Progress 10/01/07 to 09/30/13
Outputs
Target Audience: Academic audience:Other researchers/PI's, faculty, educators,graduate students, post-docs. Industry audience: Meat processors, food processors,foodworkers. Others: Food safety conscious consumers, teachers, extension educators Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Work done at the Robert M. Kerr Food & Ag Products Center (i.e., FAPC) constantly involves food companies as most research is done in a manner addressing real world problems, whereby graduate students become aware of these problems and their research is directed in an attempt to address, correct, and solve those problems; opportunities exist for students to present their research findings locally at OSU research symposia and at national meetings. Problems are both practical (students get experience with how they affect food processors) as well as applied (students can apply molecular/biochemical methods to make food safe). Students are also encouraged to attend and participate in workshops (HACCP, Basic and Advanced; GFSI; and others); some students have been given the opportunity to present workshop seminars. Therefore, training is directly involved in applied research activities performed at FAPC and our students are desired by many food manufacturing companies in Oklahoma and elsewhere. How have the results been disseminated to communities of interest? Data has been presented by either professional symposia (OSU Research week presentation competitions, FAPC Research Symposium, etc), seminars, and data provided during general food microbiology presentations at HACCP and FDA Food Safety workshops. Several manuscripts are currently in preparation for submissionto researchjournals forpublication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Food safety is an important concept forthe health and well-being of consumers. In order to protect their health and safety, research is needed to establish methods to better detect pathogens and toxins in foods.Although some testing of foods is done after manufacture (prior to commercial distribution), notevery portion of food can be tested,and some food testing isalso performed after an outbreak/illness has occurred to help identify the responsible agent. However,you cannot test your way to safety (because not every bit of food can be tested),and therefore agreatcomplement to testing is to manufacture foods that are safe from growth of pathogenic microorganisms should they be (accidentally) present during manufacture by identifying antimicrobial interventions (antimicrobial processes and/or antimicrobialingredients) that can be applied, oradded, to foods to make them safe.This helps to reduce the likelihood of outbreaks and illness whenincidental pathogen contamination occurs that may escape detection when manufactured foods are tested. Therefore, theintention ofmy research program has been to play a hand in both detection methodsandintervention/prevention strategiesfor foodborne pathogens/toxins that could lead to illness. 1. To examine methods for rapid detection of microorganisms or toxins from food (completed previously; still working on final publications resulting from the research). 2. To characterize the surface-adherence trait in strains of L. monocytogenes isolated from food processing plants, raw meats, and ready-to-eat meats. Several extraction methods were examined to best extract proteins from the surface of Listeria monocytogenes, especially because the extractions would be submitted for analysis by mass spectrometry. Various extractions methods were determined to be better or worse, based on the extent of molecular protein species identified after extraction. Ultimately, a urea-based method was found acceptable for use in identifying protein species by Orbitrap Mass Spectrometry. We utilized the results of the proteins identified as confirmation of protein expression, comparing proteins extracted and identified from both strongly- and weakly-adherent strains, as well as comparison of proteins obtained from planktonic cells (free floating in solution) vs those that are adhered to surfaces (glass beads) in an attempt to identify the molecular basis for attachment of Listeria monocytogenes. Significant differences in protein levels extracted from the surface of L. monocytogenes when subjected to different conditions (attached vs unattached, strongly-adherent vs weakly-adherent) were viewed as potential protein targets contributing to attachment. These analyses lead to identify 'internalin' as involved with strongly-adherent strains. When the internalin gene from weakly adherent strains was examined by PCR amplification and sequence analysis, we found sequence deletions within the gene that could account for weakened attachment to surfaces. Internalin is a surface virulence protein that induces intestinal epithelial cells (non-professional phagocytic cells) to take in L. monocytogenes via receptor mediated endocytosis to begin an intracellular existence. We also used quantitative real-time PCR to examine expression of various protein-coding genes based on the differences in surface presence demonstrated by Orbitrap analysis from the various phenotypes and conditions examined. Hopefully, the identity of various proteins involved in surface adherence may provide additional information with which to eliminate L. monocytogenes as surface-adhering contaminants in food processing facilities. 3. To develop methods to eliminate pathogenic & spoilage organisms from RTE/raw meats, produce, processing environments, and processed foods: a) Antimicrobial interventions for raw meats (prior to being made into non-intact beef such as ground beef or mechanical tenderization). We completed work on evaluating anti-E. coli O157:H7 and anti-Salmonella bacteriophage as antimicrobial interventions on beef and fat trim for possible reduction of E. coli and Salmonella in ground beef. The bacteriophage were produced by a commercial manufacturer of bacteriophage preparations for the food industry.Our study of the effectiveness of bacteriophage preparationwas examinedusing 4 strains of E. coli O157:H7 that were susceptible to the E. coli bacteriophage (EcoShieldTm) and 5 strains/serotypes of Salmonella that were susceptible to the Salmonella bacteriophage (SalmoFreshTm). We examined the individual sensitivities of the test strains used to inoculate beef and fat and obtained efficiencies of plaqueing (EOP’s) to be very high, 0.8-0.9 (ideal = 1.0), even though we didn’t have the original strain’s upon which the bacteriophage cocktail was obtained (plaque counts of EcoShieldTm gave plaque forming units/pfu against each of our test strains in the order of 1010 pfu/ml, which is whatthe manufacturerhad indicated they were; however, plaque counts for SalmoFreshTm gave counts around 109 pfu/ml). Despite the plaque counts on each of the individual strains being at, or near, what the manufacturer of the bacteriophage preparations had indicated, we obtainedlow reductions against Salmonella (i.e., 0.2-0.4 log cfu/cm2). However, we obtained higher reductions against E. coli O157:H7 (i.e., reduction of ~1.0 log cfu/cm2 on beef and fat) that might allow this technology to develop food safety products for the beef industry. b) Application of bacteriocins from bacteriocinogenic (Bac+)strains of lactic acid bacteria (LAB)as biopreservatives for food. We have examined several hundred strains of Bac+ LAB for inhibition of mainlyListeria monocytogenes,but also against other pathogens, including Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, E. coli O157:H7, and Salmonella. Bac+ strains were also identified by 16S rRNA PCR amplificaiton and sequencing to provide definitive identification of the organism (data obtained with API identification of prior Bac+ strains proved to be incorrect as much as 50% of the time as many LAB were shown to be Enterococcus spp.). We have demonstrated that cell-free Bac+ preparations are able to inhibit L. monocytogenes when added to meat matrix of hotdogs prior to cooking, however, a greater effectiveness against L. monocytogenes can be achieved by added significantly lower amounts on the surface of hotdogs prior to packaging, not only reducing L. monocytogenes at the time of application, but preventing re-growth during >12 weeks of shelf life testing when held at 5oC.
Publications
- Type:
Websites
Status:
Other
Year Published:
2013
Citation:
Food Microbiology Lab - Facebook Page (topics are grouped under 'albums'): https://www.facebook.com/FAPCFoodMicroLab
- Type:
Other
Status:
Other
Year Published:
2013
Citation:
3. Tiong, H.K., and P.M. Muriana. 2013. Orbitrap mass spectrometry and qRT-PCR analysis of gene expression to identify the molecular basis of bacterial adherence in Listeria monocytogenes. FAPC Research Symposium/OSU Research Week, Feb. 19, Oklahoma State Univ., Stillwater, OK.
- Type:
Other
Status:
Other
Year Published:
2013
Citation:
4. Montalvo, C., and P.M. Muriana. 2013. Antimicrobial interventions for reducing E. coli 0157:H7 and Salmonella in Raw Meat. FAPC Research Symposium/OSU Research Week, Feb. 19, Oklahoma State Univ., Stillwater, OK.
- Type:
Other
Status:
Other
Year Published:
2013
Citation:
5. Vijayakumar, P., and P.M. Muriana. 2013. Bacteriocins of lactic acid bacteria as potential food biopreservatives: the effectiveness of a multiple mode-of-action approach. FAPC Research Symposium/OSU Research Week, Feb. 19, Oklahoma State Univ., Stillwater, OK.
- Type:
Other
Status:
Other
Year Published:
2013
Citation:
6. Adhikari, R., and P.M. Muriana. 2013. Screening of Bac+ Lactobacillus strains isolated in the lab for activity against E. coli O157:H7, Staphylococcus aureus and Salmonella species. FAPC Research Symposium/OSU Research Week, Feb. 19, Oklahoma State Univ., Stillwater, OK.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Muriana, P. 2013. Antimicrobial activity of bacteriocin-producing lactic acid bacteria. Presentation made to the Antimicrobial Group round-table at the OSU Veterinary School organized by Dr. Ken Clinkenbeard (Ken Clinkenbeard, Melanie Boileau, Glenn Zhang, John Gustafson, William Barrow, Christina Bourne, Phil Bourne, Jerry Malayer, and P. Muriana).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Muriana, P. 2013. Two presentations made at the FAPC Basic-HACCP Workshop, March 6-7, 2013.
- Type:
Websites
Status:
Published
Year Published:
2013
Citation:
SUNUP TV Program - "Food Whys" - GloGerm and Probiotics. An informative news segment for SunUp on the Glo-Germ kit that facilitates educational programs on handwashing.
Glo-Germ: http://www.youtube.com/watch?v=LAfCjQaQyCo
- Type:
Websites
Status:
Published
Year Published:
2013
Citation:
SUNUP TV Program - "Food Whys" - Probiotics. I did a segment for SunUp on Probiotics and Beneficial Bacteria.
http://www.youtube.com/watch?v=YuZqVFqr1fc&feature=share&list=UU8YmKQOMZdq5-X7u--snBXQ
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2013
Citation:
Christian Montalvo, M.S. (July, 2013). Evaluation of ozone, electrolyzed water, and bacteriophage as antimicrobial interventions for raw beef.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Consortium of Food Process Validation Experts (CFPVE: J. Dickson*, G. Acuff, M. Singh, C. Cutter, H. Thippareddi, R. Phebus, J. Luchansky, J. Sofos, J. Sindelar, M. Brashears, P. Muriana, and S. Ricke). 2013. Validation of antimicrobial interventions for small and very small processors: A how-to guide to develop and conduct validations. Food Prot. Trends. 33:95-104.
- Type:
Other
Status:
Other
Year Published:
2013
Citation:
1. Jagannathan, B., and P.M. Muriana. 2013. Optimizing conditions for bacteriocin production by lactic acid bacteria using MRS broth, whey, and non-fat dry milk. FAPC Research Symposium/OSU Research Week, Feb. 19, Oklahoma State Univ., Stillwater, OK.
- Type:
Other
Status:
Other
Year Published:
2013
Citation:
2. Henning, C., and P.M. Muriana. 2013. Phylogenetic analysis and screening of bacteriocins of lactic acid bacteria that are inhibitory to foodborne pathogens. FAPC Research Symposium/OSU Research Week, Feb. 19, Oklahoma State Univ., Stillwater, OK.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: 1. Evaluation of ozone as an antimicrobial intervention for mechanically-tenderized beef. Ozone is allowed for use in processing environments and on raw and processed meats. It is considered a green solution because it turns into water and oxygen. 2. Isolation and evaluation of antimicrobial bacteriocin-producing (Bac+) lactic acid bacteria (LAB) for inhibition of Listeria monocytogenes on ready-to-eat meats. 3. Advanced and Basic HACCP workshops, FAPC Food Research Symposium. PARTICIPANTS: a. Project director: Peter Muriana b. Graduate students: Preetty Pranatharthiharan, Paul Vijayakumar, Christian Montalvo, Chris Henning, Raj Adhikari, Badrinath , Lena Lopez, Sunita Macwana. c. Collaborator: Wayne Spillner, Christina DeWitt ; Jason Young & Jake Nelson (HACCP Workshops); Chuck Willoughby, Mandy Gross (FAPC Res. Symposium) d. Partner organization: Ross Industries, Del Ozone (equipment support). TARGET AUDIENCES: Meat processors, food processors, graduate students, faculty, food safety workers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1. Ozone. A Del Ozone generator (~6 ppm ozone; 2-3 gallons per minute; with digital ozone meter) was obtained and plumbed into the integrated spray system of a Ross Industries blade tenderizer machine. Ozone solutions as high as 6.5 ppm were not effective in generating significant microbial reductions (i.e., 0.2-0.3 log reduction) when applied to E. coli O157:H7-inoculated beef wafers. Approached to bypass the inboard tank reservoir and plumb directly to the spray nozzles were equally ineffective. The fine mist stainless steel nozzles were found to reduce ozone levels (i.e., de-gassing) and even when re-fitted with plastic course mist nozzles, ozone levels were significantly lower (3-4 ppm) than were produced by the ozone generator output (6.5 ppm). Efforts will be examined if ozone in combination with other antimicrobials could be an effective antimicrobial intervention. 2. Bacteriocins. Bacteriocin-producing (Bac+) lactic acid bacteria (LAB) were isolated from a number of retail food products (fruits, vegetables, raw meats, cheese) and examined for their ability to inhibit Listeria monocytogenes and several bacteriocin-resistant variants. The ability to inhibit specific bacteriocin-resistant variants of L. monocytogenes allows us to identify those with different mechanisms of action. The use of a cocktail mixture of bacteriocins of different modes of action provides synergistic antagonism against susceptible foodborne pathogens. We are currently evaluating the cultures and/or the bacteriocins in food applications as antimicrobial bioperservatives. 3. HACCP Workshops. Approximately 100 participants in Basic and Advanced HACCP workshops were trained in the essential tenants of HACCP and accredited with the Texas HACCP Alliance. 4. Annual FAPC Food Science Research Symposium. This is advertised to all Oklahoma companies, food workers, students, faculty and staff to attend. Keynote speaker was Dr. Roger Clemens (Pres., IFT) and consisted of research oral and poster presentations from many FAPC food researchers.
Publications
- Research papers: 1. Macwana, S., and P.M. Muriana. 2012. Spontaneous bacteriocin resistance as a susceptibility screen in Listeria monocytogenes for identifying cross-resistance and mode of action in bacteriocins from lactic acid bacteria. J. Microbiol. Meth. 88:7-13. 2. Macwana, S., and P.M. Muriana. 2012. A bacteriocin PCR array for identification of bacteriocin-related structural genes in lactic acid bacteria. J. Microbiol. Meth. 88:197-204.
- Lay articles: 1. Muriana, P.M., FAPC Research Symposium, FAPC Flash, January 2012. 2. Muriana, P.M.,Food Safety: Antimicrobial Interventions, FAPC.Biz Magazine, p.16-17, Spring/Summer, 2012. 3. Muriana, P.M., Food Safety: Unexpected Bugs, FAPC.Biz Magazine, p.16-17, Fall/Winter, 2012. 4. Muriana, P.M., Ensuring Food Safety, STATE Magazine, p.2-3, Spring, 2012.
- Presentations: 1. Muriana, P.M. 2012. Nevada Food Safety Task Force (invited speaker). Microbial issues with mechanically-tenderized beef. April 10-11, 2012. 2. Lopez, L.R., P.M. Muriana, C.A.M. DeWitt. 2012. Antibacterial activities of phenolic compounds isolated from grape pomace (Vitis aestivalis ) on E. coli O157:H7, S. aureus , and L. monocytogenes. Ann. Meet. Inst. Food Technol., Las Vegas, NV (June 25-28), Abst. # 03523. 3. Montalvo, C., and P. Muriana. 2012. Ozone solution and chemical antimicrobial interventions for reducing E. coli O157:H7 on raw meats prior to blade tenderization to prevent translocation in non-intact beef. FAPC Res. Symp., Feb. 22, Stillwater, OK. 4. Vijayakumar, P., Henning, C., and P.Muriana. 2012. Isolation and characterization of bacteriocin producing lactic acid bacteria from foods and their potential use as biopreservatives. FAPC Res. Symp., Feb. 22, Stillwater, OK. 5. Pranatharthiharan, P., and P. Muriana. 2012. Attachment of Listeria monocytogenes to surfaces is enhanced by the presence of nonviable cells remaining after treatment with Bi-Quat sanitizer. FAPC Res. Symp., Feb. 22, Stillwater, OK. 6. REYAP (June 27, 2012). Demonstration of blade tenderization issues and research for the REYAP group. 7. FAPC Industry Advisory Committee (May 25, 2012). Tour and discussion for new FAPC-IAC member, Roger Clements. 8. FAPC Industry Advisory Committee (June 7, 2012). Update on blade tenderization and bacteriocin antimicrobial research.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Title: Evaluation of antimicrobials that could be applied by spray treatment during blade tenderization to minimize risk from translocated E. coli O157:H7. Mechanically-tenderized beef is a less obvious form of 'non-intact' beef that can result in translocation of surface pathogens (i.e., E. coli O157:H7) into the interior portions which may not get killed during cooking because of consumer preference for rare or medium-rare cooked beef. The USDA considers non-intact beef a greater health risk than similar cuts of intact beef. We therefore examined whether antimicrobial spray interventions applied prior to blade tenderization could reduce surface populations of E. coli O157:H7 and result in reduced translocation to the interior of mechanically tenderized non-intact beef. We evaluated 14 antimicrobial chemicals applied to the surface of beef for their ability to reduce surface populations of E. coli O157:H7 and examined 7 of the best performing antimicrobials for ability to reduce internalization of E. coli O157:H7 when spray intervention was performed just prior to blade tenderization. Outputs have resulted in 3-4 presentations at national meetings involving the meat industry, several student dissertations, and a research manuscript is being prepared for submission. PARTICIPANTS: Individuals: Peter Muriana (co-PI); Brad Morgan (co-PI); Kalpana Kushwaha (post-doc); Jackie Eager (graduate student); Brent Wellings (graduate student). Partner organizations: Ross Industries (Midland, VA) Collaborators and contacts: Wayne Spillner (Ross Industries); Dennis Smithyman (Advanced Food Technologies, Shreveport, LA) TARGET AUDIENCES: Target audiences were meat industry personnel, academia, and USDA regulatory agency personnel. Presentations were given at multiple meat-industry conferences that were well attended with academics, meat industry workers, and USDA regulatory agency personnel. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Antimicrobials (n=14) were tested for effectiveness on inoculated lean beef surfaces within a Ross TC 700MC blade tenderizer (Ross Industries, Midland, VA) equipped with a Dosatron (Clearwater, FL) custom-built spray cabinet. Lean beef wafers (n=80) for each antimicrobial, which were fabricated from boneless top butt sirloins (IMPS #184), were subjected to spray treatment within this piece of equipment. Prior to treatment, samples were inoculated with 0.1 mL of 2 X 10*8 CFU/ml of E. coli O157:H7 cocktail (ATCC 43890, ATCC 43894, ATCC 43895, ATCC 35150). After processing samples were plated in order to achieve surface reduction effectiveness of each antimicrobial at 1 h, 1 d, 7 d, and 14 d post treatment. BeefXide was the most effective (P < 0.05) antimicrobial at 1 hr post processing. AvGard-Xp, AFTEC 3000, and Cytoguard Plus were the most effective (P < 0.05) antimicrobials at surface reduction after 1 day of vacuum-sealed, refrigerated storage (2*C). After 7 days of storage (2*C) under the same conditions AvGard-XP was the most effective (P < 0.05) at reduction of E. coli O157:H7, AvGard-XP remained the most effective (P < 0.05) antimicrobial tested after 14 days of storage. The effectiveness of 7 effective antimicrobial intervention strategies (AvGard-XP, Stabilized NA-Chlorite, Cytoguard Plus, Lactic Acid FCC 88%, AFTEC 3000, Citrilow, and HB2) were further examined to control E. coli O157:H7 in mechanically tenderized beef subprimals. Boneless top sirloin beef subprimals were inoculated on the lean side with 2 x 10*4 CFU/cm2 of a four-strain cocktail of E. coli O157:H7 and passed through a blade tenderizer. Inoculated subprimals that were water sprayed without tenderization served as the negative control and inoculated subprimals that were water sprayed with tenderization served as the positive control. Four core samples (20.25 cm2) were obtained from each subprimal and cut into four consecutive 2.54 cm sections: sections A and B comprised the top 5.08 cm and sections C and D comprised the deepest 5.08 cm. The recovered cores were passed through an Infrared Grill to further eliminate surface contamination. After aseptic separation into A, B, C, and D sections, each individual core section was blended at a volume of 2.5:1 with enrichment broth and incubated for 18 h at 37*C. After incubation 1 ml of the blended samples was extracted with E. coli specific immunomagnetic beads, allowing for selective recovery. The entire amount of recovered immunomagentic beads was plated onto Levine's eosin methylene blue agar containing Rifamycin. Presence of E. coli O157:H7 (positive or negative) was recorded for each individual core section (A-D). The presence of translocated E. coli O157:H7 correlated with the levels of reduction obtained with the surface intervention portion of the study. The positive controls (W+BT) consistently showed the highest microbial presence across sections. Similarly, the negative controls (W-BT) consistently showed the lowest microbial presence across sections.
Publications
- Morgan, J.B., J. Eager, B. Wellings, K. Kushwaha, P. Muriana, J. Nelson. 2010. Evaluation of 14 antimicrobials by spray treatment against a 4-strain cocktail of E. coli O157:H7-inoculated lean beef disks. AMSA, Reciprocal Meats Conference, Texas Tech University, Lubbock, TX.
- Morgan, J.B., J. Eager, B. Wellings, K. Kushwaha, P. Muriana, J. Nelson. 2010. Evaluation of 14 antimicrobials by spray treatment against a 4-strain cocktail of E. coli O157:H7-inoculated lean beef disks. North American Meat Processors Association conference, Chicago, IL.
- Spillner, W., P.M. Muriana, and D. Smithyman. Evaluation of 14 antimicrobial intervention solutions sprayed against E. coli O157:H7 inoculated onto beef (Phase 1) and of 7 solutions during blade tenderization (Phase 2). National Meat Association MEATXPO 2011, Las Vegas, NV, Feb. 14, 2011.
- Cerruto-Noya, C.A., C.L. Goad, B. Morgan, P.M. Muriana, and C.A. Mireles-DeWitt. 2011. Comparison of 1%, 2% and 3% ammonium hydroxide solutions to control Escherichia Coli O157:H7 on lean beef surfaces. J. Food Prot. (manuscript submitted).
- Eager, J., Morgan, J.B., Muriana, P., VanOverbeke, D., Hilton, G. Validation of various antimicrobial solutions on the microbial presence of E. coli O157:H7 on blade tenderized beef subprimals. MS thesis, Oklahoma State University, 2011.2011.1500199.
- Wellings, C.B., Morgan, B., Hilton, G., Muriana, P., VanOverbeke, D. Validation of various antimicrobial solutions on the reductions of surface microbial load of E. coli O157:H7 on lean beef. M.S. thesis, Oklahoma State University, 2011.2011.1495017.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Listeria monocytogenes is an important foodborne pathogen that is often present on raw meats and finds its way into processing plants during handling and processing of raw meat ingredients. Although there are other ways in which the pathogen can enter the food chain, this has been the main way it gets into plants and it has been a recurring problem in ready-to-eat (RTE) meat processing facilities. L. monocytogenes is capable of strong adherence to equipment and/or surfaces in meat processing facilities, resulting in the formation of biofilms. In prior experimentation, we developed a microplate fluorescence assay that was able to identify bacterial strains that adhered strongly to surfaces vs those that adhered weakly. We subsequently examined the strongly-adherent and weakly-adherent strains in tissue culture assays with Caco-2 cells and demonstrated that although both adhered equally well to tissue culture cells, those that were strongly-adherent to environmental surfaces (glass, stainless steel, plastic, rubber) were more invasive than weakly-adherent strains. We were interested in further examining and confirming our tissue culture results in live mouse assays. Select strains of L. monocytogenes isolated from raw and RTE meats were divided into weakly (CW34, CW35, CW72, and SM3) or strongly (CW50, 99-38, CW77, and CW62) adherent phenotypes based on our fluorescent microplate adherence assay. A/J mice were intragastrically inoculated with 10E8 CFU of L. monocytogenes and the invasiveness of these strains was evaluated by quantifying viable Listeria recovered from the spleen and liver of mice 3 days after inoculation. Bacterial counts recovered from the spleen were approximately 5 log CFU/gm for strongly adherent strains and ∼4 log CFU/gm for weakly adherent strains. Bacterial recovery was obtained from both spleen and liver of mice inoculated with strongly adherent strains, whereas bacteria were not recovered from the liver when using weakly adherent strains. Histopathological examination of liver tissue from mice inoculated with strongly adherent strains showed very large and effacing lesions (range 30-400 μm) when compared with lesions observed with weakly adherent strains which exhibited discrete non-effacing lesions (range 30-80 μm). These observations suggest that strongly adherent strains may possess factors that enhance their invasiveness compared to weakly adherent strains. The elimination of L. monocytogenes from the food processing environment is vital as strongly adherent strains may not only persist and present a greater risk of food contamination, but perhaps also a greater degree of invasiveness. PARTICIPANTS: Graduate students are listed as co-authors on the publications. We have collaborated with local and national meat processing industries with several of our projects with Listeria monocytogenes. TARGET AUDIENCES: Presentations were made during our local (Food Safety RoundUp web-based round table series), FAPC Research Symposium, IFT Annual Meeting poster/oral sessions, invited presentations, and in discussions with individual meat processors. In all, presentations were made to at least 400-500 (estimate)individuals in attendance at these various sessions. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Listeria monocytogenes has been a persistent contamination in meat processing plants, especially for ready-to-eat (RTE) meat plants. We believe one of the reasons for the difficulty in eliminating L. monocytogenes is that it is capable of adhering strongly to various surfaces in processing plants, making them difficult to eradicate. Since adherence to biological cells in the intestine is the first stage of human infection, we wanted to determine if the adherence properties observed to processing surfaces reflect their ability to adhere and infect biological cells. Our studies with cell culture (in vitro) and live mouse (in vivo) assays indicate that the strongly- and weakly-adherent strains adhere similarly to tissue culture cells. However, the strongly adherent strains were shown to be more invasive than weakly adherent strains, presenting a greater significance to strong adherence aside from allowing persistence in processing plants, they are more likely to contaminate food products because of their adherence, and they may be more invasive as well. We are continuing our vigilance to examine the molecular mechanism and basis for this adherence and to use practical solutions to eliminate Listeria from plant processing environments. These findings have been reported at annual meetings, invited symposia, and local presentations on food safety to the meat processing industry.
Publications
- Kushwaha, K., and P.M. Muriana. 2010. Analysis of tissue invasiveness of adherent strains of Listeria monocytogenes by in vivo mouse assay. Intl. J. Food Microbiol. 141:104-109.
- Kushwaha, K., and P.M. Muriana. 2010. Comparison of invasiveness among surface-adherent variants of Listeria monocytogenes in Caco-2 cell culture assays. Intl. J. Food Microbiol. 138:166-171.
- Muriana, P.M. 2010. Pathogen Patrol: Food recalls and the reportable food registry. FAPC.biz Magazine, Vol. 5(1/2):16-17.
- Muriana, P.M. 2010. Pathogen Patrol: Which came first, the salmonella or the egg Vol. 5(3/4):16-17.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Adherence Characteristics of Listeria Strains Isolated from Three RTE Meat Processing Plants. A microplate fluorescence assay developed was used to screen strains isolated from meat processing plants. Over 1560 non-food contact surface swabs and raw meat samples were collected from 3 RTE meat processing plants (520 from each plant) from 1998 to 1999, resulting in the recovery of 259 isolates of Listeria obtained from postprocess areas including drains, floors, garbage bins, cart wheels, walls, equipment surfaces, tables, brooms, pallet jacks, hoses, ladders, and waste chutes. We examined 246 of 259 isolates for adherence phenotype and used PCR to identity L. monocytogenes. Adherence was classified as weak, medium, or strong depending on results obtained with all Listeria isolates using a fluorescent microplate adherence assay. Among the 246 isolates, there were 61 weak, 148 medium, and 37 strongly adherent Listeria of which 130 (53%) were found to be L. monocytogenes. In plant C, we recovered 164 Listeria-positive samples (32% isolation rate) that included 80 L. monocytogenes-positive samples (49% of Listeria spp.), 52 of which were moderately adherent as well as all 9 strongly adherent isolates of L. monocytogenes obtained in this study. Adherence properties of Listeria may allow persistence and recurrence in plant environments, potentially increasing the chance of eventual product contamination. Comparison of invasiveness among surface-adherent variants of Listeria monocytogenes in Caco-2 cell culture assays. Isolates of raw meats, RTE meats, and environmental samples from RTE meat processing facilities that demonstrated differences in adherence to abiotic surfaces were tested for cellular adherence and invasion in the Caco-2 human cell line. Strains of L. monocytogenes were classified into strong (CW50, 99-38, CW77, SM5 and CW62) and weakly adherent (CW34, CW35, CW72, SM3, J7 and J126) based on a microplate adherence assay using the fluorescing substrate, 5,6-CFDA. These strains were tested for adherence and invasion in cell culture assays with Caco-2 cells. At long incubation time (2 hrs) and high multiplicity of infection (MOI, 100:1) we observed equivalent cellular adherence and invasiveness. After optimizing conditions for time of infection (15-, 30-, 60-, 90-, 120-min) and MOI (100:1, 10:1, 1:1, 0.1:1), we found that under the conditions of 15-min infection time at an MOI of 10:1 (Listeria:Caco-2 cells), we again observed equivalent cellular adherence by the two distinct surface-adherent groups, but we only recovered invading Listeria from the group that was strongly adherent to environmental surfaces. The data indicate that a surface factor that provides for strong environmental surface adherence may also be involved with internal cellular virulence and survival and implicates strongly adherent strains as possibly being of greater invasiveness than weakly adherent strains. PARTICIPANTS: Graduate students are listed as co-authors on the publications. We have collaborated with local and national meat processing industries with several of our projects with Listeria monocytogenes. TARGET AUDIENCES: Presentations were made during our local (Food Safety RoundUp web-based round table series), IFT Annual Meeting poster/oral sessions, invited symposium presentations (Toronto, Canada; Santiago, Chile), and discussions with individual meat processors. In all, presentations were made to at least 350-400 (estimate) individuals in attendance. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Listeria monocytogenes has been a persistent contamination problem for ready-to-eat (RTE) meats. One of the reasons is that L. monocytogenes is capable of adhering strongly to various surfaces in processing plants, making them difficult to eliminate. Since adherence to biological cells in the intestine is the first stage of human infection, we wanted to determine if the adherence properties observed to processing surfaces reflect their ability to adhere and infect biological cells. Our studies with cell culture (in vitro) and live mouse (in vivo) assays indicate that the strongly- and weakly-adherent strains adhere similarly to tissue culture cells, but strongly adherent strains are more invasive than weakly adherent strains, presenting a greater significance to strong adherence than simply the ability to persist in processing plants. Additional testing will be targeted at both applied approaches to reduce their presence in processing plants as well as using microarrays to understand the molecular nature for their adherent to abiotic surfaces. These findings have been reported at annual meetings, invited symposia, and local presentations on food safety to the meat processing industry.
Publications
- Kushwaha, K., and P.M. Muriana. 2009. In vitro and in vivo virulence analysis of strong- and weakly-adherent strains of Listeria monocytogenes. Ann. Meet. Inst. Food Technol., Anaheim, CA (June 6-9), Abst. # 011-05.
- P.M. Muriana. 2009. Pathogen Patrol:Salmonella in peanut butter, part 2. FAPC.biz Magazine, Vol. 4(1):10-11.
- P.M. Muriana. 2009. Pathogen Patrol: Bacterial strains: Microbial matchmaking. FAPC.biz Magazine, Vol. 4(2):10-11.
- P.M. Muriana. 2009. Pathogen Patrol: Enterobacter sakazakii (Cronobacter) and infant health. FAPC.biz Magazine, Vol. 4(3):10-11.
- Kushwaha, K., and P.M. Muriana. 2009. Adherence characteristics of Listeria strains isolated from 3 ready-to-eat meat processing plants. J. Food. Prot. 72:2125-2131.
- Rank, D., M.A. Saeed, and P.M. Muriana. 2009. Cloning of the Salmonella enteric Serovar Enteritidis fimbrial protein SefA as a surface protein in E. coli confers attachment to eukaryotic cell lines. Appl. Environ. Microbiol. 75:6622-6625.
- Panneerseelan, L., and P.M. Muriana. 2009. An immunomagnetic PCR signal amplification (iPCR-SA) assay for sensitive detection of Staphylococcus aureus enterotoxins in foods. J. Food Prot. 72: 2538-2546.
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Progress 10/01/07 to 09/30/08
Outputs
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. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
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.
Publications
- Panneerseelan, L., and P.M. Muriana. 2008. Supersensitive detection of Staphylococcus aureus enterotoxins by immunomagnetic recovery and signal amplification by real-time PCR. Ann. Meet. Inst. Food Technol., New Orleans, LA (June 29-July 2), Abst. #010-37.
- Kushwaha, K., and P.M. Muriana. 2008. Molecular typing of adherent phenotypes within Listeria monocytogenes by MLST, PFGE and ribotyping. Ann. Meet. Inst. Food Technol., New Orleans, LA (June 29-July 2), Abst. #215-07.
- Leenalitha Panneerseelan. 2008. Detection of Staphylococcus aureus enterotoxins and enterotoxins producing strains (Ph.D., Dec. 2008).
- Shawnna R. Veasey. 2008. Efficacy of electrolytically generated hypochlorous acid (electrolyzed water) on fresh and processed meats(M.S., Dec. 2008).
- P.M. Muriana. 2008. Pathogen Patrol: FDA proposes new policy for Listeria monocytogenes. FAPC.biz Magazine, Vol. 3(1):10-11.
- P.M. Muriana. 2008. Pathogen Patrol: Toxins associated with chemical foodborne illnesses. FAPC.biz Magazine, Vol. 3(2):10-11.
- P.M. Muriana. 2008. Pathogen Patrol: You say tomato, I say Serrano (Salmonella). FAPC.biz Magazine, Vol. 3(3): 10-11.
- P.M. Muriana. 2008. Pathogen Patrol: Gone Viral ! FAPC.biz Magazine, Vol. 3 (4):10-11.
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