Objective 1: Develop rapid and effective means to separate and concentrate targeted pathogens from food matrices that can be coupled to very rapid detection methods such as real-time PCR. 1A. Develop filtration/centrifugation methods for separating and concentrating pathogenic Escherichia, Salmonella, Listeria, and Campylobacter spp. from a variety of food matrices. Optimize reagents, apparatus and conditions to achieve maximum speed and recovery with minimum detection limits. 1B. Develop DNA extraction methods providing rapid, efficient, unbiased recovery of inhibitor-free DNA from a variety of pathogens.
Objective 2: Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens. 2A. Detection of foodborne threat agents (model system- pathogenic Yersinia spp.). 2B. Isolation and detection of foodborne pathogens maintaining mobile genetic elements. 2C. Enrichment of pathogens while maintaining mobile genetic elements.
Objective 3: Develop protein- and nucleic acid-based methods for the multiplexed detection and characterization of food-borne pathogens. 3A. Protein-based microarray and other multiplexed methods for the analysis of foodborne pathogenic bacteria. 3B. Oligonucleotide-based microarray for multiple pathogen detection and characterization. 3C. Multiplex real-time PCR for multiple pathogen identification and quantification.
Objective 4: Develop typing methods for pathogens of concern to associated food regulatory agencies. 4A. Develop Restriction Fragment Sequence Polymorphism method for typing. 4B. Fractionation of a naÃ¯ve library of biorecognition elements for bacterial typing-An alternative to "molecular typing."
Approach: This project plan has multiple goals that are distinct yet may be combined to generate improved, rapid techniques for the analysis of foodborne pathogenic bacteria (e.g., Campylobacter, E. coli, Listeria, Salmonella, and Yersinia spp.). Compared to traditional plate culture techniques, rapid methods for bacterial detection and typing [identification] primarily suffer from relatively poor sensitivity and/or selectivity. To improve detection limits for oligonucleotides using DNA microarray or multiplex RT-PCR, improved methods for DNA extraction, including an optimized alkaline/detergent reagent, will be developed for efficient extraction of nucleic acid from bacteria. Leukocyte removal filters will be used to separate bacteria from food matrices and concentrate the cells allowing for improved limits of detection by antibody microarray and/or time-resolved fluorescence. Culture enrichment conditions (e.g., slightly acidic pH, millimolar concentrations of calcium ion, with or without Irgasan) will be initially optimized for a model pathogenic bacterium (Yersinia) with the intent of concentrating the bacteria from the sample while maintaining mobile genetic elements [plasmids] required for expression of key genotypic and phenotypic markers. Prior to detection/typing with biosensor platforms, enriched Yersinia spp. will be carefully isolated and assessed for maintenance of virulence plasmids using organic dyes (crystal violet and/or Congo red) in conjunction with low calcium plating media. Novel biorecognition elements (initially, single chain variable fragment antibodies fractionated from naive phage display libraries) will be custom generated to improve accuracy of biosensor-based detection or phenotyping platforms (e.g., microarrays) for targeted pathogens. In addition, an abbreviated restriction fragment sequence polymorphism method will be developed and assessed as a novel genotyping method. Promising technologies will be directed towards usage by food producers and regulatory agencies for food safety monitoring and follow-up investigations.
Radiation resistance and loss of crystal violet binding activity in Yersinia enterocolitica suspended in raw fround pork exposed to gamma radiation and modified atmosphere
Bhaduri S, Sheen S, Sommers CH.
J Food Sci. 2014 May;79(5):M911-6.
A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins
Gehring A, He X, Fratamico P, Lee J, Bagi L, Brewster J, Paoli G, He Y, Xie Y, Skinner C, Barnett C, Harris D.
Optimization and application of a custom microarray for the detection and genotyping of E. coli O157:H7 in fresh meat samples
Suo B, He Y, Irwin PL, Gehring AG
Food Anal Methods. 2013 Oct;6(5):1477-84.
Rapid O serogroup identification of the six clinically relevant Shiga toxin-producing Escherichia coli by antibody microarray
Hegde NV, Praul C, Gehring A, Fratamico P, Debroy C.
J Microbiol Methods. 2013 Jun;93(3):273-6.
A high-throughput antibody-based microarray typing platform
Gehring A, Barnett C, Chu T, Debroy C, D'Souza D, Eaker S, Fratamico P, Gillespie B, Hegde N, Jones K, Lin J, Oliver S, Paoli G, Perera A, Uknalis J
Sensors. 2013 May 3;13(5):5737-48.
Growth of a pYV-bearing Yersinia pestis KIM5 in retail raw ground pork
Bhaduri S, Phillips JG.
Foodborne Pathog Dis. 2013 May;10(5):467-71.
Comparison of antibodies raised against heat- and gamma radiation-killed bacteria
Gehring AG, Boyd G, Brewster JD, Irwin PL, Thayer DW, Houten LJV.
J Microb Biochem Technol. 2012 Dec 26. [Epub ahead of print]
Rapid identification and classification of Staphylococcus aureus by attenuated total reflectance fourier transform infrared spectroscopy
Xie Y, Xu S, Hu Y, Chen W, He Y, Shi X.
J Food Safety. 2012 May;32(2):176-83.
Genotypes and toxin gene profiles of Staphylococcus aureus clinical isolates from china
Xie Y, He Y, Gehring A, Hu Y, Li Q, Tu SI, Shi X.
PloS One. 2011 Dec 15. [Epub ahead of print]
Modification of an acetone-sodium dodecyl sulfate disruption method for cellular protein extraction from neurotoxigenic Clostridium botulinum
Foodborne Pathog Dis. ;9(2):172-4.
A robust multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes in fresh fruits and vegetables - (Abstract Only)
Simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes in a variety of cheeses and spinach using a multiplex real-time PCR method - (Abstract Only)
Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates - (Book / Chapter)
He, Y., Xie, Y., Reed, S.A. 2013. Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates. In: Walker, John M. Methods in Molecular Biology. New York, NY:Humana Press. p. 103-111.
High-throughput antibody microarray for bacteria and toxins - (Abstract Only)
Yersinia enterocolitica - (Book / Chapter)
Bhaduri, S. 2014. Yersinia enterocolitica. Encyclopedia of Food Microbiology. In: Batt, C.A., Tortorello, M.L. (Eds.), Elsevier Ltd, Academic Press. Vol 1. pp. 838-848.
A multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes in soft cheese - (Abstract Only)
Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for....food - (Review Article)