Colonic methane production modifies gastrointestinal toxicity associated with adjuvant 5-fluorouracil chemotherapy for colorectal cancer.

GOALS: To investigate the association of colonic methane, formed by methanogenic achaea, and pH with gastrointestinal symptoms during colorectal cancer chemotherapy. BACKGROUND: Adjuvant 5-fluorouracil chemotherapy reduces recurrences in colorectal cancer, but causes severe gastrointestinal toxicity, partly related to disturbed intestinal microbiota. STUDY: Resected colorectal cancer patients (n=143) were analyzed for colonic methanogenesis and pH before and during the 24 weeks of 5-fluorouracil chemotherapy and for gastrointestinal symptoms during chemotherapy. This study was performed within the setting of an intervention study on the effects of Lactobacillus on chemotherapy-related gastrointestinal toxicity. The site of resected cancer, resection type, stoma, chemotherapy regimen, hypolactasia, and Lactobacillus intervention were considered as possible confounding factors, and multivariate models were constructed. RESULTS: Baseline methane producers had less frequent diarrhea (more than or equal to moderate) during chemotherapy than nonproducers [odds ratio (OR), 0.42; 95% confidence interval (CI), 0.20 to 0.88; P=0.022] and more frequent constipation (OR, 4.56; 95% CI, 2.01 to 10.32; P<0.001). Baseline fecal pH was also associated with symptoms during chemotherapy; higher the pH, the lower the risk of diarrhea (OR, 0.56; 95% CI, 0.31 to 1.02; P=0.058) and higher the risk of constipation (OR, 2.23; 95% CI, 1.35 to 3.68; P=0.002). In multivariate stepwise models, methanogenesis was a significant explaining factor with inverse association with diarrhea and positive association with constipation. Fecal pH, which was significantly associated with methane production, was no longer a significant explaining factor when methanogensis was included in the model. CONCLUSIONS: Methane producer status has a role in determining whether patient experiences diarrhea or constipation during 5-fluorouracil therapy. This underscores the importance of intestinal microbiota in the development of intestinal toxicity during 5-fluorouracil therapy.

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.

Constipation is relieved more by rye bread than wheat bread or laxatives without increased adverse gastrointestinal effects.

Rye bread and lactobacilli modify the colonic environment and have the potential to relieve constipation and could be a safe and convenient alternative to laxatives. The effects of rye bread and cultured buttermilk with Lactobacillus rhamnosus GG (LGG) on bowel function and colon metabolism were investigated and compared with laxatives in 51 constipated adults. They were randomized to receive whole-grain rye bread (minimum 240 g/d), LGG (2 x 10(10) colony-forming units/d), whole-grain rye bread (minimum 240 g/d) + LGG (2 x 10(10) colony-forming units/d), white wheat bread (maximum 192 g/d), or laxatives (as usual for a participant) for 3 wk. Participants recorded their dietary habits, bowel function, and gastrointestinal symptoms. Fecal weight, pH, SCFA and bacterial enzyme activities, total intestinal transit time (TITT), and breath hydrogen were determined. Rye bread, compared with white wheat bread, shortened TITT by 23% (P = 0.040), increased weekly defecations by 1.4 (P = 0.014), softened feces [odds ratio (OR) 3.98; P = 0.037], eased defecation (OR 5.08; P = 0.018), increased fecal acetic acid and butyric acid contents by 24% (P = 0.044) and 63% (P <0.001), respectively, and reduced fecal beta-glucuronidase activity by 23% (P = 0.014). Compared with laxatives, rye bread reduced TITT by 41% (P = 0.006), fecal beta-glucuronidase activity by 38% (P = 0.033), and fecal pH by 0.31 units (P = 0.006). LGG did not relieve constipation or significantly affect colonic metabolism. Gastrointestinal adverse effects did not significantly differ among the study groups. In conclusion, rye bread relieves mild constipation and improves colonic metabolism compared with white wheat bread and commonly used laxatives without increasing gastrointestinal adverse effects.

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 rhamnosus LC705 together with Propionibacterium freudenreichii ssp shermanii JS administered in capsules is ineffective in lowering serum lipids.

OBJECTIVE: The aim of this study was to examine the effect of the administration of Lactobacillus rhamnosus strain LC705 and Propionibacterium freudenreichii ssp shermanii strain JS in capsules on serum cholesterol and triglyceride levels in mildly or moderately hypercholesterolemic men. METHODS: Thirty-eight basically healthy men, mean age 42 years (range 24-55), mean cholesterol 6.2 mmol/L (5.3-8.2 mmol/L), participated in this double-blind, randomised, placebo-controlled, two-period crossover study with 4-week treatment periods. The subjects consumed daily two probiotic capsules containing viable Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii ssp shermanii JS (2 x 10(10) colony forming units of each strain daily) or two placebo capsules. Serum lipids were assessed before the intervention, at the end of both 4-week treatment periods, and 2 weeks after the second treatment period. Dietary and lifestyle habits were carefully monitored. RESULTS: All the subjects completed the study, and the probiotic capsules were well tolerated. Dietary habits and the intake of energy and nutrients, such as saturated fatty acids and cholesterol, did not differ between the treatment groups. No changes in total cholesterol, HDL cholesterol, LDL cholesterol or triglyceride levels were observed during the consumption of the probiotics compared to placebo. CONCLUSIONS: The administration of Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii ssp shermanii JS did not affect serum lipids.

The influence of Lactobacillus rhamnosus LC705 together with Propionibacterium freudenreichii ssp. shermanii JS on potentially carcinogenic bacterial activity in human colon.

The bacterial enzymes beta-glucosidase, beta-glucuronidase, and urease may contribute to the development of colon cancer by generating carcinogens. A reduction in the activity of these enzymes by certain lactic acid bacteria is considered to be beneficial. This study examined fecal beta-glucosidase, beta-glucuronidase, and urease activities during administration of Lactobacillus rhamnosus LC705 (LC705) together with Propionibacterium freudenreichii ssp shermanii JS (PJS). Thirty-eight healthy men participated in this randomized, double-blind, placebo-controlled, two-period crossover study with treatment periods of 4 weeks. Subjects consumed daily bacterial or placebo capsules. Bacterial capsules contained viable LC705 and PJS (2x10(10) CFU of each strain daily). The activities of beta-glucosidase, beta-glucuronidase and urease, recovery of LC705 and PJS, and counts of total lactobacilli and propionibacteria were determined from feces. The mean fecal counts of total lactobacilli and propionibacteria as well as strains LC705 and PJS were significantly increased during the administration of bacteria (3.5-, 13-, 80- and 11-fold, respectively). beta-glucosidase activity decreased by 10% (P=0.18) and urease activity by 13% (P=0.16) during bacterial supplementation versus placebo. The change in beta-glucosidase activity was negatively correlated with the change in propionibacteria counts (R=-0.350, P=0.039), being -2.68 versus 0.94 nmol/min/mg protein in subjects with increased and unchanged/decreased propionibacteria, respectively (P=0.003). To conclude, the administration of LC705 and PJS was followed by an increase in the fecal counts of lactobacilli and propionibacteria and a decrease in the activity of beta-glucosidase with increasing counts of propionibacteria.

Probiotic formulations and applications, the current probiotics market, and changes in the marketplace: a European perspective.

Europe is a multicultural continent where the consumption of fermented milks has traditionally been high in several countries. Thus, it is no wonder that the market for fermented dairy products with probiotic bacteria has been successful. The market for food applications of probiotics is clearly larger than that for probiotics sold in capsules, sachets, and other pharmaceutical forms. Yogurt-type drinks are the fastest-growing product category, but the diversity of probiotic food applications is not limited to milk-based products. Probiotic fruit juices, berry soups, and soy- and cereal-based fermented products are also sold. Some probiotic strains that are successful in Europe are marketed globally, but there is also a variety of local probiotic strains that are key players in their limited markets. The health messages of probiotic products have focused mainly on general well-being and support of gastrointestinal microbiota. However, because of newly accepted regulation from the European Union regarding health claims, such claims will be evaluated officially, which may substantially change their content. However, more-specific product claims may also appear on the market. This article describes trends and phenomena in mainly the food sector, which covers the most-successful probiotic applications, but food supplements are also discussed.

Safety assessment of Lactobacillus strains: presence of putative risk factors in faecal, blood and probiotic isolates.

The widespread use of immunosuppressive therapy and antimicrobial agents as well as novel probiotics without a long history of safe use has increased requirements for safety assessment of probiotic lactobacilli. In this study 44 faecal, 52 blood and 15 probiotic isolates (including 3 dairy strains) of Lactobacillus were assayed for their adhesion properties to extracellular matrix proteins and mucus, hemolysis, ability to avoid the induction of respiratory burst in peripheral blood mononucleocytes (PMN) and resistance to human serum. Among tested strains adhesion to collagen, fibrinogen and mucus was isolate-specific and no statistically significant differences were obtained between faecal, blood and probiotic isolates. However, blood isolates showed a trend for higher adhesion to mucus than probiotic strains (P=0.07). Probiotic strains induced lower respiratory burst in PMN when compared to the blood isolates (P<0.05). Moreover, there was a positive correlation between adhesion to collagen and induction of respiratory burst for faecal isolates (P<0.05). In the determination of serum resistance, probiotic strains showed a trend for lower sensitivity to human serum-mediated killing when compared to the faecal isolates (P=0.07). None of the measurable virulence factors were found to be present at statistically higher level in clinical blood isolates when compared to faecal and/or probiotic isolates indicating that these factors do not cause risk when safety of probiotics is considered. However, the significance of adhesion to mucus, low induction of respiratory burst in PMN and resistance to human serum-mediated killing may need further evaluation in experimental animal models and in epidemiological data.

Probiotic and other functional microbes: from markets to mechanisms.

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.

Phenotypic differences between commercial Lactobacillus rhamnosus GG and L. rhamnosus strains recovered from blood.

The isolation of clinical isolates that are indistinguishable, by molecular methods, from the probiotic strain of Lactobacillus rhamnosus GG has been reported. We compared the virulence potential of these clinical isolates with that of the probiotic strain. It was observed that all isolates differed significantly in 1 or more phenotypic properties.

The efficacy and safety of probiotic Lactobacillus rhamnosus GG on prolonged, noninfectious diarrhea in HIV Patients on antiretroviral therapy: a randomized, placebo-controlled, crossover study.

PURPOSE: The aim of this placebo-controlled, crossover study was to evaluate the efficacy and safety of probiotic Lactobacillus rhamnosus GG (LGG) in ameliorating gastrointestinal symptoms in HIV-infected patients on antiretroviral therapy. METHOD: Infectious causes for diarrhea (bacteria, ova, parasites, and viruses including cryptosporidium, microsporidia, and cyclospora) were excluded with fecal samples before the study. HIV-infected patients with diarrhea for more than 1 month received in randomized order probiotic LGG preparation (containing viable LGG 1-5 x 1010 cfu/dose) and placebo twice a day for 2 weeks. Gastrointestinal symptoms were assessed daily and included the daily number of bowel movements, classification of stool consistency (watery, semi-watery, loose, firm, or foaming), and Visual Analog Scale (VAS) of gastrointestinal symptoms (flatulence, stomach pain, bloating disorders, general well-being). RESULTS: Seventeen HIV-infected patients completed the study. There were no significant differences between the treatment groups in the frequency or the consistency of diarrhea. In the VAS assessments of gastrointestinal symptoms, no difference between LGG and placebo could be detected. No adverse events were reported. The number of HIV RNA copies in the blood and CD4 cell counts remained stable during the study. CONCLUSION: Probiotic LGG preparation was well-tolerated in HIV infected patients. No significant differences in noninfectious diarrhea or gastrointestinal symptoms compared to placebo could be observed in this crossover study.

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.

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.