Probiotics and cancer: from in vitro to human studies

Studies in cell cultures and animal models provide evidence that probiotics can beneficially influence various stages in development of colon cancer including tumor initiation, promotion and metastasis. For example, oral administration of Lactobacillus and Bifidobacterium strains can prevent genotoxic damage to the colonic epithelium (considered to be an early stage of the carcinogenic process). Administration to rats of probiotics reduced the incidence of carcinogen-induced pre-cancerous lesions (aberrant crypt foci) in the colon. Furthermore a combination of Bifidobacterium longum and inulin (a prebiotic) was more effective than either treatment alone. In this latter study, the dietary treatments were given after exposure to the carcinogen, which suggests that the protective effects were being exerted at the promotional phase of carcinogenesis. L. acidophilus feeding has been shown to decrease the incidence of colon tumors in rats challenged with a carcinogen and B. longum reduced the incidence of carcinogeninduced colon, liver and mammary tumors. There is limited evidence from epidemiological studies for protective effects of products containing probiotics in humans, but a number of recent dietary intervention studies in healthy subjects and in polyp and cancer patients have yielded promising results on the basis of biomarkers of cancer risk and grade of colorectal tumors.

Caseinoglycomacropeptide inhibits adhesion of pathogenic Escherichia coli strains to human cells in culture

Caseinoglycomacropeptide (CGMP) derived from kappa-casein was investigated for its ability to inhibit the adhesion of 3 strains of verotoxigenic Escherichia coli (VTEC) and 3 strains of enteropathogenic Escherichia coli (EPEC) to human HT29 tissue cell cultures. Effects on adhesion of Desulfovibrio desulfuricans, Lactobacillus pentosus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus gasseri were also investigated. Generally, CGMP exerted effective anti-adhesive properties at a dose of 2.5 mg/mL, albeit with a high degree of strain specificity. The CGMP reduced adhesion of VTEC strains to < 50% of the control and reduced adhesion of EPEC strains to between 80 and 10% of the control. The CGMP also reduced the adhesion of L. pentosus and L. casei to 44 and 42%, respectively. A slight but significant reduction of L. acidophilus, to 81%, was observed, but no significant effects were detected with either Dsv. desulfuricans or L. gasseri. Further investigation of the dose response relationships with the E. coli strains gave IC50 values ranging between 0.12 and 1.06 mg/mL.

Oligosaccharide-mediated inhibition of the adhesion of pathogenic Escherichia coli strains to human gut epithelial cells in vitro

The aim of the study was to investigate the ability of pectic oligosaccharides (POS) to inhibit adhesion of three strains of verotoxigenic Escherichia coli, three strains of enteropathogenic E. coli, and one nonclinical strain of Desulfovibrio desulfuricans to human intestinal epithelial cell cultures. Lactobacillus acidophilus and Lactobacillus gasseri were included for comparison. Attachment wits determined in the human HT29 cell line by viable Count of adherent bacteria. POS in buffer at pH 7.2 were antiadhesive at a dose of 2.5 mg ml(-1), reducing adhesion of enteropathogenic E. coli and verotoxigenic E. coli strains to less than 30% of control values. Concentrations resulting in 50% inhibition ranged from 0.15 to 0.46 mg ml(-1). L. acidophilus was not significantly affected. but adhesion of L. gasseri was reduced to 29% of the control value. POS reduced the adhesion of D. desulfuricans to 0.33% of the control value. POS also had a protective effect against E. coli verocytotoxins VT1 and VT2 at concentrations of 0.01 and 1 mu g ml(-1), respectively.

Inhibition of the adhesion of enteropathogenic Escherichia coli strains to HT-29 cells in culture by chito-oligosaccharides

The ability of chito-oligosaccharides (COS) to inhibit selected intestinal bacteria was investigated. COS at 2.5 mg ml(-1) had no significant effect on the adhesion of three strains of verotoxigenic Escherichia coli (VTEC), Lactobacillus pentosus, L. casei or L. gasseri to human HT29 cells in tissue culture. However, COS significantly inhibited adhesion of three strains of enteropathogenic E. coli (EPEC) to below 30% of the level of adhesion seen in the controls. Dose-response curves were constructed to further characterise the inhibition of EPEC strains to HT29 cells. (c) 2005 Elsevier Ltd. All rights reserved.

Bacteria in the gut: friends and foes and how to alter the balance

The activities of the bacteria resident in the colon of companion animals can have an impact upon the health of the host. Our understanding of this microbial ecosystem is presently increasing due to the development of DNA-based microbiological tools that allow identification and enumeration of nonculturable microorganisms. These techniques are changing our view of the bacteria that live in the gut, and they are facilitating dietary-intervention approaches to modulate the colonic ecosystem. This is generally achieved by the feeding of either live bacteria (probiotics) or nondigestible oligosaccharides (prebiotics) that selectively feed the indigenous probiotics. Feeding studies with a Lactobacillus acidophilus probiotic have shown positive effects on carriage of Clostridium spp. in canines and on recovery from Campylobacter spp. infection in felines. Immune function was improved in both species. Prebiotic feeding studies with lactosucrose and fructo-oligosaccharides in both cats and dogs have shown positive effects on the microflora balance. Recently synbiotic forms (a probiotic together with a prebiotic) targeted at canines have been developed that show promise as dietary-intervention tools.

Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients

Background: Animal studies suggest that prebiotics and probiotics exert protective effects against tumor development in the colon, but human data supporting this suggestion are weak. Objective: The objective was to verify whether the prebiotic concept (selective interaction with colonic flora of nondigested carbohydrates) as induced by a synbiotic preparation-oligofructose-enriched inulin (SYN1) + Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (BB12)-is able to reduce the risk of colon cancer in humans. Design: The 12-wk randomized, double-blind, placebo-controlled trial of a synbiotic food composed of the prebiotic SYN1 and probiotics LGG and BB12 was conducted in 37 colon cancer patients and 43 polypectomized patients. Fecal and blood samples were obtained before, during, and after the intervention, and colorectal biopsy samples were obtained before and after the intervention. The effect of synbiotic consumption on a battery of intermediate biomarkers for colon cancer was examined. Results: Synbiotic intervention resulted in significant changes in fecal flora: Bifidobacterium and Lactobacillus increased and Clostridium perfringens decreased. The intervention significantly reduced colorectal proliferation and the capacity of fecal water to induce necrosis in colonic cells and improve epithelial barrier function in polypectomized patients. Genotoxicity assays of colonic biopsy samples indicated a decreased exposure to genotoxins in polypectomized patients at the end of the intervention period. Synbiotic consumption prevented an increased secretion of interleukin 2 by peripheral blood mononuclear cells in the polypectomized patients and increased the production of interferon gamma in the cancer patients. Conclusions: Several colorectal cancer biomarkers can be altered favorably by synbiotic intervention.

Profiling of composition and metabolic activities of the colonic microflora of growing pigs fed diets supplemented with prebiotic oligosaccharides

It is evident that quantitative information on different microbial groups and their contribution in terms of activity in the gastrointestinal (GI) tract of humans and animals is required in order to formulate functional diets targeting improved gut function and host health. In this work, quantitative information on levels and spatial distributions of Bacteroides spp, Eubacterium spp, Clostridium spp, Escherichia coli, Bifidobacterium spp and Lactobacillus/Enterococcus spp. along the porcine large intestine was investigated using 16S rRNA targeted probes and fluorescent in situ hybridisation (FISH). Caecum, ascending colon (AC) and rectum luminal digesta from three groups of individually housed growing pigs fed either a corn-soybean basal diet (CON diet) or a prebiotic diet containing 10 g/kg oligofructose (FOS diet) or trans-galactooligosaccharides (TOS diet) at the expense of cornstarch were analysed. DAPI staining was used to enumerate total number of cells in the samples. Populations of total cells, Bacteroides, Eubacterium, Clostridium and Bifidobacterium, declined significantly (P < 0.05) from caecum to rectum, and were not affected by dietary treatments. Populations of Lactobacillus/ Enterococcus and E coli did not differ throughout the large intestine. The relative percent (%) contribution of each bacterial group to the total cell count did not differ between caecum and rectum, with the exception of Eubacterium that was higher in the AC digesta. FISH analysis showed that the sum of all bacterial groups made up a small percentage of the total cells, which was 12.4%, 21.8% and 10.3% in caecum, AC and rectum, respectively. This supports the view that in swine, the diversity of GI microflora might be higher compared to other species. In terms of microflora metabolic activity, the substantially higher numerical trends seen in FOS and TOS treatments regarding total volatile fatty acid, acetate concentrations and glycolytic activities, it could be postulated that FOS and TOS promoted saccharolytic activities in the porcine colon. (c) 2006 Elsevier Ltd. All rights reserved.

In vitro fermentation of carbohydrates by porcine faecal inocula and their influence on Salmonella Typhimurium growth in batch culture systems

The aim of this study was to evaluate in vitro the influence of fermentable carbohydrates on the activity of porcine microbiota and survival of Salmonella Typhimurium in a batch culture system simulating the porcine hindgut. The carbohydrates tested were xylooligosaccharides, a mixture of fructooligosaccharides/inulin (FIN), fructooligosaccharides (FOS), gentiooligosaccharides (GEO) and lactulose (LAC). These ingredients stimulated the growth of selected Bifidobacterium and Lactobacillus species in pure cultures. In batch cultures, the carbohydrates influenced some fermentation parameters. For example, GEO and FIN significantly increased lactic acids compared with the control (no added carbohydrate). With the exception of LAC, the test carbohydrates increased the production of short-chain fatty acid (SCFA) and modified SCFA profiles. Quantitative analysis of bacterial populations by FISH revealed increased counts of the Bifidobacterium group compared with control and, with exception of FOS, increased Lactobacillus, Leuconostoc and Weissella spp. counts. Salmonella numbers were the lowest during the fermentation of LAC. This work has looked at carbohydrate metabolism by porcine microbiota in a pH-controlled batch fermentation system. It provides an initial model to analyse interactions with pathogens.

Molecular identification and anti-pathogenic activities of putative probiotic bacteria isolated from faeces of healthy elderly individuals

One hundred and nine lactic acid bacterial strains (56 bifidobacteria-like and 53 lactobacilli-like) were isolated from faecal samples donated by healthy elderly individuals (>65 years old). Isolates were identified to species level by phenotypic analysis (by API) and by 16S rDNA sequencing. Eleven species of Lactobacillus and six species of Bifidobacterium were identified. The most frequently isolated lactobacillus was L. fermentum and the most frequently isolated bifidobacterium was closely related to B. infantis by 16S rDNA sequence alignment. The isolates were characterized for their antimicrobial activity against Clostridium difficile, enteropathogenic Escherichia coli (EPEC), verocytotoxigenic E. coli (VTEC) and Campylobacter jejuni. The lactobacilli displayed variations in their antimicrobial activity with few strains showing inhibitory activity against all pathogens. The bifidobacteria displayed higher levels of inhibitory activity against C. jejuni and Cl. difficile than against the E. coli strains. Keywords: Lactobacillus, Bifidobacterium, elderly, gastrointestinal microbiota, inhibition, Clostridium difficile, enteropathogenic Escherichia coli (EPEC), verocytotoxigenic E. coli (VTEC), Campylobacter jejuni.

A double-blind placebo-controlled study to establish the bifidogenic dose of inulin in healthy humans

Objective: To evaluate the bifidogenic efficacy of two inulin doses in healthy human adults. Design: A double-blind, placebo-controlled, crossover human study. Setting: Food Microbial Sciences Unit, The University of Reading, Reading, UK. Subjects: Thirty healthy volunteers, 15 men, 15 women ( age range 19-35). Interventions: Subjects consumed a chocolate drink containing placebo ( maltodextrin, 8 g/day), 5 g/day inulin and 8 g/day inulin for a 2-week treatment period. Each treatment was followed by a 1-week washout at the end of which volunteers progressed to the next treatment. Faecal samples were obtained at the start of the study ( baseline) and at the end of each treatment and washout period. Fluorescent in situ hybridization was used to monitor populations of Bifidobacterium genus, Bacteroides - Prevotella, Lactobacillus - Enterococcus and Clostridium perfringens - histolyticum subgroup. Results: Bifidobacterial levels increased significantly upon ingestion of both the low ( 9.78 +/- 0.29 log(10) cells/g faeces, P < 0.05) and the high inulin dose ( 9.79 +/- 0.38 log(10) cells/g faeces, P < 0.05) compared to placebo ( 9.64 +/- 0.23 log(10) cells/g faeces). Conclusions: Both inulin doses exhibited a bifidogenic effect but a higher volunteer percentage responded to the high dose. A dose response effect was not observed but the magnitude of increase in bifidobacteria levels depended on their initial numbers. The higher the initial concentrations the smaller was the increase upon ingestion of the active treatments. Sponsorship: Financial support for the completion of this project was provided by Sensus ( Roosendaal, The Netherlands).

Gut fermentation products of insulin-derived prebiotics beneficially modulate markers of tumour progression in human colon tumour cells

Insulin is a prebiotic food ingredient, which suppresses colon tumour growth and development in rats. In the gut lumen, it is fermented to lactic acid and short chain fatty acids (SCFA). Of these, butyrate has suppressing agent activities, but little is known concerning cellular responses to complex fermentation samples. To investigate the effects of fermentation products of insulin on cellular responses related to colon carcinogenesis. Fermentations were performed in anaerobic batch cultures or in a three-stage fermentation model that simulates conditions in colon-segments (proximal, transverse, distal). Substrate was insulin enriched with oligofructose (Raftilose® Synergy1), fermented with probiotics (Bifidobacterium lactis Bb12, Lactobacillus rhamnosus GG), and/or faecal inocula. HT29 or CaCo-2 cells were incubated with supernatants of the fermented samples (2.5%-25% v/v, 24-72 hours). Cellular parameters of survival, differentiation, tumour progression, and invasive growth were determined. Fermentation supernatants derived from probiotics and Synergy1 were more effective than with glucose. The additional fermentation with faecal slurries produced supernatants with lower toxicity, higher SCFA contents, and distinct cellular functions. The supernatant derived from the gut model vessel representing the distal colon, was most effective for all parameters, probably on account of higher butyrate-concentrations. Biological effects of insulin upon colon cells may be mediated not only by growth stimulation of the lactic acid-producing bacteria and/or production of butyrate, but also by other bacteria and products of the gut lumen. These newly reported properties of the supernatants to inhibit growth and metastases in colon tumour cells are important mechanisms of tumour suppression.

Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

In vitro fermentation of oat and barley derived beta-glucans by human faecal microbiota

Fermentation of beta-glucan fractions from barley [average molecular mass (MM), of 243, 172, and 137 kDa] and oats (average MM of 230 and 150 kDa) by the human faecal microbiota was investigated. Fractions were supplemented to pH-controlled anaerobic batch culture fermenters inoculated with human faecal samples from three donors, in triplicate, for each substrate. Microbiota changes were monitored by fluorescent in situ hybridization; groups enumerated were: Bifidobacterium genus, Bacteroides and Prevotella group, Clostridium histolyticum subgroup, Ruminococcus-Eubacterium-Clostridium (REC) cluster, Lactobacillus-Enterococcus group, Atopobium cluster, and clostridial cluster IX. Short-chain fatty acids and lactic acid were measured by HPLC. The C. histolyticum subgroup increased significantly in all vessels and clostridial cluster IX maintained high populations with all fractions. The Bacteroides-Prevotella group increased with all but the 243-kDa barley and 230-kDa oat substrates. In general beta-glucans displayed no apparent prebiotic potential. The SCFA profile (51 : 32 : 17; acetate : propionate : butyrate) was considered propionate-rich. In a further study a beta-glucan oligosaccharide fraction was produced with a degree of polymerization of 3-4. This fraction was supplemented to small-scale faecal batch cultures and gave significant increases in the Lactobacillus-Enterococcus group; however, the prebiotic potential of this fraction was marginal compared with that of inulin.

Dietary-based gut flora modulation against Clostridium difficile onset

Clostridium difficile infection is a frequent complication of antibiotic therapy in hospitalised patients, which today is attracting more attention than ever and has led to its classification as a 'superbug'. Disruption of the composition of the intestinal microflora following antibiotic treatment is an important prerequisite for overgrowth of C. difficile and the subsequent development of an infection. Treatment options for antibiotic-associated diarrhoea and C. difficile-induced colitis include administration of specific antibiotics (e.g. vancomycin), which often leads to high relapse rates. More importantly, both the rate and severity of C. difficile-associated diseases are increasing, with new epidemic strains of C. difficile often implicated. For the prevention and treatment of antibiotic-associated diarrhoea and C. difficile infection, several probiotic bacteria such as selected strains of lactobacilli (especially Lactobacillus rhamnosus GG), Bifidobacterium longum, and Enterococcus faecium and the non-pathogenic yeast Saccharomyces boulardii have been used. Controlled trials indicate a benefit of S. boulardii and L. rhamnosus GG as therapeutic agents when used as adjuncts to antibiotics. However, the need for more well designed controlled trials with probiotics is explicit.

Mixed culture fermentation studies on the effects of synbiotics on the human intestinal pathogens Campylobacter jejuni and Escherichia coli

Batch and continuous culture anaerobic fermentation systems, inoculated with human faeces, were utilised to investigate the antimicrobial actions of two probiotics, Lactobacillus plantartan 0407, combined with oligofructose and Bifidobacterium bifidum Bb12, combined with a mixture of oligofructose and xylo-oligosaccharides (50:50 w/w) against E coli and Campylobacter jejuni. In batch fermenters, both E coli and C jejuni were inhibited by the synbiotics, even when the culture pH was maintained at around neutral. In continuous culture C jejuni was inhibited but the synbiotic failed to inhibit E coli. Although no definitive answer in addressing the mechanisms underlying antimicrobial activity was derived, results suggested that acetate and lactate directly were conferring antagonistic action, rather than as a result of lowering culture pH. In the course of the study culturing and fluorescent in situ hybridisation (FISH) methodologies for the enumeration of bacterial populations were compared. Bifidobacterial populations were underestimated using plating techniques, suggesting the non-culturability of certain bifidobacterial species. (C) 2003 Elsevier Ltd. All rights reserved.