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.

From probiotics to prebiotics and a healthy digestive system

Ingestion of probiotics can be recommended as a preventative approach to maintaining intestinal microflora balance and thereby enhance 'well-being'. Undoubtedly, probiotic bacteria will vary in their efficacy. The literature indicates positive results in over 50 human trials with prevention/treatment of infections the most frequently reported. In theory increased levels of probiotics may induce a 'barrier' influence against common pathogens. Mechanisms of effect are likely to include the excretion of acids (lactate, acetate), competition for nutrients and gut receptor sites, immuno-modulation and the formation of specific antimicrobial agents. An alternative, or additional, approach is the prebiotic concept. This takes the view that probiotics are present indigenous to the gut and that a rational approach towards increasing their numbers would be to consume food ingredients (carbohydrates) that have a selective metabolism in the lower gut. A prebiotic is 'a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon that can improve the host health.' In particular, the ingestion of fructo-oligosaccharides, galacto-oligosaccharides, and lactulose has shown to stimulate bifidobacteria in the lower gut.

The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota.

We have examined the gut bacterial metabolism of pomegranate by-product (POMx) and major pomegranate polyphenols, punicalagins, using pH-controlled, stirred, batch culture fermentation systems reflective of the distal region of the human large intestine. Incubation of POMx or punicalagins with faecal bacteria resulted in formation of the dibenzopyranone-type urolithins. The time course profile confirmed the tetrahydroxylated urolithin D as the first product of microbial transformation, followed by compounds with decreasing number of phenolic hydroxy groups: the trihydroxy analogue urolithin C and dihydroxylated urolithin A. POMx exposure enhanced the growth of total bacteria, Bifidobacterium spp. and Lactobacillus spp., without influencing the Clostridium coccoides–Eubacterium rectale group and the C. histolyticum group. In addition, POMx increased concentrations of short chain fatty acids (SCFA) viz. acetate, propionate and butyrate in the fermentation medium. Punicalagins did not affect the growth of bacteria or production of SCFA. The results suggest that POMx oligomers, composed of gallic acid, ellagic acid and glucose units, may account for the enhanced growth of probiotic bacteria.

Recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices

Due to the fact that probiotic cells need to be alive when they are consumed, culture-based analysis (plate count) is critical in ascertaining the quality (numbers of viable cells) of probiotic products. Since probiotic cells are typically stressed, due to various factors related to their production, processing and formulation, the standard methodology for total plate counts tends to underestimate the cell numbers of these products. Furthermore, products such as microencapsulated cultures require modifications in the release and sampling procedure in order to correctly estimate viable counts. This review examines the enumeration of probiotic bacteria in the following commercial products: powders, microencapsulated cultures, frozen concentrates, capsules, foods and beverages. The parameters which are specifically examined include: sample preparation (rehydration, thawing), dilutions (homogenization, media) and plating (media, incubation) procedures. Recommendations are provided for each of these analytical steps to improve the accuracy of the analysis. Although the recommendations specifically target the analysis of probiotics, many will apply to the analysis of commercial lactic starter cultures used in food fermentations as well.

Influence of fermentation conditions on the surface properties and adhesion of Lactobacillus rhamnosus GG

Background: The surface properties of probiotic bacteria influence to a large extent their interactions within the gut ecosystem. There is limited amount of information on the effect of the production process on the surface properties of probiotic lactobacilli in relation to the mechanisms of their adhesion to the gastrointestinal mucosa. The aim of this work was to investigate the effect of the fermentation pH and temperature on the surface properties and adhesion ability to Caco-2 cells of the probiotic strain Lactobacillus rhamnosus GG. Results: The cells were grown at pH 5, 5.5, 6 (temperature 37 °C) and at pH 6.5 (temperature 25 °C, 30 °C and 37 °C), and their surfaces analysed by X-ray photoelectron spectrometry (XPS), Fourier transform infrared spectroscopy (FT-IR) and gel-based proteomics. The results indicated that for all the fermentation conditions, with the exception of pH 5, a higher nitrogen to carbon ratio and a lower phosphate content was observed at the surface of the bacteria, which resulted in a lower surface hydrophobicity and reduced adhesion levels to Caco-2 cells as compared to the control fermentation (pH 6.5, 37 oC). A number of adhesive proteins, which have been suggested in previous published works to take part in the adhesion of bacteria to the human gastrointestinal tract, were identified by proteomic analysis, with no significant differences between samples however. Conclusions: The temperature and the pH of the fermentation influenced the surface composition, hydrophobicity and the levels of adhesion of L. rhamnosus GG to Caco-2 cells. It was deduced from the data that a protein rich surface reduced the adhesion ability of the cells.

Microencapsulation of probiotics for gastrointestinal delivery

The administration of probiotic bacteria as nutraceuticals is an area that has rapidly expanded in recent years, with a global market worth $32.6 billion predicted by 2014. Many of the health promoting claims attributed to these bacteria are dependent on the cells being both viable and sufficiently numerous in the intestinal tract. The oral administration of most bacteria results in a large loss of viability associated with passage through the stomach, which is attributed to the high acid and bile salt concentrations present. This loss of viability effectively lowers the efficacy of the administered supplement. The formulation of these probiotics into microcapsules is an emerging method to reduce cell death during GI passage, as well as an opportunity to control release of these cells across the intestinal tract. The majority of this technology is based on the immobilization of bacteria into a polymer matrix, which retains its structure in the stomach before degrading and dissolving in the intestine, unlike the diffusion based unloading of most controlled release devices for small molecules. This review shall provide an overview of progress in this field as well as draw attention to areas where studies have fallen short. This will be followed by a discussion of emerging trends in the field, highlighting key areas in which further research is necessary.

The potential mechanisms involved in the anti-carcinogenic action of probiotics

Probiotic bacteria are live microbial food ingredients that provide a health benefit to the consumer. In the past it was suggested that they served to benefit the host primarily through the prevention of intestinal infections. More recent studies have implicated probiotic bacteria in a number of other beneficial effects within the host including: *The suppression of allergies. *Control of blood cholesterol levels. *Modulation of immune function. *And the prevention of cancers of the colon. The reputed anti-carcinogenic effect of probiotics arises from in vivo studies in both animals and to a limited extent in man; this evidence is supported by in vitro studies with carcinoma cell lines and anti-mutagenicity assays. However, the mechanisms involved in any effect have thus far been difficult to elucidate; studies offer evidence for a variety of mechanisms; we have reviewed these and come to the opinion that, the anti-carcinogenic effect may not be attributable to a single mechanism but rather to a combination of events not yet fully elucidated or understood.

Human microbiota in health and disease

Each human body plays host to a microbial population which is both numerically vast (at around 1014 microbial cells) and phenomenally diverse (over 1,000 species). The majority of the microbial species in the gut have not been cultured but the application of culture-independent approaches for high throughput diversity and functionality analysis has allowed characterisation of the diverse microbial phylotypes present in health and disease. Studies in monozygotic twins, showing that these retain highly similar microbiota decades after birth and initial colonisation, are strongly indicative that diversity of the microbiome is host-specific and affected by the genotype. Microbial diversity in the human body is reflected in both richness and evenness. Diversity increases steeply from birth reaching its highest point in early adulthood, before declining in older age. However, in healthy subjects there appears to be a core of microbial phylotypes which remains relatively stable over time. Studies of individuals from diverse geopraphies suggest that clusters of intestinal bacterial groups tend to occur together, constituting ‘enterotypes’. So variation in intestinal microbiota is stratified rather than continuous and there may be a limited number of host/microbial states which respond differently to environmental influences. Exploration of enterotypes and functional groups may provide biomarkers for disease and insights into the potential for new treatments based on manipulation of the microbiome. In health, the microbiota interact with host defences and exist in harmonious homeostasis which can then be disturbed by invading organisms or when ‘carpet bombing’ by antibiotics occurs. In a portion of individuals with infections, the disease will resolve itself without the need for antibiotics and microbial homeostasis with the host’s defences is restored. The administration of probiotics (live microorganisms which when administered in adequate amounts confer a health benefit on the host) represents an artificial way to enhance or stimulate these natural processes. The study of innate mechanisms of antimicrobial defence on the skin, including the production of numerous antimicrobial peptides (AMPs), has shown an important role for skin commensal organisms. These organisms may produce AMPs, and also amplify the innate immune responses to pathogens by activating signalling pathways and processing host produced AMPs. Research continues into how to enhance and manipulate the role of commensal organisms on the skin. The challenges of skin infection (including diseases caused by multiply resistant organisms) and infestations remain considerable. The potential to re-colonise the skin to replace or reduce pathogens, and exploring the relationship between microbiota elsewhere and skin diseases are among a growing list of research targets. Lactobacillus species are among the best known ‘beneficial’ bacterial members of the human microbiota. Of the approximately 120 species known, about 15 are known to occur in the human vagina. These organisms have multiple properties, including the production of lactic acid, hydrogen peroxide and bacteriocins, which render the vagina inhospitable to potential pathogens. Depletion of the of the normal Lactobacillus population and overgrowth of vaginal anaerobes, accompanied by the loss of normal vaginal acidity can lead to bacterial vaginosis – the commonest cause of abnormal vaginal discharge in women. Some vaginal anaerobes are associated with the formation of vaginal biofilms which serve to act as a reservoir of organisms which persists after standard antibiotic therapy of bacterial vaginosis and may help to account for the characteristically high relapse rate in the condition. Administration of Lactobacillus species both vaginally and orally have shown beneficial effects in the treatment of bacterial vaginosis and such treatments have an excellent overall safety record. Candida albicans is a frequent coloniser of human skin and mucosal membranes, and is a normal part of the microbiota in the mouth, gut and vagina. Nevertheless Candida albicans is the most common fungal pathogen worldwide and is a leading cause of serious and often fatal nosocomial infections. What turns this organism from a commensal to a pathogen is a combination of increasing virulence in the organism and predisposing host factors that compromise immunity. There has been considerable research into the use of probiotic Lactobacillus spp. in vaginal candidiasis. Studies in reconstituted human epithelium and monolayer cell cultures have shown that L. rhamnosus GG can protect mucosa from damage caused by Candida albicans, and enhance the immune responses of mucosal surfaces. Such findings offer the promise that the use of such probiotic bacteria could provide new options for antifungal therapy. Studies of changes of the human intestinal microbiota in health and disease are complicated by its size and diversity. The Alimentary Pharmabiotic Centre in Cork (Republic of Ireland) has the mission to ‘mine microbes for mankind’ and its work illustrates the potential benefits of understanding the gut microbiota. Work undertaken at the centre includes: mapping changes in the microbiota with age; studies of the interaction between the microbiota and the gut; potential interactions between the gut microbiota and the central nervous system; the potential for probiotics to act as anti-infectives including through the production of bacteriocins; and the characterisation of interactions between gut microbiota and bile acids which have important roles as signalling molecules and in immunity. The important disease entity where the role of the gut microbiota appears to be central is the Irritable Bowel Syndrome (IBS). IBS patients show evidence of immune activation, impaired gut barrier function and abnormal gut microbiota. Studies with probiotics have shown that these organisms can exert anti-inflammatory effects in inflammatory bowel disease and may strengthen the gut barrier in IBS of the diarrhoea-predominant type. Formal randomised trials of probiotics in IBS show mixed results with limited benefit for some but not all. Studies confirm that administered probiotics can survive and temporarily colonise the gut. They can also stimulate the numbers of other lactic acid bacilli in the gut, and reduce the numbers of pathogens. However consuming live organisms is not the only way to influence gut microbiota. Dietary prebiotics are selectively fermented ingredients that can change the composition and/or activity of the gastrointestinal microbiota in beneficial ways. Dietary components that reach the colon, and are available to influence the microbiota include poorly digestible carbohydrates, such as non-starch polysaccharides, resistant starch, non-digestible oligosaccharides (NDOs) and polyphenols. Mixtures of probiotic and prebiotic ingredients that can selectively stimulate growth or activity of health promoting bacteria have been termed ‘synbiotics’. All of these approaches can influence gut microbial ecology, mainly to increase bifidobacteria and lactobacilli, but metagenomic approaches may reveal wider effects. Characterising how these changes produce physiological benefits may enable broader use of these tactics in health and disease in the future. The current status of probiotic products commercially available worldwide is less than ideal. Prevalent problems include misidentification of ingredient organisms and poor viability of probiotic microorganisms leading to inadequate shelf life. On occasions these problems mean that some commercially available products cannot be considered to meet the definition of a probiotic product. Given the potential benefits of manipulating the human microbiota for beneficial effects, there is a clear need for improved regulation of probiotics. The potential importance of the human microbiota cannot be overstated. ‘We feed our microbes, they talk to us and we benefit. We just have to understand and then exploit this.’ (Willem de Vos).

Microencapsulation of a synbiotic into PLGA/alginate multiparticulate gels

Probiotic bacteria have gained popularity as a defence against disorders of the bowel. However, the acid sensitivity of these cells results in a loss of viability during gastric passage and, consequently, a loss of efficacy. Probiotic treatment can be supplemented using ‘prebiotics’, which are carbohydrates fermented specifically by probiotic cells in the body. This combination of probiotic and prebiotic is termed a ‘synbiotic’. Within this article a multiparticulate dosage form has been developed, consisting of poly(d,l-lactic-co-glycolic acid) (PLGA) microcapsules containing prebiotic Bimuno™ incorporated into an alginate–chitosan matrix containing probiotic Bifidobacterium breve. The aim of this multiparticulate was that, in vivo, the probiotic would be protected against gastric acid and the release of the prebiotic would occur in the distal colon. After microscopic investigation, this synbiotic multiparticulate was shown to control the release of the prebiotic during in vitro gastrointestinal transit, with the release of galacto-oligosaccharides (GOS) initially occurred over 6 h, but with a triphasic release pattern giving further release over 288 h. Encapsulation of B. breve in multiparticulates resulted in a survival of 8.0 ± 0.3 log CFU/mL cells in acid, an improvement over alginate–chitosan microencapsulation of 1.4 log CFU/mL. This was attributed to increased hydrophobicity by the incorporation of PLGA particles.

In vitro study on the cell adhesion ability of immobilized lactobacilli on natural supports

The aim of the present study was to investigate the effect of probiotic immobilization onto wheat grains, both wet and freeze dried, on the adhesion properties of the probiotic cells and make comparisons with wet and freeze dried free cells. Lactobacillus casei ATCC 393 and Lactobacillus plantarum NCIMB 8826 were used as model probiotic strains. The results showed satisfactory adhesion ability of free cells to a monolayer of Caco-2 cells (> 1000 CFU/100 Caco-2 cells for wet cells). Cell immobilization resulted in a significant decrease in adhesion, for both wet and freeze dried formulations, most likely because immobilized cells did not have direct access to the Caco-2 cells, but it still remained in adequate levels (> 100 CFU/100 Caco-2 cells for wet cells). No clear correlation could be observed between cell adhesion and the hydrophobicity of the bacterial cells, measured by the hexadecane adhesion assay. Most notably, immobilization enhanced the monolayer integrity of Caco-2 cells, demonstrated by a more than 2-fold increase in transepithelial electrical resistance (TEER) compared to free cells. SEM micrographs ascertained the adhesion of both immobilized and free cells to the brush border microvilli. Finally, the impact of the food matrix on the adhesion properties of probiotic bacteria and on the design of novel functional products is discussed.

Differential induction of apoptosis in human colonic carcinoma cells (Caco-2) by Atopobium, and commensal, probiotic and enteropathogenic bacteria: mediation by the mitochondrial pathway

The induction of apoptosis in mammalian cells by bacteria is well reported. This process may assist infection by pathogens whereas for non-pathogens apoptosis induction within carcinoma cells protects against colon cancer. Here, apoptosis induction by a major new gut bacterium, Atopobium minutum, was compared with induction by commensal (Escherichia coli K-12 strains), probiotic (Lactobacillus rhamnosus, Bifidobacterium latis) and pathogenic (E. coli: EPEC and VTEC) gut bacteria within the colon cancer cell line, Caco-2. The results show a major apoptotic effect for the pathogens, mild effects for the probiotic strains and A. minutum, but no effect for commensal E. coli. The mild apoptotic effects observed are consistent with the beneficial roles of probotics in protection against colon cancer and suggest, for the first time, that A. minutum possesses similar advantageous, anti-cancerous activity. Although bacterial infection increased Caco-2 membrane FAS levels, caspase-8 was not activated indicating that apoptosis is FAS independent. Instead, in all cases, apoptosis was induced through the mitochondrial pathway as indicated by BAX translocation, cytorchrome c release, and caspase-9 and -3 cleavage. This suggests that an intracellular stimulus initiates the observed apoptosis responses.

Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters.

Survivability of a probiotic Lactobacillus casei in the gastrointestinal tract of healthy human volunteers and its impact on the faecal microflora

Aim: The aim of this study was to measure the gastrointestinal survival of Lactobacillus casei and its impact on the gut microflora in healthy human volunteers. Methods and Results: Twenty healthy volunteers took part in a double-blind placebo-controlled probiotic feeding study (10 fed probiotic, 10 fed placebo). The probiotic was delivered in two 65 ml aliquots of fermented milk drink (FMD) daily for 21 days at a dose of 8.6 +/- 0.1 Log(10)Lact. casei CFU ml(-1) FMD. Faecal samples were collected before, during and after FMD or placebo consumption, and important groups of faecal bacteria enumerated by fluorescent in situ hybridization (FISH) using oligonucleotide probes targeting the 16S rRNA. The fed Lact. casei was enumerated using selective nutrient agar and colony identity confirmed by pulsed field gel electrophoresis. Seven days after ingestion of FMD, the Lact. casei was recovered from faecal samples taken from the active treatment group at 7.1 +/- 0.4 Log(10) CFU g(-1) faeces (mean +/- SD, n = 9) and numbers were maintained at this level until day 21. Lact. casei persisted in six volunteers until day 28 at 5.0 +/- 0.9 Log(10) CFU g(-1) faeces (mean +/- SD, n = 6). Numbers of faecal lactobacilli increased significantly upon FMD ingestion. In addition, the numbers of bifidobacteria were higher on days 7 and 21 than on days 0 and 28 in both FMD fed and placebo fed groups. Consumption of Lact. casei had little discernible effect on other bacterial groups enumerated. Conclusions: Daily consumption of FMD enabled a probiotic Lact. casei strain to be maintained in the gastrointestinal tract of volunteers at a stable relatively high population level during the probiotic feeding period. Significance and Impact of the Study: The study has confirmed that this probiotic version of Lact. casei survives well within the human gastrointestinal tract.

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.