Progress 04/20/11 to 01/18/16
Outputs
Progress Report Objectives (from AD-416): 1: Develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self- validating system into an automated instrument. 1A: Integrate technology platforms that we have developed and individually tested into a usable technology for detecting L. monocytogenes in less than 8 h (time-to-result). 1B: Integrate technology platforms, currently being developed in our laboratory, into a usable technology for detecting Salmonella . 1C: Integrate technology platforms into a usable technology for detecting STEC. 2: Develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. 2A: Microfabricate and characterize microfluidic biochips that will direct, concentrate, and quantify living microorganisms using micro- and nano-scale electrical, mechanical, and optical methodologies. 2B: Develop light scattering technologies for rapid and high throughput detection and identification of pathogenic bacteria based on unique scattering signatures generated by concentrated colonies. 2C: Develop bacteriophages carrying reporter genes for the detection of E. coli O157:H7 and other foodborne pathogenic bacteria. 2D: Develop a highly sensitive enhanced Raman spectrosensor for field- deployable and routine benchtop in-lab identification of foodborne pathogens. Approach (from AD-416): Our approach will be to carry out our objectives using 2 important steps. The first step will be to develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self-validating system into an automated instrument. When carried out effectively, this step will enable different types of detection platforms to be more effective and accurate. The next step is to develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. An experienced multidisciplinary team of investigators from Purdue University, University of Illinois, and the USDA will engage manufacturers of commercial, off-the-shelf components to construct instruments, and the food processing industry or regulatory agencies to test them. This integrated effort will produce operational technologies that can be used to better detect and quantify microbial hazards in food. We are developing technologies for the separation, concentration, and detection of foodborne pathogens. Our bioseparation technology, winner of the Food and Drug Administration (FDA) Food Safety Challenge 2014, combines pre-filtration, enzyme treatment, and automated microfiltration to concentrate viable bacteria from foods for further analysis. Our approach reduces enrichment time, and allows for detection of low target cell numbers (1-10 CFU/g) in food. Coupled to fast detection methods, including other CFSE technologies and DuPont�s BAX PCR based detection system, the length of time from unprepared food sample to verification of pathogen has been reproducibly achieved in less than 8 hours. Prototype units are housed at USDA and an industry partner for testing and application development. Our microfluidic biochip incorporates operations to further concentrate bacteria, support bacterial growth and lysis, and then detect pathogen-specific genes. The microfluidic chip automates a labeling process (<20 min) prior to DNA amplification for detecting only viable pathogens. Our elastic light scatter �BARDOT� (Bacterial Rapid Detection using Optical Scattering Technology) system uses light scattering techniques to differentiate and classify bacterial colonies grown on Petri-dishes. The BARDOT successfully differentiated bacteria at genus, species, and serovar/serotype levels. BARDOT analysis resulted in correct classification often above 90% for foodborne pathogens (Listeria monocytogenes, Salmonella, Shiga toxin-producing E. coli (STEC), Vibrio, and Bacillus species). Additionally, a light scattering image library of indicator bacteria representing the Enterobacteriaceae family was generated, containing 1600 scatter images from 14 genera and 31 species. The technology has been licensed to Hettich GmbH, with commercial BEAM instruments recently placed at Purdue, USDA-ARS, and New Zealand. Efforts are ongoing to validate pathogen identification by BARDOT in real-world samples, emphasizing leafy greens and poultry, by coupling laser scatter patterns with DNA sequence verification. Numerous hardware and software upgrades have been made for improved data collection and analysis, and a portable instrument was developed with a prototype placed at USDA-ARS. Another platform in development is the phage-based detection of STEC wherein phage cause the target bacteria to produce light if infected. A new optical based bioluminescent reporter phage was developed using the NanoLuc luciferase derived from the deep-sea shrimp Oplophorus gracilirostris and evaluated for sensitivity and overall detection performance for E. coli O157:H7. Enrichment assays using E. coli O157:H7 grown in LB broth with a phage concentration of 1.76�102 pfu mL-1 are capable of detecting approximately 5 CFU in a 40 mL sample in 7 hours and contaminated ground beef after an overnight incubation. The nature of the phage construct also allows the recovery of the detected isolate for trace back analysis. Enhanced Raman spectroscopy and a strip-based lateral flow assay were further developed that could detect <100 CFU L. monocytogenes/mL in 30 min. Other studies established that Salmonella Heidelberg strains that were associated with a foodborne outbreak were more heat resistant and formed stronger biofilms than other Salmonella strains. Accomplishments 01 Multipathogen detection using BARDOT. Pathogen screening and detection is an integral part of mitigating outbreaks and illnesses. This process is arduous, labor-intensive, and less informed when performed separately to characterize each pathogen. Multipathogen detection from the same sample on a single platform not only saves time, but also provides more information to laboratories and regulatory agencies thereby improving informed decision making related to food safety. ARS- funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, developed a growth media that supports the growth of multiple pathogens, Salmonella enterica, E. coli O157:H7, and Listeria monocytogenes (called SEL), and used their elastic light scattering pathogen detection device (called BARDOT) to identify multiple pathogens grown on the SEL medium. This approach accurately classified (>99% classification accuracy) Listeria, E. coli, and Salmonella that had been inoculated onto foods. This cost-effective approach easily integrates into existing laboratory protocols and can be used by the food industry and regulatory agencies for multipathogen screening and detection. 02 Development of a portable BARDOT pathogen identification instrument. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, developed a small instrument that can be field deployable for pathogen identification. The instrument is small, light, and efficient and can be transported to remote sites providing accurate and fast organism identifications. The scientists miniaturized their elastic light scattering pathogen detection device (called BARDOT) which is able to differentiate bacteria at genus, species, and serovar/ serotype levels. BARDOT analysis resulted in correct classification often above 90% for foodborne pathogens (Listeria monocytogenes, Salmonella, Shiga toxin-producing E. coli (STEC), Vibrio, and Bacillus species). This technology will have significant impact on our ability to take detection technology directly to regional or remote testing facilities, as well as food production, food distribution, or packaging sites in the case of a potential contamination incident. The portable instrument will be transformational for microbial detection when normal laboratory facilities may not be available. 03 Accelerated sample preparation for pathogen detection. Current pathogen detection methods allow for detection or quantitation of bacteria in hours, but the time from sample to result is often extended by days for preparing the samples for analysis. ARS funded scientists and engineers at the Center for Food Safety Engineering in West Lafayette, Indiana, developed hollow fiber microfiltration modules and an automated instrument to rapidly concentrate and recover microorganisms from foods. The time required for microfiltering samples from spinach, milk, egg white, and chicken carcass rinse wash is one hour, compared to 16-24 hours for traditional microbiological culture enrichment. The entire procedure from sample processing, including a short enrichment step, through microfiltration followed by pathogen detection is completed within 8 hours. This rapid approach facilitates pathogen detection within one work shift, enabling informed decision making to reduce the distribution of contaminated foods. 04 Detection of pathogens using silicon bioelectronics. ARS-funded scientists at the Center for Food Safety Engineering in based in West Lafayette, Indiana, received the Singapore Challenge 2016 Prize in the Singapore Global Young Scientists Summit (GYSS) World Wide Competition. GYSS brings together young researchers with science and technology leaders from around the world for a multidisciplinary summit that covers topics ranging from chemistry, physics, and medicine to mathematics, computer science, and engineering. Participation in the summit is by invitation only. The winning technology is a silicon bioelectronics-based pathogen detection biosensor that can detect harmful food-borne pathogens in about eight hours and without the need for a trained technician. Conventional pathogen detection technologies rely on culture-based techniques that are time consuming and resource intensive. The developed biosensors could speed up diagnosis dramatically during foodborne pathogen outbreaks, thereby saving money and lives. 05 Low cost, rapid detection of E. coli O157:H7 using phage. Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, have modified a (bacterio)phage specific for E. coli O157:H7 by adding a gene from a bioluminescent shrimp which cause the live cells of the pathogenic E. coli to glow after bacteriophage infection. The developed diagnostic tool is very specific and very sensitive, detecting as little as 5 cells of the pathogen in less than 7 hours during the culture enrichment. The method also has the potential for direct detection of higher pathogen concentrations directly in ground beef. The process uses very low concentrations of phage and can be integrated into current laboratory protocols. The coupling of low phage concentrations and the ease of integration into current protocols provide a low cost method for the detection of E. coli O157:H7 potentially leading to improved pathogen surveillance and a safer food supply. 06 Low temperature short time process for extending the shelf life of fresh milk. Pasteurization has long been the standard method to extend the shelf-life of dairy products, as well as a means to reduce microbial load and the risk of food-borne pathogens. ARS-funded scientists at the Center for Food Safety Engineering (CFSE) in West Lafayette, Indiana tested a novel low temperature, short time (LTST) pasteurization method in which milk is dispersed in the form of droplets and treated with low heat/pressure variation over a short treatment time. LTST was very effective, reducing the level of microorganisms at least 10 million fold and up to 100 million fold. The CFSE-developed BARDOT technology was used to demonstrate that the only survivors of LTST were very few organisms (Bacillus spp.) that do not grow at refrigeration temperatures. The LTST process extended the shelf- life of the milk from 14-35 days to as long as 63 days. The improved shelf-life will positively impact the dairy industry in terms of shipping and overall sustainability. 07 Method for rapid detection of low pathogen numbers. The majority of biosensors and rapid detection methods require thousands of cells for detection. Because foodborne pathogens are present in low numbers, if at all, in foods, reducing the number of cells required for detection can speed up the time-to-result. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, developed sensors based on surface enhanced Raman spectroscopy (SERS) and enhanced lateral flow immunochromatography that are able to detect approximately 100 cells of a foodborne pathogens in a small food sample. The entire procedure for the assay can be performed in 30 minutes. The simplicity, analytical sensitivity, specificity, and robustness of the biosensors will enable their implementation in various sectors of the food chain in efforts to improve food safety and prevent the distribution of contaminated foods. 08 Salmonella Heidelberg outbreak strains are more likely to form biofilms than other Salmonella species. Biofilms have become widely problematic in the food industry. When bacteria form biofilms, they become more resistant to environmental conditions (such as heat and sanitizers) and more difficult to remove from food or food contact surfaces. ARS-funded scientists in the Center for Food Safety Engineering at Purdue University evaluated the biofilm forming ability of several Salmonella isolates of different (sero)types at different temperatures. The results revealed that two strains of serotype Heidelberg showed significantly stronger biofilm-formation than the other Salmonella isolates at refrigeration temperatures. These isolates represented the top two strains in the outbreak they are associated with, one of which had a 49% hospitalization rate in a recent outbreak. These results indicate that the foodborne illness-associated S. Heidelberg isolates were more likely to form biofilms under stressful conditions, such as those found in the poultry processing environment.
Impacts
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Publications
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 1: Develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self- validating system into an automated instrument. 1A: Integrate technology platforms that we have developed and individually tested into a usable technology for detecting L. monocytogenes in less than 8 h (time-to-result). 1B: Integrate technology platforms, currently being developed in our laboratory, into a usable technology for detecting Salmonella . 1C: Integrate technology platforms into a usable technology for detecting STEC. 2: Develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. 2A: Microfabricate and characterize microfluidic biochips that will direct, concentrate, and quantify living microorganisms using micro- and nano-scale electrical, mechanical, and optical methodologies. 2B: Develop light scattering technologies for rapid and high throughput detection and identification of pathogenic bacteria based on unique scattering signatures generated by concentrated colonies. 2C: Develop bacteriophages carrying reporter genes for the detection of E. coli O157:H7 and other foodborne pathogenic bacteria. 2D: Develop a highly sensitive enhanced Raman spectrosensor for field- deployable and routine benchtop in-lab identification of foodborne pathogens. Approach (from AD-416): Our approach will be to carry out our objectives using 2 important steps. The first step will be to develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self-validating system into an automated instrument. When carried out effectively, this step will enable different types of detection platforms to be more effective and accurate. The next step is to develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. An experienced multidisciplinary team of investigators from Purdue University, University of Illinois, and the USDA will engage manufacturers of commercial, off-the-shelf components to construct instruments, and the food processing industry or regulatory agencies to test them. This integrated effort will produce operational technologies that can be used to better detect and quantify microbial hazards in food. We are developing technologies for the separation, concentration, and detection of foodborne pathogens two of which were the winner and a top five finalist respectively, in the first-ever Food and Drug Administration (FDA) Food Safety Challenge 2014: the bioseparation technology and the microfluidic biochip. The bioseparation technology combines pre-filtration, enzyme treatment, and automated microfiltration to concentrate viable bacteria from foods for further analysis. By adding a short enrichment step prior to filtration, Salmonella recovery rates were improved and sample preparation accomplished within 4 hrs, leaving another 4 hrs for pathogen detection analysis to obtain results within one work shift. Prototype units are housed at USDA and an industry partner for testing and application development. A new multiplexed commercial prototype was built to increase sample throughput. The microfluidic biochip incorporates operations to further concentrate bacteria, support bacterial growth and lysis, and then detect pathogen- specific genes. Our microfluidic chip automates a labeling process (<20 min) prior to DNA amplification for detecting only viable pathogens. On- chip reference electrodes were improved and, in collaboration with a commercial partner, a novel bio-field-effect-transistor array platform with over one million transistors was fabricated, obtaining the redundancy and sensing area required for effective pathogen sensing. Our �BARDOT� (Bacterial Rapid Detection using Optical Scattering Technology) system uses light scattering techniques to differentiate and classify bacterial colonies grown on Petri-dishes. The BARDOT successfully differentiated bacteria at genus, species, and serovar/serotype levels. BARDOT analysis resulted in correct classification often above 90% for Listeria monocytogenes (91-100%), Salmonella (88-100%), Shiga toxin- producing E. coli (STEC) O157:H7 and non-O157 STECs (91-99%), Vibrio (96- 100%), and Bacillus species (93-99%). The technology has been licensed, with commercial release planned for November, 2015. Efforts are ongoing to validate pathogen identification by BARDOT in real-world samples, emphasizing leafy greens and poultry, by coupling laser scatter patterns with DNA sequence verification. Numerous hardware and software upgrades have been made for improved data collection and analysis. Another platform in development is the phage-based detection of STEC wherein phage cause the target bacteria to produce light if infected. Nine genomes of STEC-infecting phage were sequenced, anlayzed and submitted to a public database. Using the FDA procedure for enrichment of E. coli O157:H7 in leafy greens, the phage technology detected as little as 59 O157:H7 cells per gram of romaine lettuce long before the FDA-recommended DNA testing has even begun. Other studies established that most bacteria isolated from lettuce by Listeria-selective enrichment and plating on FDA- approved media are not from the genus Listeria. We now have a library of approximately 400 false positive strains, many of which are capable of growth on multiple Listeria-selective medias. Enhanced Raman spectroscopy and a strip-based lateral flow assay were developed that could detect 100 CFU L. monocytogenes/mL in 30 min. Accomplishments 01 Two CFSE technologies selected as the winner and a top 5 finalists in the first-ever FDA Food Safety Challenge 2014. Two technologies developed by ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, are among the five finalists in the first-ever FDA Food Safety Challenge 2014: the bioseparation technology and the microfluidic biochip. The innovation challenge presented by FDA was to encourage development of technologies for detecting Salmonella in minimally processed fresh produce. The grand-prize winning bioseparation technology is a physical method for concentrating Salmonella to detectable levels using a combination of enzyme treatment and prefiltration followed by automated microfiltration. The sample preparation is completed within 4 hours, leaving another 4 hours to accomplish specific pathogen detection within a work shift(8 hours). The top 5 finalist technology is a portable system for multiplexed detection of foodborne pathogens in microfluidic biochips through isothermal DNA amplification and electrical detection. The inexpensive and portable system has two core modules: one for concentration of cells, to minimize sample enrichment time, and a second for multiplexed electrical detection of pathogens by DNA amplification steps. 02 Automated microfluidic detection of viable Shiga-toxin producing E.coli. The food safety community has immensely benefited from advances in DNA amplification techniques, such as PCR, that enable rapid detection and identification of foodborne pathogens. PCR methods are faster, less expensive, and more specific than traditional bacterial culturing methods but have an important drawback: they are incapable of differentiating between living and dead pathogens. Because for many applications only viable pathogens are considered contaminants, approaches have been developed that inactivate the DNA from dead bacteria, allowing the detection of only viable pathogens; however, the DNA inactivation process is cumbersome and inefficient. To address this challenge, ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, designed, fabricated, evaluated, and optimized an automated method for detection of only viable pathogens on a microfluidic chip (about the size of regular microscope glass slide) for which the user only needs to flow the sample to be analyzed and collect labeled sample after approximately 20 min. Our experiments have demonstrated that the on- chip process is robust, enables identification of only living pathogens, and uses a fraction of the reagents used in the previous protocols. The new microfluidic device is reducing cost and complexity of a technique that will improve biomolecular analysis of food samples by enabling specific screening assays with added information. 03 Improved biochip for detection of foodborne pathogens. Electrical biosensors have several advantages over optical sensing and other current biosensing mechanisms used for the detection of foodborne pathogens in that they are inexpensive, enable multiplexed reactions, and are easily integrated with other electronics. However the use and fabrication of electrical biochip sensors requires stable voltage referencing to be able to identify signals coming from the detection reaction. Conventional reference electrodes for ion-sensitive field effect transistors (ISFETs) are bulky, fragile, and too big for small volume applications. Previous solutions have been limited by poor robustness, high cost, or complex integration with other microfabrication processes preventing the widespread use of ISFET as biological sensors. ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, created a simple method to create robust on-chip reference electrodes with demonstrated good stability, low drift, and sensitive pH response and a fabrication technique that is easily transferable from the academic laboratory to an industrial setup. This new development is important for the fabrication of electrical biosensors used in detection of foodborne pathogens. 04 Development of a 1 million field-effect transistor (FET) biosensor array for multiplexed detection of biomolecules. Challenges faced by healthcare and regulatory industries are driving the development of new miniaturized biological sensors that promise to revolutionize diagnostics and screening methods. The demand for higher quality with lower costs could be met using novel systems for automated point-of- care or in-plant diagnostics that relax workloads and improve control. After a decade of development, the FET biosensing technology is well understood; however, commercialization of this technology has proven challenging due to lack of uniformity and low yields. A possible solution to this issue relies on the highly scalable nature of FET fabrication which will allow multiplexing the detection and averaging the results from multiple independent tests. In collaboration with Taiwan Semiconductor Manufacturing Co, ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, fabricated a bio-FET array platform with over one million transistors obtaining the redundancy and sensing area required for effective biological sensing that will be used for screening of food samples with a novel concept that will result in faster, less expensive, and portable detection. The electrical biochip pathogen detector will result in more frequent testing in multiple environments improving the enforcement ability of regulatory agencies creating a safer food supply. 05 Detection of Bacillus species with BARDOT. Bacillus species are widely distributed in nature and have great significance both as industrially beneficial microbes and as public health burdens. To improve the detection of Bacillus species in foods, ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, applied their optical scatter technology (called BARDOT), a label-free nondestructive method in which a laser beam passes through the center of a bacterial colony on a Petri-plate and generates a unique scatter signature (fingerprint) that can be used for bacterial detection and identification. A total of 265 Bacillus and non-Bacillus isolates were used to develop and verify scatter image libraries including isolates from food, environmental, and clinical samples. All 118 strains of Bacillus were detected with a high positive predictive value (= 90%), and BARDOT successfully detected Bacillus from inoculated baby formula, cheese, and naturally contaminated raw milk in 7-16 h. BARDOT results were confirmed by molecular methods. This study demonstrates that BARDOT can be used as a screening tool to identify relevant Bacillus colonies from complex microbial communities present in foods. 06 Rapid detection of Salmonella from naturally contaminated chicken carcass rinse samples using a fiber optic sensor and BARDOT. Among foodborne illnesses, Salmonella is the topmost foodborne pathogen contributing to 35% of domestically acquired bacterial foodborne illnesses that lead to numerous hospitalizations and several deaths in the United States each year. ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, used a fiber optic immunosensor and their optical scatter technology (called BARDOT) to facilitate rapid detection of Salmonella from naturally contaminated poultry samples. The fiber optic sensor identified S. enterica in selective enrichment broth in less than 12 h. The colonies produced by plating the enriched samples on selective agar were scanned using BARDOT after 13�15 h, and S. enterica was identified after matching individual colony scatter patterns to the scatter image library with a sample-to-answer time of about 24 h. Both sensors revealed 4 positive samples (8%), and the results were identical to the USDA-Food Safety and Inspection Service (FSIS) Salmonella detection method which can take 72 h to yield results. The sensors used individually or in combination demonstrate potential for accurate and rapid detection of S. enterica in poultry. 07 Faster bioluminescent detection of E. coli O157:H7. Using the FDA procedure for enrichment of E. coli O157:H7 in leafy greens, ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, have shown that they can improve and reduce the time to detection of E.coli O157:H7 by applying their bioluminescent phage-based detection technology. After an initial five- hour incubation, selective antibiotics were added along with PhiV10-Lux phage solution. The bacteriophage transferred bioluminescence genes to the E. coli O157:H7 and light emission from those bacterial cells could be clearly seen. Using this approach, it was possible to rapidly detect a low number of pathogen cells (59 cells per gram) from romaine lettuce. This method offers an initial positive result indicating the presence of the pathogen during the enrichment procedure, long before the FDA recommended confirmatory DNA testing. This approach does not impact the workflow or expertise necessary for testing procedures and does not interfere with later genetic or serological diagnostic tests. 08 Growth and persistence of L. monocytogenes on and within romaine lettuce plants. Over the past 20 years, the number of foodborne illness attributed to consumption of fresh produce has increased dramatically and Listeria monocytogenes has become a major cause of both produce recalls and disease outbreaks. ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, examined the persistence of three L. monocytogenes strains on three romaine lettuce cultivars. Seeds were inoculated with L. monocytogenes and grown for up to 60 days on two soil types and on sterile soft-top agar, and then the average number of viable L. monocytogenes cells retained on seeds or persisting on growing plants was determined. Plants grown on sterile soft-top agar maintained high levels of L. monocytogenes after a 60 day period, while the pathogen was not found after 18 and 45 days on the two different soils, suggesting that soil microflora may impede pathogen persistence. When young plants grown from inoculated seeds were investigated for internalized pathogenic cells, L. monocytogenes cells were found to be internalized in all major tissues: the presence of the bacterium in the plant vasculature indicates its potential to be transported throughout the plant and reside within edible tissue. The significance of these findings is that romaine lettuce can support growth and internalization of L. monocytogenes, which could serve as a vehicle for L. monocytogenes transmission to consumers. 09 A low prevalence of L. monocytogenes associated with commercial varieties of romaine lettuce seeds. Listeria monocytogenes is a deadly foodborne pathogen and has recently been associated with outbreaks in romaine lettuce. ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, tested seeds from 16 cultivars of romaine lettuce and one cultivar of radicchio as a potential source of contamination in a 2014 commercial recall of bagged salad products. Only a single seed, out of 1,700 tested, was contaminated with Listeria monocytogenes. These data indicate that, while it is possible that the seed could be a source of potential L. monocytogenes contamination, it is unlikely that the seeds of the tested cultivars were the source of bagged salad recalls. 10 FDA-approved media for the selective isolation of Listeria doesn�t work well for lettuce. Listeria monocytogenes is a deadly foodborne pathogen and has only recently been associated with outbreaks caused by romaine lettuce. ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, determined the suitability of standard selective growth media for the isolation of Listeria from lettuce. All four media tested could be used successfully to isolate Listeria spp. from lettuce; however, DNA sequencing technologies revealed that a vast a majority (80-90%) of the bacterial colonies that appeared to be Listeria were actually other types of bacteria. A library containing approximately 400 strains of �false positives� has been assembled. These findings establish that preliminary screening of lettuce samples by the FDA-approved selective enrichment and plating technique is not very useful due to the high false positive rate, but that rapid DNA sequencing assays can be used to provide the needed accuracy. 11 Variability in bacterial communities associated with lettuce. Outbreaks of foodborne illness associated with leafy-green vegetables have increased dramatically in the last few decades. Pathogenic bacteria on leafy greens are present on leaf surfaces in association with a much larger community of harmless bacteria, which may affect pathogen association and persistence of the edible portion of the leaf. ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, have determined the composition of bacterial communities located at different locations on the same lettuce leaf as well as on different leaves on the same plant. Many sampling protocols assume that bacterial communities found on plants are fairly similar over the entire surface of a leaf or even an entire plant. Metagenomic DNA sequencing data showed that this is not the case, with a large degree of variability evident in samples taken only a few centimeters apart on the same leaf. This information is important because it provides evidence that bacterial contaminants found on the surface of plants may be extremely localized, making them much more difficult to detect by conventional sampling strategies. 12 Salmonella outbreak strains show increased heat resistance. Salmonella enterica is the most costly of all the foodborne pathogens, causes the most deaths per year, and is the most common source of bacterial foodborne illness, and Salmonella heidelberg was responsible for an outbreak in 2013 that sickened 634 people and hospitalized 200, and several of the strains involved were resistant to multiple antibiotics. Due to the extended length and scope of the outbreak, we hypothesized that the outbreak strains might have enhanced resistance to food processing stresses such as heat and acid. ARS-funded scientists at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, evaluated the heat resistance of 12 Salmonella isolates; nine related to a foodborne outbreak as well as three non-outbreak related strains. Six of the nine outbreak-related isolates showed significantly increased heat resistance compared to the Salmonella typhimurium reference strain, and two showed significantly higher heat resistance than the S. heidelberg reference strain. There was no correlation between an isolate�s antibiotic resistance profile and enhanced heat resistance. Given that enhanced heat resistance might allow an isolate to survive food processing measures and even improper cooking, the high heat resistance of some of the outbreak isolates may help explain the length and scope of the outbreak.
Impacts
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): 1: Develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self- validating system into an automated instrument. 1A: Integrate technology platforms that we have developed and individually tested into a usable technology for detecting L. monocytogenes in less than 8 h (time-to-result). 1B: Integrate technology platforms, currently being developed in our laboratory, into a usable technology for detecting Salmonella . 1C: Integrate technology platforms into a usable technology for detecting STEC. 2: Develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. 2A: Microfabricate and characterize microfluidic biochips that will direct, concentrate, and quantify living microorganisms using micro- and nano-scale electrical, mechanical, and optical methodologies. 2B: Develop light scattering technologies for rapid and high throughput detection and identification of pathogenic bacteria based on unique scattering signatures generated by concentrated colonies. 2C: Develop bacteriophages carrying reporter genes for the detection of E. coli O157:H7 and other foodborne pathogenic bacteria. 2D: Develop a highly sensitive enhanced Raman spectrosensor for field- deployable and routine benchtop in-lab identification of foodborne pathogens. Approach (from AD-416): Our approach will be to carry out our objectives using 2 important steps. The first step will be to develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self-validating system into an automated instrument. When carried out effectively, this step will enable different types of detection platforms to be more effective and accurate. The next step is to develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. An experienced multidisciplinary team of investigators from Purdue University, University of Illinois, and the USDA will engage manufacturers of commercial, off-the-shelf components to construct instruments, and the food processing industry or regulatory agencies to test them. This integrated effort will produce operational technologies that can be used to better detect and quantify microbial hazards in food. We are developing technologies for the separation, concentration, and detection of foodborne pathogens. Our bioseparation technology achieved a 500-fold increase in cell concentration in <30 min by combining pre- filtration, enzyme treatment, and automated microfiltration. Natural flora and introduced pathogens were separated and concentrated from water and complex foods (including chicken carcasses) with high pathogen recovery (= 70%). A prototype of this instrument was delivered to USDA for testing and application development. Our microfluidic biochip technology incorporates operations to further concentrate bacteria, support bacterial growth and lysis, and then detect pathogen-specific genes. Another microchip with 16,384 individual sensors was able to complete array interrogation in <15min. A system called �BARDOT� (Bacterial Rapid Detection using Optical Scattering Technology) uses light scattering techniques to differentiate and classify bacterial colonies grown on Petri-dishes. A shortened enrichment protocol enabled BARDOT detection of the 20 most common Salmonella with >80% accuracy in 24 hr. BARDOT analyses of Bacillus, non-O157:H7 pathogenic E. coli, and antibiotic resistant staphylococci and enterococci were also undertaken. A portable BARDOT was delivered to a collaborating USDA lab and improvements were made to allow the grouping of organisms based on scatter patterns for bacterial community analysis. BARDOT systems using three different wavelengths of light, reflection capability, and an integrated colony morphology analyzer improved BARDOT sensitivity. Efforts are ongoing to validate BARDOT pathogen identification in real- world samples using DNA sequence verification. Another platform in development is the phage-based detection of E. coli O157:H7 wherein the target bacteria produces light if infected by the engineered phage. When used during selective enrichment of ground beef, high levels of E. coli were detectable within 1 hr, while as few as 10 cells could be detected in 12 hr. In the retail deli environment, persistent L. monocytogenes isolates had higher biofilm forming ability and sanitizer resistance than transient isolates, and, based on virulence gene characterization, had greater potential to cause disease. Studies of plant-associated pathogens revealed: 1) L. monocytogenes in 8% of the commercial cantaloupe seeds analyzed; 2) L. monocytogenes is able to grow to high levels in cantaloupe and lettuce plants; 3) ~70% of the romaine lettuce analyzed was positive for Listeria based on conventional plating assays, but >95% of these were determined to be false positives using DNA sequencing methods; and 4) bacterial communities and pathogens have a non-uniform distribution between lettuce leaves and between different locations on the same leaf. Finally, a strip-based lateral flow immunochromatography assay was developed that could detect 100 L. monocytogenes cells per mL in 30 min. The ADODR monitored progress via e-mail, phone calls, and visits with CFSE faculty during the Annual Meetings of the MOST-USDA Joint Research Center for Food Safety (China) & the 15th Purdue CFSE/USDA-ARS Research Planning Workshop. Accomplishments 01 Nondestructive and high-throughput detection of Salmonella. Among foodborne illnesses, Salmonella is the topmost bacterial pathogen in the United States, causing 35% of domestically acquired bacterial foodborne illnesses and leading to numerous hospitalizations. There are thousands of types (serovars) of Salmonella with the �top 20� causing more than 70% of Salmonella-related outbreaks. It is important to distinguish between the different serovars to track foodborne outbreaks; however, the current USDA-Food Safety and Inspection Service (FSIS) Salmonella detection method can take 72 hours to yield results. To improve the detection of Salmonella in foods, ARS-funded researchers at the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana, applied their optical scatter technology (called BARDOT). BARDOT is a label-free nondestructive method in which a laser beam passes through the center of a bacterial colony on a Petri-plate and generates a unique scatter signature (fingerprint) that can be used for bacterial detection and identification. Fingerprint optical scatter patterns of 36 Salmonella serovars, including the �top 20�, could be differentiated from non-Salmonella bacteria within 24 hours with the same level of accuracy as the FSIS method (>80%). The technique is therefore suitable for the rapid on-plate detection of Salmonella, and the bacterial colonies are available for further testing. After an initial investment in the equipment, BARDOT can be operated with a minimum of consumables and supplies and could be an attractive tool for the food industry, regulatory agencies, and diagnostic laboratories. 02 Detection and identification of the �big six� pathogenic E. coli using a nanobiosensor. Six types of pathogenic Escherichia coli [non-O157 Shiga toxin-producing E. coli (STEC)], known as the �big six�, have been classified by the USDA Food Safety and Inspection Service (FSIS) as adulterants and require routine verification testing in raw beef trimmings. The current FSIS protocol for the detection of the �big six� STECs requires several days of microbiological growth and at least two steps requiring a relatively complex and expensive method for detection of specific genes (quantitative polymerase chain reaction or qPCR). To reduce the complexity and cost of the �big six� detection and identification process, micro-fabrication and DNA isothermal amplification techniques were combined into a nanobiosensor. Using an array of fabricated micro-wells, a primer dehydration technique, and a microinjection system, ARS-funded researchers at the Center for Food Safety Engineering, Indiana, demonstrated a method for parallel detection and identification of the �big six� STECs in extremely small volumes using a nanobiosensor. The use of simpler DNA amplification technique (loop-mediated isothermal amplification or LAMP) improved the process by allowing the reaction to be kept at a constant temperature. A microfabricated platform utilizing standard semiconductor processes enables the possibility even smaller and less expensive detection systems. The current nanobiosensor enables the simultaneous detection of multiple pathogens, including the �big six�, from a small number of template molecules in under an hour and represents a significant step towards affordable, fast, and automated on-site detection of foodborne pathogens. 03 Reducing the contamination of fresh produce using bacteriophage. Fresh produce has been linked to foodborne illness outbreaks. Produce is vulnerable to contamination from pathogens that may be present in their environment and may enter plants during germination. Once the plant is colonized with a pathogen, whether on the surface or internally, post- harvest disinfection may prove inadequate and thus the plant may be unsafe for consumption. Bacteriophages (viruses that infect bacteria) have a history of use as antibacterial agents. They function by binding to specific bacteria, and then killing the target bacteria. This creates the possibility of selecting bacteriophage that are specific to foodborne pathogens to develop treatments to reduce pathogen contamination in food. ARS-funded researchers at Purdue University's Center for Food Safety Engineering in Indiana, have developed effective and inexpensive bacteriophage based strategies for reducing pathogen contamination of seeds. Seeds are coated with bacteriophage encapsulated in a polymer. This approach is effective for reducing pre- harvest contamination of produce. Applications such as these further demonstrate the broad and powerful potential for adapting naturally- occurring bacteriophages for the purpose of making our food supply safer. 04 Detection of pathogenic E. coli using bacterial viruses. Rapid, inexpensive, and sensitive methods for detecting harmful bacteria, such as E. coli O157:H7, are needed to meet the demands for increased testing frequency. Bacteriophage (viruses that infect bacteria) are effective platforms for detection of viable foodborne pathogens because by nature they only bind to specific live bacteria. To use these bacteriophage for pathogen detection, they must elicit a easily detectable response once they have bound to the target bacteria. ARS- funded researchers at the Center for Food Safety Engineering at Purdue University in West Lafayette, Indiana, modified a bacteriophage specific for E. coli O157:H7 to transfer antibiotic resistance to the infected E. coli O157:H7 host and also elicit detectable light production. The antibiotic resistance facilitates the separation of the target bacterium during selective enrichment, facilitating detection of low numbers of pathogenic cells in the presence of complex food systems. When used during the selective enrichment of ground beef, as few as 10 cells could be detected in 12 hours. After 24 hours, positive samples containing the pathogen could be detected visually based on the amount of light produced. The technology platform built around this bacteriophage integrates into existing laboratory procedures, and therefore this could offer a useful and inexpensive rapid screening technique for the presence of E. coli O157:H7 with minimal modification of conventional sample preparation protocols. 05 Harmful bacteria found in retail delis'. Listeria monocytogenes is among the foodborne pathogens with the highest death toll in the United States, and an estimated 83% of listeriosis cases result from consumption of deli meats that are cross-contaminated at retail. In efforts to reduce the presence of harmful bacteria in retail delis, ARS- funded researchers at the Center for Food Safety Engineering at Purdue University in West Lafayette, Indiana, are working to better understand and develop control strategies for L. monocytogenes in retail food systems. L. monocytogenes can persist in retail delis for at least one year despite standard sanitation efforts. A novel deep-cleaning protocol for retail delis was implemented in an effort to more aggressively clean food- and non-food contact surfaces with the goal of eliminating or reducing niches that harbor the harmful organism; however, the aggressive cleaning did not permanently eliminate the persistent strains in many cases. The persistent strains were found to be more likely to form biofilms and exhibit sanitizer resistance than transient strains, which likely lead to the diminished effectiveness of cleaning protocols. Also of concern is the virulence potential of these strains: 442 L. monocytogenes isolates from retail deli surfaces were screened for the presence of 18 separate virulence-reducing mutations. Unfortunately, a vast majority of the isolates retained potential virulence: only <10% of isolates from food contact surfaces and <3% of all deli isolates contained the virulence-reducing mutations. Additionally, stores with high L. monocytogenes prevalence had fewer isolates with virulence-reducing mutations and these isolates were therefore more likely to cause illness if transferred into foods. Combined, these results underscore the importance of more aggressive and targeted deli cleaning procedures to reduce L. monocytogenes prevalence and prevent transfer of L. monocytogenes into ready-to-eat food products. 06 Listeria monocytogenes on cantaloupe and lettuce plants. The fresh produce industry is concerned about the contamination of plants with harmful bacteria. In 2011, cantaloupes contaminated with L. monocytogenes resulted in 146 cases of listeriosis and 3 deaths. In addition, numerous recalls of lettuce have cost the leafy greens industry millions of dollars. To better understand the association of harmful bacteria on plants, ARS-funded researchers at the Center for Food Safety Engineering at Purdue University in West Lafayette, Indiana: 1) determined the growth and persistence of L. monocytogenes on cantaloupe and romaine lettuce, 2) screened cantaloupe seeds for the presence of Listeria, and 3) tested four types of FDA-approved microbiological culture media for detection of Listeria from romaine lettuce. The results revealed that: 1) L. monocytogenes grew to very high levels over a minimum 30 days in all plants tested. Therefore, exposure of growing plants to harmful bacteria can lead to levels of bacteria that could cause health concerns. 2) Approximately 14% of cantaloupe seeds were positive for some kind of Listeria and 8% were contaminated with the human pathogen L. monocytogenes. It is therefore possible that contaminated seeds could contribute to foodborne outbreaks. 3) Four types microbiological growth media developed for the detection of Listeria from other foods were not effective in specifically detecting the presence of Listeria from lettuce. These results underscore the need to reduce the incidence of harmful bacteria in cantaloupe and lettuce by implementing methods to reduce the seed and plant exposure to harmful bacteria, effective post-harvest sanitization treatments, and improved microbiological methods detection for Listeria from produce. 07 Biosensors with enhanced resolution for electrical detection of nucleic acid amplification. Instrumentation for detecting and identifying harmful bacteria based on DNA profiles can be miniaturized, and made more portable and less expensive, by electrically detecting DNA amplification through changes in pH. Microsensors based on specialized transistors to measure pH offer advantages of low cost, label-free detection, and the potential to enable numerous simultaneous measurements; features that make them attractive option for rapid pathogen detection and point-of-care diagnostics. ARS-funded researchers at the Center for Food Safety Engineering, Indiana, in collaboration with Taiwan Semiconductor Manufacturing Company designed novel sensors that overcame several technical limitations associated with previous sensors, enabling interrogation of thousands of individual samples in very small volumes. The enhanced sensitivity and improved resolution reduced the detection time and increased the output signal for a loop-mediated isothermal DNA amplification reaction (LAMP) of a human pathogenic bacterial strain. With this micro-LAMP technology the time to result for identifying harmful bacteria is reduced by amplifying the signals coming from the amplification of the pathogenic bacterial DNA.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): 1: Develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self- validating system into an automated instrument. 1A: Integrate technology platforms that we have developed and individually tested into a usable technology for detecting L. monocytogenes in less than 8 h (time-to-result). 1B: Integrate technology platforms, currently being developed in our laboratory, into a usable technology for detecting Salmonella . 1C: Integrate technology platforms into a usable technology for detecting STEC. 2: Develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. 2A: Microfabricate and characterize microfluidic biochips that will direct, concentrate, and quantify living microorganisms using micro- and nano-scale electrical, mechanical, and optical methodologies. 2B: Develop light scattering technologies for rapid and high throughput detection and identification of pathogenic bacteria based on unique scattering signatures generated by concentrated colonies. 2C: Develop bacteriophages carrying reporter genes for the detection of E. coli O157:H7 and other foodborne pathogenic bacteria. 2D: Develop a highly sensitive enhanced Raman spectrosensor for field- deployable and routine benchtop in-lab identification of foodborne pathogens. Approach (from AD-416): Our approach will be to carry out our objectives using 2 important steps. The first step will be to develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self-validating system into an automated instrument. When carried out effectively, this step will enable different types of detection platforms to be more effective and accurate. The next step is to develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. An experienced multidisciplinary team of investigators from Purdue University, University of Illinois, and the USDA will engage manufacturers of commercial, off-the-shelf components to construct instruments, and the food processing industry or regulatory agencies to test them. This integrated effort will produce operational technologies that can be used to better detect and quantify microbial hazards in food. Progress was made on both objectives and several sub-objectives, all of which fall under National Program 108, Food Safety, contributing to Component 1: Food Contaminants and Problem Statement 1.C Technologies for the Detection and Characterization of Microbial Contaminants of the 2011- 2015 Strategic Action Plan. To prevent outbreaks of foodborne illness, food regulatory agencies and the food industry need rapid, sensitive, and specific methods to check for the presence of harmful bacteria (pathogens) in food. Our approach to develop such methods involves novel bioseparation technology to separate and concentrate pathogens from foods (a 1000-fold concentration of cells in less than 30 minute) and various methods to detect and quantify foodborne pathogens. Two prototype filtration instruments were built this year. The filtration device was used to separate and concentrate bacteria from water and foods and, when coupled to polymerase chain reaction (PCR) analysis, pathogen detection was completed within 6 hours. A variety of platforms for detection of the concentrated pathogens were studied. A novel method was developed to rapidly heat sub-nanoliter droplets on the biochips, to facilitate ultra-rapid (< 10 min) PCR assays. In addition, developments in loop mediated isothermal amplification enabled multiplexed screening of virulence genes of L. monocytogenes, E. coli, and Salmonella on the chip. A second system called BARDOT (Bacterial Rapid Detection using Optical Scattering Technology) uses light scattering techniques to differentiate and classify bacterial colonies grown on Petri-dishes. Major hardware and software updates supported the development of a portable instrument, and a prototype was delivered to USDA for further testing and application development. The BARDOT technology was augmented with different optical methods (Raman spectroscopy and a multispectral interrogation) and preliminary analyses of E.coli and Listeria are underway. Efforts are ongoing to validate pathogen identification by BARDOT in real world produce samples through DNA sequence verification. A third system employs an E. coli O157:H7- specific phage that was genetically engineered to cause the target bacteria to produce a bright yellow color if infected. This year methods were developed to encapsulate the phage into a pill format using commercially available polymers to enhance shelf-life and facilitate their addition to selective enrichments. Efforts are ongoing to expand the reporter phage specificity to other Shiga toxin-producing E. coli. Also, methods employing fluorescent immunoassays in combination with magnetic concentration enabled rapid (2 hours) and sensitive (5 CFU/mL) detection of E. coli O157:H7, Salmonella, and L. monocytogenes. Finally, as part of larger study, DNA fingerprinting of over 800 L. monocytogenes isolates from retail delis representing diverse geographic regions was completed. Accomplishments 01 Portable method for identifying harmful bacteria from food. Rapid detection of harmful bacteria in food is necessary to prevent foodborne illness and safeguard public health. The BARDOT sensor technology is easy to use and allows for rapid identification of bacteria. Developed by ARS-funded researchers at Purdue University�s Center for Food Safety Engineering (CFSE) in West Lafayette, Indiana, BARDOT involves shining a laser light through the bacterial colony on a plate and collecting images of the light that passes through. The resulting pictures of these colonies contain descriptive characteristics which can be used to identify bacteria by comparing the resulting image with a library of previously collected images. We report here the development of a portable BARDOT instrument by CFSE scientists and the delivery of an instrument to a USDA laboratory. The new instrument is able to identify known pathogenic bacteria, including pathogenic E.coli, Salmonella, and Listeria monocytogenes. The pathogen identification capabilities coupled with the portability of this new BARDOT instrument has tremendous potential for improving the response to foodborne illness outbreaks because the method can travel to the source thereby reducing the time to detection. 02 Detection of foodborne pathogens using nanobiosensors. Consumption of foods contaminated with pathogenic bacteria is a major public health concern. A key challenge in food safety is to rapidly screen foods to determine the presence of pathogens so that appropriate intervention protocols can be pursued as needed. Nanobiosensors have advantages over traditional microbiological and standard-scale biosensors for pathogen detection because of their low cost and potential for testing multiple samples simultaneously. ARS-funded researchers in collaboration with Purdue University�s Center for Food Safety Engineering at West Lafayette, Indiana, have developed a nanobiosensor for simultaneous detection of several pathogens using a method for specific detection of pathogen DNA. Unlike traditional DNA amplification which involves numerous temperature changes, the developed nanobiosensor is simplified because it uses a DNA amplification method that occurs at one temperature. Using these biochip sensors, scientists were able to rapidly and simultaneously detect L. monocytogenes, E. coli O157:H7, and Salmonella. Another nanobiosensor is based on a fluorescent nanoparticle strategy. In this sensor, nanoparticle magnetic beads are modified to bind specific pathogenic bacteria, and upon binding of the target pathogen to the beads, a fluorescent signal is generated that is easily detectable by common laboratory equipment. This nanoparticle magnetic bead method detected low numbers of E. coli O157:H7, Salmonlla Typhimurium, and L. monocytogenes in less than 2 hours. These nanobiosensors could be used for fast, portable, and inexpensive on- site testing of foodborne pathogen contamination and could therefore be used to reduce the public health impact of foodborne pathogens. 03 Identification of Listeria monocytogenes in retail delis. Listeria monocytogenes is one of the deadliest foodborne pathogens in the United States and an estimated 83% of listeriosis cases result from consumption of deli meats that are cross-contaminated at retail. Nevertheless, very few studies have investigated L. monocytogenes transmission, prevalence, and persistence in retail delis. To determine if the same L. monocytogenes strain was being recovered routinely from a deli (evidence of persistence) or if L. monocytogenes was transient in the deli environment, ARS-funded researchers at Purdue University�s Center for Food Safety Engineering at West Lafayette, Indiana, characterized over 800 strains of L. monocytogenes collected from 30 retail delis in three states. DNA fingerprinting of the revealed significant similarities between the strains and led to the conclusion that over 40% of the delis studied had evidence of persistent L. monocytogenes contamination. The substantial genetic similarity between the isolates suggests that a small number of highly related L. monocytogenes types are responsible for retail deli contamination. This study concludes that: (i) some retail delis have an increased likelihood of prevalent and persistent L. monocytogenes contamination, (ii) there is a need to develop feasible and practical approaches to control L. monocytogenes in these environments, and (iii) control of L. monocytogenes in retail delis will likely lead to a decrease in listeriosis cases in the United States. 04 Rapid concentration and detection of pathogens from vegetable wash water. Outbreaks of illness linked to consumption of leafy greens are an increasing concern, and from an industry perspective it would be ideal to avoid the high costs associated with issuing a recall. Sampling of large volumes of vegetable wash water to detect pathogens, that may be present in low numbers, is a major obstacle. New methods to recover and concentrate foodborne pathogens from large sample volumes would facilitate rapid detection leading to a reduction in the distribution of contaminated foods and the prevention of outbreaks of foodborne illness. ARS-funded researchers at Purdue University�s Center for Food Safety Engineering in West Lafayette, Indiana, identified ultra and micro-filtration methods for the rapid concentration and recovery of Salmonella and E.coli O157:H7 from commercial vegetable wash water. The developed system is a rapid (2 hours), automated operation from cell concentration and recovery to cleaning steps (ie, �hands off� operation), which minimizes the risk of cross-contamination, reduces labor intensity, and results in high recovery rates (75-80%) for the target pathogen. These concentrated samples can be analyzed by conventional or emerging pathogen detection techniques. For example, the concentration method used in conjunction with conventional microbiological plating techniques allowed detection of Salmonella and E.coli O157:H7 in about 24 hours or, in conjunction with DNA-based detection methods (real-time PCR), could be completed in about 7 hours. The combined approach of concentrating the microorganisms and then testing the concentrated samples for the presence of food pathogens will facilitate more sensitive and timely testing of large volumes of vegetable wash water and reduce the likelihood of distributing contaminated products. 05 Reduced time to detect Salmonella in peanut butter. Salmonella is an important cause of human illness in the United States. Following an outbreak of Salmonella infections linked to peanut butter in 20 states in 2012, in which over half of ill persons were children, ARS-funded researchers at Purdue University�s Center for Food Safety Engineering at West Lafayette, Indiana, improved the detection of Salmonella in peanut butter using the BARDOT bacterial identification system they developed. The method includes a brief culture enrichment prior to BARDOT-based detection of Salmonella. The BARDOT technique was able to detect Salmonella in peanut butter within 24 hours with an accuracy of 98%. This is comparable to the current USDA-FSIS method, which requires about 72 hours. This reduced time to detect Salmonella could improve the response time to foodborne illness outbreaks, as well as better facilitate analyses to prevent the distribution of contaminated foods. 06 Understanding microbial populations on fresh produce. Food safety outbreaks linked to consumption of leafy greens are an increasing concern. Produce is often consumed raw, and, while many sanitizers are highly effective on the majority of produce, they fail to effectively kill harmful and/or spoilage microorganisms on some leafy greens. To date, most studies of bacterial populations on vegetables report only population reductions following sanitation treatments but provide no information concerning which bacteria are present. ARS-funded researchers at Purdue University�s Center for Food Safety Engineering at West Lafayette, Indiana, established and optimized experimental protocols to reliably isolate and identify both total populations of bacteria as well as specific harmful bacteria from leafy green vegetables by DNA sequencing methods. Application of the developed methods revealed the bacterial communities associated with fresh produce are complex and variable. Although most of the communities examined are dominated by just two kinds of bacteria, several hundred types of bacteria are present in smaller numbers. The bacterial communities observed on spinach and lettuce are markedly different from one another, and they change in different ways following sanitization and/or cold storage. The results of these studies can be used to develop more targeted approaches for the sanitation of leafy green vegetables.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): 1: Develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self- validating system into an automated instrument. 1A: Integrate technology platforms that we have developed and individually tested into a usable technology for detecting L. monocytogenes in less than 8 h (time-to-result). 1B: Integrate technology platforms, currently being developed in our laboratory, into a usable technology for detecting Salmonella . 1C: Integrate technology platforms into a usable technology for detecting STEC. 2: Develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. 2A: Microfabricate and characterize microfluidic biochips that will direct, concentrate, and quantify living microorganisms using micro- and nano-scale electrical, mechanical, and optical methodologies. 2B: Develop light scattering technologies for rapid and high throughput detection and identification of pathogenic bacteria based on unique scattering signatures generated by concentrated colonies. 2C: Develop bacteriophages carrying reporter genes for the detection of E. coli O157:H7 and other foodborne pathogenic bacteria. 2D: Develop a highly sensitive enhanced Raman spectrosensor for field- deployable and routine benchtop in-lab identification of foodborne pathogens. Approach (from AD-416): Our approach will be to carry out our objectives using 2 important steps. The first step will be to develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self-validating system into an automated instrument. When carried out effectively, this step will enable different types of detection platforms to be more effective and accurate. The next step is to develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. An experienced multidisciplinary team of investigators from Purdue University, University of Illinois, and the USDA will engage manufacturers of commercial, off-the-shelf components to construct instruments, and the food processing industry or regulatory agencies to test them. This integrated effort will produce operational technologies that can be used to better detect and quantify microbial hazards in food. Our approach involves development of a bioseparation technology to separate and concentrate pathogenic microorganisms from food matrixes, as well as effective methods for the detection and quantification of foodborne pathogens. The bioseparation technology concentrates cells directly via a sequential filtration process, achieving a 1000-fold concentration of cells in less than 30 minutes. The filtration system was improved this year by optimizing sanitation procedures and software, such that a �hands-off� operation cycle from cell concentration and recovery to cleaning steps can be completed within 2 hours. The filtration device was used successfully to separate and concentrate pathogens from complex foods and coupled to quantitative polymerase chain reaction (qPCR) analysis for detection of very low levels of Salmonella in chicken rinse water within 7 hours. A wide variety of platforms appropriate for detection of the concentrated pathogens were also studied. One detection method uses a microfluidic biochip to further concentrate bacteria, grow and break-open the bacteria, and detect specific pathogens using PCR. This year developments in field effect transistors (FETs) for single cell lysis and localized heating on the biochip were important steps to release and denature DNA from the bacteria cells as a precursor to PCR. Electrical detection of PCR products as an essential part of monitoring the reaction�s progress was also accomplished. A second system called �BARDOT� (Bacterial Rapid Detection using Optical Scattering Technology) uses light scattering techniques to differentiate and classify bacterial colonies grown on Petri-dishes. The database of scattering images from different pathogens continued to be expanded to include a wider variety of microorganisms, including molds, yeasts, and spoilage bacteria in addition to pathogenic bacteria, as well as more types of growth media. BARDOT was able to differentiate and classify 32 different types of Salmonella and seven common types of pathogenic Shiga-toxin producing E. coli. To reduce total detection time from food samples, detection of micro-colonies was optimized. BARDOT could differentiate micro-colonies of Salmonella, E. coli, and Listeria after only 8-12 h of growth on agar plates. Furthermore, BARDOT was equipped with multiple wavelength lasers to improve discriminatory power of scatter signatures and to detect microorganisms that are difficult to detect. A third system is a phage- based detection of E. coli O157:H7. Phage are bacterial viruses that infect specific bacteria. Genetic engineering tools were developed to manipulate the bacterial E. coli O157:H7 phage to produce a phage which causes the target bacteria to glow (luminesce) if infected. Initial experiments detected 1-5 cells in less than 12 hours using observable luminescence. Finally, methods employing Raman (infrared light) spectroscopy were improved this year by developing Raman probes and magnetic nanoparticles that are able to target pathogens and enhance Raman signals such that key pathogens (E. coli O157:H7, Salmonella and L. monocytogenes) could be detected at very low concentration. Accomplishments 01 Rapid recovery, concentration, and detection of Salmonella from chicken extracts. Despite a recent decline in some bacterial foodborne illnesses foodborne infections caused by the most common strains of Salmonella hav not declined in 15 years. New methods to recover and concentrate foodbor pathogens from complex food matrices would greatly facilitate the rapid detection of Salmonella and other foodbonre pathogens leading to a reduction in the distribution of contaminated foods and the prevention o outbreaks of foodborne illness. ARS-funded researchers at Purdue University�s Center for Food Safety Engineering in West Lafayette, India identified ultra and micro-filtration methods for the rapid concentratio and recovery of Salmonella from chicken. The concentration method has been used in conjunction with microbiological plating technique for detection of Salmonella in about 24 hours or with DNA-based detection methods (real-time PCR) that can be completed in about 7 hours. The combined approach of concentrating the microorganisms and then testing t concentrated samples for the presence of food pathogens using PCR based techniques will contribute to food safety by increasing identification specificity of the target organism. 02 Nanotechnology for bacterial detection. Nanobiosensors offer advantages over traditional microbiological and standard-scale biosensors for pathogen detection because of their low cost, label-free detection, and potential for massive parallelization. ARS-funded researchers in collaboration with Purdue University�s Center for Food Safety Engineerin in West Lafayette, Indiana have made significant advances toward the development of nanobiosensors for nucleic acid-based detection of pathogens. The scientists developed a method to position tiny droplets an array of individual silicon microwave heaters, allowing precise contr the temperature of droplets-in-air and subsequently perform biochemical reactions like DNA melting. 03 Identification of unknown foodborne pathogens. Rapid detection of bacterial foodborne pathogens is necessary to prevent foodborne illness and safeguard public health. The optical light scattering sensor, BARDO is a noninvasive label-free detection system which allows identification of bacterial colonies in real-time. Developed by ARS-funded researchers Purdue University�s Center for Food Safety Engineering in West Lafayette Indiana, BARDOT involves shining a laser light through the bacterial colony and collecting images of the light that passes through. The imag collected contain descriptive characteristics of bacterial colonies, whi can be used to identify bacteria by comparing the resulting light scattering image with a library of previously collected images. This method has tremendous potential for properly classifying foodborne pathogens; even emergent pathogens such as the previously unknown type o E. coli that recently caused a serious foodborne outbreak in Europe.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) 1: Develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self- validating system into an automated instrument. 1A: Integrate technology platforms that we have developed and individually tested into a usable technology for detecting L. monocytogenes in less than 8 h (time-to-result). 1B: Integrate technology platforms, currently being developed in our laboratory, into a usable technology for detecting Salmonella . 1C: Integrate technology platforms into a usable technology for detecting STEC. 2: Develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. 2A: Microfabricate and characterize microfluidic biochips that will direct, concentrate, and quantify living microorganisms using micro- and nano-scale electrical, mechanical, and optical methodologies. 2B: Develop light scattering technologies for rapid and high throughput detection and identification of pathogenic bacteria based on unique scattering signatures generated by concentrated colonies. 2C: Develop bacteriophages carrying reporter genes for the detection of E. coli O157:H7 and other foodborne pathogenic bacteria. 2D: Develop a highly sensitive enhanced Raman spectrosensor for field- deployable and routine benchtop in-lab identification of foodborne pathogens. Approach (from AD-416) Our approach will be to carry out our objectives using 2 important steps. The first step will be to develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self-validating system into an automated instrument. When carried out effectively, this step will enable different types of detection platforms to be more effective and accurate. The next step is to develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. An experienced multidisciplinary team of investigators from Purdue University, University of Illinois, and the USDA will engage manufacturers of commercial, off-the-shelf components to construct instruments, and the food processing industry or regulatory agencies to test them. This integrated effort will produce operational technologies that can be used to better detect and quantify microbial hazards in food. This project was going through the OSQR review process and was only recently certified. This project is a continuation of previous work and related progress is included in the report for 1935-42000-061-00D.
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