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Detection of Pathogenic Bacteria by Biosensors


Develop sensitive, specific and rapid processes that require minimal culture enrichment,for the detection of pathogenic bacteria and the indication of product safety in food systems, particularly, to serve as substantiating tests for HACCP. Compare with traditional microbial detection and sampling methods.

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Specific research areas include:(1) develop means such as magnetic continuous flow systems to selectively concentrate targeted bacteria in ground beef, chicken and pork by the use of specific immuno magnetic beads,and (2) develop hardware and software to capture and electrochemically detect bacteria on millipore filters, optimize binding of ruthenium to antibodies to detect bacteria by chemiluminescence, and develop automated digital imaging process for detection of fluorescent labeled, magnetically aligned, immunomagnetic captured bacteria, (3) develop on-food sensors for rapidly indicating the safety level of food products and (4) evaluate the statistical validity of the developed detection processes for establishing their sampling criteria.
<ol> <li>What major problem or issue is being resolved and how are you resolving it? The presence of pathogenic bacteria at any stage of food production, processing, and distribution must be quickly determined to allow proper disposition prior to consumption by the general public. Detection and quantification of pathogenic microorganisms in foods is vital for ensuring a safe food supply. There is a need for rapid, sensitive, and specific tests which researchers, farmers, processors, and retailers can use to verify that foods are safe to consume. Effective tests must meet a number of criteria: speed is critical since modern processing and distribution systems operate rapidly; high sensitivity is desirable since an infectious dose may be as little as one organism; selective detection is required because pathogenic bacteria comprise a small fraction of an otherwise benign population of microorganisms. Thus, we conduct research to develop new, or modify existing technologies to increase the speed, specificity and sensitivity of detecting food-borne pathogens to meet the needs of food producers/processors and regulatory agencies.
<li>How serious is the problem? Why does it matter? Foodborne illnesses caused by pathogenic microorganisms pose a threat to public health. Over 5 million cases of foodborne bacterial diseases occur annually in the U.S. The economic impact of foodborne illnesses is significant in that time lost from work, medical care, and the costs of recalling and destroying contaminated products amount to billions of dollars a year. Traditional microbiological methods require days to detect pathogenic bacteria such as E. coli O157:H7, Campylobacter, Salmonella, etc. in foods. New approaches must be developed to allow detection of low levels of specific pathogenic bacteria within a standard 8-hour shift.
<li>How does it relate to the National Program(s) and National Component(s)? Our research is directed to address the needs of developing tests that are precise, reliable, and rapid enough to detect microbial contamination in all foods prior to their entering into commerce, a major goal of the Microbial Pathogens component of the Food Safety Program of ARS (National Program 108, 100%.)
<li>What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2000 year: Established Theory and Methodology for High Precision Enumeration of Bacteria. Enumeration of bacteria is an essential part of any research program on detection, but current methods of enumeration have serious deficiencies in terms of preparation time, throughput, and accuracy. We recently developed a new technique, the micro-growth method, for automated, accurate, high-throughput enumeration. In the past year, we have tested a range of microorganisms to include Salmonella, E. coli O157:H7, Campylobacter coli, and Lactobacilli. We have also developed and experimentally confirmed the theory for self-calibration using serial dilutions of an unknown sample. This allows the micro-growth method to be used for absolute enumeration without standards, and removes the only significant limitation of the methodology. B. Other Significant Accomplishments(s), if any: 1. Modeling the Capture of Pathogenic Bacteria by Immunomagnetic Beads (IMB). IMBs capture targeted bacteria using the specific antibody coated on the bead surface. The captured bacteria can then be separated from other components on/in the food matrix with a magnet. However, there is no quantitative information on the conditions to achieve maximum capture. We have developed a systematic approach to mathematically model the capture of any (target or non-target) bacterial species with any IMB. This model allows us to predict the quantity of IMB and mixing time needed to achieve desired capture and thus develop standard procedures for using IMB techniques in high throughput laboratories. 2. Determining the cause of the apparent loss of bacterial activity during magnetic separation (MS) of IMBs. There is a loss of approximately 5-15% of the cellular activity associated with IMB-captured bacteria per MS. Our research has shown that this value varies mainly as a function of the number of cells captured per IMB and is not due to the death of captured cells. These data argue that the loss is related to an increased buoyancy of the bacteria-IMB complex. This work is significant in as much as it indicates that repeated MS steps (e.g., as in washing) should be avoided and that maximum efficiency is attained when the ratio of IMBs to cells is much greater than one. 3. Developed a rapid, sensitive and 96-well microplate Reade-based method for detecting Escherichia coli O157:H7 in beef hamburgers. The 96-well microplate reader is widely used in food microbiology laboratories for applications such as enzyme linked immunosorbent assay (ELISA) of bacterial cultures after overnight enrichment. To maximize the practical value of the 96-well apparatus, we have incorporated immunomagnetic bead capture and separation of E. coli O157:H7 from artificially spiked beef hamburger and an alkaline phosphatase linked ELISA method for detection. We found that this approach detected the presence of 1 cell of the E. coli per gram of hamburger after a four-hour incubation. This result demonstrated that proper modification of testing procedures could provide rapid and sensitive pathogen detection using existing laboratory apparatus.
<li>Enhanced stability of bacterial samples on biotin coated membranes. Current practice of bacterial analysis requires shipping of large chucks of meat from remote processing locations to central laboratories. The practice is costly and may introduce additional contaminations. In the process of developing new detection method for E. coli O157:H7 in beef hamburger using a device called Light Addressable Potentiometric Sensor (LAPS), we used streptavidin coated magnetic beads conjugated with proper antibody to capture the E. coli. The beads and captured bacteria were then locked on a biotin-coated membrane, and exhibited a relatively long stability toward LAPS detection at refrigerated temperatures. We have now improved the stability to about three days at room temperature by the use of smaller magnetic beads. This information is useful for food processing institutions relying on external bacterial testing support.
<li>Developed time-resolved-fluorescence assay for Pathogenic Bacteria Detection. Fluorescence detection of pathogens is often compromised by similar fluorescence associated with other components in foods. We have utilized a method called Time-Resolved-Fluorescence (TRF) in which targeted bacteria were tagged with antibody containing europium, a rare-earth metal ion that maintains the ability to emit specific fluorescence long after interfering fluorescence fades away. Thus, by delaying the start of measurement we could specifically correlate fluorescence intensity to the quantity of pathogens in foods. With this fluorescence measurement method and immunomagnetic capture approach, we were able to detect about 10 E. coli O157:H7 spiked in one milliliter of apple cider after a four-hour enrichment and about 1 cell of E. coli O157:H7 spiked in 1 gram of beef hamburger after four and half hour enrichment. 6. Improved the process safety of the immunomagnetic electro-chemiluminescent method for E. coli O157:H7 detection. The immunomagnetic-electrochemiluminescent method we developed to detect E. coli 0157:H7 in food required heat-killing and filtering steps to alleviate any concern about potential aerosols generated during a mixing step and eliminate large particles that could block the fluidics system of the detection instrument. These steps are labor intensive and time consuming, so we examined various chemical techniques to kill the cells and ultimately eliminated the heat-killing step by incubating the cells in a reagent that disrupts the cell membrane. The disruption of the cell membrane releases additional cell membrane associated antigen which increases the sensitivity of the assay and solubilizing the membrane eliminates the need to filter the sample. Eliminating the heat-killing and filtering steps allows robotics to be used to automate the assay thereby reducing cost and increasing throughput. C. Significant Accomplishments/Activities that Support Special Target Populations: N/A. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. Since the inception of our current CRIS in 1996, we have adopted the use of IMBs to separate and concentrate pathogens for detection by a variety of biosensors. Some examples described below, could detect E. coli O157:H7 at a level of 1 CFU g-1 (beef hamburger) after enrichment at 37 oC for 5 6 hrs. (1). We have developed an immunomagnetic-electrochemiluminescent (IM-ECL) method to detect Escherichia coli O157 in ground beef, using a commercially available analytical instrument and have also developed a confirmatory method based on immunomagnetic cell capture (IMCS) with plating on chromogenic media. We have collaborated with a government inspection agency to adapt the method to the procedures used in their laboratories. (2). A government inspection agency has incorporated our approaches for immunomagnetic cell separation and plating on Rainbow(R) agar O157 media into their official methods. The IM-ECL assay is 10 to 100 times more sensitive than the test they currently use. However, they have not adopted this presumptive assay because of the physical manipulations required for heat killing and carousel loading. The inspection agency has also requested that the IM-ECL assay be put in a 96 well format which can then be incorporated into a robotic system. (3). We have used IMBs to capture E. coli O157:H7, treated the organism with a fluorescent nucleic acid stain (DAPI), and used fluorescent microscopic imaging for detection. In this work, we applied a magnet to concentrate and align the IMB on the microscope slides for faster counting of fluorescent area and enumerated the bacteria by the use of an automated 2-dimensional microscope stage and CCD camera. This process should reduce the enumeration time and eliminate the operator fatigue associated with manual microscopic counting. (4). We have devised a new procedure to determine the viability of detected E. coli O157:H7. In this approach, the energetic status of the bacteria was adjusted by the addition of glucose, a carbon nutrient source, and carbonyl cyanide meta-chlorophenyl hydrazone (CCCP), a membrane protonophore. The addition of glucose slightly increased the ATP content of the bacteria. On the other hand, CCCP depleted the ATP content. None of the glucose and CCCP effects could be detected with heat-killed and gamma-ray irradiated E. coli O157:H7. Thus, immunomagnetic capture of the E. coli followed by described tests would confirm the presence of viable E. coli O157:H7. Bioluminescence determination of ATP has been widely adopted by the food industry to monitor sanitary conditions. The approach developed by us can increase the value of this screening technique. To minimize the need of enrichment, methods that can concentrate small quantities of targeted pathogens in samples with large volume, must be developed. To meet this goal, we have developed micro-immunoaffinity columns for capture and concentration of bacteria from food samples. Initial experiments used porous, low-density polyacrylamide particles to capture Salmonella enteriditis, while effective in capture, these particles required centrifugation to separate from the sample. Attempts to visualize or detect bacteria bound to the particles with fluorescent or enzyme-conjugated antibody was difficult due to high background from antibody trapped in the pores of the particles. We have now prepared columns using solid, high-density glass beads that exhibit low background binding and settle rapidly to allow simple separation of the particles from the sample. These columns have been used to capture E. coli O157:H7 and Campylobacter coli with >90% efficiency.
<li>What do you expect to accomplish, year by year, over the next 3 years? Over the next three years, we intend to accomplish the following: (1). To develop magnetic columns using immunomagnetic beads as column packings to rapidly concentrate targeted food pathogens in food systems. The milestones for each year are: Year 1. To complete characterizing the interactions between magnetic column materials and immunomagnetic beads; Year 2. To complete the design of automatic control of the magnetic column and application to pure bacterial cultures.; and Year 3. To test the assembly in food systems. (2). To automate our developed detection processes to use a 96-well format. The milestones are: Year 1. To complete the reagent injection sequence study on the confirmation test of E. coli O157:H7 using ATP luminescence measurement. Year 2. To complete the integration of sequence study to a commercially available 96-well plate reader and Year 3. To expand the automation work to other pathogenic bacteria.
<li>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 & durability of the technology product? In FY 2000, there are a couple of technology transfer activities worthy of mention. First, we have worked with a federal regulator agency to implement the assay in its laboratories. The procedure for the assay was briefly adopted and was then put on-hold for certain laboratory safety issues. We are currently addressing these concerns. Because of our development of the assay for E. coli, our CRADA partner has formed a business division to develop and market kits to detect enteric pathogens, non-enteric pathogens (Listeria, Salmonella, Campylobacter, etc.) and other organisms such as parasites, mycotoxins, and mycoplasma. They have completed the conversion of the laboratory assay we developed for E. coli O157 into testing kits and are being evaluated by a few major fast food chains. Our partner has also redesigned the assay into a 96 well format high-throughput instrument. We have formed a collaborative arrangement with an instrument company to develop procedures for detecting pathogenic bacteria in foods. The methodology included the applications of time-resolved fluorescence measurements. Because the non-specific background fluorescence was minimized, the developed procedure would exhibit very low false-positive measurement and would also allow multiplex measurement of a single sample. We anticipate this collaboration would lead to the development of a highly desirable and automated pathogen detection procedure for the food industry and inspection laboratories.

Tu, Shu-I
USDA - Agricultural Research Service
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