Soile Tynkkynen

Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein

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.

Lactobacillus Bacteremia during a Rapid Increase in Probiotic Use of Lactobacillus rhamnosus GG in Finland

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.

Lactobacillus Bacteremia, Clinical Significance, and Patient Outcome, with Special Focus on Probiotic L. Rhamnosus GG

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.

Probiotic supplementation improves tolerance to Helicobacter pylori eradication therapy--a placebo-controlled, double-blind randomized pilot study.

Background : H. pylori is the major cause of chronic gastritis, and a risk factor for peptic ulcer and gastric cancer.

Mucosal Adhesion Properties of the Probiotic Lactobacillus rhamnosus GG SpaCBA and SpaFED Pilin Subunits

ABSTRACT 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.

The effect of age and non-steroidal anti-inflammatory drugs on human intestinal microbiota composition

Ageing has been suggested to cause changes in the intestinal microbial community. In the present study, the microbiota of a previously well-defined group of elderly subjects aged between 70 and 85 years, both non-steroidal anti-inflammatory drugs (NSAID) users (n 9) and non-users (n 9), were further compared with young adults (n 14) with a mean age of 28 years, by two DNA-based techniques: percentage guanine+cytosine (%G+C) profiling and 16S rDNA sequencing. Remarkable changes in microbiota were described with both methods: compared with young adults a significant reduction in overall numbers of microbes in both elderly groups was measured. Moreover, the total number of microbes in elderly NSAID users was higher than in elderly without NSAID. In 16S rDNA sequencing, shifts in all major microbial phyla, such as lower numbers of Firmicutes and an increase in numbers of Bacteroidetes in the elderly were monitored. On the genus level an interesting link between reductions in the proportion of known butyrate producers belonging to Clostridium cluster XIVa, such as Roseburia and Ruminococcus, could be demonstrated in the elderly. Moreover, in the Actinobacteria group, lower numbers of Collinsella spp. were evident in the elderly subjects with NSAID compared both with young adults and the elderly without NSAID, suggesting that the use of NSAID along with age may also influence the composition of intestinal microbiota. Furthermore, relatively high numbers of Lactobacillus appeared only in the elderly subjects without NSAID. In general, the lowered numbers of microbial members in the major phyla, Firmicutes, together with changes in the epithelial layer functions can have a significant effect on the colon health of the elderly.

Lactobacillus bacteremia, species identification, and antimicrobial susceptibility of 85 blood isolates.

BACKGROUND Data regarding antimicrobial susceptibility of clinical Lactobacillus isolates are scarce, and appropriate interpretation criteria for susceptibility tests are not available. METHODS We examined 85 cases of Lactobacillus bacteremia, of which 47 cases have been included in our previous studies. Overall, 14 antimicrobial agents were evaluated by the E-test method, and these results were compared with disk diffusion test findings. The clinical outcomes of the patients and their antimicrobial treatments were registered. RESULTS The antimicrobial susceptibility of Lactobacillus strains was species dependent. The considerable number of Lactobacillus rhamnosus (n=46), Lactobacillus fermentum (n=12), and Lactobacillus casei (n=12) strains available for testing made it possible to compare the susceptibilities within 1 species, as well. Of the 46 L. rhamnosus isolates, 22 were identified as L. rhamnosus GG type by pulsed-field gel electrophoresis. All Lactobacillus isolates demonstrated low minimum inhibitory concentrations (MICs) of imipenem, piperacillin-tazobactam, erythromycin, and clindamycin. MICs of vancomycin were high (>256 microg/mL) for all other species except Lactobacillus gasseri and Lactobacillus jensenii. Disk diffusion and E-test results were concordant. The MICs of cephalosporins varied; cefuroxime demonstrated a higher level of activity than did ceftriaxone. Benzylpenicillin and ampicillin MICs had variable ranges between different species. Combination therapy was given to 83% of the patients, but, in 54% of them, therapy included only 1 microbiologically active agent, according to results of the susceptibility tests. Mortality at 1 week was 12% among patients who presumably were receiving adequate treatment and 27% among patients who were receiving inadequate treatment (P=.131, by E-test). CONCLUSION Most clinical Lactobacillus blood isolates demonstrated low MICs of imipenem, piperacillin-tazobactam, erythromycin, and clindamycin, but they had variable susceptibility to penicillin and cephalosporins.

Proteomics and Transcriptomics Characterization of Bile Stress Response in Probiotic Lactobacillus rhamnosus GG

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.

Vancomycin resistance factor of Lactobacillus rhamnosus GG in relation to enterococcal vancomycin resistance (van) genes

Lactobacillus rhamnosus GG (ATCC 53103) is a probiotic strain used in fermented dairy products in many countries and is also used as a food supplement in the form of freeze-dried powder. The relationship of the vancomycin resistance factor in L. rhamnosus GG and the vancomycin resistance (van) genes of Enterococcus faecalis and E. faecium were studied using polymerase chain reaction (PCR), Southern hybridization and conjugation methods. Our results show that the vancomycin resistance determinant in L. rhamnosus GG is not closely related to enterococcal van genes, since no PCR product was amplified in L. rhamnosus GG with any of the three sets of vanA primers used, and enterococcal vanA, vanB, vnH, vanX, vanZ, vanY, vanS and vanR genes did not hybridize with DNA of L. rhamnosus GG. This strain does not contain plasmids and transfer of chromosomal vancomycin resistance determinant from L. rhamnosus GG to enterococcal species was not detected. Our results are in accordance with previous findings of intrinsically vancomycin-resistant lactic acid bacteria.

Persistence of probiotic strains in the gastrointestinal tract when administered as capsules, yoghurt, or cheese

Most clinical studies of probiotics use freeze-dried, powdered bacteria or bacteria packed in capsules. However, probiotics are commercially available in various food matrices, which may affect their persistence in the gastrointestinal tract. The objective of the study was to compare oral and faecal recovery during and after administration of a combination of Lactobacillus rhamnosus GG and LC705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium animalis subsp. lactis Bb12 as capsules, yoghurt, or cheese. This randomized, parallel-group, open-label trial (n=36) included a 4-week run-in, 2-week intervention, and 3-week follow-up period. Participants consumed 10(10)cfu/day of probiotic combination and provided saliva and faecal samples before, during, and after the intervention. Strain-specific real-time PCR was used to quantify the strains. L. rhamnosus GG was the only probiotic strain regularly recovered in saliva samples. During the intervention period it was recovered in the saliva of 88% of the volunteers at least once. No difference was found between the yoghurt and cheese groups. At the end of the intervention, L. rhamnosus GG and LC705 counts were high in faecal samples of all product groups (8.08 and 8.67log(10) genome copies/g, respectively). There was no matrix effect on strain quantity in faeces or the recovery time after ceasing the intervention. For P. freudenreichii subsp. shermanii JS and B. animalis subsp. lactis Bb12, a matrix effect was found at the end of the intervention (P<0.01 and P<0.001, respectively) and in the recovery time during follow-up (P<0.05 for both). Yoghurt yielded the highest faecal quantity of JS and Bb12 strains (8.01 and 9.89log(10) genome copies/g, respectively). The results showed that the administration matrix did not influence the faecal quantity of lactobacilli, but affected faecal counts of propionibacteria and bifidobacteria that were lower when consumed in cheese. Thus, the consumption of probiotics in yoghurt matrix is highly suitable for studying potential health benefits and capsules provide a comparable means of administration when the viability of the strain in the capsule product is confirmed.