<p>The goals are for isolation, identification, and characterization of bacterial primary and opportunistic pathogens that are present on retail poultry products in Orangeburg County, SC. Monitoring the dominance of specific pathogens will be assessed after characterization of the bacterial isolates from the poultry products. The determination of antibiotic resistant patterns and profiles for characterized poultry bacterial isolates from retail poultry products will be investigated after identification has been established. Biosensor technology will be applied to detect bacterial pathogen presence in poultry products. This technology can track changes in presence of pathogens from different sources of retail poultry products and during seasonal timeframes. The project objectives of the first year are to </p>
<p>1) set up the laboratory; </p>
<p>2) collect retail poultry products; </p>
<p>3) isolate and identify poultry bacterial isolate and screen for known pathogens present on retail poultry products. Stock bacterial cultures will be prepared and stored using ultra low freezer to maintain cultures for future use. </p>
<p>The project objectives of the second year are to characterize the antibiotic resistance properties of poultry bacterial isolates (pathogens and non-pathogens) that were collected and identified during year one. Antibiotic resistance characterization will be analyzed initially by using the Biolog Microstation reader (Micronaut System) and antibiotic resistance genes will be analyzed using a Real Time Polymerase Chain Reaction (RT-PCR) to fully profile the bacterial isolates especially the primary pathogens. The project objective of the third year will be using biosensing technology to detect and monitor pathogens present in retail poultry products. Real Time PCR can be used for quantitative and qualitative detection of pathogens on poultry product samples.</p>
<p>NON-TECHNICAL SUMMARY:<br/> Food-borne infections remain a major public health problem in the United States. Food-borne bacterial pathogens affect tens of millions of people and kill thousands annually. Tracking single cases of food-borne illness and investigating outbreaks are critical public health functions. Some resources of antibiotic resistant bacteria stem from animal feed with therapeutic antibiotic supplements. Retail poultry products have been shown to have food pathogens still present on the chicken surface after purchase by the consumer. Depending on the poultry farm and processing plant in which these products originate, pathogen prevalence and antibiotic resistance can show different trends. Therefore, different regions of poultry products may have different risks for the consumer depending on where the retail poultry product is bought. This proposed project
will investigate the prevalence, antibiotic resistance susceptibilities and the use of biotechnology for quick detection of food pathogens on retail poultry products from retail food stores within the Orangeburg county area. Microbial identification systems, antibiotic susceptibility assays and Real Time PCR assays will be the major methodologies used to accomplish the objectives of this project.
<p>APPROACH:<br/> Initially, retail poultry products will be collected from various food retail stores within the Orangeburg County, SC region. Samples will be prepared from the poultry products for analyzation with the various following assays. The methodologies that will be used in the project will be the following: Biolog Microbial Identification system - This system will include a computer, software, databases, Microstation reader instrument that will read samples for identification and a printer. It also comes with accessory instruments that will be use for sample preparation. Micronaut software to use with Microstation instrument to analyze bacterial antibiotic resistance patterns for each bacterial isolate. Real Time PCR-This instrument can confirm antibiotic resistance and different genotypic patterns of bacterial isolates. This technology can be used to as a
biosensor, as well, to detect targeted bacterial pathogens in samples by detecting the specific DNA presence of these organisms. Fluorescence microscopy that will use fluorescent probes to help screen for presence of targeted bacteria in a sample for initial screening of food samples. Fluorescent antibody probes and other stains can be used to characterize bacterial organisms.
<p>PROGRESS: 2012/01 TO 2012/12<br/>OUTPUTS: During the period between May 16, 2012 and December 31, 2012, student research assistants were being trained in various microbiological protocols. They included the following: (Laboratory safety procedures and practices were reviewed with the students before any training began.) -Aseptic techniques in laboratory preparations and maintenance-Before handling any bacterial culture, students were required to wear proper lab coat attire, gloves, and protective eye wear. Students were then taught how to properly sanitize surfaces before and after handing bacterial cultures on work surfaces in the lab. They were also shown how to properly transport bacterial cultures by putting them in appropriate transport vessels to prevent contamination. -Different media preparations for both liquid and solid media-Students were shown how to
properly calculate and measure powder media mixes for liquid and solid media forms, autoclave (sterilize); and properly pour into petri plates for solid media agar or dispense liquid media in culture tubes or bottles. After media preparations, they were shown how to label and store the media. -Bacterial culturing techniques-transferring cultures-Students were shown how to transfer bacterial cultures aseptically from one plate of growth to a new solid media plate to keep cultures viable; bacterial isolation-students were shown how to use streaking techniques on solid media (agar) plates to obtain isolated colonies (CFUs-colony forming units) to purify bacterial cultures; bacterial isolate collection- students were shown how to collect bacteria from food samples and shown how to properly enrich cultures after collection; preparation of bacterial stock cultures-students were shown how to
make stock cultures of different bacteria strains and preserve them in ultra-low freezing storage. -Various laboratory instruments included proper use of pipettors and pipettes; use of autoclaves and sterilizers; use of peristaltic instruments for liquid media dispensing; use of incubators for culturing bacteria; hotplates with stirrers used for media preparations; refrigerator and freezer-use for storage of media, chemicals, supplies, bacterial cultures, samples etc.; and use of compound microscope. -Bacterial staining techniques-students were taught simple staining and gram-staining techniques that will be needed for preliminary screening of bacterial cells for the Biolog microbial identification system and other assays. -Record keeping-Students were given each a lab notebook to record daily laboratory assignments and results from training protocols. Students helped with organizing a
Standard Operating Procedures Manual for the research lab. -Literature search-Students were assigned specific literature-search topics from the PI to assist with collection of background information on related research topics and techniques. Equipment Set-Up All necessary equipment had not been received by Dec. 31, 2012. The equipment that was received was certified and calibrated. The PI did the set-up, along with the contracting of equipment certification specialists. <p>PARTICIPANTS:<br/> Principal Investigator (PI), Dr. Sheriase Sanders, managed this project. The PI was responsible for managing the budget of the project, ordering supplies and equipment, setting up equipment, and training student research assistants on the equipment; organizing work schedules and managing timesheets for students. The PI also trained the students in learning laboratory techniques and monitoring progress of
this training. Also, the PI was responsible for creating daily laboratory assignments for student assistants in relation to the project. Undergraduates- William Dumpson, Alyssa Murray, Adisa Julien (3- Student Research Assistants) Three undergraduate students assisted the PI with the project. Each of the students had equal duties in the lab. Each were responsible for learning and performing lab techniques taught by the PI, handling, and maintaining bacterial cultures, as well as keeping the laboratory space maintained. They also performed computer-search assignments to help build a database of background knowledge for the project. <p>TARGET AUDIENCES:<br/> Nothing significant to report during this reporting period. <p>PROJECT MODIFICATIONS:<br/> Project progress has been severely delayed due to lab space renovation delays and equipment order processing and delivery delays. This has pushed back the
progress of initial planning of results which were based upon lab space renovations and equipment being completed and delivered by Summer 2012/early Fall 2012 Semester (June-October2012). Laboratory renovations were still ongoing by December 31, 2012.