Location: Quality & Safety Assessment Research2018 Annual Report
1a. Objectives (from AD-416):
The goal of the research is to develop and validate early, rapid, sensitive and/or high-throughput methods and techniques for detecting biological and physical hazards in poultry (food) products with optical sensing methods and instruments. Thus, the nature of the research is to combine chemistry and engineering disciplines (optical, agricultural, and food) with microbiological techniques to solve food safety detection problems in poultry (food). Specific objectives are: Objective 1: Develop high-speed imaging methods for rapid detection of pathogens in live poultry flocks, and foodborne hazards, including foreign materials, in processed poultry products. Sub-objective 1A: Develop Salmonella surveillance system for early detection of diseased birds. Sub-objective 1B: Develop high-speed hyperspectral imaging methods and system for foreign material detection. Objective 2: Develop rapid methods and protocols for early detection, identification, and quantification of pathogens in poultry products (foods) using imaging spectroscopy. Sub-objective 2A: Develop hyperspectral microscope imaging (HMI) methods and system for early detection and identification of pathogen at the cellular level. Sub-objective 2B: Develop fluorescence in-situ hybridization (FISH) imaging methods to identify pathogenic bacteria at the cellular level. Sub-objective 2C: Develop nanobiosensor for pathogen detection with surface enhanced Raman spectroscopy (SERS) at the cellular level. Sub-objective 2D: Develop methods for intervention carryover for Salmonella detection. Sub-objective 2E: Develop methods for plate detection with optimized agar media at the colony level. Objective 3: Develop methods to detect biofilms in poultry processing facilities with optical technologies. In developing the methods assess if any biomarkers can be identified to enhance or improve the detection sensitivity or specificity.
1b. Approach (from AD-416):
Ensuring poultry meat is safe to eat is of utmost importance to producers and consumers alike and rapid and early detection of foodborne pathogenic bacteria and foreign material in poultry products is needed. This research, which is divided into three objectives, primarily investigates optical sensors for rapid or improved detection of pathogenic bacteria with imaging and spectroscopic methods. Obj. 1A: an early-warning imaging surveillance system will be developed to detect bile in poultry droppings from laying hens in their cages. These higher levels of bile have been linked to birds with very high levels of Salmonella. Spectra will be collected to optimize key wavelengths and then a color-imaging system will be optimized for wireless real-time monitoring. Obj. 1B: Building on the success of a high speed hyperspectral imaging system developed within the unit, research will be expanded to detect foreign materials in various processed poultry products. Spectral libraries of normal meat features (muscle, fat, skin) and foreign material (rubber, metal, plastics, bone) will be used to develop algorithms suitable for high-speed use and then tested in real time. Obj. 2A: Hyperspectral microscope imaging (HMI) will be used to classify and quantify pathogens commonly found in poultry and other meats. The focus will be on identifying single bacteria cells from chicken rinsate by combining cell morphology and spectral profiles into an automated method for counting and classifying pathogenic bacteria. Additionally, markers will be used to enhance detection or means to separate and concentrate the bacteria will be implemented (immunomagnetic beads). Obj. 2B: Multiplex fluorescence in-situ hybridization (m-FISH) will be combined with HMI to further enhance detection with new protocols that will combine multiplexed probes and enhanced HMI detection resulting in broader, more robust methods of identification. Obj. 2C: Surface enhanced Raman spectroscopy (SERS), utilizing aptamers or antibodies and nano-enhanced surfaces, will be studied for Salmonella detection in broiler meat. Both labeled and label-free SERS will be evaluated. Obj. 2D: At FSIS’s request, research to neutralize sanitizers, frequently used to reduce pathogens while processing poultry meat, will be conducted to prevent interference of those sanitizers on bacterial analysis. Four potential neutralizing agents (quaternary ammonium, peroxyacetic acid, acidified sodium chlorite, acid solution, and dibromodimethylhydantoin sanitizers) will be screened for efficacy. Obj. 2E: Hyperspectral imaging (HI) systems will be used to classify pathogenic serovars growing in agar plates and collaborations will explore additional agar additives (both chromogenic and non-chromogenic) that will help differentiate serovars of E. coli O157:H7 and other shiga-toxin producing E. coli (STEC). Obj. 3: First in the lab, and then in processing plants, HI systems will be used and paired with spray-on markers to enhance the detection of biofilms on equipment surfaces. This research is potentially collaborative with ARS Beltsville and will help to discriminate biofilms from other organic material.
3. Progress Report:
A red dye, F, D, & C Red #3, has been determined to be a suitable candidate for use in poultry processing establishments to disclose bacterial biofilms and other contaminants. The dye is approved by FDA for use in food, and a methodology has been developed by which the dye can be used as a stain to visually disclose the presence of biofilm extracellular polymeric substances (EPS), including proteins and carbohydrates, and individual gram-positive and gram-negative cells. In addition, the dye will disclose the presence of other proteins, carbohydrates, and fats on processing equipment resulting from inadequate cleaning and sanitation procedures. The new methodology will be of value to food plant managers as a tool to monitor cleaning and sanitation effectiveness. Listeria innocua and Pseudomonas putida biofilms were produced as models for pathogenic Listeria and Pseudomonas biofilms. Numerous cleaning and sanitizing agents currently utilized in food processing facilities were investigated for their abilities to both inactivate and remove biofilm matrices and cells. Biofilms were grown on stainless steel coupons and the coupon was subjected to cleaning/sanitizing treatments. Following each cleaner/sanitizer treatment on the model biofilms, a red dye methodology was used to stain the residual biofilm matrix material. Results indicate that most cleaning/sanitizing agents are able to inactivate the cells residing in the biofilm, but that the matrix and dead cells were not removed from the coupons. Only treatment of the biofilm using a mixture of enzymes was capable of completely removing the biofilms, but this treatment did not affect cell inactivation. The results suggest that in order for poultry processing plants to effectively inactivate and remove biofilms, a two stage process consisting of enzyme treatment followed by disinfectant treatment may be necessary. Additional data of E. Coli O157:H7 and other Shiga-Toxin producing E. Coli (STEC) strains, which have been identified by the Food Safety Inspection Service as adulterants, were collected. Both high-resolution digital images and hyperspectral images of pathogenic E. Coli growing on either Rainbow agar or a modified MacConkey agar, provided by ARS scientists at the Meat Animal Research Institute, Nebraska, were collected. Results from data analysis indicated that images of E. Coli pathogens growing on the modified MacConkey agar were no better at classifying the pathogens than images of the pathogens on Rainbow agar. Very high resolution digital images and high-resolution hyperspectral imaging both showed some promise at increasing prediction accuracies, especially the high resolution hyperspectral images of Rainbow agar, compared to lower resolution hyperspectral images collected in the past. However, accuracies were around 90 percent and not sufficiently better to warrant additional research in this direction. Next, high-resolution DSLR and hyperspectral images of E. Coli O157:H7 and the other STEC strains described above were also collected on R & F E. coli O157:H7 Chrome Medium and CHROMagar O26/O157 agars. Multiplex and Label-Free Screening of Foodborne Pathogens Using Surface Plasmon Resonance Imaging. In order to protect outbreaks caused by foodborne pathogens, more rapid and efficient methods are needed for pathogen screening from food samples. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for label-free screening of multiple targets simultaneously with minimum or no sample preparation. ARS researchers at Athens, Georgia evaluated the feasibility of SPRi in simultaneous detection of four most important foodborne pathogens, Salmonella spp., Shiga-toxin producing E. coli, Listeria monocytogenes, and Campylobacter jejuni. The SPRi-biochip was functionalized with corresponding polyclonal antibodies and blocking agents. Bacterial cells were tested for performance of the multiplex SPRi method. The influence of antibody concentration and immobilization pH was optimized, and the specificity was evaluated. The cells were also fragmentized thermally and ultrasonically, whose SPRi signals were compared with intact cells. The results suggest that the new SPRi technique demonstrated the potentials in multiplex pathogen screening, and with further improvements in sensitivity, this platform could provide a flexible and automated means to pathogen detection in food matrices. Identification and detection of Campylobacter from poultry rinsate with hyperspectral microscope imaging. Campylobacter are the major group of bacteria responsible for foodborne gastroenteritis in humans. Most of the cases of campylobacteriosis have revealed their poultry origins so that various control measures have been employed both at the farm and processing levels. Campylobacter jejuni is the most common cause of bacterial foodborne illness in the United States. Current method for isolation and detection of Campylobacter from foods are culture-based techniques with several selective agars designed to isolate Campylobacter colonies. In addition, several immunological and molecular techniques are commercially available for the detection and identification of Campylobacter. However, most significant drawbacks of current commercially available methods include laborious, time-consuming, and expensive. ARS researchers at Athens, Georgia develop a hyperspectral microscope imaging (HMI) method to demonstrate the potential to identify Campylobacter from other foodborne pathogens including E. coli, Listeria, Staphylococcus, and Salmonella with high accuracy. Also, HMI method was able to classify C. coli., C. fetus, and C. jejuni with over 95% accuracy. Hyperspectral imaging for detection of bile on droppings from poultry. When the gallbladder becomes infected, inflammation can result in excretion of large amounts of bile in a manner that changes the color of excreta from brown to green. One such discoloration of poultry droppings results from hens infected with S. enteritidis. This occurrence raises the possibility that machine-vision techniques could be useful for detection of infected birds or those with other gastrointestinal illnesses. ARS researchers at Athens, Georgia developed a hyperspectral imaging (HSI) method to detect bile in droppings for possibly early detection of broilers infected by Salmonella. Using key wavelengths identified earlier with visible/near-infrared spectroscopy, HSI was able to identify small amounts of bile in poultry droppings. Additional image processing methods, including background separation, noise removal, and segmentation of bile contaminants, were used to identify potentially infected poultry flocks. The methods developed with HSI was further evaluated with low-cost color cameras and associated image processing for potential use as an early indicator of bird illness and/or infection.
1. Multiplex and label-free screening of foodborne pathogens using surface plasmon resonance imaging. In order to protect outbreaks caused by foodborne pathogens, more rapid and efficient methods are needed for pathogen screening from food samples. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for label-free screening of multiple targets simultaneously with minimum or no sample preparation. ARS researchers at Athens, Georgia, evaluated the feasibility of SPRi for simultaneous detection of four important foodborne pathogens, Salmonella spp., Shiga-toxin producing E. coli, Listeria monocytogenes, and Campylobacter jejuni. The SPRi-biochip was functionalized with corresponding polyclonal antibodies and blocking agents. Bacterial cells were tested for performance of the multiplex SPRi method. The influence of antibody concentration and immobilization pH was optimized, and the specificity was evaluated. The results suggest that the new SPRi technique demonstrated the potential for multiplex pathogen screening, and with further improvements in sensitivity, this platform could provide a flexible and automated means to pathogen detection in food matrices.
Berrang, M.E., Harrison, M., Meinersmann, R.J., Gamble, G.R. 2017. Self-contained chlorine dioxide generation and delivery pods for decontamination of floor drains. Journal of Applied Poultry Research. 26(3):410-415. doi: 10.3382/japr/pfx009.
Berrang, M.E., Gamble, G.R., Hinton Jr, A., Johnson, J. 2018. Neutralization of residual antimicrobial processing chemicals in broiler carcass rinse for improved detection of Campylobacter. Journal of Applied Poultry Research. doi:10.3382/japr/pfx071.
Jiang, H., Yoon, S.C., Zhuang, H., Wang, W., Yang, Y. 2017. Evaluation of factors in development of Vis/NIR spectroscopy models for discriminating PSE, DFD and normal broiler breast meat. British Poultry Science. 58(6):673-680.
Eady, M.B., Park, B., Yoon, S.C., Haidekker, M., Lawrence, K.C. 2018. Methods for hyperspectral microscope calibration and spectra normalization from images of bacteria cells. Transactions of the ASABE. 61(2): 437-448.
Eady, M.B., Park, B. 2018. Unsupervised classification of Salmonella Typhimurium from mixed bacteria cultures with hyperspectral microscope imaging. Journal of Spectral Imaging. DOI:10.1255/jsi.2018.a6.
Seo, Y., Park, B., Yoon, S.C., Lawrence, K.C., Gamble, G.R. 2018. Morphological image analysis for foodborne bacteria classification. Transactions of the ASABE. 61(1): 5-13.