Identification and sub-typing of food-born bacteria from animal feed ingredients, animal feed & pet foods samples using Matrix-Assisted Laser Desorption & Ionization Time-of-Flight Mass Spectrometry.

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FDA CVM VET-LRN PROGRAM (U18) GRANT PROPOSAL FOR FOA # PA-13-244, CFDA 93, 103 Project Summary/Abstract This project will use Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) for identification of human foodborne pathogens, including Salmonella, E. coli, Campylobacter species, Listeria monocytogenes, Clostridium perfringens, and Staphylococcus aureus in animal feed ingredients, pet foods and animal feed. The first year of the project will focus on identification and subtyping of Salmonella bacteria using MALDI- TOF MS. The same approach will be used for other foodborne pathogens in the following four years. Salmonella contamination of pet foods/animal feeds has resulted in outbreaks of human Salmonellosis. However, contamination of animal feed ingredients, animal feed, and pet treats and foods have not been systematically studied. Routine culture is the "gold standard" method for isolation and identification. This method is labor intensive, expensive, and time consuming, which does not provide timely information before the distribution of the contaminated animal food matrices. Therefore, alternative methods for rapid and reliable identification and subtyping of Salmonella bacteria are urgently needed to improve safety and integrity of animal feed supply, which in turn will limit human infections. In this application we propose to develop methods using MALDI-TOF MS for rapid and specific identification and typing of Salmonella bacteria grown on preliminary culture plates from animal feed matrices. Microbial identification using this technology is based on the comparison of spectral patterns obtained directly from cells or cell extracts against reference spectra using a pattern recognition algorithm. This technology is currently available in the form of commercial platforms, and a considerable amount of studies have been generated demonstrating its utility for identification and typing of Salmonella bacteria. We have successfully used this technology for identification of Salmonella bacteria in fecal and animal tissue samples. There is, however, a lack of knowledge regarding the use of MALDI-TOF MS for identification of bacterial isolates from pet food and animal feed matrices. The proposed study will address these issues by the following three specific aims: Specific aim 1: Identification and subtyping of foodborne bacterial isolates from various animal food matrices using MALDI-TOF MS using a novel library. Specific aim 2: Identification and subtyping of foodborne bacteria directly from colonies grown on various culture media from animal feed and food matrices. Specific aim 3: Transferring and training of this new methodology to other Vet-LIRN laboratories. Successful completion of this project will expand the current application of this technology from fecal and animal tissue samples to animal feed matrices to improve the safety and integrity of the US animal feed supply. In addition, the multi-laboratory collaboration validation of this methodology will increase Vet-LIRN laboratory capability and capacity in order to serve CVM's need for rapid turnaround times when responding to animal food contamination events.