<OL> <LI> To examine methods for rapid detection of microorganisms or toxins from food: <BR>a. Signal-amplification in combination with immunomagnetic bead detection of toxins. <BR>b. Amplimer-capture fluorescence-based PCR methods for detection of foodborne pathogens (i.e., L. monocytogenes).
<LI>To characterize the surface-adherence trait in strains of L. monocytogenes isolated from food processing plants, raw meats, and ready-to-eat meats: <BR>a. To identify the relationship of adherence to the formation of biofilms on abiotic surfaces.<BR> b. To characterize the molecular basis of adherence in strains of L. monocytogenes found to be strongly-adherent (in contrast with those showing weak adherence). <BR>c. To examine the virulence characteristics of strongly- vs. weakly-adherent strains of L. monocytogenes in tissue culture virulence assays.
<LI>To develop methods to eliminate pathogenic & spoilage organisms from RTE/raw meats, produce, processing environments, and processed foods: <BR>a. Using natural bacteriocins as biopreservatives. <BR>b. Using antimicrobial chemicals (i.e., liquid smoke extracts, acid) <BR>c. Using thermal process (surface pasteurization) in combination with chemicals d. Using electrolytically-generated hypochlorous acid.
NON-TECHNICAL SUMMARY: Food processing facilities are prone to contamination from raw materials. Some contamination is acquired during processing (i.e., during slaughter of animals) that may enter processing plants, become established as biofilms or in hard to clean areas, and possibly recontaminate finished, ready-to-eat products. Some products (produce) may be contaminated directly in the field and need special processing to render them safe for consumption. A. One purpose of the project is to identify and examine the application of new approaches to rapid detection of foodborne pathogens and toxins. Methods often designed for medical/clinical trials do not always work in the complex medium of foods to detect foodborne pathogens. Rapid detection is important for industry in reducing commercial 'test-and-hold' periods before food is shipped and important for consumers to quickly detect causes of illness. B. Another purpose of the project is to characterize unique features of foodborne pathogens. One such feature we have identified is the strong adherence of certain strains of Listeria monocytogenes to equipment surfaces in processing plants, attaching at 100,000-fold greater levels than weakly-adhering strains. We are interested in the molecular basis of adherence and whether this has any relationship to the adherence which is the first stage of intestinal infection by this pathogen. C. Lastly, another purpose is to examine various microbial interventions to prevent growth or inhibit the viability of pathogenic bacteria on raw/processed foods and in processing environments. <P>
APPROACH: Objective 1a/Approach. Paramagnetic particles (beads) will be used that allow recovery from food slurries using magnet capture devices. The magnetic beads will be coated with toxin-specific (Staphylococcal enterotoxin A, SEA) antibodies (i.e., anti-SEA). Recovery of such particles can be done manually with tubes placed in magnetic holders that fix magnetic particles along the sides of the tube while the non-essential materials are removed by aspiration. We currently have a BeadretrieverTm that is designed to accomplish this automatically. Once recovered, the toxin antigen will be detected with toxin-specific antibodies. A secondary antibody will carry a reporter molecule (i.e., DNA strand) that can provide enhanced detection by typical methods of DNA amplification.
Objective 1b/Approach. Incorporation of biotinylated primer or nucleotides that would allow magnetic bead separation using immobilized streptavidin on a magnetic support. The captured amplimer can then be used in end-point fluorescence assay with minimal fluorescence contributed by unutilized fluorogenic primers to give greater signal-to-noise ratios than currently observed.
Objective 2/Approach. We have collected data on the re-occurrence of strains isolated in meat processing facilities over the course of 1.5 yrs of sampling and collecting isolates of L. monocytogenes from 3 large commercial processing facilities. In addition, we have isolates of L. monocytogenes recovered from raw and processed meats over a 1-1.5 year period. These strains have been examined by our previously developed fluorescence adherence assay (Wright and Muriana, 2004a, 2004b) and many strains have been found to be strongly- or weakly-adherent to abiotic surfaces. Since cellular adherence is the first stage of infection with L. monocytogenes, we are interested to see whether the strong adherence observed with abiotic surfaces would also facilitate cellular adherence and aid virulence. The potential ramifications of this would be highly significant if the retention of strains in meat processing plants (due to their adherence) were to provide for retention of strains that are more virulent.
Objective 3/Approach. Bacteriocins (antimicrobial peptides) have been proposed as potential food preservatives because of their widespread production by food-grade lactic acid bacteria (LAB) used in the manufacture of cultured and fermented foods. Nisin, produced by Lactococcus lactis is currently well established as the only bacteriocin allowed as a direct food additive; other bacteriocins are used by virtue of the use of GRAS ingredients incorporating cultured or fermented milk or whey using bacteriocinogenic LAB cultures. We have identified a novel approach to screen bacteriocins into functional groups based on different resistance mechanisms and a PCR array system to quickly identify sequence information to determine the relatedness of new bacteriocins.
PROGRESS: 2007/10 TO 2008/09<BR>
OUTPUTS: Development of an Immunomagnetic PCR Signal Amplification (iPCR-SA) Assay for Sensitive Detection of Staphylococcus aureus Enterotoxins in Foods. Antibodies to staphylococcal enterotoxin A (anti-SEA) and enterotoxin B (anti-SEB) were coated onto the surface of paramagnetic beads as the capture agent. An oligonucleotide was then sequestered to secondary anti-SEA or anti-SEB antibodies using a bifunctional linker (SMCC) as the detection agent. The signal was then amplified by PCR amplification of an internal portion of the tethered oligonucleotide and visualized by SYBR green fluorescence chemistry during real-time PCR. The antibody-coated beads were used to capture staph enterotoxin spiked into various foods, after heating in foods, and from production by enterotoxigenic strains. The method was sensitive to as low as 7.5 femtograms SEA or SEB/ml and had 103-106 improvement in detection over commercial assays. A "PCR Primer Array" for Detection and Typing of Staphylococcus aureus Toxigenic strains. A PCR "primer array" was developed for all 17 staph enterotoxin gene sequences found in GenBank. Enterotoxigenic strains were obtained from ATCC cultures as well as isolates from humans, cattle (hides, utters, raw milk), and fermented apples. All Staphylococcus aureus strains isolated from various sources were assayed against the PCR array of primers by real-time PCR, indicating what toxins may be present. Sequence analysis of the amplimers allowed us to use (single) Toxin Locus Sequence Typing (TLST) in order to determine similarity or relatedness of the enterotoxigenic sequences and potentially, of the strains themselves. All S. aureus strains were confirmed by 16S rRNA PCR analysis. One strain had as many as 8 toxin genes, while others had 1 to 6, and still others had as few as none. <BR><BR>IMPACT: 2007/10 TO 2008/09<BR>
Staphylococcus aureus enterotoxins are the world's leading cause of foodborne intoxication. Staph enterotoxins are also heat stable and can remain active even if present before cooking. Therefore, improved, rapid, and highly-sensitive testing methods are important in helping to identify if staph enterotoxins are involved in a foodborne illness and what toxin(s) may have been involved. The methods employed here for detection demonstrate such high sensitivity that they may also be used for detection of other foodborne toxins as well as those that are considered potential biological warfare agents. The PCR Array is able to distinguish the specific toxin that might be present from the isolated strain(s) and sequence determination of the specified region is used to correlate with those of others to determine if there is similarity or identity to other toxins and/or toxin producing strains.