Microbiology Animal Food Project Testing Track 3 Accelerated Sample Preparation for Microbial Pathogen Detection in Pet Food Samples

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Accelerated Sample Preparation for Microbial Pathogen Detection in Large Number of Pet Food Samples Abstract We are submitting a proposal for Discipline A: Microbiology, track 3 for Animal Food Product testing with Dog treat food/Salmonella as a hazard to Commodity pairs. Here we combine different expertise from 2 laboratories, Laboratory of Renewable Resources Engineering (LORRE) and the Office of Indiana State Chemist (OICS), to assemble a proposal consisting in animal food sample collection, accelerated food sample preparation to bring microorganisms of interest to detection levels, FDA approved loop mediated isothermal amplification (LAMP) for Salmonella screening in animal food, and whole genome sequencing as a method for confirmation of pathogen isolates. We have further optimized our sample preparation approach, as a follow up to 2015 FDA challenge award, and we now can simultaneously concentrate and recovery microorganisms of interest in 4 samples, bringing their numbers from initially not detectable, due to be present at initial levels of 1CFU/g or lower, to detection levels. This accelerated processing of samples is complete in a time frame of about 4 hours. It consists of a microbial short enrichment in selective medium combined with an enzyme treatment, followed by a pre-filtration step to remove large particles and microfiltration to concentrate and recovery selected microorganisms to detection level. The enzyme treatment helps to break down possible biofilms formation and particles that may clog pores in the hollow fiber membrane during microfiltration. Enzymes selected are based on food matrices. In the case of the samples proposed here, proteases possible combined with lipases will be used. We have shown the enzymes do not affect microbial viability, when in the presence of their respective substrates. While we have focused on Salmonella as a microorganism of interest, our developed approach can also be applied to other microorganisms. The Office of Indiana State Chemist (OISC) microbiology laboratory has major expertise in sample collection for animal food testing. It has also many years of experience in microbiological method development, testing for antibiotic concentration, and bacteria enumeration and identification, including several years of pathogen screening for Salmonella, Listeria monocytogenes, Campylobacter and Escherichia coli (STEC) in animal feed. Last year, the OISC microbiology laboratory contributed for testing in a national wide Salmonella outbreak in pet treat pig ear in Indiana. The proposed number of samples to be tested here is 250 samples pairing dog treat/Salmonella. Expected outcomes are accelerated sample preparation, increased speed and sensitivity of sample testing, and initial insights of potential recovering and detection of difficult to culture microorganisms.