(Naar Nederlandse samenvatting)Timmerman H.M.
Summary
The main task of our gastrointestinal tract is to function as a biological food processor and the GI-tract could therefore easily be envisaged as ‘the inner tube of life’. In the process of food digestion the gut microbiota play an important role, such as degradation of complex carbohydrates. As many as 1014 bacteria (10 x more than the number of cells in the body) are contained within our digestive tract with the majority being beneficial to host health. However, a minority consists of gut bacteria which potentially are harmful or even pathogens. The intestine exploits a wide variety of defense systems to protect the host against aggressions from the external environment (i.e. pathogenic gut bacteria and viruses). This defensive task is based on 3 constituents that are in permanent contact and dialogue with each other: the microflora, the mucosal barrier, and the immune system. The presence of beneficial microbes (commensals) in the intestine prevents colonization or overgrowth by potential pathogenic microbes, referred to as colonization resistance. Constant ‘cross-talk’ between the commensal microflora and the host epithelium triggers a wide array of host genes involved in intestinal maturation and mucosal barrier fortification. This deliberate host-microbe cross-talk may aid in the colonization of the intestine by commensal bacteria that keep unwanted microbes at bay. Furthermore, the presence of a commensal microflora elicits anti-inflammatory systems protecting the intestinal epithelium from uncontrolled inflammation.
One way to reinforce or preserve the symbiotic relationship between mammals and their bacterial partners is through administration of beneficial microbes, i.e. probiotics. Probiotics are defined as live microbial food supplements that beneficially affect the host animal by improving its intestinal microbial balance. Although the concept of improving the intestinal balance with probiotics is already more than 100 years old, robust documentation of their specific health promoting effects and underlying mechanism of action is limited. In order to improve probiotic effectiveness and to gain insight into its associated mechanisms of action, this thesis addresses: 1) the design of probiotics for optimalization of gut health or treatment of specific diseases, 2) the effects of different probiotic designs on growth performance and general health variables in production animals, and 3) the possible mechanisms of action underlying the probiotic effects.
Chapter 1 is a general introduction describing the current state of knowledge on development of the intestinal microflora and its symbiotic interrelationships with the host. A multilevel view by which the microflora influences the 3 major constituents of intestinal defense is being discussed. Also the concept of probiotics and the most widely used probiotic strains are summarized. Furthermore, the aims and outline of this thesis are presented.
In Chapter 2 a literature review was performed to make a comparison of functionality and efficacy between monostrain, multistrain and multispecies probiotics. A monostrain probiotic is defined as containing a single strain of a certain species and consequently multistrain probiotics contain more than one strain of the same species or, at least of the same species or closely related species, for instance Lactobacillus acidophilus and Lactobacillus casei. Arbitrarily, the term multispecies probiotics is used for preparations containing strains that belong to one or preferentially more genera, e.g. L. acidophilus, Bifidobacterium longum, Enterococcus faecium and Lactococcus lactis. Analysis of all available literature shows that multispecies preparations have advantages when compared to monostrain probiotics or, to a lesser extent, multistrain probiotics. Well-designed multispecies probiotics could lead to synergistic effects when different probiotic effects of different probiotic species are combined. Probiotic activity can possibly also be stimulated through symbiosis among strains in the preparation. It was concluded that probiotic design should aim at finding combinations which show synergistic and symbiotic activities towards each other to maximize the chance of providing clinically more effective probiotic preparations.
Chapter 3 describes a series of experiments with 1-week old veal calves to test the effect of different probiotic concepts on growth rate and health indicators. The probiotics used were a multispecies probiotic (MSPB) containing different probiotic species of human origin, or a calf-specific probiotic (CSPB) containing 6 Lactobacillus species isolated from calf feces and selected on basis of their probiotic properties. When the data for all experiments were pooled, the probiotics enhanced the growth rate during the first 2 weeks. During the total 8-weeks experimental period, average daily weight gain and feed efficiency were significantly improved in the probiotic-treated groups. The MSPB-induced increase in weight gain was greater when the control calves were less healthy based on a general health score (expressed as an index of diarrhea and therapeutic treatments). Apart from the effects on general health, probiotic treatment also tended to diminish overall mortality. The CSPB treatment reduced the incidence of diarrhea and the fecal counts of coliforms. Most interestingly, probiotic treatment reduced the percentage of calves that required antibiotic therapy and the amount of treatments needed against digestive or respiratory diseases.
Chapter 4 reports on the application of a multispecies and a chicken-specific probiotic preparation in broiler production. The multispecies probiotic (MSPB) contained different probiotic species of human origin, whereas the chicken specific probiotic (ChSPB) consisted of seven Lactobacillus species isolated from the digestive tract of chickens. In a field trial with broilers, MSPB treatment did not result in significant improvement of broiler performance. However, ChSPB treatment improved feed efficiency, reduced mortality and increased total final body weight in two independent, consecutive field trials. In a controlled trial with broilers kept under optimal conditions, the treatment with probiotics did not influence feed efficiency, but it lowered mortality and increased total final body weight. Overall, probiotic treatment induced a clear reduction in mortality. Based on comparison with data in literature it was concluded that there is a tendency of probiotic treatment being more effective when broilers are kept under sub-optimal conditions and/or high infection pressure.
In Chapter 5 a functional differentiation is made for probiotics strains with regard to their metabolic activity. One of the potential mechanisms of action for probiotic bacteria may be their capacity to ferment mono- and disaccharides (glucose, lactose etc.). By doing so they reduce available substrates for other bacteria, and simultaneously they create an excess of fermentation products which may affect the non-probiotic flora. Regarding the capacity to generate these fermentation products (lactic acid, ethanol, acetic acid and CO2), specific probiotic strains can be selected on basis of their assumed potential to remove unwanted bacteria from the small intestine involved in ammonia production, a process thought to be relevant in the pathogenesis of hepatic encephalopathy. It is argued that especially CO2-producing (facultative) heterolactic lactobacilli, that produce both lactic acid and CO2 from sugars, are preferred in the treatment of hepatic encephalopathy.
Stabilization of the normal gut flora by probiotic bacteria as described above has health promoting effects. Next to the use of probiotics to prevent or treat gastrointestinal infections, probiotics also have profound immunomodulatory properties. This makes them of prime importance for diseases caused by a dysregulated immune system. Dysregulation of the Th1 (T helper 1)/Th2 (T helper 2) balance, in favor of Th2, might lead to the development of diseases like asthma and allergy. At birth, the immune system has been developed but not matured yet and is skewed towards Th2. Development of the gut microbiota is essential for driving the postnatal maturation of the immune system into a balanced Th1/Th2 immunity. Potentially, modulation of the gut microflora may generate appropriate immunoregulatory responses that evoke the necessary switch to Th1/Th2 equilibrium. Recent clinical trials suggest that probiotic bacteria may decrease and prevent allergic symptoms, but which species or strains are most effective is poorly understood.
In Chapter 6 potential probiotic strains were selected on basis of their capacity to skew immune responses to a less allergic phenotype. Therefore, peripheral blood mononuclear cells (PBMCs), purified monocytes, and lymphocytes from healthy donors were co-cultured with 13 different strains of probiotic bacteria. The effect of lactic acid bacteria on different cell populations and effects on cytokine production induced by the polyclonal T cell stimulator phytohaemagglutinin was evaluated by measuring Th1, Th2 and regulatory cell cytokines in culture supernatants by multiplex assay. Production of the regulatory cytokine IL-10 by in vitro cultured mononuclear cells was strain-specifically upregulated. In PHA stimulated PBMC cultures, the tested strains decreased the production of cytokines associated with atopic disease (Th2). Neutralization of IL-10 production with a blocking antibody resulted in partial to full restoration of Th2 cytokine production and concurred with an increase in pro-inflammatory cytokines such as IL-12p70 and TNF-α. It was suggested that probiotic bacteria may divert the immune system into a regulatory mode by inducing IL-10. Based on these data, Bifidobacterium bifidum, Bifidobacterium infantis, and Lactococcus lactis were selected to be applied as a multispecies probiotic in a clinical trial (the PANDA study) on primary prevention of allergic diseases by probiotic bacteria.
Chapter 7 addresses the potential role of probiotics in the prevention of infectious complications in surgical patients. Under normal circumstances the mucosal epithelium of the intestine forms a strict barrier between the luminal content preventing microorganisms from entering the body and causing infectious complications. In these patients the three major constituents of intestinal defense (the commensal microflora, the mucosal barrier, and the immune system) are impaired. This can be caused directly by the underlying disease (such as pancreatitis), antibiotic treatment or surgical intervention. In the critically ill patient, small bowel motility is disturbed, leading to bacterial overgrowth and subsequent bacterial translocation due to dysfunction of the gut mucosal barrier and the immune system. For several decades antibiotic prophylaxis to prevent translocation of pathogenic bacteria has been used but with variable or even conflicting results. Selective decontamination of the digestive tract (SDD) has shown promising results, but the risk of bacterial multiresistance has prevented widespread implementation. In recent years, probiotics have shown promising results in several randomized placebo-controlled trials.
Based on pathophysiological events in critically ill patients, selection procedures of specific strains were undertaken to design a disease specific multispecies probiotic (Chapter 8).From a strain collection of 69 different lactic acid bacteria a primary selection was made of the 14 species showing superior survival in a simulated gastrointestinal environment. Functional tests like antimicrobial activity against a range of clinical isolates and cytokine inducing capacity in cultured human peripheral blood mononuclear cells (PBMCs) were used to further identify potentially useful strains. Lactobacillus plantarum, Bifidobacterium infantis and Lactobacillus rhamnosus superiorly inhibited growth of the clinical isolates whereas Bifidobacterium infantis, Lactococcus lactis and Lactobacillus acidophilus were the strongest inducers of the anti-inflammatory cytokine IL-10. Production of the pro-inflammatory cytokines IL-2 and IL-6 in phytohaemagglutinin stimulated PBMCs was inhibited by the majority of the probiotic strains tested. Based on these results and general criteria regarding probiotic design and safety a selection of the following 6 strains was made (Ecologic 641); Bifidobacterium bifidum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius and Lactococcus lactis. Combination of these strains with specific probiotic effects resulted in a wider antimicrobial spectrum, superior induction of IL-10 and silencing of pro-inflammatory cytokines as compared to the individual components.
The potential of this specifically designed multispecies probiotic mixture (Ecologic 641) for reduction of bacterial translocation or improvement of clinical outcome in a rat model of acute pancreatitis was tested in Chapter 9. To that end, probiotics or placebo were administered daily via a permanent gastric cannula, from five days prior to until seven days after induction of pancreatitis. It was found that probiotic treatment significantly reduced bacterial overgrowth of potential pathogens in the duodenum (10 5 and10 3 CFU/g in placebo and probiotic treated animals, respectively), resulting in significantly reduced bacterial translocation to extra-intestinal organs, including the pancreas (10 5 and 10 3 CFU/g tissue in placebo and probiotic treated animals, respectively). Accordingly, health scores of surviving rats treated with probiotics were better throughout the experiment and late phase mortality was significantly reduced by 44%.
In Chapter 10 it is assessed whether plasma levels of various cytokines and chemokines correlated with clinical outcome in experimental pancreatitis, and secondly, how the cytokine levels were affected by probiotic treatment. Therefore, plasma cytokine levels were determined before, and 6 hrs, 24 hrs and 7 days after induction of pancreatitis. We showed that specific ‘cytokine signatures’ can be identified during the course of acute pancreatitis which are predictive of either early mortality due to the severity of acute pancreatitis (IL-6, IL-10, CXCL1) or bacteraemia potentially causing late mortality (TNF-a and IL-1ß). An early and self-resolving IL-10 response was found in animals protected from early mortality, whereas sustained elevated levels of IL-10 predisposed to the development of bacteraemia. Probiotic treatment resulted in marginally lower levels of pro-inflammatory cytokines, followed by improved clinical outcome (reduction of mortality or bacteraemia).
To increase our insight in the role of the gut flora on the process of bacterial translocation during experimental pancreatitis a more detailed analysis of the bacterial communities in the various parts of the intestine was made (Chapter 11). By means of molecular techniques based on the 16S ribosomal RNA (rRNA) gene analysis it has become possible to detect unculturable bacteria as well. The applied technique, microbial community profiling and characterization (MCPC), allowed us to identify pancreatitis and probiotic induced changes in intestinal microflora, and secondly, to correlate probiotic induced changes in microbial communities to clinical outcome. During acute pancreatitis the host-specific microflora is replaced by an ‘acute pancreatitis associated microflora’. Although probiotic treatment did not reverse this situation, the presence of an unidentified commensal rat ileal bacterium (CRIB) was significantly upregulated. The amount of CRIB was positively and significantly correlated with improved pancreas histology, reduced bacterial counts in duodenal, mesenteric lymph nodes, spleen, liver and pancreatic necrosis, and reduced plasma levels of pro-inflammatory cytokines. It was concluded that protection and stimulation of an as yet unidentified commensal bacterium in the ileum of rats by probiotic therapy resulted in a reduced severity of pancreatitis and associated sepsis. We anticipate that our current findings and future studies involving the currently unidentified commensal bacterium will be of key importance in unraveling the mechanisms of probiotic action.
Conclusions and perspectives
Rational grounds for strain selection criteria have been provided to develop disease-specific multispecies probiotics. The presented method could also be used as a template for other disease entities. Finally, in vivo experiments and clinical trials are needed to confirm the in vitro observed synergism between individual strains outranking the probiotic activity of individual strains.
Probiotic treatment improved the general health score in different animal models of mild (intensive animal production systems) and severe stress (acute pancreatitis). A probiotic-induced improvement of growth performance was found only when health was (severely) compromised. Surprisingly, the health-promoting effects of probiotics are not only restricted to the gastrointestinal tract, but may also extend to extra-intestinal organs like the lungs. In all experimental models studied, probiotic treatment reduced the incidence of mortality.
Probiotic treatment alters the host physiology at multiple levels, implying that a unifying mechanism of action may not be found. Preliminary data, presented in the General Discussion (Chapter 12), indicate that probiotics can interfere with pathogenic biofilm formation, a mechanism with great potential for chronic and/or catheter associated infections. To that end, the current view that probiotics are bactericidal or bacteriostatic agents should be extended towards sociomicrobiology in which pathogenic biofilm formation can be inhibited and in which the commensal microflora is maintained or even stimulated. The data presented in Chapter 11 indicate that stimulation of an as yet unidentified commensal has great impact on the well-being of the host. This probiotic induced effect may indirectly overrule its own potential to positively influence the immune system. Identification of this commensal and its beneficial interrelationship with the host and the applied probiotic could provide a key mechanism of probiotic action and will extend or improve therapeutic possibilities dramatically.
A central mechanism of action of probiotic bacteria may be preservation or reinforcement of the mucosal barrier. Indeed, administration of probiotics resulted in a significant reduction of bacterial translocation in experimental pancreatitis. The cellular and molecular pathways by which probiotics interact with the immune system have not been fully elucidated yet. It is attractive to postulate a central role for dendritic cells in this respect and receptors such as DC-SIGN (or other C-type lectins) at the interface. Dendritic cells in the gut-associated lymphoid tissue probe the bacterial composition with dendrites reaching into the lumen. Dendritic cells are major IL-10 producers and can dictate the direction of ensuing immune responses. Thus, probiotic strains with the capacity to induce regulatory T-cells in vivo through modulation of dendritic cell function might point towards new therapies for autoimmune diseases like diabetes and rheumatoid arthritis.
Broadening of the scientific basis for probiotic action should set new selection criteria, allowing for the development of improved probiotic concepts. Furthermore, clinical trials are warranted to demonstrate the effectiveness of probiotics in vivo. Once probiotic action and functionality are more widely scientifically proven, probiotic therapy can become part of general medical practice.
thesis University of Utrecht 2006