Progress 02/01/99 to 02/01/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The presence of microbial and chemical pathogens at any stage of food production, processing, and distribution must be quickly determined in order to allow proper treatments before food consumption by the general public. There is a need for rapid, sensitive tests and effective intervention processes which researchers, farmers, processors, and retailers can use to verify that foods are safe to consume. To accelerate the development of the detection and intervention technologies, we have formed a collaborative research agreement with the Food Safety Engineering Center, Purdue University, Indiana (1935-42000-035-01S). Under the agreement, Purdue faculty members have developed specific projects in the high priority ARS program areas to address those issues. ARS scientists who may directly contribute to the
collaborations are being identified to team together with Purdue researchers. We believe that the cooperation will facilitate the achievement of enhancing food safety for the public. Food safety has been an increasing concern for consumers, retailers, and all production and processing areas of the food industry. An estimated 25-81 million cases of foodborne illness and an estimated 9000 death are associated with consumption of contaminated foods each year. In controlling the risk of foodborne illness, there are two important questions that must be answered. First, where is the source of microbiological and chemical foodborne contaminants? And, secondly, how can control be elicited to prevent hazards during food production to better assure a safe, high quality, wholesome food for consumers? To identify the source of microbiological and chemical contaminants is extremely challenging because the contaminants may be present in low levels and/or the complex food system may interfere
with detection methods. Determining the source is important because it helps to ultimately determine what the best method of control may be. 2. List the milestones (indicators of progress) from your Project Plan. During the reporting period of 2003-2004, 11 multi-disciplinary research projects (listed below as "A" through "K") have been supported. The project titles and primary investigator (shown in parenthesis) are listed by project alphabetically in the section below. Milestones for each project are listed numerically. A. "Bioamplification using phage display for the detection of Salmonella spp. and its evaluation as a technology platform for the simultaneous detection of multiple pathogens in the same sample" (Applegate) 1. Construct and propagate modified bacteriophages (MB) in host cells consisting of E. coli and Salmonella spp. 2. Purify modified bacteriophages using affinity chromatography. 3. Develop an assay using the modified bacteriophages to detect food samples
infected with Salmonella and/or non-pathogenic organisms, capture, concentrate, and detect using affinity chromatography and other forms of liquid chromatography. B. "Biosensor-based approaches for rapid and sensitive detection of Listeria monocytogenes from food". 1. Develop or evaluate cell line that would grow on the interdigitated microsensor electrode chip. 2. Measurement of pathogenic potential of Listeria off and on chip containing RAW cells. 3. Adhesion and invasion properties of Listeria monocytogenes serotypes on adherant Caco-2 cells. 4. Use of frozen Ped-2E9 cells or cell analog (liposome) for fluorescence based or colorimetric based detection. 5. Test stressed but viable Listeria cells (heat, cold, salt and acid induced stress) with the two step detection method. The two-step method consisted of, (a) capture of Listeria by antibody-coated beads and followed by (b) cytotoxicity testing on mammalian cell lines. 6. Develop a rapid cytotoxicity assay for Bacillus cereus
toxin. C. "Detection of specific foodborne pathogens using a two component bacteriophage-bioluminescent reporter system in conjunction with a hand held luminometer" (Applegate) 1. Genetically engineer a lux-based bioreporter cell line responsive to acyl-homoserine quorum sensing signal induction. 2. Develop four luxI-based bacteriophage specific for infection of L. monocytogenes, E. coli O157:H7, Salmonella spp., and Campylobacter spp. 3. Test bacteriophage/bioreporter systems in pure culture studies, emphasizing hand-held luminometer format. 4. Lyophilize bacteriophage/bioreporter systems for long-term storage assessment. 5. Comprehensively test bioreporter systems on lettuce and tomato with introduced pathogens. 6. Utilizing hand-held photomultiplier units and microtiter plate format. 7. Continue evaluating lyophilized bacteriophage/bioreporter systems for long-term storage assessment. 8. Evaluate and incorporate MicroTox strain into assay. D. "Development of immunocapture
real-time PCR to detect Fusarium species in grains and foods" (Cousin) 1. Capture Fusarium species with antibodies. 2. Combine immunoassay with PCR to specifically detect Fusarium verticillioides that produce fumonisins and Fusarium graminearum that produce trichothecenese (04-05). 3. Capture Fusarium species with antibodies. E. "Engineering of biosystems for the detection of Listeria monocytogenes in foods" (Ladisch) 1. Recover viable microorganisms in fluid derived from stomached hotdog sample. 2. Gain a fundamental understanding of how sensitivity and specificity of immuno-interactions are affected by the environment (buffer, salts, heat) to which the pathogen is exposed during food process processing or during sampling for purposes of interrogation for detecting the presence of pathogens. 3. Make progress towards development of a biochip that integrates the various functions needed to rapidly detect microorganisms. F. "Infrared sensors for rapid detection of select microbial
foodborne contaminants" (Mauer) 1. Create a library of FT-IR spectra of bacterial cell wall components and whole cells (from Salmonella, Campylobacter jejuni, and Escherichia coli O157:H7) needed for cell identification and differentiation. 2. Develop FT-IR methods to identify and quantify cells in water, cultural media, and food. 3. Develop a limited wavelength approach for cell identification. 4. Build and validate an IR sensor based on the most promising few- wavelength algorithm developed using FT-IR techniques developed in the first two milestones. G. "Light scattering sensory method for rapid assessment of foodborne bacterial contaminants" (Hirleman) 1. Determine how angle resolved elastic light scattering can be used to detect bacteria and distinguish particular foodborne pathogenic bacteria from nonpathogenic bacteria. Target pathogens were Listeria and enterohemorrhagic E. coli. 2. To determine angle-dependent scattering can rapidly detect and identify bacterial microcolonies
on selective agar plate. 3. To generate scattering characteristics based on growth time, density, strains and pathogencity. H. "Multi-pathogen screening and/or donfirmation via microarray detection" (Bhunia) 1. Develop antibody specific for L. monocytogenes, Salmonella enterica and E. coli O157:H7. 2. Identify specific unique target antigens for antibody development. 3. Determine the effect of environmental and physiological stresses on antigen expression. 4. Develop sandwich ELISA for each pathogen. I. "Multiplexed detection of pathogens using FRET in a spatial format" (Applegate) 1. Design of molecular beacon incorporating appropriate FRET pairing to provide maximum emission separations with excitation overlap. 2. Design of molecular beacon target sequence and corresponding amplicon primer pairs for identified target sequences to use in a multiplex PCR. 3. Construction of the molecular beacon array and optimization of hybridization conditions with the PCR amplicons. J. "Optical
biosensor for food pathogen detection" (Bhunia) 1. Develop polyclonal and monoclonal antibodies against Listeria monocytogenes and Salmonella enteritidis. 2. Combine PAb for capture and MAb for detection produced the best signal for L. monocytogenes. 3. The milestones mentioned above for Listeria monocytogenes will be done for Salmonella and E. coli. K. "Rapid detection of total PCBs and toxicity equivalence quotient (TEQ) in fish tissue from Indiana waters and use of a novel device to predict contaminant load in fish" (Santerre) 1. Validate the extraction and cleanup methodology for fish tissue that is required prior to PCB analysis by ELISA. 2. Analyze PCBs in fish tissue using ELISA. 3. Milestones: A. List the milestones (from the list in Question #2) that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. This
section is addressed in the report of the Specific Agreement 1935 - 42000-035-01S. List the milestones (from the list in Question #2) that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This project was terminated in January of 2004. All the future research milestones are described in related new projects (1935-42000-049-00D and 1935-42000-049-01S). 4. What were the most significant accomplishments this past year? This project received a small fund increase ($40 K) in FY 2004. Purdue and the ARS team developed a new program to replace this project in FY 2004. In addition to the accomplishments reported under CWU 1935-42000- 035-01S, this program has provided important training opportunities for Purdue Graduate Students to work on pathogen detection and intervention. Altogether, 8 graduate students have completed their graduate training (4 Ph.D and 4 MS) under this
program during 2002-2004. A. Single most significant accomplishment during FY 2004 (one per Research (OOD) Project): Biochip function is a key part of pathogen detection. A second generation silicon-based microfluidic biochip was fabricated that enables collection and concentration of cells accompanied by impedance-based detection of their presence on the chip. This work combines results from Food Science, Bio-Medical Engineering, and Laboratoty of Renewable Resources Engineering, and packages them into a prototype suitable for further testing. The chip was designed, fabricated, and tested in a manner that integrates off-chip processing with on-chip microfluidic handling and interrogation of a small sample volume. This is an important step forward towards achieving the goal of systems integration for a laboratory prototype for L. monocytogenes detection in samples derived from hotdog (Milestone.E-3, NP 108 Postharvest Pathogen Food Safety Action Plan 2.1.1.2). B. Other
significant accomplishment(s), if any: Research showed how environmental stress reduces binding of L. monocytogenes to its bioreceptor and consequently reduces sensitivity for detection of the pathogen. This work showed how enrichment media might be formulated to optimize the physiological status of the microorganism and to enhance its binding to a pathogen specific antibody while minimizing "cloaking," thereby reducing probability of a false negative result. The outcome of this work will ultimately enhance reliability and sensitivity of biochip-based detection of L. monocytogenes by formulating special media or buffers for this purpose (Milestone H-3, NP 108 Postharvest Pathogen Food Safety Action Plan 2.1.1.2). Bacillus cereus produces emetic (1.2 kDa) and diarrheal (38 - 43 kDa) toxins, and the latter toxin is most commonly associated with foodborne illnesses in Europe and North America. Current toxin assay methods are time consuming. Therefore, a highly sensitive, rapid and
quantitative assay was developed using a B cell line, Ped-2E9. B. cereus cell-free supernatants containing toxins were added to Ped-2E9 cell suspensions, incubated for 15 min - 1 h and analyzed for cell cytotoxicity with an alkaline phosphatase release assay. Cytotoxicity analysis indicated that several B. cereus strains were found to produce positive cytotoxicity results in as early as 15 min. The highly cytotoxic MS1-9 strain showed a dilution as high as 1:32 (toxin:buffer) was still able to produce a cytotoxic effect. The supernatant from MS1-9 strain was fractionated and the cytotoxicity was associated with fractions greater than 30 kDa. SDS- PAGE revealed this fraction to contain proteins with MW in the range of 22 - 66 kDa. PCR analysis results showed a strong correlation between the number of toxin genes in a strain and its cytotoxic potential. When compared to the HEp-2-based emetic assay and the CHO-cell assay and commercial diarrheal toxin kits, the Ped-2E9-based
Bacillus enterotoxin assay was found to be a more rapid and sensitive alternative to current methods (Milestone B-4, NP 108 Action Plan Toxic Chemicals 2.1.2.2). We completed construction of a P22/luxI Salmonella Bacteriophage/Bioluminescent Reporter System for the detection of Salmonella and integrated the MicroTox assay strain into the assay. This construct allows the testing and validation of the overall project objectives of the luxI phage based approach for pathogen detection. The phage was constructed using an amber P22 phage mutant which can not propagate in wild type Salmonella spp. and a recombination vector which restores P22 wild type phenotype while inserting desired DNA (constitutively expressed luxI). Detection limits in the developed assay were around 103 cfu (pure culture) in approximately 6 hours. The assay developed has the potential of immediate impact in the food safety arena as it can be incorporated with equipment already deployed and used routinely for the
ATP hygiene test and utilize the MicroTox assay strain as the bioluminescent reporter, which also is commercially available (Milestone C-8, NP 108 Action Plan Postharvest Pathogen Food Safety 2.1.2. 2). Rapid concentration and recovery of cells from foods is needed to reduce the time for sample preparation from the current 1 to 3 days to less than an hour. A systematic study of membrane separations that not only concentrate cells, but also enable cell recovery in sub-microliter volumes was carried out as a collaborative effort among collaborating laboratories. GFP E. coli were prepared to rapidly and accurate track the movement of living microorganisms through the concentration and detection steps. We have now demonstrated capture of L. monocytogenes from a background of E. coli by anti-Listeria antibodies immobilized on a derivatized surface of a nanoporous, polycarbonate membrane. The impact of combined selective capture and CCR is that L. monocytogenes, if it is present, is
concentrated before interrogation of the sample on the biochip, therefore enhancing the probability (sensitivity) of detecting the pathogen (Milestone E-3, NP 108 Action Plan Postharvest Pathogen Food Safety 2.1.1.2). We successfully developed an approach for sample preparation, FTIR spectral collection, and data analysis that is able to both quantify and identify Salmonella and E. coli O157:H7 from cultural media. This approach will be used as the format for development of the portable IR sensor. To develop this approach, we evaluated sample preparation methods (including preenrichment, selective enrichment, filtration and immunomagnetic separation techniques), FTIR data collection methods (ATR, transmission, reflection, Continuum IR microscope), and analysis of raw spectra. Further development of this approach will enable the design of a sensor that can be used in a production or retail facility to characterize a food sample as contaminated or free of select pathogenic bacteria in
less time than current methods for detection (Milestone F-2, NP 108 Action Plan, Postharvest Pathogen Food Safety 2.1.1.2). We demonstrated that differentiation and identification of Salmonella and E. coli strains are possible using FTIR spectra and chemometric analysis. Analysis of FTIR spectra of crude lipopolysaccharide extracts was better at classifying these strains than lipopolysaccharide pattern analysis using deoxycholic acid-polyacrylamide gel electrophoresis (DOC-PAGE) in terms of speed and sensitivity. Spectra of intact cells and crude lipopolysaccharide extracts from the strains were collected and analyzed using canonical variate analysis and Mahalanobis distances. Spectra collected identified structural regions of similarity and difference between the strains. The FTIR method and analytical approach used can be useful for taxonomical or epidemiological studies and can be expanded to focus on the similarities and differences in spectra to aid in future investigations
(Milestone F-3, NP 108 Action Plan Postharvest Pathogen Food Safety 2.1.1.2). A scatterometer was developed to measure the angel-resolved forward scattering characteristics of surfaces of bacterial colonies and was successfully adapted to measure and differentiate specific patterns of different Listeria cultures (Milestone G-1, NP 108 Action Plan Postharvest Pathogen Food Safety 2.1.1.4). After genomic/proteomic analysis, nine rabbit polyclonal antibodies (PAb) were developed against different target peptide antigens of L. monocytogenes. All but one showed specific reaction with the target peptide. Of which, only PAb Lm404 (internalin B) and LmC639 (ActA) (actin polymerization protein) showed specific reactions for the proteins from L. monocytogenes and not from any other Listeria species. However, the remaining PAbs reacted with multiple protein bands and showed cross- reactions with other Listeria species. The lack of specific binding could be due to the denaturation and altered
folding of the antigen on the cell surface. InlB and ActA peptides appeared to be the most promising targets for L. monocytogenes specific antibody production. The major drawback for these antibodies was, high background reactions with E. coli and Salmonella and this is because the rabbits already had high titers of antibodies against several common microorganisms in the serum. Specific pathogen-free rabbits are currently being considered for antibody development for these antigens (Milestone H-1, NP 108 Action Plan Postharvest Pathogen Food Safety 2.1.1.4). Reaction patterns of several monoclonal and polyclonal antibodies, routinely used for detection of Escherichia coli O157:H7, Salmonella Enteritidis and L. monocytogenes, were tested in ELISA for their reaction with cells subjected to different stress environments for short duration (3 h) or for extended period (7 days). The stress conditions employed for short duration include high temperature (45DGC), acidic environment (pH 5. 5),
salt (5.5% NaCl), ethanol (5%) and oxidative stress (15 mM H2O2). The results indicate that temperature salt and alcohol-induced stress appeared to repress antigen expression for anti E. coli O157:H7 and anti Salmonella antibodies, whereas most stresses caused increased reaction for anti L. monocytogenes PAb. The ELISA reaction of antibodies to each culture grown for extended period under the combined stress conditions (12DGC or 4DGC, pH 5.5 and NaCl 3.5%), likely to be encountered during food processing and storage, were studied. Stress conditions manifested variable ELISA reaction; however, low temperature affected the reactions most. This phenomenon was apparent in pathogens, which were spiked in hotdog samples subjected to combined stress conditions Milestone H-3, NP 108 Action Plan Postharvest Pathogen Food Safety 2.1.1.4). Proof of principle experiments were successful in showing FRET using the previously identified fluorescein and JOE fluorophore pairs which were previously
determined. A molecular beacon was synthesized with fluorescein and JOE and examined on a fluorescence spectrophotometer for the appropriate wavelength shift. Heat induced denaturation of the beacon showed the wavelength shift from the hybridized to the nonhybridized and reverse. This information will be utilized in the design of the field deployable detection unit (Milestone I-2, NP 108 Action Plan Postharvest Pathogen Food Safety 2.1.1.2). A fiber optic sensor was developed for L. monocytogenes that can detect about 4000 to 40,000 CFU/ml after 2.5 h of sampling even in the presence of common food contaminants or stress conditions. This sensor was able to detect L. monocytogenes from hotdog or bologna naturally contaminated or artificially inoculated with 10 to 1000 CFU/g after enrichment in buffered Listeria enrichment broth in less than 24 h. This is an important achievement since this sensor is capable of detecting target pathogen from a real-world food sample in less than 24 h
(Milestone J-2, NP 108 Action Plan Postharvest Pathogen Food Safety 2.1.1.4). C. Significant activities that support special target populations: Since many of the projects involve detection and control of foodborne pathogens that effect immuno-compromised populations (i.e. L. monocytogenes for infants and pregnant women, E. coli O157:H7 in small children, Salmonella in the elderly, there are several benefits for preventing foodborne illness for special target populations. D. Progress Report: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The project's goals are to contribute to the fundamental scientific and engineering knowledge base for rapid detection of microbial pathogens that present significant risks to public health. A wide variety of approaches, undertaken from engineering and non-engineering scientists, are being investigated to improve samples preparations, target separation, and detection.
Fundamental advances and basic research findings have been submitted to and published in refereed journals, presented as papers at national professional society meetings, seminars at universities, and cooperative research meetings. The impact of this research has manifested itself in the development of various laboratory prototypes and information packaged for purposes of testing in a preliminary manner leading to commercialization for food industry samples. This research has advanced knowledge in the field of detecting food pathogens by applying fundamental research to address practical challenges for food pathogen detection. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The research of this program has resulted in two issued US patents: Bashir,
R., Gomez,R., Robinson, P., Bhunia, A., Ladisch, M.R. Biosensor and Related Method, US Patent 6,716,620 (April , 2004). A microscale biosensor for use in the detection of target biological substances including molecules and cells is a microfluidic system with integrated electronics, inlet-outlet ports and interface schemes, high sensitivity detection of pathogen specificity, and processing of biological materials at semiconductor interfaces. A fabrication process includes an all top- side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the non-planar topography of the chip using spin-on-glass as an intermediate bonding layer. Detection sensitivity is enhanced by small fluid volumes, use of a low-conductivity buffer, and electrical magnitude or phase measurements over a range of frequencies. Sayler, G. S., Ripp, S. A., Applegate, B. Bioluminescent Biosensor Device.
U.S. Patent 6,544,729 (April,2003). Disclosed are methods and devices for detection of bacteria based on recognition and infection of one or more selected strains of bacteria with bacteriophage genetically modified to cause production of an inducer molecule in the bacterium following phage infection. The inducer molecule is released from the infected bacterium and is detected by genetically modified bacterial bioreporter cells designed to emit bioluminescence upon stimulation by the inducer. Autoamplification of the bioluminescent signal permits detection of low levels of bacteria without sample enrichment. Also disclosed are methods of detection for select bacteria, and kits for detection of select bacteria based on the described technology. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Reported under CWU 1935-42000-035-01S.
Impacts
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Publications
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