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Detection, Characterization, and Inhibition of Foodborne Pathogens, Toxins, and Spoilage Microorganisms

Muriana, Peter
Oklahoma State University
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Thelong termgoals of this project are broadly stated as 'detection andinhibition offoodborne pathogens and spoilage organisms'. Some indication of what I can accomplish can be described by summarizing what I have done in the past. We haveexamined sensitive detection of toxins (staph enterotoxin; aflatoxin) usingimmunomagnetic bead-based toxin capture combined with secondary antibody-tethered oligonucleotides andPCR for detection down to femtogram levels (SEA/SEB)or quantification at picogram levels (aflatoxin).I have developeda fluorescent microplate assay for screening adherence characteristics ofListeria monocytogenes isolated from processing plants (and othersources), showing that various isolates could be distinguished by strong- vs weak-adherence phenotypes; further studies demonstrated that thestrongly-adherent strains were more invasive in tissue culture and live mouse assays. We are currently continuing to study the molecular basis for adherence in L. monocytogenes.This may require studies to enhance their detectionand/or studytheir phenotypic characteristics. We are also involved with antimicrobial interventions against foodborne pathogens (STEC E. coli and Salmonella)and spoilage organisms (Leuconostoc sp.) in/on meat products by either processing interventions (pre- and post-package pasteurization of deli meats), antimicrobial spray treatments of meat surfaces (for carcass,ground beef, and mechanical tenderization/non-intact beefinterventions), orfermented metabolitesproduced bylactic acid bacteria (bacteriocins) as biopreservatives. Also, since we use 16S rRNA sequence identification of isolates, we are interested in combining this sequence with other pathogen/spoilage-specific loci (i.e., virulence genes, spoilage-based genes) to elicit multi-locus sequence typing of the various isolates. This serves the purpose of providing positive sequenceinformation on isolatesthat is portable (usable by various sequence analysis programs)as compared to DNA restriction pattern images (minimal information) as well as typing strains based on phenotypes that arespecificto the isolates. Our interest in spoilage organisms comes from our work in the R.M. Kerr Food & Ag Products center with numerous processors who may feel reluctant to allow interactions targetting pathogens (i.e., their current processes are 'sufficient'), however, all/most processors feel the pinch and suffer economic losses from spoilage organisms and are more likely to engage interactions based on antimicrobial interventions against spoilage organisms. And efforts that address and reduce spoilage organisms more than likely also address reduction of pathogens.
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The proposed projectis specifically involved with various facets offood safety:the evaluation of food safety measures (i.e., antimicrobial interventions)that could beimplemented to enhance the safety of foods either byaddressing the food itself or the processes by whichfoodis manufactured;the characterization of those organisms that are human pathogens (foodborne pathogens) or affect the quality of foods (spoilage organisms)such that by understanding and knowing the characteristics of these organisms, we can better apply interventions to foods in order to reduce or eliminate them. Antimicrobial interventions that are applied areusually applied against food samples inoculated with challenge microorganisms, often the targeted pathogens themselves (i.e., E. coli O157:H7, Salmonella, Listeria monocytogenes). Inmany cases,the challenge organisms that are used are preferablyantibiotic resistant (for research testing purposes). Thisallows theenumeration of survivingchallenge organismson media thatcontains those antibioticsto insurethat we arerecovering onlythoseorganisms we inoculatedand not any other organisms that are normallypresent innon-sterile foods (i.e.,ground beef). Also, since we spray some of these antimicrobials, there is the possibility of a 'displacement' reduction, so as a control measure, we will often compare a sprayed antimicrobial (lethal)solution with spray treatmentusing water (non-lethal)for comparison. Some research is practical (i.e., antimicrobial interventions against pathogens), while other aspects of the research are basic research (understanding the molecular basis for adherence) which may lead to practical solutions for food safetyproblems. The premiss is that we can better provide a solution only once the pathogen/problem is well understood and that involves obtaining answers to questions such as 'how do pathogens get into food processing environments?', 'how do they persist despite sanitation programs?', 'how do they get onto foods?', 'how do we eliminate them from foods?'.After understanding various characteristics of problematic foodborne pathogens and spoilage organisms, we can better apply antimicrobial interventions to result in their reduction and increase the safety of foods.

Methods. The general scientific methods involved are basic food microbiology protocols. Protocols for isolating and identifying bacteria (pathogens, spoilage microorganisms) are standard microbiological methods. Once obtained, isolates of interest will be subject to 16S rRNA PCR amplification using bead disruption of cellsfrom fresh-grown colonies on petri plates, PCR amplification with universal 16S rRNA primers, amplimer cleanup, submission to OSUMolecular Biology Core facility for sequence determination,manual evaluation of ABI sequencer-derived sequence data to use only the bestsequence data, and BLAST search of sequences against the NCBI databases for identity. Methods for antimicrobial interventions has put us in contact with industry who have provided us with unique equipments that we would not be able to have obtained otherwise. It is understood that the research data obtained with such 'loaned' or 'donated' equipment would be required to be disseminated for the greater benefit of all. For instance, we have pilot-plant scale steam-injected commercial water pasteurizers, a radiant heat oven, blade tenderizer with uniquely integrated spray system for spraying antmicrobial solutions onto beef prior to mechanical tenderization, hypochlorous acid and digitally-monitored ozonegenerators. The 'efforts' to deliever science-based knowledge to audiences include formal class room teaching whereby actual examples of food industry problems we work on in the lab are presented at appropriate topics during the semester and provide unique, real-life solutions to current problems and issues; students benefit from having someone with an active food microbiology lab importing and weavinginformation from laboratory research into classroom teaching giving a real-life experience to the course. Similar approaches are used in various seminars,workshops, and local symposia. 'Evaluation' of efforts is obtained by reviewers ofpeer reviewed publications. Most of our workshops we haveprovide pre- and post-workshop testing to quantify the impact of the workshop as indicators of success.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Project number
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Viruses and Prions
Natural Toxins
Escherichia coli
Bacterial Pathogens
Meat, Poultry, Game