict well the treatment responses in humans. The survival of PRO was presumably enhanced by the use of prebiotic supplementation and advocates the use of particular combinations of pro- and prebiotics.
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