Source: TEXAS A&M UNIVERSITY submitted to
ROLE OF LACTIC ACID BACTERIA IN HUMAN AND ANIMAL HEALTH, NUTRITION AND FOOD SAFETY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0222650
Grant No.
(N/A)
Project No.
TEX09405
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jun 10, 2010
Project End Date
Jun 9, 2015
Grant Year
(N/A)
Project Director
Duong, T.
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Poultry Science
Non Technical Summary
Lactic Acid Bacteria (LAB) are gram-positive, acid tolerant, non-sporulating bacteria that include the species Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, and Streptococcus. LAB are naturally found in a wide range of environments including dairy products, meats, and plant materials and are associated with animal and human mucosal surfaces. LAB are important in the production and preservation of foods serving as starter cultures for fermented dairy products, meat, and vegetables and are important for the production of coffee, chocolate and wine. In addition to their role in food production, LAB are important in the production of renewable, low-carbon foot print energy and materials. l-lactic acid is an important building block for the synthesis of biodegradable polymers and is the substrate for the production of other compounds such as acrylic acid, acetic acid, propylene glycol, and ethanol LAB have long been thought to be beneficial to animal and human health. While LAB, particularly probiotic lactobacilli, have been shown to confer a number of beneficial health effects, the physiological and genetic basis of these is largely unknown. Recently, the genomes of several species of LAB have been sequenced including L. acidophilus (1), L. gasseri, L. johnsonii and others. The availability of these genome sequences will provide much insight as to the physiology, metabolism and biosynthetic capabilities of these organisms and will contributed to an improved understanding of the molecular mechanisms underlying the probiotic functionality of LAB. Because of their record of safety, importance in human health, genetic tractability and wide range of potential applications, LAB sit at the cutting edge of microbial biotechnology. Utilizing functional genomic and molecular genetic techniques we will investigate the physiology and genetics of these organisms which will be important in their development of microbially based biotech solutions for bioprocessing, human heath, and animal agriculture with particular focus on the use of lactic acid bacteria.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123220104020%
7123220110020%
7124010104020%
7124010110020%
7224010104010%
7224010110010%
Knowledge Area
722 - Zoonotic Diseases and Parasites Affecting Humans; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3220 - Meat-type chicken, live animal; 4010 - Bacteria;

Field Of Science
1100 - Bacteriology; 1040 - Molecular biology;
Goals / Objectives
The objective of our research program is to investigate the role of the gastrointestinal (GI) microflora, particularly LAB, in human and animal health, nutrition, and food safety. The proposed research objectives are (1) To identify mechanisms of adherence and colonization of probiotic bacteria in the GI tract using in vitro and avian animal models (2) Identify and understand host-microbe interactions at mucosal surfaces in the GI-tract, particularly those of probiotic (beneficial) bacteria. (3) Identify applications and mechanisms of LAB in food safety. (4) Develop recombinant lactic acid bacteria for the expression gene products important in bioprocessing/fermentations, improved human/animal nutrition and vaccine delivery.
Project Methods
Objective 1. Identification of mechanisms of adherence and colonization of probiotic bacteria in the GI tract. In order to understand specific microbial factors involved in adherence and colonization (1) we will adapt the LMH cell culture model as an assay for the determination of microbial adherence to chicken epithelial cells, (2) determine the adherence of a number of difference probiotic LAB, (3) use functional genomic and molecular genetic techniques to identify specific microbial gene/gene products involved in adherence, and (4) apply and correlate knowledge gained from in vitro studies back into the live animal system. Objective 2. Indentify and understand host-microbe interactions at mucosal surfaces in the GI-tract. In these studies, we will create and employ a transgenic commensal Lactobacillus strain expressing model antigens. We will use these recombinant bacteria to colonize conventional and germ-free mice to minimize competition from other intestinal bacteria and track antigen-specific adaptive immune responses to this bacterium using common immunologic tools. Objective 3. Identify applications and mechanisms of LAB in food safety. One potential major area of application is the use of probiotic LAB, and similar strategies, as countermeasures to reduce the threat of foodborne infection. One strategy with strong potential is the reduction of the carriage of foodborne pathogens, including Campylobacter and Salmonella, in chickens using probiotics. Particular focus would on be four potential mechanisms: competitive exclusion through binding and occupation of attachment sights preventing colonization, in situ production of antagonistic molecules including bacteriocins and organic acids, modulation of host immunity using probiotics, and vaccine delivery via probiotic LAB. Objective 4. Develop recombinant lactic acid bacteria for the expression gene products important in bioprocessing/fermentations, improved human/animal nutrition and vaccine delivery Recombinant expression of proteins will be important in the future applications of LAB in bioprocessing/fermentation and human and animal health. We have previously developed a number of vectors for the recombinant expression of gene products by LAB. We will identify potential areas of application of LAB and use these molecular genetics tools to apply recombinant expression of proteins to a number of different areas including biotherapeutic and vaccine delivery, immune modulation, and improved digestion and nutrition.

Progress 06/10/10 to 06/09/15

Outputs
Target Audience:1. Scientific audiences for gram positive commensal bacteria and their roles in the GI tract. 2. Members of the community of scientists working with LAB important to bioprocessing and health. 3. Scientists and industries working with probiotic bacteria to promote human and animal health and define mechanisms important to their functionality. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate and graduate students were trained in poultry science, microbiology, and biotechnology principals and technologies while conducting experiments. PARTICIPANTS: Tyler Askelson, Ph.D. student in Poultry Science Andrew Rehkopf, M.S. Student in Poultry Science Timothy Broderick, B.S. Student in Poultry Science Madison Becka, B.S. Student in Biology Alyssa Domingue, B.S. Student in Poultry Science; Sadie Horrocks, staff research scientist providing technical platform support How have the results been disseminated to communities of interest?Results of our research have been disseminated to communities of interest through publication of scientific research articles and conference presentations as listed in the products/outcomes of the project. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Primary accomplishments of this project over the reporting period are as follows: Objective #1:Persistance ofProbiotic Bacteria in Poultry Characterization of bile tolerence in L. crispatusand L. gallinarumcultures as a potential colonization factor Identification of putative bile tolerence factors inL. crispatus Objective #2: Host microbe interactions in the Gastroinestinal tract of Poultry Characterized the effect ofcommercial feed additives (DFMs, enzymes, etc...) on growth performance and gastrointestinal microbiota of broiler chickens Objective #4: Recombinant Lactic Acid Bacteria Adapted chromosomal integration system for targeted mutagensis inL. gallinarum

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Askelson, T. E., A. K. Barker, and T. Duong. 2015. Application of a targeted chromosomal integration system in Lactobacillus gallinarum ATCC 33199. Poultry Science Association Annual Meeting, Louisville, KY
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Broderick, T. J., T. E. Askelson, and T. Duong. 2015. Characterization of putative bile salt hydrolases in Lactobacillus crispatus and Lactobacillus gallinarum. Poultry Science Association Annual Meeting, Louisville, KY.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Flores, C. A., Y. Dersjant-Li, T. E. Askelson, K. Gibbs, T. Duong, A. Awati, and J. T. Lee. 2015, Effect of a feed additive containing multi-enzymes and direct fed microbial on growth performance in broilers fed US commercial-type diets with or without AGP. Poultry Science Association Annual Meeting, Louisville, KY.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Jasek, A., J. Barnes, R. Carpenter, T. Duong, and J. T. Lee. 2015. Effects of mixed-species direct fed microbial on male broiler growth performance. Poultry Science Association Annual Meeting, Louisville, KY.
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Askelson, T.E. and T. Duong. 2015. Perspectives on differences between human and livestock animal research in probiotics and prebiotics. p. 457-458. In K. Venema and A. P. do Carmo (ed.), Probiotics and Prebiotics: Current Research and Future Trends. Horizon Scientific Press, Poole, U.K.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: 1. Scientific audiences for gram positive commensal bacteria and their roles in the GI tract. 2. Members of the community of scientists working with LAB important to bioprocessing and health. 3. Scientists and industries working with probiotic bacteria to promote human and animal health and define mechanisms important to their functionality. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Undergraduate and graduate students were trained in food science, poultry science, microbiology, and biotechnology principals and technologies while conducting experiments. PARTICIPANTS: Tyler Askelson, Ph.D. student in Poultry Science; Andrew Rehkopf, M.S. Student in Poultry Science; Lindy Froebel, B.S. student in Poultry Science; Andrew Rehkopf, B.S. student in Poultry Science; Timothy Broderick, B.S. Student in Poultry Science; Alyssa Domingue, B.S. Student in Poultry Science; Science; Sadie Horrocks, staff research scientist providing technical platform support. How have the results been disseminated to communities of interest? Results of our research have been disseminated to communictes of interest through publication and submission of scientific journal articles as listed under the products/outcomes of the project. What do you plan to do during the next reporting period to accomplish the goals? We plan to conduct experiments which will (1) Characterize the role of genes of Lactobacillus species putatively important in the gastrointestinal colonization of poultry, (2) Evaluate the effects of DFM/probiotic treatments on colonization by humanfoodborne pathogens in the poultry gastrointestinal tract and, (3) Evaluate the effects of DFM/probiotic treatments on the performance of poultry.

Impacts
What was accomplished under these goals? Primary accomplishments of this project over the reporting period are as follows: (1) Identification of putative adhesion and colonization factors of Lactobacillus gallinarum; (2) Characterization of the stability of commericial direct fed microbial(DFM)/probiotic microorganisms in animal feed; (3) Evaluation of the effects of commercial DFM/probiotic culture administration pre-harvest food safety in poutlry

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Askelson, T.E., A. Campasino, J.T. Lee, and T. Duong. 2014. Evaluation of phytate-degrading Lactobacillus administration in broiler chickens. Appl Environ Microbiol 80:943-950. PMID: 24271165.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Spivey, M.A., S.L. Dunn-Horrocks, and T. Duong*. 2014. Evaluation of Lactobacillus adherence to epithelial cells in vitro and gastrointestinal colonization of poultry. Poult Sci 93:2910-2919 PMID: 25239531
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: 5Askelson, T.E. and T. Duong*. 2014. Perspectives on differences between human and livestock animal research in probiotics and prebiotics. In K. Venema and A. P. do Carmo (ed.), Probiotics and Prebiotics: Current Research and Future Trends. Horizon Scientific Press, Poole, U.K.


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

Outputs
Target Audience: 1. Scientific audiences for gram positive commensal bacteria and their roles in the GI tract. 2.Members of the community of scientists working with LAB important to bioprocessing and health. 3. Scientists and industries working with probiotic bacteria to promote human and animal health and define mechanisms important to their functionality. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training Activities:Undergraduate and graduate students were trained in food science, poultry science, microbiology, and biotechnology principals and technologies while conducting experiments. PARTICIPANTS: Tyler Askelson, M.S. student in Poultry Science; Megan Spivey, M.S. student in Food Science; Alex Wood, M.Biot student in Biotechnology; Anna Barker, B.S. student in Biotechnology; Lindy Froebel, B.S. student in Poultry Science; Andrew Rehkopf, B.S. student in Poultry Science; Sadie Horrocks, staff research scientist providing technical platform support. How have the results been disseminated to communities of interest? Results of our research have been disseminted to communities of interest through poster and oral presentations made at local, national and international conferences as listed under the products/outcomes of the project. What do you plan to do during the next reporting period to accomplish the goals? We plan to conduct experiments which will (1) Identify genes of Lactobacillus species putatively important in the gastrointestinal colonization of poultry, (2) Evaluate the effects of DFM/probiotic treatments on colonization by human-foodborne pathogens in the poultry gastrointestinal tract and, (3) Evaluate the effects of DFM/probiotic treatments on the performance of poultry.

Impacts
What was accomplished under these goals? Primary accomplishments of this project over the reporting period are as follows: (1) The adherence of Lactobacillus crispatus and Lactobacillus gallinarum to poultry epithelial cells in vitro was evaluated using the LMH cell line, (2) Colonization/persistance of Lactobacillus crispatus and Lactobacillus gallinarum in the gastrointestinal tract of broiler chickens was evauated and, (3) Phytate-degredation in the gastrointestinal tract of poultry by Lactobacillus species was characterized.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Menon, R., M. Shields, T. Duong, and J.M. Sturino. 2013. Development of a carbohydrate-supplemented semidefined medium for the semiselective cultivation of Lactobacillus spp. Lett. Appl. Microbiol. 57:248-57
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Duong, T., S. Tomar, T.E. Askelson, M.A. Spivey. 2013. Mechanisms of Probiotic Functionality in Poultry. Indian Poultry Science Association Conference. Izatnagar, India.
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Douglas, G. L., E. A. Pfeiler, T. Duong, and T. R. Klaenhammer. 2013. Genomics and Proteomics of Foodborne Microorganisms p. 975-996. In M. P. Doyle and L. R. Beuchat (ed.), Food Microbiology: Fundamentals and Frontiers, 4th ed. ASM Press, Washington, DC.
  • Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Spivey, M.A. and T. Duong*. Evaluation of Lactobacillus adherence to epithelial cells in vitro and gastrointestinal colonization of poultry.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Askelson, T.E. 2013. Effect of phytate-degrading probiotics on broiler performance. Texas A&M University
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Spivey, M.A. 2013. Development of an in vitro model of probiotic adherence and colonization in poultry. Texas A&M University
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Askelson, T.E., J.T. Lee, A. Campasino, and T. Duong. 2013. Administration of phytate-degrading Lactobacillus improves growth of broiler chicks, Texas A&M University Student Research Week, College Station, TX. Oral Presentation
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Askelson, T.E., J.T. Lee, A. Campasino, and T. Duong*. 2013. Administration of phytate-degrading Lactobacillus improves growth of broiler chicks, Texas A&M University Poultry Science Department Student Research Symposium, College Station, TX.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Askelson, T.E., J.T. Lee, A. Campasino, and T. Duong*. 2013. Administration of phytate-degrading Lactobacillus improves growth of broiler chicks, Poultry Science Association Annual Meeting, San Diego, CA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Duong, T.*, M. A. Spivey, S. L. Dunn-Horrocks, A. K. Barker , and A. T. Wood. 2013. Analysis of putative Lactobacillus crispatus adherence proteins important in gastrointestinal colonization of poultry, Poultry Science Association Annual Meeting, San Diego, CA.


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

Outputs
OUTPUTS: Over this period, this project began conducting experiments investigating the mechanisms important in the probiotic functionality of Lactobacillus species in broiler chickens. In the conduct of these experiments, students were trained in poultry science, food science, microbiology, and biotechnology principles and technologies. Primary output events included conferences where research outputs from this project were reported on (1) adherence of Lactobacillus cultures to poultry epithelial cells, (2) biocatalytic degradation of indigestible feed components by Lactobacillus cultures, (3) the development of novel strain specific transformation protocols for poultry Lactobacillus isolates. The project has developed the tools and technology for efficient functional characterization of the roles of various enzymes, cell surface proteins and structural components in mechanisms that underlie probiotic functions and attributes PARTICIPANTS: Tyler Askelson, M.S. student working in understanding the role of probiotic lactic acid bacteria in poultry nutrition. Megan Spivey, M.S. student working in understanding mechanisms important to colonization of poultry by probiotic lactic acid bacteria. Anna Barker, B.S. student working in developing ransformation protocols for use with poultry Lactobacillus isolates. Sadie Horrocks, staff research scientist providing technical platform support. Patricia Baynham, visiting faculty/researcher receiving sabbatical training TARGET AUDIENCES: 1. Scientific audiences for gram positive commensal bacteria and their roles in the GI tract. 2. Members of the community of scientists working with LAB important to bioprocessing and health. 3. Scientists and industries working with probiotic bacteria to promote human and animal health and define mechanisms important to their functionality. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Lactobacillus species are used extensively as probiotic cultures in human health and animal agriculture. The techniques we are developing will be important to investigations of mechanisms important to the functionality in humans and animals. Understanding the mechanisms through which these probiotic bacteria impact health and nutrition is drive expansion of their use to improve human health and animal agriculture.

Publications

  • 1. Calix-Lara, T.F., T. Duong, and T.M. Taylor. 2012. Addition of a surfactant to tryptic soy broth allows growth of a Lactic Acid Bacteria food antimicrobial, Escherichia coli O157:H7, and Salmonella enterica. Lett Appl Microbiol 54:392-397.
  • 2. Neal-McKinney, J.M., X. Lu, T. Duong, C.L. Larson, D.R. Call, D.H. Shah, and M.E. Konkel. 2012. Production of organic acids by probiotic lactobacilli can be used to reduce pathogen load in poultry. PLOS One 7:e43928.
  • 3. Barker, A.K., L.W. Morgan, and T. Duong. 2012. Application of a targeted chromosomal integration system for use in Lactobacillus crispatus. Texas A&M University Student Research Week, College Station, TX. Poster Presentation.
  • 4. Askelson, T.E., J.T. Lee, and T. Duong. Recombinant expression of Bacillus subtilis phytase improves phytate degradation of Lactobacillus. 2012. Poultry Science Association Annual Meeting, Athens, GA. Poster Presentation
  • 5. Spivey, M.A., and T. Duong. Development of an in vitro model of probiotic adherence and colonization for poultry. 2012. Poultry Science Association Annual Meeting, Athens, GA. Poster Presentation


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

Outputs
OUTPUTS: Over the reporting period, significant progress was made in establishing technical platform and core competencies important to achieving the stated objectives. Experiments were performed in developing an in vitro platform for studying adherence and colonization of bacteria in poultry and in the in situ production and delivery of digestive enzymes in the gastrointestinal tract using recombinant probiotic lactic acid bacteria. In the conduct of these experiments students were trained in food science, poultry science, microbiology, and biotechnology principals and technologies. PARTICIPANTS: Tyler Askelson, M.S. student working in understanding the role of probiotic lactic acid bacteria in poultry nutrition. Megan Spivey, M.S. student working in understanding mechanisms important to colonization of poultry by probiotic lactic acid bacteria. Sadie Horrocks, staff research scientist providing technical platform support. Simmi Tomar, visiting faculty/researcher receiving sabbatical training through the Borlaug Institute's international scholar's program. TARGET AUDIENCES: 1. Scientific audiences for gram positive commensal bacteria and their roles in the GI tract. 2. Members of the community of scientists working with LAB important to bioprocessing and health. 3. Scientists and industries working with probiotic bacteria to promote human and animal health and define mechanisms important to their functionality. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Primary outcomes of this project over the reporting period are as follows: (1)an in vitro assay for studying adherence and colonization of poultry using the LMH cell line was adapted for use with lactic acid bacteria; (2) protocols for electrotransformation of poultry LAB isolates were developed; (3) the composition of the gastrointestinal microbiota of broiler chickens fed various diets was characterized.

Publications

  • McFarland, A.P., R. Savan, S. Wagage, A. Addison, K. Ramakrishnan, M. Karwan, T. Duong, and H.A. Young. 2011. Localized Delivery of Interferon-β by Lactobacillus Exacerbates Experimental Colitis. PLOS One 6:e16967.
  • Duong, T., M. J. Miller, R. Barrangou, M. A. Azcarate-Peril, and T. R. Klaenhammer. 2011. Construction of vectors for inducible and constitutive gene expression in Lactobacillus. Microbial Biotechnology 4:357-367.