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

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.