Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): To improve analytical methods to enhance and validate detection of a wide spectrum of biological and chemical threats against food supply systems. Approach (from AD-416): A collection of category B bacterial pathogens are maintained at the USDA- ARS, Richard B. Russell Research Center, Poultry Microbiological Research Unit. Bacterial cultures were isolated from poultry samples consisting of either whole carcass rinses or fecal/cecal specimens from conventionally- reared broiler chickens or processing plants. The collection of category A bacterial pathogens are maintained at the USDA-ARS, Richard B. Russell Research Center, FSIS, Federal Response Emergency Network (FERN) Laboratory. We are collaborating with FERN and they maintain ownership of the pathogens. All work will be conducted at the Bio-Safety Level 2 and a Standard Operation Procedure (SOP) has been written and reviewed by Ms. Frankie J. Beacorn, Biosecurity Officer, USDA, FSIS, OPHS, FERN Division, Athens, GA and Joseph P. Kozlovac, ARS Biosafety Officer, Beltsville , MD. Stock cultures of non-virulent strains of four pathogen (two category B) and bio-threat agents (two category A) will be grown in solution and enriched to at least three concentrations. Additionally, common background microflora will also be grown and enriched. The pathogens, bio-threat agents, and background microflora will all be spot plated on appropriate growth media. Hyperspectral imaging systems (400-900 nm, 1000-2500 nm, and hyperspectral microscope) will be used to collect spectral libraries of the organisms and growth media. Multivariate models will then be developed to classify the various organisms. Additionally, the enriched solutions of the organisms will also be measured with a Fourier-transform Infrared (FTIR) spectrometer and a Raman spectrometer and multivariate models once again developed. Further modeling will then be used to determine the minimum detection level for all pathogens and bio-threat agents. Once reasonable results are obtained for pure cultures, important food matrices will be spiked with pathogens and bio-threat agents at varying concentration levels and hyperspectral images will be collected on both the total contaminated food matrices and on a rinsate from the infected food matrices. Multivariate calibration models, based on the spectral libraries of the bio-threat agents and food matrices, will then be developed and the lower limit of detection for each pathogen and bio- threat agent will be determined. This research is related to inhouse objective 2. Detect and characterize foodborne pathogens, toxins, and bacterial threat agents with rapid optical methods. In this project, five optical detection systems were evaluated for detection of foodborne pathogens including E. coli in ground beef, Salmonella in poultry carcass rinse. They are: Visible Near Infrared (VNIR) Hyperspectral Imaging System � 400 to 1000 nm pushbroom hyperspectral imager Visible Near Infrared (VNIR) Hyperspectral Microscope Imaging System (HMI) � 400 to 850 nm acousto-optic tunable filter (AOTF) hyperspectral imager with Nixon light microscope Short-Wave Near Infrared (SWIR) Hyperspectral Imaging System � 900 to 1700 nm pushbroom hyperspectral imager Fourier-transform infrared (FTIR) micro-spectrometer � 4000 to 6000 reciprocal cm Surface Enhance Raman Spectroscopy (SERS) � 785 nm excitation confocal Raman Spectroscopy Each system had both strengths and weaknesses when compared to the other systems. In the final analysis, no attempt was made to compare these systems to other non-optical detection methods. Therefore, all comparisons were described relative to these five systems, and their corresponding detection methods were presented with their relative strengths and weaknesses. Early in the assessment of the systems, the Short-Wave Near Infrared (SWIR) hyperspectral imaging system was determined to be unsuitable for further evaluation as the high moisture content of the agar media produced a broad water absorption peak which effectively masked any pathogen related response. The VNIR and SWIR systems were easier to use (relatively) than the HMI, FTIR, and SERS systems. Likewise, sample preparation was easier and required no sample contact for the VNIR and SWIR systems while additional sample preparation and sample removal was required for the HMI, FTIR, and SERS systems. Thus, the VNIR and SWIR systems were also nondestructive. Although samples had to be removed for the HMI and the FTIR systems, the samples were still alive on the slide (HMI) and in the cell (FTIR). The cells could later be immersed in a broth and would grow. Even though the SERS method did not kill the bacteria, they were bound by the aptamer and thus, the SERS method was considered destructive. All systems required a significant amount of training and intimate knowledge to operate with the SWIR, FTIR, and SERS systems requiring more knowledge and training to use. In our studies for specificity, the VNIR system was able to provide the highest classifications of bacteria, while the HMI, FTIR, and SERS systems provided good classification rates. As mentioned, the SWIR system was not suitable and had a very poor specificity. For sensitivity, both the FTIR and SERS systems had lower detection limits while the VNIR and HMI systems were slightly higher. Although capable of high signal response, the FTIR suffered from inconsistencies in sample preparation that could not be overcome and reduced the repeatability of its measurements. For imaging foodborne pathogens of moderate sample size, the VNIR and SWIR systems were faster in data acquisition than the others yet none of them were considered very fast. In summary, comparing all the characteristics of the five systems described above, and from the data collected on the various systems within this study with the described methodologies, the VNIR system had the best feasibility for implementation and had the best commercial potential of the five systems. The VNIR system is rather expensive and requires significant knowledge to operate and process the data. A key element in using such a system is automation of all preprocessing steps. It can take a substantial amount of time to manually perform all the steps need for analysis. Therefore programs to automate the data analysis are critical. Furthermore, it would be very advantageous if the method could be reduced to a simpler format. It is hypothesized that a color-balanced digital SLR camera might have potential for identifying the pathogenic bacteria from the non-pathogenic background microflora found in ground beef or poultry carcass rinse. The results for these preliminary tests were encouraging but would require improvement before a technology could be developed. Thus, significant additional research will be required to confirm or reject this hypothesis.
Impacts
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): To improve analytical methods to enhance and validate detection of a wide spectrum of biological and chemical threats against food supply systems. Approach (from AD-416): A collection of category B bacterial pathogens are maintained at the USDA- ARS, Richard B. Russell Research Center, Poultry Microbiological Research Unit. Bacterial cultures were isolated from poultry samples consisting of either whole carcass rinses or fecal/cecal specimens from conventionally- reared broiler chickens or processing plants. The collection of category A bacterial pathogens are maintained at the USDA-ARS, Richard B. Russell Research Center, FSIS, Federal Response Emergency Network (FERN) Laboratory. We are collaborating with FERN and they maintain ownership of the pathogens. All work will be conducted at the Bio-Safety Level 2 and a Standard Operation Procedure (SOP) has been written and reviewed by Ms. Frankie J. Beacorn, Biosecurity Officer, USDA, FSIS, OPHS, FERN Division, Athens, GA and Joseph P. Kozlovac, ARS Biosafety Officer, Beltsville , MD. Stock cultures of non-virulent strains of four pathogen (two category B) and bio-threat agents (two category A) will be grown in solution and enriched to at least three concentrations. Additionally, common background microflora will also be grown and enriched. The pathogens, bio-threat agents, and background microflora will all be spot plated on appropriate growth media. Hyperspectral imaging systems (400-900 nm, 1000-2500 nm, and hyperspectral microscope) will be used to collect spectral libraries of the organisms and growth media. Multivariate models will then be developed to classify the various organisms. Additionally, the enriched solutions of the organisms will also be measured with a Fourier-transform Infrared (FTIR) spectrometer and a Raman spectrometer and multivariate models once again developed. Further modeling will then be used to determine the minimum detection level for all pathogens and bio-threat agents. Once reasonable results are obtained for pure cultures, important food matrices will be spiked with pathogens and bio-threat agents at varying concentration levels and hyperspectral images will be collected on both the total contaminated food matrices and on a rinsate from the infected food matrices. Multivariate calibration models, based on the spectral libraries of the bio-threat agents and food matrices, will then be developed and the lower limit of detection for each pathogen and bio- threat agent will be determined. This research is related to inhouse project objective Objective 2: Detect and characterize foodborne pathogens, toxins, and bacterial threat agents with rapid optical methods. Specifically: Sub-objective 2A. Develop methods and techniques for detection of bacterial pathogens and bacterial threat agents on agar plates by hyperspectral imaging; and Sub-objective 2B. Develop hyperspectral microscope imaging techniques for enhancing pathogen detection limits. Previous work described the creation of spectral libraries of pure Shiga- Toxin producing Escherichia Coli (STEC) serogroups other than E. coli O157:H7 (non-O157) which are better known as non-O157 STEC. These spectral libraries were then used for classification model development with hyperspectral imaging. In this study, we compared the spectra of pure non-O157:H7 STEC serogroups to the spectra of non-O157:H7 STEC serogroups contaminated (i.e. spiked) in ground beef. The mean spectra of pure non-O157:H7 STEC serogroups presented earlier and the same serogroups spiked into the broth of ground beef had similar spectral responses. Serogroup O111 also showed a distinctive spectral signature linearly increasing from 500 to 650 nm and was less absorptive in the blue and green portions of the spectrum (esp. 475-575 nm) than all the other serogroups. Serogroups O26, O103, and O145 showed similar spectral responses with purple and/or gray-blue centers surrounded by more reddish perimeters. Serogroup O45 was highly absorptive across the visible spectrum, with the 500 to 700 nm regions demonstrating the greatest absorption, while the 400 to 500 nm region (blue) was somewhat less absorptive. This resulted in a dark blue appearance to the human eye and rendered it spectrally unique relative to the other serogroups. The pair-wise comparisons of each pure serogroup versus the spiked serogroups showed difference in the spectra of pure O26 versus spiked O26. The average pure spectra spiked O103 and O111 were similar but the pure spectra were slightly more absorptive (less reflectivity) than the spiked spectra. However, the greatest difference between pure and spiked spectra was for serogroup O45. In general, the pure O45 had a higher absorption than the spiked O45 across the entire spectral range, with 500 to 700 nm region demonstrating the greatest difference. This results in the pure O45 having a darker blue appearance to the human eye than the spiked O45 colonies. Nevertheless, it is still spectrally unique relative to the other serogroups. Futhermore, spectral libraries and classification models developed from the pure serogroups are applicable to detect and classify the serogroups in ground beef and also in the presence of background flora. Earlier research reported the development of classification models for pure Bacillus anthracis Stern from spectral libraries of hyperspectral imaging data. In this research, the spectra of pure B. anthracis Stern was compared to the spectra of B. anthracis Stern contaminated (i.e. spiked) in hot dog. The hotdog samples spiked with B. anthracis Stern were plated on sheeps blood agar (SBA) and incubated for 15 to 24 hours without any additional background microflora. There was a replicate affect do to different types of hotdogs tested but no difference in the B. anthracis Stern spectrum. Salmonella Typhimurium (ST) was spiked in poultry carcass rinses and plated on brilliant green sulfa (BGS) agar with and without Nalidixic acid. In general, ST colonies grew with reddish color and circular shape in BGS agar. The spectral signatures of the hyperspectral data from pure ST were similar to each other between samples from the plates with and without Nalidixic acid. However, some other non-ST colonies had similar color and shape to ST. These colonies had spectral signatures that increased at lower wavelengths but then decrease between 400 nm and 560 nm (blue and green color bands). Confirmation of each colony type is currently being conducted with a real-time polymerase chain reaction (PCR) . Similar studies were conducted with ST growing on Xylose Lysine Terigtol 4 (XLT4) agar. In this research, it was difficult to discriminate the ST from the background microflora. In XLT4, colonies that changed to a black color are supposed to be ST. However, some black- colored colonies were identified as different pathogens than ST. Further experiments are being conducted to validate the results. In general, the green and red color bands from the colonies growing in XLT4 agar plates were more important in identifying Salmonella in the spectral domain. In addition to spectral information, different morphological characteristics (between circular and irregular shape) of the colonies growing on the agar plate need to be incorporated into future classification models.
Impacts
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) To improve analytical methods to enhance and validate detection of a wide spectrum of biological and chemical threats against food supply systems. Approach (from AD-416) A collection of category B bacterial pathogens are maintained at the USDA- ARS, Richard B. Russell Research Center, Poultry Microbiological Research Unit. Bacterial cultures were isolated from poultry samples consisting of either whole carcass rinses or fecal/cecal specimens from conventionally- reared broiler chickens or processing plants. The collection of category A bacterial pathogens are maintained at the USDA-ARS, Richard B. Russell Research Center, FSIS, Federal Response Emergency Network (FERN) Laboratory. We are collaborating with FERN and they maintain ownership of the pathogens. All work will be conducted at the Bio-Safety Level 2 and a Standard Operation Procedure (SOP) has been written and reviewed by Ms. Frankie J. Beacorn, Biosecurity Officer, USDA, FSIS, OPHS, FERN Division, Athens, GA and Joseph P. Kozlovac, ARS Biosafety Officer, Beltsville , MD. Stock cultures of non-virulent strains of four pathogen (two category B) and bio-threat agents (two category A) will be grown in solution and enriched to at least three concentrations. Additionally, common background microflora will also be grown and enriched. The pathogens, bio-threat agents, and background microflora will all be spot plated on appropriate growth media. Hyperspectral imaging systems (400-900 nm, 1000-2500 nm, and hyperspectral microscope) will be used to collect spectral libraries of the organisms and growth media. Multivariate models will then be developed to classify the various organisms. Additionally, the enriched solutions of the organisms will also be measured with a Fourier-transform Infrared (FTIR) spectrometer and a Raman spectrometer and multivariate models once again developed. Further modeling will then be used to determine the minimum detection level for all pathogens and bio-threat agents. Once reasonable results are obtained for pure cultures, important food matrices will be spiked with pathogens and bio-threat agents at varying concentration levels and hyperspectral images will be collected on both the total contaminated food matrices and on a rinsate from the infected food matrices. Multivariate calibration models, based on the spectral libraries of the bio-threat agents and food matrices, will then be developed and the lower limit of detection for each pathogen and bio- threat agent will be determined. This project is related to in-house Objective 1A: To improve analytical methods to enhance and validate detection of a wide spectrum of biological and chemical threats against food supply systems. A visible and near-infrared (VNIR) hyperspectral imaging technique was developed to detect and differentiate Campylobacter in Petri dishes from five primary contaminants commonly found in commercial poultry carcass rinses. A protocol for imaging the spots of bacteria growing in agar media and processing their hyperspectral images was developed such that spectral libraries of the pure bacteria were collected for development of classification and validation models. From the research based on spot- plate imaging, the research was extended to image spread plates using the known mixture formula of pure bacteria. Currently, the protocol for imaging spread plates of pure pathogenic bacteria has been established and the performance of detection is being validated with results obtained by phase contrast microscopic tests. A short-wavelength infrared (SWIR) hyperspectral imaging system was developed to image Petri dishes in the wavelength range from 1,000 to 2, 500 nanometers (nm). The developed SWIR hyperspectral imaging system was designed to extract spectral fingerprints beyond the wavelength range most commonly used for hyperspectral imaging (400 to 1000 nm). The SWIR imaging system contains a Mercury Cadmium Telluride (MCT) camera with a spectrograph, a moving stage with precision motion and location control, a sample holder, and custom application software for image acquisition via motion and camera controls. The system is also capable of imaging a Petri dish either in the reflectance or transmittance mode. Next, the potential of the system for detection of pathogens in agar media was investigated by conducting a study with a spectrometer and the SWIR imaging system. The study found the spectral region from 1,000 to 2,500 nm was not viable for detecting pathogen colonies on agar plates because the water in the agar media dominated the spectral responses.
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