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Goal: The long-term goal of this research is to introduce a paradigm shift for identifying microbial pathogens in diverse sample types (soil, water) by deploying a portable sniffer technology to "smell" the volatile organic compounds (VOCs) emitted by the pathogens growing in soil or water to effect on-site, in situ, sensitive (100 to 1000 colony forming units, cfu, per gram of soil/ml of water) and rapid (< 30 minutes) diagnostics, by exploiting the unprecedented sub-parts-per-billion (ppb) to parts-per-trillion (ppt) sensitivity levels of our portable, micro-pulsed discharge helium ionization detector (?PDHID) integrated with an onboard micro-gas chromatograph (?GC) systems package, and thereby eliminating the need for analysis using bacterial culture. Alternatively, the approach can be used to prioritize samples that will be selected for more labor-intensive and expensive tests where bacterial culture is necessary for confirmation and source tracking. analysis using bacterial culturing. We will deploy our suitcase-sized sniffer unit between years 3-4 of this proposal for testing soil/water field samples and compare its performance to the reference gold standards (culture confirmation and commercial molecular diagnostic kits of nucleic acid amplification and/or antibody-based immunoassays). <P>Hypothesis: The sniffer technology will be able to detect unique voc patterns that will allow it to rapidly and accurately identify foodborne pathoghes in soil and water. First set of Objectives for year 1/5: Our future set of objectives will mirror these as we progress through the target organism list we provided previously. Objective 1: To determine the VOC profiles of E. coli O157:H7 vs Salmonella enterica grown in TSB. Objective 2: To determine the VOC profiles of our STEC-7 panel in TSB. Objective 3: To determine the VOC profiles of our STEC-7 panel in selective media slants (rainbow media) Objective 4: To determine the VOC profiles of the STEC-7 panel in spiked soil and water using rainbow media. Objective 5: To determine the detection limits of the Sandia Sniffer Technology (SST) for each serotype of our STEC-7 panel. Objective 6: Optimize time vs detection limit of SST for each serotype of our STEC-7 panel in spiked soil and water Objective 7: Optimize time vs detection limit of SST utilizing a cocktail of the STEC-7 panel in soil and water. The overall group of organisms we project to study during this 5 year plan will be the following: E. coli (STEC) O157:H7, non-O157 STEC, Salmonella, Shigella, Campylobacter jejuni, and Listeria monocytogenes.
Non-Technical Summary:<br/>
When foodborne outbreaks of illnesses occur, it is important to have the capability to rapidly and accurately do traceback investigations. In this case, the project is focused upon going to the field and identify or rule-out soil and water sources that may have contributed to the origins of the pathogen. The benefits will be shortening the time required to rule in or out any ecosystem sources of soil or water that may be harboring the foodborne pathogen(s) of interest.
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Approach:<br/>
We propose an integrated systems approach for the rapid, specific, sensitive, point-of-use pathogen detection with a broad range of plant, veterinary, and food industry applications. Briefly, the technology is based upon the emission characteristics of volatile organic compounds (VOCs) that are natural metabolites of the microorganisms or arise from the interactions of the growing bacteria with the growth media or matrix (for reviews please see, Effmert et al, 2012, Boots et al, 2012, Thorn and Greenman, 2012, Nicholson et al, 2012, Dymerski et al, 2011, Brattoli et al, 2011, Baldwin et al, 2011, Velusamy et al, 2010, Wilson and Baietto, 2009, Peris and Escudere-Gilabert, 2009, Casalinuovo et al, 2006, and Ampuero and Bosset, 2003). Also, the bacteria need not be separated or enriched from the sample matrix or culture media. Furthermore, sample preparation is minimized or even eliminated since the pathogen need not be physically isolated from the matrix and separated from inhibitory compounds, prior to initiating culture or molecular testings. Instead, the air above the growing bacteria ("head space") is sniffed and identification is effected through a combination of specific volatiles and their emission pattern. Refinement and implementation of this sniffer technology will allow more rapid. accurate field investigations to take place; thus, enhancing the traceback capabilities of foodborne illness investigations. This will allow important, timely communication activities to take place between investigators, regulators, public health officials, producers and consumers.