Progress 09/01/07 to 08/31/11
Outputs
OUTPUTS: Activities: most of the efforts of this project have been conducting laboratory-based experimental research intended to assess the use of neutral electrochemically activated (NECA) water and bacteriophages against pure cultures of food-borne pathogenic bacteria. The NECA experiments have compared different technologies of electrolyzed water solutions evaluating their effect against pure cultures and biofilms of Listeria monocytogenes, Salmonella and Escherichia coli O157:H7. The bacteriophage experiments have involved the isolation, screening and selection of bacteriophages against specific strains of the same pathogenic bacteria. The bacteriophages specific against L. monoctoygenes have been tested for their efficacy in killing many strains and the effectiveness of a mixture of 6 phages has been tested in different food matrices. In addition to the experimental work, manuscript preparation and presentation at scientific meetings has also been conducted. One Ph. D. student was guided and mentored and three undergraduate students were taught experimental research techniques. Events: researchers attended the International Association for Food Protection Annual Meeting in Milwaukee, WI, to present the results of the bacteriophage treatment against Listeria. Products: research yield a collection of bacteriophages against Listeria referred as LP1 and it is currently being evaluated for patenting. Hongshun Yang successfully passed his Ph. D. oral preliminary exam. Dissemination: one poster was presented at by Dr. Luna Akhtar at the IAFP meeting on the Listeria phages. Researchers discussed their findings with audiences as well as distributed copies of the poster to attendees. PARTICIPANTS: PI: Dr. Francisco Diez-Gonzalez; Co-PI: Dr. Joellen Feirtag; Graduate Research Assistant: Hongshun Yang; Post-doctoral Research Associate: Dr. Mastura Akhtar, Undergraduate students: Phillip Kraemer, Grace Nelson and Kyle Christensen. Collaborators: Dr. Todd Callaway, ARS/USDA, Drs. Andy Brabban, Betty Cutter, Evergreen State University, Dr. Larry Goodridge, Colorado State University, and Dr. Martin Loessner, Switzerland. Mike Magee, Wastewater Treatment Facility, Rice Lake, WI; Scott Joseph and Angella Craft-Reardon, Seneca Wastewater Treatment Plant, Eagan, MN contributed by providing sewage samples. TARGET AUDIENCES: Organic producers and processors, food safety scientists, as well as food producers interested in natural alternatives to artificial sanitizers. PROJECT MODIFICATIONS: The efforts intended to develop other bacteriophages in addition to Salmonella, E. coli O157 and L. monocytogenes was not pursued given the complexity of identifying effective types. The work on NECA water has been focused on studying the mechanism of inactivation, given the lack of success in other approaches and on extension efforts.
Impacts
Change in knowledge: 1) NECA water project: A few simpler antimicrobial technologies that are also based on electrolysis or ozonation have been recently marketed by their manufacturers claimed to have sanitizing properties for controlling pathogens. The objective of this study was to determine the sanitizing effect of solutions from some of these commercial technologies (Ionator, Salt Ionator and Lotus) on Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella and compare them with a non-portable ECAW. The results showed that 100 mg/L ECAW generated from STEL 80 ECT US had sanitizing effects of at least 5 log CFU/ml reductions on liquid culture and more than 4 log CFU/coupon reductions for E. coli O157:H7, L. monocytogenes and Salmonella dried on stainless steel surface, respectively. No bacterial cells were detected by direct plate counting post-ECAW treatment. In contrast, the other commercial technologies tested were not effective in sanitizing. These results would be helpful for guiding consumers when choosing a right sanitization to ensure food safety. 2) Bacteriophages against Listeria monocytogenes: Phage treatments were most effective at 10C and 4C in TSB, as viable cells were not detected (<1 log CFU/mL) after 24 h at 100 and 1000 MOI. Phage cocktail completely lysed L. monocytogenes cells at 1000 MOI after 1 h compared with controls. Phage cocktail, LP1 lysed L. monocytogenes mix cultures (N=5) in liquid medium (TSB) at different temperaures, 4 , 10 and 30C. LP1 had optimum lytic effect at MOI (rate of PFU of phage to CFU of bacteria) of 1,000 at all experimental incubation temperatures, and no viable cells of L. monocytogenes were detected after 1 h of treatment. TEM photographs indicated the LP1 phages belonged to Order Caudovirales, Family Myoviridae. Treatment of milk samples inoculated with mixtures of L. monocytogenes strains with LP1 was able to reduce the count to undetectable levels when incubated at 4C in less than 2 days. Similar results were observed in ham and turkey meats. These findings suggest that LP1 can be a promising alternative to control this pathogenic bacterium in ready-to-eat foods. 3) Bacteriophages against E. coli O157:H7: The objective of this study was to determine the effect of a previously characterized collection of bacteriophages, BEC8, on the inactivation kinetics of a mixture of EHEC O157:H7 strains applied on surfaces of materials commonly found in food processing plants. D-values on any surface material ranged from 3.9 min at 37C to 46.7 min at 12C. Z-values calculated for stainless steel, ceramic tile, and high-density polyethylene resulted in 26.1, 23.7, and 26.7C, respectively for dry cells and 23.2, 23.7, and 24.5C, respectively for liquid cells. There was a significant difference on the effect the phage cocktail had on the E. coli O157:H7 mixture between the two lower temperatures (12 and 23C) vs. the two higher temperatures (30 and 37C). These results indicated that bacteriophage cocktails can be used as an effective antimicrobial against E. coli O157:H7 present in dry or liquid form on hard surfaces.
Publications
- Viazis, S., M. Akhtar, A. Brabban, J. Feirtag, and F. Diez-Gonzalez. 2011. Isolation and host range characterization of lytic bacteriophages against enterohemorrhagic Escherichia coli. J. Appl. Microbiol. 110:1323-1331.
- Viazis, S., M. Akhtar, J. Feirtag, and F. Diez-Gonzalez. 2011. Reduction of Escherichia coli O157:H7 viability on hard surfaces by treatment with a bacteriophage mixture. Intl. J. Food Microbiol. 145: 37-42.
- Viazis, S., M. Akhtar, J. Feirtag, and F. Diez-Gonzalez. 2011. Reduction of Escherichia coli O157:H7 viability on leafy green vegetables by treatment with a bacteriophage mixture and trans-cinnamaldehyde. Food Microbiol. 28: 149-157.
- Viazis, S., and F. Diez-Gonzalez. 2011. Enterohemorrhagic Escherichia coli: The 20th. centurys emerging foodborne pathogen. A Review. Adv. Agron. 111: 1-50.
- Posters: Viazis, S., T. P. Labuza, F. Diez-Gonzalez. 2011. Inactivation kinetics of Escherichia coli O157:H7 on hard surfaces by use of a bacteriophage mixture. IAFP Annual Meeting, Sept. 31-August 3, Milwaukee, WI.
- Akhtar, M., S. Viazis, K. Christensen, P. Kraemer and F. Diez-Gonzalez, 2011. Isolation of virulent bacteriophages for bio-control of Listeria monocytogenes. IAFP Annual Meeting, Sept. 31-August 3, Milwaukee, WI.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Activities: most of the efforts of this project have been conducting laboratory-based experimental research intended to assess the use of neutral electrochemically activated (NECA) water and bacteriophages against pure cultures of food-borne pathogenic bacteria. The NECA experiments have evaluated different concentrations of free chlorine against biofilms of Listeria monocytogenes, Salmonella and Escherichia coli O157:H7. The bacteriophage experiments have involved the isolation, screening and selection of bacteriophages against specific strains of the same pathogenic bacteria. The bacteriophages specific against E. coli O157:H7 have been tested for their efficacy in killing bacteria on specific application conditions that involved solid surfaces typical of food processing equipment and vegetable (lettuce and spinach) leaves. In addition to the experimental work, other activities involved manuscript preparation and presentation at scientific meetings. Two Ph. D. students were guided and mentored and two undergraduate students were taught experimental research techniques. Events: researchers attended the American Society for Microbiology Annual Meeting in San Diego, CA to present the work on E. coli O157 and Salmonella bacteriophages, and the International Association for Food Protection Annual Meeting in Anaheim, CA, to present the NECA water treatment findings. Products: research yield a collection of bacteriophages (BEC8) that show promising results against E. coli O157 and Salmonella bacteriophages. Stelios Viazis, a Ph. D. candidate defended his dissertation and graduated in July. Dissemination: two posters were presented at the ASM meeting, one by Dr. Mastura Akhtar on the selection of Salmonella bacteriophages and another one by Stelios Viazis on the application of E. coli O157 phages on solid surfaces. One poster was presented by Hongshun Yang at the IAFP meeting on the NECA water project. At both meetings, researchers discussed their findings with audiences as well as distributed copies of the posters to attendees. PARTICIPANTS: PI: Dr. Francisco Diez-Gonzalez; Co-PI: Dr. Joellen Feirtag; Graduate Research Assistants: Stelios Viazis and Hongshun Yang; Post-doctoral Research Associate: Dr. Mastura Akhtar, Undergraduate students: Phillip Kraemer and Kyle Christensen. The members of the advisory panel are: Jim Riddle, an organic policy consultant and former Chair of NOSB; Emily Brown Rosen, Policy Director of Pennsylvania Certified Organic and consultant for Organic Research Associates; Dr. Carl Rosen, Professor and Extension Soil Scientist at the University of Minnesota; and Dr. Jennifer Ryder Fox, Dean of the College of Agriculture of the California State University-Chico. Collaborators: Dr. Todd Callaway, ARS/USDA, Drs. Andy Brabban, Betty Cutter, Evergreen State University, Dr. Larry Goodridge, Colorado State University, and Dr. Martin Loessner, Switzerland. Mike Magee, Wastewater Treatment Facility, Rice Lake, WI; Scott Joseph and Angella Craft-Reardon, Seneca Wastewater Treatment Plant, Eagan, MN contributed by providing sewage samples. TARGET AUDIENCES: Organic producers and processors, food safety scientists, as well as food producers interested in natural alternatives to artificial sanitizers. PROJECT MODIFICATIONS: The efforts intended to develop other bacteriophages in addition to Salmonella, E. coli O157 and L. monocytogenes will not be pursued given the complexity of identifying effective types. The work on NECA water has been focused on studying the mechanism of inactivation, given the lack of success in other approaches and on extension efforts.
Impacts
Change in knowledge: 1) NECA water project: the culture viability of 34 strains of Listeria monocytogenes, Salmonella and Escherichia coli O157:H7 on coupon surfaces was reduced more than 4 log CFU by the treatment of NECAW. NECAW treatment of biofilms reduced bacterial counts by 1 to 5 log CFU per coupon with an average of 2.4. S. Newport B4442CDC and E. coli O157:H7 ATCC 43895 were the most resistant strains to NECAW for surfaces and biofilms, respectively. The biofilm structures and microbial cells on stainless steel (SS) were destroyed by the treatment with NECAW. Different species and strains of foodborne pathogens had variable sensitivity to NECAW. NECAW could be effective in controlling surface contamination with pathogenic bacteria and biofilm growth. 2) Bacteriophages against E. coli O157:H7: At 37C and 12C on SS chips, no culture survivors were detected (detection limit 10 CFU/chip) after treatment with a bacteriophage cocktail (BEC8) at ratios of viral to bacterial counts of 100 after 10 min and at 23C after 1 h on SS chips. These results indicated that the phage cocktail was effective within an hour against low levels of the EHEC mixture at above room temperature on solid surfaces. Spinach and lettuce leaves treated with BEC8 and trans-cinnamaldehide (TC) individually at low inoculum levels after 24 h at 23 and 37C had no detectable viable cells. When the EHEC inoculum size increased and/or incubation temperature decreased, the efficacy of BEC8 and TC decreased. However, when the two treatments were combined, no survivors were detected after 10 min at all temperatures and inoculum levels on both leafy greens. These results indicated that the BEC8/TC combination was highly effective against EHEC on both leafy greens. This combination could potentially be used as an antimicrobial to inactivate EHEC O157:H7 and reduce their incidence in the food chain. 3) Bacteriophages against Salmonella: A total of 51 phages were isolated , and fifteen phages were purified and selected for further analysis. Phages lysed three different Salmonella Typhimurium strains with high efficiency of plaquing values (almost 1.0). Four phages cross infected serovars. Results of positive spot testing had broad specificity against different serovars. Phage SEA1 infected most Typhimurium, Newport, and Enteritidis strains, and a Tennessee and a Saintpaul serovars. Treatment of tomato surfaces with SEA1 phages previously inoculated with S. Enteritidis caused a reduction of 2.4 logs CFU/g at 23C after 2 h incubation. Phage genome sizes ranged from 45 to 190 kb. These findings suggested that phages can serve as an effective and natural control strategy to reduce the incidence of foodborne Salmonella. 4) Bacteriophages against Listeria monocytogenes: A total of 37 phages were isolated against Listeria monocytogenes from sewage samples. The most efficient phages were tested for different serotypes of L. monocytogenes (N=24). Based on the spot test and EOP data, ten phages had higher efficiency and broader specificity against the tested strains of serotypes 4a, 4b, 3a, 3b, 1/2a, 1/2b, 1/2c; interestingly, none of the isolated phages were effective against 3c serotype.
Publications
- Abstracts: Yang, H., Feirtag, J. and Diez-Gonzalez, F. 2010. Effect of neutral electrochemically activated water on the viability and biofilms of foodborne pathogens on stainless steel surfaces. IAFP Annual Meeting, August 1-4, Anaheim, CA.
- Viazis, S., Akhtar, M., Feirtag, J. and Diez-Gonzalez, F. 2010. Reduction of Escherichia coli O157:H7 viability on hard surfaces by treatment with a bacteriophage mixture. ASM Annual Meeting, May 23-27, San Diego, CA.
- Akhtar, M., Viazis, S. Thompson, H., Erbach, C., Feirtag, J. and Diez-Gonzalez, F. 2010. Efficacy of bacteriophages for biocontrol of Salmonella serovars. ASM Annual Meeting, May 23-27, San Diego, CA.
- Dissertation: Viazis, S. 2010. Control of enterohemorrhagic Escherichia coli using bacteriophages. Ph.D., University of Minnesota. 239 pp.
- Peer-review papers: Viazis, S., Akhtar, M., Feirtag, J. and Diez-Gonzalez, F. 2010. Reduction of Escherichia coli O157:H7 viability on hard surfaces by treatment with a bacteriophage mixture. Intl. J. Food Microbiol. 10.1016/j.ijfoodmicro.2010.11.021.
- Viazis, S., Akhtar, M., Feirtag, J. and Diez-Gonzalez, F. 2010. Reduction of Escherichia coli O157:H7 viability on leafy green vegetables by treatment with a bacteriophage mixture and trans-cinnamaldehyde. Food Microbiol. doi:10.1016/j.fm.2010.09.009.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: The overall goal of this project is the development of sanitizers that can be used in organic food production. Specifically we are exploring the use of electrochemically activated (ECA) water and bacteriophages capable of inhibiting foodborne pathogens. We have continued working with our collaborators as well as with our advisory panel. We have been able to receive samples from different companies including Bix, Inc., Seward Coop., as well as sewage samples from different cheese companies and water processing plants to isolate bacteriophages. An expert in Listeria phages has been providing valuable input to obtain these type of phages. The first findings obtained from this project were presented at the 2009 annual meeting of the International Association for Food Protection. PARTICIPANTS: PI: Dr. Francisco Diez-Gonzalez; Co-PI: Dr. Joellen Feirtag; Junior Scientist/Technician: Leena Griffith; Graduate Research Assistants: Stelios Viazis and Hongshun Yang; Post-doctoral Research Associate: Dr. Mastura Akhtar. The members of the advisory panel are: Jim Riddle, an organic policy consultant and former Chair of NOSB; Emily Brown Rosen, Policy Director of Pennsylvania Certified Organic and consultant for Organic Research Associates; Dr. Carl Rosen, Professor and Extension Soil Scientist at the University of Minnesota; and Dr. Jennifer Ryder Fox, Dean of the College of Agriculture of the California State University-Chico. Collaborators: Dr. Todd Callaway, ARS/USDA, Drs. Andy Brabban, Betty Cutter, Evergreen State University, Dr. Larry Goodridge, Colorado State University, and Dr. Martin Loessner, Switzerland. TARGET AUDIENCES: Organic producers and processors as well as food producers interested in natural alternatives to artificial sanitizers. PROJECT MODIFICATIONS: The extension efforts have been delayed because of several reasons: the change of supplier of ECA water technology, the departure of the technician that had been working on the project and the inconsistency of effectiveness to be able to effectively deploy the technology. As a replacement for the sodium carbonate-based ECA water, a number of other alternatives have been evaluated.
Impacts
CHANGE IN KNOWLEDGE: 1) ECA water research: A variety of commercially available brands of electrolyzed water were evaluated for their effectiveness against pathogenic microorganisms. In experiments that assess their antimicrobial effect in liquid culture, as well as on surfaces, the only electrolyzed water capable of causing significant viable count reductions was electrochemically activated water with 50 ppm free chlorine. However, in pure culture, some strains of Salmonella and E. coli O157:H7 were not completely killed and residual populations remained after 30 s of exposure. 2) Bacteriophage research: Specific objectives included isolation, identification, characterization and evaluation of phages for controlling Salmonella Typhimurium and Escherichia coli O157:H7. The bacteriophage isolation method included enrichment, spot test, filtration, plaque assays, purification and amplification. Salmonella Typhimurium strains were used as host strains in the isolation process. Isolated phages were tested against pure cultures of different Salmonella serovars. The efficiency of plaquing (EOP) method was used to measure lytic activity of phages. Transmission electron microscopy (TEM) was used to observe phage morphology. From twenty four manure samples, a total of nineteen extracts had some lytic activity against Salmonella and ten phages were further purified. Phages previously isolated were provided. Additional phages were isolated from dairy and feedlot manure using EHEC O157, O26 and O111 strains as hosts. Manure was enriched using tryptic soy (TS) broth and exponentially growing cultures of specific bacterial hosts incubated overnight at 37C. The enriched extracts were centrifuged, filtered, combined with the host strain in tryptone top agar and plated on TS agar. Plaques were purified and screened against additional strains (14, O157; 10, O26; 10, O111) using the efficiency of plaquing method (EOP). Those phages were capable of lysing three different Salmonella Typhimurium strains including one multidrug resistant isolate with high EOP values (range: 0.05 to 1.0). None of the phages were active against four Salmonella Newport, one S. Montevideo, and two S. Enteritidis. The phages showed very specific lytic activity against Salmonella Typhimurium strains. Phage CEV2 and seven other phages previously isolated were able to lyse all 14 O157 strains with EOP values consistently above 0.001. Four out of six phages isolated from a cattle feedlot were effective against all O157 strains and one O26 strain with EOP values greater than 0.001. Based on TEM, some of the phages were classified as Group 1 Caudovirales. These results indicated that the isolated bacteriophages were highly effective against multiple strains of two EHEC serotypes. This collection of phages can be grouped and potentially used as an antimicrobial cocktail to inactivate Salmonella and E. coli O157 in the food processing environments and on fresh produce. These findings suggest that the use of bacteriophages could be a viable antimicrobial alternative to control foodborne pathogens in the organic food processing environment.
Publications
- Diez-Gonzalez, F. and Mukherjee, A. 2009. Produce safety in organic vs. conventional crops. In: X. Fan, B. A. Niemira, C. J. Doona, F. E. Feeherry, and R. B. Gravani (eds.), Microbial Safety of Fresh Produce: Challenges, Perspectives and Strategies, Institute of Food Techonologist Press-Wiley Blackwell, Hoboken, N. J.
- Akhtar, M., Viazis, S., Feirtag, J. and Diez-Gonzalez, F. 2009. Isolation and identification of bacteriophages against Salmonella Typhimurium, International Association of Food Protection annual meeting, July 12-15, Dallas, TX.
- Viazis, S., Akhtar, M., Feirtag, J. and Diez-Gonzalez, F. 2009. Isolation and characterization of lytic bacteriophages against enterohemorrhagic Escherichia coli. International Association of Food Protection annual meeting, July 12-15, Dallas, TX.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: 1) Hiring The single most important activity conducted during the first year of the project was to fill the positions of researchers who would be conducting most of the work. 2) Advisory panel meetings On January 29th, the first meeting with our advisory panel was held in St. Paul, MN. This meeting served as the kick-off event of the project in which we informed the panel about the progress, changes in the activities schedule as well as plans for the rest of the grant. Both PI and Co-PI as well as most of the research and extension team participated in this meeting. On November 14th, 2008, a conference call/webcast was conducted with the same advisory panel to discuss the latest results and outcomes of the project. We received excellent feedback from the panel members at both of these events and we look forward to continue interacting and collaborating with them. 3) Interactions with collaborators have been very supportive of our efforts in developing our expertise in phage techniques and obtaining initial phage stocks. A new company, IET, Inc., that manufactures electrochemically activated (ECA) water generators has agreed to provide expertise and facilitate the use of their units. 4) Extension efforts The application of water has been tested in three of our industry collaborators facilities for the disinfection of fresh vegetables. These companies include Bix Produce, Inc., Seward Coop and SuperMom's Commissary. A group of organic farmers has been selected in order to identify testing sites for ECA water application. PARTICIPANTS: Researchers: PI: Dr. Francisco Diez-Gonzalez; Co-PI: Dr. Joellen Feirtag Junior Scientist/Technician: Leena Griffitth; Graduate Research Assistants: Stelios Viazis and Hongshun Yang; Post-doctoral Research Associate: Dr. Mastura Akhtar. The members of the advisory panel are: Jim Riddle, an organic policy consultant and former Chair of NOSB; Emily Brown Rosen, Policy Director of Pennsylvania Certified Organic and consultant for Organic Research Associates; Dr. Carl Rosen, Professor and Extension Soil Scientist at the University of Minnesota; and Dr. Jennifer Ryder Fox, Dean of the College of Agriculture of the California State University-Chico. Collaborators: Dr. Todd Callaway, ARS/USDA, Drs. Andy Brabban, Betty Cutter, Evergreen State University and Dr. Larry Goodridge, Colorado State University. TARGET AUDIENCES: Organic producers and processors as well as food producers interested in natural alternatives to artificial sanitizers. PROJECT MODIFICATIONS: In addition to exploring the use of ECA water and bacteriophages as potential organic sanitizers we will also investigate the utilization of other natural approaches such as chitosan and citrus extracts. We will be discontinuing exploring the use of sodium carbonate as antimicrobial because of its limited antimicrobial effect. We will, however, continue to study the use of sodium bicarbonate as an alternative salt for ECA water treatment.
Impacts
Change in Knowledge 1)ECA water research and extension. In order to determine the antimicrobial efficacy of ECA water we used the AOAC method for testing sanitizers against, Escherichia coli, Staphylococcus aureus, Salmonella species and Listeria monocytogenes. The ECA water parameters were 30 ppm free available chlorine, ORP at 800 mV and pH 6.2-7.4. There was a 5 to 6 log reduction of these pathogens when exposed to the above ECA solution. In addition, there was a 1 to 2 log reduction in of aerobic and coliform microorganisms when lettuce was treated. We have also established a 3 month shelf-life of ECA water with respect to the efficacy against the above pathogens. 2)Bacteriophage research A total of 40 strains of bacteriophages were obtained from two of our collaborators. We have identified that at least three of them have broad spectrum against 11 Escherichia coli O157:H7 strains and their morphological characteristics using electron microscopy have been determined. At least 10 additional coliphages have been directly isolated from cattle manure and three of those phages are capable of infecting as many as 10 E. coli O26 strains. Eleven bacteriophages specific against Salmonella have also been isolated from cattle, swine and poultry manure. We have characterized the morphology of 5 of them which they appear to be highly infectious for S. Typhimurium, the serovar responsible for most of the salmonellosis cases.
Publications
- No publications reported this period
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