DISCOVERY OF NEXT GENERATION PROBIOTICS FOR HUMAN HEALTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0209576
• Project Start Date: May 1, 2012
• Project End Date: Dec 31, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Microbiology & Molecular Genetics

Non Technical Summary
THIS IS AN END DATE CHANGE ONLY! THIS PROJECT NEEDS TO BE TERMINATED EARLY DUE TO THE PD LEAVING THE UNIVERSITY. IT IS URGENT THIS END DATE CHANGE BE APPROVED QUICKLY AS THE PD'S FINAL REPORT CANNOT BE SUBMITTED UNTIL THE END DATE CHANGE HAS BEEN APPROVED!The reduced efficacy of antibiotics, coupled with the increase in diseases with an intestinal microbial community dysbiosis, indicates there is an increased need for probiotic therapy. This highlights a need to explore the use of probiotics in ameliorating a variety of diseases. Although many probiotic strains are available on the market today, most were not selected for their ability to cure or alleviate the symptoms of human disease. The projects highlighted here are aimed at identifying and characterizing next-generation probiotics that are targeted for specific human diseases. If successful we will be bringing to the public valuable alternative therapies for the betterment of human health.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
724	4010	1100	100%

Knowledge Area
724 - Healthy Lifestyle;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

probiotics

microbiota

clostridium difficile

lactobacillus reuteri

Goals / Objectives
THIS IS AN END DATE CHANGE ONLY! THIS PROJECT NEEDS TO BE TERMINATED EARLY DUE TO THE PD LEAVING THE UNIVERSITY. IT IS URGENT THIS END DATE CHANGE BE APPROVED QUICKLY AS THE PD'S FINAL REPORT CANNOT BE SUBMITTED UNTIL THE END DATE CHANGE HAS BEEN APPROVED!Objective 1. Probiotic amelioration of bone loss in various disease models. Our long-term goal is to understand how probiotic Lactobacillus reuteri can suppress bone loss in response to menopause, diabetes, aging, and inflammatory bowel disease. Utilizing mouse models and in vitro approaches we will attempt to uncover the mechanisms by how probiotic bacteria impact bone health with the ultimate goal of bringing these therapies to people for the improvement of osteoporosis. Objective 2. Identification of next-generation probiotics effective against enteric disease. Our long-term goal is to understand the contribution of the microbial community to gastrointestinal health and disease. In this objective we will use high-throughput human fecal bioreactors to identify key characteristics and functions of pathogen resistant and susceptible communities. Using this knowledge we will identify individual strains of bacteria that are effective at inhibiting a variety of pathogenic organisms from invading the intestinal tract. We expect to present this work at international meetings and publish in peer reviewed journals.

Project Methods
THIS IS AN END DATE CHANGE ONLY! THIS PROJECT NEEDS TO BE TERMINATED EARLY DUE TO THE PD LEAVING THE UNIVERSITY. IT IS URGENT THIS END DATE CHANGE BE APPROVED QUICKLY AS THE PD'S FINAL REPORT CANNOT BE SUBMITTED UNTIL THE END DATE CHANGE HAS BEEN APPROVED!Objective 1. Probiotic bacteria often display systemic effects, often far from the intestinal site they were delivered. While this may seem counterintuitive, recent work indicates that secreted factors from the intestinal microbiota can enter the bloodstream and may be delivered to other sites in the body. Because our preliminary data suggests that increased bone density is likely due to increased bone formation rather than bone resorption, we will utilize a human osteoblast cell line to study the effects of L. reuteri on osteoblast activation. Several parameters can be easily monitored, including osteocalcin expression and secretion, bone matrix production and bone matrix mineralization. Increases in one or more of these parameters will be consistent with a direct effect of a factor produced by L. reuteri that is stimulating osteoblast activity. This will be coupled with the assessment of L. reuteri in animal models of osteoporosis to identify mechanisms active in vivo and in vitro. Objective 2. This study will utilize human fecal bioreactors in which the microbial community is generated from a fecal slurry obtained from healthy volunteers. People will be required to not have had antibiotic use or a diarrheal episode in the previous six months. Fecal samples will be screened for C. difficile prior to use in the study. Initially we will pool several fecal samples to create a uniform sample with which to populate the bioreactors. Although it may be interesting to probe individual variation of fecal microbiotas from healthy volunteers we are not pursuing this line of investigation at this time. Once the bioreactors are populated with a human fecal microbiota we will assess how long the reactors take to stabilize. Stability will be monitored by the analysis of volatile fatty acids. In general stabilization of the community takes approximately 2-3 weeks. Once stabilized, communities will be challenged with C. difficile spores, ranging from 102-107 spores/ml. Several strains of C. difficile are present in Dr. Britton's laboratory and are available for testing including two commonly used laboratory strains, CD630 and CD196. Additional clinical isolates from the NAP1 lineage (hypervirulent C. difficile) and non-NAP1 strains have been obtained and characterized. Spores from each of these strains have been purified and are available. C. difficile vegetative cells and spores will be monitored in the bioreactors daily. The appearance of C. difficile vegetative cells and an increase in total cell numbers will indicate that C. difficile has invaded the community. Production of toxins A and B, which participate in disease, will be monitored by ELISA and quantitative RT-PCR. Dr. Britton's laboratory has developed primers for the quantitative monitoring of tcdA (toxinA) and tcdB (toxin B) mRNA production.

Progress 05/01/12 to 12/31/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two graduate students and two postdoctoral researchers were provided with professional development opportunities such as various types of workshops hosted by the graduate school. In addition, a subset of these individuals attended an international conference and presented work there. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Dr. Britton moved to Baylor College of Medicine on Aug. 1, 2014, so these comments were provided by Interim Chair Robert Hausinger. Dr. Britton has two major accomplishments during the project period as summarized in the following two abstracts: Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post-menopausal bone loss. Recent studies suggest an important role for gut-bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 (L. reuteri) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri-treated mice. Consistent with this, L. reuteri suppressed Ovx-induced increases in bone marrow CD4+ T-lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identified that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost-effective approach to reduce post-menopausal bone loss. Osteoporosis can result from intestinal inflammation, as is seen with inflammatory bowel disease. Probiotics, microorganisms that provide a health benefit to the host when ingested in adequate amounts, can have anti-inflammatory properties and are currently being examined to treat inflammatory bowel disease. Here, we examined if treating healthy male mice with Lactobacillus reuteri ATCC PTA 6475 (a candidate probiotic with anti-TNFα activity) could affect intestinal TNFα levels and enhance bone density. Adult male mice were given L. reuteri 6475 orally by gavage for 3×/week for 4 weeks. Examination of jejunal and ileal RNA profiles indicates that L. reuteri suppressed basal TNFα mRNA levels in the jejunum and ileum in male mice, but surprisingly not in female mice. Next, we examined bone responses. Micro-computed tomography demonstrated that L. reuteri 6475 treatment increased male trabecular bone parameters (mineral density, bone volume fraction, trabecular number, and trabecular thickness) in the distal femur metaphyseal region as well as in the lumbar vertebrae. Cortical bone parameters were unaffected. Dynamic and static histomorphometry and serum remodeling parameters indicate that L. reuteri ingestion increases osteoblast serum markers and dynamic measures of bone formation in male mice. In contrast to male mice, L. reuteri had no effect on bone parameters in female mice. Taken together our studies indicate that femoral and vertebral bone formation increases in response to oral probiotic use, leading to increased trabecular bone volume in male mice.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Britton RA, Irwin R, Quach D, Schaefer L, Zhang J, Lee T, Parameswaran N, McCabe LR. (2014). Probiotic L. reuteri treatment prevents bone loss in a menopausal ovariectomized mouse model. J Cell Physiol. 229(11):1822-30
• Type: Journal Articles Status: Published Year Published: 2013 Citation: McCabe LR, Irwin R, Schaefer L, Britton RA. (2013). Probiotic use decreases intestinal inflammation and increases bone density in healthy male but not female mice. J Cell Physiol. 228(8):1793-8

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two graduate students are participating in these projects with two postdocs actively being recruited. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We plan on continuing to explore both of these research areas.

Impacts
What was accomplished under these goals? There are two projects underway for this particular proposal. In the area of probiotic amelioration of osteporosis we were able to obtain NIH funding to pursue bone loss in a menopause model and we have one manuscript under review in this area. In the second project we are actively screening bacteria isolated from the human body for key activities that would impact obesity and type II diabetes. We have nothing to report at this time for this project other than assays are underway.

Publications

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: In the past year we have made significant strides in the identification of next generation probiotics for the treatment of bone health and obesity. First, we have developed recombineering technology in Lactobacillus reuteri and Lactococcus lactis that will enable us to perform powerful genetic assays to identify key probiotic features in these organisms. We have now generated 135 mutants that are defective in expressing cell surface or secreted proteins, which are those most likely to be modulating host functions. Second, we have demonstrated that L. reuteri 6475 has a positive effect on bone health in three different models of osteoporosis in mice. Lastly, we are isolating and cataloguing over 2000 intestinal bacterial isolates from healthy human volunteers to be screened for beneficial properties in obesity, type II diabetes, and antibiotic associated diarrhea. The first two findings were presented at international meetings during the past calendar year. PARTICIPANTS: Laura McCabe. Professor of Physiology, Michigan State University Nara Parameswaran. Associate Professor of Physiology, Michigan State University Darin Quach, DO/PhD candidate. Attended an international conference to present his work on probiotic effects on bone health. Jan Peter van Pijkeren. Postdoctoral Associate. Attended international conference to present his work on recombineering. Recently accepted a faculty position at the University of Wisconsin. Jane Zhang. Postdoctoral Associate. Attended two international conferences to present her work on probiotic effects on bone health in diabetes. TARGET AUDIENCES: People that suffer from osteoporosis. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Recombineering technology was used to identify histamine as a key anti-inflammatory component of L. reuteri. We were able to knockout the genes responsible for histamine production and show the organism was reduced in its ability to suppress TNF production in activated monocytes. The finding that L. reuteri can suppress bone loss in a number of osteoporosis models has provided enough insight into how an ingested bacterium can affect bone health that we are now attempting to pursue studies in humans.

Publications

• 3. Gut-bone signaling: a link between type 1 diabetes and osteoporosis. Jing Zhang , Regina Irwin, Robert Britton, Narayanan Parameswaran, Laura R. McCabe. American Society of Bone and Mineral Research 2012.
• 4. Probiotic treatment prevented type 1 diabetic bone loss by restoring dysregulated calcium metabolism J. Zhang, R. Irwin, R. Britton, LR McCabe. American Society of Bone and Mineral Research 2012.
• 5. Elucidating key features required for optimized recombineering in Lactobacillus reuteri ATCC PTA 6475. Jan-Peter van Pijkeren, Kar Mun Neoh and Robert A. Britton. American Society for Microbiology Beneficial Microbes Conference 2012.
• 1. van Pijkeren, JP and Britton RA. (2012). High-efficiency recombineering in lactic acid bacteria. Nucleic Acids Research. 2012 May 1;40(10):e76. Epub 2012 Feb 10.
• 2. Thomas, C.M., Hong, T., van Pijkeren, J.P., Hemerajata, P., Trinh, D.V., Hu, W., Britton, R.A., Kalkum, M., and Versalovic, J. (2012). Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling. PLoS One. 2012;7(2):e31951. Epub 2012 Feb 22.
• 3. van Pijkeren JP, Neoh KM, Sirias D, Findley AS, Britton RA. (2012) Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri. Bioengineered Bugs. ( Jul 1;3(4):209-17. doi: 10.4161/bioe.21049. Epub 2012 Jul 1. PMID: 22750793 [PubMed - in process]
• 4. De Weirdt R, Crabbe A, Roos S, Vollenweider S, Lacroix C, van Pijkeren JP, Britton RA, Sarker S, Van de Wiele T, Nickerson CA. (2012). Glycerol supplementation enhances L. reuteri's protective effect against S. Typhimurium colonization in a 3-D model of colonic epithelium. PLoS One. ;7(5):e37116. doi: 10.1371/journal.pone.0037116. Epub 2012 May 31. PMID: 22693569.
• 5. Britton RA, Young VB. (2012). Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance. Trends Microbiol. 2012 Jul;20(7):313-9. doi: 10.1016/j.tim.2012.04.001. Epub 2012 May 15. Review. PMID: 22595318.
• 1. Estrogen Deficiency Alters Intestinal Physiology To Promote Bone Loss. Jing Zhang, R Irwin,S Raehtz, R Britton, LR McCabe. Experimental Biology Meeting 2012.
• 2. Prevention of menopause related osteoporosis in mice by treatment with probiotic Lactobacillus reuteri. R. A. Britton, R. Irwin, J. Zhang, L. Schaefer, S. Raehtz, D. Quach, N. Parameswaran, L. McCabe. American Society for Microbiology Beneficial Microbes Conference 2012.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Over the past year we have made significant progress in three areas regarding the functional genomics of probiotic Lactobacillus reuteri. First, we have continued to optimize recombineering in L. reuteri and Lactococcus lactis and now have the ability to change individual base pairs in the genome without the need for antibiotic selection. This represents a major advance in the genetics and genomics of these medically and industrially important microorganisms. This research was presented as platform presentations at two international meetings this summer (Functional Genomics of Gram-positive Bacteria and the 10th Conference on Lactic Acid Bacteria). We have been approached by several academic and industrial partners about the technology and have begun to disseminate the system to various groups. Second, we have incorporated this technology into an undergraduate course that I teach in Advanced Microbial Genomics. Thus we have brought this new technology into the hands of undergraduates and have successfully been able to have them modify genomes with this new technology. Finally, we have had two major breakthroughs in the implementation of probiotic L. reuteri and its impact on health and disease. We showed that L. reuteri was capable of protecting mice from lethal challenge of E. coli O157:H7, an important food-borne pathogen. We also demonstrated that L. reuteri has potent anti-inflammatory activity that is modulated by the composition of the bacterial membrane. These advances have led to new areas of research in how L. reuteri impacts health and disease, and the development of recombineering will help us answer these new questions. PARTICIPANTS: Robert Britton, PI. Kathryn Eaton, University of Michigan James Versalovic, Baylor College of Medicine TARGET AUDIENCES: Advanced Microbial Genomics Laboratory 434 undergraduate students - laboratory instruction. Colleagues in the lactic acid bacterial field that will benefit from recombineering - meeting presentations. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Clearly the development of recombineering in lactic acid bacteria has elicited quite a response from our colleagues in both academia and industry. Since July we have shared the technology with five academic laboratories and have come to agreements with two industrial partners. Note this is all before publication of the work. Thus we are committed to getting important tool into the hands of researchers as soon as possible without compromising our publication on the work. The ability to alter the genome without the need for antibiotic selection is clearly going to be a major breakthrough in the field of lactic acid bacteria.

Publications

• Cyclopropane fatty acid synthase mutants of probiotic human-derived Lactobacillus reuteri are defective in TNF inhibition. Jones SE, Whitehead K, Saulnier D, Thomas CM, Versalovic J, Britton RA. Gut Microbes. 2011 Mar-Apr;2(2):69-79. Epub 2011 Mar 1.
• Probiotic Lactobacillus reuteri ameliorates disease due to enterohemorrhagic Escherichia coli in germfree mice. Eaton KA, Honkala A, Auchtung TA, Britton RA. Infect Immun. 2011 Jan;79(1):185-91. Epub 2010 Oct 25. PMID: 20974822 [PubMed - indexed for MEDLINE] Free PMC Article
• Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm. Walter J, Britton RA, Roos S. Proc Natl Acad Sci U S A. 2011 Mar 15;108 Suppl 1:4645-52. Epub 2010 Jun 25. Review.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We presented results from our findings at two international conferences. The first was at the DDW meeting in New Orleans in May 2010. The abstract "Lactobacillus Reuteri Ameliorates Disease Due To Enterohemorrhagic E. Coli In Germ Free Mice" was selected for a platform presentation that was delivered by my collaborator on this project, Kathryn Eaton (U of Michigan). The second was at the 3rd ASM Conference on Beneficial Microbes in Miami in October 2010. The abstract "LACTOBACILLUS REUTERI PTA ATCC 6475 AS A MODEL FOR RECOMBINEERING IN LACTIC ACID BACTERIA" was presented by my postdoctoral associate Jan Peter van Pijkeren. We have also applied a new technology for disrupting genes in lactic acid bacteria in an efficient manner. We have shared recombineering technology with three other academic laboratories and will make this widely available to the academic community once published. The laboratories we have sent the technology to are Dr. Jens Walter, University of Nebraska, Dr. James Versalovic, Baylor College of Medicine, and Dr. Stefan Roos, Swedish Agricultural University. PARTICIPANTS: In this project Dr. Robert Britton serves as PI and has directed the research outlined in the project. We have collaborated with Dr. Kate Eaton at the University of Michigan on the work assessing probiotic effects on EHEC disease. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The main outcomes this year were the establishment that Lactobacillus reuteri can ameliorate disease caused by EHEC infection in mice and the development of recombineering in medically and industrially important lactic acid bacteria. EHEC is a disease that is not responsive to antibiotic therapy and there is no effective therapy for preventing long-term complications that are associated with some infections. Thus the identification of a therapy that is working in mice may pave the way for the use of probiotic therapies in humans. The development of recombineering in L. reuteri and Lactococcus lactis, which now allows us to modify essentially any base pair in the genome, will greatly enhance our study of these organisms as probiotics and in industry. We expect that many groups around the world will use the technology we have developed for studies of these and other lactic acid bacteria.

Publications

• Eaton KA, Honkala A, Auchtung TA, Britton RA. (2010). Probiotic Lactobacillus reuteri ameliorates disease due to Enterohemorrhagic Escherichia coli in germ free mice. Infection and Immunity. Oct 25. [Epub ahead of print]
• Walter J, Britton RA, and Roos S. (2010) Microbes and Health Sackler Colloquium: Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm. PNAS. June 25, 2010. Epub ahead of print.
• Schaefer L, Auchtung TA, Hermans KE, Whitehead D, Borhan B, Britton RA. (2010). The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative stress via interaction with thiol groups. Microbiology. 156:1589-1599.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The work in this project was disseminated at two international conferences. The first was the 17th Annual Microbial Genomics Conference and the second was the National Academy of Sciences Sackler Symposium on Microbes and Health. I also participated in the International Scientific Association of Probiotics and Prebiotics workshop on Probiotics and Mucosal Immunity. A graduate student on this project, Kristi Whitehead, published her doctoral thesis and is in the process of preparing two manuscripts for publication in scientific journals. I presented two departmental seminars on this work (Bowling Green University and Northeast Ohio Medical College). I currently supervise two graduate students, three postdoctoral researchers, and two undergraduates as part of this project. PARTICIPANTS: Robert Britton - Principal investigator. Directed all aspects of research in the current project. Dr. Kathryn Eaton (University of Michigan) collaborated on the ability of Lactobacillus reuteri to ameliorate EHEC disease in mice. Dr. James Versalovic (Baylor College of Medicine) is a collaborator on the development of genetic tools for Lactobacillus reuteri. TARGET AUDIENCES: Nothing to report during this period. PROJECT MODIFICATIONS: Nothing to report this period.

Impacts
We had two major breakthroughs this past year in our research. The first was demonstrating that probiotic Lactobacillus reuteri could reduce disease in a mouse model of Enterohemmorhagic E. coli (EHEC) infection. EHEC disease is a currently untreatable form of diarrheal E. coli that can result in life threatening complicatons, mainly in young children. We expect this research to be a first step in identifying alternative therapies for this disease. The second major breakthrough was the establishment of recombineering in probiotic bacteria. Recombineering is a genetic technique that allows researchers to alter a single base pair in the chromosome without leaving behind an antibiotic resistance marker. This will lead to increased use of genetics to study probiotic bacteria as well as a safe way to eliminate undesirable characteristics in probiotics currently marketed to consumers.

Publications

• Britton, RA and Versalovic, J. (2009). Probiotics and gastrointestinal infections. Interdisciplinary Perspectives in Infectious Disease. PMCID: PMC2648624.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The work in this project were disseminated at an international conference and an international workshop. In August two posters were presented at the 9th International Lactic Acid Bacteria conference. In November I participated in the International Scientific Association for Probiotics and Prebiotics workshop that addressed emerging issues regarding the science and commercial use of probiotics. I was also plenary speaker at the American Society for Microbiolgy Michigan Chapter Fall Meeting. Lastly, I presented departmental seminars at Michigan State University and the University of Michigan. PARTICIPANTS: Robert Britton - Prinicpal Investigator - Directed all aspects of research in the current project. Mentored students and postdoctoral associates in helping them achieve their research goals. This was done by aiding in the design of experiments, analysis of experimental data, and dissemination of research findings to colleagues. Vincent Young - Collaborator - University of Michigan. Dr. Young is attempting to help us establish a mouse model of colitis in which probiotic bacteria have a beneficial effect. Kathryn Eaton - Collaborator - University of Michigan. Dr. Eaton is conducting experiments to determine if L. reuteri has the ability to ameliorate disease in a EHEC model of colitis. James Versalovic - Collaborator - Baylor College of Medicine. Dr. Versalovic is the director of the L. reuteri genome project that we also participate in. We are currently working with his lab to generate genetic tools for use in L. reuteri. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The first objective of this work is to identify how Lactobacillus reuteri can withstand exposure to bile salts in the small intestine. Previous work identified approximately 100 genes that were differentially regulated upon exposure to bile. We have found three of these genes to be critical for survival upon the initial exposure to bile and a fourth gene that is required for L. reuteri to grow in the presence of bile. This work was accepted for publication this year in the journal Applied and Environmental Microbiology. We are now characterizing bile salt hydrolase activities in various L. reuterin strains. The second objective was to determine how the antimicrobial compound reuterin impacts growth and physiology of pathogens. We have determined that reuterin interacts with thiol groups in proteins and small molecules, leading to an oxidative stress response in Escherichia coli. Therefore reuterin likely targets many cellular targets in mediating its bactericidal effects. A publication is planned in 2009. The third objective is to develop a mouse model to study the effects of probiotics against pathogenic E. coli strains that cause diarrhea. We have not yet been successful in achieving this objective yet but experiments are ongoing.

Publications

• Whitehead, K.J., Versalovic, J., Roos, S. and Britton, R.A. (2008). Genomic and genetic characterization of the bile stress response of probiotic Lactobacillus reuteri ATCC 55730. Applied and Environmental Microbiology. 74:1812-19.
• Cadieux P, Wind A, Sommer P, Schaefer L, Crowley K, Britton RA, Reid G. (2008). Evaluation of Reuterin Production in Urogenital Probiotic Lactobacillus reuteri RC-14. Applied and Environmental Microbiology. 74:4645-9.
• Hufner E, Britton RA, Roos S, Jonsson H, Hertel C. (2008). Global transcriptional response of Lactobacillus reuteri to the sourdough environment. Syst Appl Microbiol. 31:323-38.
• Whitehead, K.J., Versalovic, J., Roos, S. and Britton, R.A. (2008). Characterization of the bile stress response of probiotic Lactobacillus reuteri ATCC 55730. 9th International Lactic Acid Bacteria Meeting. Egmond an Zee, The Netherlands.
• Schaefer, L., Auchtung, T. and Britton, R.A. (2008). Genetic and genomic characterization of the antimicrobial compound reuterin. 9th International Lactic Acid Bacteria Meeting. Egmond an Zee, The Netherlands.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: During the past year I have been involved in mentoring two graduate students and three undergraduate students directly related to this project. As part of their training one graduate student and one undergraduate attended the 2007 ASM General Meeting and presented their work in poster form to the research community. Kristi Whitehead presented her work on how L. reuteri responds to bile acids that are encountered by the bacterium in the small intestine. Karley Hermans presented her work on the antimicrobial compound reuterin and how this molecule plays a role in the beneficial effects exerted by L. reuteri. I have also incorporated part of this project into an undergraduate course that I teach - Microbial Genomics 433. Students in this course helped annotate the genome of a Lactobacillus reuteri strain being investigated in several laboratories around the world. Thus over 40 undergraduate students were able to participate directly in a research project within the classroom setting. Lastly, the genome sequence of a newly released probiotic strain, L. reuteri ATCC 6475, was completed. DNA microarrays for this strain are planned for early 2008. PARTICIPANTS: Robert Britton - Principal Investigator. Conceived and directed all aspects of the research proposed in the current project. Mentored students and postdoctoral associates in helping them obtain their research goals. This was achieved by aiding in the design of experiments, analysis of experimental data, and dissemination of research findings to colleagues. Vincent Young, University of Michigan - Collaborator. Dr. Young has conducted experiments using a murine model of colitis to identify the role of reuterin in probiosis. His laboratory has handled all aspects of the animal work. He has since moved his laboratory to the University of Michigan and we will continue to collaborate on this project. Kathryn Eaton, Universtiy of Michigan - Collaborator. Dr. Eaton is conducting experiments that will determine the ability of L. reuteri to ameliorate EHEC disease in a newly developed mouse model. James Versalovic, Baylor College of Medicine - Collaborator. Dr. Versalovic is the principal investigator on the L. reuteri ATCC 6475 genome project. We will be developing DNA microarrays for this organism in the near future.

Impacts
The first objective of this project is to understand how Lactobacillus reuteri can withstand exposure to bile salts in the small intestine. Previously, we had discovered nearly 100 genes that are altered in expression when L. reuteri is exposed to physiological concentrations of bile. This past year we have identified four of these genes as being important for properly adapting to bile stress using a genetic strategy. These findings were the basis of a poster presented at the 2007 ASM General Meeting and for a paper in submission to the journal Applied and Environmental Microbiology. We are currently investigating the mechanisms by which these genes impact that ability of L. reuteri to survive bile stress. The second objective is aimed at identifying how the antimicrobial compound reuterin impacts the ability of L. reuteri to provide beneficial health effects to the host. Using an infectious mouse model of colitis we have preliminary evidence that indicates reuterin does play an important role in probiosis. We have engineered a mutant L. reuteri strain that lacks the ability to produce reuterin. We have found that a wild-type L. reuteri strain is more capable of preventing intestinal hyperplasia than a strain unable to produce reuterin. This is the first demonstration that this antimicrobial compound is providing a beneficial effect to an animal in vivo. We are currently attempting to determine the mechanism of action of reuterin in probiosis within this model. The third objective of the study is to identify L. reuteri strains that are potentially effective against enterohemmorhagic E. coli (EHEC) or Enteropathogenic E. coli (EPEC). Both of these types of organisms cause significant morbidity and mortality in children worldwide. We have established in the laboratory that reuterin producing L. reuteri can effectively kill both EHEC and EPEC strains. We are now testing the ability of L. reuteri to ameliorate disease in a new murine model of EHEC disease.

Publications

• Wall, T., Bath, K., Britton, R.A., Jonsson, H., Versalovic, J., and Roos, S. (2007). The early response to acid shock in Lactobacillus reuteri involves the ClpL chaperone and a putative cell wall-altering esterase. Applied and Environmental Microbiology. 73:3924-35
• Whitehead, K., Versalovic, J., Roos, S. and Britton, R.A. (2007). Genomic and genetic characterization of the bile stress response of probiotic Lactobacillus reuteri ATCC 55730. Applied and Environmental Microbiology. Submitted.
• Storm, M., Xiang, Q., Roos,S., Spinler, J.K., Janecki, M., Dillon, M.G., Britton, R.A., Petrosino, J.F., Highlander, S.K., Jonsson, H., Weinstock, G.M., Versalovic, J. (2007). Comparison of Two Lactobacillus reuteri Genomes Reveal Divergence Within a Probiotic Species. 2007 American Society for Microbiology General Meeting. Toronto, Canada.
• Hermans, K., Schaefer, L., and Britton, R. (2007) Characterization of the production of reuterin, an antimicrobial compound secreted by probiotic Lactobacillus reuteri strains. 2007 American Society for Microbiology General Meeting. Toronto, Canada.
• Whitehead, K.J., Roos, S., Versalovic, J. and Britton, R.A. (2007) Characerization of the bile stress response of Lactobacillus reuteri ATCC 55730. 2007 American Society for Microbiology General Meeting. Toronto, Canada.