The innovative potential of Lactobacillus rhamnosus LR06, Lactobacillus pentosus LPS01, Lactobacillus plantarum LP01, and Lactobacillus delbrueckii Subsp. delbrueckii LDD01 to restore the "gastric barrier effect" in patients chronically treated with PPI: a pilot study.

BACKGROUND: Gastroesophageal reflux disease is a very widespread condition. In Europe, it is estimated that about 175 million people suffer from this disease and have to chronically take drugs to increase gastric pH. The proton pump inhibitors (PPIs) such as omeprazole, lansoprazole, and esomeprazole are the most widely used drug typology in this regard. However, the inhibition of normal gastric acid secretion has important side effects, the most important being bacterial overgrowth in the stomach and duodenum with a concentration of >105 viable cells/mL. As a major consequence of this, many harmful or even pathogenic bacteria contained in some foods could survive the gastric transit and colonize either the stomach itself, the duodenum, or the gut, where they could establish acute and even chronic infections with unavoidable consequences for the host's health. In other words, the "gastric barrier effect" is strongly reduced or even disrupted. To date, there are no real strategies to deal with this widespread, although still relatively little known, problem. The aim of this study was to confirm the gastric bacterial overgrowth in long-term PPI consumers and to assess the efficacy of some probiotic bacteria, belonging to both genera Lactobacillus and Bifidobacterium, in the reduction of gastric and duodenal bacterial overgrowth, therefore partially restoring the gastric barrier effect against foodborne pathogenic bacteria. METHODS: For this purpose, probiotics with a strong demonstrated inhibitory activity on gram-negative bacteria, such as Escherichia coli, were tested in a human intervention trial involving a total of 30 subjects treated with PPIs for either 3 to 12 consecutive months (short-term) or >12 consecutive months (long-term). An additional 10 subjects not taking PPIs were enrolled and used as a control group representing the general population. Four selected probiotics Probiotical SpA (Novara, Italy), namely Lactobacillus rhamnosus LR06 (DSM 21981), Lactobacillus pentosus LPS01 (DSM 21980), Lactobacillus plantarum LP01 (LMG P-21021), and Lactobacillus delbrueckii subsp. delbrueckii LDD01 (DSM 22106) were administered for 10 days to 10 subjects treated with PPIs for >12 months (group B). In the 60 mg formulation, N-acetylcysteine was included as well in light of its well-known mechanical effects on bacterial biofilms. Gastroscopies were performed at the beginning of the study (d0) in all the groups (A, B, C, and D) and after 10 days (d10) in group B only; that is, at the end of probiotics intake. The total viable cells and total Lactobacillus were quantified in gastric juice and duodenal brushing material from all subjects. The results were compared among all the groups and with the control subjects (group D) to confirm the bacterial overgrowth. A comparison was made also between d0 and d10 in group B to quantify the efficacy of the 4 probiotics administered for 10 days. Fecal samples were collected from all groups at d0, including subjects not treated with PPIs, and in group B only at d10. Specific bacterial classes, namely enterococci, total coliforms, E. coli, molds, and yeasts were quantified in all fecal specimens. RESULTS: The results collected confirmed the strong bacterial overgrowth in the stomach and duodenum of people treated with PPIs compared with subjects with a normal intragastric acidity. It is also worth noting that the bacterial cell counts in subjects who underwent a long-term treatment with a PPI were greater than the results from subjects taking these drugs for 3 to 12 months. The intake of 4 specific probiotic strains with a marked antagonistic activity towards 5 E. coli bacteria, including the enterohaemorrhagic O157:H7 strain, and an effective amount of N-acetylcysteine (NAC) was able to significantly reduce bacterial overgrowth in long-term PPI-treated subjects. Total lactobacilli represented the major percentage of bacterial counts, thus demonstrating the ability of such bacteria to colonize the stomach and the duodenum, at least temporarily, and to consequently restore the gastric barrier effect. A significant decrease in fecal enterococci, total coliforms, E. coli, molds, and yeasts in subjects treated with PPIs was recorded at the end of probiotics supplementation (d10) compared with baseline (d0) in group B. This is a further confirmation of the barrier effect also exerted at the stomach level. CONCLUSIONS: PPIs are the most widely sold and used drugs in the world. However, the chronic use of these pharmacological molecules exposes the subject to the risk of foodborne infections as most pathogens are able to survive the gastric transit in a condition of significantly decreased acidity.

Assessment of the in vitro inhibitory activity of specific probiotic bacteria against different Escherichia coli strains.

BACKGROUND: Lactobacilli and bifidobacteria are often associated with health-promoting effects. These live microorganisms, defined as probiotics, are commonly consumed as part of fermented foods, such as yoghurt and fermented milks, or as dietary supplements. Escherichia coli is a gram-negative, rod-shaped bacterium commonly found in the lower intestine of warm-blooded organisms. As a part of the normal gut microbiota, this microorganism colonizes the gastrointestinal tract of animals and humans within a few hours after birth. All E. coli strains can produce a wide variety of biogenic amines responsible for potentially harmful systemic intoxications. Enterohemorrhagic E. coli serotype O157:H7 is a pathotype of diarrhoeagenic strains with a large virulence plasmid pO157 able to produce 1 or more Shiga toxins. METHODS: The overall aim of this study was to determine the inhibitory effects of different strains of probiotics on E. coli serotypes, including E. coli O157:H7 (CQ9485). In particular, the antagonistic activity of 4 Bifidobacterium strains (Probiotical SpA, Italy) and 16 lactic acid bacteria, more specifically 14 Lactobacillus spp. and 2 Streptococcus spp., was assessed against selected E. coli biotypes (ATCC 8739, ATCC 10536, ATCC 35218, and ATCC 25922). The diarrhoeagenic serotype O157:H7 was also tested. RESULTS: The experimental data collected demonstrated an in vitro significant inhibitory effect of 6 Lactobacillus strains, namely L. rhamnosus LR04, L. rhamnosus LR06, L. plantarum LP01, L. plantarum LP02, L. pentosus LPS01, and L. delbrueckii subsp. delbrueckii LDD01, and 2 Bifidobacterium strains, B. breve BR03 and B. breve B632. The inhibiting extent was slightly different among these strains, with L. delbrueckii subsp. delbrueckii LDD01 showing the highest activity on E. coli O157:H7. CONCLUSIONS: Most of the probiotics studied are able to antagonize the growth of the 5 strains of E. coli tested, including the O157:H7 biotype, well known for their characteristic to produce a wide variety of biogenic amines considered responsible for dangerous systemic intoxications.

Comparison of the kinetics of intestinal colonization by associating 5 probiotic bacteria assumed either in a microencapsulated or in a traditional, uncoated form.

BACKGROUND: Beneficial findings concerning probiotics are increasing day by day. However, one of the most important parameters able to significantly affect the probiotic value of a microorganism is its survival during the transit through the stomach and the duodenum. Some techniques may be applied that aim to improve this parameter, but microencapsulation of bacterial cells remains one of the most important. A recent study assessed the kinetics of intestinal colonization by a mixture of 2 probiotic strains, given either in a microencapsulated or in a traditional, uncoated form. METHODS: A comparison between the intestinal colonization by associating 5 microencapsulated bacteria and the same uncoated strains was performed by a double-blind, randomized, cross-over study. The study (December 2007 to January 2009) involved 53 healthy volunteers. In particular, subjects were divided into 2 groups: group A (27 subjects) was given a mix of probiotic strains Probiotical S.p.A. (Novara, Italy), Lactobacillus acidophilus LA02 (DSM 21717), Lactobacillus rhamnosus LR04 (DSM 16605), L. rhamnosus GG, or LGG (ATCC 53103), L. rhamnosus LR06 (DSM 21981), and Bifidobacterium lactis BS01 (LMG P-21384) in an uncoated form, whereas group B (26 subjects) received the same strains microencapsulated with a gastroprotected material. The uncoated strains were administered at 5×109 cfu/strain/d (a total of 25×109 cfu/d) for 21 days, whereas the microencapsulated bacteria were given at 1×109 cfu/strain/d (a total of 5×109 cfu/d) for 21 days. At the end of the first period of supplementation with probiotics, a 3-week wash-out phase was included in the study setting. At the end of the wash-out period, the groups crossed over their treatment regimen; that is, group A was administered the microencapsulated bacteria and group B the uncoated bacteria. The administered quantities of each strain were the same as the first treatment. A quantitative evaluation of intestinal colonization by probiotics, either microencapsulated or uncoated, was undertaken by examining fecal samples at the beginning of the study (time 0), after 10 days and after 21 days of each treatment period. In particular, fecal total Lactobacilli, heterofermentative Lactobacilli, and total Bifidobacteria were quantified at each checkpoint. A genomic analysis of an appropriate number of colonies was performed to quantify individual L. rhamnosus strains among heterofermentative Lactobacilli. RESULTS: A statistically significant increase in the fecal amounts of total Lactobacilli, heterofermentative Lactobacilli, and total Bifidobacteria was registered in both groups at the end of each supplementation period compared with d0 or d42 (group A: P=0.0002, P=0.0001, and P<0.0001 at d21, P=0.0060, P=0.0069, and P<0.0001 at d63 for total Lactobacilli, heterofermentative Lactobacilli, and Bifidobacteria, respectively; group B: P=0.0002, P=0.0006, and P<0.0001 at d21, P=0.0015, P=0.0016, and P<0.0001 at d63 for total Lactobacilli, heterofermentative Lactobacilli, and Bifidobacteria, respectively), confirming the ability of each strain in the administered composition to colonize the human gut, whether supplemented in a gastroprotected or in a traditional freeze-dried form. On the contrary, subjects receiving microencapsulated bacteria reported a kinetics of intestinal colonization that was entirely comparable with those who were given uncoated strains at a 5 times higher amount. CONCLUSIONS: The microencapsulation technique used in this study is a valid approach aimed to significantly improve the survival of strains during gastroduodenal transit, thus enhancing their probiotic value and allowing the use of a 5 times lower amount.

Is microencapsulation the future of probiotic preparations? The increased efficacy of gastro-protected probiotics.

In a recent publication we assessed the kinetics of intestinal colonization by microencapsulated probiotic bacteria in comparison with the same strains given in an uncoated form. It's well known, in fact, that microencapsulation of probiotics with specific materials is able to confer a significant resistance to gastric juice, thus protecting the cells during the gastric and duodenal transit and enhancing the probiotic efficacy of any supplementation. In any case, this was the first study reporting the fecal amounts of probiotics administered in a coated, protected form compared with traditional, uncoated ones. Here we discuss additional in vitro data of resistance of the same bacteria to gastric juice, human bile and pancreatic secretion and correlate them with the results of in vivo gut colonization.

Evaluation of the intestinal colonization by microencapsulated probiotic bacteria in comparison with the same uncoated strains.

BACKGROUND: Beneficial findings concerning probiotics are increasing day by day. However, one of the most important parameter which affects the probiotic activity of a microorganism is its survival during the gastroduodenal transit. Some microencapsulation techniques could be applied to bacterial cells to improve this parameter. METHODS: A comparison between the intestinal colonization by microencapsulated bacteria and the same not microencapsulated strains has been conducted in a double blind, randomized, cross-over study. The study (April to July 2005) involved 44 healthy volunteers. In particular, participants were divided into 2 groups: group A (21 participants) received a mix of probiotic strains Lactobacillus plantarum LP01 (LMG P-21021) and Bifidobacterium breve BR03 (DSM 16604) in an uncoated form, group B (23 participants) was given the same strains microencapsulated with a gastroresistant material. The not microencapsulated strains were administered at 5 x 10(9) colony forming units/strain/d for 21 days, whereas the microencapsulated bacteria were given at 1 x 10(9) colony forming units/strain/d for 21 days. At the end of the first period of treatment with probiotics a 3 weeks washout phase has been included in the study protocol. At the end of the washout period the groups were crossed: in detail, group A had the microencapsulated and group B the uncoated bacteria. The administered amounts of each strain were the same as the first treatment. The quantitative evaluation of intestinal colonization by strains microencapsulated or not microencapsulated was made by fecal samples examination at the beginning of the clinical trial, after 10 and 21 days of each treatment period. In particular, fecal heterofermentative Lactobacilli and Bifidobacteria have been counted. RESULTS: A statistically significant increase in the fecal amounts of Lactobacilli and Bifidobacteria was recorded in both groups at the end of each treatment compared with d0 or d42 (P<0.0001 and P<0.0001 at d21, P<0.0001 and P<0.0001 at d63 for Lactobacilli and Bifidobacteria, respectively), confirming the ability of the 2 strains to colonize the human gut, either in a gastroprotected form or not. Participants treated with the microencapsulated bacteria reported a kinetics of intestinal colonization quite similar to participants who received not coated strains. CONCLUSIONS: Probiotics are able to exert many different beneficial effects on the human host. These effects are mediated by the number of viable cells which reach the gut. The microencapsulation technique used in this study is a valid strategy to significantly improve gastroresistance of strains, thus enhancing their probiotic activity and allowing the use of a 5 times lower amount.

The use of probiotics in healthy volunteers with evacuation disorders and hard stools: a double-blind, randomized, placebo-controlled study.

BACKGROUND: Evacuation disorders and hard stools are common in industrialized countries, affecting on average 12% to 17% of the adult healthy population at any age. Dietary supplementation with probiotic microorganisms may be useful in reducing the disorder. METHODS: We performed a double-blind, randomized, placebo-controlled study to evaluate the effectiveness of 2 different probiotic blends, either mixed Lactobacillus plantarum LP01 (LMG P-21021) and Bifidobacterium breve BR03 (DSM 16604) or Bifidobacterium animalis subspecies lactis BS01 (LMG P-21384), in the management of evacuation disorders and intestinal discomfort. In a period of 5 years (2003 to 2008), the study involved 300 healthy volunteers (151 males and 149 females; age 24 to 71 y) with evacuation disorders and hard stools. In particular, subjects were divided into 3 groups: 80 subjects in the group A received placebo, 110 subjects in the group B received mixed L. plantarum LP01 and B. breve BR03 (2.5 x 10 colony-forming units/d of each strain), and 110 subjects in the group C received B. animalis subsp. lactis BS01 (5 x 10 colony-forming units/d) for 30 days. At the beginning of the observational study, the healthy status of volunteers was evaluated by a complete, laboratory and ultrasound study of the abdomen. The physical examination was repeated after 15 and 30 days. In particular, the main troubles typically associated with evacuation disorders and hard stools as well as abdominal bloating were considered as parameters of interest. Exclusion criteria were items of gastrointestinal diseases and antibiotics intake. RESULTS: Subjects treated with the mixed probiotic strains L. plantarum LP01 and B. breve BR03 or B. animalis subsp. lactis BS01 reported a significant improvement in the number of weekly bowel movements and in the main troubles associated with evacuations, particularly consistency of feces and ease of expulsion. Discomfort items such as abdominal bloating and anal itching, burning, or pain also registered a relevant improvement in the active groups receiving probiotics. CONCLUSIONS: The intake of an effective amount of mixed L. plantarum LP01 and B. breve BR03 or B. animalis subsp. lactis BS01 for 30 days is able to significantly relieve the evacuation disorders and hard stools, thus providing a useful tool for the management of such condition, which is particularly widespread in industrialized countries at any age.

Clinical experience with probiotics in the elderly on total enteral nutrition.

BACKGROUND: Recent data support that after 2 years of age, intestinal microflora remains relatively constant over time, except in elderly people, who harbor fewer bifidobacteria and a higher population of fungi and enterobacteria than young adults. Diet supplementation with probiotics may improve the nutritional status and reduce the impaired immunity associated with aging. The goal of this study was to establish the effect on bifidobacteria fecal counts, and some clinical parameters, of bifidobacteria supplementation to elderly patients in total parenteral nutrition. METHODS: Thirteen patients (6 men and 7 women; mean age, 69 years; range, 65-76 years) affected by permanent vegetative status (PVS) and fed by total enteral nutrition (TEN) were studied. Bifidobacteria and clostridia were investigated by microbiologic and molecular biology methods in stool specimens collected twice at basal time (T-2 and T0) and after 12 and 15 days (T12 and T15, respectively). Seven patients with basal bifidobacteria counts less than 10 were supplemented with Bifidobacterium longum W 11 for 12 days. The remaining 6 patients were used as control subjects. For 1 month diarrhea and fever episodes, use of antibiotics, and nutritional status (BMI) were assessed. RESULTS: In the 7 patients with bifidobacteria counts less than 10, the administration of B. longum W 11 resulted in a 1 log increase in 6 of 7 patients at T12. No statistically significant difference in episodes of fever or diarrhea, use of antibiotics, or BMI was observed between the treatment and control groups. CONCLUSION: The administration of B. longum W11 in PVS patients fed by TEN is effective in increasing the population of bifidobacteria. Larger studies with longer follow-up could demonstrate the influence of these microbiologic changes in a clinical setting.