Progress 12/15/01 to 12/31/04
Outputs
Salmonellosis is one of the most common foodborne diseases in the United States. There is a tremendous need to apply molecular approaches to develop better and more specifically targeted control measures at the preharvest stage of food production. The main goal of this project was to use a novel approach for screening the genome of Salmonella Typhmurium for the virulence genes that are required for survival within live host animals. We devised a simple method, termed transposon footprinting, for functional screening of the bacterial genome. In this project, we used transposon mutants of S. Typhimurium as a model system to take advantage of the feasibility of transposon footprinting for a thorough identification of the essential genes required for in vitro growth as well as the virulence genes required for in vivo survival. In addition, this approach can also be used to conduct a genome-wide comparison of the virulence genes for in vivo survival between different host
animals or tissues. Our procedure has revealed some of genes that are potentially important in basic cellular processes and the virulence genes that are involved in various features of the bacterial pathogenesis including host-adaptation or tissue-tropism. These studies should provide insights into the molecular mechanisms governing the pathogenesis for foodborne S. Typhimurium and also have practical food safety applications for the development of vaccine, antimicrobial agents, and preventive strategies.
Impacts
Foodborne salmonellae continues to be difficult to limit in preharvest food production. New control strategies will require more extensive genetic analysis. Transposon footprinting is expected to facilitate the comprehensive functional analysis of bacterial genome for the genes that are required to increase fitness to a variety of selective conditions. When this technique is conducted with a transposon that harbors an inducible promoter facing outwardly, it would allow efficient identification of genes for Salmonella adherence and invasion. The elucidation of the proteins encoded by the genes identified in this study could provide new targets for novel vaccine development and preventative strategies. This approach is technically very simple and does not require any prior sequence information of the genome to be analyzed. Now that bacterial genome sequencing databases are available for S. Typhimurium this may help to facilitate identification of the genes that
correspond to optimal targets to design vaccine strains.
Publications
- Cox, M., R.L. Ziprin, L.F. Kubena, D.J. Nisbet, S.C. Ricke, and Y. Kwon. 2004. Genome screening for virulence genes by transposon signature profiling. American Society for Microbiology Conference on Functional Genomics and Bioinformatics Approaches to Infectious Disease Research, Oct. 6-9, Portland, OR.
- Krabasil, N. S. Bulajic, W.K. Kim, K.D. Dunkley, T.R. Callaway, T.L. Poole, S.C. Ricke, R.C. Anderson, D.J. Nisbet. 2004. Comparison of antibiotic resistance frequency of Salmonella Typhimurium growth in glucose-limited continuous cultures at slow and fast dilution rates. Poultry Science Society Annual Meetings, St. Louis, MO.
- Dunkley, K.D., M.M. Kundinger, C.S. Dunkley, T.R. Callaway, R.C. Anderson, D.J. Nisbet, and S.C. Ricke. 2004. Cell yield and genetic response in Salmonella Typhimurium in a continuous culture during shifts in pH. Poultry Science Society Annual Meetings, St. Louis, MO.
- Chalova, V.I., W.K. Kim, I.B. Zabala-Diaz, C.L. Woodward, and S.C. Ricke. 2004. Development of a whole cell sensor - green fluorescent protein based method for estimating lysine bioavailability in poultry feed proteins. Poultry Science Society Annual Meetings, St. Louis, MO.
- Ricke, S.C.. 2004. Anaerobic animal-waste environments: Salmonella species ecology . pp. 13-17. In: A. Pandey (Ed.), Concise Encyclopedia of Bioresource Technology, The Haworth Press, Binghamton, NY.
- Maciorowski, K.G., F.T. Jones, S.D. Pillai, and S.C. Ricke. 2004. Incidence and control of foodborne Salmonella spp. in poultry feeds- A review. Worlds Poultry Sci. J. 60: 446-457.
- Nutt, J.D., C.L. Woodward, L.F. Kubena, D.J. Nisbet, Y.M. Kwon, and S.C. Ricke. 2004. Potential for rapid in vitro assays to measure foodborne Salmonella virulence in foods - A review. J. Rapid Meths. Auto. Microbiol. 12: 234-246.
- Zabala Diaz, I.B. and S.C. Ricke. 2004. Influence of short chain fatty acids and lysine on Salmonella typhimurium cadA expression. Antonie von Leeuwenhoek 85: 45-51.
- Ricke S.C., M.E. Hume, S.Y. Park, R.W. Moore, S.G. Birkhold, L.F. Kubena, and D.J. Nisbet. 2004. Denaturing gradient gel electrophoresis (DGGE) as a rapid method for assessing gastrointestinal tract microflora responses in laying hens fed similar zinc molt induction diets. J. Rapid Meth. Automation Microbiol. 12: 69-81.
- Woodward, C.L., S.Y. Park, D.R. Jackson, X. Li, S.G. Birkhold, S.D. Pillai, and S.C. Ricke. 2004. Optimization and comparison of sampling systems for bioaerosol detection of bacterial load and sampling time in a poultry layer house. J. Appl. Poultry Res. 13: 433-442.
- Rishi, P., C.L. Woodward, W-K. Kim, and S.C. Ricke. 2004. Salmonella enterica serovar Typhimurium hilA-lacZY fusion gene response to iron chelation or supplementation in rich and minimal media. J. Environ. Sci. Health B39: 861-870.
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Progress 01/01/03 to 12/31/03
Outputs
This study was conducted to test if transposon footprinting could be used to identify transposon mutants of Salmonella typhimurium with growth defects in a media containing short-chain fatty acids (SCFA) as the test selective condition. High concentrations of SCFA are one of the characteristic conditions in the animal intestine that has been suggested to play a role in inhibiting colonization by nonindigenous bacterial pathogens. When the mutant pools containing 25 Tn5 mutants/pool were analyzed for transposon footprints before and after selection, a polymerase chain reaction (PCR) product could be identified that was present in an input pool, but not in a corresponding output pool. The results indicate that transposon footprinting can be used for negative screening of genes sensitive to SCFA in the S. typhimurium bacterial genome.
Impacts
Foodborne salmonellae continues to be difficult to limit in preharvest food production. New control strategies will require more extensive genetic analysis. Transposon footprinting is expected to facilitate the comprehensive functional analysis of bacterial genome for the genes that are required to increase fitness to a variety of selective conditions. When this technique is conducted with a transposon that harbors an inducible promoter facing outwardly, it would allow efficient identification of genes for Salmonella adherence and invasion. The elucidation of the proteins encoded by the genes identified in this study could provide new targets for novel vaccine development and preventative strategies. This approach is technically very simple and does not require any prior sequence information of the genome to be analyzed. Now that bacterial genome sequencing databases are available for S. Typhimurium this may help to facilitate identification of the genes that
correspond to optimal targets to design vaccine strains.
Publications
- Churi, A., I.B. Zabala-Diaz, and S.C. Ricke. 2003. PCR application for rapid detection of virulence state in Salmonella Typhimurium. Poultry Sci. 82 (Suppl. 1): 41.
- Cox, M.M, R.L. Ziprin, L.F. Kubena, D.J. Nisbet, S.C. Ricke and Y.M. Kwon. 2003. Functional analysis of Salmonella genome for virulence genes using transposon sequence tag profiles. Poultry Sci. 82 (Suppl. 1): 61.
- Kundinger, M.M., I.B Zabala-Diaz, R.W. Moore, and S.C. Ricke. 2003. Salmonella Typhimurium growth and virulence response to Maillard reaction products. Poultry Sci. 82 (Suppl.1): 61.
- Kwon, Y.M., L.F. Kubena, D.J. Nisbet, and S.C. Ricke. 2003. Genetic screening for identification of Salmonella typhimurium Tn5 mutants with hypersensitivity to short-chain fatty acids. J. Rapid Meth. Automation Microbiol. 11: 89-95.
- Li, X. and S.C. Ricke. 2003. A suicide vector for constructing a lysA insertion mutation in Escherichia coli. J. Rapid Meth. Automation Microbiol. 10: 281-290.
- Li, X. and S.C. Ricke. 2003. Characterization of Escherichia coli lysA insertion targeted mutant using phenotype arrays. Bioresource Technol. 89: 249-253.
- Li, X. and S.C. Ricke. 2003.Generation of an Escherichia coli lysA targeted deletion mutant by double cross-over recombination for potential use in a bacterial growth-based lysine assay. Letts. Appl. Microbiol. 36: 458-462.
- Nutt, J.D., S.D. Pillai, C.L. Woodward, K.L. Sternes, I.B. Zabala-Diaz, Y.M. Kwon, and S.C. Ricke. 2003. Use of a Salmonella Typhimurium hilA fusion strain to assess effects of environmental fresh water sources on virulence gene expression. Water Research 37:2219-3326.
- Nutt, J.D., X. Li, I.B. Zabala-Diaz, C.L. Woodward, and S.C. Ricke. 2003. Development of a microtiter-based assay for assessment in Salmonella Typhimurium virulence response exposed to fresh produce extracts. J. Rapid Meth. Automation Microbiol. 11:1-9.
- Zabala-Diaz, I.B. and S.C. Ricke. 2003. Influence of short chain fatty acids and lysine on Salmonella Typhimurium cadA expression. Antonie von Leeuwenhoek (In press).
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Progress 01/01/02 to 12/31/02
Outputs
Salmonellosis is one of the most common foodborne diseases in the United States. There is a tremendous need to apply molecular approaches to develop better and more specifically targeted control measures at the preharvest stage of food production. With more specific targets Salmonella it becomes easier to control these organisms without compromising beneficial microflora. The main goal of this project is to use a novel approach for screening the genome of Salmonella typhmurium for the virulence genes that are required for survival within live host animals. We have devised a simple method, termed transposon footprinting, for functional screening of the bacterial genome. In this project, we will use mariner-based transposon mutants of S. typhimurium as a model system to take advantage of the feasibility of transposon footprinting for a thorough identification of the essential genes required for in vitro growth as well as the virulence genes required for in vivo
survival. In addition, this approach is being used to conduct a genome-wide comparison of the virulence genes for in vivo survival between different host animals or tissues. It is expected that our procedure would reveal the essential genes that are important in basic cellular processes and the virulence genes that are involved in various features of the bacterial pathogenesis including host-adaptation or tissue-tropism. These studies will provide insights into the molecular mechanisms governing the pathogenesis for foodborne S. typhimurium and also have practical food safety applications for the development of vaccine, antimicrobial agents, and preventive strategies.
Impacts
The idea here is to screen the genome of S. typhimurium for the virulence genes that are required for survival within live host animals using a novel approach. In this project we will use mariner-based transposon mutants of S. typhimurium as a model system to exploit the feasibility of footprinting in the comprehensive identification of essential genes that are required for in vitro growth as well as the virulence genes that are required for in vivo survival.
Publications
- Nutt, J.D.*, L.F. Kubena, D.J. Nisbet, and S.C. Ricke. 2002. Virulence response of a Salmonella Typhimurium hilA fusion strain to spent media from pure cultures of selected bacteria and poultry cecal mixed culture. J. Food Safety 22: 169-181.
- Nutt, J.D.*, K.L. Medvedev, C.L. Woodward, S.D. Pillai, and S.C. Ricke. 2002. Assessment of laboratory media controls for determining Salmonella virulence potential of poultry water sources using a hilA:lacZY fusion strain. J. Rapid Meth. Automation Microbiol. 10: 173-184.
- Park, S.Y.*, Y.M. Kwon, S.G. Birkhold, L.F. Kubena, D.J. Nisbet, and S.C. Ricke. 2002. Application of polymerase chain reaction and a novel transposon footprinting technique for rapid identification of Salmonella typhimurium Tn5 mutants required for survival under desiccation stress. J. Rapid Meth. Automation Microbiol. 10: 197-206.
- Kwon, Y.M., L.F. Kubena, D.J. Nisbet, and S.C. Ricke. 2003. Isolation of Salmonella typhimurium Tn5 mutants defective for survival on egg shell surface using transposon footprinting. J. Environ. Sci. Health B38: 103-110.
- Kwon, Y.M., L.F. Kubena, D.J. Nisbet, and S.C. Ricke. 2002. Functional screening of bacterial genome for virulence genes by transposon footprinting. Meths. Enzymol., Bacterial Pathogenesis, Part C, V.L. Clark and P.M. Bavoil (eds.), Vol. 358: 141-152.
- Ricke, S.C.. 2002. Concepts for understanding Salmonella spp. ecology in anaerobic animal waste environments. In: Concise Encyclopedia of Bioresource Technology, A. Pandey (Ed.), (Submitted).
- Ricke, S.C. 2002. The gastrointestinal tract ecology of Salmonella Enteritidis colonization in molting hens. Poultry Sci. (Accepted).
- Ricke, S.C. 2002. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry Sci. (Accepted).
- Kundinger, M.M.*, R.W. Moore, I.B. Zabala-Diaz*, J.A. Byrd, D.J. Nisbet, and S.C. Ricke. 2003. Effect of spent media from a PREEMPT(TM) continuous flow culture on Salmonella Typhimurium virulence expression. Southern Poultry Science Society Annual Meeting, Atlanta, GA.
- Howard, Z.R.*, K.L. Medvedev,*, R.W. Moore, S.G. Birkhold, and S.C. Ricke. 2003. Affect of storage time on growth of Salmonella Typhimurium in egg components. Southern Poultry Science Society Annual Meeting, Atlanta, GA.
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