RAPID DETECTION OF FOODBORNE PATHOGENS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0181571
• Project Start Date: Oct 1, 2008
• Project End Date: Sep 30, 2013
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Food Science

Non Technical Summary
Foodborne diseases cause approximately 76 million illnesses with 5000 deaths annually in the US. Of these, L. monocytogenes causes about 2,500 illnesses and 500 deaths while E. coli O157:H7 causes 73,500 illnesses and 61 deaths annually. Salmonella enterica causes gastroenteritis in humans and nearly 1.4 million people in the US are infected annually, resulting in approximately 16,000 hospitalizations and over 500 deaths. The economic impact of salmonellosis in the US is substantial, with annual costs of 0.5 to 2.3 billion dollars. Immunologically challenged individuals, children and pregnant women, are most susceptible to L. monocytogenes infection while E. coli affects mostly the children. Furthermore, these three are among the top five foodborne pathogens that are of significant concern and currently the US government has implemented a zero tolerance policy for these pathogens in ready-to-eat foods. Much of these foodborne illnesses could be avoided if sensitive and dependable accurate detection tools are available. Furthermore, the "bottleneck" of a rapid detection method is sample preparation. Often the concentration of the target organisms are very low and due to the complex nature of food, it is difficult to extract bacterial cells or toxins from food without exhaustive sample preparation. We propose to aid in the sample preparation strategy by improving our sample preparation device, PED system and by developing improved medium for enrichment of multipathogens and thereby facilitating detection by biosensors. A majority of the biosensor tools reported in the literature are capable of detecting pure bacterial cultures in an artificial media; however, their usefulness with real-world food samples has not been thoroughly investigated yet. From our ongoing project we have shown that fiber-optic, cell-based, and optical light scattering sensors can be used with food samples and these sensors will be modified to allow detection of multipathogens on a single device to provide total microbiological safety of a product in a cost effective manner.

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
712	3260	1100	20%
712	3270	1100	20%
712	3320	1100	20%
712	3450	1100	20%
712	4010	1040	20%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3320 - Meat, beef cattle; 3260 - Poultry meat; 4010 - Bacteria; 3270 - Eggs; 3450 - Milk;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

listeria monocytogenes

e. coli o157:h7

salmonella enterica

multipathogen detection

biosensors

sample preparation

media development

light scattering sensor

fiber optic sensor

microfluidic biochip

cell based sensor

Goals / Objectives
To develop multipathogen biosensor tools for sensitive detection of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella and others from food to ensure total microbiological safety of a product. Methods for rapid detection of low concentrations of bacterial pathogens or toxins are highly desirable. Current trend emphasizes multipathogen detection on a single assay platform. This would allow analysis of a single product for the presence of multiple pathogens to ensure total microbiological safety. Moreover, multipathogen detection approach would reduce cost per test as well as assay time. Certain biosensor platforms developed over the years in our laboratory are currently being modified to allow multipathogen detection. These sensors include mammalian cell-based sensor to detect varieties of live pathogens or active toxins, fiber optic sensor and protein biochip to detect pathogens with the aid of antibody or receptor protein, and optical forward light scattering sensor for label-free detection of live bacterial cells. Currently several pathogens are being targeted for detection including Listeria monocytogenes, Escherichia coil including O157:H7, Salmonella, Bacillus spp., Staphylococcus spp., and Vibrio spp. In addition, we continue to improve the sample preparation method to allow separation and concentration of target pathogens from food using immunomagnetic separation and a self-contained pathogen enrichment device (PED). Once completed this research would allow us to develop multipathogen detection sensors to ensure total microbiological safety of a product.

Project Methods
Sample preparation and media development: In the last 5 years, we have designed and built a simple, inexpensive, hand-held sample preparation device called, PED (Pathogen Enrichment Device) to allow bacterial separation from complex food matrices. Our goal is to continue to improve the device to accommodate larger sample sizes (25-65 g) and also to separate bacteria from more complex samples like milk, soil and fecal maters for application with multiplex biosensor platforms. Fiber optic biosensor: The fiber optic sensor instrument is equipped with four sample testing channels thus would allow the detection of four separate pathogens from a given sample. The optical fibers will be coated with pathogen specific antibody or receptors to capture target pathogens and exposed to samples. Subsequently, pathogen-specific second antibody previously conjugated with a fluorophor (Cy5 or Alexa-Flour 647) will be added. The formation of antigen-antibody sandwich will emit fluorescence light that will generate evanescent wave, which will be detected by a laser detector. Cell-based sensor: As part of infection process, pathogens/toxins interact with mammalian cells in body resulting in cell damage. As a result, intracellular enzyme such as alkaline phosphatase is released, which could be sensitively detected in a plate reader. Recently, we have demonstrated that collagen matrix can be used to immobilize mammalian cells in a 96-well plate and this could be used to test a large number of samples at a time. We plan to continue this research by optimizing the assay by testing with samples of food, water and beverages artificially spiked with various pathogens and toxins. Beside food safety applications, this technology could potentially be used by first responders in food defense to test for the presence of potential toxic agents in food or water. Microfluidic biochips: Microfabricated electronic devices, such as a semiconductor chips are often referred to as biochips and is used for monitoring the presence of target pathogen in a sample. In the past, we have used antibody for capture of bacteria on the chip; however to improve sensitivity and specificity we plan to use specific receptor such as Hsp60 that is commonly used by pathogens during infection of the host to capture and detect on biochip. Optical light scattering sensor: We have developed a laser-based sensor called BARDOT (BActerial Rapid Detection using Optical Technology) for detection and identification of bacterial colonies growing on a Petri dish. BARDOT was able to discriminate different bacterial species within a genus. Our goal is to create a robust scatter image library of bacterial colonies from different genera including Listeria, Staphylococcus, Salmonella, Vibrio and Escherichia, Campylobacter, Enterococcus and others. In addition, we plan to optimize procedure for detection of foodborne bacterial pathogens from food using BARDOT.

Progress 10/01/08 to 09/30/13

Outputs
Target Audience: Food safety professionals, educators, studentsand industry personnel Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduateand undergraduate students, postdoctoral scientists, and visiting international scientistis and scholars received training in this project. This project also helpeddevolop collaboration and scientific exchange between our group andseveral national and internationalinstitutions and biotechnology companies. How have the results been disseminated to communities of interest? Through peer-reviewed journal article publication, conference presentation, demo of equipment and device to scientist and users. The light scattering sensor has been patented and it is licensed to a company who is currently marketing this technology for pathogen testing for food safety application. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the past five years our team focused on improving the sensitivity and specificity of biosensor tools for detection of foodborne pathogens. We also developed three high throughput sensor platforms for multiple pathogens and toxins at a time to allow for total microbiological safety assessment and potentially to make the microbiological testing less expensive. To accomplish such goals we developed (i) a multipathogen enrichment broth, SEL (Salmonella, E. coli and Listeria), for simultaneous growth of pathogens and (ii) various pathogen-specific biorecognition molecules including, antibodies, aptamers, and mammalian cell receptor proteins. SEL was used for enrichment of L. monocytogenes, E. coli O157:H7 and Salmonella enterica from food matrices and detected using various sensor platforms. The biorecognition molecules were used in both pathogen concentration using paramagnetic beads and on biosensor platforms. In addition, the pathogen enrichment device (PED) was successful in separating pathogens from food matrices before analysis by biosensors. The sensor platforms that were developed and optimized during this period include (i) fiber optic immunosensor, (ii) light scattering sensor and (iii) mammalian cell-based sensor. Fiber optic sensor was developed for detection of the three major pathogens using specific biorecognition molecules. The laser based light scattering sensor can detect specific bacterial pathogens while growing on an agar plate based on their scattering properties without the need for any biorecognition molecules. Cell-based sensor allows the detection of varieties of pathogens and toxins for rapid screening of food and water. This system monitors interaction of live pathogens or active toxins with live mammalian cells and highly efficient for detection of low amounts of harmful target analytes. In addition, we also developed methods to control pathogens using antimicrobial coated nanoparticles on products and probiotics in mammalian host.

Publications

• Type: Book Chapters Status: Published Year Published: 2013 Citation: Bhunia, A.K. 2013. Forthcoming New Technologies for Microbial Detection. In Guide to Foodborne Pathogens, Second Edition, R.G. Labbe and S. Garcia (ed), Wiley-Blackwell, Oxford, UK. Chapter 25; pp 414-421.
• Type: Book Chapters Status: Published Year Published: 2013 Citation: Burkholder, K.M., and Bhunia, A.K. 2013. Listeria monocytogenes and Host Hsp60 - an Invasive Pairing. In Moonlighting Cell Stress Proteins in Microbial Infections, B. Henderson (Ed), Chapter 17, Springer, pp 267-282.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Amalaradjou, M.A.R., Singh, A.K., Dikshit, T., and Bhunia, A. K. 2013. Designer probiotic expressing Listeria adhesion protein protects mice from Listeria monocytogenes infection. Institute of Food Technologists (IFT) Annual Meeting, Chicago, IL, July 13-16, 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Abdelhaseib, M., Amalaradjou, M.A.R., Singh, A.K., Bae, E., and Bhunia, A. K. 2013. Optical sensor and immunoassay-based detection of Salmonella Enteritidis and Salmonella Typhimurium. Institute of Food Technologists (IFT) Annual Meeting, Chicago, IL, July 13-16, 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Singh, A.K., Abdelhaseib, M., Bae, E., and Bhunia, A. K. 2013. Real-Time, on-plate rapid detection and identification of Bacillus cereus-group using light scattering sensor. 113th General Meeting of American Society for Microbiology, May 18-21, 2013, Denver, CO. Abst# 1161
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Krishna K. Mishra, T. Bailey, H. Kim, T. Dikshit, Arun K. Bhunia. 2013. SecA2 aids in secretion of proteins involved in both pathogenesis and house-keeping function in pathogenic and nonpathogenic bacteria.113th General Meeting of American Society for Microbiology, May 18-21, 2013, Denver, CO. Abst # 1341
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Ohk, S-H., and Bhunia, A.K. 2013. Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to- eat meat samples. Food Microbiol.33:166-171.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Ahmed, W.M., Bayraktar, B., Bhunia, A.K., Hirleman, E.D., Robinson, J.P., and Rajwa, B. 2013. Classification of Bacterial Contamination Using Image Processing and Distributed Computing. IEEE Journal of Biomedical and Health Informatics 17:232-239.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Amalaradjou, M.A.R., and Bhunia, A.K. 2013. Bioengineered probiotics, a strategic approach to control enteric infections. Bioengineered 4 (6):379-387
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Kim, H., Bai, N., Bhunia, A.K., King, G.B., Hirleman, E.D., and Bae, E. 2013. Development of an integrated optical analyzer for characterization of growth dynamics of bacterial colonies. Journal of Biophotonics 6:929-937
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Kim, H., and Bhunia, A.K. 2013. Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier. Gut Pathogens 5:16.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Li, X., Ximenes, E., Amalaradjou, M. A. R., Vibbert, H.B., Foster, K., Jones, J., Liu, X., Bhunia, A.K. and Ladisch, M.R. 2013. Rapid Sample Processing for Foodborne Pathogen Detection via Crossflow Microfiltration. Applied and Environmental Microbiology 79:7048-7054
• Type: Book Chapters Status: Published Year Published: 2013 Citation: Bae, E. and Bhunia, A.K. 2013. Nano Optical Sensors for Food Safety and Security, In Optochemical Nanosensors, Editor, A. Cusano, F. J. Arregui, M. Giordano, and A. Cutolo. CRC Press, Taylor and Francis Group, Boca Raton, FL., Chapter 19, pp 497-512.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Improvement in sensor developments and control of pathogens were the two major focus of our group. Ability of light scattering sensor and fiber-optic sensors to detect and identify different serovars of Salmonella, Listeria monocytogenes and Shiga-toxin producing E. coli (STEC) separately or together were evaluated. In addition, we also developed and evaluated antibody and other recognition molecules for specific detection of L. monocytogenes from food using paramagnetic beads (PMB), sensors, and control of this pathogen using probiotics and antimicrobial coated nanoparticles. PARTICIPANTS: Titiksha Dikshit Rishi Drolia Mohammed Elfaki Atul Singh Mary Anne Roshni Amalaradjou Marcelo Mendoca Euiwon Bae Huisung Kim Bartek Rajwa J. Paul Robinson TARGET AUDIENCES: Scientists and food safety researchers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The light scattering sensor was able to identify and classify the colonies of 32 serovars of Salmonella and seven most important sorovars of STEC strains growing on selective media in real-time. Serovar classification based on scatter pattern was in total agreement with genetic fingerprints. Light scattering sensor also successfully detected Salmonella from food samples. With improved antibody and recognition molecules, PMB coupled with fiber-optic sensor was able to detect very low levels (102 cfu) of L. monocytogenes. Furthermore, L. monocytogenes-specific biorecognition molecules also provided novel strategies for its control using probiotics or antimicrobial peptide coated nanoparticles.

Publications

• Ohk, S-H., and Bhunia, A.K. 2013. Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to- eat meat samples. Food Microbiol. 33:166-171
• Huff, K., Aroonnual, A., Fleishman Littlejohn, A.E., Rajwa, B., Bae, E., Banada, P.P., Patsekin, V., Hirleman, E.D., Robinson, J.P., Richards, G.P., and Bhunia, A.K. 2012. Light-scattering sensor for real-time identification of Vibrio parahaemolyticus, V. vulnificus, and V. cholerae colonies on solid agar plate. Microb. Biotechnol. 5(5):607-620.
• Bhunia, A.K. 2012. Bioengineered Probiotics-A Solution to Broaden Probiotics Efficacy! J. Nutr. Food Sci. 2:3: 1000e105
• Mialon, M., Tang, Y., Singh, A.K., Bae, E., and Bhunia, A.K. 2012. Effects of preparation and storage of agar media on the sensitivity of bacterial forward scattering patterns. Open J. Appl. Biosens. 1: 26-35.
• Amalaradjou, M. A. R., and A. K. Bhunia. 2012. Modern approaches in probiotics research to control foodborne pathogens. Adv. Food Nutr. Res. 67:185-239
• Salm, E., D. Marchwiany, D., Liu, Y.S., Morisette, D., He, Y., Bhunia, A., and Bashir, R. 2011. Electrical detection of dsDNA and polymerase chain reaction amplification. Biomed. Microdev. 13 (6): 973-982
• Koo, O.K., Amalaradjou, M.A.R., and Bhunia, A.K. 2012.Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro. PLoS One 7 (1): e29277
• Gehring, A.G., Albin, D.M., Bhunia, A.K., Kim, H., Reed, S.A., and Tu, S-I. 2012. Mixed culture enrichment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, and Yersinia enterocolitica, Food Control 26:269-273.
• Bhunia, A.K., Bae, E., Rajwa, B., Robinson, J.P. and Hirleman, E.D. 2011. Utilization of optical forward scatter image biological database: food-borne pathogen colony differentiation and detection. In Omics Technologies and Microbial Modeling in Food-borne Pathogens Study. Editors: Yan, X., Juneja, V., Huang, L. DEStech Publications, Inc. Lancaster, PA. pp 599-624.
• Bae, E. and A. K. Bhunia. 2012. Nano Optical Sensors for Food Safety and Security, In Optochemical Nanosensors, Editor, M. Giordano, Wiley Interscience, pp 491-506.
• Kim, H., M.A.R. Amalaradjou, K.H. Kim, and A.K. Bhunia. 2012. Listeria Adhesion Protein Induces Epithelial Tight Junction Compromise through Activation of NF-κB and Down Regulation of Tight Junction Proteins. 112th American Society for Microbiology General Meeting, San Francisco, CA. June 16-19, 2012, Poster # 1745.
• Amalaradjou, M.A.R., Singh, A.K., Bhunia, A.K. 2012. Label-free detection of Escherichia coli O157:H7 and Listeria monocytogenes from food samples using light-scattering sensor. Institute of Food Technologists (IFT) Annual meeting, Las Vegas, NV, June 25-28, 2012. Abst # 262-05.
• Amalaradjou, M.A.R., Singh, A.K., Bhunia, A.K. 2012. Application of a Listerial housekeeping protein for its detection and targeted inactivation. Institute of Food Technologists (IFT) Annual meeting, Las Vegas, NV, June 25-28, 2012. Abst # 179-04
• Mendonca, M., N.L. Conrad, K.S. Mendonca, F.R. Conceicao, A.N. Moreira, W.P. da Silva, J.A. Guimaraes Aleixo, and A.K. Bhunia. 2012 Confirmation of immunomagnetic captured Listeria monocytogenes by immunomagnetic separation using an optical light-scattering sensor. IUFoST, Foz do Iguacu, Parana State, Brazil, Aug 5-9, 2012.
• Mendonca, M., W.P. da Silva, N.L. Conrad, R.C. Franca, F.R. Conceicao, A.N. Moreira, H. Kim, and A.K. Bhunia, J.A. Guimaraes Aleixo. A novel monoclonal antibody with high specificity towards genus Listeria. IUFoST, Foz do Iguacu, Parana State, Brazil, Aug 5-9, 2012.
• Mendonca, M., Conrad, N.L., Conceicao, F.R., Moreira, A.N., da Silva, W.P., Aleixo, J.A.G., and Bhunia, A.K. 2012. Highly specific fiber optic immunosensor coupled with immunomagnetic separation for detection of low levels of Listeria monocytogenes and L. ivanovii. BMC Microbiol. 12:275
• Bae, E., Patsekin, V., Rajwa, B., Bhunia, A.K., Hirleman, E.D., Holdman, C., and Robinson, J.P. 2011. Development of a microbial high-throughput screening instrument based on elastic light scatter patterns. Rev. Scientific Instruments, 83:044304.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Improvement in sensor developments and control of pathogens were major focus of our group. Ability of light scattering sensor and fiber-optic sensors to detect and identify different serovars of Salmonella, Listeria monocytogenes and Shiga-toxin producing E. coli (STEC) were evaluated. In addition, we also developed and evaluated antibody and other recognition molecules for specific detection of L. monocytogenes from food using immunomagnetic beads (IMB), sensors, and control of this pathogen using probiotics and antimicrobial coated nanoparticles. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Food scientists and food safety experts. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The light scattering sensor was able to identify and classify the colonies of top eight serovars of Salmonella and seven most important sorovars of STEC strains growing on selective media in real-time. Serovar classification based on scatter pattern was in total agreement with genetic fingerprints. With improved antibody and recognition molecules, IMB coupled with fiber-optic sensor was able to detect very low levels (102 cfu) of L. monocytogenes. Furthermore, L. monocytogenes-specific biorecognition molecules also provided novel strategies for its control using probiotics or antimicrobial peptide coated nanoparticles.

Publications

• Rajwa, B., Dundar, M.M., Akova, F., Bettasso, A., Patsekin, V., Hirleman, E.D, Bhunia, A.K., Robinson, J.P. 2010. Discovering unknown: detection of emerging pathogens using label-free light scattering system, Cytometry Part A,77A: 1103-1112.
• Burkholder, K.M. and Bhunia, A.K. 2010. Listeria monocytogenes uses LAP to promote bacterial transepithelial translocation, and induces expression of LAP receptor Hsp60. Infect. Immun. 78(12): 5062-5073
• Bae, E., Aroonnual, A., Bhunia, A.K., and Hirleman, E.D. 2011. On the sensitivity of forward scattering patterns from bacterial colonies to media composition. J. Biophoton 4:236-243.
• Bae, E., Bai, N., Aroonnual, A., Bhunia, A.K., and Hirleman, E.D., 2011. Label-free identification of bacterial microcolonies via elastic scattering. Biotechnol. Bioeng. 108(3):637-644.
• Goodridge, L.D., Fratamico, P., Christensen, L.S., Hoorfar, J., Griffiths, M., Carter, M., Bhunia, A.K., O Kennedy, R. 2011. Detection technologies: strengths and shortcomings. In Rapid Detection, Characterization and Enumeration of Foodborne Pathogens. Editor, J. Hoorfar. ASM Press, Washington DC.
• Bai, N., Y. Tang, A. K. Bhunia, E. D. Hirleman, E. Bae. 2011. Characterization of optical properties of bacterial micro-colonies via the comprehensive morphology analyzer. SPIE Proceedings.
• Rajwa, B., M. M. Dundar, F. Akova, V. Patsekin, E. Bae, Y. Tang, J.E. Dietz, E. D. Hirleman, J. P. Robinson, and A.K. Bhunia. 2011. Digital microbiology: detection and classification of unknown bacterial pathogens using a label-free laser light scatter-sensing system. SPIE proceedings.
• Mendonca, M., Mishra, K.K., and Bhunia, A.K. 2011. Acetaldehyde Dehydrogenase Domain of the Listeria Adhesion Protein in L. monocytogenes is Responsible for Adherence to ileocecal HCT-8 cell line. American Society for Microbiology General Meeting, New Orleans, LA. Abstract # 11-GM-A-3379-ASM.
• Tang, Y., Singh, A.K., Bae, E., Hirleman, E.D., and Bhunia, A.K. 2011. Differentiation of Serovars of Escherichia coli Colonies Using Optical Forward-scattering Sensor. American Society for Microbiology General Meeting, New Orleans, LA. Abstract # 11-GM-A- 3717 -ASM.
• Amalaradjou, M. A .R., Koo, O.K., and Bhunia, A.K. 2011. Lactobacillus paracasei Expressing LAP Reduces Cell Invasion, Translocation and Cytotoxicity of L. monocytogenes in Caco-2 Cells. American Society for Microbiology General Meeting, New Orleans, LA. Abstract #11-GM-A-2431-ASM
• Fleishman Littlejohn, A., Bigando, A., Bae, E., and Bhunia, A.K. 2011. Light-scattering sensor for real-time identification of Vibrio parahaemolyticus and V. vulnificus colonies. American Society for Microbiology General Meeting, New Orleans, LA. Abstract #11-GM-A-2378-ASM
• Kim, H., Christina, B., and Bhunia, A.K. 2011. Influence of secreted Listeria Adhesion Protein (LAP) on transepithelial translocation and cytokine release during Listeria infection in Caco-2 cell model. Institute of Food Technologist Annual Meeting, New Orleans, LA. Abstract # 037-01, page #19.
• Singh A.K., Amalaradjou M.A.R., Bhunia A.K. 2011. Synthesis and assessment of peptide functionalized gold nanoparticles as a Biocompatible antilisterial agent. 51st Interscience conference on Antimicrobial Agents and Chemotherapy. Sept 17-20, 2011, Chicago. Poster no. F1-1356/229
• Medina, S. 2011. Effect of Listeria monocytogenes pre-exposure to mammalian cells under anaerobic environment in LAP-mediated pathogenesis in subsequent infection under anaerobic conditions. Purdue University. MS Thesis, p. 60.
• Bhunia, A.K. 2011. Rapid pathogen screening tools for food safety. Food Technol. 65(2):38-43.
• Sirsat, S.A., Burkholder, K.M., Dowd, S.E., Muthaiyan, A., Bhunia, A.K., and Ricke, S.C. 2011. Influence of microbial and host cell sublethal heat stress on S. Typhimurium gene expression and adhesion to Caco-2 cell line. J. Appl. Microbiol. 110: 813-822.
• Bi, L., Yang, L., Narsimhan, G., Bhunia, A.K., and Yao, Y. 2011. Carbohydrate nanoparticle-mediated delivery for prolonged efficacy of antimicrobial peptide. J. Control. Release 150: 150-156.
• Bi, L., Yang, L., Bhunia, A.K., and Yao, Y. 2011. Carbohydrate nanoparticle-mediated colloidal assembly for prolonged efficacy of bacteriocin against food pathogen. Biotechnol. Bioeng. 108 (7): 1529-1536.
• Koo, O.K., Aroonnual, A., and Bhunia, A.K. 2011. Human heat shock protein 60 receptor coated paramagnetic beads show improved capture of Listeria monocytogenes in presence of other Listeria in food. J. Appl. Microbiol. 111: 93-104.
• Jagadeesan, B., Fleishman Littlejohn, A.E., M.A.R. Amalaradjou, A.K. Singh, Mishra, K, La, D., Kihara, D., and Bhunia, A.K. 2011. N-terminal Gly224-Gly411 domain in Listeria Adhesion Protein interacts with host receptor Hsp60. PLoS One.6 (6):e20694.
• Jaradat, ZW., Rashdan, A.M., Ababneh, Q.O., Jaradat, S.A., and Bhunia, A.K. 2011. Characterization of surface proteins of Cronobacter muytjensii using monoclonal antibodies and MALDI-TOF Mass spectrometry. BMC Microbiol 11:148.
• Mishra, K.K., Mendonca, M., Aroonnual, A., Burkholder, K.M. and Bhunia, A.K. 2011. Genetic organization and molecular characterization of secA2 locus in Listeria species. Gene 489:76-85.
• Hoorfer, J., Bhunia, A.K., Pagotto, F., Rudi, K., O Kennedy, R. 2011. Future trends in rapid methods. In Rapid Detection, Characterization and Enumeration of Foodborne Pathogens. Editor, J. Hoorfar. ASM Press, Washington DC.
• Malorny, B., Bhunia, A.K., Aarts, H.J.M., Lofstrom, C., and Hoorfar, J. 2011. Salmonella in pork, beef, poultry and egg. In Rapid detection, characterization and enumeration of food-borne pathogens. Editor, J. Hoorfar, ASM Press, Washington DC.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Improvement in sensor design and performance evaluations for pathogens and toxin continue to be the focus of our group. Ability of sensors to detect and identify multiple pathogens and toxins on a single sensor platform is essential to provide total microbiological quality of products and to reduce cost for product testing. During this reporting period, we optimized mammalian cell-based sensor and light scattering sensors for their ability to detect and identify multiple pathogens including Salmonella, E. coli, and Listeria, and various toxins from food products. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Microbiologists and food scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Mammalian cell-based sensor was able to detect nano-to pico-gram quantities of various microbial toxins or bacteria from inoculated meat, rice and dairy foods in 2-4 h. The light scattering sensor was able to identify and classify the colonies of different serovars of Salmonella and pathogenic Escherichia coli growing on Petri-dish in real-time. These multipathogen detection tools demonstrate promising application in rapid and specific detection of foodborne pathogens including Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella serovars in a cost-effective manner.

Publications

• Bueno, V.F.F., Banerjee, P., Banada, P.P., Mesquita, A.J., Marques, E.G.L. and Bhunia, A.K. 2010. Pathogenic potential and molecular characterization of L. monocytogenes isolates of food and clinical origin from Brazil. Int. J. Environ. Health Res. 20(1):43-59.
• Ohk, S.H., Koo, O.K., Sen, T., Yamamoto, C.M. and Bhunia, A.K. 2010. Antibody-aptamer functionalized fiber-optic biosensor for specific detection of Listeria monocytogenes from food. J. Appl. Microbiol. 109:808-817.
• Akova, F., Dundar, M., Jo Davisson, V., Hirleman, E.D., Bhunia, A.K., Robinson, J.P. and Rajwa, B. 2010. A machine-learning approach to detecting unknown bacterial serovars. Stat. Anal. Data Mining 3(5):289-301.
• Banerjee, P. and Bhunia, A.K. 2010. Cell-based biosensor for rapid screening of pathogens and toxins. Biosens. Bioelectron. 26:99-106.
• Banerjee, P., Franz, B. and Bhunia, A.K. 2010. Mammalian cell-based sensor system. Adv. Biochem. Eng. Biotechnol. 117:21-55.
• Jagadeesan, B., Koo, O.K., Kim, K.P., Burkholder, K.M., Mishra, K.K., Aroonnual, A. and Bhunia, A.K. 2010. LAP, an alcohol acetaldehyde dehydrogenase enzyme in Listeria promotes bacterial adhesion to enterocyte-like Caco-2 cells only in pathogenic species. Microbiology-SGM. 156 (9):2782-2795.
• Bae, E., N. Bai, Bhunia, A.K., Robinson, J.P. and Hirleman, E.D. 2010. Modeling light propagation through bacterial colonies and its correlation with forward scattering patterns. J. Biomed. Opt. 15(4):045001.
• Bhunia, A.K. 2010. Mammalian cell-based sensors: a rapid screening tool for pathogens and toxins in food. Food Eng. Ingred. 10: 15-17.
• Bhunia, A.K., Nanduri, V., Bae, E. and Hirleman, E.D., 2010. Biosensors, Foodborne Pathogen Detection. In Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology. Flickinger, M.C. (Ed), John Wiley & Sons, Inc., Hoboken, NJ.
• Dundar, M. M, Hirleman, E.D., Bhunia, A.K., Robinson, J.P., and Rajwa, B. 2009. Learning with a non-exhaustive training dataset: a case study: detection of bacteria cultures using optical-scattering technology. In Proceedings of the 15th ACM SIGKDD international conference on Knowledge discovery and data mining, 279-288. Paris, France: ACM. doi:10.1145/1557019.1557055.
• Bi, L., Yang, L., Bhunia, A.K. and Yao, Y. 2010. Emulsion-based system for prolonged efficacy of nisin against L. monocytogenes. Institute of Food Technologist Annual Meeting. Chicago, IL. Abstract # 130-01.
• Koo, O. and Bhunia, A.K. 2010. Selection of probiotic lactic acid bacteria for prevention of L. monocytogenes. Institute of Food Technologist Annual Meeting. Chicago, IL. Abstract # 072-02.
• Banerjee, P. and Bhunia, A.K. 2010. Hand-held biosensor for rapid detection of hemolytic and cytolytic toxins in foods and beverages. Institute of Food Technologist Annual Meeting. Chicago, IL. Abstract # 036-10.
• Kim, H., Burkholder, K.M., and Bhunia, A.K. 2010. Influence of secreted LAP on Listeria infection in Caco-2 cell model. Institute of Food Technologist Annual Meeting. Chicago, IL. Abstract # 037-30.
• Mishra, K., Kim, H., Mendonca, M., Aroonnual, A., and Bhunia, A.K. 2010. Study of cell structure and colony morphology in secA2 deletion mutant of Listeria monocytogenes 4b (F4244). Midwest Microbial Pathogenesis Conference, St Louis, MO. Sept 11-12, 2010.
• Koo, O.K. 2010. Listeria adhesion protein and heat shock protein 60: application in pathogenic Listeria detection and implication in listeriosis prevention. Purdue University. PhD Dissertation, p. 165
• Burkholder, K.M., 2010. The role of Listeria adhesion protein (LAP) during the intestinal phase of Listeria monocytogenes pathogenesis. Purdue University. PhD dissertation. p. 134
• Bettasso, A. M., 2010. Application of light scattering technology in microorganism detection and classification. Purdue University. MS Thesis, p. 98.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Improving sensitivity and specificity of biosensor tools is continued to be a major focus of our group. In addition, broadening sensor application to simultaneous detection of multiple pathogens and toxin using a single sensor platform is becoming a priority. This approach would not only allow total microbiological assessment of a product for the presence of certain foodborne pathogens but also make the microbiological testing less expensive. During this reporting period, we optimized fiber optic based immunosensor, mammalian cell based sensor and light scattering sensors for their ability to detect multiple pathogens including Salmonella, E. coli, Listeria, and Vibrio and various toxins from food products. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
In laboratory setting, the biosensor tools demonstrated their suitability in detection of various foodborne pathogens from inoculated food samples. These multipathogen detection tools would cut down assay steps to facilitate improved and specific detection of foodborne pathogens including Listeria monocytogenes, Escherichia coli O157:H7, Vibrio vulnificus, V. parahaemolyticus and Salmonella serovars in a cost-effective manner.

Publications

• Bae, E., Aroonnual, A., Bhunia, A.K., Robinson, J.P., and Hirleman, E.D. 2009. System automation for bacterial colony detection and identification instrument via forward-scattering. Measure. Sci. Technol. 20:015802
• Banada, P.P., Huff, K., Bae, E., Rajwa, B., Bayraktar, B., Aroonnual, A., Adil, A., Robinson, J.P., Hirleman, E.D., and Bhunia, A.K. 2009. Label - free detection of multiple bacterial pathogens using light scattering sensor. Biosens. Bioelectron. 24: 1685-1692.
• Banerjee, P., and Bhunia. A.K. 2009. Mammalian cell-based biosensors for pathogens and toxins. Trends Biotechnol. 27 (3): 179-188.
• Kim, G., Moon, J.H., Hahm, B.K., Morgan, M., Bhunia, A., and Om, A.S. 2009. Rapid detection of Salmonella enteritidis in pork samples with impedimetric biosensor: Effect of electrode spacing on sensitivity. Food Sci. Biotechnol. 18: 89-94.
• Burkholder, K.M., Kim, K-P., Mishra, K., Medina, S., Hahm, B-K., Kim, H., and Bhunia, A.K. 2009. Expression of LAP, a SecA2-dependent secretory protein, is induced under anaerobic environment. Microbes Infect. 11:859-867.
• Burkholder, K.M. and Bhunia, A.K. 2009. Salmonella enterica serovar Typhimurium adhesion and cytotoxicity during epithelial cell stress is reduced by Lactobacillus rhamnosus GG. Gut Pathog. 1:14.
• Valadez, A.M., Lana, C.A., S.I., Morgan, M.T., and Bhunia, A.K. 2009. Evanescent wave fiber-optic biosensor for detection of Salmonella from food. Sensors 9: 5810-5824.
• Bae, E., Bai, N., Aroonnual, A., Bhunia, A.K., Robinson, J.P., and Hirleman, E.D. 2009. Prediction of the light scattering patterns from bacteria colonies by a time-resolved Reaction-diffusion model and the scalar diffraction theory. Proceedings of SPIE. 7315: 73150.
• Bai, N., Bae, E., Aroonnual, A., Bhunia, A.K., Robinson, J.P., and Hirleman, E.D. 2009. Development of a real-time system of monitoring the bacterial growth and registering the forward-scattering pattern. Proceedings of SPIE. 7315: 73150.
• Koo, O.K., Aroonnual, A., Bhunia, A.K. 2009. Competitive capture of Listeria monocytogenes using Heat shock protein 60 coated paramagnetic beads. Institute of Food Technologist annual meeting. Anaheim, CA. Abstract # 152-12
• Koo, O.K., Liu, Y., Shuaib, S., Bhattacharya, S., Ladisch, M.R., Bashir, R., and Bhunia, A.K. 2009. Targeted capture of pathogenic bacteria using mammalian cell receptor coupled with dielectrophoresis on biochip. Anal. Chem. 81:3094-3101.
• Ohk, S.H., Koo, O.K., Bhunia, A.K. 2009. Concurrent detection of Listeria monocytogenes, E. coli O157 and Salmonella enterica from artificially inoculated food samples with fiber-optic biosensor. Institute of Food Technologist Annual Meeting, Anaheim, CA. Abstract # 152-18.
• Jagadeesan, B., La, D., Kihara, D., and Bhunia, A.K. 2009. N terminal Gly224-Gly411 Domain in Listeria adhesion protein (LAP) interacts with receptor Hsp60. American Society for Microbiology General Meeting, Philadelphia, PA. B-107/248.
• Liu, Y., Koo, O., He, Y., Ladisch, M., Bhunia, A., Bashir, R. 2009. Label Free Detection of PCR Amplification. 215th ECS Meeting - San Francisco, CA: Abstract # 1395, May 28, 2009
• Banerjee, P., Merkel, G.J., and Bhunia, A.K. 2009. Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells. Gut Pathog. 1:8.
• Bi, L., Narsimhan, G., Bhunia, A.K., and Yao, Y. 2009. Phytoglycogen-based dendritic polysaccharide binds nisin and offers prolonged inhibitory activity against Listeria monocytogenes. Institute of Food Technologist Annual Meeting. Anaheim, CA. Abstract # 250-26.
• Burkholder, K and Bhunia, A.K. 2009. Listeria monocytogenes uses Listeria Adhesion Protein (LAP) to Promote Bacterial Translocation, and Induces Expression of LAP Receptor Hsp60. Midwest Microbial Pathogenesis Conference, West Lafayette, IN Oct 10, 2009.
• Mishra, K., Aroonnual, A., Burkholder, K.M., and Bhunia, A.K. 2009. Presence of functional accessory secretory system (SecA2) in non pathogenic Listeria. Midwest Microbial Pathogenesis Conference, West Lafayette, IN Oct 10, 2009.
• Jagadeesan, B., 2009. Molecular characterization of Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase homologue involved in the adhesion of Listeria monocytogenes to intestinal epithelial cells. PhD dissertation. Purdue University. 127 p

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Improving sensitivity and specificity of biosensor tools have continued to be a major focus of our group. In addition, we are also developing sensor platforms for simultaneous detection of multiple pathogens. This approach would not only allow microbiological assessment of the presence of certain foodborne pathogens but also could potentially make the microbiological testing less expensive. In support of multipathogen detection, a multipathogen enrichment broth, SEL, was formulated. This medium allowed simultaneous growth of Salmonella, E. coli and Listeria. This media is currently being used for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from various artificially inoculated food samples using fiber optic-based immunosensor, light scattering sensor and mammalian cell-based sensor. Cell-based sensor allows the detection of varieties of pathogens and toxins while, the light scattering sensor detects and identifies bacterial colonies growing on a Petridish without the need for any labeling reagents or probes. The identification success rate is 99.6% for Listeria, 95.8% for Staphylococcus spp., 94% for Vibrio spp., 87% for E. coli virotypes and 78% for Salmonella serovars. This system has been currently evaluated for its ability to detect and identify pathogens from various food samples. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Microbiologists PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The biosensors tools currently are being used in the laboratory and have demonstrated their suitability in detection of various foodborne pathogens from food samples. These multipathogens detection tools would reduce assay steps and thus facilitate improved and specific detection of several foodborne pathogens including Listeria monocytogenes, E. coli O157:H7 and Salmonella in a cost-effective manner.

Publications

• Lathrop, A.L., P.P. Banada, and A.K. Bhunia. 2008. Differential expression of InlB and ActA in Listeria monocytogenes in selective and nonselective enrichment broths. Journal of Applied Microbiology. 104:627-639.
• Banerjee, P., Lenz, D., Robinson, J.P, Rickus, J.L., and Bhunia, A.K. 2008. A novel and simple cell-based detection system with collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins. Laboratory Investigation 88:196-206.
• Bae, E., Banada, P.P., Huff, K., Bhunia, A.K., Robinson, J.P., and Hirleman, E.D. 2008. Analysis of time-resolved scattering from macroscale bacterial colonies. Journal of Biomedical Optics 13(1): 014010.
• Bao, N., Jagadeesan, B., Bhunia, A.K., Yao, Y., and Lu, C. 2008. Enumeration of bacterial cells based on autofluorescence on a microfluidic platform. Journal of Chromatography A 1181:153-158.
• Ngamwongsatit, P., Banada, P.P., Panbangred, W., and Bhunia, A.K. 2008. WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line. Journal of Microbiological Methods 73: 211-215.
• Liu, Y.-S., Banada, P.P., Bhattacharya, S., Bhunia, A.K. and Bashir, R. 2008. Electrical characterization of DNA molecules in solution using impedance measurements. Applied Physics Letters 92:143902.
• Yang, L., Banada, P.P., Bhunia, A.K., Bashir, R. 2008. Effects of dielectrophoresis on growth, viability, and immuno-reactivity of Listeria monocytogenes. Journal of Biological Engineering 2:6.
• Kim, H. and Bhunia, A.K. 2008. SEL, a selective enrichment broth for simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Applied and Environmental Microbiology 74 (15): 4853-4866.
• Bhattacharya, S., Salamat, S., Morisette, D., Banada, P., Akin, D., Liu, Y.-S., Bhunia, A.K., Ladisch, M. and Bashir, R. 2008. PCR-based detection in a micro-fabricated platform. Lab on a Chip 8:1130-1136.
• Kim, G., Morgan, M., Hahm, B.K., Bhunia, A., Mun, J.H. and Om, A.S. 2008. Interdigitated microelectrode based impedance biosensor for detection of Salmonella Enteritidis in food samples. Journal of Physics: Conference Series 100:052044.
• Banada, P.P., Huff, K., Bae, E., Rajwa, B., Bayraktar, B., Aroonnual, A., Adil, A., Robinson, J.P., Hirleman, E.D., and Bhunia, A.K. 2008. Label - free detection of multiple bacterial pathogens using light scattering sensor. Biosensors and Bioelectronics (published online).
• Bhunia, A.K. 2008. Biosensors and bio-based methods for the separation and detection of foodborne pathogens. In Advances in Food and Nutrition Research. Editor: S. Taylor, Vol. 54, Academic Press, pp 1-44.
• Banada PP and AK Bhunia. 2008. Antibodies and immunoassays for detection of bacterial pathogens. In Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems, Zourob, Mohammed; Elwary, Souna; Turner, Anthony (Eds.) Cambridge University, Manchester, UK.
• Bae, E., Lesmana, A., Bhunia, A.K., Robinson, J.P., and Hirleman, E.D. 2008. Development and optimization of two-dimensional centering algorithm for bacterial rapid detection system using forward scattering. Proceedings of the Society for PhotoOptical Instrumentation Engineers 6849:684905 (1-8).
• Mishra, K., Burkholder, K.M., Medina-Maldonado, S., and Bhunia, A.K. 2008. Cloning, genomic organization and expression of SecA2 gene in Listeria species. American Society for Microbiology General Meeting. Boston, MA. June 1-5, 2008, B-202.
• Jagadeesan, B., Raizman, E., Nanduri, V., Bannantine, J., and Bhunia, A.K. 2008. Rapid label free sero-diagnosis of Johnes disease using surface plasmon resonance biosensor. American Society for Microbiology General Meeting. Boston, MA. June 1-5, 2008, U-096.
• Koo, O.K., Shuaib, S., Ladisch, M.R., Bashir, R, and Bhunia, A.K. 2008. Targeted capture of pathogenic Listeria using a Listeria Adhesion Protein (LAP) specific mammalian cell receptor, Hsp60 for detection of bacteria on biosensor platforms. American Society for Microbiology General Meeting. Boston, MA. June 1-5, 2008, P-095.
• Ngamwongsatit, P, Banada, P.P., A. K. Bhunia, A.K., and Panbangred, W. 2008. Study on correlation between enterotoxin genes and cytotoxicity in Bacillus cereus isolated from patient, food and soil in Thailand. American Society for Microbiology General Meeting. Boston, MA. June 1-5, 2008, P-074.
• Rajwa, B., Bayraktar, B., Banada, P.P., Huff, K., Bae, E., Hirleman, E.D., Bhunia, A.K., Robinson, J.P. 2008. Phenotypic analysis of bacterial colonies using laser light scatter and pattern-recognition techniques. Proceedings of SPIE 6864:686405 (1-7).
• Burkholder, K.M., Kim, K.-P., Hahm, B.-K. Mishra, K., Medina-Maldonado, S., and Bhunia, A.K. 2008. Anaerobic environment increases surface localization of Listeria Adhesion Protein (LAP) and promotes infectivity of Listeria monocytogenes. American Society for Microbiology General Meeting. Boston, MA. June 1-5, 2008, B-101.
• Kim, K., and Bhunia, A.K. 2008. Performance evaluation of a multiplex selective enrichment broth, SEL by proteomic analysis and immunoassay. Institute of Food Technologist Annual Meeting, New Orleans, LA. June 29-July 2, 2008. Abs 176-12, p 225.
• Jagadeesan, B, and Bhunia, A.K. 2008. Binding characterization of Listeria Adhesion Protein from different Listeria species to its eukaryotic receptor Hsp60 using a surface plasmon resonance biosensor. International Association of Food Protection annual meeting, Columbus, OH. P3-49.
• Koo, O.K., Jagadeesan, B., Burkholder, K., Liu, Y-S., Ladisch, M.R., Bashir, R., Linton, R., and Bhunia, A.K. 2008. Mammalian cell receptor, Hsp60 on microfluidic biochip allows improved capture and detection of Listeria monocytogenes. FoodMicro2008. Aberdeen, Scotland. Sept 1-4, 2008, P I57.
• Bae, E., Banada, P.P., Huff, K., Aroonnual, A., Rajwa, R., Robinson, J.P., Hirleman, E.D., and Bhunia, A.K., 2008. Label - free detection of bacterial pathogens using light scattering sensor from food. FoodMicro2008. Aberdeen, Scotland. Sept 1-4, 2008, P I58.
• Kim, H. 2007. A selective enrichment medium for simultaneous growth and detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Enteritidis from food. MS Thesis. Purdue University, West Lafayette.
• Huff, K. 2008. The light scatterometer BARDOT as a non-invasive sensor for the identification of common foodborne bacteria. MS thesis, Purdue University, West Lafayette.
• Banerjee, P. 2008. Mammalian cell-based biosensor for rapid screening of pathogenic bacteria and toxins. PhD Thesis. Purdue University, West Lafayette.

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Improving sensitivity and specificity of biosensor tools has continued to be a major focus of our group. In addition, detection of multipathogens using a single sensor platform is of major importance because it can provide safety assessment of a product very rapidly and can save money for pathogen testing. In support of multipathogen detection, a multiplex enrichment broth, SEL, has been formulated that allows simultaneous growth of Salmonella, E. coli and Listeria. This media is currently being evaluated for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from various artificially inoculated food samples using fiber optic-based immunosensors. A cell-based sensor that employs collagen-trapped mammalian cells for onsite detection of Listeria and Bacillus toxins is shown to be promising and the results could be obtained in 4-6 h with inoculated food samples. An automated laser light scattering system has been built that allows rapid (in seconds) identification of bacterial colonies growing on the plate without destroying the sample. The identification success rate is 99.6% for Listeria, 95.8% for Staphylococcus spp., 94% for Vibrio spp., 87% for E. coli virotypes and 78% for Salmonella serovars. This system has been currently evaluated for its ability to detect and identify pathogens from food samples. TARGET AUDIENCES: Food scientists interested in food safety and rapid biosensor detection

Impacts
The biosensors tools currently under development would reduce assay steps and thus facilitate improved and specific detection of three major pathogens, Listeria monocytogenes, E. coli O157:H7 and Salmonella from food in a cost-effective manner.

Publications

• Banada, P.P., Bernas, T., Robinson, J.P., and Bhunia, A.K. 2007. Proteomic analysis of cytotoxic factors from Bacillus cereus. American Society for Microbiology General Meeting. Toronto, ON. May 21-25, 2007, Abstr. P-097, p206.
• Hahm, B.K., Kim, H., and Bhunia, A.K. 2007. Enrichment and detection of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes using a sample preparation device, PEDD (Pathogen Enrichment and Detection Device). American Society for Microbiology General Meeting. Toronto, ON. May 21-25, 2007, Abstr. P-118.
• Liu, Y., Banada, P., Bhattacharya, S., Akin, D., Bhunia, A.K., and Bashir, R. 2007. Electrical characterization of DNA molecules in fluids using impedance measuremenst. Biomedical Engineering Society (BMES) Annual Meeting, Los Angeles, CA, Sept 27-29, 2007. P2-109.
• Bhunia, A.K., Banada, P.P., Banerjee, P., Valadez, A., and Hirleman, E.D. 2007. Light scattering, fiber optic and cell-based sensor for sensitive detection of foodborne pathogens. Journal of Rapid Methods and Automation in Microbiology. 15:121-145.
• Kim, G., Morgan, M.T., D.R. Ess, B.K. Hahm, A. Kothapalli, and A.K. Bhunia. 2007. An automated fiber-optic biosensor based binding inhibition assay for the detection of Listeria monocytogenes. Food Science and Biotechnology. 16(3):337-342.
• Akin, D., Sturgis, J., Ragheb, K., Sherman, D., Burkholder, K., Robinson, J.P., Bhunia, A.K., Mohammed, S. and Bashir, R. 2007. Bacteria-mediated delivery of nanoparticles into cells. Nature Nanotechnology. 2:441-449.
• Nanduri, V., A.K. Bhunia, S.-I Tu, G.C. Paoli, and J.D. Brewster. 2007. SPR biosensor for the detection of L. monocytogenes using phage displayed antibody. Biosensors and Bioelectronics. 23:248-252.
• Amass, S., A. K. Bhunia, A. Chaturvedi, D. Dolk, S. Peeta and M. Atallah 2006. Advances in Homeland Security, 2006. Vol. 1, The Science of Homeland Security, Editors: Purdue University Press, West Lafayette, IN.
• Rajwa B., Bayraktar, B., Banada, P.P., Huff, K., Bae, E., Hirleman, E.D., Bhunia, A.K., Robinson, J.P. 2006. Noninvasive forward scattering system for rapid detection, characterization, and identification of Listeria colonies: Image processing and data analysis. Proc. Soc. PhotoOpt. Instrument. Eng. Optics East. 6381:638105-1-638105-8.
• Bae, E., Banada, P.P., Bhunia, A.K., Hirleman, E.D. 2006. Bio-physical modeling of time-resolved forward scattering by Listeria colonies. Proceedings of the Society for PhotoOptical Instrumentation Engineers. Optics East. 6381:638107-1-638107-8.
• Tu, S-I., Geng, T., Uknalis, J., and Bhunia, A.K. 2006. Fiber optic biosensor employing Alexa-Fluor conjugated antibodies for detection of Escherichia coli O157:H7 and Shiga-like toxins. Proceedings of the Society for PhotoOptical Instrumentation Engineers. Optics East. 6381: 638106-1 - 638106-6.
• Bhunia, A.K. 2006. Control and prevention of Listeria monocytogenes in the dairy industry. 2006. Perspectivas E Avancos Da Qualidade Do Leite No Brasil. pp 259-260.
• Zhao, J., Xue, Z., Berhane, N.H., Corvalan, C., Stanciu, L., Bhunia, A.K., Rickus, J.L. 2007. A computational model of listeriolysin O pore formation in liposome-doped sol-gel nanocomposites. Institute of Biological Engineering Annual Meeting, St. Louis, MO; March 29-April 1, 2007.
• Banerjee, P., Lenz, D., Sherman, D.L., Robinson, J.P., and Bhunia, A.K. 2007. Cell-based sensor with collagen entrapped Ped-2E9 cells in multi-well plate for rapid detection of viable pathogenic Listeria and Bacillus cells or toxins in food samples. Institute of Food Technologist Annual Meeting, Chicago, IL, July 28-Aig 1, 2007, Abstr. 165-04, pp. 230.
• Koo, O.K., Jagadeesan, B., Burkholder, K., and Bhunia, A.K. 2007. Targeted capture of pathogenic Listeria using a Listeria adhesion protein (LAP) specific mammalian cell receptor, Hsp60 for detection of bacteria on biosensor platforms. Institute of Food Technologist Annual Meeting, Chicago, IL July 28-Aig 1, 2007, Abstr. 058-19., pp 91.
• Valadez, A.M., and Bhunia, A.K. 2007. Development of a fiber-optic waveguide biosensor assay for the detection of Salmonella Enteritidis from poultry products. Institute of Food Technologist Annual Meeting, Chicago, IL, July 28-Aig 1, 2007, Abstr. 098-31, pp. 144.
• Kim, J.M.., Koh, S., Bhagat, A., Bhunia, A.K., and Linton, R.L. 2007. Selecting surrogate microorganism for evaluation of pathogens on chlorine dioxide gas treatment. American Society for Microbiology General Meeting. Toronto, ON. May 21-25, 2007, Abstr. P-058.
• Burkholder, K.M., and Bhunia, A.K. 2007. Listeria adhesion protein facilitates L. monocytogenes translocation through intestinal epithelial cell lines. American Society for Microbiology General Meeting. Toronto, ON. May 21-25, 2007, Abstr. B-004.
• Bhattacharya, S., Salamat, S., Banada, P., Liu, Y., Morisette, D., Bhunia, A.K., Akin, D., and Bashir, R. 2007. Integrated detection of microorganisms in a microfluidic biochip. Biomedical Engineering Society (BMES) Annual Meeting, Los Angeles, CA, Sept 27-29, 2007. P4-83.
• Banada, P.P., S. Guo, B. Bayraktar, E. Bae, B. Rajwa, J. Paul Robinson, E.D. Hirleman, and A.K. Bhunia. 2007. Optical forward scattering for detection of Listeria monocytogenes and other Listeria species. Biosensors and Bioelectronics 22:1664-1671.
• Banerjee, P., M.T. Morgan, J.L. Rickus, K. Ragheb, C. Corvalan, J. P. Robinson, and A.K. Bhunia. 2007. Hybridoma Ped-2E9 cells cultured under modified conditions can sensitively detect Listeria monocytogenes and Bacillus cereus. Applied Microbiology and Biotechnology 73:1423-1434.
• Kim, K.P., B.K. Hahm, and A.K. Bhunia. 2007. The 2-cys peroxiredoxin deficient Listeria monocytogenes displays impaired growth and survival in the presence of hydrogen peroxide in vitro but not in mouse organs. Current Microbiology 54:382-387.
• Bae, E., Banada, P.P., Huff, K., Bhunia, A.K., J.P. Robinson, and Hirleman, E.D. 2007. Bio-physical modeling of forward scattering from bacterial colonies using scalar diffraction theory. Applied Optics. 46: 3639-3648.

Progress 10/01/05 to 09/30/06

Outputs
Improving sensitivity, specificity and the applicability of biosensors with food samples were the major focus for the last year. Fiber optic - based immunosensor was able to detect E. coli O157:H7 at an initial contamination level of 1 cfu/g of ground beef after 4-6 h and Listeria monocytogenes with a sensitivity of 100,000 cells/ml in meat samples after 20 h enrichment employing the automated RATOR system. A laser light scattering system was used to demonstrate its ability to distinguish bacterial colonies grown on solid agar plates. This noninvasive simple system was able to differentiate different species of Listeria with 90-99% accuracy and was also able to distinguish closely related bacterial strains in minutes. In a blind test, this system was able to detect viable and stress exposed L. monocytogenes accurately from meat samples. This system was also demonstrated to be useful for identification of bacterial colonies belonging to genus, Salmonella, Escherichia, and Vibrio.

Impacts
The light scattering sensor can identify bacterial colonies growing on solid agar plates in a noninvasive manner in seconds thus would reduce assay time and cost of bacterial detection/identification from food.

Publications

• Yang, L., P.P. Banada, Y.S. Liu, A.K. Bhunia, and R. Bashir. 2005. Conductivity and pH dual detection of growth profile of healthy and stressed Listeria monocytogenes. Biotechnology and Bioengineering 92(6):685-693.
• Kim, K.-P., B. Jagadeesan, K. Burkholder, Z.W. Jaradat, J.L. Wampler, A.A. Lathrop, M.T. Morgan, and A.K. Bhunia. 2006. Adhesion characteristics of Listeria adhesion protein (LAP)expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor. FEMS Microbiology Letters 256:324-332.
• Banada, P.P., Y.S. Liu, L. Yang, R. Bashir, and A.K. Bhunia. 2006. Performance evaluation of a low conductive growth medium (LCGM) for growth of healthy and stressed Listeria monocytogenes and other common bacterial species. International Journal of Food Microbiology 111:12-20.
• Lathrop. A., K. Huff, and A.K. Bhunia. 2006. Prevalence of antibodies reactive to pathogenic and nonpathogenic bacteria in preimmune serum of New Zealand white rabbits. Journal of Immunoassay and Immunochemistry 27:351-361.
• Bayraktar, B., P.P. Banada, E. D. Hirleman, A.K. Bhunia, J. Paul Robinson, and B. Rajwa. 2006. Feature extraction from light-scatter patterns of Listeria colonies for identification and classification. Journal of Biomedical Optics 11(3):034006-1-034006-8.
• Geng, T., B.K. Hahm, and A.K. Bhunia. 2006. Selective enrichment media affect the antibody-based detection of stress-exposed Listeria monocytogenes due to differential expression of antibody-reactive antigens identified by protein sequencing. Journal of Food Protection 69(8):1879-1886.
• Yang, L., P.P. Banada, R. Chatni, K.S. Lim, A.K. Bhunia, M. Ladisch, and R. Bashir. 2006. A multifunctional micro-fluidic system for dielectrophoretic concentration coupled with immuno-capture of low number of Listeria monocytogenes. Lab-On-A-Chip 6:896-905.
• Geng, T., J. Uknalis, S.-I. Tu, and A.K. Bhunia. 2006. Fiber optic biosensor employing Alexa-Fluor conjugated antibody for detection of Escherichia coli O157:H7 from ground beef in four hours. Sensors 6:796-807.
• Nanduri, V., G. Kim, M.T. Morgan, D. Ess, B.-K. Hahm, A. Kothapalli, A. Valadez, T. Geng, A.K. Bhunia. 2006. Antibody immobilization on waveguides using a flow through system show improved Listeria monocytogenes detection in an automated fiber optic biosensor: RAPTOR. Sensors 6:808-822.
• Morgan, M.T., G. Kim, D.R. Ess, A. Kothapalli, B.K. Hahm, and A.K. Bhunia. 2006. Binding inhibition assay using fiber optic based biosensor for the detection of foodborne pathogens. Key Engineering Materials 321-323:1145-1150.
• Huff, K. R., P. P. Banada, E. Bae, B. Bayraktar, B. Rajwa, J. P. Robinson, E. D. Hirleman, G. P. Richards, A. K. Bhunia. 2006. Detection and identification of foodborne pathogens in genus and species levels using a noninvasive modified light scatterometer, BARDOT. American Society for Microbiology General Meeting. Orlando, FL. May 21-25, 2006, Abstr. P-075, p455-456.
• Kim, H., and A.K. Bhunia. 2006. A selective enrichment medium for simultaneous detection of Escherichia coli, Listeria monocytogenes and Salmonella. Institute of Food Technologist Annual Meeting, June 24-28, 2006, Orlando, FL Abstr. 003A-12.
• Bhunia, A.K. 2006. Detection of significant bacterial pathogens and toxins of interest in homeland security. In Advances in Homeland Security, Vol. 1, The Science of Homeland Security, Editors: S. Amass, A. K. Bhunia, A. Chaturvedi, D. Dolk, S. Peeta and M. Atallah, Purdue University Press, West Lafayette, IN, pp 109-149.
• Geng, T., and A.K. Bhunia. 2006. Optical biosensors in foodborne pathogen detection. In Smart Biosensor Technology, Editors: G. K. Knopf and A.S. Bassi, Taylor and Francis, Boca Raton, FL. pp 505-519.
• Banerjee, P., P. P. Banada, J.L. Rickus, M.T. Morgan, and A.K. Bhunia. 2005. A portable cell based optical detection device for rapid detection of Listeria and Bacillus toxins. Proceeding of SPIE, 5996:599602-1- 7.
• Wang, H.-Y., C. Lu, P.P. Banada, B. Jagadeesan, and A.K. Bhunia. 2005. Microfluidic pretreatment of bacterial cells for analysis of intracellular contents. Proceeding of SPIE 5996:599603-1- 10.
• Bayraktar, B., Banada, P.P., Hirleman, E.D., Bhunia, A.K., Robinson, J.P., Rajwa. B. 2006. Bacterial phenotype identification using Zernike moment invariants. Proceeding of SPIE. 6080:155-162.
• Kim, G., Morgan, M.T., D.R. Ess, B.K. Hahm, A. Kothapalli, A. Valadez, and A.K. Bhunia. 2006. Detection of Listeria monocytogenes using an automated fiber optic biosensor: Raptor. Key Engineering Materials 321-323:1168-1171.
• Jagadeesan, B. K. M. Burkholder, J.L. Wampler, and A.K. Bhunia. 2006. Interaction of Listeria adhesion protein (LAP) with human Hsp60 on the surface of stressed epithelial cells. American Society for Microbiology General Meeting. Orlando, FL. May 21-25, 2006, Abstr. B-103, p48.
• Banerjee, P., D. Lenz, D.M. Sherman, J.L. Rickus, M.T. Morgan, J. P. Robinson, and A.K. Bhunia. 2006. Cell-based sensor with collagen encapsulated Ped-2E9 cells for rapid detection of Listeria and Bacillus toxin. American Society for Microbiology General Meeting. Orlando, FL. May 21-25, 2006, Abstr. P-083, p457.
• Zhao, J., S. Jedlicka, J. Lannu, A.K. Bhunia, and J.L. Rickus. 2006. Liposome-doped nanocomposites as artificial-cell-based biosensors: Detection of listeriolysin O. Biotechnology Progress 22:32-37.
• Hahm, B. K and A.K. Bhunia. 2006. Effect of environmental stresses on antibody-based detection of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes. Journal of Applied Microbiology 100:1017-1027.

Progress 10/01/04 to 09/30/05

Outputs
A fiber optic sensor has been developed for Listeria monocytogenes with a sensitivity of 1000 cells/ml in a pure culture setup. An automated microfluidic controlled fiber optic system called RATOR is also shown to be able to detect Listeria from hotdog samples. A cell-based sensor that employs collagen trapped mammalian cells for onsite detection of Listeria and Bacillus toxins is shown to be promising and the results could be obtained in 4-6 h. A laser light scattering system is being used to demonstrate its ability to distinguish bacterial colonies grown on solid agar plates. This noninvasive simple system is able to differentiate different species of Listeria with 90-99% accuracy and is also able to distinguish closely related bacterial strains in minutes. This system is currently being evaluated for its ability to differentiate Salmonella species.

Impacts
The biosensors tools currently under development would reduce assay steps and thus facilitate improved and specific detection of Listeria monocytogenes from food.

Publications

• Lathrop, A.A., and A.K. Bhunia. 2005. Expression of Listeria monocytogenes InlB and ActA surface proteins under different growth media. American Society for Microbiology General Meeting. Atlanta, GA. June 5-9, 2005, Abstr. P-046, p446.
• Banada, P., L. Yang, R. Bashir, T. Bernas, J. Robinson, and A.K. Bhunia. 2005. Performance evaluation of a low conductive growth medium for Listeria monocytogenes, by conventional and proteomics approaches. American Society for Microbiology General Meeting. Atlanta, GA. June 5-9, 2005, Abstr. P-059, p449.
• Bhunia, A.K., and Wampler, J.L. Animal and cell culture models for foodborne pathogens. Fratamico, P., Bhunia, A.K., Smith, J.L. editors. Horizon Scientific Press, Norfolk, UK. Foodborne Pathogens: Microbiology and Molecular Biology. 2005. p. 15-32.
• Paoli, G.C., Bhunia, A.K., Bayles, D.O. Listeria monocytogenes. In Foodborne Pathogens: Microbiology and Molecular Biology, Fratamico, P., Bhunia, A.K., Smith, J.L. editors. Horizon Scientific Press, Norfolk, UK. Foodborne Pathogens: Microbiology and Molecular Biology. 2005. p. 295-339.
• Gray, K.M., and A.K. Bhunia. 2005. Specific detection of cytopathogenic Listeria monocytogenes using a two-step method of immunoseparation and cytotoxicity analysis. Journal of Microbiological Methods, 60:259-268.
• Chen, W.-T., R.L. Hendrickson, C.-P. Huang, D. Sherman, T. Geng, A.K. Bhunia, and M.R. Ladisch. 2005. Mechanistic study of membrane concentration and recovery of Listeria monocytogenes. Biotechnology and Bioengineering. 89:261-273.
• Maldonado, Y., J. Fiser, C. H. Nakatsu, and A.K. Bhunia. 2005. Cytotoxicity potential and genotypic characterization of Escherichia coli isolates from environmental and food sources. Applied and Environmental Microbiology 71 (4): 1890-1898.
• Burkholder, K.M, and A.K. Bhunia. 2005. Interaction of Salmonella with cultured intestinal cell lines during heat stress. American Society for Microbiology General Meeting. Atlanta, GA. June 5-9, 2005, Abstr. P-041, p445.

Progress 10/01/03 to 09/29/04

Outputs
Improving sensitivity and specificity of biosensor tools has continued to be a major focus. A Fiber-Optic sensor has been developed for Listeria monocytogenes. This sensor is sensitive and can detect 1000 cells/ml in a pure culture setup. Sensitivity diminishes when the cells are stressed or present with natural microflora in food. A buffered selective sample enrichment step helps resuscitation of stressed cells and eliminates interference with other microflora. Food containing an initial load of 10-1000 cells/g could be detected in less than 24 h from the point of food sampling. A two-step detection system specific for pathogenic L. monocytogenes has also been developed. In step one, Listeria cells from enriched ready-to-eat food samples are captured on antibody-coated immunobeads and tested for their ability to kill mammalian cells. This assay is sensitive and extremely specific for cytopathogenic L. monocytogenes and results could be obtained in less than 28 h starting with the food sample. A laser light scattering system has also been developed to distinguish bacterial colonies grown on solid agar plates. This system is able to differentiate closely related bacterial species in minutes. Further testing with contaminated food products is in progress.

Impacts
The detection tools developed here are sensitive and specific and will enable us to detect only the pathogenic Listeria monocytogenes in 24 h from ready-to-eat food products where this organism is a major concern. Early detection would reduce ware-house holding time for products, and prevent potential foodborne Listeria monocytogenes related outbreaks and mortality.

Publications

• Minocha, U., R. Jennings, A. Bhunia, and B. Applegate. 2004. Genome sequence analysis and evaluation of strain specificity of E. coli O157:H7 bacteriophase phi V10. American Society for Microbiology General Meeting. New Orleans, LA. May 23-27, 2004, Abstr. M-028, p396.
• Bhunia, A.K., Geng, T., A. Lathrop, A. Valadez, and M.T. Morgan. 2004. Optical immunosensors for detection of Listeria monocytogenes and Salmonella Enteritidis from food. Proceeding of Society for PhotoOptical Instrumentation Engineering 5271: 1-6.
• Lathrop, A. and K.P. Kim, and A.K. Bhunia. 2004. Development of Listeria monocytogenes specific antibodies through the use of genomics/proteomics. American Society for Microbiology General Meeting. New Orleans, LA, May 23-27, 2004, Abstr. P-070, p494.
• Hahm, B.K., T. Geng, and A.K. Bhunia. 2004. Effect of environmental stress of antibody-based detection of Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes cells. American Society for Microbiology General Meeting. New Orleans, LA. May 23-27, 2004, Abstr. P-099, p499.
• Gray, K.N., E. ONeal, and A.K. Bhunia. 2004. Quantitative detection of Bacillus cereus enterotoxin in 15 minutes using a mammalian cell enzyme release assay. American Society for Microbiology General Meeting. New Orleans, LA. May 23-27, 2004, Abstr. P-094, p498.
• Geng, T., M.T. Morgan, and A.K. Bhunia. 2004. Detection of low levels of Listeria monocytogenes cells by using a fiber-optic immunosensor. Applied and Environmental Microbiology. 70: 6138-6146.
• Wampler, J.L., K-P. Kim, Z.W. Jaradat, and A.K. Bhunia. 2004. Heat-shock protein 60 acts as a receptor for the Listeria adhesion protein in Caco-2 cells. Infection and Immunity 72(2): 931-936.
• Jaradat, Z.W., J.H. Bzikot, J. Zawistowski, and A.K. Bhunia. 2004. Optimization of a rapid dot-blot immunoassay for detection of Salmonella enterica serovar Enteritidis in poultry products. Food Microbiology 21: 761-769.
• Ariefdjohan, M.W., P.E. Nelson, R.K. Singh, A.K. Bhunia, V.M. Balasubramaniam, and N. Singh. 2004. Efficacy of high hydrostatic pressure treatment in reducing Escherichia coli O157 and Listeria monocytogenes in alfalfa seeds. Journal of Food Science 69(5): M117-120.
• Zhang, P., J.L. Wampler, A.K. Bhunia, K.M. Burkholder, J.A. Patterson, and R.L. Whistler. 2004. Effects of arabinoxylans on activation of murine macrophages and growth performance of broiler chicks. Cereal Chemistry. 81(4): 511-514.

Progress 10/01/02 to 09/30/03

Outputs
We are continuing our efforts in developing biosensor tools for the detection of Listeria monocytogenes. Antibodies are critical for immunosensor applications. We are using a L. monocytogenes-reactive monoclonal antibody (MAb-C11E9) and polyclonal antibodies for our biosensor assays. These antibodies were characterized in detail to determine their reactivity spectrum with strains of L. monocytogenes and L. innocua (a nonpathogenic species). About 88% of L. monocytogenes strains showed strong reactions with C11E9 while only 23% of L. innocua gave equivalent results. This antibody was used in a resonant mirror immunosensor and the antibody was able to detect surface protein extracts from L. monocytogenes and L. innocua and showed no reaction with other Listeria species. However, this biosensor instrument was unable to detect whole cells of Listeria because of the configuration of the sensing layer that was not suitable for detection of intact bacterial cells. In the fiber optic sensor, rabbit polyclonal antibody was used to capture bacteria on the fiber wave-guide and the fluorescent-labeled C11E9 was used to detect bacteria. Using this setup the sensitivity limit of this sensor was determined to be 1000 cells/ml. This sensor could detect L. monocytogenes in the presence of other Listeria species, or other common food contaminants (Enterococcus faecalis, Escherichia coli, Salmonella typhimurium). Furthermore, this sensor was able to detect L. monocytogenes from naturally contaminated or spiked hotdog at cell concentrations of 10, 100 or 1000 cfu/ml after 20 h of enrichment.

Impacts
Fiber optic sensor shows promise in detecting low levels of Listeria monocytogenes from spiked hotdog samples after 20 hour of enrichment. This method would allow early and sensitive detection of L. monocytogenes from processed products thus reduce holding time for processed products and prevent potential foodborne Listeria infection. Efficacy of this sensor to detect L. monocytogenes from other meat products is currently under investigation.

Publications

• Bhunia, A.K. and A. Lathrop. 2003. Pathogen Detection, Foodborne. McGraw- Hill 2003 Year Book of Science and Technology, pp.320-323. McGraw-Hill Professional, New York, NY.
• Singh, N., R.K. Singh, and A.K. Bhunia. 2003. Sequential disinfection of Escherichia coli O157:H7 inoculated alfalfa seeds before and during sprouting using aqueous chlorine dioxide, ozonated water and thyme essential oil. Lebensmittel-Wissenschaft & Technologie 36: 235-243.
• Huang, T., J. Sturgis, R. Gomez, T. Geng, R. Bashir, A.K. Bhunia, J.P. Robinson, and M.R. Ladisch. 2003. Composite surface for blocking bacterial adsorption on protein biochips. Biotechnology and Bioengineering. 81:618-624.
• Huang, T., T. Geng, D. Akin, W.J. Chang, J. Sturgis, R. Bashir, A.K. Bhunia, J.P. Robinson, and M.R. Ladisch. 2003. Micro-assembly of functionalized particulate monolayers on C18-derivatized SiO2 surfaces. Biotechnology and Bioengineering. 83: 416-427.
• Hahm, B.-K., Y. Maldonado, A.K. Bhunia, and C.H. Nakatsu. 2003. Subtyping of clinical and environmental isolates of Escherichia coli by multiplex PCR, AFLP, REP-PCR, Box-PCR, PFGE and ribotyping. Journal of Microbiological Methods. 53: 387-399.
• Shroyer, M. and A.K. Bhunia. 2003. Development of a rapid 1-h fluorescence-based cytotoxicity assay for Listeria species. Journal of Microbiological Methods 55:35-40.
• Menon, A., M. Shroyer, J. L. Wampler, C.B. Chawan, and A.K. Bhunia. 2003. In vitro study of Listeria monocytogenes infection to murine primary and human transformed B cells. Comparative Immunology Microbiology and Infectious Diseases 26 (3): 157-174.
• Jaradat, J.W., J.L. Wampler, and A.K. Bhunia. 2003. A Listeria adhesion protein-deficient Listeria monocytogenes strain shows reduced adhesion primarily to intestinal cell lines. Medical Microbiology and Immunology 192:85-91.
• Jaradat, J.W. and A.K. Bhunia. 2003. Adhesion, Invasion, and translocation characteristics of Listeria monocytogenes serotypes in a Caco-2 cell and mouse model. Applied and Environmental Microbiology 69 (6): 3640-3645.
• Davis, K.C., C.H. Nakatsu, R. Turco, S. Weagent, and A.K. Bhunia. 2003. Analysis of environmental Escherichia coli isolates for virulence genes using the TaqMan system. Journal of Applied Microbiology 95:612-620.
• Geng, T., K.P. Kim, D. Sherman, R. Gomez, R. Bashir, M. Ladisch, and A.K. Bhunia. 2002. Expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies C11E9 and EM-7G1 under acid, salt and temperature-induced stressed environment. Journal of Applied Microbiology 95: 762-772.
• Lathrop, A.L., Z.W. Jaradat, T. Haley, and A.K. Bhunia. 2003. Characterization and application of a Listeria monocytogenes reactive monoclonal antibody C11E9 in a resonant mirror biosensor. Journal of Immunological Methods 281:119-128.
• Wampler, J.L., K.P. Kim, and A.K. Bhunia. 2003. Identification of Hsp60 as a putative receptor for the Listeria adhesion protein in Caco-2 cells. American Society for Microbiology General Meeting, Washington, DC. May 18-22, 2003. Abstr. P-03, p490.
• Kim, K.P., T. Geng, and A. K. Bhunia. 2003. Negative regulation of LepA in Listeria monocytogenes survival and growth in stress conditions. Institute of Food Technologist General Meeting, Chicago, IL. July 12-16, 2003. Abstr. 29G-13, p83.
• Geng, T., K.P. Kim, M.T. Morgan, and A. K. Bhunia. 2003. Antibody-based rapid detection of low levels of Listeria monocytogenes cells using a fiber optic biosensor. Institute of Food Technologist General Meeting, Chicago, IL. July 12-16, 2003. Abstr. 76E-3, p195.
• Weller, J., A.A. Lathrop, S. Federspiel, M.A. Cousin, and A. K. Bhunia. 2003. Study of Listeria monocytogenes survival / inactivation on a new conveyor design. Institute of Food Technologist General Meeting, Chicago, IL. July 12-16, 2003. Abstr. 29G-23, p85.
• Kim, K.P., J.W. Jaradat, and A.K. Bhunia. 2003. Alcohol acetaldehyde dehydrogenase homologue of Listeria monocytogenes is a Listeria adhesion protein (LAP) involved in pathogenesis. American Society for Microbiology General Meeting, Washington, DC. May 18-22, 2003. Abstr. P-04, p490.
• Davis, K.C. and A.K. Bhunia. 2003. Detection of pathogenic Escherichia coli in food and environmental samples using PCR. American Society for Microbiology General Meeting, Washington, DC. May 18-22, 2003. Abstr. P-031, p494-495.
• Naschansky, K.N., S. Budiarty, A. Sharma, M. Morgan, R. Bashir, and A.K. Bhunia. 2003. Rapid detection of Listeria monocytogenes in naturally contaminated food samples through enzymatic and biosensor-based cytotoxicity analysis. American Society for Microbiology General Meeting, Washington, DC. May 18-22, 2003. Abstr. P-044, p497.
• Hahm, B.K., A.K. Bhunia, and C.H. Nakatsu. 2003. Application of AFLP for discriminating Escherichia coli isolated from livestock, wildlife and humans. American Society for Microbiology General Meeting, Washington, DC. May 18-22, 2003. Abstr. Q-343, p576.

Progress 10/01/01 to 09/30/02

Outputs
Developing a sensitive detection tool for viable and virulent Listeria monocytogenes is the major focus of this project. (1) We are continuing to improve our cell-based sensor that measures L. monocytogenes interaction with mammalian cells. We were able to grow mammalian cells (Ped-2E9, Caco-2, Vero and RAW) on the interdigitated microsensor electrode (IME)-chip; and demonstrated that Ped-2E9 cell death on the chip could be detected within two hours. We also have demonstrated that all 13 serotypes of L. monocytogenes were capable of adhering and invading Caco-2 cells suggesting that Caco-2 cells could be used as a potential candiate cell line for use in the cell-based sensor. (2) We have completed our enzyme-fluorescence-based cytoxicity assay to measure Listeria interaction with Ped-2E9 cells. Data showed that L. monocytogenes at 10 million/ml could be detected in 1 hour. (3) In the antibody-coupled fiber optic biosensor, we are evaluating the cross reaction of fiber optic sensor with non-Listeria organisms and optimizing conditions for detection of L. monocytrogenes cells only. (4) In the past year we have initiated a project on detection of Listeria by using a surface plasmon resonance immunosensor (IAsys sensor). This optical sensor measures the binding event of antigens to the antibody on a sensing layer at real-time. We found out that this system is unable to detect intact bacterial cells, however can detect soluble antigens such as toxins or surface proteins. We have demonstrated that L. monocytogenes proteins at 5 microgram quantities could be detected in less than 15 minutes. Although the testing is very expensive but shows great promise in real-time detection of L. monocytogenes. (5) In addition, TaqMan PCR system was used to confirm the presence of toxin genes (Stx I and II) in food and environmental E. coli isolates. All food isolates were confirmed to be O157:H7 containing stx I or stx II genes. None of the environmental isolates contained stx genes, however 15% of 604 isolates were identified as O157:H7. This data indicates that TaqMan system is capable of generating false results and may not be suitable for testing environmental E. coli isolates.

Impacts
Cell-based sensor shows promise in the developemnt of Listeria monocytogenes detection tool. Mammalian cell-based enzyme fluorescence assay can detect L. monocytogenes at levels of 10 million in one hour. Surface plasmon resonance immunosensor can detect soluble L. monocytogenes antigens in 15 minutes but is unable to detect intact cells.

Publications

• Jaradat, Z., G. E. Schutze, and A.K. Bhunia. 2002. Genetic homogeneity among Listeria monocytogenes strains from infected patients and meat products from two geographic locations determined by phenotyping, ribotyping and PCR analysis of virulence genes. International Journal of Food Microbiology 76:1-10.
• Jaradat, Z.W. and A.K. Bhunia. 2002. Glucose and nutrient concentrations affect the expression of a 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes. Applied and Environmental Microbiology 68 (10): 4876-4883.
• Gomez, R., R. Bashir, and A.K. Bhunia. 2002. Microscale electronic detection of bacterial metabolism. Sensors and Actuators B: Chemical. 86: 198-208.
• Chang, H., A. Ikram, F. Kosari, G. Vasmatzis, A. Bhunia, and R. Bashir. 2002. Electrical characterization of microorganisms using microfabricated devices. Journal of Vacuum Science and Technology B. 20 (5): 2058-2060.
• Gomez, R., R. Bashir, A.K. Bhunia, and M.R. Ladisch. 2002. Microfabricated device for impedance-based detection of bacterial metabolism. Proceedings of the Spring MRS 2002. San Fransisco, CA.
• Nakatsu, C.H. B.-K. Hahm, J. Fiser, S. Yeary, and A.K. Bhunia. 2002. Comparison of environmental and clinical isolates of Escherichia coli using various genetic fingerprinting methods. In the Abstracts of the USEPA Microbial Source Tracking Workshop. Irvine, CA. Feb 5-7, 2002.
• Lathrop, A.L., Z.W. Jaradat, and A.K. Bhunia. 2002. Increased antibody specificity for Listeria monocytogenes by partially masking of antigen binding site in MAb C11E9 with L. innocua antigens. American Society for Microbiology General Meeting, Salt Lake City, UT. May 19-23, 2002. Abstr. P-42, p370.
• Jaradat, Z.W., and A.K. Bhunia. 2002. Variation in adhesion and invasion behavior among different serotypes of Listeria monocytogenes to Caco-2 cells. American Society for Microbiology General Meeting, Salt Lake City, UT. May 19-23, 2002. Abstr. P-76, p376.
• Kim, K-P., T. Geng, F. Soyer, and A.K. Bhunia. 2002. Increased transcription of lepA homologue, a GTP binding protein, in Listeria monocytogenes during heat stress. Institute of Food Technologist General Meeting, Anaheim, CA. June 15-19, 2002. Abstr. 100A-4.
• Naschansky, K.M., and A.K. Bhunia. 2002. Detection of low levels of pathogenic Listeria monocytogenes in 14 - 20 h using immunoseparation and cytotoxicity techniques. Institute of Food Technologist General Meeting, Anaheim, CA. June 15-19, 2002. Abstr.100A-22.
• Lathrop, A. 2001. Characterization of monoclonal and polyclonal antibodies to Listeria species: Reaction spectrum, partial blocking of epitope binding sites and cross absorption. MS Thesis. Purdue University.
• Shroyer, M.L. 2001. Fluorescence-based cytotoxicity assay for Listeria monocytogenes and analysis of mammalian cell death. MS Thesis. Purdue University.

Progress 10/01/00 to 09/30/01

Outputs
One of our goals is to develop methods that would be able to detect very low numbers of Listeria monocytogenes rapidly from food. Two-step methods were developed: First, concentration of Listeria cells from food and second specific detection by biosensor-based probes such as (a) interdigitated microsensor electrode (IME)-chip, (b) enzyme-fluorescence assay to measure Listeria interaction with animal cells, and (c) antibody-coupled fiber optic biosensor. In addition, various genomic typing methods (amplified fragment length polymorphism (AFLP) and repetitive PCR (Rep-PCR) methods were used to generate genomic fingerprint patterns for E. coli O157:H7 strains. [1] Immunoseparation and cytotoxicity assay with Ped-2E9 cell: Immunobeads (magnetic and nonmagnetic) were used to capture Listeria cells from naturally contaminated or spiked hotdog samples after a selective enrichment step, and bead-captured bacterial cells were directly tested in a cytotoxicity assay. Results showed that L. monocytogenes at a concentration of about 1-100 CFU/100 ml of food extract could be captured in 12-18 h and subsequently be detected in additional 1-2 h. Advantages of this current assay; (i) it is specific, (ii) it detects only viable cells and (iii) most importantly, it detects virulent Listeria. [2] Interdigitated microsensor electrode (IME)-chip based cytotoxicity assay: Mammalian cells (Ped-2E9) exposed to L. monocytogenes for 1-2 h was placed on the IME-chip, and the Ped-2E9 damage was measured by an Impedance Analyzer. The data showed that IME-chip is capable of distinguishing Listeria induced damaged Ped-2E9 cells from the healthy ones. This IME-chip based cytotoxicity assay could be used in conjunction with immunobead separation method for direct detection of L. monocytogenes. [3] Fiber optic biosensor: Polystyrene wave-guides were immobilized with anti-Listeria antibody and were used to capture Listeria cells. Specific detection was achieved by fluorescein-labeled anti-Listeria mAb and a laser detector. Using a pure culture of L. monocytogenes, we were able to detect as low as 38 CFU/ml with the fiber optic probe. [4]Fluorescence based cytotoxicity assay using mammalian Ped-2E9 cells: Various potential foodborne contaminants such as E. coli O157:H7, Salmonella, Citrobacter, Bacillus, Staphylococcus, Corynaebacterium and L. monocytogenes were examined with Ped-2E9 cells in a fluorescence-based cytotoxicity assay. Results showed that Citrobacter, Bacillus, Corynaebacterium and L. monocytogenes produced positive cytotoxicity, whereas other test organisms did not. Selective enrichment media was used to inhibit non-Listeria organisms prior to the assay and the data showed that Ped-2E9 -based cytotoxicity assay could be used for specific detection of L. monocytogenes. [5] Genomic fingerprinting of E. coli O157:H7: Preliminary results indicated that genomic fingerprint patterns generated by AFLP and Rep-PCR could effectively separate E. coli O157:H7 strains from other E. coli serotypes. Furthermore, these methods appeared to be more sensitive than the Ribotyping and pulsed-field gel electrophoresis (PFGE) methods.

Impacts
A rapid Listeria detection method was developed that could sensitively detect 1-100 CFU of L. monocytogenes cells in 14-20 h. Advantages of this current assay; (i) it is specific for L. monocytogenes, (ii) it detects only viable cells and (iii) most importantly it detects virulent Listeria. Currently, we are testing this method with naturally contaminated hotdog and other food samples.

Publications

• Naschansky, K.M. 2001. Development of an immunoseparation and impedance spectroscopy-based cytotoxicity assay to isolate and detect Listeria monocytogenes from hotdog samples. MS Thesis, Purdue University.
• Singh, N., R.K. Singh, A.K. Bhunia, R.L. Stroshine and J.E. Simon. 2001. Different surface inoculation methods with Escherichia coli O157:H7 affect the effectiveness of sanitizers on shredded lettuce. Food Microbiol. (in press).
• Gomez, R., R. Bashir, A. Sarikaya, M.R. Ladisch, J. Sturgis, J.P. Robinson, T. Geng, A.K. Bhunia, H.L. Apple and S.T. Wereley. 2001. Microfluidic biochip for impedance spectroscopy of biological species. Biomedical Microdevices: BioMEMS and Biomed. Nanotechnol. J. (in press).
• Nebeker, B.M., B. Buckner, E. D. Hirleman, A. Lathrop, and A.K. Bhunia. 2001. Identification and characterization of bacteria on surfaces using polarized light scattering. Proceedings of SPIE, 4206: 224-234.
• Naschansky, K.M., M. Morgan, and A.K. Bhunia. 2001. Interdigitated microsensor electrode-chip for detection of cytotoxicity effect of Listeria monocytogenes from food. American Society for Microbiology General Meeting, Orlando, FL, May 20-24, 2001. Abstr. P-32, p563.
• Geng, T., R. Gomez, R. Bashir, M. Ladisch, and A.K. Bhunia. 2001. Reaction patterns of monoclonal antibodies C11E9 and EM-7G1 to stressed or injured Listeria monocytogenes cells for use in the biochip. American Society for Microbiology General Meeting, Orlando, FL, May 20-24, 2001. Abstr. P-33, p563.
• Gomez, R., T. Geng, R. Bashir, M.R. Ladisch, and A.K. Bhunia. 2001. Micro-fabricated biochip for the electronic detection of Listeria cells. American Society for Microbiology General Meeting, Orlando, FL, May 20-24, 2001. Abstr. P-91, p575.
• Shroyer, M.L., A. Menon, and A.K. Bhunia. 2001. A sensitive fluorescence-based cytotoxicity assay to differentiate Listeria monocytogenes from other common foodborne bacteria. American Society for Microbiology General Meeting, Orlando, FL, May 20-24, 2001, Abstr. P-11, p559.
• Singh, A, R.K. Singh, and A.K. Bhunia. 2001. Use of plant essential oils as antimicrobial agents against Listeria monocytogenes in hotdogs. Institute of Food Technologist Annual Meeting. Abstr. 59E-21, p144.
• Singh, N, R.K. Singh, A.K. Bhunia, and R.L. Stroshine. 2001. Effect of inoculation methods for evaluating the efficacy of different sanitizers against E. coli O157:H7 on lettuce. Institute of Food Technologist Annual Meeting. Abstr. 59E-19, p143.

Progress 10/01/99 to 09/30/00

Outputs
Direct detection of Listeria monocytogenes: We have developed fiber optic biosensor to directly detect Listeria from food samples. Polystyrene fibers (wave-guide) were coated with Listeria species specific polyclonal antibody and exposed to different Listeria species. Subsequently fibers were exposed to L. monocytogenes-specific monoclonal antibody (C11E9)-conjugated to a fluorescene dye (Cy5) and signals were obtained from a laser photodiode detector (Analyte 2000). Preliminary data showed that signals generated by L. monocytogenes is about 2.5 times higher than the nonpathogenic L. innocua. Ability of this fiber optic biosensor to detect very low number of L. monocytogenes cells is in progress. Indirect detection of Listeria monocytogenes: L. monocytogenes positively interact with hybrid B-lymphocytes (Ped-2E9) from humans and mice. Sensitive measurement of this interaction is currently used for detection of Listeria in 1-2 h. An Interdigitated Microsensor Electrode (IME)-chip was employed to measure Listeria interaction with Ped-2E9 cells using an impedance analyzer. Preliminary results indicated that this technology could be used for sensitive detection of L. monocytogenes. Also, we measured alkaline phosphatase (AP) release from infected Ped-2E9 cells using a substrate (methylumbelliferyl phosphate) that produces fluorescence end product. We have observed that fluorescence based detection is at least 10 times more sensitive than the previously described colorimetric based assay. Additionally, we found that a panel of Gram-negative foodborne pathogen did not show any cytotoxicity effect on Ped-2E9 cells. Several Gram-positive bacterial strains will also be tested with Ped-2E9 cells. Based on this preliminary result it appears that Listeria interaction with Ped-2E9 cell is specific and could be used to detect L. monocytogenes only. Genomic typing: Genetic diversity and virulence gene expressions of 30 Listeria monocytogenes isolates from human patients and foods originated from two different geographic locations without any epidemiological relations were analyzed. All strains contained virulence genes, inlA, inlB, actA, hlyA, plcA and plcB with expected product size in PCR assay except for the actA gene. Some strains produced actA gene product of 268 and others 385 bp. Phenotypically all were hemolytic but showed variable expressions of phospholipase activity. Ribotyping classified isolates into 12 different groups, which consisted primarily of clinical or food isolates or both. Cluster analysis also indicated possible existence of clones of L. monocytogenes that are maintained in food or human hosts or are evenly distributed in nature. Two isolates (F1 from food and CHL1250 from patient) had unique ribotype patterns that were not previously reported in the RiboPrinterO database. This study indicates distribution of diverse L. monocytogenes strains in clinical and food environments and some of which carry a remarkably high genetic homogeneity in spite of their origins from two different geographic locations and environments.

Impacts
Various biosensor based methods employed in this project show great promise for direct or indirect detection of Listeria monocytogenes from food. These assays would reduce time for overall microbial analysis of ready-to-eat food products thus save lives and reduce economic losses. Genetically diverse L. monocytogenes strains exist in clinical and food environments and some of which carry a remarkably high genetic homogeneity in spite of their origins from different geographic locations or environments.

Publications

• Roberts, P.H., K.C. Davis, W.R. Garstka, and A.K. Bhunia. 2000. Lactate dehydrogenase release assay from vero cells to distinguish verotoxin producing Escherichia coli from non-verotoxin producing strains. Journal of Microbiological Methods (in press).
• Bhunia, A., Z.W. Jaradat, K. Naschansky, M. Shroyer, M. Morgan, R. Gomez, R. Bashir, and M. Ladisch. 2000. Impedance spectroscopy and biochip sensor for detection of Listeria monocytogenes. Proceedings of SPIE, 4206: (in press).
• Singh, R.K., A. Bhunia, A. Singh. 2000. Light emission based biosensors for detection of food pathogens: a review. Proceedings of SPIE, 4206: (in press).
• Williams, L.L. and A.K. Bhunia. 2000. Random amplified polymorphic DNA analysis and antibiotic resistance patterns of verotoxin producing Escherichia coli from ground beef. Inst. Food Technol. Gen. Meet. Dallas, TX. Abstr. 51H-21, p. 117.
• Menon, A. and A.K. Bhunia. 2000. Listeria monocytogenes induces cytopathogenic effect on human B-lymphoma cell line in vitro. Am. Soc. Microbiol. Gen. Meet. Los Angeles, CA. Abstr. D-58, p.238.

Progress 10/01/98 to 09/30/99

Outputs
1. Listeria monocytogenes positively interact with hybrid B-lymphocytes from humans and mice, which could be used as an indicator for L. monocytogenes cytotoxicity in 5-6 h. Addition of dithiothreitol (0.5 mM) to the cell suspension can expedite L. monocytogenes mediated lymphocyte death in 2 h or less thus allowing rapid detection of L. monocytogenes. Monoclonal antibody (C11E9) specific for L. monocytogenes are harvested in large quantities from hybridoma C11E9 cells and purified using Protein-A affinity chromatography. These antibodies will be used to capture Listeria cells from meat samples with antibody coated magnetic beads. Furthermore, experiments are currently underway to conjugate this antibody to fiber optic cables for sensitive detection of Listeria cells. 2. We have isolated several enterohemorrhagic E. coli (EHEC) strains from naturally contaminated ground beef (an ongoing project from Alabama A&M University). Thirteen percent beef samples are found to be positive for E. coli O157:H7. About 76% of those isolates showed 0-45% Vero cell toxicity by lactate dehydrogenase (LDH) assay and 24% exhibited 45-83% toxicity. The cytotoxicity patterns were comparable to those with EHEC obtained from various outbreaks. Additionally, these isolates were shown to be more resistant to multiple antibiotics than the previously reported outbreak strains. 3. EHEC isolates were also examined for genomic fingerprint profiles using randomly amplified polymorphic DNA fragmentation (RAPD) assay. RAPD indicated that isolates from same sample, as expected, had similar RAPD patterns while very distinct patterns for isolates from different samples. RiboPrinter analysis of selected strains confirmed the isolates to be E. coli O157:H7.

Impacts
1. Dithiothreitol (DTT) can enhance L. monocytogenes mediated lymphocyte toxicity thus can significantly reduce the cytotoxicity assay time from 6 h to 2 h or less. 2. About 13% of beef samples were found to be positive for E. coli O157:H7 strains. 3. These E. coli isolates showed enhanced antibiotic resistance patterns than the previously published reports.

Publications

• Amoril, J.G. and Bhunia, A.K. 1999. Immunological and cytopathogenic properties of Listeria monocytogenes isolated from naturally contaminated meats. J. Food Safety 19: 195-207.
• Williams, L., Bhunia, A.K. and Rao. D.R. 1999. Antibiotic resistance profile and PCR characterization of enterohemorrhagic Escherichia coli isolates from ground beef. Inst. Food Technologist Gen. Meet. Chicago, IL. Abtr. 79C-4. p 214-215.
• Bhunia, A.K. and Feng, X. 1999. Examination of cytopathic effect and apoptosis in Listeria monocytogenes-infected hybridoma B-lymphocyte (Ped-2E9) line in vitro. Microbiol. Biotechnol. 9: 398-403.