DIET AND THE GUT MICROBIOTA: NEW DIRECTIONS The 2nd Workshop of the European Network for Gastrointestinal Health Research (ENGIHR) Research Networking Programme, European Science Foundation

Helsinki (Finland), 2nd-4th May 2012

Contents

Programme

1

List of Participants

2

List of Posters

4

Speaker Abstracts

5

Poster Abstracts

8

Personal Profiles

16

The European Network for Gastrointestinal Health Research (ENGIHR) The European Network for Gastrointestinal Health Research (ENGIHR) is a European Science Foundation Research Networking Programme (RNP) which promotes interactions between researchers interested in gut health research in Europe. This is done through a series of scientific meetings organised over a four-year period. The Network has a multidisciplinary nature, encompassing food manufacturers, food scientists, nutritionists, microbiologists, and clinicians. It promotes the training and development of young scientists through short visits grants, and encourages the integration of new partners. Workshop aims: This workshop aims to build on the first exploratory workshop in Braga (Portugal) by bringing together experts in the field as well as younger researchers to identify needs and challenges in the Gut Health field with the aim of translating these findings into new research proposals. The workshop will include introductory talks and will then focus on Working Groups which will address specific topics related to Diet and the Gut Microbiota.

Programme WEDNESDAY 2nd MAY 7,0( 19:00

7,7/( Registration, Welcome & Evening Buffet (Raddison Blu)

THURSDAY 3rd MAY 7,0( 09:00 09:30-12:40

7,7/( Introduction: Maria Saarela, VTT, Finland (host) Seve Pandiella, University of Manchester, UK (Chair, ENGIHR) Introductory Seminars:

63($.(5

7,7/(

Jose Lagaron (IATA-CSIC, Spain)

Nanotechnology for the design of novel functional foods, encapsulation of bioactives and efficient gut delivery

Michael Blaut (DIFE, Germany)

Impact of host and nutrition factors on intestinal bacteria

10:50

12:40-14:00

Coffee Break

Tor Lea (UMB, Norway)

Regulatory interactions between gut microflora and the intestinal immune system

Sam Possemiers (University of Ghent, Belgium)

The use of integrated in vitro models for the combined study of intestinal microbiota modulation and host response

Lunch and Poster Session

Parallel Working Groups 14:00-17:00

GROUP

1. 2. 3. 4. 17:15-18:00

Keynote Talk:

TITLE

Development and Maintenance of the Microbiota Effect of the Diet on Shaping the Intestinal Microbiota Current and Future Technologies to Investigate the Intestinal Microbiota Bioactives: Discovery and Delivery Dusko Ehrlich (MetaHIT, INRA, France): Gut microbiome in health & disease assessed by the MetaHIT consortium

Evening: Workshop Dinner: Tarvaspaa Cafe and Restaurant

FRIDAY 4th MAY TIME 09:00 09:30-12:30

12:30

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TITLE Dirk Hadrich (EU Commission): European funding in personalised medicine Reports from Working Groups & Expert Panel Discussion 09:30-10:30

Working Groups 1&2

10:30-11:00

Coffee Break

11:30-12:30

Working Groups 3&4

Closing Comments and Lunch

List of Participants Working Group 1: Development and Maintenance of the Intestinal Microbiota Facilitators:

Caroline Karlsson Julian Marchesi

Herbert Tilg Willem de Vos Matej Oresic Annick Mercenier Billy Hargis Ali Oguz Buyukkileci Maria Carmen Collado Marie-France de la Cochetière Lawrance Nyoni Arjan Narbad Bengt Bjorksten Bianca-Maria Exl-Preysch Nathalie Juge Ekaterina Avershina Maria Jenmalm Viola Strompfová

University of Lund, Sweden University of Cardiff, UK

Innsbruck University Hospital, Austria Helsinki University, Finland VTT, Finland Nestle, Switzerland University of Arkansas, USA Izmir Institute of Technology, Turkey IATA-CSIC, Spain INSERM, France University of Manchester, UK Institute of Food Research, UK Karolinska Institute, Sweden Exl-lent Nutrition Consultants, Switzerland Institute of Food Research, UK University of Life Sciences, Norway Linköping University, Sweden Slovak Academy of Sciences, Slovakia

Working Group 2: Effect of the Diet on Shaping the Intestinal Microbiota Facilitators:

Bernhard Corfe Anne Salonen Johanna Maukonen

Paul Ross Tor Lea Marika Mikelsaar Göran Molin Marion Priebe Bernhard Watzl Harry Flint Bruno Pot Clara G. de los Reyes-Gavilán Atsushi Yokota Charles Franz Jean-Michele Antoine Catarina Simões Maria João Fraqueza Maria Lima Kirsi Vaali Dominika Swiatecka Minja Miettinen Tore Midtvetd Torkel Wadstrom

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University of Sheffield, UK University of Helsinki, Finland VTT, Finland

Alimentary Pharmabiotic Centre, Ireland Norwegen University of Life Sciences, Norway Universtity of Tartu, Estonia University of Lund, Sweden University of Groningen, Netherlands Max-Ruben Institute, Germany Rowett Institute, UK INSERM, France IPLA-CSIC, Spain Hokkaido University, Japan Max Rubner Institute, Germany Danone, France VTT, Finland UTL, Portugal IASMA, Italy University of Helsinki, Finland Polish Academy of Sciences, Poland Valio, Finland Karolinska Institute, Sweden University Hospital of Lund, Sweden

List of Participants Working Group 3: Current and Future Technologies to Investigate the Intestinal Microbiota Facilitators:

Velitchka Gotcheva Karen Wright

Michael Blaut John McLaughlin Reet Mändar Maria Saarela Dusko Ehrlich Severino Pandiella Koen Venema Alfonso Clemente Sabina Leanti La Rosa Adele Costabile Merve Samli Lorenza Conterno Nick Chadwick Signe Adamberg Baltasar Mayo Carolin Kolmeder Anna Lyra

University of Food Technologies, Bulgaria University of Lancaster, UK

DIFE, Germany University of Manchester, UK University of Tartu, Estonia VTT, Finland INRA, France University of Manchester, UK TNO, The Netherlands University of Granada, Spain UMB, Norway The University of Reading, UK Izmir Institute of Technology, Turkey IASMA, Italy University of Manchester, UK Tallin University, Estonia IPLA-CSIC, Spain University of Helsinki, Finland Danisco Sweeteners Oy, Finland

Working Group 4: Bioactives: Discovery and Delivery Facilitators:

Rachael Rigby Patricia Ruas-Madiedo

Ailsa Hart Didier Dupont José Maria Lagarón José Teixeira Aldona Miezeliene Andrea Lauková Sam Possemiers Sebnem Harsa Amparo López Rubio Riitta Korpela Renáta Szabóová Ana Cristina Freitas Catherine Stanton José Carlos Andrade Ana Gomes Ernesto Hernandez Nagendra Shah

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Lancaster University, UK IPLA-CSIC, Spain

Imperial College, UK INRA, France CSIC-IATA, Spain University de Minho, Portugal Kaunas technological university, Lithuania Slovak Academy of Sciences, Slovakia University of Ghent, Belgium Izmir Institute of Technology, Turkey IATA-CSIC, Spain University of Helsiki, Finland Slovak Academy of Sciences, Slovakia University of Aveiro, Portugal Alimentary Pharmabiotic Centre, Ireland ISCSC, Portugal Portuguese Catholic University, Portugal University of Manchester, UK University of Hong Kong, Hong Kong

List of Posters Signe Adamberg: Survival of probiotic cultures of Bifidobacterium and Lactobacillus in the presence of multiple prebiotics in vitro: a strategy to develop new synbiotics Jose Andrade: Isolation of lactic acid bacteria strains with conjugated linoleic acid-producing ability from Portuguese cheeses Ekaterina Avershina: Succession and correlation-networks of Bifidobacteria in a large unselected cohort of mothers and their children Marie-France de La Cochetière: A Specific Intestinal Microbiota Profile predisposes to Severe ChemotherapyInduced Diarrhoea Alfonso Clemente: Monomer and linkage type of galacto-oligosaccharides affects their resistance to ileal digestion and bifidogenic effect in rats Lorenza Conterno: Role of food matrix in gastrointestinal survival of probiotic microorganisms using in vitro digestion of a model cheese Clara de los Reyes-Gavilán: Establishment and Development of Intestinal Microbiota in Preterm Neonates. A possible target for the probiotic action Billy Hargis: Successes and Failures in Development of Effective Commercialized Probiotics and Direct Fed Microbials (DFM) for Enteric Health: A 20 Year Overview Maria Jenmalm: Pre- and postnatal administration of Lactobacillus reuteri reduces TLR2 responses in infants Carolin Kolmeder: Effects of a probiotic intervention on the human intestinal metaproteome Andrea Laukova: Can bioactive compounds (bacteriocins) play beneficial role in health status of animals? Amparo Lopez-Rubio: Viability enhancement of probiotics through microencapsulation in electrosprayed structures Johanna Maukonen: Characterization of the gut microbiota of healthy adults experiencing gastrointestinal discomfort related to cereal-based food products Baltasar Mayo: Microbial diversity within the human stomach by culturing and culture-independent methods Aldona Miezeliene: Probiotics and prebiotics in daily food – consumer standpoint Marion Priebe: Anti-inflammatory properties of short-chain fatty acids relevant for the prevention of type 2 diabetes Patricia Ruas-Madiedo: Behaviour in real time of cellular lines in the presence of bioactive compounds: interaction of surface components from Bifidobacterium with colonocyte-like HT29 cells Merve Samli: Simulation of human colon system using potential probiotic yoghurt cultures from Toros region of Turkey Anne Salonen: Intestinal cleansing and vegan diet as a novel modality for treatment of irritable bowel syndrome (IBS) Caterina Simões: Nutritional intake affects the gut microbiota of Finnish monozygotic twins Viola Strompfová: Experiences with the use of a new canine-derived probiotic strain Lactobacillus fermentum AD1 (CCM 7421) in dogs Renata Szabóová: Effect of bioactive strain Enterococcus faecium CCM 4231 in rabbits Koen Venema: Use of 13C labelled substrates to trace microbial metabolism in the colon; light in the tunnel Atsushi Yokota: Bile acid is a host factor that regulates the composition of the cecal microbiota in rats

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Speaker Abstracts Nanoencapsulation to Enhance the Efficacy of Gut Health Promoting Bioactives Jose Maria Lagaron (Group Leader, Novel Materials and Nanotechnology for Food Applications, IATA,CSIC, Spain)

The current paper highlights some recent advances carried out within the research community, but with special emphasis in the pioneering activities of our research group in the field, in which various encapsulation applications that make use of nanofabrication techniques and of food hydrocolloids will be reviewed, which aim to enhance bioactives stability, storability, handling, novel foods design control, lactic bacteria viability, bioavailability and control delivery. These include examples in which proprietary nanostructured materials have been designed by the high voltage spinning technique for the encapsulation of bioactive food ingredients such as antioxidants, marine oils and also prebiotics and probiotics of interest in functional foods and specially in dairy products.

Impact of host and nutrition factors on intestinal bacteria Michael Blaut (Head of Department of Gastrointestinal Microbiology, German Institute of Human Nutrition)

There is an intricate relationship between the human host and its intestinal microbiota. Considerable efforts have been made to better define the role of the intestinal microbiota in host physiology and to unravel the underlying mechanisms. While the knowledge on specific functions of intestinal microbes in the intestinal tract has considerably increased very little is known whether and to which extent host and nutrition factors affect intestinal bacteria and possibly their reaction toward the host. To study the impact of host factors on gut microbes a simplified model of host-bacteria interaction was created by associating germfree mice with commensal Escherichia coli. We investigated how dietary composition influences bacterial activities in the intestine and how this in turn affects the host. We used mice monoassociated with Escherichia coli MG1655 as a simplified model for host-microbiota interaction to investigate the influence of dietary factors on bacterial protein expression in the intestine. The mice were fed three different diets: a carbohydrate (lactose)-rich diet, a protein-rich diet and a diet rich in starch. Two-dimensional difference gel electrophoresis followed by electro-spray ionization-tandem mass spectrometry was used to identify proteins differentially expressed in E. coli cells recovered from the mouse intestinal tract. The lactose-rich diet led to an induction of proteins involved in E. coli’s oxidative stress response (FUR, AhpCF, DPS). The corresponding genes are under control of the OxyR transcriptional dual regulator. Luciferase reporter gene assays demonstrate that osmotic stress activates genes of the oxyR regulon. We propose that feeding the mice the lactose rich diet increased the intestinal osmolality which in turn triggered the upregulation of OxyR dependent proteins, which enable intestinal E. coli to better cope with diet induced osmotic stress. To identify Escherichia coli (E. coli) proteins involved in the adaptation to intestinal inflammation germfree mice were monoassociated with the colitogenic E. coli UNC (UNC) or with the probiotic E. coli Nissle (EcN). Intestinal inflammation was induced by treating the mice with 3.5% dextran sodium sulfate (DSS). Differentially expressed proteins were identified by two-dimensional difference gel electrophoresis in E. coli collected from cecal contents. In both strains acute inflammation led to a downregulation of pathways involved in carbohydrate breakdown and energy generation. Accordingly, DSStreated mice had lower concentrations of bacterial fermentation products in their cecal contents than control mice. Differentially expressed proteins also included the Fe/S cluster repair protein NfuA, the tryptophanase (TnaA). Expression of NfuA was 3-fold higher in E. coli from inflamed than control mice. Reporter experiments confirmed the induction of nfuA in response to superoxide stress, a condition characteristic of inflammation. EcN isolated from DSS and control mice had 4 to 7-fold higher levels of TnaA than UNC. Indole resulting from the TnaA reaction was higher in control animals associated with EcN. Because of its anti-inflammatory functions it is hypothesized to be involved in extension of the remission phase in ulcerative colitis described for EcN.

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Speaker Abstracts Regulatory interactions between gut microflora and the intestinal immune system Tor Lea (Institute of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences)

There is an increasing awareness of the significance of the intestinal microflora in the modulation of mucosal immune responses. Furthermore, in addition to inflammatory diseases of the digestive tract, changes in gut microbial communities are also associated with more systemic diseases like autoimmune diseases, type I diabetes, allergy and metabolic syndrome. Thus, there is now great interest in exploring the possible therapeutic potential of both commensal bacteria and probiotics in the treatment of a range of immune-mediated disorders. During the last decade there has been an enormous development in our understanding of the complexity of the indigenous microbiota. At the same time, studies of the mucosal immune system have unraveled novel regulatory mechanisms operating between the epithelium and cells belonging to both innate and adaptive immunity. Currently, balanced interactions between microflora, epithelium and the mucosal immune system are essential to maintain homeostatic conditions and a healthy gut. An updated view of the organization of the intestinal mucosal immune system of will be presented. Furthermore, molecular details in the interactions between luminal bacteria and the epithelium are described, and the functional consequences thereof. Also, the significance of the microflora in regulating both innate and adaptive immunity will be thorougly scrutinized.

The use of integrated in vitro models for the combined study of intestinal microbiota modulation and host response Sam Possemiers (Group Leader, Facility of Bioscience Engineering, Ghent University, Belgium)

In vitro simulation technologies offer a useful complementary approach for human or animal studies to study the interaction between dietary ingredients, the gut microbiota and human health. However, typical in vitro models are limited to study either intestinal processes (gut simulation models) or host response (cell culture/tissue models), whereas both are in reality closely linked and should be combined to study host-related endpoints of gut microbiota activity. Gut models allow to study the intestinal fate, metabolism and bioavailability of bioactives and their intestinal metabolites, while cell culture/tissue models typically investigate the host response to isolated bioactives. In this presentation, some currently existing dynamic gut models will be discussed and recent developments will be shown which integrate expertise from different disciplines to improve the modeling capacity of such systems. By using experiments with pre- and probiotics as model dietary interventions, the relevance will be shown of combining well-designed gut models with a mucus environment to model bacterial adhesion to the gut lining and cell culture models to evaluate host response to intestinal processes. Finally a novel technology will be presented which allows bi-directional interactions between intestinal microbiota and the (simulated) gut epithelium/host response in health and disease. Summarized, the development of such novel integrated technologies offers a promising strategy to improve the modeling capacity of in vitro systems and therefore the predictive value and final relevance of the outcome of studies which aim to unravel the link between our diet and host response through interaction with the gut microbiota.

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Speaker Abstracts Gut microbiome in Health and Disease assessed by the MetaHIT consortium Dusko Ehrlich (Group Leader, INRA, MICA Division, Jouy en Josas, France)

One of the major questions in human biology is the role of gut microbial communities in health and disease. The MetaHIT consortium has developed a new approach, which we term quantitative metagenomics, to visualise the gut microbial communities. A central element is a reference catalogue of the intestinal microbial genes (Qin et al., Nature, 2010, on which we map a high number of short sequences generated from total stool DNA of an individual, thus determining presence and abundance of each catalogue gene harboured by that individual. Use of this approach has led to detection of three gut enterotypes to which humans belong (Amurugam et al. Nature, 2011), that are characterized by different bacterial communities. This basic feature of human biology remains to be elucidated, but the enterotypes will be crucial to stratify individuals and assess the microbial communities associated with health and disease. We used the approach to study obese and lean individuals or IBD patients and healthy controls and revealed considerable differences in the microbial communities, in terms of overall diversity and the prevalence of particular bacterial species. This new view opens avenues to better understanding of the role of microbes in health and disease.

European funding in Personalised Medicine Dirk Hadrich (Scientific officer for Personalised Medicine, European Commission, Brussels, Belgium)

Personalised medicine emerged from the problem that 90% of common drugs work in only 40% of the patients. Apart from unsatisfied patients causes this inefficiency an unacceptable financial burden for the health systems. Personalised medicine aims to bring more tailored medical interventions and individual treatments based on personal characteristics. The systematic analysis of the function of all genes was one of the first crucial research priorities but now more and more data on epigenetic changes, metagenomics, protein modifications, biomarkers, immunomonitoring, xenobiotics etc become available and make the health picture very complex. Very interesting ideas come from various research disciplines and promise to bring advanced treatments. The challenge will be to use all these data in order to predict drug reactions in different individuals and to modify treatments accordingly so that the right patient is treated at the right time. Future funding decisions will require evaluating how much research proposals could modernise medical treatments, how close they could come to clinical outcomes and how much they stimulate the whole innovation chain from the basic idea to the market. For this purpose it's indispensable to join forces from a wide range of countries, partner profiles and scientific disciplines.

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Poster Abstracts Survival of probiotic cultures of Bifidobacterium and Lactobacillus in the presence of multiple prebiotics in vitro: a strategy to develop new synbiotics Adamberg, S.(1), Sumeri, I.(2), Uusna, R.(2), Ambalam, P.(3), Kanthi Kiran, K.(3) and Wadström, T.(3) (1) Institute of Food technology, Tallinn University of Technology, Tallinn, Estonia; (2) Competence Center of Food and Fermentation Technologies, Tallinn, Estonia; (3) Department of Clinical Microbiology, Lund University hospital, Lund, Sweden

A single vessel Gastro-Intestinal-Tract Simulator (GITS) was used to study the survival of multistrain probiotics containing bifidobacteria and lactobacilli. The environmental conditions of stomach, small and large intestine were simulated during 24 hours fermentation experiments. Two different substrate combinations in equal proportions (total concentration 1%) were used in the dilution medium: a) galacto-oligosaccharides, fructo-oligosaccharides and xylo-oligosaccharides, b) GOS and soluble starch. Survival of bacteria was evaluated by plate counts and the proportion of each strain was found on the basis of rep-PCR patterns. Concentrations of organic acids and ethanol in the culture medium were determined by HPLC. The strains of lactobacilli - Lactobacillus plantarum F44 and Lactobacillus paracasei F8 survived well in all experiments as shown per recovery numbers. The bifidobacteria remained subdominant in most experiments, and only one strain - Bifidobacterium breve 46 showed good recovery consistently. Based on the current study, the strains B. breve 46, L. plantarum F44 and L. paracasei F8 can be potential candidates for development of synbiotic formulations. The results also suggest that the population dynamics on a certain substrate can be characterized by the metabolic products. Higher content of lactate correlated with higher numbers of lactobacilli in the population while the growth of bifidobacteria was typically accompanied with higher concentrations of acetate, formate and ethanol. Currently, the growth and carbohydrate metabolism of pure cultures on single oligosaccharide substrates is studied. The project has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 232087 (www.Qualvivo.eu)

Behaviour in real time of cellular lines in the presence of bioactive compounds: interaction of surface components from Bifidobacterium with colonocyte-like HT29 cells Sánchez, B., Hidalgo, C., Margolles, A. and Ruas-Madiedo, P. Department of Microbiology and Biochemistry. Dairy Research Institute of Asturias –Spanish National Research Council (IPLA-CSIC: Instituto de Productos Lácteos de Asturias –Consejo Superior de Investigaciones Científicas) Villaviciosa, Asturias, Spain.

The definition of a “healthy” human microbiota is unclear; but, it is known that a dysbiosis, or imbalance, of the microbial population inhabiting our digestive tract could be cause of, or is related to, several disorders. This indicates that the interaction between microorganisms and host is paramount to keep our wellbeing. The use of probiotics for prevention and treatment of intestinal disorders, as well as for restoration of microbiota after drastic treatments, is becoming more popular and the scientific evidence of efficacy is also increasing. However, not all probiotics have the same capability to confer health benefits on the host. Therefore, it is crucial the use of reliable, reproducible and fast methods monitoring the host response in the presence of probiotics. We have recently implemented in our group a technique based on the use of a “Real Time Cell Analyser” apparatus to test the behaviour of cellular lines (in proliferative or in confluent state) from human (colon) origin in the presence of different Bifidobacterium strains, extracellular polymers (exopolysaccharides) and bacterial surface extracts, among others. Preliminary results showed that, for a given bacterial compound at different doses, the behaviour of the eukaryotic cells was very much time-dependent. One of the compounds tested had cytotoxic effect at high dose on HT29 cells; whereas, at lower doses, we have detected an initial anti-proliferative effect ending with cell death. Besides, this behaviour was retarded when the bacterial compound concentration decreased. This method is a valuable tool that could be extended for the screening of different bioactive compounds obtained from different origins.

Succession and correlation-networks of Bifidobacteria in a large unselected cohort of mothers and their children Avershina, E.(1,3), Storrø, O.(2), Øien, T.(2), Johnsen, R.(2), Wilson, R.(1), Egeland, T.(3) and Rudi, K.(1,3) (1) Faculty of Education and Natural Sciences, Hedmark University College, 2317 Hamar, Norway; (2) Department of Public Health and General Practice, Norwegian University of Science and Technology, 7491 Trondheim, Norway; (3) Department of Chemistry, Biotechnology and Food Science, University of Life Sciences, Ås, Norway

Bifidobacteria are a major microbial component of infant gut microbiota which is believed to promote health benefits for the host and stimulate the maturation of the immune system. Despite their importance, we know little about the natural development of bifidobacteria in human populations. To address this question, we analyzed mixed Bifidobacterium clpC gene sequences from IMPACT (The Immunology and Microbiology study in the Prevention of Allergy among Children in Trondheim study) stool samples of 83 infants and their mothers using a multivariate statistical approach. Fecal material was sampled during the pregnancy, at 3 and 10 days, 4 months, 1 and 2 years after birth. Five dominant Bifidobacterium species were identified and verified by amplicon cloning, real-time PCR and culturing. Stool samples were predicted to be rich in the species B. adolescentis, B. bifidum, B. dentium, B. breve and B. longum. Due to high variation we did not identify a clear age-related structure at the individual level. Within the population as a whole, however, there were clear age-related successions. The percentage of B. adolescentis in infant samples reached adult levels by the age of 10 days and then remained stable, regardless of the change in the amount of Bifidobacteria. Negative correlations between the B. longum group and B. adolescentis were detected in adults and 1- and 2-year old children, whereas negative correlations between B. longum and B. breve were characteristic for newborns and 4-month-old infants. B. longum longum was detected in the majority of stool samples irrespective of age, and B. longum infantis was mostly identified in 4-month-old individuals. The highly structured age-related development of and correlation-networks between bifidobacteria during the first two years of life mirrors probably their different biological functions in the development of a healthy gut.

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Poster Abstracts A Specific Intestinal Microbiota Profile predisposes to Severe Chemotherapy-Induced Diarrhoea Montassier, E.(1), Batard, E.(1), Hardouin, J.B.(4), Caillon, J.(1), Le Fresne, S.(3), Carton, T.(3), Caroff, N.(1), Gastinne, T.(2), Moreau, P.(2), Potel, G.(1), Le Vacon, F.(3) and De La Cochetière, M.F.(5) (1) Université de Nantes, EA 3826 Thérapeutiques cliniques et expérimentales des infections. Faculté de médecine, 1 Rue G Veil, 44000 Nantes, France; (2) Service d’hématologie clinique. Centre Hospitalier Universitaire de Nantes, 1 Place A Ricordeau, 44000 Nantes, France; (3) Biofortis, Mérieux Nutrisciences.3 route de la Chatterie 44800 Saint Herblain, France; (4) Université de Nantes, EA 4275 Biostatistique, recherche clinique et mesures subjectives en santé. Faculté de médecine, 1 Rue G Veil, 44000 Nantes, France; (5) INSERM, Université de Nantes, Thérapeutiques cliniques et expérimentales des infections. Département des Maladies Infectieuses, Faculté de médecine, 1 Rue G Veil, 44000 Nantes, France.

Objectives: The role of the intestinal microbiota (IM) in the pathophysiology of chemotherapy-induced diarrhoea (CID) remains poorly understood. The objectives of our study were to describe the IM during chemotherapy and to investigate pre-chemotherapy patterns that could predispose to CID. Methods: Patients undergoing BEAM conditioning chemotherapy for bone marrow transplantation were eligible. Exclusion criteria were inflammatory bowel disease, intake of probiotics, steroids, immunosuppressants, antibiotics during 1 month prior to study or during treatment. Fecal samples were collected before (S1) and after (S2) chemotherapy. We looked for Escherichia coli, Enterococcus, Streptococcus, Lactobacillus, Bifidobacterium, total aerobic and anaerobic bacteria. For culture-independent molecular analyses, total DNA was extracted using the bead-beating method coupled with QIAmp DNA stool mini kit. The V6 to V8 region of the 16S rRNA gene was amplified. Purified PCR products were separated by dHPLC on a DNASep®HT cartridge (Transgenomic). Results: Eight patients were included. Significant increase in bacterial counts between pre-chemotherapy and post-chemotherapy were observed for Escherichia coli (p=0.002), Streptococcus spp (p=0.02) and anaerobic bacteria (p=0.009). Using dHPLC, hierarchic cluster analysis showed that fecal samples collected before chemotherapy clustered separately from those collected after. A Principal Component Analysis was performed on S1 samples to investigate differences in pre-chemotherapy fecal samples between patients who developed CID and patients who didn’t. The score plot showed that 2 patients with the most severe CID were separated from the 6 others. Conclusion: IM rapidly alters in patients during BEAM conditioning chemotherapy. A specific initial distribution of dominant microbiota may predispose to severe CID.

Establishment and Development of Intestinal Microbiota in Preterm Neonates. A possible target for the probiotic action Arboleya, S.(1), Salazar, N.(1), Fernández, N.(2), Solís, G.(3), Margolles, A.(1), Hernández-Barranco, A.(1), de los Reyes-Gavilán, C.G.(1) and Gueimonde, M.(1) (1) Department of Microbiology and Biochemistry of Dairy Products. Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Villaviciosa, Asturias, Spain; (2) Paediatrics Service, Hospital de Cabueñes, SESPA, Gijón, Asturias, Spain; (3) Paediatrics Service, Hospital Universitario Central de Asturias, SESPA, Oviedo, Asturias, Spain

Microbial colonization of the infant gut plays an essential role in the development of the intestine and the immune system of newborns. The intestinal microbiota of full-term breast-fed (FTBF) infants is currently considered as the health standard for newborns. In contrast, the immature intestine, the frequent use of antibiotics and formula milk and the long stay at hospitals jeopardize the proper microbiota development in premature infants. The establishment of the gut microbiota in preterm neonates during the first three months of life was assessed and compared with that of FTBF infants. Microbial composition was determined in faeces by qPCR, and metagenomic analyses; short chain fatty acids (SCFA) were quantified by Gas-Chromatography-Flame Injection Detector/Mass Spectrometry (GC-FID/MS). All techniques allowed clearly differentiating preterm from FTBF infants. Premature infants showed higher levels of facultative anaerobes and lower levels of anaerobes such as Bifidobacterium, Bacteroides and Atopobium as well as lower levels of SCFA during the first days of life. The deep alterations found in the process of microbiota establishment in preterm infants, indicated the need for intervention strategies to counteract them. Then, 16 Bifidobacterium strains were tested in fecal batch slurry cultures from preterm babies for their ability to modulate in vitro the intestinal microbiota. Those bifidobacteria that in fecal cultures counteracted better the aberrancies previously found in feces of preterm babies as compared with FTBF infants, were selected. Three Bifidobacterium bifidum strains from infant feces (IF23/, IF10/10, and IF10/20) as well as two Bifidobacterium breve strains from breast milk (BM 13/14 and BM 23/20) promoted the most suitable shift in SCFA profiles and in the population of facultative anaerobes and anaerobes, representing promising candidates for further in vitro and in vivo studies.

Isolation of lactic acid bacteria strains with conjugated linoleic acid-producing ability from Portuguese cheeses Ascenção, K.(1), de Marco, P.(1), Moreira, P.(2) and Andrade, J.(1) (1)Centro de Investigação em Ciências da Saúde (CICS), Instituto Superior de Ciências da Saúde Norte/CESPU, Rua Central de Gandra, 1317/4585-116 Gandra PRD, Portugal; (2) Faculdade de Ciências da Nutrição e Alimentação da Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal.

Conjugated Linoleic Acid (CLA) is a mixture of positional and geometric isomers of linoleic acid (C18:2) in which double bonds are conjugated. Several studies realized in animal models and/or cell cultures have shown antitumor, antiobese, antiatherogenic, antidiabetic and immunomodulatory activities. The CLA are produced through the isomerization of linoleic acid or vaccenic acid by animal, but various studies show that they can be also synthesized by microorganisms in milk or in different cultural substrates. The aim of this study was to identify lactic acid bacteria (LAB) able to synthesize CLA from cheeses commercialized in Portugal. Seventy-two CLA-producing lactic acid bacterial strains were isolated in the study. Two of these isolates, designated OAL2 and OCL1, have shown, on synthetic medium (MRS broth) added with free linoleic acid (LA), the highest CLA production (31.2 and 22.9 μg of CLA mL-1 of medium, respectively). Both strains were identified as Lactobacillus plantarum by API 50 CHL system and full-length 16S rDNA sequence analysis. CLA production by these strains was assessed in different conditions (microaerophilic and anaerobic) and various LA initial concentrations in the culture medium. Under the best conditions studied, 181 and 196 μg mL-1 of CLA were obtained, respectively by L. plantarum OAL2 and L. plantarum OCL1.

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Poster Abstracts Successes and Failures in Development of Effective Commercialized Probiotics and Direct Fed Microbials (DFM) for Enteric Health: A 20 Year Overview Hargis, B.M.(1), Tellez, G.I.(1), Bielke, L.R.(1), Wolfenden, R.E.(2), Wolfenden, A.D.(1), Faulkner, O.T.(1), Pumford, N.R.(1), Morgan, M.(1) and Menconi, A.(1) (1) JKS Poultry Health Laboratory, University of Arkansas, Fayetteville, Arkansas, USA 72701; (2) Pacific Vet Group USA Inc., Johnson, Arkansas, USA 72703

Increasing regulatory pressures and consumer preferences in Europe and North America are driving a marked increase in poultry production with limited or no antibiotic usage. Enteric disease and poultry-origin human food-borne pathogens are among the greatest challenges for efficient monogastric animal production when traditional chemicals are not used. For more than 20 years, our laboratory has been highly active in development of probiotic/DFM products for improved enteric health and reduced Salmonella and Campylobacter carriage in poultry. Defined cultures, providing that they consist of generally-recognized-as-safe (GRAS) isolates, are largely unregulated in the United States, leading to a plethora of “probiotic” products that are largely ineffective and which also have tended to reduce the acceptance of proven technologies. Importantly, undefined cultures, even those that are produced from semidefined and quality-controlled batch fermentations are not allowed for treatment of poultry in the United States, thus necessitating the development of highly effective and defined products, and for rigorous research proving efficacy both in the laboratory and under field conditions. Our experiences with this area of research will be summarized in this presentation.

Pre- and postnatal administration of Lactobacillus reuteri reduces TLR2 responses in infants Forsberg, A.(1,2), Abrahamsson, T.(1), Jimenez E.(1), Björkstén B.(3) and Jenmalm M.C.(1,2) (1) Division of Pediatrics, (2) Autoimmunity and Immune Regulation, Department of Clinical and Experimental Medicine Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden; (3) Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

Background: Mice models indicate that intact Toll like receptor (TLR) signaling may be essential for the allergy protective effects of diverse bacterial exposure observed in clinical and epidemiological studies. We have previously shown that supplementation with the Gram positive probiotic strain Lactobacillus reuteri from pregnancy week 36 and to the infant through the first year of life decreased the prevalence of IgE-associated eczema at two years. We explored the possibility that the supplementation affected innate immune responses to bacterial products and the expression of associated TLRs. Methods: Blood mononuclear cells were collected at birth, 6, 12 and 24 months from 61 infants and cultured with the ligands for TLR2, 4 and 9, i.e. lipoteichoic acid (LTA) from Gram positive and lipopolysaccharide (LPS) from Gram negative bacteria and unmethylated bacterial CpG DNA. Cytokine and chemokine secretion was determined using Luminex and mRNA expression of TLR2, 4 and 9 by real time RT-PCR. Results: Probiotic supplementation was associated with reduced LTA induced chemokine (CCL4, p