XVI Symposium of the International Association on Gnotobiology & XXXI Congress of the Society of Microbial Ecology and Disease

Animal models for gastrointestinal pathological and funtional studies

Shigeru Kamiya

Department of Infectious Diseases, Kyorin University School of Medicine, Tokyo, Japan

Germ-free animals are an excellent tool for investigating bacterial interactions, because the effects of bacterial flora can be ruled out in such animals. We have examined the pathogenesis in various bacteria such as Clostridium difficile, enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and Helicobacter pylori. Gnotobiotic mice (86%) mono-associated with C. difficile were dead within 2 days of infection, although all the SPF mice infected with C. difficile survived without any intestinal symptoms. Gnotobiotic mice mono-associated with EHEC developed ruffled fur and convulsion of limbs or hindleg weakness within 3 days afther the challenge, and all the mice were dead within 5 days of infection. Colonization of H. pylori with inflammatory changes in gastric mucosa was observed in gnotobiotic mice mono-associated with H. pylori.

Probiotics are viable cell preparations that have been beneficial effects on the health of host. Clostridium butyricum is a butyric-acid producing gram-positive anaerobe, and C. butyricum M588 strain has been used as a probiotic for the treatment and prevention of diarrheal diseases. Using the above experimental animal models, we have examined the effects of the probiotic agent, Clostridium buttyricumM588 strain. Protective and prophylactic effect of the M588 strain on C. difficile, EHEC and H. pylori were demonstrated. In addition, detection of H. pylori in fecal samples by a immunomagnetic-bead separation technique was reported by using gnotobiotic mice mono-associated with H. pylori. We also examined the effect of bacterial flora on postimmunization gastritic following oral vaccination of mice with H. pylori heat shock protein 60 (HSP60) by using germ-free mice. It was concluded that the presence of bacterial flora appeared to contribute to the induction of postimmunization gastritis.

The infection model for H. pylori using Mongolia gerbils is considered to be a useful, as gastric mucosal changes in stomach of Mongolian gerbils were similar to those in human stomachs following H. pylori infection. We have reported microbiological, histopathological and serological analyses after the long-term infection of Mongolian gerbils with H.pylori. In this model, marked atrophy of gastric mucosa, multiple cysts in the submucosa and squamous cell papilloma with hyperkeratosis were observed after long-term infection with H. pylori for 18 months. By using gerbil model, we also analyzed the significance of quorum sensing (QS) system in H. pylori. QS-defective, luxS mutant strain showed decreased motility and decreased colonization activity in the stomach of Mongolian gerbil, suggesting the involvement of QS in pathogenesis following H. pylori infection.

Contact e-mail: [email protected]

Intestinal microbiota composition

Erwin G. Zoetendal

Laboratory of Microbiology, Wageningen University, The Netherlands

Background: The microbial community in the gastrointestinal (GI) tract consists of a very diverse collection of microbes from which the majority cannot yet be cultivated. Despite the advantages of using Small SubUnit ribosomal RNA (SSU rRNA) sequence information in the characterization of the human GI tract microbiota, establishing links between the microbiota and intestinal disorders is hampered by this exceptional complex diversity which composition differs between individuals and GI tract location. In order to pinpoint microbial groups that can be linked to intestinal disorders, high throughput characterization of the microbiota at various phylogenetic levels is needed.

Material and Methods: In our laboratory, a comprehensive and high-throughput phylogenetic microarray, the Human Intestinal Tract Chip (HITChip) was developed and validated. The HITChip enables studying the intestinal microbiota at high spatiotemporal resolution, and combines the power of SSU rRNA-based phylogenetic fingerprinting and relative quantification.

Results and discussion: High throughgput profiling of the GI tract microbiota using the HITChip has provided novel insights into the impact of host- and environment-related factors on its composition. In addition, correlations between specific phylogenetic groups of microbes in the intestine and certain intestinal diseases were identified. These results will be presented and discussed at the symposium.

Contact e-mail: [email protected]

Molecular fingerprinting techniques of the intestinal microbiome

Lars Engstrand

Department of Bacteriology, Swedish Institute for Infectious Disease Control & Department of Microbiology, Tumor and Cell Biology, MTC, Karolinska Institutet, Stockholm, Sweden

Isolation of bacterial strains by microbial culture is still the golden standard for diagnosis and characterisation of pathogens as well as commensal bacteria. The need to analyse bacterial communities or complex microbial ecosystems, rather than examine single bacterial strains or members of a bacterial genus, has attracted more and more interest during the last decade since alterations in the human microbiota have been associated with disease development. Determining the microbial composition in patients and healthy controls is currently a time-consuming and expensive process. Large scale-studies have therefore been prohibited. However, new high-throughput culture-independent molecular tools have been developed that provide the scientific community with an array of new methods for such studies.

The most commonly utilized target for such analysis is the ubiquitous gene coding for small subunit ribosomal RNA. The 16S rRNA gene (16S rDNA), has historically proved to be the most accurate gene for studies of bacterial diversity, evolution, as well as phylogenetic analyses. The 16S rDNA consists of consensus sequences, universal for all procaryotes, and variable sequences that are specific for particular groups or species of bacteria. Hypervariable sequences, that may be unique for certain strains within a species, are contained within the variable areas.

Several sequencing based methods can be used to study the diversity of the intestinal microbiome by an unbiased sequencing approach of the 16S rDNA population in the clinical sample. The next generation sequencing techniques will probably allow us to study human intestinal microorganism communities by using metagenomic approaches on a large-scale level.

Contact email: [email protected]

Metaproteomics of the human gut microbiota reveals previously undescribed pathways and interactions

J. K. Jansson^1,2, A.Godzik³, A.L. Russell⁴, M. Shah⁴, M. Rosenquist², J. Halfvarson⁵, M. G. Lefsrud⁴, J. Apajalahti⁶, C. Tysk⁵, R. L. Hettich⁴, N. C. VerBerkmoes⁴

¹Ecology Department, Lawrence Berkeley National Laboratory, Berkeley, CA

²Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala

³Burnham Institute for Medical Research, La Jolla, CA

⁴Oak Ridge National Laboratory, Oak Ridge, TN

⁵Örebro University Hospital, Örebro, Sweden

⁶Alimetrics Ltd, Helsinki, Finland

Background: Recently the human gut microbiota has been investigated using a combination of molecular fingerprinting approaches and metagenomics sequencing. This information provides an idea of the composition of the gut microbiota and potential functions, but not about which genes are expressed and functioning in the gut environment.

Method: A novel shotgun metaproteomics approach was used to determine the identities of thousands of proteins in fecal samples. Bacteria were enriched from feces collected from two healthy individuals by differential centrifugation, the cells were lysed, proteins digested and separated by liquid chromatography and infused directly into rapidly scanning tandem mass spectrometers to obtain highly accurate mass predictions. Proteins were identified by matching the mass data to deposited genome and metagenome sequence data. Bioinformatics tools were used to elucidate the identities of hypothetical proteins.

Results: Thousands of proteins were identified and many of the hypothetical proteins were assigned to previously undescribed pathways. In addition, several human proteins were identified that have a postulated role in host interaction with the gut microbiota, including immune response. The metaproteome was significantly skewed by comparison to average metagenomic data with a higher representation of proteins involved in carbohydrate metabolism and some other functions indicative of the importance of these processes in the gut environment.

Conclusions: Shotgun metaproteomics has revealed for the first time the microbial functions that are most prevalent in the healthy human intestine and suggest the importance of interactions between the gut microbiota and the human host. This study lays the groundwork for future studies of the function of the gut microbiota in health and disease.

Contact email: [email protected]

Padlock probe detection of lactobacillus reuteri in fecal samples

Mia Thorsélius¹, Jenny Göransson², Joakim Bjerketorp¹, Klara Båth¹, Mats Nilsson² and Magnus Rosenquist³

¹Dept of Microbiology, Swedish University of Agricultural Sciences, Box7025, SE-750 07 Uppsala, Sweden. ²Dept of Genetics and Pathology and ³Dept of Clinical Immunology Radiology and Oncology, Uppsala University, Rudbeck laboratory, SE-751 85 Uppsala, Sweden

Background: We intended to set up a rapid, sensitive and unbiased method for relative quantification of bacteria in unprocessed feces. The initial aim was to study transition time of Lactobacillus reuteri ATCC55730 in an artificial stomach.

Methods: We used a three-stage continuous culture system, mimicking the gastrointestinal tract, to which L.reuteri was administrated at time zero and 24 hours. The amount of L.reuteri was analyzed with CFU count, padlock probes combined with circle-to-circle-amplification (C2CA) and Q-PCR. In C2CA and Q-PCR, specific L.reuteri and general probes or primer pairs targeting 16S were used.

Result: CFU count showed an increase in the number of L.reuteri 8 hours after the first administration, and a maximum count at 32 hours (8h after second dose) after which it gradually decreases. C2CA and Q-PCR analyses showed the highest levels of L.reuteri after 8 hours.

Conclusion: The bulk of viable L.reuteri passed through the artificial colon in 8 hours, with subsequent steady decrease to baseline levels, indicating that no colonization occurred. DNA from the cells was detected at the same time-points but with a more rapid decrease to baseline levels suggesting that components of disrupted cells have a shorter transition time. Preliminary data also show promising results using C2CA for direct analysis of fecal samples without prior DNA extraction.

Contact e-mail: [email protected]

Probiotics and gut comfort – a new challange

Dr J.M. ANTOINE MD, Ph.D. Danone Research Daniel Carasso Centre

Background: Gut comfort is a key element of well-being, and the micro-biota is part of the gut physiology. Therefore gut comfort should be sensitive to the gut microbiota including transient living micro-organisms, specifically probiotics. This can be achieved either by a modulation of the local pressure, and this can be the result of a change in motor activity, or the sensitive mechanisms or the gas production.

Different probotics are used in humans with reported impact on gut functions and comfort.

The most classical one is the improvement of lactose digestibility. It must be noticed that improving lactose digestion in lactose malabsorbers is not 100% related with an improvement of gut comfort. It looks like the colonic microbiota as a role in it.

Improvement of gut transit can have an impact of the well being and the comfort of daily stools, as well as easiness of passing stools, including improved shape of stools can be improved with specific probiotics in different populations.

Modulation of local inflammation is another commonly targeted benefits by different probiotics even for pouchitis.

Finally, pain can be modulated by probiotics as recently reported in the literature.

Some specific probiotics can have an impact of gut comfort through different mechanisms, opening more topics for fruitful research.

Contact e-mail: [email protected]

Gut microbial-host metabolic interactions in health and disease

Jeremy K. Nicholson

Department of Biomolecular Medicine, Imperial College London, London SW7 2AZ UK

Post-genomic technologies are being widely applied to improve the understanding of the molecular basis of human disease. Metabonomics is a top-down systems biology approach that enables multivariate profiling of the integrated metabolic responses of complex systems to patho-physiological stress, and so involves understanding the way the whole metabolic regulatory system varies with interventions [1]. Mammalian biochemistry is strongly influenced by gut microbial symbionts that can alter drug metabolism and toxicity; the study of such transgenomic and gene-environment interactions is termed “global systems biology” [2]. It has been shown that gut microbial population structure and metabolic activities influence calorific bioavailablity and the development of hepatic steatosis (in non-alcoholic fatty liver disease) and experimentally-induced insulin resistance [3] and may contribute to changing patterns of obesity and related pre-pathologic states in human populations. Metabolic interactions occur at multiple levels including bile acid co-metabolism which affects lipid absorption and hepatic processing via endocrine stimulation [4]. The influence of altered microbial-mammalian metabolic interactions and their possible contributions to the development of obesity, type 2 diabetes and insulin resistance will be discussed in relation to individual health and changes patterns of disease in human populations.

Contact e-mail: [email protected]

Bile acids metabolism by intestinal bacterial: pathological implications

P.B. Hylemon, J.M. Ridlon and D.J. Kang.

Department of Microbiology/Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Va

Deoxycholic acid (DCA) is produced from cholic acid solely by a small population of intestinal Clostridium species. DCA can accumulate to high levels in the bile of some individuals and may contribute to the pathogenesis of colon cancer, cholesterol gallstones, and other gastrointestinal (GI) diseases. During their enterohepatic circulation, bile salts are exposed to the gut microflora. Bacteria in the GI tract can deconjugate bile salts, epimerize the hydroxy groups at C3, C7 and C12 and 7α-dehyroxylate cholic acid and chenodeoxycholic acid forming deoxycholic acid and lithocholic acid, respectively. A unique multi-step biochemical pathway has been described in intestinal bacteria for removing the 7α-hydroxy group from primary bile acids. Many of the enzymes/transporters in the 7-dehydroxylation pathway have been isolated and characterized. The rate limiting enzyme in this pathway appears to be bile acid 7α-dehydratase. This pathway may serve as an ancillary electron acceptor in the anaerobic environment of the colon. Unlike most other animals, the human liver can not 7α-hydroxylate deoxycholic acid forming cholic acid. Hence, it can accumulate to high levels in some individuals. The level of deoxycholic acid in bile of humans appears to be controlled by: 1) levels and enzymatic activity of 7-dehydroxylating bacteria in the colon; 2) intestinal transit time; and 3) colonic pH. It is unclear what controls the levels of bile acid 7α-dehydroxylating bacteria; however, it is hypothesized that diets high in protein may favor the growth of these bacteria in the colon as most Clostridium species are capable of fermenting amino acids via the Stickland reaction. High levels of DCA have been found in the feces, blood and bile of colon cancer patients as compared to controls. Moreover, high levels of DCA have been reported in a subset of cholesterol gallstone patients. Antibiotic treatment of these patients decreased DCa and the cholesterol saturation index of bile, a major risk factor for cholesterol gallstone formation. These data implicate diet, intestinal bacteria and DCA in the pathogenesis of two major diseases of the gastrointestinal system.

Contact e-mail: [email protected]

Nitrate, bacteria and human health

Jon Lundberg

Department of Fysiology and Farmacology (FyFa), Karolinska Institutet, Stockholm, Sweden

Nitrate is generally considered a water pollutant and an undesirable fertilizer residue in the food chain.

Research in the 1970s indicated that, by reducing nitrate to nitrite, commensal bacteria might be involved in the pathogenesis of gastric cancers and other malignancies, as nitrite can enhance the generation of carcinogenicN-nitrosamines.

More recent studies indicate that the bacterial metabolism of nitrate to nitrite and the subsequent formation of biologically active nitrogen oxides could be beneficial. I will here consider the evidence that nitrate-reducing commensals have a true symbiotic role in mammals and facilitate a previously unrecognized but potentially important aspect of the nitrogen cycle.

Contact e-mail: [email protected]

Effect of luminal bacteria on the circulatory and gastrointestinal nitrate metabolism

Sobko T¹, Midtvedt T², Kamiya S¹

¹Dept of Infectious Diseases, Kyorin University School of Medicine, Tokyo

²Dept of Cell and Molecular Biology, Karolinska Institutet, Stockholm

Recent reports show extensive positive effects of dietary nitrate emphasizing the biological importance of the nitrate-nitrite-nitric oxide (NO) pathway. NO generation by gastrointestinal (GI) bacteria differ profoundly from the classical mammalian, via NO synthases, pathway as bacteria use nitrate and nitrite as substrates. Using germ free (GF) animals we investigated the effect of luminal bacteria on nitrate metabolism in the GI tract.

Male GF rats were pre-treated with sodium nitrate (NaNO₃,1 mmol/kg/day) and thereafter either monoinnoculated with Citrobacter (C), Bifidobacterium (B) or diinnoculated with these two strains (CB). All rats received nitrate-containing water until they were killed and examined 4 weeks after. Colonization rate, gastrointestinal NO, nitrate/nitrite and ammonia levels in serum, tissues, and contents have been analysed.

Bacterial strains were established in high number as mono- and dicontaminants. Nitrate increased significantly in the B group (588,1µM), while nitrite levels were highest in blood of the dicontaminated group, CB (1.7µM) followed by C (0.8µM) group. Luminal NO levels increased significantly in all parts of the GI tract in nitrate-fed CB group, mostly due to the presence of Citrobacter.

We conclude that gastrointestinal bacteria could modify the circulatory and gastrointestinal nitrite and nitrate levels in GF animals. Citrobacter but not Bifidobacterium posses nitrate and nitrite reductases and if given together, increased nitrite levels significantly. Future studies will clarify the significance of this alternative NO source.

Contact e-mail: [email protected]

Usefulness of germfree rats and dihydro-vitamin k1 for the clarification of physiological importance of tissue- accumulated menaquinone-4 (mk-4), a possible tissue-active form of vitamin k

Michio KOMAI¹, Yuta KOMANO¹, Toshiro SATO², and Hitoshi SHIRAKAWA¹

¹Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan

²J-Oil Mills, Inc., Fukuroi, Shizuoka 437-1111, Japan

Background: Phylloquinone (vitamin K1 = VK1) and the menaquinones (MK-n, or vitamin K2 = VK2) are naturally occurring forms of vitamin K. Most of the menaquinone series are synthesized by microorganisms, but we have reported that MK-4 is usual in being synthesized by the conversion of orally ingested VK1 or MK-n in the major tissues of germfree rats and mice which lack their intestinal microflora. In addition to the liver, VK is found in the bone, brain, heart, testis, kidney, pancreas and salivary glands mainly as MK-4, and it has been reported that MK-4 itself has specific biological activities in these tissues beside Gla-protein formation. However, the physiological role of MK-4 in these organs has not been fully understood yet. Recently MK-4 has been attracted the attention of researchers due to its activities such as apoptotic activity on the osteoclast cells and leukemia cells, SXR/PXR ligand, and so on.

Methods: In the present study, we used germfree Wistar rats that is very ready to become lower level of tissue vitamin K status, and dihydro-vitamin K1 that has Gla-protein formation activity but no conversion activity into MK-4 in most of tissues.

Results and Conclusion: We found out that MK-4 concentrations in testis and bone (femur) were decreased by dihydro-vitamin K1 feeding under the germfree circumstance than ever observed before, so that we could analyze the possible important physiological role of MK-4 in bone and testis without hypoprothrombinemia. Possible new functions of MK-4 will be presented from the viewpoint of the gene expression in these tissues.

Contact e-mail: [email protected]

Intestinal flora functions

Tore Midtvedt

MTC, Karolinska Institutet, Stockholm, Sweden

With a slight travesty of terms first used by Claude Bernhard, the host's side of the ecosystem can be defined as milieu interieur (MI), the microbial side as milieu exterieur (ME) and MI and ME together as milieu total (MT). Studies in germfree (GF) mammals, birds, fish, insects, and reptiles have established basal values for anatomical structures and physiological, biochemical and immunological variables in MI. When such basal values or baselines are established, the normal functions of the flora, as well as alterations in these functions, can be worked out.

At the VIII IAG in Notre Dame, Indiana in 1984, two terms – microflora-associated characteristics (MACs) and germfree animal characteristics (GACs) was introduced. A MAC was – and is – defined as the recording of any anatomic structure or physiological, biochemical or immunological function in a macroorganism that has been influenced upon by the microflora. When microganisms that influence the variable under study are absent, as in GF organisms, or newborns and sometimes in relation to ingestion of antibiotics, the structure and functions are defined as GACs. Consequently, a collection of GACs describes a MI, and a similar collection of MACs describes a MT. A simple equation MT minus MI gives ME.

Over the years, many sets of MACs/GACs have been presented and hopefully, so will also be the case under this IAG meeting.

Contact e-amil; [email protected]

Helicobacter pylori and enterohepatic helicobacter – present state of the art

T. Wadström, H.O. Nilsson and Å. Ljungh

Sect. Medical Microbiology, Lund University, Sweden

H. pylori was accepted as a Class 1 carcinogen already in 1994 and became the topic for the Nobel Prize in medicine 2005. Nonetheless, final acceptance of this human gastric pathogen and related species causing infections in laboratory rodents and other animal species and man has been slow. This despite a great interest to reveal all steps in early and chronic H. pylori gastritis in childhood, and stomach cancer in about one percent of the infected persons in the western world. Studies of H. pylori in mouse and gerbil models have revealed important aspects on co-carcinogenesis with other carcinogens in gut and liver cancer processes with aspects on how the gastrin and ghrelin may be linked to gut malignancies such as colon cancer and MALT lymphoma in the gut and hepatobiliary system. The evolution of the Helicobacter and related genera from mice to man will be discussed and how to discriminate gut commensals from pathogens among these fastidious microbes colonizing the stomach and gut of the gastric versus intestinal species in rodents; man and other primates. Finally, a recent report on possible gut microflora contributory role for breast cancer will be discussed.

Contact e-mail; [email protected]

Disturbed gut microbiota in haart-naive hiv-positive adults: effect of intervention with a specific prebiotic oligosaccharide mixture

Kaouther Ben Amor¹, Giuliano Rizzardini², Carlo Torti³, Tiziana Quirino⁴, Mauro Moroni², Jan Knol¹, Dorothy Bray⁵, Aldwin Vriesema¹, Mario Clerici⁶, and Andrea Gori⁷

Numico Research, Wageningen, The Netherlands¹, “Luigi Sacco” Hospital, Milan Italy², University of Brescia, Brescia, Italy³, Busto Arsizio Hospital, Busto Arsizio, Italy ⁴; ImmunoClin, Paris, France⁵; University of Milan, Milan, Italy ⁶ and “San Gerardo” Hospital, University of Milano-Bicocca, Monza, Italy⁷

Background: HIV-1 infection is characterized by massive destruction of CD4⁺ cells in the gut and chronic immune activation. Exposure of peripheral immune cells to translocated microbial products from the gut was recently suggested as a cause of the chronic immune activation. The microbial components enter the bloodstream upon alterations of the GI barrier function that occur with progressive HIV infection. The role of the composition of the gut microbiota in the immune activation process is however unknown.

Aim: The aim of this study was to assess the gut microbiota of HIV positive adults and investigated the effect of a specific prebiotic oligosaccharide (OS) mixture.

Methods: In a double blind, placebo controlled pilot study (COPA trial), 57 HAART-naive HIV-1 positive adults (baseline median (range) of CD4⁺ T-lymphocyte count 451 (300-1080) cells/µl and HIV RNA levels 11014 (49-196524) copies/ml) were randomized to receive either placebo (maltodextrin) or a specific prebiotic OS mixture (15g or 30g per day) consisting of Galactooligosaccharides (GOS), long-chain Fructooligosaccharides (lcFOS) and Acidic Oligosaccharides (AOS) for 12 weeks. Fecal samples were examined for the microbiota composition at week 0 and 12 using Q-PCR and FISH. Bacterial levels were calculated as percentage of the total bacterial load. Statistical analyses were performed using ANOVA or Mann-Whitney.

Results: Low levels of bifidobacteria and lactobacilli, species known to have a positive health effect, and high prevalence of opportunistic pathogens were observed in the HIV-positive adults. Twelve weeks of intervention with GOS/lcFOS/AOS resulted in a significant increase of bifidobacteria in both prebiotic groups, either compared to placebo (15g/d, p = 0.009; 30g/d, p = 0.015) or to baseline (15g/d, p = 0.007; 30g/d, p = 0.010). This was accompanied by a decrease from baseline of the Clostridium coccoides/Eubacterium rectale cluster in the 15g/d (p = 0.035) and 30g/d group (p = 0.005). The intervention also induced a significant (30g/d; p = 0.009) decrease from baseline in the Clostridium histolyticum/Clostridium lituseburense group.

Conclusion: This is the first study to show a clear disturbance of the gut microbiota in HIV patients, even at early stages of infection. Ingestion of a specific prebiotic OS mixture results in restoration of a normal gut microbiota and a decreased fecal pathogenic load. This improved gut microbiota might beneficially affect immune activation in HIV infection, a link which warrants further research.

Contact e-mail: [email protected]

Long-term consequences of treatment with claritromycin and metronidazole on the human gut microbiota

Hedvig Jakobsson^1,2, Cecilia Jernberg¹, Anders Andersson¹, Maria Sjölund³, Janet Jansson^4,5, and Lars Engstrand^1,2

¹Department of Bacteriology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden, ²Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden, ³CDC, Georgia, USA, ⁴Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden, ⁵Ecology Department, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

Background: Resistance to antibiotics is a major public health problem and long-term ecological effects of antibiotic treatment on the human indigenous microbiota have not been well established. We examined how a common therapy containing clarithromycin and metronidazole affects the indigenous microbiota in feces and throat.

Method: The bacterial compositions in fecal samples and throat passages were monitored by terminal-restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing. The abundance of the antibiotic resistance gene erm(B) in DNA extracted from feces, was measured by real-time PCR.

Results: While the untreated control microbial communities displayed high stability over time, the antibiotic treatment resulted in a dramatic shift in the bacterial community structure in feces as well as in the throat. In both communities, phylotypes within the Bacteroidetes and Actinobacteria phyla declined sharply, while members of Proteobacteria increased in relative abundance. Interestingly, the composition of the indigenous microbiota was perturbed in different ways in the analyzed individuals. We also show a high abundance of the erm(B) gene, which can cause high-level macrolide resistance in enterococci, after treatment and this high abundance persisted for up to 4 years without any additional antimicrobial treatment.

Conclusions: In conclusion, we show that a common one-week antimicrobial treatment regimen results in marked ecological disturbances in the indigenous microbiota and has long-term consequences.

Contact email: [email protected]

Metaproteomics of the human gut microbiota reveals previously undescribed pathways and interactions

J. K. Jansson^1,2, A.Godzik³, A.L. Russell⁴, M. Shah⁴, M. Rosenquist², J. Halfvarson⁵, M. G. Lefsrud⁴, J. Apajalahti⁶, C. Tysk⁵, R. L. Hettich⁴, N. C. VerBerkmoes⁴

¹Ecology Department, Lawrence Berkeley National Laboratory, Berkeley, CA

²Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala

³Burnham Institute for Medical Research, La Jolla, CA

⁴Oak Ridge National Laboratory, Oak Ridge, TN

⁵Örebro University Hospital, Örebro, Sweden

⁶Alimetrics Ltd, Helsinki, Finland

Background: Recently the human gut microbiota has been investigated using a combination of molecular fingerprinting approaches and metagenomics sequencing. This information provides an idea of the composition of the gut microbiota and potential functions, but not about which genes are expressed and functioning in the gut environment.

Method: A novel shotgun metaproteomics approach was used to determine the identities of thousands of proteins in fecal samples. Bacteria were enriched from feces collected from two healthy individuals by differential centrifugation, the cells were lysed, proteins digested and separated by liquid chromatography and infused directly into rapidly scanning tandem mass spectrometers to obtain highly accurate mass predictions. Proteins were identified by matching the mass data to deposited genome and metagenome sequence data. Bioinformatics tools were used to elucidate the identities of hypothetical proteins.

Results: Thousands of proteins were identified and many of the hypothetical proteins were assigned to previously undescribed pathways. In addition, several human proteins were identified that have a postulated role in host interaction with the gut microbiota, including immune response. The metaproteome was significantly skewed by comparison to average metagenomic data with a higher representation of proteins involved in carbohydrate metabolism and some other functions indicative of the importance of these processes in the gut environment.

Conclusions: Shotgun metaproteomics has revealed for the first time the microbial functions that are most prevalent in the healthy human intestine and suggest the importance of interactions between the gut microbiota and the human host. This study lays the groundwork for future studies of the function of the gut microbiota in health and disease.

Contact email: [email protected]

Thursday session abstracts

The hygiene hypothesis – revised

Bengt Björkstén

Karolinska Institutet, 171 77 Stockholm, Sweden

In 1976, the Canadian paediatrician suggested that recurrent infections protected the indigenous population in Northern Canada from allergies. More than a decade later, an inverse relationship between allergic rhinitis in adults growing up with older siblings was reported. It was suggested that childhood infections could protect against allergy. Since then at least 30 studies have confirmed the relationship. Subsequent studies showed that growing up on a farm during the first years of life, high levels of endotoxin in house dust and pet exposure are also associated with protection against respiratory allergies. More recent studies indicate however, that respiratory tract infections do not protect against allergies as originally suggested, but rather increase the risk.

In the 1990ies, comparative studies in Estonia, with a low prevalence of allergies and juvenile diabetes, and Sweden revealed pronounced differences in the composition of the gut microbiota in healthy infants. The Estonian gut flora was similar to what was described in Sweden and eastern Germany in the 1960:ies. Further studies revealed that the composition also differed between allergic and non-allergic children in Sweden and subsequently also that differences were present already during the neonatal period, i.e. at the time of gut colonisation and before any clinical manifestations of allergy. Such differences have now been confirmed in several studies from other countries. The most consistent findings in these studies seem to be less diversity of the gut microbiota in allergic infants and also possibly less colonisation with bifidobacteria.

A number of diseases related to a modern, affluent life style may be associated with an altered gut microbiota via an effect on the developing immune system early in life. These “immunologically mediated diseases of affluence” include allergies, IBD, insulin dependent diabetes mellitus and other autoimmune diseases. The fact that variations over time and region seem to be similar for both Th1- and Th2 mediated cast doubt over the initial hypothesis that infections would protect against allergies by enhancing Th1 type immune responses.

The gut microbiota are the quantitatively most important source of microbial stimulation and provide a primary signal for driving the postnatal maturation of the immune system and the development of a balanced immunity. Thus, there is mounting evidence that commensal microbes acquired during the early postnatal period are required for the development of oral tolerance. Interaction with the normal microbial flora of the gastrointestinal tract seems to be the principal environmental signal for postnatal maturation of T-cell function in humans, in particular immune regulation. Comparative prospective studies show that postnatal maturation of immune responses, including secretory IgA occur more rapidly in Estonia than Sweden and in healthy as compared to subsequently allergic infants.

We have very recently observed differences in the composition of the gut microbiota between infants with and without older siblings. These observations may be relevant for the lower incidence of allergies in the former group.

Conclusions: The term “hygiene hypothesis” is unfortunate, since it would suggest that poor hygiene is the cause of allergies and other immune mediated diseases. It would be inconceivable for society to go back to primitive conditions. Rather, the road forward may involve enhancing a more diverse gut microbiota. Several recent controlled studies have shown some allergy preventive effects by probiotic lactobacilli given to pregnant women and their babies after birth.

Contact e-mail; [email protected]

Atophy and allergy in 5 different eu countries – functional aspects

Edwards CA¹, Gil A², Scott J¹, Amarri S³, Norin E⁴, Adam R⁵, Schmitt J⁶, Doré, J⁷, Fons M⁸, Hamelmann E⁹ and other members of INFABIO project

¹Human Nutrition Section, Division of Developmental Medicine, University of Glasgow, Scotland, ²Department of Pediatrics University of Granada, Spain, ³Azienda Santa Maria Nuova di Reggio Emilia, Italy, ⁴ Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden, ⁵ Department of General Pediatrics University Children's Hospital, Heinrich-Heine Universitat, Dusseldorf, Germany, ⁶ Numico Research, Germany, Milupa, Friedrichsdorf. Germany, ⁷ Unité d’écologie et de Physiologie du Systême Digestif. INRA, Jouy en Josas, Cedex, France., ⁸ Institut Mediterrneen de Recherche en Nutrition, Faculte des Sciences et Techniques de Marsailles St Jerome, Marseilles, France, ⁹Department of Pediatric Pneumology/ Immunology, Hospital Charite’, Humboldt-University Berlin (HUB), Augustenburger Platz 1, 13353 Berlin, Germany

Background: The increasing rate of allergy in Europe may be related to environmental exposure and infant microflora.

Method: A cohort of 700 infants, in 5 European countries, was followed for 1 year. Faecal samples were collected with health, diet and environment data. A case control study of infants with diagnosed cows’ milk protein allergy compared similar parameters.

Results: Infant feeding practice varied between countries. Stockholm, had the highest breast-feeding rate and Glasgow the lowest. Parents in Granada and Deusseldorf weaned early. Infants at high risk of atopy were less likely to have a plastic mattress covers. There was no association with infant feeding, parental smoking, or household pets. Those who had cows’ milk before 12 months were less likely to report eczema by 12 months. As age of introduction of solids increased the odds of reporting eczema increased. There were significant inter-country differences in infant microflora at 6 weeks allowing for feeding method, caesarean, and antibiotics. Spanish infant had more predominant Bacteroides, greater diversity and less acetic and lactic acid. Northern European infants had a flora more dominated by bifidobacteria. Infants with parent-reported eczema had different microflora. In the case control study, cases had higher proportions of C Coccoides, Atopobium and C perfringens+ C difficile.

Conclusion: Infant microflora differs across Europe and may be related to allergy risk.

Contact email: [email protected]

This study was carried out with financial support from the commission of the European Communities, specifically the RTD program ‘Quality of life and Management of Living Resources’, QLRT-2001 02606, ‘INFABIO’,. It does not necessarily reflect its views and in no way anticipates the commission's future policy in this area. Other members of the INFABIO project involved: Benoit Thezé⁷, Sheila Khanna¹, Khalid M Khan¹, Agneta Uusijarvi⁴, Francesca Benatti³, Rosa Maria De Mola², Horst Schroten⁵, José Maldonado², Oscar Thompson² José Maria Vieites², Marco Ferrer² Birgit Marsing⁹

Effects of probiotics against infections

Leda Quercia Vieira¹, Liliane Martins dos Santos¹, Elisabeth Neumann¹, Anderson Paulo da Silva¹, Lilian Nobre Moura², Luís Carlos Crocco Afonso³, Jacques Robert Nicoli²

¹Departamento de Bioquímica e Imunologia and ²Departamento de Microbiologia, Instituto de Ciências Biológicas, UFMG, ³Departamento de Ciências Biológicas e Núcleo de Pesquisa em Ciências Biológicas, Instituto de Ciências Biológicas e Exatas, UFOP.

Background: Our group has concerned itself with the study of the effect of probiotics on the resistance to infections, using experimental models. Here, we will mainly focus on evidence that the UFVH2b20 strain of Lactobacillus delbrueckii var. bulgaricus may be considered a probiotic and has protective effects on mice against a variety of bacterial infections.

Method: Germ free, mono-associated and conventional mice were used. Mice were treated with probiotics and challenged with Escherichia coli, Salmonella enterica serovar Typhimurium or Listeria monocytogenes and the outcome of infection was measured as mortality, quantification of bacteria in target organs and systemic of local cytokine production. Human peripheral blood mononuclear cells were used to investigate the production of cytokines stimulated by L. delbrueckii.

Results: L. delbrueckii increased clearance of E. coli and production of systemic inflammatory cytokines. This strain also protected mono-associated and conventional mice against infection with S. enterica serovar Typhimurium. Mono-associated mice were more resistant to L. monocytogenes as measured by mortality and the number of bacteria in spleens and liver. In addition, mono-associated mice challenged with L. monocytogenes showed increased production of inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) and nitric oxide. Interestingly, IL-10 levels were not altered by mono-association or infection. Accordingly, human peripheral blood cells produced inflammatory cytokines in response to L. delbrueckii, but not IL-10.

Conclusion: L. delbrueckii triggers a pro-inflammatory response that is beneficial to the host when resisting infections.

Support: CNPq, FAPEMIG and CAPES.

Contact e-mail: [email protected]

Influence of overweight on intestinal microbiota of pregnant women

Riikka Rantanen1, M. Carmen Collado1, Kirsi Laitinen2,3, Erika Isolauri2,3, Seppo Salminen1

¹University of Turku, Department of Biochemistry and Food Chemistry, Functional Foods Forum, 20014 University of Turku, Finland

²Department of Paediatrics, University of Turku, 20014 Turku, Finland

³Department of Paediatrics, Turku University Central Hospital, 20014 Turku, Finland

Background: Several studies have demonstrated that some commensal gut bacteria harvest energy from food more efficiently than others. Studies by Gordon et al have shown that obese mice have a different intestinal microbiota compared to lean mice, and that these deviations may predispose towards obesity. The main findings are that obese mice have a higher proportion of firmicutes and a lower proportion of bacteroidetes than lean mice, and a recent study by the same group suggests that the same applies to humans.

Objective: To characterise the intestinal microbiota composition of pregnant women with normal and high BMI. We were also interested in how weight gain over pregnancy affects the microbiota.

Design: Overweight women (n = 18) were selected according to their pre-pregnancy body mass index (BMI 30) from a prospective follow-up study. Normal weight (BMI 18-25) women (n = 36) were selected as controls. Women were followed over the course of pregnancy, and faecal samples were collected at first and third trimester of pregnancy. Faecal microbiota composition was analysed by fluorescent in situ hybridisation coupled with flow cytometry (Flow-FISH) and by quantitative real-time PCR (qPCR).

Results: Bacteroides group levels were significantly higher in overweight than normal weight women. In addition, the levels of Bacteroides group were significantly higher in women with excessive weight gain over pregnancy.

Conclusions: Our results demonstrate that there are differences in faecal microbiota composition between normal weight and overweight pregnant women.

Key words: Intestinal microbiota, energy harvest, overweight

Contact e-mail: [email protected]

The development of intestinal microbiota in infancy and the relationship between microbiota and atopic diseases

Lotta Jalonen, Reetta Satokari, Mirjana Rajilic-Stojanovic, Hans Heilig, Marko Kalliomäki, Erika Isolauri, Seppo Salminen and Willem M de Vos

Functional Foods Forum, University of Turku, Department of Basic Veterinary Sciences, University of Helsinki, Department of Pediatrics, University of Turku, FINLAND and Laboratory of Microbiology, Wageningen University and Research Centre, The Netherlands

Background: Previous studies have shown that intestinal microbiota may have a role in the development of allergic diseases. These studies have mainly focused on the microbiota during early infancy (from birth to three months of age) and/or to the analysis of few bacterial groups.

Methods: Fecal samples of twelve infants that developed atopy and ten infants who did not develop atopic disease were analysed at the age of 6 and 18 months. A global overview of the microbiota composition was assessed by using the HITChip (Human Intestinal Tract Chip) high density microarray. Quantitative PCR (qPCR) analysis focused on individual bacterial groups and species, particularly on different Bifidobacterium species.

Results: The results show that there are differences in specific bacterial groups between the microbiota of infants developing and not developing atopy.

Conclusion: The results confirm the earlier findings that the composition of infant microbiota can precede the development of atopy later in life.

Contact e-mail: [email protected]

Some possible pathways of probiotic action

John Bienenstock

McMaster University, Brain-Body Institute, St. Joseph's Healthcare Hamilton

While the accepted definition of a probiotic is that it be live, increasingly it is becoming apparent that the effects of various forms of killing of these organisms be at the very least investigated and compared to those obtained with live bacteria. Several mechanisms of possible action will be explored including those on the immune system, the nervous system and the inhibitory actions upon visceral pain pathways. The role of Toll receptors, especially Toll 9, in systemic effects will be noted, the effects on the HPA axis and the brain as well as modulation of tryptophan metabolism. The talk will mostly focus on areas and pathways which are not most frequently studied and it is hoped that it will stimulate interest and discussion and open up new areas for study.

Contact e-mail: [email protected]

The antimicrobial barrier of the small intstine

Katrin Pütsep¹, Ulf Meyer-Hoffert1^1,2, Mathias Hornef³, Lars-Gunnar Axelsson¹, Tore Midtvedt¹, Jenny Karlsson¹, Birgitta Henriques-Normark² and Mats Andersson¹.

¹Department of Microbiology Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden;Swedish ²Institute for Infectious Disease Control, Stockholm, Sweden; Institute for Medical Microbiology and ³Hospital Epidemiology, Hannover Medical School, Hannover, Germany

Background: Antimicrobial peptides play an important role in the mammalian body barrier defense and most likely in limiting the natural microflora of the small intestine. Secretory cells of the intestinal crypt, the Paneth cells, produce and secrete high levels of antimicrobial proteins and peptides such as the α-defensins (in mouse cryptdins) and CRS-peptides. This strong bactericidal armament contributes to the defense of the stem cells, which reside in the crypt of the small intestine.

Methods: Using HPLC, mass-spectrometry and assays for antimicrobial activity we have characterized antimicrobial components of the small intestine and the spatial distribution of these components in extracts from small intestine, crypt/epithelial cells, mucus and lumen. We have by qRT-PCR determined the distribution of CRS-peptides versus cryptdins along the small intestine.

Results: Lumen was almost devoid of antimicrobial peptides/proteins while the antimicrobial activity was retained by the mucus layer. Bacteria were efficiently killed when forced into the mucus, in vitro. Certain CRS-peptides varied along the small intestinal longitudinal axis.

Conclusions: Antimicrobial peptides coat the intestinal mucus layer and thus create a physical and antibacterial barrier that prevents bacteria from invasion and the natural microflora from close adhesion to the epithelial layer.

Contact e-mail: [email protected]

Expression of lactobacillus sp. antimicrobial compounds in different ecosystems

Marika Mikelsaar

Department of Microbiology, University of Tartu, Tartu, ESTONIA

Lactobacillus sp. are among the most well-studied bacteria in regards to microbial antimicrobial compounds. In vitro sudies have shown their antimicrobial action against various enteric and sexual pathogens,facilitating their use as probiotics. The safest way to prevent food-borne infections is through the application of bacteria that have remarkable antimicrobial activity in food production. Therefore an important use for Lactobacillus sp. is as food preservatives. Their main antimicrobial factors include different non-proteinaceous and proteinaceous compounds with high-grade strain specificity. Still, the main problem is not due to the limited discovery of new strains with new antimicrobial properties but in proving their action in vivo. The mechanisms by which these compounds grant the survival and competiteveness of the producers in different microbial ecosystems have not been well explored.

To test the effectivity of different antimicrobial compounds several experimental models have been applied, such as cultivation in conditions resembling the particular microbiotope and modulating the conditions of cultivation (anaerobiosis, aerobiosis, differences in pH, content of cultivation media). Cell lines like the Caco-2, LOVO, Hep-2 have been successfully applied to reveal the suppression of some pathogens (e.g. Clostridium difficile) by particular strains of lactobacilli. However, under these experimental conditions, different confounding factors are emerging. In some systems the adhesive properties of lactobacilli may be even more determinative than the strict antimicrobial compounds of the strain. For instance, in rhodent models of infection (Salmonella sp., Listeria sp., E. coli, etc.), it is difficult to achieve sufficient colonization with some Lactobacillus. The specificity of the lactobacilli colonizing animals may be an important confounding factor. On the other hand, the high count of lactobacilli is decisive when relying on their antimicrobial activity. In addition, in animal models it is difficult to determine if the positive suppressive effect was gained due to the antimicrobial compounds or some other indices of the strain, e.g. by the induction of the antioxidative activity. The latter may improve the state of mucosa, preventing the translocation and the generalization of the experimental infection. In volunteer models even more complications arise, like problems with differentiation of the local influence due to antimicrobial compounds of the applied strain as compared to the influence from the other microbiota and their combined impact on host humoral and cellular immunity. The role of individual differences in the microbiota composition for the temporary colonization of new strains of lactobacilli has not yet been assessed.

In this presentation, the expression of the antimicrobial activity of the two strains of Lactobacillus plantarum will be compared using different in vitro and in vivo models for their application.

Contact e-mail: [email protected]

Protective effect of probiotic strain escherichia coli nissle 1917 on the development of intestinal inflammation induced in gnotobiotic models

T. Hudcovic, R. Štepánková, H. Kozáková, T. Hrncír, M. Schwarzer, H. Tlaskalová-Hogenová

Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Nový Hrádek

Background: The participation of the intestinal microflora, mainly role of E. coli strains, in the development of the acute and chronic intestinal inflammation (model of ulcerative colitis) was studied in mice.

Methods: Experimental colitis was evoked by an administration of 2.5% dextran sodium sulphate (DSS) in drinking water (7 days). Germ-free mice, mice monoassociated by (E. coli Nissle 1917 and E. coli O6K13) and conventional mice of SCID and BALB/c strains were used in our experiments. One week prior to DSS exposure the conventional mice received daily E. coli Nissle 1917 by intragastrical tubing or intrarectally and bacteria were continuously given during DSS drinking. Colon morphology and mucin production were evaluated.

Results: Mice monoassociated with E. coli O6K13 developed intestinal inflammation in colon whereas colonization with E. coli Nissle 1917 strain protected mice against inflammation. Intragastrically administrated E. coli Nissle had only a mild effect on the intestinal inflammation, whereas mice treated per rectum remained healthy. In this group, the level of pro-inflammatory cytokine TNF-alpha and IL-6 were reduced markedly in colon descendens compared with controls.

Conclusion: We conclude that E. coli Nissle 1917 colonization protects mice against intestinal inflammation induced by DSS treatment.

Supported by grants 303/08/0367 of the Czech Science Foundation and by grants 2B06 155 and 2B06 053 of the Ministry of Education, Youth and Sports of the Czech Republic.

Contact e-mail: [email protected]

Development of mucosal tolerance to birch pollen in germ-free, lactobacillus plantarum-monoassociated and conventional mice

¹Kozakova H., ^2,3Repa A., ¹Schwarzer M., ¹Hrncir T., ¹Stepankova R., ¹Hudcovic T., ¹Tlaskalova-Hogenova H., ²Wiedermann U.

¹Institute of Microbiology of the Academy of Sciences of the Czech Republic, Novy Hradek, Czech Republic,

²Department of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Austria,

³Department of Neonatology, University Children's Hospital Vienna, Austria

Background: The aim of our study was to investigate the impact of the presence of the intestinal microflora on allergic sensitization and induction of mucosal tolerance to main component of birch pollen allergen Bet v 1.

Methods: Inbred BALB/c mice reared in germ-free or conventional conditions were three times pre-treated intragastrically or intranasally with Bet v 1 before sensitization by subcutaneous injections of Bet v 1. Allergen-specific serum antibodies, as well as Th1/Th2 cytokine production were examined. In subsequent experiments germ-free mice were colonized either with probiotic bacteria Lactobacillus (Lb.) plantarum or recombinant Lb. plantarum with inserted gene for Bet v 1 production. Sensitization to Bet v 1 was done after colonization.

Results: Allergen-specific antibody levels were comparably high in Bet v 1-sensitized germ-free and conventional mice. Oral as well as intranasal tolerance induction led to a significant reduction of allergen-specific antibody levels and Th1/Th2 cytokine production shift to non-allergic responses in both germ-free and conventional mice. Decreased level of pro allergic IgE response was found in mice colonized with rec. Lb. plantarum produced Bet v 1.

Conclusions: Mucosal tolerance to Bet v 1 was established in germ-free, conventional and rec. Lb. plantarum-colonized mice. Supported by grants IAA500200710 and 2B06053 (Czech Republic).

Contact e-mail: [email protected]

Obesity – pediatric aspects

Prof Claude Marcus

Department of Pediatrics and The National Childhood Obesity Centre B57, Karolinska University Hospital, Huddinge, Stockholm, Sweden.

Obesity – microbial aspects

Fredrik Bäckhed

The Wallenberg Laboratory and the Center for Cardiovascular and Metabolic Research at the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

Obesity is a worldwide epidemic threatening both industrialized and developing countries and is accompanied by a dramatic increase in obesity-related disorders, including type II diabetes, hypertension, cardiovascular diseases, and non-alcoholic fatty liver disease. The incidence of obesity is increasing at alarming rates and there are no successful treatments beside bariatric surgery, thus novel drug targets are needed.

The human intestine harbours an immense collection of microbes (microbiota) that have co-evolved with its host and performs numerous functions that we have not had to evolve our selves. However, recent studies have shown that the gut microbiota is an environmental factor that regulates obesity by increasing energy harvest from the diet. While microbial fermentation of dietary polysaccharides has been extensively explored, less is known about microbial regulation of lipid metabolism. By taking a lipidomics approach we identified the gut flora as an important regulator of host serum lipid metabolism. Germ-free mice have elevated levels of triglycerides in their serum, which may be explained by increased absorption of dietary lipids. In addition the gut flora regulates the expression of several genes in the intestinal mucosa that affect lipid metabolism and/or metabolic inflammation (e.g. serum amyloid A, fasting-induced adipose factor). In combination these factors suggest that the gut microbiota may be a suitable target for interventions of obesity and type 2 diabetes.

Contact e-mail; [email protected]

Gut microbiota modulation improved glucose tolerance in mice with insuline resistance

F. Blancher¹, M. Membrez¹, R. Bibiloni¹, E. Rezzonico¹, M. Moser², K. Macé¹, C. J. Chou¹

Nestlé Research Centre, ¹Department of Nutrition & Health, ²Department of Bioanalytical Science, Lausanne, Switzerland.

Background: Recent reports indicate that certain bacterial groups that dwell in the gut may be responsible for the development of adiposity. The potential influence of gut microbes on obesity may also be associated with other metabolic disorders like type II diabetes. The aim of the study was to assess the impact of gut microbiota on obesity and insulin resistance in two animal models.

Methods: Gut microbiota of ob/ob and diet-induced obese (DIO) mice was modulated by means of antibiotic cocktails. Glucose tolerance in mice, metabolic markers as well as ceacal bacterial composition were evaluated using molecular methods.

Results: Norfloxacin/Ampicilin improved glucose tolerance and fasting glycaemia in both ob/ob and DIO mice, and dramatically reduced ceacal bacterial contents. Two-week treatment with Polymixin B/Neomycin had a modest effect in improving glucose tolerance of DIO mice, but the treatment created a new bacterial profile in the ceaca that persisted after a 4-week wash out period. Interestingly, glucose tolerance and fasting glycaemia of DIO mice were further improved after the washout period, suggesting the new profile of ceacal bacteria is beneficial for improving glucose management in mice.

Conclusions: Our results indicate that the intestinal microbiota is involved in the regulation of blood glucose in mice. Modulation of gut microbiota could be a novel therapeutic strategy for managing Type II diabetes.

Contact e-mail: [email protected]

Human flora-associated mice with different metabolic activities against soy isoflavones

K. Hirayama, M. Tamura*, K. Itoh

Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, The University of Tokyo and *National Food Research Institute, Japan

Background: Beneficial effects of soy isoflavones on human health have been demonstrated in both epidemiological and experimental studies. It is know that soy isoflavones are metabolized by intestinal microbiota to more bioactive or inactive metabolites. There are great inter-individual differences in metabolic activities against soy isoflavones due to the difference of the composition of microbiota.

Method: Genistein and daidzein were anaerobically incubated with human feces in vitro and the isoflavones and their metabolites, particularly equol, were analyzed. Individuals with different metabolic activities against isoflavones were selected and their fecal suspensions were inoculated to germfree mice to produce human flora-associated (HFA) mice. Fecal metabolic activities of HFA mice against soy isoflavones were analyzed and compared with those of inocula.

Results: The metabolic activities of human feces against soy isoflavones were successfully transferred into germfree mice and HFA mouse groups with different metabolic activities against soy isoflavones were established. Although bacteria responsible to isoflavone metabolism have not been identified, our results suggest that the bacteria producing equol are probably in sub-dominant group.

Conclusions: The HFA mouse groups colonizing intestinal microbiota of human origin with different metabolic activities against soy isoflavones produced in this study should be a useful tool for studying the role of human intestinal microbiota on the health effects of soy isoflavones in human health and diseases in vivo.

Contact e-mail: [email protected]

Oxalobacter formigenes: a potential treatment for primary hyperoxaluria

Harmeet Sidhu¹and Bernd Hoppe²

¹Oxthera, 13709 Progress Blvd., Alachua FL,USA and Smedsgränd 2A, 753 20 Uppsala, Sweden; ² Division of Pediatric Nephrology, University Children's Hospital, Cologne, Germany.

Background: Primary Hyperoxaluria (PH) is a rare genetic disease associated with increased synthesis of oxalate in liver. Extremely high urinary oxalate levels cause renal calculi, neprocalcinosis, declining renal function and renal failure. Current therapeutic options are scarce and not successful in majority of patients. Hence we aimed to develop a new therapeutic tool and speculated that colonic degradation by O. formigenes may enhance the enteric elimination of endogenously produced oxalate. O. formigenes is an anaerobic bacterium that utilizes oxalic acid as its sole source of energy and several epidemiological studies have confirmed its role in oxalate homeostasis in humans.

Methods: O. formigenes as frozen cell paste and formulated as Oxabact ™(enteric-coated capsule) was administered for four weeks to PH patients in two clinical trials. The safety of treatment was followed through adverse events and safety lab testing. Efficacy of treatment was monitored through determination of 24h urine oxalate and plasma oxalate.

Results: Seven patients with normal renal function completed the Oxabact™ treatment. All the subjects showed a decrease in urinary oxalate excretion and the mean urinary oxalate excretion at the end of four week treatment was 60% lower than the baseline level (0.74 vs 1.85 mmole/day/1.73m²). In addition three patients in end stage renal failure were treated with O. formigenes cell paste. Two out of three patients showed a very dramatic decrease in plasma oxalate during treatment providing first proof of enteric elimination of oxalate in humans. In both studies the treatment was well tolerated by the patients and no safety concerns were observed.

Conclusion: Results from these clinical studies has shown that O. formigenes is safe and can lead to significant reduction in urinary and plasma oxalate in PH patients. Oxabact™ is currently in a Phase III randomized, multicentric, double blind placebo controlled trial in PH patients.

Contact e-mail; [email protected]

Cancer and steroid receptors – state of the art

Jan-Åke Gustafsson

Division of Medical Nutrition, Dept of Biosciences and Nutrition, Karolinska Institutet, SE-141 86 Stockholm, Sweden

Nuclear receptors are ligand activated transcription factors which control a plethora of physiological and pathophysiological processes in the organism. They are involved in regulation of e g the CNS, immune system, metabolic homeostasis, bone metabolism, hematopoiesis and cardiovascular system. In addition, some of them play important roles in control of development of many tissues and organs and thereby proliferation and differentiation of cells. Even when proliferation gets out of control, as in carcinogenesis, nuclear receptors are in many cases essential for the prognosis/development of the disease. This lecture will address some of these aspects, with an emphasis on the antiproliferative steroid receptor estrogen receptor β.

Contact; [email protected]

Prostate cancer- status of animal models

Morris Pollard

The University of Notre Dame, Notre Dame, IN

Prostate cancer (PC) in the U.S. develops spontaneously by endogenous mechanisms with the highest recorded morbidity and mortality of cancers in men: 90% of new cases of PCs are local (in situ) and patients survive symptom-free for 5 years, but 10% of new cases die of hormone refractory prostate cancer (HRPC). In one group of models, immune -deficient nude mice, rats, and SCID mice, do not reject transplants of human HRPCs, for in vivo studies. The second group of models for prevention of PC involves genetically engineered mice and inbred laboratory rats. Few PCs in the mice are metastatic. Fischer and Wistar rats develop low levels of endogenous testosterone (T), do not develop HRPC spontaneously nor by induction. The PC model in Lobund Wistar (LW) rats, with high levels of T, replicate spontaneously the natural history of HRPC, as in man.

Conclusions: Models for prevention must develop HRPC. Immune deficient models propagate refractory tumors. Engineered mice need more refinement to determine their relevance to human PC, or are they artifacts? PCs in Fischer and Wistar rats are not models of HRPC. The LW rat model replicates HRPC characteristics as in man and is preventable.

Contact e-mail: [email protected]

Microbial products and colorectal cancer

Ian Rowland

Department of Food Biosciences, University of Reading, Reading RG6 6AP UK

Background: Colorectal cancer is one of the major causes of death from malignant disease in Western Europe, USA, Australasia. It constitutes around 14% of new cases of cancer in these countries

Results: There is considerable evidence from laboratory animals and human studies that the colonic microflora is involved in the aetiology of colon cancer. For example, germ-free rats treated with the carcinogen 1,2-dimethylhydrazine have a lower incidence of colon tumours than similarly treated rats having a normal microflora. We have recently demonstrated potent DNA-damaging activity in faecal extracts from a proportion of healthy human subjects. Intestinal bacteria possess a range of xenobiotic metabolizing enzymes such as nitrate reductase and β-glucuronidase which enable them to produce, from dietary components, substances with genotoxic, carcinogenic and tumour-promoting activity. They can synthesize carcinogens such as N-nitroso compounds, activate carcinogens to reactive DNA-damaging metabolites and deconjugate detoxified carcinogens in the colon releasing the active carcinogen. In addition they can produce tumour promoters such as ammonia and bile acids. In contrast products of fermentation of carbohydrates in the gut, namely short chain fatty acids, appear to exert anti-carcinogenic effects via stimulation of apoptosis and inhibition of promotion. Furthermore the gut microflora is implicated in the metabolism of ingested phytochemicals, such as flavonoids, which may modulate their anti-cancer activity

Conclusions: Evidence implicating gut microbial metabolites in the initiation and development of colorectal cancer suggests that dietary modulation of the microflora could beneficially influence cancer risk.

Contact email: [email protected]

The efficacy of immunogrobulin for infection

Toru Masaoka MD

Osaka medical center for cancer and cardiovascular Diseases

Intravenously injectable immunogrobulin (IVIG) is approved for severe infection since 30 years in Japan, while it is not yet approved in USA. At first approval was obtained by showing several cases in whom fever came down by administration of IVIG after ineffective administration of antibiotics for long period, such as 6-10days. At present 10 preparations of IVIG were approved for the indication of infection by similar data. However it was considered not a convincing evidence for the efficacy. We have started the following studies and written a table and shown the days from antibiotics to IVIG varied 0–12 days, the days from antibiotics to defervescence varied 3–14.5 days and the days from IVIG to defervescence. remained almost constant of 3–4 days. The next study was conducted on a hypothesis that IVIG is not effective, then the efficacy was caused by antibiotics. The hypothesis was rejected because of the discrepancy of significantly small number of non responders in the real trial of antibiotics with IVIG compared with the postulated trial of antibiotics alone. Finally ministry and welfare Japan has ordered reevaluation of IVIG by a prospective randomized controlled study. It is allowed to conduct one study using one representative IVIG preparation for the 10 preparations. We have conducted a randomized controlled study of 141 center. Evaluable 504 patients with suspicious of sepsis were entered in the study who were non responders of 3 day treatment of cephem and aminoglucoside, were randomized on 4th day into 2 groups; IPM/CS + AMK and IPMS/CS + AMK with IVIG 5g/day for 3 days.

Results: response rate was 54.8% in IVIG group and37.2% in control group.(p= 0.002)

The indication of IVIG for infection was further approved in Japan.

Contact e-mail; [email protected]

Friday session abstracts

Gmos and their unmodified counterparts: substantially equivalent or different?

Terje Traavik

GenØk-Centre for Biosafety & Faculty of Medicine, University of Tromsø, Tromsø, Norway

The dynamic and interconnected regulation of the genome is now slowly being revealed. The genome does not function in a constant, stable and linear fashion, but is instructed by and fine-tunes its activities according to networks of signals received from the external ecosystem and the internal environment of the organism. The genomic signal pathways may be modified by ecosystem variation as well as by physiological changes in the organism. Thus, the chromatin structure, the genome, the epigenome, the transcriptome, the proteome, the metabolome, and the interactome are interlinked and intertwined in various ways with information transfer in multiple directions.

Integration of foreign DNA into an established genome may have unanticipated side-effects, e.g. in terms of chromatin changes, genome instability, unexpected protein products from the transgene(s), and influence on overall organismal gene expression patterns, in quantitative as well as qualitative terms, of the recipient organism.

A number of studies have not detected any major differences in GMO chemical composition. However, there are published examples of statistically significant differences between GM plants and their isogenic unmodified counterparts grown side by side, planted and harvested at the same time (e.g. Knudsen & Poulsen 2007)

Transgene and genome rearrangements have been reported for already commercialized transgenic crop plant varieties (for recent reviews see Traavik and Lim Li 2007). The nature of these rearrangements and their significance to “substantial equivalence” considerations will be further discussed.

There are at least two well-documented examples of unanticipated RNA transcripts in commercialized transgenic plants (Rang et al. 2005, Rosati et al 2008). In both cases it is uncertain to which extent the novel mRNAs are translated into proteins. But are the transcriptomes of such plants substantially equivalent to or different from their unmodified counterparts?

The proteomes, interactomes, metabolomes and epigenomes of GMOs may be quantitatively as well as qualitatively changed. The relative and absolute expression levels for endogenous genes may have changed, and metabolic pathways may be influenced. The transgene protein product(s) may be different in the recipient organism as compared to the donor organism (for recent reviews see Traavik and Lim Li 2007 and references therein).

Do these types of unanticipated changes imply adverse health or environmental effects? No general answer can be given. A number of recent studies have, however, indicated unintended negative effects of GMO consumption on macro-organisms in aquatic (e.g. Rosi-Marshall et al. 2007, Bøhn et al. 2008) and terrestric ecosystems (e.g. Hilbeck & Schmidt 2006, Lövei and Arpaia 2005, Knudsen and Poulsen 2007, Kroghsbo et al 2008).

Contact e-amil: [email protected]

Intestinal gene transfer – gnotobiology and intestinal gene transfer

Tore Midtvedt

MTC, Karolinska Institutet, Stockholm, Sweden

Our intestinal microbes have always been exposed to DNA. However, in the last few years, this also include genetically modified DNA, from plants, animals and microbes. Therefore, the fate of orally delivered chromosal and extrachromosomal DNA is crucial to know more about and pre4sent knowledge about orally delivered DNA will be outlined.

Additionally, present knowledge about intestinal microbial gene transfer can be summarized as follows:

Trnsduction occurs equally in vitro and in vivo

Conjugation seems to occur more easily in vivo

Tranformation is difficult to demonstrate in vivo

Contact e-amil: [email protected]

Antimicrobial agents and alterations in microflora composition

Carl Erik Nord

Department of Laboratory Medicine, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden

The normal microflora acts as a barrier against colonisation of potentially pathogenic microorganisms and against overgrowth of already present opportunistic microorganisms. Administration of antimicrobial agents, therapeutically or as prophylaxis, causes disturbances in the ecological balance between the host and the normal microflora. Most studies on the impact of antimicrobial agents on normal microflora have been carried out on the intestinal flora. Less is known on the effects on oropharyngeal, skin, and vaginal microflora. Knowledge about the interaction between antimicrobial agents and the normal microflora gives the clinician the possibility to choose agents associated with lesser degrees of ecological disturbances. Consequently, the risk of development of resistant strains and transfer of resistance elements between microorganisms is reduced. Consideration of the ecological consequences is also an important step to prevent distribution of resistant strains between patients in hospital settings. Resistant microorganisms, which are now well recognized pathogens isolated from the normal microflora during antimicrobial administration, include methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, quinolone-resistant Clostridium difficile, extended-spectrum betalactamase-producing enterobacteria and metronidazole-resistant Bacteroides fragilis. The dimension of disturbances is not only dependent on the spectrum of the agents but also is influenced by the degree of absorption of the agent, the route of elimination, and the enzymatic activity of the microflora. Individual pharmacokinetic variations as well as the character of composition and susceptibility of the normal microflora are variables that also influence the interactions.

Contact e-mail: [email protected]

T-cell activation by bacterial superantigen

Professor Agnes Wold

Department of Clinical Bacteriology, Göteborg University, Sweden

Biofilm in the oral cavity

Ingar Olsen

Institute of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway

The oral microflora is rich and diversified and consists of bacteria, virus, yeasts and mycoplasmas, occasionally protozoa. Most of the bacteria are anaerobic. The ecology in the mouth is unique. Imbalance in the ecology can promote caries and periodontitis, both of which are directly induced by dental biofilm. The number of cells in a dental biofilm can be extraordinary high, sometimes exceeding the US population (300 million). Dental plaque is a typical biofilm as found elsewhere in nature with voids, channels and cell clusters. Biofilm formation includes attachment, colonization and growth of microorganisms. Coaggregation is an important step during biofilm development together with cell-to cell communication and signaling. There is also site specificity of recognized biofilm species, now counting more than 700. Only half of these bacteria have been cultivated so far. 400 species have been isolated from the periodontal pocket. 9 bacterial phyla have been detected. The closeness of bacterial cells in biofilm promotes horizontal gene transfer between the same or different species. House-keeping genes are transferred between P. gingivalis isolates in the periodontal pocket indicating that transfer of virulence-associated elements occur. The prevalence of β-lactamases is high in subgingival biofilm from “refractory” periodontitis (68-72%). Checkerboard DNA-DNA hybridization has revealed that the red complex (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) is most related to development of periodontitis. Also, not-yet-cultivated species may be involved. Periodontal treatment involves the removal of multiple species in supra- and subgingival biofilm. Physical removal of biofilm, previously considered the cornerstone of treatment, is enhanced by simultaneous administration of systemic agents such as metronidazole, amoxicillin, plus metronidazole, or azithromycin.

Contact e-mail: [email protected]

The role of biofilm formation and cyclic DI-gmp signaling on phenotypes of salmonella enterica serovar typhimurium and eschericia coli related to virulence properties in the gastrointestinal tract

A. Lamprokostopoulou*, C. Monteiro* and U. Römling

*contributed equally

Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, 171 77, Sweden

Cyclic di-GMP (c-di-GMP) is a novel secondary signaling molecule present in most bacteria. Biofilm formation and c-di-GMP signalling are tightly linked, indeed regulation of biofilm formation is a major target for c-di-GMP signalling. We are investigating the impact of biofilm formation and c-di-GMP signalling on virulence and colonisation properties of bacteria in the gastrointestinal tract.

We have studied the effect of c-di-GMP signaling on virulence phenotypes of Salmonella enterica serovar Typhimurium concentrating mainly on the phenotype of invasion of the gastrointestinal epithelium by S. Typhimruium. A di-guanylate cyclase was overexpressed to saturate the cell with c-di-GMP (biofilm-positive phenotype), while a phosphodiesterase was overexpressed to deplete the cell from c-di-GMP (biofilm-negative phenotype). Our results show that under our experimental conditions c-di-GMP signaling, mainly the overexpression of c-di-GMP synthesizing di-guanylate cyclase, has a profound effect on S. Typhimurium phenotypes related to the invasion of the gastrointestinal epithelium. This effect is partially mediated by some of the extracellular matrix components of the biofilm and the major regulator of biofilm formation CsgD.

In contrast to many strains of S. Typhimurium and Escherichia coli, where cellulose production is highly regulated, the probiotic strain E. coli Nissle 1917 produces cellulose constitutively. We could show that cellulose production in Nissle 1917 is dependent of the allosteric activator c-di-GMP. Investigating the biological impact of cellulose production in Nissle 1917, we could show that cellulose production affects adhesion of Nissle 1917 to the gastrointestinal epithelial cell line HT-29. The consequences of the cellulose-dependent adhesion phenotype will be discussed.

Contact e-mail: [email protected]

Novel strategies to control biofilms on medical devices

Gianfranco Donelli

Istituto Superiore di Sanità, Department of Technologies and Health, Rome, Italy.

The development of new technologies of surface coating based on controlled adsorption and/or long-lasting release of antimicrobial agents, represents a promising target for the prevention of medical device-associated infections. In fact, the antiseptic- or antibiotic-loaded medical devices already on the market, mainly suffer from a relatively short persistence of their antimicrobial action as consequence of an early and rapid drug release. On the other hand, the most frequently implicated bugs in device colonisation, such as Staphylococcus, Pseudomonas and Candida spp., are known to exhibit an higher antibiotic resistance due to their sessile mode of growth.

Thus, the increasing number of device-associated infections as well as the higher antibiotic-resistance of causative microorganisms, are critical issues for the management of patients in clinical settings. Therefore, innovative and safer medical devices refractory to microbial adhesion and biofilm formation need to be developed.

To this aim, we recently designed and successfully tested in vitro two different experimental models based on the adsorption on functionalised polymers of: i) pore-forming agents promoting the controlled release of rifampin, cefamandole nafate and fluconazole from polyurethanes;

ii) Dispersin B (a β-N-acetylglucosaminidase produced by the Gram-negative periodontal pathogen Aggregatibacter actinomycetemcomitans), able to dissolve mature biofilms produced by S. epidermidis as well as, when combined with cefamandole nafate, to synergistically promote an enhanced antibiotic killing activity.

The entrapping within our functionalised polyurethanes of antibiotic/antifungal drugs, when needed in conjunction with porogens, as well as of biofilm-dissolving agents alone or combined with antibiotic molecules, seem to offer promising strategies in the prevention of bacterial colonisation and biofilm formation.

Contact e-mail: [email protected]

Screening of molecular markers of quorum sensing in pseudomonas aeruginosa and staphylococcus aureus strains isolated from biofilm associated infections

*Ani Cotar, Mariana-Carmen Chifiriuc, **Otilia Banu, Sorin Dinu, Carmen Iordache, Marcela Bucur, Olguta Dracea, Veronica Lazar

University of Bucharest, Faculty of Biology, Microbiology Immunology Department, Ale. Portocalelor 1-3, Bucharest 060101, Romania

*National Institute for Research in Microbiology and Immunology Cantacuzino, Bucharest, Romania

**Institute for Cardiovascular Diseases Prof. C.C. Iliescu, Bucharest, Romania

Introduction: Biofilm associated chronic infection is a frequent and major complication associated with the use of the medical devices.

The purpose of this study was the molecular characterization of quorum-sensing (QS) systems of 10 Pseudomonas (P.) aeruginosa and 9 Staphylococcus (S.) aureus strains isolated from hospitalized patients with cardiovascular devices associated infections.

Material and methods: The strains were firstly characterized at phenotypic level for the presence of pathogenicity and virulence hallmarks and subsequently analyzed by PCR assay for lasI, lasR, rhlI and rhlR genes encoding for the two QS las and rhl regulatory systems in P. aeruginosa and the global regulatory locus agr, that encodes the QS and the global regulatory loci sar, srrAB, arlRS and rot in S. aureus strains. PCR assays for detection of agrP2 and agrP3, the promoters of agr QS locus were used to demonstrate the presence of a functional agr QS system in the analyzed strains.

Results: All P. aeruginosa and S. aureus strains isolated from biofilm associated infections harbor functional QS systems, known to be implicated in the coordinate expression of bacterial virulence factors of adherent and biofilm forming cells on different substrata, including medical devices. Six S. aureus strains belonged to agr group I and three to agr group III.

Conclusion: This study is demonstrating the necessity of functional QS systems for the initiation of an infectious process by opportunistic bacteria, highlighting their utility in the development of new ecologic anti-infectious strategies, based on the use of QS antagonists.

Contact e-mail: [email protected]; [email protected]

Potential probiotic isolated from dadih for human health promotion

Ingrid S. Surono

SEAMEO-TROPMED RCCN-University of Indonesia,

Salemba Raya 6 Jakarta 10430, Indonesia

Dadih, an Indonesian traditional fermented buffalo milk in Indonesia has been believed to have a beneficial impact on human health due to probiotic properties of the natural lactic acid bacteria (LAB) involved in its fermentation process. In previous studies the adhesion capacity of specific LAB isolates from dadih to intestinal mucus and the ability to inhibit human pathogens and displace them from human mucus showed that L. plantarum IS-10506 is a potential probiotic bacteria. The effect of the most potential strains on immune system as well as viability as fecal lactic acid bacteria in vivo were assessed in Sprague Dauley rats as well as Balb/c mice. Lactobacillus plantarum IS-10506 significantly increased fecal lactic acid bacteria of Sprague Dauley rats as well as Balb/c mice. Fecal sIgA in both Sprague Dauley rats and Balb/c mice was significantly higher as compared to control. A three week intervention before-after study to HIV/AIDS patients showed significant increase of CD4 in probiotic group as compared to control group at 95% confident level. The Lactobacillus plantarum IS-10506 supplemented to the HIV/AIDS subjects was also found in the stool of subjects observed by PCR. As a conclusion, L. plantarum IS-10506 is a potential probiotic bacteria having good properties in modulating immune system.

Keywords: Lactobacillus plantarum, dadih, probiotic, viability, immune system

Contact e-mail; [email protected]

Regulation and scientific substantiation of health claims – europe

Susanne Bryngelsson and Nils-Georg Asp, SNF Swedish Nutrition Foundation,

Ideon Science Park, SE-223 70 LUND, Sweden

A European Community (EC) Regulation on nutrition and health claims made on foods [1] came into force in 2007 and is implemented during a three-year period. There are two principal types of health claims: Article 13-claims including ‘Health claims other than those referring to the reduction of disease risk and to children's development and health’, and Article 14-claims including ‘Reduction of disease risk claims and claims referring to children's development and health’. The Article 13.1-claims shall be based on generally accepted scientific evidence, whereas claims based on newly developed scientific evidence will be handled according to Article 13.5. Use of the terms probiotics or prebiotics will be regarded as health claims in themselves.

Member States have provided the Commission with lists of Article 13.1-claims by 31 January 2008, including suggestions for claims on probiotic microorganisms and substances with perceived prebiotic effects. After consulting the European Food Safety Authority (EFSA) the Commission shall adopt a Community list of permitted claims by 31 January 2010 at the latest.

Both Article 13.5- and Article 14-claims will be subject to a more rigorous scientific evaluation by EFSA. This will be based on extensive applications according to guidance from EFSA [2].

The European Commission concerted action project PASSCLAIM (Process for the assessment of scientific support for claims on foods) [3] developed a set of criteria for the scientific substantiation of claims. As in the guidance from EFSA [2] randomised controlled studies (RCT) are required showing the effect to be claimed.

Some European countries have had voluntary Codes of Practice allowing some health claims to be used. The Swedish Code approved claims related to alleviation of everyday gastrointestinal complaints for two probiotic products, based on studies in IBS patients. Expert reports are available from the Swedish Nutrition Foundation′s homepage (www.snf.ideon.se).

E-mail: [email protected]

Prebiotics and probiotics – rules from regulatory agencies in Japan

Shigeru Kamiya

Department of Infectious Diseases, Kyorin University School of Medicine, Tokyo, Japan

In Japan, probiotics are used as medical drugs, and can be used as foods without claiming medical effects. When probiotics are used as foods, there is no specific restriction. However, fermented milk or lactic acid drinks are required to have a lower limit of lactic acid bacteria.

Twenty-eight kinds of probiotic drugs which are often used for stimulating of intestinal functions and decreasing of severity of diarrheal symptoms are prescribed by medical doctors according to the strict regulation by the Pharmaceutical Affairs Law in Japan. Good manufacturing practice (GMP) is required for the probiotic drugs. These probiotics should be scientifically clarified for their beneficial functions and their safety to human.

If one wants to have efficacy on the label, it is required to obtain special permission from the Ministry of Health, Welfare and Labor (MHWL) in Japan as foods for specific health uses (FOSHU). For FOSHU, certain efficacy claims can be put on the product label. Necessary tests and requirements are listed in the announcement from MHWL.

Foods with health claims (FHC) are grouped in two categories; FOSHU and foods with nutrient function claims (FNFC). FNFC contain nutrients such as vitamins (Vit. A, B1, B2, B6, B12, C, D, E and folic acid) or minerals (Zn, Ca, Fe, Cu, Mg etc). The dose of the nutrient in FNFC should be clearly indicated on the label of the product.

Recently, there have been much more concerns on probiotics and functional foods in not only medical researchers but also ordinary people in Japan. Scientific evaluation of the functions and safety of probiotics and functional foods is required to be performed according to the concept of evidence-based medicine (EBM).

Contact e-mail: [email protected]

Rules concerning probiotics and prebiotics from russia regulatory agencies

Shenderov B.A.*, Sheveleva S.A.

Moscow Research Institute Epidemiology and Microbiology after G.N.Gabrichevsky*;

Institute of Nutrition of Russian Academy of Medical Science

First living bacteria of human origin for restoration of human microbial ecology and for other health claims had been used at the Soviet Union at the first half of last century (“Vagosan”-on the base of vaginal lactobacilli) -1929; E. coli sour milk -1931; Bifidobacterium sour milk -1945; E.coli/Bifidobacterium sour milk -1946; Enterococci sour milk –1952). Industrial production of probiotics was being organized in the USSR during 1960-1982. Dry forms of probiotics were subject to regulation as a drug, fermented milk-as a food under the USSR Government Regulation Acts. During the last 25 years many tens probiotics, prebiotics and synbiotics on the base of selected strains of living bifidobacteria, lactobacilli, propionibacteria, escherichia, enterococci, bacilli, saccharomyces or their cell structural components, metabolites, as well as different oligosaccharides and polysaccharides taken separately or in the complexes in different forms (medicals-tablets, capsules, aerosol, suppositories, oitments, creams, and nutritionals- food supplements, functional and dietary foods) were being developed and produced in Russia.

Now in Russia there are two main state sanitarian rules and norms (2001; 2003) that contain information about Specifications, Quality Assurance and Regulatory Issues of probiotic- and prebiotic containing foods and dietary supplements. Official definitions of probiotic, prebiotic and synbiotic terms were presented in state sanitarian riles (2.3.2.1078-2001 “Hygienic requirements of safety and value of foods”) and Russian National Standard (N 52349-2005) “Functional Foods. Terms and Definitions”.

Two years ago new Draft of National Guidelines for Evaluation of Probiotics were worked out by Russian specialists. This Guidelines take into account both the Russian experience accumulated and data presented in the Draft Guidelines for the Evaluation of Probiotics (Joint FAO/WHO Working Group, London Ontario, Canada, 2002), Draft Guidance for Industry “Complementary and Alternative Medicine Products and Their Regulation by the Food and Drug Administration” (U.S., December 2006) and other International Regulation Rules. Some main principles of the last Russian Draft Guidance will be presented for the discussion.

Contact e-mail: [email protected]

Metchnikoff-a lesson to learn

Tore Midtvedt

MTC, Karolinska Institutet, Stockholm, Sweden

The Russian scientist Elie Metcnikoff (1845–1916, Nobel Prize 1908) was the first to discuss the concept of creating beneficial health effects through ingestion of living bacteria. As a young scientist he has observed that the “tartars” in Eastern Russia had a daily intake of fermented milk and he assumed that this (the term probiotics was not invented) could counteract toxic bacteria in the large intestine. Some years later, he wrote a about his observations and consumption of sour milk became very popular. However, he was not happy with the English translation “The Prolongation of life: Optimistic Studies”. It created an over-optimistic opinion that was not fulfilled. Usage of lactic acid bacteria declined and Metchnikoff died as a disappointed man.

Nowadays we are again observing an increased usage of probiotics worldwide – together with an increasing number of health claims and articles about usage. At May 11, 2008, the number of articles was 4270. In most fields, it is a disproportion between claims and proven facts and one example will be given: The Cochrane Central Register of Controlled Trials investigated trials dealing with the effect of probiotics given to infants for the prevention of allergic disease or food hypersensitivity, and the conclusion was clear: “there is insufficient evidence to recommend the addition of probiotics to infants feeds for prevention of allergic disease or food hypersensitivity”.(CD006475)

Obviously, time has come to take a closer look into many of the claims. Otherwise, the consumption of fermented milk might decline again.

Contact e-mail: [email protected]

Poster abstracts

Effectiveness of ozone against microorganisms colonizing raw vegetables

Stefania Ceciu¹, Athanasios Alexopoulos², Veronica Lazar¹, Eugenia Bezirtzoglou²

¹University of Bucharest, Faculty of Biology, Department of Microbiology, 76201, Bucharest, Romania

²Democritus University of Thrace, Department of Food Science and Technology, Laboratory of Microbiology, Biotechnology and Hygiene. GR68200, Orestiada, Greece

Background: Raw vegetables are usually contaminated by different microorganisms. Post-harvesting the microflora is very different reflecting environmental and human habits for their procedure. Before consumption in ready to eat restaurants, raw vegetables and fruits are immerged in diluted chlorine solutions in an attempt to destroy the colonizing microflora. However, since chlorine producing harmful by-products, ozonation was studied as an alternative approach.

Method: Lettuce and pepper samples were examined for microbial contamination and were plated onto a range of selective media. The microorganisms were enumerated before and after exposure in ozonated and chlorinated solutions.

Results: The total number of microorganisms isolated varied considerably from 2.3 to 4.8 logs CFU/g as well as ozone and chlorine decontamination efficiencies. In average, increased concentrations of ozone reduced the total viable microbial counts by 1.1 logs for every 5 minutes of contact time. Moreover, a loss in the reduction rate was recorder and complete sanitation wasn't observed even after 35 min of exposure. In contrast, chlorine has an immediate effect and a constant reduction rate.

Conclusions: The bacterial growth was directly correlated with the nature of the vegetable and the contamination rate. In all samples, the maximum efficacy of ozone was observed after 30 minutes while exposure for short time periods seems to be inefficient.

The study was sustained by Democritus University of Thrace, Faculty of Agricultural Development, Department of Food Science and Technology, Laboratory of Microbiology, Biotechnology and Hygiene (Greece) and a European ERASMUS Program.

Contact e-mail: [email protected] (Prof E. Bezirtzoglou)

Targeted impact of coffee consumption on gut microbiota

R. Bibiloni¹, I. Rochat¹, M. Jaquet¹, J. Moulin², M. Beaumont², S. Oguey-Araymon², A. Blondel-Lubrano², C. Cavin³

Nestlé Research Centre, ¹Department of Nutrition & Health, ²Department of Bioanalytical Science, ³Department of Quality and Safety, Lausanne, Switzerland

Background: Epidemiological studies indicate an association between coffee consumption and prevention of some disorders like liver disease, type II diabetes, and some types of cancer. A newly developed coffee preparation resulting from a co-extraction of green and roasted coffee beans contains a particular composition of high-molecular-weight carbohydrates. This makes for an attractive source of potentially active oligosaccharides with prebiotic properties. The aim of the study was to assess the impact of a moderate consumption of a coffee product on the general composition of the human intestinal bacterial population.

Methods: Seventeen (17) healthy adult subjects volunteered for this study. They consumed a daily dose of 3 cups of coffee during 3 weeks, after a 3-week period of diet restriction. Faecal samples were collected before and after the consumption of coffee. The impact of the ingestion of the product on the intestinal bacteria as well as the quantification of specific bacterial groups was assessed using nucleic acid-based methods.

Results: Although faecal profiles of the dominant microbiota were not significantly affected after the consumption of the coffee (S_Dice= 92%, n = 16), the population of Bifidobacterium spp. increased after the 3-week test period (P = 0.02). Moreover, in some subjects, there was a specific increase in the metabolic activity and/or numbers of B. adolescentis.

Conclusions: Our results show that the consumption of the coffee preparation resulting from a co-extraction of green and roasted coffee beans produce an increase in the metabolic activity and/or numbers of the Bifidobacterium spp. population, a bacterial group of reputed beneficial effects, without major impact on the dominant microbiota.

Contact e-mail: [email protected]

Gut microbiome modulates hepatic glutathione and drug metabolizing enzymes

Sandrine P. Claus¹, Sandrine Ellero², Yulan Wang¹, Elisabeth Perdu³, Jérôme Molina³, Alain Paris³, Serge Rezzi⁴, Alastair Ross⁴, Isabelle de Waziers², Sunil Kochhar⁴, Elaine Holmes¹ and Jeremy K. Nicholson¹

¹Department of Biomolecular Medicine, SORA Division, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, United Kingdom

²U775-Bases moléculaires de la réponse aux xénobiotiques, INSERM Paris V University of medicine, 75270 Paris Cedex 6, France

³UMR 1089-Xénobiotiques, INRA, BP 3, 31931 Toulouse Cedex 9, France

⁴Nestlé Research Center, Lausanne, NESTEC Limited, Vers-Chez-les-Blanc, 1000 Lausanne 26, Switzerland

Background: In a previous multicompartmental study, we investigated the effect of the germ-free (GF) status on host metabolism in mice. We observed strong modifications of the enterohepatic cycle of bile acids, putatively associated with lower levels of glutathione in livers of GF animals, suggesting a potential impact of the gut microbiome on drug metabolism in liver.

Method: The aim of this study was to investigate liver metabolic perturbations induced by the acclimatization process in GF mice for 20 days. Intact liver metabolic profiles were assessed every 5 days by ¹H NMR technique coupled with pattern recognition methods. Global cytochrome P450 (CYP) activity was assessed by monitoring testosterone oxidative metabolism in liver microsomes. Transcriptomics analyses on selected CYPs, main nuclear receptors and Gclm, the regulatory subunit of the rate-limiting enzyme of glutathione synthesis, completed the enzymatic assay.

Results: Testosterone 6β-hydroxylase activity was significantly reduced in liver of GF animals and did not recover after 3 weeks of colonization. This observation was supported by the lower expression levels of cyp3a11 and cyp2c29 (respectively 40% and 50% of control level) in GF livers. These CYPs have both 6β-hydroxylase activity and are the main CYPs involved in xenobiotic metabolism in mouse liver. Furthermore, liver metabolic profiles revealed a significantly lower amount of reduced glutathione in GF livers that may be attributed to the observed lower expression of Gclm in the same tissues. Glutathione level in ex-GF metabolic profiles recovered after 3 weeks of colonization, a result confirmed by the similar level of Gclm RNA in ex-GF and in conventional livers.

Conclusion: Thus, this original study demonstrates that gut microbiota play an essential role on liver homeostasis and drug-metabolizing capabilities influencing both phase I (CYP) and phase II (glutathione) detoxification reactions.

Contact email: [email protected]

Molecular characterization of lactobacillus helveticus strains producing antihypertensive peptides

Lucia Aquilanti¹, Andrea Carbini², Rebecca Strappati¹, Gloria Silvestri¹, Francesca Clementi¹

¹Department of Food Science (DiSA) and ²Department of Science Applied to Complex Systems (SASC), Polytechnic University of Marche, via Brecce Bianche, 60131 Ancona, Italy

Background: Lactobacillus helveticus is a thermophilic lactic-acid-producing species that is traditionally used as a starter culture in the manufacture of Swiss and Italian cheeses. Recently, the ability of selected L. helveticus strains to hydrolyse caseins and release anti-hypertensive tripeptides that have angiotensin-I converting enzyme (ACE) inhibitory activity has been assessed. This latter trait makes these strains of great interest for the manufacture of fermented milks with blood-pressure-lowering effects.

Method: In the persent study, the relatedness of four L. helveticus isolates of dairy origins producing peptides with ACE inhibitory activities was assessed by analysing their genomes with random primers and by investigating the distribution of six insertion sequences, namely ISLhe1, ISLhe6, ISLhe5, ISL2, IS15807 and IS1201.

Results: The combined approach based on the random, whole- genome analysis and insertion sequence distribution allowed the molecular discrimination of these four dairy isolates. Furthermore, strain-specific molecular markers for the identification of the strains under study and their monitoring during the fermentation process and in the end products have been identified.

Conclusions: The combined use of different typing techniques has allowed the genetic relatedness among L. helveticus strains of technological interest to be revealed.

E-mail address: [email protected]

Glycopeptide antibiotic effects on host immune response

Dennis J. Kitz, Omara Ousley, Sharitha Anderson, Robert Bone, Jennifer Lesko,

Sarah Busalaki, *Sadegh Khazaeli.

Departments of Biological Sciences and *Chemistry, Southern Illinois University Edwardsville, IL 62026-1651 USA

Background: Vancomycin Eli Lilly is a tricyclic glycopeptide that expresses bactericidal activity thru inhibition of cell wall biosynthesis, disruption of cell membrane permeability and blockage of RNA synthesis. Vancomycin is used to treat drug- resistant Staphylococcus aureus and intestinal overgrowth of Clostridium difficile causing pseudomembranous enterocolitis. Dalbavancin Vicuron(now)Pfizer is a lipoglycopeptide derivative of the glycopeptide molecule and approved for treating hospital-acquired infections including those by vancomycin-resistant bacteria.

Methods: A contact sensitizing assay ( J Immunol 112:115, 1974) was used to determine drug effects on T cell mediated (DTH) function. Phagocytic cells (macrophages and neutrophils) were harvested from the peritoneal cavity of mice following thioglycolate elicitation. Organ clearance assays were done by i.v. yeast challenge of mice with or without drug i.p., and mice were later sacrificed to determine yeast CFU in spleen, liver and kidneys.

Results: The glycopeptide molecules had little effect on the killing ability of phagocytic cells. Conversely both drugs boosted murine DTH to DNFB Sigma. Organ clearance assays are in progress and will be reported at the SOMED meeting.

Conclusion: These glycopeptide drugs have become part of the hosts’ last defenses against microbes including drug-resistant S. aureus and the enterococci; perhaps this immune enhancement by drugs may contribute to their successful antimicrobial activity. Supported in part by the NSF-funded LS-AMP Research Scholar's Program.

Contact e-mail: [email protected]

Imidazole effects on murine immune responses

Dennis J. Kitz, Robert Bone, S. Janel Purlee, Lydia Freed, Tara Bradstreet, Jennifer Barton.

Department of Biological Sciences, Southern Illinois University Edwardsville, IL 62026-1651 USA

Background: Antifungal molecules including amphotericin B (Fungizone) Squibb are known to have profound effects on many aspects of host immune response. Our laboratory has extended this study to include two imidazole antifungal molecules: fluconazole (Diflucan) Pfizer and a derivative molecule voriconazole (Vfend) Pfizer. Both azole drugs inhibit cytochrome P450 and 14a-demethylase, an enzyme acting on fungal membrane sterols.

Method: A contact sensitizing assay (J. Immunol 112:115, 1974) employing dinitrofluorobenzene (DNFB) Sigma belly sensitization of shaved mice in groups with or without drug i.p. on day 0,1 of assay followed by ear-painting with DNFB on day 13 and measurements made of ear thickness before and 24,48 & 72hr after ear painting. Histology of these thickened ears are consistent with accumulation of T cells and macrophages as seen with DTH.

Results: Fluconazole enhanced DTH in mice at dosages of up to 6.0 mg total. Fluconazole dosages and time-points for sensitization as related to optimal DTH response was determined. Fluconazole was also found to reverse tolerization of mice to DNFB elicited by prior intravenous injection of DNBSO3 Kodak. Studies on vorconazole's effects on DTH response are in progress and seem to parallel much of our findings with fluconazole.

Conclusions: Our findings suggest that patients receiving these imidazole drugs may not benefit just from their direct antifungal activity, but also benefit from the boost of their T cell and macrophage immune function. This work was supported in part by the Max Baer Heart Fund, Fraternal Order of Eagles (Granite City, IL).

Contact e-mail: [email protected]

Detection and specific enumeration of probiotic lactobacillus acidophilus, lactobacillus rhamnosus and streptococcus salivarius k12 in rat intestinal contents

Hee Ji Lee¹ J. Paul Fawcett¹ Momir Mikov¹ David Orlovich² and John R. Tagg^3,4

¹School of Pharmacy, ²Department of Botany, ³Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand, and BLIS Technologies Limited, ⁴Centre for Innovation, University of Otago, Dunedin, New Zealand

Background: Probiotics increasingly attract commercial interest. Monitoring of probiotic viability in stool samples is important to verify their in vivo survival and persistence. Selective growth on conventional bacteriological media cannot discriminate probiotic strains from normal microflora. In this study, rats were pretreated with three antibiotic-resistant probiotics after which DGGE molecular fingerprinting and plating on antibiotic-containing media enabled specific monitoring of the total and viable populations of all three strains in the intestinal contents.

Methods: Rats were pretreated with a mixture of antibiotic resistant strains of the intestinal probiotics Lactobacillus acidophilus and Lactobacillus rhamnosus and the oral probiotic Streptococcus salivarius K12. Faecal samples were collected before, during and after the treatment and analysed by DGGE and by plating on antibiotic-containing media.

Results: Sequencing of selected DGGE bands appearing post-treatment identified the probiotic strains in faecal specimens. Detection of the probiotic strains on antibiotic-supplemented medium was more sensitive than DGGE and provided differential viable counts of each strain.

Conclusions: Oral treatment of rats with antibiotic-resistant probiotics has shown that intestinal probiotics survive passage through the intestinal tract better than an oral probiotic. The plating method has high sensitivity and specificity for the differential counting of viable cells in probiotic mixtures whereas DGGE detects both viable and non-viable cells.

Contact e-mail: [email protected]

The role of c-di-gmp in virulence phenotypes if salmonella enterica serovar typhimurium

A. Lamprokostopoulou, C. Monteiro, U. Römling

Karolinska Inst., Stockholm, SWEDEN

Background: Cyclic di-GMP (c-di-GMP) is a novel secondary signalling molecule present in most bacteria. Recently, we have investigated the effect of c-di-GMP signalling on biofilm development in Salmonella enterica serovar Typhimurium. The biofilm in S. Typhimurium is characterized by the expression of proteinaceous and polysaccharide extracellular matrix components, which are regulated by CsgD, a transcriptional modulator. Expression of CsgD and the exopolysaccharide cellulose is regulated by c-di-GMP signalling. However, S. Typhimurium is mainly known as a pathogen causing self-limiting gastroenteritis in humans and typhoid fever in immunocompromised mice. It is already shown that c-di-GMP is involved in virulence phenotypes of several bacteria like Pseudomonas aeroginosa and Vibrio cholerae. In this work, we therefore investigate the effect of c-di-GMP signalling on virulence phenotypes of S. Typhimurium

Methods: A di-guanylate cyclase was overexpressed to saturate the cell with c-di-GMP, while a phosphodiesterase was overexpressed to deplete the cell from c-di-GMP. With this system we studied some of the basic phenotypes that are major determinants of Salmonella virulence.

Results: Our results show that c-di-GMP signalling, mainly the overexpression of c-di-GMP synthesizing di-guanylate cyclase, has a profound effect on S. Typhimurium phenotypes related to virulence. CsgD affected S. Typhimurium virulence phenotypes by different pathways, partially involving the expression of extracellul.ar matrix components.

Conclusions: C-di-GMP indeed has a profound effect in S. Typhimurium virulence, which is not exclusively due to one single pathway involving c-di-GMP and CsgD

Contact e-mail:[email protected]

Cytokinic profile as a tool in the investigation of bacterial –eukaryotic crosstalk

Veronica Lazar, Mariana Carmen Chifiriuc, *Coralia Bleotu, Carmen Iordache, Mariana Lixandru, Marcela Bucur, Olguta Dracea, Cristina Larion

University of Bucharest, Faculty of Biology, Microbiology Immunology Department, Ale. Portocalelor 1-3, Bucharest 060101, Romania

*Institute of Virology St. Nicolau, Bucharest, Romania

Introduction: The human opportunistic pathogen Pseudomonas (P.) aeruginosa employs a complex hierarchy of QS signalling systems, which not only coordinate expression of virulence factors by the bacterium, but also have immunomodulatory effects on the host organism, including modulation of proinflammatory cytokines synthesis, phagocytosis and induction of apoptosis.

Purpose: To investigate by ELISA assay the changes of pro/anti-inflammatory status of the immune cells in the presence of the P. aeruginosa native and purified QS molecules, in order to identify the potential functional responses of the mammalian cells and the roles of these molecules in the beneficial or pathogenic effects on host cells.

Results: Eventhough the cytokines patterns of macrophages and lymphocytes were similar, however the human lymphocytes responded more intensively than macrophages to the tested microbial products. The cell free supernatants of heat–inactivated P. aeruginosa logphase cultures reproduced the cytokine profile induced by purified mediators in concentrations similar to those reported to be accumulated in log phase cultures, inducing a slight anti-inflammatory effect (stimulating the anti-inflammatory IL-10 and inhibiting the main pro-inflammatory IL-1-alpha).

Conclusion: This study demonstrates the ability of bacterial soluble QS signal molecules to modulate the expression of a variety of cytokines and inflammatory molecules produced by the immune cells and the necessity to find QS inhibitors.

Contact e-mail: [email protected]; [email protected]

The influence of usnic acid on the p. aeruginosa and s. aureus biofilms development

Veronica Lazar, Mariana Carmen Chifiriuc, Eliza Oprea, Marcela Bucur, Mirela Iacob, Cristina Larion, Osakwe Chiukwunomso Ekene, Cornelia Cercasov

University of Bucharest, Faculty of Biology, Microbiology Immunology Department, Ale. Portocalelor 1-3, Bucharest 060101, Romania

University of Bucharest, Faculty of Chemistry, Organic Chemistry Department, 4-12 Regina Elisabeta, Bucharest 030018, Romania

Introduction: The pathogenicity of biofilms formed by opportunistic pathogens is magnified by the increased resistance or tolerance to antimicrobials. It is already known that some lichens, algae and plants are producing soluble metabolites interfering with QS mechanisms developed by sessile bacteria grown in biofilms. Usnic acid, a secondary lichen metabolite, possess antimicrobial activity against a number of planktonic bacteria, and, as many other secondary lichen metabolites, offers protection to lichen communities against the adherent microorganisms.

The purpose of this study was to investigate the efficiency of usnic acid on the P. aeruginosa and S. aureus biofilm development on inert substrata (plastic wells, cardiovascular catheters) and cellular (HeLa cell line) substrata.

Material and methods: The usnic acid was extracted from the lichen Usnea barbata and tested by two simple models for the development of artificial, monospecific biofilms in liquid and solid media were used: 1) the bacterial cells were immobilized in an agar matrix similar to the natural biofilm polysaccharidic mathrix, adapted for the study of biofilm sensitivity to different antimicrobial products; 2) the biofilms were grown in microplates with/without catheter sections and different usnic acid concentrations, the biofilms being thereafter fixed, colored, resuspended and quantified by spectrophotometry.

Results: Although a quantifiable antimicrobial activity against planktonic Gram-negative bacteria was not observed, usnic acid inhibited the development of both P. aeruginosa and S. aureus in biofilms on all tested substrata, rendering them sensitive to usual doses of antimicrobials, such metabolites being studied as QS inhibitors.

Contact e-mail: [email protected]; [email protected]

Helicobacter pylori and lactobacilli in the human stomach

Mathilda Lindberg^1,2*, Stefan Roos³*, Hans Jonsson³, Cecilia Jernberg¹, and Lars Engstrand^1,2

¹Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden; ²Department of Microbiology, Cell and Tumor Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden ³Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden

*ML and SR contributed equally to this work

Background and methods: 170 non-symptomatic individuals were screened for lactobacilli and Helicobacter pylori by culturing of stomach biopsies. A subset of individuals was re-endoscoped after 4 years. To determine if clonally related lactobacillus strains were present at both occasions Repetitive Sequence Based PCR was used. Such strains were further examined for binding to human gastric mucus and survival at low pH.

Results: Lactobacilli were found in 20% of the individuals and H. pylori in 24%, co-existence was found in 9%. 13 different lactobacilli species were identified and all co-existed with H. pylori. No decrease in inflammation could be seen when lactobacilli were present. We found four clonally related strains, two of those showed mucus binding and survived at low pH. The binding increased at pH 2.5 compared to pH 6. In comparison H. pylori had an increase in binding at pH 6. When adding H. pylori and lactobacilli simultaneously to the mucus both bacteria showed significant decrease in binding.

Conclusions: Lactobacilli seem to be able to survive or colonize the human stomach by binding to gastric mucus. Furthermore, H. pylori and lactobacilli can co-exist in the same stomach however they negatively impact each others binding capabilities.

Contact e-mail: [email protected]

Impact of colon bacteria on dietary fiber: results from a model system

Steven McCommas, Elizabeth Alton, Jeremy Benedick, Keith Robinson, Shaival Patel, Joel Kruse, and Melissa Kelley

Dept. of Biological Sciences, Southern Illinois Univ. Edwardsville, USA

Background: Dietary soluble fiber may be beneficial in lowering risks of colon cancer, but normal colon bacteria might mutate and be selected for increased ability to metabolize soluble fiber molecules, thus reducing their protective effect. We are studying this possibility using a greatly simplified model system.

Method: Our model system uses E. coli and galactose as the “novel” carbohydrate. We have isolated 8 mutant strains of wildtype MG1655 which are distinguishable as white colonies on MacConkey plates. Our basic experiment is to compete wildtype and mutant strains in liquid culture with galactose being the only carbon source. Periodically, samples are plated on MacConkey medium and transferred to fresh galactose medium.

Results: The relative numbers of both strains in the competition experiment is determined by the numbers of red (wildtype) and white (mutant) colonies on MacConkey plates. Although these experiments are started with a slight preponderance of wildtype bacteria, after several passages the mutants greatly outnumber, or even replace, the wildtype.

Conclusions: The idea that mutant gut bacteria could arise which would be better at utilizing dietary fiber for food and ultimately replace their wildtype ancestors is supported by our initial findings with our simple model system.

Contact e-mail: [email protected]

Competition for proline between indigenous Escherichia coli and E. coli O157:H7 in gnotobiotic mice associated with infant intestinal microbiota and its contribution to the colonization resistance against E. coli O157:H7

Yoshika Momose, Kazuhiro Hirayama and Kikuji Itoh

Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

Background: Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to E. coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 mice became carriers.

Methods: Concentrations of amino acids in the caecum of GB-3 and GB-4 mice were measured by HPLC. Proline determination in the test medium was carried out by reacting with isatin. Competition for amino acids between intestinal microbiota of GB-3 and GB-4 mice and E. coli O157:H7 was examined in vitro.

Results: The concentrations of amino acid, especially proline, in the caecal contents of GB-3 were different from those of GB-4. The supplementation of proline into GB-3 caecal suspension decreased the lag time of E. coli O157:H7 growth in vitro. When E. coli O157:H7 was cultured with each of the strains used to produce GB-3 mice in vitro, 2 strains of E. coli (proline consumers) out of 5 enterobacteriaceae strains strongly suppressed E. coli O157:H7 growth and the suppression was attenuated by the addition of proline into the medium.

Conclusions: Competition for proline with indigenous E. coli affected the growth of E. coli O157:H7 in the intestine and may contribute to E. coli O157:H7 elimination from the intestine.

Contact e-mail: [email protected]

Role of cellulose production in the probiotic strain nissle 1917

C.Monteiro, X.Wang, D.Nobles, R. M. Brown and U.Römling

Karolinska Institute, Stockholm, Sweden

Introduction: The beneficial effects of the probiotic microorganisms are still poorly understood, they may result from complex interactions between the microorganisms, the intestinal microflora and the gut epithelium. Nissle 1917, is one of the best studied probiotics and seems to be very effective in the treatment of several gut pathologies like: ulcerative colitis and Crohn's disease. It is known that some E.coli strains produce cellulose as an extracellular matrix component. In this work we investigated the biological functions of cellulose in the interaction between Nissle 1917 and an epithelial cell line.

Methods: To detect cellulose expression Congo Red plates and Electron microscopy were used. The role of cellulose in the interaction between the probiotic strain Nissle 1917 with the intestinal epithelial cell line HT-29 was studied.

Results: Our results show that Nissle 1917 produces cellulose. We show that IL-8 production is reduced in the cellulose knockout strain while invasion seems to be enhanced. In addition adherence is very much reduced in the absence of cellulose.

Conclusions: Cellulose production is a conserved feature in Nissle 1917 and moreover we show that cellulose production alters the interaction of Nissle 1917 with HT-29 cells.

Contact e-mail: [email protected]

Asymptomatic inflammatory prostatitis: prevalence and etiology

Paul Korrovits^1,2, Silver Türk¹, Kristo Ausmees², Margus Punab², Reet Mändar¹

¹Department of Microbiology, University of Tartu, Estonia; ²Andrology Centre, Tartu University Hospital, Estonia

Objectives: To determine the prevalence of asymptomatic inflammatory (NIH category IV) prostatitis (AIP), and to prove the need for quantitative full-microflora semen analysis for determining the role of microorganisms in its etiology.

Material and methods: In 565 young men cytologic examination of semen, analysis for basic semen parameters, prostate-specific antigen (PSA) in serum and IL-6 in seminal plasma were performed. In 37 men with AIP and 32 controls microbiological analysis of semen was performed.

Results: The prevalence of AIP was 6.0% (>10⁶ WBC/ml, according to WHO guidelines). When we used lower threshold suggested by our previous studies (>0.2×10⁶ WBC/ml), the prevalence was 19.0%. IL-6 and PSA levels were significantly higher in persons with AIP than those without. One to 8 different species were found per semen sample, and their total concentration ranged from 2.0 to 7.5 log₁₀ CFU/ml. Both parameters were significantly higher in AIP patients than controls (median 5 vs 3, P = 0.004; median 4.8 vs 3.9 log₁₀ CFU, P < 0.001). In most of the specimens, the counts of anaerobic bacteria were equal to or outnumbered the aerobic ones.

Conclusions: AIP has a notable prevalence among healthy young males. Detection of IL-6 and/or PSA serves as additional tool for diagnosing AIP. Unlike the controls the AIP patients harbour abundant polymicrobial microbiocenosis in their semen, containing anaerobic, microaerophilic and aerobic bacteria.

Contact e-mail: [email protected]

Changes of viability and composition of the escherichia coli flora in fecal sampples during long time storage

Roland Möllby, Sara Achá & Inger Kühn

MTC, Karolinska Institutet, Stockholm, Sweden

Background: Long-time storage of faecal samples is necessary for investigations of intestinal micro floras. The aim of the present study was to evaluate how the viability and the composition of the Escherichia coli flora are affected in faecal samples during different storage conditions.

Methods: Fresh faecal samples from calves and infants were divided into sub-samples and stored in four different ways: with and without addition of glycerol broth at −20°C and at −70°C. The viability and the phenotypic diversity of the E. coli flora in the sub-samples were evaluated after repeated thawings and after storage during 1 year. The samples stored for one year without thawing were also kept at room temperature for five days and subsequently analysed.

According to phenotyping of 32 isolates per sample on day zero, all samples contained two dominating strains of E. coli each, and between one and eight less common strains.

Results: Samples stored at −70°C in glycerol broth showed equal or even higher bacterial numbers as the original samples, even after repeated thawings, whereas samples stored at −20°C showed a considerably lower survival rate, also with addition of glycerol. Sub-samples containing glycerol broth that were kept at room temperature after storage for one year showed a clear increase in viability as well as in diversity. The diversities in each sub-sample showed a tendency to decrease after several thawings as well as after storage.

Conclusion: We conclude that for long time storage of faecal samples, storage at −70°C is preferable. If samples have to be thawed repeatedly, addition of glycerol is preferable both for samples stored at −70°C and for samples stored at −20°C.

Contact e-mail: [email protected]

Intestinal metabolism of cholesterol in children with cows-milk allergy-the infabio study

Anna-Karin Persson, Agneta Uusijärvi, Johan Alm and Elisabeth Norin

Department of Microbiology, Tumor and Cell Biology and Department of Clinical Science and Education, Section for Pediatrics, Sachs′ Children's Hospital, Karolinska Institutet

Background: The prevalence of allergy in children has until recently increased markedly, especially in children. Cow's milk protein allergy is one of the earliest manifestations of allergy. In Sweden, approximately 2% of children develop allergy to cow's milk protein. The aim of the present study was to evaluate if there were any biomarkers of functionally active microbes in fecal samples that differed between the children with newly diagnosed cow's milk allergy and sex and age matched controls.

Method: Twenty-six Swedish cow's milk allergic children, 16 males and 10 females – aged 6,7 months and 31 controls matched according to age and sex were identified at Sachs′ Children's Hospital, and included in the study. Frozen fecal samples were analyzed for intestinal conversion of cholesterol to coprostanol using gas-chromatography.

Results: Preliminary results show that twenty-one (81%) of the cow's milk allergic children had already established a flora with functionally active microbes able to convert cholesterol to coprostanol, in contrary to what was found in the controls, where no child had coprostanol present in the fecal sample at the same age. The difference was statistically significant, p = 0.000-Mann-Whitney Test.

Conclusion: Establishment of some functionally active microbes differed between these two groups of children. In healthy children this function is earlier shown to be established later. The cause and the consequence of these differences have to be further evaluated.

Contact e-mail: [email protected]

Polypropylene reuseable shipping container for defined flora non-laboratory animals

Razzaboni F¹, Fitzgerald S²

¹Park Bioservices, LLC, Groveland, MA, USA, ²Millennium Plastics Inc. Groveland, MA, USA

Background: Shipping traditional gnotobiotic laboratory animals has become facile and the supplies necessary are readily available from commercial breeders and other vendors. Shipping non-traditional laboratory species such as gnotobiotic cats presents a challenge to find a suitable shipping container. Commercial breeders, laboratory staff and university research staff have been left to their own device to find appropriate shipping vessels. Further complicating the shipping scenario are stringent local government, EU, North American and IATA regulations and requirements. To be economically feasible and environmentally sound a reusable, easily disinfected and inexpensive container was sought by a research department of a commercial animal diet company.

Method: Polypropylene was selected as an ideal material. Polypropylene is light weight, cleanable, can be formed and welded into the appropriate configuration. IATA regulations specify the area needed for various species (the animal must be able to freely turn around) and also fixes the amount of open space needed in the walls to allow for ventilation. The four side walls were fabricated with a perforated section and a frame to sandwich layers of filter media (DW4 and a new non-woven rigid filter media (unicore S250 air permeability 330 cfm/ft²).

Results: A prototype transport container was constructed that successfully transported cats by air carrier and were then disinfected with a cold sterilant and readied for reuse. Conclusion: It is possible for a commercial or academic entity to work with a isolation technology specialist and a plastic fabricator to build a transport container that could be reused multiple times with an initial cost well under $1000 USD. Whereas the number of reuses possible are very high he cost per use is very affordable even for cash strapped university budgets.

The core facility for germfree research (cfgr)

Annika Samuelsson, Britta Björkholm, Joseph Rafter, Alexandra Are, Velmurugesan Arulampalam, Gediminas Greicius, Shugui Wang,Sebastian Pott, Chek Mei Bok, Linda Aronsson, Yinghui Li and Sven Pettersson

Karolinska Institutet, MTC/CFGR

Microbiota, host function and utilization of animal models. In the scientific community, there is a renaissance of interest in the role of the gastrointestinal microbial flora in maintenance of health and to sustain unwanted effects of potentially harmful bacteria. This awakening stems from the accumulated reports and expanding evidence that the gut microbiota can impose beneficial effects on local chronic inflammatory processes as well as impact on the growing organism. The more recent data indicate that gut flora appear also to be able to exert hitherto unsuspected systemic effects, on the liver and adipose tissue, angiogenesis, development of allergic disorders and lately also on cognitive functions. Thus, a substantial part of the gut microbiota is still to be described and its relevance and impact on host's normal physiology are waiting to be explored. Resident bacteria outnumber human somatic and germ cells tenfold and represent a combined microbial genome well in excess of the human genome. KI have a unique platform for the handling and derivation of mice kept under germ-free conditions. In its present form it is organized like a core facility in order to assist the scientific community within KI as well as across the world. The access to genome information, high through put technology has dramatically changed the utilization of this facility. In addition, gnotobiotic models has opened for sophisticated studies to investigate underlying molecular mechanisms of diseases connected chronic inflammation (IBD, Psoriasis, SLE), metabolic disorders like type II diabetes and obesity. In addition, accumulating evidence also connects the gut microbiota to participate in chronic inflammation and colorectal cancer. The facility can tailor make germ free lines upon request and to a competitive price. Control animals kept under stringent SPF conditions is enclosed in the generation of lines. The access to cryogenic freezing capacities facilitates this expansion and we are new able to take orders by shipment of frozen embryos. Assistance in experimental planning and execution of experiments is available and access to mouse pathologist, simpler tissue analysis and a state of the art molecular biology laboratory is available at KI.

Contact e-mail: [email protected], [email protected]

Immunohistochemcal analysis of colitis developing in SCID mice reconstituted with CD4 + CD45RB^high T cells. The effect of intestinal bacteria

R. Stepankova¹, H. Kozakova¹, F. Powrie³, O Kofronova², T. Hudcovic ¹, T. Hrncir¹, H. Tlaskalova-Hogenova¹

¹Department of Immunology and Gnotobiology and ²Laboratory of Electro Microscopy, Institute of Microbiology, Prague, Czech Republic, ³Sir William Dunn School of Pathology, University of Oxford, UK

Background: The aim of work was to analyze the effect of gut bacteria on the development of of colitis in transfer model.

Method: The subpopulation of T cells CD4 + CD45RB^high was purified from spleen of conventional BALB/c mice by magnetic separation (MACS) and cells were transferred. i.p. into immunodeficient SCID mice. Recipient SCID mice were colonized by defined cocktail of bacteria isolated from SPF (Specific Pathogen Free) mice (Oxford Laboratory). Other recipient SCID mice were monoassociated with SFB (Segmented Filamentous Bacteria) bacteria; control SCID mice were either germfree or conventional. 8-12 weeks, after the cell transfer morphological changes in colon and terminal ileum were evaluated by histology (H&E, PAS-Schiff) and scanning electron microscopy. Immunohistological analysis of expression of α-defensin and TJs structural proteins (ZO-1 and Claudin -1) on ileal mucosa was performed. In situ hybridization (FISH) using 16S rRNA oligonucleotide probes was performed to detect bacteria attached to mucosal layer.

Results: We found in inflammated colon decreased production of mucus by goblet cells as an important feature of intestinal inflammation. Simultaneously impairment of tight junctions (TJs) detected by changes in ZO-1 and Claudin-1 expression was found in terminal ileum. We did not detect α- defensin (Cryptdin- 4) expression on inflammed ileal mucosa.

Conclusions:Colitis was present only in SCID recipients colonized by defined SPF cocktail microflora plus SFB bacteria. Monoassociation of SCID mice with SFB, Enteroccus faecalis, Fusobacterium mortiferum and Bacteroides distasonis and after the transfer T cells CD4 + CD45RB^high did not lead to intestinal inflammation. The principal inflammatory changes were found in terminal ileum of SCID mice with colonized SPF + SFB bacteria or in conventional mice.

Dihydrodaidzein-producing clostridium-like intestinal bacterium, strain tm-40: effects on IN VITRO metabolism of daidzein by fecal flora from human male equol producers and non-producers

Motoi Tamura^a,*, Hiroyuki Nakagawa^a,, Mayumi Ohnishi-Kameyama^a, Kazuhiro Hirayama^b, Kikuji Itoh^b

^aNational Food Research Institute, Tsukuba 305-8642, and ^bLaboratory of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

Background: Much attention has been focused on the biological effects of equol, a metabolite of daidzein produced by intestinal flora. Recently, we isolated a dihydrodaidzein (DHD)-producing Clostridium-like bacterium, strain TM-40, from human feces. We investigated the effects of strain TM-40 on in vitro daidzein metabolism by human fecal flora from two male equol producers and two male equol non-producers.

Method: We selected the feces from four adult men (from 21 to 45 years old) previously checked for equol productivity. Feces from the male equol producers and non-producers were collected and immediately used for in vitro incubation of daidzein with the fecal flora. Daizein, DHD and equol were measured by HPLC.

Results: In the fecal suspension from the male equol non-producers (DHD non-producer or DHD producer), DHD was detected by adding a high concentration of the DHD-producing bacteria strain TM-40. In the fecal suspension from the low and high equol producers, in vitro fecal equol production was increased by adding strain TM-40. The equol/daidzein ratio increased with the increased concentration of strain TM-40.

Conclusions: Studies on the interactions among the equol-producing flora and DHD-producing bacteria might lead to clarifying some regulating mechanisms of equol production by fecal flora.

Contact E-mail: [email protected]

“The Self Centred Bug”- The Possible Role of Enteric Short Chain Fatty Acids on the Pathogenesis and Characteristics of Autism Spectrum Disorders

Derrick F. MacFabe

Director-The Kilee Patchell-Evans Autism Research Group, Departments of Psychology and Psychiatry (Division of Developmental Disabilities), University of Western Ontario, London, Canada, N6A 5C2. ([email protected])

Autism spectrum disorders (ASD) are an increasing problem in Western society. Recent research is redefining ASD as a systemic encephalopathic condition involving immune, digestive and metabolic dysfunction exacerbated by environmental triggers in genetically sensitive subpopulations. Clinical observations suggest that certain gut and dietary factors may transiently worsen symptoms in ASD. Propionic acid (PPA) is a short chain fatty acid and an important intermediate of cellular metabolism. PPA is also a fermentation by-product of a subpopulation of opportunistic enteric bacteria (i.e clostridia), a putative risk factor for ASD, and is also a common food preservative. PPA and other short chain fatty acids (i.e butyrate and acetate), effect diverse physiological processes such as cell signaling, neurotransmitter synthesis and release, mitochondrial function, cholesterol synthesis, immune function, and gene expression. We are examining the behavioral, electrophysiological, neuropathological, biochemical and epigenetic effects of PPA and related compounds in a novel rodent model of ASD. Intraventricular infusions of PPA in adult rats induces predictable and enduring changes on brain neocortical, hippocampal and striatal electrical activity. PPA infusion immediately produces reversible repetitive dystonic behaviors, hyperactivity, turning, retropulsion, caudate spiking, the progressive development of limbic kindled seizures, and impairs social behavior, suggesting that this compound has central effects. Examination of brain tissue from PPA treated rats reveals an innate neuroinflammatory response (reactive astrogliosis and activated microglia), an increase in oxidative stress markers, and a reduction of glutathione, a broad spectrum xenobiotic detoxifier. Collectively, these central effects of PPA suggest a possible link between diet, seizure, movement disorder, social impairment, neuroinflammation, increased oxidative stress, mitochondrial dysfunction and environmental sensitivity found in ASD. We propose that some types of ASD may be partial forms of genetically inherited or acquired disorders of altered short chain fatty acid metabolism, resulting in increased exposure to these enteric metabolites at critical times during the life cycle. This study also suggests the novel concept that gut bacteria may have evolved to utilize production of these compounds to influence behavior in the host to ensure survival.