6
Manipulating Host-Microbe Interactions: Probiotic Research and Regulations

OVERVIEW

As defined by an expert panel convened in 2002 by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), probiotics are “live microorganisms administered in adequate amounts that confer a beneficial health effect on the host.” (FAO/WHO, 2002). Contributors to this chapter discuss the meaning of “beneficial health effects” in this context, and more importantly, how such overtly qualitative notions can be replaced by quantifiable variables. The first two papers explore genetic and molecular mechanisms specific to interactions between probiotic bacteria and their hosts; two additional papers describe how the health benefits of probiotic bacteria are currently understood and how their safety and efficacy could be characterized in the future.

Recently developed genomic technologies provide a promising route to evaluating the effects of probiotics on the host-microbe relationship, according to Michiel Kleerebezem of the Wageningen Centre for Food Sciences in the Netherlands. 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) tract, and that are currently being applied to probiotic bacteria, may permit the construction of site-directed bacterial vehicles for delivering beneficial molecules to the human GI tract: the next generation of probiotics.



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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary 6 Manipulating Host-Microbe Interactions: Probiotic Research and Regulations OVERVIEW As defined by an expert panel convened in 2002 by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), probiotics are “live microorganisms administered in adequate amounts that confer a beneficial health effect on the host.” (FAO/WHO, 2002). Contributors to this chapter discuss the meaning of “beneficial health effects” in this context, and more importantly, how such overtly qualitative notions can be replaced by quantifiable variables. The first two papers explore genetic and molecular mechanisms specific to interactions between probiotic bacteria and their hosts; two additional papers describe how the health benefits of probiotic bacteria are currently understood and how their safety and efficacy could be characterized in the future. Recently developed genomic technologies provide a promising route to evaluating the effects of probiotics on the host-microbe relationship, according to Michiel Kleerebezem of the Wageningen Centre for Food Sciences in the Netherlands. 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) tract, and that are currently being applied to probiotic bacteria, may permit the construction of site-directed bacterial vehicles for delivering beneficial molecules to the human GI tract: the next generation of probiotics.

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary Research in Suzanne Cunningham-Rundles’ lab concerns the possible role of probiotic bacteria in the modulation of the host immune response. In their contributed paper, Cunningham-Rundles and coworkers describe recent studies characterizing the initial formation of mucosal immunity through interaction with the GI microflora—an interaction that may induce specific and persistent immune response patterns in the host, and which could potentially be manipulated with probiotics. 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) application for probiotic-fortified formula intended for use in low birth-weight infants. The IND application process and its implications for probiotic development are described in the subsequent essay on U.S. probiotics regulatory issues by workshop participant Julienne Vaillancourt of the FDA’s Office of Vaccine Research and Review in the Center for Biologics Evaluation and Research, which has regulatory authority over the development and marketing of probiotics for clinical treatment indications. To date, all probiotics on the U.S. market fit the FDA definition of a dietary supplement (“a product taken by mouth that contains a dietary ingredient intended to supplement the diet”). Although this situation is expected to change, there is little incentive for manufacturers of probiotics—currently marketed as dietary supplements—to develop them as biotherapeutics given the rigors and expense of the associated review and regulation process. A similar situation currently exists in Europe, where several countries are currently considering legislation to require proof for health claims by manufacturers of dietary supplements. In the United States, the dietary supplement/biotherapeutic dichotomy is likely to remain a part of probiotic regulations for some time. However, as Vaillancourt notes, the regulatory process for biotherapeutics is likely to expand and change to reflect new knowledge; she identifies several issues that need to be addressed through collaborative efforts between all involved parties. It is not only clear that guidelines and regulations governing probiotics must be revised to reflect recent research findings, but also that this goal is a fast-moving target. In the final contribution to this chapter, workshop presenter Lorenzo Morelli and coworkers describe the process and considerations that produced the recent FAO/WHO guidelines for the evaluation of probiotics in food and raise a variety of cutting-edge issues that need to be addressed in subsequent guidelines, including: new genomic techniques that allow enhanced characterization of the gut microbiota, evaluation of emerging methods that allow assessment of bacteria-epithelial interactions,

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary potential for targeted probiotic or biotherapeutic methods, such as to enhance the production of a specific cytokine or to suppress a specific pathogen. The authors emphasize the need for such guidelines to be flexible and to reflect ongoing communication between regulatory bodies and the scientific community. 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. These advances can ultimately be exploited to develop novel and designer probiotics with a predestined impact on consumer gut health. Introduction The term probiotics was coined in the 1960s, although, the probiotic concept has existed for a much longer time. The definition of the term has changed through the years, but perhaps the most appropriate definition was published by an expert consultation at a meeting convened by the FAO and the WHO in October 2001, which states, “probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2001). Although relatively simple, it defines six aspects that must be fulfilled by probiotic culture applications, while it encompasses probiotic applications outside the food market (Sanders, 2003): Probiotics must be alive, which redirects reference to physiological effects in hosts of the administration of dead cells or cellular fractions to an alternative term. Probiotics must deliver a measured physiological benefit that requires substantiation by studies performed in the target host organism. 1   Wageningen Centre for Food Sciences, NIZO Food Research. P.O. Box 20, 6710 BA Ede, The Netherlands, Phone: 31-318-659629, Fax: 31-318-650400, E-mail: Michiel.Kleerebezem@nizo.nl.

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary Probiotics are not necessarily administered as food products via the oral route, but could encompass other applications. Pharmaceutical and therapeutic applications of probiotics are not excluded. Restrictions in terms of mode of action are not defined; therefore, aspects such as survival of the gastrointestinal (GI) tract passage or affect on the normal microflora are not required. Because it is scientifically untenable to envision validated host-physiology effects convened by undefined microbial preparations, the definition also implies that probiotics are defined strains. Despite this definition of the term probiotics, regulatory restrictions for the use of the term in specific applications are lagging. Moreover, probiotic health claims on products in general tend to be vaguely phrased. As a consequence, in most cases it is virtually impossible for consumers to perceive what to expect from a product carrying the probiotic designation. 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). The species most commonly encountered in these products are specific strains of bifidobacteria and lactobacilli. The health benefit(s) attributed to these products is highly diverse, and the quality of the scientific evidence underlying these benefits is highly variable. The evidence is mostly descriptive and frequently lacks comparison of different strains of bacteria to validate the probiotic’s specificity. In addition, the molecular basis of the effects on host physiology of specific probiotics remains largely unexplored. Therefore, correlating specific physiological effects measured in the target host to a specific characteristic of a bacterial strain is not yet possible. Such correlations could provide avenues toward second-generation probiotics with predestined or improved health effects. Probiotics and the Human GI Tract Microbiota The human GI tract is colonized by a vast and complex consortium of mainly bacterial cells. This microbiota consists of at least 1013 microbes, dominated by anaerobic bacteria, comprising over 1,000 species, of which the majority cannot yet be cultured under laboratory conditions (Vaughan et al., 2000; Zoetendal et al., 2004). Culture-independent molecular approaches that utilize the 16S ribosomal RNA (rRNA) genes as a universal bacterial biomarker have been used to monitor the composition of the dominant GI tract microbiota in different individuals at different time points in their lives. These approaches revealed a relatively stable composition in individual adults, but appeared to vary considerably when differ-

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary ent individuals were compared (Zoetendal et al., 1998). Moreover, host development, host genotype, and environmental factors influence the composition of the microbiota, illustrating how microbiologically challenging this environmental niche is (Zoetendal et al., 2004). Many functions are associated with the bacterial GI tract communities and their interaction with the host system including roles in host nutrition, intestinal epithelial development and activity, education of the immune system, maintenance of the integrity of the mucosal barrier, and contribution to drug and xenobiotic metabolism. However, we are only beginning to understand the dimensions of these activities and interactions. Next to the global microbiota-composition profiling efforts described above, dedicated 16S rRNA-based methods have been developed to track and trace specific species within the GI tract (Vaughan et al., 2005). These studies not only enable profiling the microbiota at the level of a specific species that is endogenously present in the GI tract, but also allow more detailed analyses of the fate of orally administered (probiotic) bacteria during their transit through this complex system. Nevertheless, these technologies do not allow the study of microbiota or probiotic in situ activity, and thus do not provide insight in their mode of action in relation to host functionality. In contrast, genomics-based approaches should allow the functional evaluation of host and microbe interactions at the molecular level. Such molecular studies should reveal the molecules involved in these interactions, which would allow the development of molecular models that underlie host-microbe interactions. Such mechanistic insight into the way probiotic bacteria affect health will not only contribute to novel, improved functional foods but also to the support of their health claims that will most likely be subject to an increased scientific scrutiny in the future. Therefore, these developments will not only provide benefits for the scientific or industrial community, but will also meet the interest of the consumer (de Vos et al., 2004) (Figure 6-1). Bacterial Genomics Over the past decade, the sequences of more than 200 bacterial genomes have become available in the public domain. Considerable attention has focused on pathogenic bacteria, including food-borne pathogens. However, over the last years, sequencing the genomes of GI tract commensals and symbionts as well as food-grade bacteria has received considerable attention. 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). Among these are the published complete genome sequences of species associated with probiotic health effects in humans, including Lactobacillus plantarum (Kleerebezem et al., 2003; van Kranenburg et al., 2005), Lactobacillus acidophilus (Altermann et al., 2005), Lactobacillus johnsonii (Pridmore et al., 2004), and Bifidobacterium longum (Schell et al., 2002). Additionally, the release of genomic sequences of commensal human GI bacteria such as Bacteroides thetaiotaomicron (Xu et al., 2003)

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary FIGURE 6-1 Schematic representation of pure culture genomics in relation to GI tract behavior, and corresponding molecular analyses of host responses. 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. SOURCE: Kleerebezem (2005). will add valuable information to this field. Moreover, rapid expansion of this knowledge base is anticipated upon publication of the genomic sequences of several other species that can be categorized as (potential) probiotics (Table 6-1). The availability of these bacterial genome sequences and their annotated functions provides valuable clues towards the survival strategy of these bacteria during their residence in the human GI tract. Moreover, in silico comparative

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary TABLE 6-1 Overview of Genome Sequences of Food-Grade Bacteria. Adapted from Klaenhammer et al., 2005 Genus Species Strain(s) Genome size (Mbp) Bifidobacterium longum NCC2705, DJ010A 2.3, 2.4   breve NCIMB 8807 2.4 Brevibacterium linens ATCC9174 4.4 Enterococcus faecalis V583 3.2 Lactobacillus acidophilus NCFM 2.0   gasseri ATCC333323 1.8 johnsonii NCC533 2.0 plantarum WCFS1 3.3 casei ATCC334, BL23 2.5, 2.6 rhamnosus HN001 2.4 helveticus CM4, CNRZ32 2.0, 2.4 sakei 23K 1.9 delbrueckii ATCCBAA365, ATCC11842, DN-100107 2.3, 2.3, 2.1 reuteri   2.0 salivarius UCC118   brevus ATCC367 2.0 Lactococcus lactis spp. lactis IL1403 2.3   lactis spp. cremoris SK11, MG1363 2.3, 2.6 Leuconostoc mesenteroides ATCC8293 2.0 Oenococcus oeni ATCCBAA331, IOEB84.13 1.8, 1.8 Pediococcus pentacosus ATCC25745 2.0 Propionibacterium freundereichii ATCC6207 2.6 Streptococcus thermophilus LMD9, LMG 18311, CNRZ1066 1.8, 1.9, 1.8   SOURCE: Kleerebezem, 2005. genomics can provide important insight in diversity, evolutionary relationship, and functional variation between bacteria (Boekhorst et al., 2004, 2005), which might eventually generate a comprehensive prediction of microbe behavior during residence in the human GI tract. 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. A clear example where this could experimentally be established is the mannose-specific adherence capacity observed in L. plantarum that is proposed to be involved in inhibition of pathogenic Escherichia coli infections via a mechanism of competitive exclusion (Adlerberth et al., 1996; Pretzer et al., 2005). It had been reported earlier that the capacity to adhere specifically to mannose moieties is a variable phenotype among L. plantarum strains. Nevertheless, thanks to the availability of the L. plantarum WCFS1 template genome and the corresponding DNA microarrays, this relevant phenotype could be correlated to a genotypic diversity

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary that corresponded to a single gene that could subsequently be shown to encode the mannose-specific adhesion (therefore designated: msa gene) in this species by mutation analysis (Molenaar et al., 2005; Pretzer et al., 2005). The functional proof of the E. 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). Monitoring Bacterial Responses to Intestinal Conditions: In Vitro and In Vivo Approaches Severe barriers are met by bacteria that we consume as part of our diet. These include several physical and chemical conditions, such as gastric acidity, the presence of bile salts, and stress conditions associated with oxygen gradients that are steep at the mucosal surface while the intestinal lumen is virtually anoxic. In addition, bacterial competition is high throughout the intestinal tract, but reaches a climax in the colon where bacterial densities are highest. Because the GI tract environment is highly complex and differs in different regions of this niche, many studies describe the in vitro response of bacteria to a simplified model that mimics a single host GI tract parameter. Most of these studies have focussed on intestinal pathogens, including studies describing the response toward acid and bile stress in bacteria such as Salmonella species, Escherichia coli, Listeria monocytogenes, and Enterococcus faecalis. More recent studies describe the molecular responses of food-grade and probiotic bacteria to similar stress conditions. Only a few studies describe genomewide approaches aiming at the identification of genes and proteins important for acid- and bile-resistance in lactobacilli or bifidobacteria. With Lactobacillus plantarum, a genetic screen resulted in the identification of more than 30 genes that are induced by bile-stress in vitro, and for a selection of these genes it could be established that induction of these genes also occurs in vivo in the duodenum of mice (Bron et al., 2004a). Additionally, a recent DNA microarray-based study in the same species revealed a number of genes and gene clusters of which the expression is significantly affected by the presence of bile acids (Bron et al., 2006). Many of these genes are predicted to play a role in the cell membrane or cell wall, which is in good agreement with several physiological studies in GI tract bacteria, including Lactobacillus plantarum and Propionibacterium freudenreichii, demonstrating how bile salts induce severe changes in the morphology of the cell membranes and/or cell walls of these organisms (Bron et al., 2004a; Leverrier et al., 2003). Overall, these in vitro studies have generated valuable insight in the responses of food-grade and probiotic bacteria to the individual parameters that are encountered in the host GI

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary tract. However, the extrapolation of these results to the real-life situation in this highly complex niche is certainly not straightforward and requires a substantial amount of additional research to establish the importance of the in vitro responses in terms of the full response repertoire triggered in vivo (Bron et al., 2004a). To experimentally approach the elucidation of in vivo responses of bacteria to environmental parameters in complex niches such as the GI tract, more sophisticated methods have been developed. Three main strategies have been employed for the identification of genes that are highly expressed in vivo as compared to laboratory conditions: in vivo expression technology (IVET) and recombination-based in vivo expression technology (R-IVET), signature-tagged mutagenesis (STM), and selective capture of transcribed sequences (SCOTS) (Bron et al., 2005). These in vivo gene-identification strategies were initially used exclusively in pathogenic bacteria, aiming at the identification of genes important to the virulence of these species. Recently, the first two reports appeared that describe the utilization of R-IVET strategies in food-grade or commensal microorganisms in order to determine the specific induction of gene expression in these bacteria after introduction in the GI tract of animal models. With L. reuteri an IVET strategy based on in vivo selection of an antibiotic-resistant phenotype led to the identification of three in vivo induced (ivi) genes during colonization of the GI tract of Lactobacillus-free mice (Walter et al., 2003). One of these genes encodes a peptide methionine sulfoxide reductase that has previously been identified using IVET in Streptococcus gordonii during endocarditis. 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. plantarum (Bron et al., 2004b). This study revealed 72 L. plantarum genes that were ivi during passage of the GI tract of conventional mice. Functional classification of these genes indicated that prominent in vivo responses include functions associated with nutrient acquisition, intermediate and/or cofactor biosynthesis, stress response, and cell surface proteins. However, also a number of (conserved) hypothetical proteins were identified among the L. plantarum ivi genes (Bron et al., 2004b). Remarkably, one of the latter group of genes displayed significant homology (32 percent identity) to the conserved hypothetical protein that was identified with IVET in L. reuteri (Bron et al., 2004b; Walter et al., 2003), indicating conservation of GI tract responses in different lactobacilli. Moreover, a striking number of the ivi functions and pathways identified by R-IVET in L. plantarum were previously found among the in vivo response in pathogens, suggesting that survival rather than virulence is the explanation for the importance of these genes during host residence (Table 6-2; Bron et al., 2004b). Following the above-mentioned ivi gene-identification strategies, the actual in situ expression of a selection of the L. plantarum ivi genes could be obtained by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using RNA preparations from mouse intestinal samples that had consumed

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary TABLE 6-2 Functional Classification of Predicted ivi Gene Functions in L. plantarum and Pathogenic Bacteria. Adapted from Bron et al., 2004b; de Vos et al, 2004 Functional Classes Number of Functionally Classified ivi Genes Identified by R-IVET L. plantarum Pathogenic Bacteria Transport and binding proteins 12 7 Regulatory functions 2 1 Energy metabolism 7 1 Cell envelope 4 0 Protein synthesis 3 0 Cellular processes 3 2 Purines, pyrimidines, nucleosides and nucleotides 2 1 DNA metabolism 1 1 Amino acid metabolism 1 2 Biosynthesis of co-factors, prosthetic groups and carriers 2 2 Fatty acid and phospholipid metabolism 3 0 Central intermediary metabolism 2 2 Protein fate 2 1 Transcription 0 0 Other categories 1 1 NOTE: The number of functionally classified ivi genes identified by R-IVET in L. plantarum during passage (left column) compared with those obtained in similar screening of pathogenic bacteria (right column). Hypothetical proteins are excluded in this comparison. SOURCE: Kleerebezem, 2005. L. plantarum containing food. Time-range analysis in a series of these mice revealed geographical differentiation of the induction patterns displayed by these ivi genes in the various regions of the mouse intestine (Unpublished observations, M. Marco, R. Bongers, and M. Kleerebezem. Wageningen Centre for Food Sciences, The Netherlands, 2005). This information could be exploited to construct dedicated, site-directed bacterial delivery vehicles aiming to deliver health beneficial (therapeutic) 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. (2004). In addition to in situ transcript detection, the importance for bacterial functionality in the host GI tract of the identified ivi genes can be established by comparative persistence and survival analysis of wild-type and isogenic mutant analysis in vivo. Such approaches have been performed in both lactobacilli. In L. reuteri, mutation of the methionine sulfoxide reductase-encoding msrB gene (one of the ivi genes) resulted in reduced ecological performance in the mouse GI tract as compared to the wild-type strain. Analogously, mutation of the or—the gene encoding the Lsp surface protein of L. reuteri that is implicated in adherence of this bacterium to epithelial cells—also resulted in similar in vivo performance defects (Walter et al., 2005). In L. plantarum, nine isogenic ivi mutants

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary were constructed, mainly focusing on genes that encode proteins with a predicted role in cell envelope functionality, stress response, and regulation. Quantitative PCR experiments were performed to monitor the relative population abundance of the group of the L. plantarum ivi mutants in fecal samples after competitive passage through the GI tract of mice; the experiments revealed that the relative abundance of three of the ivi gene mutants was reduced by 100- to 1,000-fold compared to other mutant strains and the wild-type strain, suggesting a critical role for these ivi genes in GI tract survival and persistence (Bron et al., 2004c). Global bacterial expression profiling of Bacteroides thetaiotaomicron residing in the cecum of monoassociated mice has recently been reported for the first time. By varying the mouse diet from a polysaccharide-rich diet to isocaloric diets that only contained simple sugars, this pioneering study revealed that B. thetaiotaomicron adapts its enzyme expression patterns to the sugars available; furthermore, upon paucity of the polysaccharides, the commensal microbe shifted its glycan-foraging behavior to the host mucous as a nutritive source, a property that aids its stability in the intestine (Sonnenburg et al., 2005). This work establishes that—although technically difficult—it is certainly feasible to monitor bacterial gene expression in the GI tract at a global level. The experiments described above were all performed using mouse model systems, and an obvious question that should be raised is whether these bacterial responses to the residence in the mouse GI tract can be extrapolated to bacterial activities in the human GI tract. This question was addressed in a study aiming to measure L. plantarum gene expression in the human GI tract by analyzing RNA extracted from mucosa-associated cells and hybridising to L. plantarum WCFS1 microarrays or gene-specific qRT-PCR. Appropriate controls were performed and included the absence of specific hybridization in biopsies from a subject that had not consumed the L. plantarum cells, the absence of interference by human nucleic acids on the microarray, and confirmation of the specificity by sequence analysis of the gene-specific qRT-PCR (de Vries MC, Marco M, Kleerebezem M, Mangell P, Ahrne S, Molenaar D, de Vos Wm, Vaughan EE, Unpublished data). The ingested L. 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. These findings support an at least partially conserved response of this bacterium to the GI tract conditions encountered in different host systems (de Vries et al., unpublished data). The studies highlighted above give a first glimpse of what the near future could bring in terms of understanding probiotic activity in situ in the GI tract of host organisms. This information will be of critical importance to construct molecular host-microbe interaction models. However, the responses by the host to probiotic encounter should also be addressed to fill in both sides of the interaction model.

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary Due to the relevance of this problem, further research is suggested for antibiotic resistance of lactobacilli and bifidobacteria. Research is required to relate the antibiotic resistance of lactobacilli and bifidobacteria and the potential for transmission of genetic elements to other intestinal and/or food-borne microorganisms. The European Union has recently funded a research project, named ACE-ART (Assessment and Critical Evaluation of Antibiotic Resistance Transferability in Food Chain). The project (www.aceart.net) started one year ago and aims to provide a critical evaluation of the presence of antibiotic resistance genes in nonpathogenic bacteria belonging to Lactobacillus, Bifidobacterium, Lactococcus, and Streptococcus thermophilus. One of us (Morelli) is the coordinator of this project, involving 14 laboratories spread all over Europe. At the moment more than 1,300 strains belonging to 20 species have been studied; a little more that 100 strains with “atypical” profiles of antibiotic resistance have been detected. Prebiotics or the Modification of the Host Nutritional Environment Prebiotics were not assessed by FAO/WHO consultation and only the following remarks were made: Prebiotics are generally defined as “nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already established in the colon, and thus improve host health” (Gibson and Roberfroid, 1995). The concept of prebiotics essentially has the same aim as probiotics: to improve host health via modulation of the intestinal flora, although by a different mechanism. 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). If a synbiotic relationship is intended, then it should be verified scientifically, following the guidelines outlined in section 5 of this report. Conclusion More than a dozen years of intense microbiological, genetic, and clinical research have provided enough sound science to convince regulatory bodies that it is necessary to provide suggestions and guidelines for this evolving field; however, it is paramount to realize the difficulty in establishing guidelines for a field which is in fast progress. New techniques that allow enhanced characterization of the gut microbiota are now a long way from “culture and phenotype” and we are

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary approaching a no-culture, genotype-only approach. Moreover, emerging methods that allow assessment of bacteria-epithelial interactions are also to be evaluated. As it is more a possibility that lifelong cross-talk between the host and the gut microbiota determines whether health is maintained or disease intervenes, it is clear that understanding of these bacteria-bacteria and bacteria-host immune and epithelial cell interactions is likely to lead to a greater insight of disease pathogenesis. It is highly possible that a new generation of probiotic or biotherapeutic could be used for obtaining a more targeted use, such as enhancing the production of a specific cytokine or suppressing a specific pathogen. If guidelines are prepared to provide suggestions and regulatory boundaries, it is important to take note that flexibility as well as constant dialogue between regulatory bodies and the scientific community is necessary. To manage this new war we must look for new allies, but we need also to write down new “alliance treaties” or guidelines for an optimal use of these alliances. Acknowledgments We would like to thank Mary Ellen Sanders, president of the International Scientific Association for Probiotics and Prebiotics (ISAPP), for critically reading and editing the manuscript. REFERENCES Abreu MT, Fukata M, Arditi M. 2005. TLR signaling in the gut in health and disease. Journal of Immunology 174(8):4453–4460. Adlerberth I, Ahrne S, Johansson ML, Molin G, Hanson LA, Wold AE. 1996. A mannose-specific adherence mechanism in Lactobacillus plantarum conferring binding to the human colonic cell line HT-29. Applied Environmental Microbiology 62(7):2244–2251. AFSSA (Agence Francaise de Sécurité Sanitaire des Aliments/French Food Safety Agency). 2005 (February). Effects of probiotics and prebiotics on flora and immunity in adults. [Online]. Available: http://www.usprobiotics.org/docs/AFFSA%20probiotic%20prebiotic%20flora%20immunity%2005.pdf [accessed January 12, 2006]. Agostoni C, Axelsson I, Braegger C, Goulet O, Koletzko B, Michaelsen KF, Rigo J, Shamir R, Szajewska H, Turck D, Weaver LT (ESPGHAN Committee on Nutrition). 2004a. Probiotic bacteria in dietetic products for infants: A commentary by the ESPGHAN Committee on Nutrition. Journal of Pediatric Gastroenterology and Nutrition 39(4):365–374. Agostoni C, Axelsson I, Goulet O, Koletzko B, Michaelsen KF, Puntis JW, Rigo, J, Shamir R, Szajewska H, Turck D (ESPGHAN Committee on Nutrition). 2004b. Prebiotic oligosaccharides in dietetic products for infants: A commentary by the ESPGHAN Committee on Nutrition. Journal of Pediatric Gastroenterology and Nutrition 39(5):465–473. Ahrne S, Lonnermark E, Wold AE, Aberg N, Hesselmar B, Saalman R, Strannegard IL, Molin G, Adlerberth I. 2005. Lactobacilli in the intestinal microbiota of Swedish infants. Microbes and Infection 7(11–12):1256–1262. Altermann E, Russell WM, Azcarate-Peril MA, Barrangou R, Buck BL, McAuliffe O, Souther N, Dobson A, Duong T, Callanan M, Lick S, Hamrick A, Cano R, Klaenhammer TR. 2005. Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM. Proceedings of the National Academy of Sciences USA 102(11):3906–3912.

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