Ending the War Metaphor The Changing Agenda for Unraveling the Host-Microbe Relationship: Workshop Summary (2006) / Chapter Skim
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6 Manipulating Host-Microbe Interactions: Probiotic Research and Regulations
6 Manipulating Host-Microbe Interactions: Probiotic Research and Regulations
Pages 207-260
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From page 207... ...
In the first contribution to this chapter, he notes that comparative genomic studies of probiotic bacteria may lead to insights on the nature of molecular mechanisms that confer probiotic effects -- findings that could be complemented by DNA microarray technology that analyzes host responses to probiotic microbes. Further, Kleerebezem describes how techniques previously used to elucidate in vivo responses of pathogenic bacteria to environmental parameters in such complex niches as the gastrointestinal (GI)
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In particular, they explore the potential of probiotic lactic acid bacteria to enhance systemic, as well as mucosal, immune response in infants and young children and the use of probiotic bacteria as antigen delivery vehicles or adjuvants in HIV-1-positive patients. Based on the results of their efforts, Cunningham-Rundles noted in workshop discussion that she and her coworkers are preparing an investigational new drug (IND)
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MOLECULAR ANALYSIS OF PROBIOTIC-HOST INTERACTIONS IN THE GASTROINTESTINAL TRACT Michiel Kleerebezem1 Abstract Recent years have seen an explosion in the number of complete, and almost complete, genome sequences of lactic acid and other food-grade bacteria, including probiotic strains that are applied as functional food ingredients to increase the health of the consumer. This information is crucial for the development of functional, comparative, and other postgenomic approaches to unravel the in situ functionality of these bacteria in the human intestinal tract and how they affect consumer health at the molecular level.
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Most probiotic preparations currently marketed aim at functional modulation of specific physiological aspects of the GI tract of the consumer. In this respect, it is important to note that these probiotic cultures are to exert their effects on host physiology within this highly complex ecosystem that is colonized by a myriad of endogenous microbes (microbiota, as described below)
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This progress includes elucidation of the (partial) genome sequences of more than 20 lactic acid bacteria and bifidobacteria (Table 6-1; Klaenhammer et al., 2005)
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The molecular interaction models should allow the identification of bacterial and host markers involved in probiotic-mediated host health benefits. Such information can subsequently be employed to construct or select specific strains or mutants that have improved or enhanced health of the host, which would generate second-generation probiotic cultures with science-based health claims and validated modes of action.
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Based on the probiotic definition, it is postulated that probiotic features can be attributed to specific strains rather than to any particular species as a whole. This notion suggests that activities proposed to confer probiotic effects are expected to be variable among different strains of a species.
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coli inhibitory, probiotic character mediated by this msa gene can now be established using isogenic variants that either lack or overexpress a functional copy of this gene. In analogy, application of host and microbe postgenomics tools in the actual in vivo setting enables new avenues towards more exploratory research aiming to unravel the molecular interactions between host and microbe that underly the health benefits mediated by consumption of probiotic bacteria (Figure 6-1)
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Although not noticed by the authors at the time of publication, this finding already hinted at overlap in the genetic response triggered in the pathogenic and nonpathogenic bacteria upon contact with the host intestine. This notion was further exemplified by an R-IVET approach in L
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molecules in the GI tract of humans. The potential of lactic acid bacteria in this type of application has recently been reviewed by Hanniffy et al.
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plantarum cells were found to be metabolically active in all subjects, and differences between gene expression between the individuals and intestinal location were apparent. Moreover, significant parallels were observed in the mouse intestine-induced ivi genes and those that were detected as being highly expressed in the human intestinal system.
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thetaiotaomicron colonization of gnotobiotic mice provided valuable information on the influence of one particular member of the microbiota on the host. However, the host response during colonization by more complex mixtures of microbes, and/or the host response in other animal systems, remained to be investigated at that time.
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These results suggest that the gut microbiota have a major impact on food-derived energy harvest and storage in the host (Bäckhed et al., 2004)
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Besides the application of DNA microarray technology to reveal the bacterial side of host-microbe interactions, this technology also allows host response analyses. Combination of these knowledge datasets should enable scientists to unravel the mechanisms that are underlying the effects of intestinal and food-derived bacteria on host physiology, and will provide the probiotic arena with new and more scientifically solid consumer health benefits.
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Probiotic lactic acid bacteria offer a possible approach for stimulating the GI immune system thereby enhancing systemic as well as mucosal immune response. Rigorous studies are needed to assess whether the microbial microenvironment can be modulated by the introduction of defined probiotic bacteria and to analyze the long-term potential for host defense.
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. The gut microbiota as a whole is essential for production of short-chain fatty acids from polysaccharides and has been shown to regulate host metabolism through direct effects on fat storage (Bäckhed et al., 2004)
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However, efforts to improve IBD and irritable bowel syndrome with probiotic bacterial supplementation have only had limited success, perhaps because the central mechanism of these disorders appears to involve altered host response to commensal flora (Schultz et al., 2004b)
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Breast-fed infants have higher levels of lactic acid bacteria and enhanced mucosal immune response (Martin et al., 2003)
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. Probiotic bacteria have been given to infants primarily for prophylaxis and alleviation of diarrheal disease, reduction in atopic disease, reduction in necrotizing enterocolitis, and reduction in infection, with generally beneficial effects (Kliegman, 2005; Schrezenmeir et al., 2004)
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These results indicate that the innate immune response in newborns to commensal bacteria is strong, and they also suggest that specific bacterial strains may have differential effects on the maturation of the immune system. Response to Lactobacillus in HIV-1 Immune Deficiency Primary immune deficiency is associated with sinopulmonary or GI infections, autoimmunity, and neoplasia (Kalha and Sellin, 2004, CunninghamRundles, 1994)
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plantarum 70 60 Activated 50 40 Percent 30 20 10 0 IL-6 IL-8 FIGURE 6-3 Intracellular cytokine response of cord blood monocytes from term infants to bacterial antigens. Data are shown as percent of monocytes producing the denoted cytokines after 18 hours of culture.
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In summary, commensal bacteria have a modulatory effect on GI immune function that is critical for host interaction with neoantigens and also for the development and maintenance of systemic immune response. Probiotic bacteria have been shown to support gut defense through immune exclusion, immune elimination, and immune regulation in several settings and have potential future use as immune modulators.
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has regulatory authority over the development and marketing of probiotics for clinical treatment indications. FDA/CBER's Experience in Regulating Probiotics as Biological Drug Products As interest has grown in the United States over the last few years in studying the effects of probiotics on specific diseases and conditions, CBER/OVRR has provided much regulatory advice to individuals including practitioners interested in conducting clinical research to evaluate probiotics, recipients of government funding for such research, as well as probiotic manufacturers.
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In this case, the prebiotic substance is subject to regulation by CBER as a component or ingredient of a biological drug product. Nonregulatory Product Terms It is important to clarify for regulatory purposes that currently probiotic, prebiotic, and live biotherapeutic are not regulatory terms, even though these terms are used to describe particular products that if introduced into interstate commerce within the United States would be regulated by the FDA.
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As noted above in this section, this term is not currently used or defined in any law or regulation pertaining to food and/or drugs. Live biotherapeutic products differ from traditional vaccine products because their dosing regimens more closely resemble those of drugs (i.e., they are typically administered on a daily basis for a given period of time or indefinitely)
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Of note, GRAS has been frequently misused as a regulatory term, concerning probiotic products that are considered biological drug products, because use of the term GRAS is limited to food regulation.
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based on limited claims such as those that pertain to affecting the structure and function of the human body or general well-being,10 but not based on drug or disease claims. Probiotic products intended for intravaginal or topical use are not dietary supplements by definition, because they are not intended for ingestion.
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· A probiotic manufacturer who intends to market a probiotic product based on a drug claim must seek approval for that claim. The Biological Drug Development Pathway for Live Biotherapeutics Data to support approval of a biological product are provided in a BLA to CBER for review.
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data that are required to support the clinical evaluation of a live biotherapeutic product in human subjects.16 In this regard, probiotic manufacturers may not be familiar with biological product manufacturing requirements, which greatly exceed those for foods or dietary supplements.
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Specifically, CBER's OVRR has gained tremendous experience in this area in recent years. In general, the development of a live biotherapeutic product as a biological drug product should proceed along the established product and clinical development pathway for drugs and biologics, with safety being continuously 17Possible approaches to defining and evaluating the potency of live biotherapeutic products might include the quantitation of specific, live organisms or the production of particular substances (e.g., lactic acid)
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regulatory standards for biological products and clinical study design that can be applied to the development of live biotherapeutic products. Finally, in its recent experience in regulating these products, CBER's OVRR has encountered numerous challenges and issues that impact valid evaluation of these products for therapeutic uses.
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To evaluate the potential of good bacterium to proliferate into a gut already heavily inhabited is not an easy task. Technical difficulties abound in analysing the gut microbiota as a whole.
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. This statement is in full opposition with "conventional wisdom," and even with many recent scientific reviews on the selection of probiotic bacteria stating that probiotic bacteria must be selected among "bacteria isolated from human beings." Even acknowledging the difficulty with determining the natural source of a particular strain, species including Saccharomyces boulardii and Bifidobacterium animalis, which are not considered to have humans as their natural habitat, are documented as very effective probiotics (see also comments on the efficacy of Saccharomyces boulardii, in the section "Good Bugs: Data with Consensus")
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Among those microorganisms that are culturable, species identification by traditional biochemical identification methods is often difficult. Assessment of gut microbiota using genetic techniques, applicable also to unviable bacterial cells, is a relatively new approach able to overcome at least some of these problems.
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The Subdominant Population If human beings are looking for allies in the war against pathogens, they should not forget the so-called subdominant population bacteria that are detected at levels below 108 per gram of faeces. Among these groups we can encounter the most studied and used "healthy bacteria," the lactic acid bacteria, mainly belonging to the genus Lactobacillus.
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TABLE 6-3 Lactobacillus Species Recovered and Genetically Identified from Biopsies of Seven Adults Subjects Lactobacillus species A L rhamnosus; all sites B L
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. Recently we identified an abundant Lactobacillus microbiota in honeybees, and, reinforcing the concept of host specificity, the four Lactobacillus species identified were all new species never before isolated (Bessi and Morelli, 2005)
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For example, probiotic bacteria currently on the market persist for weeks, at a maximum, in the gut of fed volunteers. Probiotics able to persist long term in the human gut may need to be reassessed for safety (Fujiwara et al., 2001)
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· The probiotic activity was originally quite similar to a feed supplement for bacteria, then it turned into an ecological action towards the intestinal microbiota. Nowadays, there is increasing evidence that the presence of specific bacterial strains in the human gut could have a favourable action per se, without any detectable change in the overall composition of the gut microbiota.
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Probiotic Activities with Consensus The expert consultation reviewed · disorders associated with the GI tract, · prevention of diarrhea caused by certain pathogenic bacteria and viruses, · Helicobacter pylori infection and complications, · inflammatory diseases and bowel syndromes, · cancer, · constipation, · mucosal immunity, · allergy, · cardiovascular disease, · urogenital tract disorders, · bacterial vaginosis, · yeast vaginitis, · urinary tract infections, and · use of probiotics in otherwise healthy people. What seems relevant to a microbiologist in this section of the presentation is that the above evidence of probiotic activities have been obtained with bacterial probiotic populations detected in fecal samples at a level less than 1/100 of the total bacterial population.
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. The important role of commensal bacteria in mucosal immunity development was first demonstrated in a germ-free animal model; colonization of germ-free zebrafish with individual members of its microbiota revealed the bacterial species specificity of selected host responses.
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FAO/WHO constructed a decision tree (Figure 6-6) to obtain data with consensus for new probiotic products (FAO/WHO, 2002)
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· There is concern over the use in foods of probiotic bacteria that contain specific drug resistance genes. · The consultation recognized the need for the development of standardized assays for the determination of drug insensitivity or resistance profiles in lactobacilli and bifidobacteria.
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However, there are some cases in which prebiotics may be beneficial for the probiotic, especially with regard to bifidobacteria; that is the synbiotic concept. Synbiotics are defined as "mixtures of probiotics and prebiotics that beneficially affect the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract of the host" (Andersson et al., 2001)
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2005. Com plete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM.
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2002. Molecular biological meth ods for studying the gut microbiota: The EU human gut flora project.
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2004. Post-genomics of lactic acid bacteria and other food grade bacteria to discover gut functionality.
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. Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria.
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1998. Taxonomy and physiology of probiotic lactic acid bacteria.
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2003. Case study of the distribution of mucosa-associated Bifidobacterium species, Lactobacillus species, and other lactic acid bacteria in the human colon.
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2002. Gastrointestinal tract and the elderly: Functional foods, gut microflora and healthy ageing.
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2005. Diversity, vitality and activities of intes tinal lactic acid bacteria and bifidobacteria assessed by molecular approaches.
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Applied Environmental Microbiology 68(7)
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