BAX-Q7-system to Increase Efficiency and Accuracy of Microbial Detection in Environmental and Food Matrices

<p>NON-TECHNICAL SUMMARY:<br/> In the past two decades, numerous foodborne outbreaks have been associated with pathogens such as shiga-toxigenic Escherichia coli (STECs), Salmonella, Campylobacter, and Listeria monocytogenes. A large number of these outbreaks have been traced back to the farm environment, making pre-harvest food-safety an area of increased focus. Research conducted at Oklahoma State University (OSU) focuses on developing effective pre-harvest strategies to reduce pathogen contamination. However, a reliable, quantitative system to efficiently and accurately characterize various microorganisms in food and environmental samples is critical to the success of such studies. Currently, lack of such a system has become a major limitation in carrying out studies with large sample sizes. The purpose of this equipment grant is to purchase the BAX-Q7-system which is a
gene-based method for detection and confirmation of microorganisms in various sample types. It has the capacity to test large sample sizes for multiple target microorganisms, simultaneously. Acquisition of such a system will aid with the following studies: (1) Prevalence of and risk factors associated with STECs and Salmonella on cattle farms and development and validation of effective mitigation strategies; and (2) Assessment of pastured poultry farms for contamination of Salmonella spp. Aforementioned projects will also provide a venue for educational segments regarding molecular methods for qualitative as well as quantitative detection of microorganisms. These will be incorporated into the food-safety courses offered at OSU as well as in the research carried out by students, thereby enhancing the graduate and undergraduate programs at the institution.
<p>APPROACH:<br/> The BAX®-Q7 System is a rapid method for detecting pathogens or other organisms in food and environmental samples. The system breaks down samples at the genetic level, using the real-time polymerase chain reaction for detection and confirmation of bacteria and other microbes. It has the capacity to test multiple target microorganisms in the same run with up to 96 samples per batch providing information beyond presence or absence, such as quantification and species differentiation. The BAX®-Q7 System was chosen because of its flexibility with regard to the types of samples and the number of microorganisms that could be characterized and due to the large number of samples it can process in short periods of time. The immediate use of the proposed equipment is to carry out and bring to completion several food safety projects funded through USDA
programs specifically AFRI, Southern SARE and OREI. Currently, several studies are in progress requiring the isolation, enumeration and molecular confirmation of pathogenic bacteria such as Escherichia coli O157:H7, non O157 STEC, Salmonella, Campylobacter, and Listeria monocytogenes from environmental and food samples. Intended Research Activities Experiment 1. Objectives: 1. Determine the prevalence of STEC and Salmonella on small-scale cow/calf operations 2. Determine contamination sources for STEC and Salmonella in small-scale cow/calf environment 3. Develop and validate effective mitigation strategies. Prevalence of E. coli O157:H7, nonO157 E. coli and Salmonella will be determined on cow/calf farms located in Oklahoma by taking into account the relevance of controlling this pathogen under different conditions of herd, farm and environment. We will assess on-farm practices; develop
guidelines for best management practices; and validate the effectiveness of these best practices in reducing the pathogen burden on cow/calf operations, thereby resulting in a decrease in pathogen loads on calves entering the feedlots and abattoirs. Validation will be carried out in multiple locations in Oklahoma by training cow/calf producers on pre-harvest food safety and by following changes in pathogen loads on their farms after implementation of control strategies. At least 30 small-scale cow/calf operations located in Oklahoma will be selected for sample collection to determine E. coli O157:H7, non-O157 STEC and Salmonella populations on the farm. Farm samples will include fecal (n=15), water (n=5), sediments (n=5) and water equipment swabs (n=5). During the summer and fall months (May-November), samples will be collected from each farm in 2 separate sampling intervals. Immediately
after collection, the samples will be transported on ice to the laboratory and processed for enrichment of target microorganisms. Detection, isolation and characterization of test pathogens will be done using the BAX®-Q7-System. Results will be used to determine on-farm contamination sources and to establish mitigation strategies that need to be implemented on these farms. These will include basic principles of cattle management such as provision of clean feed and water, sanitary handling of water and waste, proper drainage and maintenance of the environment, and freedom from vermin and pests, along with decontamination strategies, disinfection programs, and possible use of probiotic therapies. Following the preparation of guidelines, several training workshops will be held for cattle producers across Oklahoma. We will validate these strategies by follow-up surveys and microbial
sampling of selected farms for pathogen loads after their implementation. Experiment 2. Objectives: 1. Assess the food safety risk (concentration of Salmonella) of on-farm processed pastured poultry chicken carcasses. 2. Assess the biosafety risk of pathogens in waste disposal (wastewater and solid waste) of on-farm processed pastured poultry. Over a one-year period, three pastured poultry farms will be visited five times. During each visit, ten pastured broiler carcasses will be collected after on-farm processing is completed at the farms. Furthermore, ten carcasses will be collected from farmer who processes his birds at the USDA-inspected facility. Chicken carcass samples will be placed in sterile plastic bags and shipped on ice to the laboratory within 24 hours for Salmonella analyses. We will also assess the pathogen concentration in wastewater and solid waste (i.e., feathers,
heads, fecal matter, and viscera) disposal when processing pastured bird on-farm. Survival of Salmonella in soil, where wastewater is disposed, and in compost, where solid waste is treated before using as fertilizer, will be assessed.Influent wastewater samples at the time of processing will also be collected and processed as follows: a 1 L sample of processing water will be collected before processing begins. Thereafter, a 1 L composite sample of the influent wastewater will be diverted from the waste stream (runoff) for every 10-25 birds processed. For example, if a farm processes 100 birds at a time, then a total of 4 samples will be collected during processing. At the farms where wastewater is disposed (runoff) onto the field, soil samples will be collected monthly for six months. For microbial analysis in soil samples, 25 g composite surface soil samples will be collected from 3
different areas within which wastewater has been applied. Solid waste such as intestine/viscera, feather, and fecal matter are commonly composted at the farm. Sampling of the compost will be performed at the farms once a month for 6 month. Three samples (25 g) will be collected from the compost at each collection time. The 25 g soil or compost samples will be added to the respective enrichment broths and quantification done by the BAX®-Q7-System.