Source: PURDUE UNIVERSITY submitted to
IDENTIFICATION AND CLASSIFICATION OF FOODBORNE PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1008516
Grant No.
(N/A)
Project No.
IND020627
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Dec 1, 2015
Project End Date
Sep 30, 2020
Grant Year
(N/A)
Project Director
Robinson, J..
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Veterinary Basic Medical Sciences
Non Technical Summary
Foodborne pathogens cause significant economic losses across all levels of industry and society, and a number of very dangerous pathogens are deadly. Even pathogens that cause no significant stress to normal healthy individuals can result in death for individuals who are immunocompromised, very young, or elderly. Thus, maintaining the integrity of the food chain is a critical part of our well-developed agricultural system. While there are many tools and technologies available for testing foods, reagents, liquids, etc. for pathogens, many tests are time consuming and/or expensive. Some can only be performed within high-technology environments, and some cannot be adapted to all types of samples.One of the important goals of this application is to design and implement test systems that are low cost and easy to perform, and therefore provide a huge return on the investment to society at large. While the USA is a highly technological society with tremendous infrastructure, many countries cannot afford a lot of current technology. Our proposed technology will not be expensive to manufacture, and because it is reagent-free, the cost of pathogen detection is significantly reduced. Therefore we hope that in future as the technology matures and becomes available, low-cost versions will be available in resource-poor environments.
Animal Health Component
50%
Research Effort Categories
Basic
40%
Applied
30%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3060210110010%
5030210110010%
7120199110030%
7233320202010%
5035010208010%
7237299110010%
7230210110020%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 306 - Environmental Stress in Animals; 503 - Quality Maintenance in Storing and Marketing Food Products; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
3320 - Meat, beef cattle; 0210 - Water resources; 0199 - Soil and land, general; 7299 - Research equipment and methods, general/other; 5010 - Food;

Field Of Science
2020 - Engineering; 1100 - Bacteriology; 2080 - Mathematics and computer sciences;
Goals / Objectives
Microbial identification is essential in biosecurity, food safety, and in the clinical environment that would be relevant to this technology might be the monitoring and preventing nosocomial infections. In general, three steps are needed to deliver correct identification of a species: sample acquisition/preparation, microbe detection, and microbe identification. Throughout the years, most effort in instrument development using optical technology has been focused on the development of a colony counter, which is essentially a simple detection device; further testing is required for identification of organisms however. To identify and classify bacterial colonies, morphological methods, which observe the morphological characteristics of the bacterial colony via visual inspection, are widely studied.The objectives of this project are:Provide low-cost, reagent-free pathogen-identification technologies that enhance all aspects of food safetyProvide a low-cost method for monitoring microbial species in a variety of situations, and 3Develop a series of software suites that allow direct and rapid analysis of the results. In this project we will develop the technology, expand the software for advanced classifications, and build a database of know organisms that can be used to track and identify species from different locations
Project Methods
The incoming wavefront from the light source interacts with the microscopic features of the bacterial colony and propagates to the imaging point to provide a unique scatter pattern that does not require a labeling agent to discriminate among different types of bacteria. The first reported forward-scatterometer system required manual positioning of the incident beam and motion control to record the forward-scatter signatures, which is tedious, laborious, and inefficient. To increase the efficiency of the data-acquisition process, Bae et al. (Bae, Aroonnual, Bhunia, Robinson, & Hirleman, 2009) further developed a new version of the instrument that was fully automated. We have done preliminary work on a portable forward scatterometer that is lighter in weight, smaller in footprint, and provides wireless data-transmission capability. Our key objectives include trying to reduce the size and weight as well as the power requirement for such a portable system. Implementation of the above characteristics make the portable forward scatterometer a perfect instrument for field-deployable microbial identification as suggested by Robinson et al. (Robinson et al., 2011). Several designs are possible, but we propose an instrument with rotation-linear movements rather than two linear movements, resulting in lighter weight and a smaller footprint for the instrument. To achieve these goals several image-processing schemes must also be improved. Third, wireless communication capability enabling access to the scatter-pattern database will facilitate detection and identification from remote locations. We hope to implement a GPS chip into the instrument to allow determination of its exact location, something of particular value in doing environmental studies. A considerable amount of engineering and design is required to implement this first specific objective; this will be achieved in collaboration with Professor Euiwon Bae and his graduate students from Mechanical Engineering.The second objective is to expand and develop new analytical approaches to analysis of the scatter patterns produced when the laser strikes the bacterial colony growing on the agar. The laser beam creates an interference pattern (the fingerprint) on the CCD array immediately below the colony. The analysis of this fingerprint requires a considerable amount of advanced math integrated within an easy-to-use operating system. Thus the software development requires a significant attention to make these technologies user-friendly. Clearly not every user will be familiar or comfortable with advanced math to a user, so this aspect of the technology has to be "under the hood" so to speak. To achieve this, we must ensure that the user is able to intelligently and accurately apply the right approaches to the particular organisms being evaluated. It is easy to make these sorts of toolsets overly complex; it is very difficult to reduce very complex tools to simple navigation tasks. This, however, is a critical aspect of our technology development and must be accomplished properly to make this a true "user-friendly" system. We will engage Prof. Bartek Rajwa in the area of algorithm development. He has a long track record of excellence in this area and we will utilize many of the tools he has already developed (Bayraktar, Bernas, Robinson, & Rajwa, 2007; Grégori et al., 2012; Rajwa et al., 2010; Rajwa et al., 2007, 2008; Robinson et al., 2011).Another major objective of this program will be the design and application of a managed database that can contain all of the classifications necessary for efficient operation of this technology. This requires an advanced engine to operate the database as well as the ability to access the database from almost anywhere. This is critical if the database is to contain the subset of trained classifications that are used to determine the presence or absence of a pathogen. We propose to expand our currently developed database so that it can become a nationally accessible resource. In order to achieve this we will attempt to create as many classifiers as possible for common organisms, particularly pathogens. The classifiers must be tested for accuracy; this process will be a long-term component of this program. We will also engage a variety of colleagues in academia and industry to participate in testing the efficacy of this database and the classifiers contained within it.

Progress 12/01/15 to 09/30/20

Outputs
Target Audience:Food Inspectors, USDA employees, food processing companies, feed lot companies, government agencies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Pubiications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Microbial identification is essential in biosecurity, food safety, and in the clinical environment that would be relevant to thistechnology might be the monitoring and preventing nosocomial infections. In general, three steps are needed to delivercorrect identification of a species: sample acquisition/preparation, microbe detection, and microbe identification. Throughoutthe years, most effort in instrument development using optical technology has been focused on the development of a colonycounter, which is essentially a simple detection device; further testing is required for identification of organisms however. Toidentify and classify bacterial colonies, morphological methods, which observe the morphological characteristics of thebacterial colony via visual inspection, are widely studied.The objectives of this project are: 1. Provide low-cost, reagent-free pathogen-identification technologies that enhance all aspects of food safety 2. Provide a low-cost method for monitoring microbial species in a variety of situations, and 3Develop a series of software suites that allow direct and rapid analysis of the results. In this project we will develop thetechnology, expand the software for advanced classifications, and build a database of know organisms that can be used totrack and identify species from different locations

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: . Iyll-Joon Doh, Jennifer Sturgis, Diana Vanessa Sarria Zuniga, Robert E. Pruitt, J. Paul Robinson, and Euiwon Bae, Generalized spectral light scatter models of diverse bacterial colony morphologies, Journal of Biophotonics, Published on line August 22, 2019 https://doi.org/10.1002/jbio.201900149
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: . Gondhalekar, C., Rajwa, B., Bae, E., Patsekin, V., Sturgis, J., Kim, H., Doh, I-Y., Diwikar, P., Robinson, J. Paul; Multiplexed detection of lanthanides using laser-induced breakdown spectroscopy: a survey of data analysis techniques; SPIE Paper No. 11016-8 Tracking No.SI19-SI217-20 https://Doi:10.1117/12.2521453
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Valery Patsekin, Stephen On, Jennifer Sturgis, Euiwon Bae, Bartek Rajwa, Aleksandr Patsekin, and J. Paul Robinson, Classification of Arcobacter species using variational autoencoders, Published April 11, 2019 online, SPIE Paper No.11016-7, Tracking No.SI19-SI217-24 https://doi.org/10.1117/12.2521722, CID:1101608 .


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Food Inspectors, USDA employees, food processing companies, feed lot companies, government agencies Changes/Problems:The key development for us was a major infux of new instrumentation which in the first instance slowed our progress for several months, but is now a fully operational system and we have already seen a significant increase in research output, and submissions to journals. What opportunities for training and professional development has the project provided?We have several undegrad students working on this project which gives them knowledge and skills in research. In addition, one student was an co-author on a publication. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A key acomplisment in the present cycle was the submission of a new patent based on developing an alternative approach to lateral flow assay development. We believe that this new technology will be highly advantageous in the future for multiplexed assays, something that is a major goal of our technology development.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Iyll-Joon Doh, Jennifer Sturgis, Diana Vanessa Sarria Zuniga, Robert E. Pruitt, J. Paul Robinson, and Euiwon Bae, Generalized spectral light scatter models of diverse bacterial colony morphologies, Journal of Biophotonics, Published on line August 22, 2019 DOI: 10.1002/jbio.201900149
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Gondhalekar, C., Rajwa, B., Bae, E., Patsekin, V., Sturgis, J., Kim, H., Doh, I-Y., Diwikar, P., Robinson, J. Paul; Multiplexed detection of lanthanides using laser-induced breakdown spectroscopy: a survey of data analysis techniques; SPIE Paper No. 11016-8 Tracking No.SI19-SI217-20 https://Doi:10.1117/12.2521453 181. J. Paul Robinson, Spectral Flow cytometry: Quo Vadimus?, Cytometry A, published online April 30, 2019, https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.23779
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Valery Patsekin, Stephen On, Jennifer Sturgis, Euiwon Bae, Bartek Rajwa, Aleksandr Patsekin, and J. Paul Robinson, Classification of Arcobacter species using variational autoencoders, Published April 11, 2019 online, SPIE Paper No.11016-7, Tracking No.SI19-SI217-24 https://doi.org/10.1117/12.2521722, CID:1101608
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: . Iyll-Joon Doh, Carmen Gondhalekar, Valery Patsekin, Bartek Rajwa, Keegan Hernandez, Euiwon Bae, and J. Paul Robinson; A Portable Spark-Induced Breakdown Spectroscopic Instrument and its Analytical Performance. Applied Spectroscopy, Vol. 73, Issue 6, pp. 698-708 https://www.osapublishing.org/as/abstract.cfm?uri=as-73-6-698
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 397. Carmen Gondhalekar, Bartek Rajwa, J. Paul Robinson; Modeling multiplexed detection of lanthanides using laser-induced breakdown spectroscopy  techniques in data analysis., SPIE Meeting Baltimore, Sensing for Agriculture and Food Quality and Safety XI, April 12-18, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 396. Carmen Gondhalekar, Ewa Biela, Pablo Martinez Ulloa, Bartek Rajwa, Euiwon Bae, Valery Patsekin, Jennifer Sturgis, Huisung Kim, Iyll-Joon Doh, Prasoon Diwakar, Larry Stanker, Vassilia Zorba, Richard Russo Xianglei Mao, J. Paul Robinson Detection of heavy-metal conjugated antibodies on paper bioassay material using laser-induced breakdown spectroscopy and laser ablation inductively coupled optical emission spectroscopy, SCIX Annual Conference, 21-25 October, 2018 Atlanta GA, USA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 395. D.V. Sarria-Zuniga, E. Bae, A. Deering, M. Aime, J.P. Robinson, R. Pruitt; Culturable Bacterial Communities of Romaine Lettuce Leaves and Application of a New Optical Scattering Technology (BEAM) for their Characterization, ASM, June 7-11,2018 Atlanta GA, USA
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: 393. Carmen Gondhalekar, Pablo Martinez Ulloa1, Bartek Rajwa, Euiwon Bae, Valery Patsekin, Jennifer Sturgis, Iyll-Joon Doh, Larry Stanker, Vassilia Zorba, Richard Russo, Xianglei Mao, J. Paul Robinson; Detection of Anti-Toxin Heavy-Metal Conjugated Antibodies on Lateral Flow Assay Material using Laser-Induced Breakdown Spectroscopy (LIBS), Interagency SME & Program Manager Research Operations Division, RD-CB-CBC Defense Threat Reduction Agency, Fort Belvoir, Virginia May 23, 2018
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: 399. Carmen Gondhalekar, Euiwon Bae, Jennie Sturgis, Bartek Rajwa, J. Paul Robinson Portable and bench-top atomic spectroscopy for food contamination testing (Bio-PAS), USDA-FSIS Conference, USDA Virgina Feb 20-23, 2019


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Food inspectors, USDA employees, food processing companies, and government agencies. Changes/Problems:Major problem we had this year was the instability of our bench based LIBs instrument that we are trying to use for our gold-standard analysis. The failure of the present instrument was a major concern, however this has been rectified by the availability of a new laser, new spectrometer and new dete tion camera. We are excited about this - these instruments arrived in Early December 2018 and will be assembled in January 2019. What opportunities for training and professional development has the project provided?We had 3 undergraduate students work on this project and this enable them to learn new techniques, particuipate in team based research and learn the fundamental aspects of research. How have the results been disseminated to communities of interest?Yes we have presented several papers at conferences as noted above. What do you plan to do during the next reporting period to accomplish the goals?Key goals 1. the development of the multi-layer Lateral flow assay. 2. Finalizing the building af a new bench based LIBS instrument using new laser, spectrometer and camera we obtained this year 3. publishing at least 2 key papers on our work in oeer reviewed publications 4. Present at least 3 papers at the RAFA meeting in Prague November 2019

Impacts
What was accomplished under these goals? Key work achieved in the recent year has been the building of a new gold-standard bench based instrument to create a basline for the portable instrument. During the past year we designed several components of the portable instrument using 3D printing technologies. The goal of this was to identify the possible specifications for internal components. We tested one small portable laser, but found it insufficent power to use and so we are waiting for new higher power modles to be released. The second key success of our project this year was an emphasis on the assay design. We developed a new concept of multi-layerd lateral flow for whcih we have filed a patent. WE will begin to test this concept over then next 6 months. Initial tests for proof of conce\pt enable us to submit a provisional patent.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: 394. Maryam Salehi, Jennie Sturgis, Christian Ley, Euiwon Bae J. Paul Robinson, Andrew J Whelton; Application of Label Free Optical Scattering Technology for Rapid Detection of Bacteria in Drinking Water Samples ACE 18 (Annual Conference & Exposition Innovating the Future of Water) at Las Vegas June 11-14, 2018.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Carmen Gondhalekar, Ewa Biela, Pablo Martinez Ulloa, Bartek Rajwa, Euiwon Bae, Valery Patsekin, Jennifer Sturgis, Huisung Kim, Iyll-Joon Doh, Prasoon Diwakar, Larry Stanker, Vassilia Zorba, Richard Russo Xianglei Mao, J. Paul Robinson Detection of heavy-metal conjugated antibodies on paper bioassay material using laser-induced breakdown spectroscopy and laser ablation inductively coupled optical emission spectroscopy, SCIX Annual Conference, 21-25 October, Atlanta GA, USA
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: ul Robinson,  Utilizing Laser Induced Breakdown Spectroscopy for the determination of pathogens using lanthanide conjugated antibodies:, Lincoln University, Christchurch, New Zealand, April 5, 2018


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Scientific groups and associations in the area of food science. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Through journal articles and at conferences through papers and presentations. What do you plan to do during the next reporting period to accomplish the goals?During the next period we are putting together two new components: 1 - a new lateral flow test strip that we are developing that will be used for LIBs detection. 2. New hand held hardware (dependent on additional funding from some new applications)

Impacts
What was accomplished under these goals? We have been expanding the software developed for this project to solve a few key issues that have been of concern over the past couple of years. First, the issue of documenting the algorithms has been a difficult and demanding issue. We have now accomplished this. This has allowed us to review the algorithms in a much more detailed way and determine levels of accuracy. We have also developed and perfected an automated barcoding system that allows users to ensure that the samples identified are accurate. This required the development of code to produce barcodes, a database structure that manages the data, and an output system to print the barcodes for dishes and a reader to read the barcode.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: J. Paul Robinson, Kenitra Hamack, Euiwon Bae, Rachael P. Eberle, Jill Osterhus, Sabeen Nadir, Jennie Sturgis, Valery Patsekin, and Bartek Rajwa; Elastic Light Scatter and MALDI-TOF: Complementary technologies for reducing false negatives; 8th International Symposium on Recent Advances in Food Analysis, Nov 7-10, 2017 Prague, Czech Republic
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Carmen Gondhalekar, Ewa Biela, Bartek Rajwa, Euiwon Bae, Valery Patsekin, Jennifer Sturgis Huisung Kim, Iyll-Joon Doh, Larry Stanker, J. Paul Robinson; Detection of anti-toxin heavy-metal tagged antibodies using laser induced breakdown spectroscopy (LIBS), SCIX Congress, Reno Nevada, Oct 8-14, 2017
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: J. Paul Robinson, Carmen Gondhalekar, Euiwon Bae, Bartek Rajwa; Field-Deployable Laser-Induced-Breakdown-Spectroscopy for Multiplexed Detection of Food Contaminants Using Rare-Earth Labeled Antibodies Embedded in a Lateral Flow Strip; 8th International Symposium on Recent Advances in Food Analysis, Nov 7-10, 2017 Prague, Czech Republic


Progress 12/01/15 to 09/30/16

Outputs
Target Audience:Target audience is scientists studying or working with food, identification of pathogens, government agencies, etc. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?There is a graduate student and a postdoc on the project and both have learned a great deal. In addition there are two undergraduate students who have been using this project to learn about research processes. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are continuing to develop the technology.

Impacts
What was accomplished under these goals? We have been developing several different versions of instruments that will be used for microbial detection. These have been tested and evaluated to some extent. We are constantly updating these instruments with new components and new software. Our goal is to extend the technology so that it is highly accurate and inexpensive to operate.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Carmen Gondhalekar, Eva Biela, Bartek Rajwa, Euiwon Bae, Valery Patsekin, Jennifer Sturgis, Huisung Kim, Iyll-Joon Doh, Larry Stanker, J. Paul Robinson, Multiplexed detection of biological and chemical food contaminants using spark-induced breakdown spectroscopy (SIBS), CYTO2016, Congress of the International Society for Advancement of Cytometry, Seattle, WA, June 18-23, 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: J. Paul Robinson, Euiwon Bae, Jennifer Sturgis, Bartek Rajwa; Valery Patsekin; Maria Bogdanov, A rapid, label-free technology for microbial identification for food-borne pathogens, RAFA2015 Conference, Prague Nov 3-6, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Bartek Rajwa, Allison Irvine, Murat Dundar, Euiwon Bae, Valery Patsekin, J. Paul Robinson Application of machine-learning methods to recognition and classification of foodborne pathogens on the basis of elastic light scattering characteristics, RAFA2015 Conference, Prague Nov 3-6, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bartek Rajwa, Allison Irvine, Euiwon Bae, Valery Patsekin, J. Paul Robinson, Murat Dundar Machine-learning methods for screening, recognition and classification of bacteria on the basis of elastic light scattering characteristics; SLAS meeting, 2016 Jan 23-27, San Diego, CA, USA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: J. Paul Robinson, Euiwon Bae; Jennifer Sturgis; Bartek Rajwa, Valery Patsekin; Rapid Identification of Bacteria using Elastic Light Scatter: Label-free, low cost and fast; Purdue University, West Lafayette, IN, SLAS meeting, 2016 Jan 23-27, San Diego, CA, USA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Valery Patsekin; Jennifer Sturgis; Bartek Rajwa, Euiwon Bae, J. Paul Robinson, A software-hardware system for detection and Identification of pathogens, RAFA2015 Conference, Prague Nov 3-6, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Euiwon Bae; Huisung Kim, Jennifer Sturgis, Bartek Rajwa, Valery Patsekin, J. Paul Robinson, Phenotyping of bacterial colonies from the biophotonics perspective: The fundamentals of pathogen detection, RAFA2015 Conference, Prague Nov 3-6, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Huisung Kim; Iyll-Joon Doh; Ewa Biela; Carmen Gondhalekar; Bartek Rajwa; Valery Patsekin; Prasoon Diwakar; Euiwon Bae; Larry Stanker, J. Paul Robinson, Laser induced breakdown spectroscopy (LIBS) using lanthanide tagged bio-molecules, CYTO2016, Congress of the International Society for Advancement of Cytometry, Seattle, WA, June 18-23, 2016