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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 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.

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.

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.

Elderly individuals are more susceptible to gastrointestinal problems such as constipation than young adults. Furthermore, the common use of non-steroidal anti-inflammatory drugs (NSAID) among the elderly is known to further increase such gastrointestinal ailments. To describe the specific changes in elderly, intestinal microbes, their metabolites and immune markers were measured from faecal samples obtained from fifty-five elderly individuals (aged 68–88 years), using either NSAID or not, and fourteen young adults (aged 21–39 years). The faecal DM content increased with age but was significantly lower among the elderly NSAID users. The microbial metabolism was especially influenced by NSAID use and/or ageing, although fewer changes were observed in the composition of the microbial community, whilst the level of aerobes was increased in the elderly and the level of Clostridium coccoides–Eubacterium rectale reduced in the elderly NSAID users as compared with young adults. An increase in the concentrations of some branched SCFA and l-lactate but a decrease in some major SCFA concentrations were observed. Evidently, the decreased defecation frequency in the elderly directed colonic fermentation toward an unfavourable microbial metabolism but this was partially offset by the use of NSAID. Irrespective of the use of NSAID, the elderly subjects had significantly lower concentrations of faecal PGE2 than the young adults, reflecting possibly a reduced immune response. According to the present study more attention should be paid to the development of dietary products that seek to enhance bowel function, saccharolytic fermentation and immune stimulation in the elderly population.

Summary The growth phase during which probiotic bacteria are harvested and consumed can strongly influence their performance as health‐promoting agents. In this study, global transcriptomic and proteomic changes were studied in the widely used probiotic Lactobacillus rhamnosus GG during growth in industrial‐type whey medium under strictly defined bioreactor conditions. The expression of 636 genes (P ≤ 0.01) and 116 proteins (P < 0.05) changed significantly over time. Of the significantly differentially produced proteins, 61 were associated with alterations at the transcript level. The most remarkable growth phase‐dependent changes occurred during the transition from the exponential to the stationary growth phase and were associated with the shift from glucose fermentation to galactose utilization and the transition from homolactic to mixed acid fermentation. Furthermore, several genes encoding proteins proposed to promote the survival and persistence of L. rhamnosus GG in the host and proteins that directly contribute to human health showed temporal changes in expression. Our results suggest that L. rhamnosus GG has a highly flexible and adaptable metabolism and that the growth stage during which bacterial cells are harvested and consumed should be taken into consideration to gain the maximal benefit from probiotic bacteria.

The present randomised, double-blind, placebo-controlled study was conducted to determine whether consumption of probiotic Lactobacillus rhamnosus GG (GG) would lead to the recovery of GG in tonsil tissue. After 3 weeks' daily consumption of GG as a single strain (n 20), GG as a part of a multispecies combination (n 17) or placebo (n 20), tonsil tissue samples were collected from fifty-seven young adults during tonsillectomy due to chronic or recurrent tonsillitis. Strain-specific real-time PCR was used to detect GG in the tonsil tissue. GG was recovered in the tonsil sample of 40 % of the subjects in the GG group, 41 % in the multispecies group and 30 % in the placebo group (P value between groups 0·79). In all subjects with positive recovery of GG in the tonsil tissue, GG was also recovered in the faecal sample taken at the start of the intervention and at the time of the tissue sample collection, which indicates more persistent adherence of the probiotic. To conclude, GG can be recovered from tonsil tissue after oral administration as a single-strain probiotic or as a part of a multispecies probiotic combination. The present results suggest that individual variation exists in the ability of GG to adhere to tonsil tissue. Persistence of GG appears to be high in tonsil tissue as well, in addition to persistence in faecal samples, which has been demonstrated previously. Further clinical trials are warranted to evaluate whether probiotic adherence in the tonsil tissue could have a role in respiratory symptom prevalence.

The aim of this study was to compare a combination of Lactobacillus GG (LGG) and galacto-oligosaccharides (GOS) with LGG on its own, and their effects on the intestinal microbiota in school-aged children. The randomized, double-blinded, crossover study comprised 30 healthy children. There were two 3-week study periods with a 4-week wash-out period in between. The children ingested daily 65 ml of milk-based fruit juice containing either LGG alone (6.5 x 10(9) CFU) or LGG plus 2 g of GOS. Symptom diaries were filled during the study periods. Fecal samples were collected at the beginning and end of both study periods. At the end of both study periods, the amount of bifidobacteria was significantly greater after the ingestion of LGG + GOS compared with LGG alone (geometric mean 9.33 x 10(9) vs. 4.28 x 10(9) CFU/g, p < 0.001). No significant differences were seen in the amount of lactobacilli or LGG, nor did gastrointestinal symptoms, defecation frequency, consistency of stools or ease of defecation differ between the two study periods. Ingestion of LGG combined with 2 g of GOS increased the bifidobacteria more than LGG on its own and thus GOS clearly has a prebiotic effect in children. The children tolerated well a daily intake of 2 g of GOS.