Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Target audience was industry professionals, students, and other goverment and academic researchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided graduate education forten students who completed their degrees during the projects duration (5 PhD, 5 MS). Additionally, sevenother graduate students (6 PhD and 1 MS) and 15 undergraduate students were involved in the project. How have the results been disseminated to communities of interest? The results were disseminated through presentations at national meetings, research papers, invited lectures and workshops. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Bioluminescent reporter phagewere constructed for detection ofE. coliO157:H7 using the temperate bacteriophage Phi V10. The simple platform allows the insertion of different genes which allow detection of the pathogen in various food matrices and is applicable to environmental samples. The resultant assay requires minimalmodification of existing protocols and allowsplus minus detection using visual analysis. Along with the construction of the phages acost effective and safe method of production was developed as well allowing the production cost tobe minimal (< 10 cents per assay) which would allow increased monitoring. Immobilization procedures and immobilizationmatrices were also evaluated. Currently work is focusing on the use of pharmaceutical coatings for actual application providing a very amenable assay. The mechanism ofnanoemulsions using essential oils (Thymol etc.) wasinvestigated using bioluminescence and the datasuggested the uncoupling of oxidative phosphorylation. Investigation of chlorine dioxide inactivation mechanisms continued to point to the reduction of bacterial metabolic reducing potential. The previous resultswill allow theefficient use of these interventionsto controlfoodborne pathogen contamination.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Horton, J. L., J. D. McGlothlin, B. M. Applegate, and J. F. Schweitzer. 2014. Evaluation and Control of Airborne Pathogens for Health Care Workers in the Post Anesthesia Care Unit (PACU) and Intensive Care Unit (ICU). American Industrial Hygiene Association (student night). Chicago, IL. (First Place in Graduate Poster Competition)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Ismail, H., T. Zhu, and B. M. Applegate. 2014. Effect of microbiological media and food matrix on phage infectivity. American Society for Microbiology. Boston, MA.
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Farrokhzad, K., C. Rosenfield, and B. Applegate. 2014. Bacteriophage technology in high throughput screening for detecting pathogens in food. In High throughput screening for food safety assessment. Arun Bhunia, Moon Kim, Chris R. Taitt (eds.).
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2014
Citation:
Myer, P., M. del Busto Ramos, L. Hartono and B. Applegate. 2014. Bioluminescent biosensors in non-fiber optic formats. In Luminescent Microbial Biosensor Devices: Design, Construction and Implementation. Gerald Thouand and Robert Marks (eds.) In Press.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Zhang, D., S. P. Thomson, A. M. Liceaga, M. S. Martin-Gonzalez, and B. Applegate. 2014. Immobilization of bacteriophage using Asian carp proteins. American Society for Microbiology General Meeting. Boston, MA.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Rosenfield*, C., U. Minocha, K. Farrokhzad, P. Romero, M. Morgan, and B. Applegate. 2014. Bacteriophage ?V10-mediated bioluminescent detection of E. coli O157:H7. American Society for Microbiology General Meeting. Boston, MA. (*recipient of PULSe Travel Award)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Fleishman Littlejohn, A., T. Lim, A. Broady, K. Farrokhzad, A. Bhunia, M. Morgan, and B. Applegate. 2014. Efficacy of low level chlorine dioxide gas treatment on romaine lettuce and cantaloupe as indicated by microbial diversity. American Society for Microbiology General Meeting. Boston, MA.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Lim, T., A. Broady, J. Jackson, B. Anderson, A. Fleishman Littlejohn, J. Jensen, K. Keener, A. Bhunia, and B. Applegate. 2014. Microbial diversity as an indicator of the efficacy of atmospheric cold plasma treatments of produce. American Society for Microbiology General Meeting. Boston, MA.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Rosenfield, C., M. Martinez, F. Zhu, K. Farrokhzad, G. Paoli, M. Morgan, L. Csonka, and B. Applegate. 2014. Detection of E. coli O157:H7 with a reporter phage containing the luxCDABE cassette. ASsured, SafE and Traceable Food (ASSET) Conference 2014. Queens University Belfast, Ireland. (Awarded Best Poster Prize in the area of: New Analytical means of verifying the integrity of the agri food supply chain)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Horton, J. L., J. D. McGlothlin, B. M. Applegate, and J. F. Schweitzer. 2014. Pilot Laboratory Study to Control Airborne Pathogens Using a New Scavenging Mask to Protect Health Care Workers in the Post Anesthesia Care Unit (PACU) and Intensive Care Unit (ICU). American Industrial Hygiene Conference. San Antonio, TX
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Phage mediated luminescent detection/identification of E. coli O157:H7 Emerging Sensor Technologies for Food Safety. Baltimore, MD. 2014.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
"New low temperature/short time pasteurization method yields better tasting, longer shelf life fresh milk Aseptipak Europe.Warsaw, Poland. 2014.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Duarte-G�mez, E. E., D. Graham, M. Budzik, B. Paxson, L. Csonka, M. Morgan, B. Applegate, and M. F. San Mart�n-Gonz�lez. 2014. High hydrostatic pressure effects on bacterial bioluminescence. LWT - Food Science and Technology 56(2):484-493.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Cho, I. H., A. D. Radadia, K. Farrokhzad, E. Ximenes, E. Bae, A. K. Singh, H. F. Oliver, M. Ladisch, A. Bhunia, B. Applegate, L. Mauer, R. Bashir, and J. Irudayaraj. 2014. Nano/micro and spectroscopic approaches to food pathogen detection. Annual Reviews of Analytical Chemistry. 7: 65-88.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Thomson, S., B. Applegate, R. Martyn, and A. Liceaga. 2014. Analysis of seed vigor responses in soybean to invasive silver carp protein hydrolysate treatments. American Journal of Experimental Agriculture. In press
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Recent research developments were presented at the American Society of Microbiology in Denver Colorado in which the target audience was industry professionals, students, and other goverment and academic researchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Results were prsented at the Annual Meeting of the American Society of Microbiology. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The goals of this research are facilitated by the specific objectives listed below and accomplishments of these portions of these objectives are discussed below: Construct pathogen-specific recombinant bacteriophage to identify the infected host and develop large scale production strategies for the resultant phage During this project period significant effort was dedicated to the construction of a bioluminescent reporter phage for E. coli O157:H7. E. coli O157:H7 containing a kan-luxCDABE fusion in pGEM were plaqued with ΦV10, the plates were scraped and phage were eluted in LB. Filtered supernatants were subsequently used to infect WT E. coli O157:H7. The kanamycin resistant luminescent colonies were selected for further study in which it was determined that the putative transductants were lysogens carrying the lux containing plasmid. These strains were capable of releasing lux transducing phage for multiple generations. One isolate was chosen and used for production of lux transducing phage. The transducing particles were then evaluated for light based detection of E. coli O157:H7. Dilutions of cells were prepared and mixed with transducing particles in 1 mL volumes, incubated at 37ºC and analyzed using a Zylux luminometer. A positive signal (3x control) was detected after 1 h for 106, 105 in 2 h, 104 in 4 h, and all lower concentrations showed a positive result after 24 h. Previously we have shown that ΦV10 can infect O157:H7 in selective enrichment broths allowing the use of this approach for detection during the enrichment step of current detection protocols. The use of luxCDABE as opposed to previously developed luxAB reporters has numerous advantages including primarily the elimination of the addition of n-decanal which reduces the complexity and the cost of the assay. investigate factors affecting survival and control of foodborne bacteria in fresh produce, minimally-processed foods, or processing equipment Fresh produce has been implicated in major outbreaks of food borne pathogens; therefore, non-thermal processing strategies for the reduction of microbial loads on temperature sensitive products are a concern. Chlorine dioxide (ClO2) gas has proven an effective surface sterilizer of produce; however, at higher concentrations the loss of fresh attributes and consumer acceptance occurs in leafy greens. Consequently, treatment of leafy greens, such as Romaine lettuce, necessitates the use of lower concentration ClO2 gas than other produce, such as cantaloupes which have a different surface structure. In an effort to compare the efficacy of on treatment on produce as indicated by microbial diversity, cantaloupe surfaces and Romaine lettuce leaves were divided into control and treatment groups which were subjected to the same treatment of 200 ppm ClO2 gas for 2 minutes. All samples were blended in a sterile blender in PBS to obtain a 1:10 dilution then serially diluted, plated onto Yeast Extract Peptone Glucose plates and incubated at room temperature for 24-51 hours. Colonies (~1 mm) were illuminated by a 625-nm laser beam creating scatter-image signatures subsequently acquired by a CCD camera in an automated BARDOT (BActerial Rapid Detection using Optical light-scattering Technology) system. Scatter signatures were then grouped by their distinctive attributes. Untreated lettuce resulted in 21 unique scatter signatures while only 13 unique signatures after treatment were found, nine of which matched untreated images meaning those microorganisms survived treatment. Untreated cantaloupes yielded 62 unique scatter signatures while the treated group consisted of 40 signatures, giving a 35.5% reduction in diversity, however there was not significant reduction in microbial load. The BARDOT images exclusively in the treated groups are thought to be a product of reduced competition. The decrease in unique images from the untreated to treated group indicates the low level ClO2 gas treatment effectively killed certain native microbial populations on both products and illustrated product differences. evaluate methods to immobilize bacteriophage (or other antimicrobials) for detection and control of pathogens. Use of bacteriophages specifically against E. coli could be an effective and inexpensive strategy to eradicate pathogens from seeds or seedlings and was investigate under this objective. This research uses a bioluminescent E. coli and the lytic bacteriophage T4 in an agar based model system to examine the efficacy of using phage as a prophylactic. The results from the agar model system showed there was a decline in bioluminescence with increasing concentrations of phage applied to seeds prior to germination. These data suggest bacteriophage-based bio-control of pathogens from seed application is feasible. The agar based system also allowed the monitoring of inactivation below the surface as the plants grow. To increase concentrations of bacteriophage associated with the seed, a polymer-based coating system was also investigated. Plaque assays from the immobilized phage showed a 700-fold reduction in the titer of T4 immobilized in the dried coating polymer compared to a 10-fold reduction in the polymer prior to drying. These results suggest that the polymer does affect infectivity, however the drying process results in a greater loss. This approach has the potential to be applied to other leafy greens which could become contaminated during sprouting. .
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Zhang, D., A. Opoku, U. Minocha, P. Myer, R. Turco, J. Youngblood, F. M. San Martin-Gonzalez, and B. Applegate. 2013. Use of bacteriophage coating on seeds to prevent pathogen contamination during germination. American Society for Microbiology, Denver CO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
96. Myer, P., K. Parker, A. Kanach, W. Dominguez, R. Turco, and B. Applegate. 2013. Application of the Solvent Effect on Bioluminescent Reporter Bacteria as a Real-Time Membrane Toxicity Assay. American Society for Microbiology, Denver CO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Broady, A., P. Turner, K. Farrokhzad, K. Parker, B. Applegate, and M. Morgan. 2013. Efficacy of Chlorine Dioxide Gas at Penetrating Romaine Lettuce Tissue. American Society for Microbiology. Denver CO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Vasquez-Mejia, C.M., J. G. Auer, E. E. Duarte-Gomez, V. Rodriguez-Martinez, W. A. Peer, B. Applegate, and F. San Martin. 2013 The Effect of 5-isopropyl-2-methylphenol on a Bioluminescent Strain of E. coli O157:H7. American Society for Microbiology, Denver CO.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Tong, Z., M. Bischoff, L. F. Nies, P. Myer, B.M. Applegate and R.F. Turco. 2012. Response of Soil Microorganisms to As-Produced and Functionalized Single-Wall Carbon Nanotubes (SWNTs). Environmental Science & Technology. 46(24): 13471-13479.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Serrano-Ni�o,J. C., A. Cavazos-Gardu�o, A. Hernandez-Mendoza, B. Applegate, M.G. Ferruzzi, M.F. San Martin-Gonz�lez, H.S. Garc�a. 2013. Assessment of probiotic strains ability to reduce the bioaccessibility of aflatoxin M1 in artificially contaminated milk using an in vitro digestive model. (IF 2.849) Food Control 31 (2013) 202-207.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Duarte-G�mez, E. E., E.E. Duarte-G�mez, D. Graham, M. Budzik, B. Paxson, L. Csonka, M. Morgan, B. Applegate, M.F. San Mart�n-Gonz�lez. High hydrostatic pressure effects on bacterial bioluminescence, LWT - Food Science and Technology.
|
Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Research on this project period focused on a) development of a model bacteriophage-bioluminescent host system to be used in an in-situ approach for monitoring phage inactivation in food matrices and b) development of antimicrobial bioactive polymers. The overall goal for part a) was to use bioluminescence as an indicator of cell viability to monitor the kinetics of phage inactivation. Due to the prominence of the genus Pseudomonas as a commonly identified spoilage organism, the host-bacteriophage pair of Pseudomonas fluorescens M3A and the lytic bacteriophage phi-S1 was chosen as the test system. Pseudomonas fluorescens M3A was rendered luminescent by biparentally mating with an E. coli strain harboring the MiniTN5 nahRG::luxCDABE transposon and the associated delivery vector. The resultant bioluminescent P. fluorescens M3A was evaluated for fitness and growth kinetics in milk. P. fluorescens M3A was grown to a specific cell density and mixed with phi-S1 phage in a matrix of decreasing multiplicity of infections and absolute concentrations. The assay is based on the decrease in bioluminescence, indicating P.fluorescens M3A inactivation by phi-S1. The assay was continuously monitored using an automated luminometer in standard media and model liquid food systems consisting of: Skim, 1 percent, 2 percent, and whole milk. Assays were performed at 25 degrees C and monitored every 140 s. Experiments were also undertaken to determine the effect of milk on bioluminescence monitoring to determine the extent of quenching of the luminescent signal. The overall goal for part b) was to investigate the effects of trimethylolpropane triacrylate (TMPTA) and hydroxyethylmethacrylate (HEMA) combinations on the antimicrobial activity of immobilized lysozyme in various ratios of UV curable resin mixtures against E. coli O157:H7 and Listeria innocua. All the components were mixed and spread on low density polyethylene strips. UV polymerization was performed in an UV chamber with nitrogen atmosphere for 5 minutes in order to obtain a solid coating. A plate overlay assay was used to qualitatively determine the antimicrobial activity of immobilized lysozyme on UV cured LDPE against E. coli and L. innocua. PARTICIPANTS: This project has supported two graduate students and several undergraduates in laboratory methods, research experiences, and professional development. TARGET AUDIENCES: Food safety experts in industry. Presentations as professional meetings such as the Institute of Food Technologists, International Association for Food Protection and American Society of Microbiology have been used to disseminate results along with journal publications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The use of luminescence as an insitu monitor for cell inactivation in food matrices using bacteriophage showed promising results. Evaluation of the effect of milk on bioluminescence showed an approximately 25 percent reduction in luminescence compared to a translucent media. The colloidal properties of the milk caused light scattering however the photons ultimately were able to escape the solution and reach the detector. The lack of luminescent quenching also allowed an indirect method of monitoring of growth kinetics within the milk matrix. Interestingly, the kinetics showed that increases in multiplicity of infection lead to sharp reductions in bioluminescence, whereas decreases in multiplicity of infections lead to slight augmentation in light, followed by sharp reductions, demonstrating prolonged cell survival as phage are propagated by infected cells. Matrices of varying MOIs (1x10e9 to 1x10e1) also showed changes in the phage/host kinetics between different media (between milks and between milk and Luria broth). It was also shown that P. fluorescens M3A/phi-S1 kinetics of inactivation is altered by the increase in concentration of milk fat. This work suggests that a bioluminescence-based assay in a milk model system can be used as an effective tool to quantitatively monitor, in situ, phage infectivity as a method to control foodborne pathogens and spoilage organisms which can be extrapolated to other matrices and surface applications. For the antimicrobial, bioactive polymer study, all combinations of TMPTA (0-30%) and HEMA (100-70 %) showed antimicrobial activity against L. innocua and E. coli O157:H7, but with increasing HEMA ratios, a significant enhancement in antimicrobial activity was observed (p< 0.05). No statistical differences were noticed among different mixtures, when opacity was measured, even after adding the antimicrobial compound. After 4 hours of contact time, approximately 2 log CFU/sq cm reduction was obtained in all treated samples, as compared to the control. After 24 hours, the microbial population was reduced to less than detectable values with the mixture containing 100% HEMA. The study demonstrated that resin types influenced the performance of bioactive packaging and the combinations selected have potential applicability for immobilizing lysozyme on packaging surfaces in order extend produce shelf-life, storage time, and enhance food safety and quality.
Publications
- Trinetta V, R. Linton, G. Sadler, M. Morgan. 2011. Antimicrobial activity of non-migratory bioactive polymers against L. monocytogenes and E. coli O157:H7. IFT annual meeting, New Orleans, LA (poster abstract). Myer,P., J. Mieher, A. Kanach, U. Minocha, M.T. Morgan, and B. Applegate. 2012. In situ Monitoring of Bacteriophage/Host Interactions in Milk Using Bioluminescence. International Food Technologists Annual Meeting, Las Vegas Nevada.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Research for the project period focused on development and evaluation of a bacteriophage lysogen assay for detection of E. coli O157:H7 and the effectiveness of UV immobilized bacteriophage and glucose oxidase as an antimicrobial agents against E. coli O157:H7. The goal was to characterize the bacteriophage "Phi"V10 and evaluate its use as a bioreporter for detection of E. coli O157:H7. The temperate bacteriophage "Phi"V10 was characterized for its ability to infect ~300 different strains of E. coli O157:* and its stability in distilled water and buffer at 4 deg C. The complete genome of "Phi"V10 was sequenced and determined to be 39104 bp containing 55 predicted genes. One predicted gene at open reading frame (ORF) 26 was tentatively identified to belong to the acyltransferase family associated with seroconversion. ORF 26 was amplified from the phage genome and cloned into the expression plasmid pBad-TOPO to determine if it was a factor in specific phage infection of E. coli O157:H7 Lipopolysaccharide (LPS) was isolated from the strain expressing ORF 26, analyzed using nuclear magnetic resonance spectroscopy, and evaluated for its impact on E. coli' s immunity to "Phi"V10 infection. In order to improve the quantification of "Phi"V10 phage, a lysogen assay was investigated to determine if incubation temperature or media type affected the size and visibility of the "Phi"V10 plaques. A Reporter phage "Phi"V10cobA-kan was constructed by replacing the non-essential recET with a cobA-kanamycin gene cassette. E. coli O157:H7 infected with "Phi"V10cobA-kan were detected as colonies on plates containing kanamycin due to the integration of "Phi"V10cobA-kan into the chromosome. This assay allowed for detection of E. coli O157:H7 due to the specificity of the phage infection and the kanamycin resistance in the bacteria after infection. PARTICIPANTS: This project has provided training opportunities for two undergraduate and three graduate students druing the current reporting period. TARGET AUDIENCES: Food scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Bacteriophage "Phi"V10 was determined to be most stable when stored in distilled water rather than phage dilution buffer. One log reduction of phage activity was lost after 4 months storage in distilled water and 2 log reductions after only six weeks in phage dilution buffer. Expression of ORF26 in O157:H7 was determined to confer "Phi"V10 immunity using plaque assays. Lipopolysaccharide (LPS) isolated from the strain expressing ORF 26 was found to inactivate "Phi"V10. Further analysis of the LPS using nuclear magnetic resonance spectroscopy showed the acetylation of the O-antigen suggesting it is the receptor for "Phi"V10. However, when using a commercial test kit for E. coli O157 on previously "Phi"V10-infected E. coli, the strain still tested positive for O157. Therefore, the polyclonal mixtures employed by the commercial assay were able to detect the acetylated O157 antigen, even though "Phi"V10 was no longer able to bind to these previously infected cells. This has ramifications when designing pathogen detection methods as a phage-based method could result in a false negative under these circumstances. This research was also successful in developing a lysogen assay for detecting E. coli O157:H7 based on bacteriophage-specific infection. Lysogen formation increased with increasing temperatures but plateaued at temperatures above 20 C. Results from experiments with different selective media were fairly similar suggesting that any of the selective media tested could be used in the lysogen assay. Although the assay works well in bacterial growth media, using it in food applications, e.g., produce and meat, still needs to be evaluated. In stomached food matrices there may be naturally occurring inhibitors that non-selectively bind phage likely due to electrostatic interaction. This study suggested that incorporating freeze dried bacteriophage into packaging films could produce antimicrobial packaging material specifically targeting Escherichia coli. Ultraviolet light did not exhibit inhibitory effect on the bacteriophage. However, the freeze drying process and the UV-crosslinked polymer formula reduced the activity of bacteriophage by 2-3 logs. Also, immobilized glucose oxidase in UV-crosslinked polymers can be used to inactivate Escherichia coli O157:H7 Lux in apple juice during refrigerated storage.
Publications
- Stratton T.R., B.M. Applegate, and J.P. Youngblood. 2011. Effect of Steric Hindrance on the Properties of Antibacterial and Biocompatible Copolymers. Biomacromolecules. 12(1): 50-56.
- Shehan,C., E. Duarte Gomez, F. San Martin Gonzalez, and B. Applegate. 2011.The effect of pressure on bioluminescent reporter strains. American Society for Microbiology, New Orleans, LA.
- Minocha, U., P. Romero, J. Bourn, C. Rosenfield, and B. Applegate. 2011. Detection of Escherichia coli O157:H7 using ΦV10cobA-kan lysogens. American Society for Microbiology, New Orleans, LA.
- Duarte Gomez, E.E., D. Graham, M. Budzik, B. Paxson, M.T. Morgan, L. Csonka, B. Applegate, and M.F. San Martin-Gonzalez. 2011. High Hydrostatic Pressure Effect on E. coli Heat Stable and Heat Sensitive lux Proteins. IAFP Annual Meeting, Milwaukee, WI
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Research for this project period focused on two main areas related to packaging and high pressure inactivation of bacteria which could potentially be interfaced with active packaging to provide longer shelf life for food products. Increasing consumer demand for safer food and extended shelf life has led to the development of new and novel packaging techniques. Antimicrobial packaging represents an innovative way to enhance the safety and quality of food products by controlling and/or reducing the growth of inherent foodborne microorganisms. One concept for antimicrobial packaging is to incorporate an active component into the food contact surface without migration into the product. UV polymerization is an innovative technique for immobilizing bioactive compounds in energy curable food contact resins. This research focused on the use of three natural antimicrobial substances: lysozyme, nisin and lactoferrin. Each antimicrobial agent offers the advantages of being generally recognized as safe, specific in its antimicrobial action, and active at low concentrations. The effect of UV immobilization on the antimicrobial activity of these molecules was studied against Listeria monocytogenes using a plate overlay assay. Low density polyethylene (LDPE) and aluminum were used as polymer support materials; while trimethylolpropane triacrylate and hydroxyethyl methacrylate were chosen as monomers based on their cure characteristics (curing time, polarity of the cured film, and flexibility of the cured film). The final concentration of lysozyme, nisin and lactoferrin in the film was 1 mg square cm. High Hydrostatic Pressure (HHP), or High Pressure Processing (HPP), is used in the food industry to inactivate bacterial pathogens and spoilage microorganisms along with enzymes that can shorten a food products shelf life. It is an attractive alternative to conventional thermal treatments due to consumer's demands for safe products which retain organoleptic characteristics and nutrition. The technology is attractive as pressure is applied uniformly and instantaneously throughout the food without permanent deformation in size and shape. However a detailed understanding of the mechanism of bacterial inactivation by this process is not known. To examine the effect high hydrostatic pressure a system consisting of a bioluminescent strains of bacteria integrated with a stainless steel vessel with a sapphire window (1.3 cm dia.) to which a Photomultiplier Tube to monitor light emission was constructed. A pressure sensor was also attached to the pressure vessel. The system was integrated with a computer and software was adapted to allow real time monitoring of both pressure and light. PCR vials containing 350 microliters of bioluminescent bacteria were placed inside the pressure chamber. Three different pressure treatments, 69, 103 and 138 MPa were evaluated over three cycles. Each cycle consisted of exposure to the treatment pressure for a duration of 5 minutes followed by 5 minutes decompression at 0 MPa. Completion of three consecutive cycles provided a total treatment time of 30 minutes at 25 degrees C. Three replicates were performed per each treatment pressure. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Food scientists and food safety experts PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Immobilization of antimicrobials showed promising results. After immobilization, lysozyme showed a significant antimicrobial activity in all the conditions tested; lactoferrin enhanced its activity in HEMA but a decrease was observed when TMPTA was used. Nisin demonstrated a strong antimicrobial activity with TMPTA, while low activity was detected when HEMA was added. Both LDPE and aluminum were suitable packaging material supports for film activity following UV immobilization. Based on our preliminary results, matrix changes ensuing from binding of antimicrobial compounds to a variety of resin types influenced the performance of bioactive packaging. This work was presented at the annual meeting of International Food Technologists in Chicago Illinois. Using bioluminescence to monitor the effect of high pressure provided valuable insight into the relationship between the thermal stability of enzymes and their pressure stability. Escherichia coli expressing heat-sensitive lux proteins from Vibrio fischeri (E. coli VF lux) were constructed and exposed to 69, 103 and 138 MPa cyclic treatments for 30 minutes at 25 degrees C to assess the effect of pressure treatments on these isogenic plasmids and host cells. Bioluminescence was monitored as a function of the pressure applied and the results were compared to bacterial enumeration before and after pressure treatments. Light patterns in photons per second for each strain suggested that there was a reversible protein unfolding when compression cycles were applied. The decrease in light emission was determined by increments in pressure applied and background light readings were observed in E. coli VF lux when exposed to the highest pressure treatment. Bioluminescence loss after 138 MPa for E. coli XL lux and E. coli VF lux was 70% and 93% respectively, suggesting that lux protein heat stability is positively correlated to pressure stability. Plate counts before and after pressure treatments did not show a decrease in bacterial viability for any of the two strains. The results from this study have shown that reversible protein and enzyme inactivation occurred in the bioluminescent strains exposed to the different pressure treatments. The decline and increase in light emission also suggests that bacterial metabolic activity was suppressed under HHP treatments. The results of this work are currently being prepared for publication as both a journal article and a Masters Thesis.
Publications
- Stratton, T.R., Howarter, J. A., Allison, B. C., Applegate, B. M., and Youngblood, J.P. 2010. Structure−Activity Relationships of Antibacterial and Biocompatible Copolymers. Biomacromolecules. 11(5): 1286-1290.
- Choi, J. H., Lee, S., Kang, H., Lee, J.H., Kim, J., Yoo, H., Stratton, T.R., Applegate, B.M., Youngblood, J.P., and Kim, H.K. 2010. Synthesis of Water-soluble chitosan-g-PEO and its application for preparation of superparamagnetic iron oxide nanoparticles in aqueous media. Macromolecular Research 18(5): 504-511.
- Farrokhzad, K., Tanner, P., Radcliffe, J., and Applegate, B.M. 2010. Studying the effect of plant extracts on attachment of pathogenic bacteria to intestinal tissue using a porcine model. American Society of Microbiology Annual Meeting, San Diego California.
- Habteselassie M., Bischoff, M., Applegate, B.M., Reuhs, B., and Turco, R.F. 2010. Understanding the Role of Agricultural Practices in the Potential Colonization and Contamination by E. coli in Rhizosphere of Fresh Produce. Journal of Food Protection. Volume 73, Number 11, November, pp. 2001-2009(9).
- Stratton T.R., Applegate, B.M. and Youngblood, J.P. 2010. Effect of Steric Hindrance on the Properties of Antibacterial and Biocompatible Copolymers. Biomacromolecules 11, 1286-1290.
- Minocha, U., Welkie, D., Tanner, P., Orr,M-J., McCarthy, A.T., Bettasso, A., Wickham, G., Thompson, D., Bhunia, A., Turco, R.F., Cousin, M., and Applegate, B.M. 2010. Use of natural microbial flora to identify parameters associated with pathogen transference to leafy greens during primary production. American Society of Microbiology Annual Meeting, San Diego California.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: OUTPUTS: During the project period 10/01/2008-9/30/2009 work was performed in two major areas of the research objectives in the continued proposal. Work was continued on the development of the reporter bacteriophage for the detection and concentration of the foodborne pathogen E. coli O157:H7. Assays were developed to determine the efficiency of the formation of lysogens using the modified bacteriophage phiV10 containing the cobA reporter gene. For phi V10 to be a functional diagnostic tool in real pathogen detection scenarios, the modified reporter phage must retain its specificity, infectivity, and ability to form lysogens. Following recET replacement in the laboratory, the team harvested viable phage and demonstrated potent and specific E. coli O157:H7 cell infectivity and lysogen formation. Testing with these reporter phages was then undertaken and preliminary results were obtained. It is important to note that since lysogens are being detected the strain can be recovered to facilitate the epidemiology of an outbreak. Immobilization assays were also begun using water soluble components to simplify the process. Initial work was also begun evaluating the use of bacteriophage to coat seeds of leafy greens to prevent contamination during germination. Alfalfa, the virulent bacteriophage T4, and a bioluminescent E. coli were used in a model system to determine the efficacy of the phage as a prophylactic. Alfalfa seeds were soaked in different concentrations of T4 bacteriophage and the seeds were dried for 48 hours. Subsequently, the seeds were placed on the surface of agar tubes containing the bioluminescent E. coli. Bioluminescence was measured from seeds on agar surfaces with varying concentrations of bacteriophage and compared to the bioluminescence from seeds without bacteriophage. Initial experiments suggest that there is a decline in the bioluminescence with increasing concentrations of phage prior to germination. Research was also continued on evaluating the mechanism of chlorine dioxide inactivation of bacteria. I mentored 4 graduate students on this project and prepared manuscripts for publication. The results of this work was disseminated at the annual meeting of the American Society of Microbiology and the Annual Rapid Methods and Automation in Microbiology Workshop. TARGET AUDIENCES: Food safety experts for fruits and vegetables and Food producers which monitor their products for foodborne pathogens. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Due to the small and unclear plaques formed by the recombinant phi V10, difficulties enumerating viable phage when applied for detection of E. coli O157:H7 (for example, immobilized onto a stomacher bag) are expected. For this purpose, a quantification method was developed taking advantage of the ability of V10 to form lysogens, by correlating the number of lysogens to the number plaques formed. Purified recombinant phiV10 reporter phage lysate was serially diluted in LB broth and used to perform a plaque assay as described previously. A linear correlation was observed (.9960) Additionally, 100 microliter aliquots of the diluted phage were used to transduce new E. coli O157:H7 cells in decreasing concentrations. It was determined as few as 100 cells of E. coli O157:H7 could be detected with 10 million phage. Research also showed that the water soluble immobilization components resulted in less inactivation than previous ly utilized solvent based. Assays showed less than 50% loss of acivity compared to previous immobilization which resulted in 99% loss. These results suggest a very robust detection technology. Several provisional patents on this technology were converted to a utility patent and licensed by a startup company (Intelliphage). Preliminary experiments performed varying the ratio of phage coating to bacteria to determine efficacious concentrations were performed. Lack of luminescence in the rhizosphere (around the root) of the phage coated seeds indicated a positive result. Vials imaged using an Andor low light camera during germination and growth showed a minimum concentration of 1 million phage per seed would result in the inactivation of E. coli in the surrounding media. A major of this approach is that phage are released into their surrounding environment upon killing the host cell. It is important to note that bacteria have to be active for phage replication and lysis. However phage can still attach and inject there DNA into the host cell then when the bacteria are active they undergo lysis. Therefore infected E. coli O157:H7 not in the rhizosphere would be susceptible upon metabolic activation. If succesfull this low cost phage-based biocontrol strategy can impact food safety. The application of phage for coating of seeds is only one of the possible future applications for this technology.
Publications
- Stratton, T., R. E. Garc, B.M. Applegate and J. P. Youngblood. 2009. Application of a High Throughput Bioluminescence-Based Method and Mathematical Model for the Quantitative Comparison of Polymer Microbicide Efficiency. Biomacromolecules. 10 (5), 1173-1180.
- Perry, L. L., P. SanMiguel, Udit Minocha, Anton I. Terekhov, M.L. Shroyer, L. A. Farris, N. Bright, B. L. Reuhs, and Bruce M. Applegate. 2009. Sequence analysis of Escherichia coli O157:H7 bacteriophage ΦV10 and identification of a phage encoded immunity protein that modifies the O157 antigen. FEMS Microbiology Letters. 292(2):182-186.
- Kim S. , B. Schuler, A. Terekhov, J. Auer, L.J. Mauer, L. Perry, B. Applegate. 2009. A bioluminescence-based assay for enumeration of lytic bacteriophage. Journal of Microbiological Methods. 79, pp. 18-22.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: During the project period 10/01/2007-9/30/2008 work was performed in two major areas of the research objectives in the original proposal. Work was performed to develop a reporter bacteriophage that can be used for the detection and concentration of the foodborne pathogenE. coli O157:H7. Bacteriophage phiV10 a lysogenic, non-virulent phage specific for E. coli O157:H7 strains was modified. The phage was modified by replacing a non-essential genetic component of its genome with DNA comprised of a kanamycin resistance marker fused to reporter gene cobA. cobA, found in Propionibacterium freudenreichii when expresed results in the formation trimethylpyrrocorphin a fluorophore. When exposed to UV light at 302 nm, strains expressing CobA emit a strong red fluorescence which can be detected visually. A phiV10 lysogen harboring the inducible λ Red recombination genes on a temperature sensitive plasmid (pKD46) was used to generate the recombinant phage. The subsequent reporter phage were subjected to a transduction assay to determine if kanamycin resistance could be conferred to E. coli O157:H7 cells. A rapid protocol for purification of recombinant phage using a scalable liquid gradient column chromatography was also developed. An associated area of investigation was also performed on examining the immobilization of the lytic bacteriophage T4 which lyses E. coli. Experiments were performed which substantially demonstrated the effectiveness of UV immobilization of phage onto plastic surfaces. Research was also performed to determine if E. coli 0157:H7 (curli) ATCC 43895 expressing the curli phenotype could attach and be internalized by lettuce. These experiments were performed using a bioluminescent E. coli 0157:H7 (curli) which was generated by inserting a constitutively expressed luxCDABE gene cassette into the bacteria. Analysis was performed by attaching lettuce leaves to the rear plate of a flow cell using cyanoacrylate . Leaves were attached in both dorsal and ventral configurations and incubated with 100000 CFU/ml of the bioluminescent E. coli O157:H7 (curli) for 60 min. The flow cell was drained and the lettuce was rinsed twice with fresh media following and incubated for 10 min at 37˚C prior to imaging with a photon counting camera to determine spatial patterns of attachment. I mentored 2 graduate students on this project and prepared manuscripts for publication. The results of this work were disseminated at the annual meeting of the American Society of Microbiology and the International Association of Food Protection. TARGET AUDIENCES: Food safety experts for fruits and vegetables and Food producers which monitor their products for foodborne pathogens. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Recombinant phiV10 bacteriophage was constructed to confer a red fluorescence phenotype upon infection of E. coli O157:H7. The phage was capable of transforming E. coli O157:H7 cells transducing kanamycin resistant to new lysogens which could subsequently enter the lytic cycle and release progeny carrying the modified phage genome. Although biologically active, changes in the phenotype of the modified phiV10 were observed both plaque size and appearance were affected by the modification which resulted in small plaques which was in contrast to large clear plaques of the non modified phiV10. A scalable protocol for phage purification was developed which removed proteins and DNA impurities in a single step yielding approximately 50% of the initial phage concentration. This is a rapid and effective method for phage purification that can be scaled up for purification of large volumes of phage lysate. Successful immobilization of phage on plastic surfaces using UV polymerization was also demonstrated. However there was significant loss of phage activity (99%) during the lyophilization procedure which precedes the polymer immobilization. This loss seems substantial however since we routinely obtain 100 trillion phage per mL during their production the loss is insignificant. This bacteriophage-based fluorescent bioreporter can become a valuable tool for the detection of E. coli O157:H7, being economical, easy, and most importantly rapid since long enrichment and incubation periods are unnecessary. The work performed on lettuce showed the bioluminescent E. coli O157:H7 (curli) attached to and followed the veins of lettuce leaves, moreover they were more prevalent along the portions connected to the main vein of the lettuce. E. coli O157:H7 (curli) lux also traveled to the interior of the leaves and continued to bioluminesce after the lettuce was sprayed with 70% ethanol. These results show E. coli 0157:H7 (curli) lux attached to the veins of the lettuce and remained active after decontamination which may provide possible explanations of foodborne illness associated with leafy greens and suggest ways to improve food safety during processing.
Publications
- del Busto-Ramos, M., M. Budzik, C. Corvalan1, M. Morgan1, R. Turco, D. Nivens and B. Applegate. 2008. Development of an on-line biosensor for in situ monitoring of chlorine dioxide gas disinfection efficacy. J Appl Micro and Biotech. 8:573-580
- Habteselassie, M., M. Bischoff, E. Blume, B. Applegate, B. Reuhs, S. Brouder, and R. F. Turco. 2008. Environmental controls on the fate of Escherichia coli in soil. Water, Air and Soil Pollution. 190:143-155. Bakhmutova-Albert, E. V., D. W. Margerum, J. G. Auer and B. M. Applegate. 2008. Chlorine Dioxide Oxidation of Dihydronicotinamide Adenine Dinucleotide (NADH). Inorganic Chemistry. 47: 2205-2211.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: During the project period 10/01/2006-9/30/07 work was performed in three major areas of research previously outlined in the original proposal. Experiments were performed in analyzing the mechanisms of internalization and metabolic activity of E. coli O157:H7 in romaine lettuce leaves using bioluminescence. Post harvest internalization by transpiration/capillary action were examined using romaine lettuce. Intact leaves were submerged in reservoirs of O157:H7-lux and allowed to respire overnight at room temperature. . Mechanical entry of E. coli O157:H7 into lettuce tissue via injury by lettuce processing were recreated in the laboratory. Romaine lettuce leaves were chopped and washed in suspensions of bioluminescent E. coli O157:H7. Leaves were subsequently visualized for internalization using the photon counting camera. Internalization was determined using a photon counting camera. Similar work was also performed involving surface contamination of cantaloupes with Salmonella
Poona. Cantaloupes were inoculated with a bioluminescent S. Poona at the following sites: stem, manual wounds, peeling, and intact rind. The inoculated cantaloupes were placed at room temperature ( 1 or 4 days) or 4 degrees C (1, 4, 7, 11, 14 days). Canteloupe inoculation sites were imaged using a CCD camera to determine activity and depth of penetration. After imaging samples of the inoculated tissue were enumerated. I mentored two graduate students associated with the projects. The students disseminated the findings at the annual meetings of the American Society of Microbiology and International Food Technologists. Work was also performed on the fundamental understanding of how chlorine dioxide inactivates bacterial pathogens utilizing bioluminescence to examine insitu inactivation. The research used a real-time online photon monitoring system (PMS) to quantify the bioluminescence produced by a bioluminescent derivative of E. coli O157:H7 during chlorine dioxide treatments in order
to investigate the antimicrobial mechanism. Bioluminescence is only presnt in viable bacterial cells enabling immediate observation of sub-lethal or injurious effects of chlorine dioxide exposure not detected using traditional treatment and plating methods. The response to sub-lethal doses was monitored with and without chloramphenicol. I mentored one graduate student on this project and prepared a manuscript. The results of this work were disseminated at the annual meeting of the American Society of Microbiology.
TARGET AUDIENCES: Food safety experts for fruits and vegetables
Impacts
The internalization of O157:H7 via transpiration/capillary action was shown in Romaine lettuce. Local entry was observed in each case but translocation to distal parts of the plant was not seen suggesting transpiration is not responsible for O157:H7 internalization into plant tissues. However, attachment and entry of O157:H7-lux the cut edges of lettuce leaves was demonstrated. The results did not allow the determination of the exact mechanism by which O157:H7-lux enters the plants although capillary action is a definite possibility. The appearance of O157:H7-lux throughout the leaf pieces raises concern. Lettuce processing appears to expose multiple sites of pathogen entrance into the plant/leaf vascular system. Exposure of cut leaves to an aqueous suspension containing O157:H7 may result in total colonization of a leaf vascular system. Bagged salad greens have been identified as the vehicle for the transmission of E. coli O157:H7. The processing of the leaves prior
to packaging results in cut edges and exposed vascular tissue which can result in contamination as determined above. Imaging of bagged romaine lettuce leaves revealed areas of internalization and intense microbial. The bagged salad environment seems to provide the perfect niche not only for internalization, but also for the growth of O157:H7. This finding points to a convenience (precut and packaged) in terms of lettuce consumption increases the risk. Cantaloupe contamination experiments showed an increase in colony forming units (CFU) in the manual wounds especially at depths of 1-2 cm or 2-3 cm, but the cell numbers showed a rapid reduction for the stem and intact rind, whereas a small increase and then reduction for the peeling rind. Results indicated that S. Poona may get water and nutrients from the mesocarp of cantaloupe to stay metabolically actively, whereas availability of water and nutrients is lacking in the stem and intact rind resulting in a rapid reduction of CFU. S.
Poona was detected up to 14 days in the wounds where mesocarp may provide nutrients, but not on the stem and intact surface. These results show the effect of both depth of wound and temperature are crucial to the survival and amplification of Salmonella on cantaloupe. Previous reports suggest the primary mode of chlorine dioxide inactivation may be attributed to the oxidation of protein or inhibition of protein synthesis. Oxidation has been observed during in vitro reactions involving the amino acids tryptophan and tyrosine and cystine in proteins. The bioluminescent derivative of E. Coli O157:H7 demonstrate the primary mode of action for chlorine dioxide disinfection is not oxidation of proteins but rather reaction with high energy electron donors within the bacterial cell such as NADH, NADPH, and FMNH and subsequent interruption of the proton motive force and membrane integrity.
Publications
- Perry, L., P. Heard, M. Kane, H. Kim, S. Savikhin, W. Dominguez and B. M. Applegate . 2007. Application of multiplex pcr to the detection of pathogens in food. Journal of Rapid Methods. 15 (2) : pp. 176-198.
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Progress 10/01/05 to 09/30/06
Outputs
A bioluminescent derivative of E. coli O157:H7 C7297, designated E. coli O157:H7-lux, was constructed and is highly bioluminescent when grown in rich media. The level of bioluminescence is dependent on several factors, including oxygen, high energy compounds, and a fatty acid substrate. The resultant bioluminescence E. coli O157:H7 cells can be conditioned in minimal salt media to minimize bioluminescence, followed by the addition of test carbon sources, which increase bioluminescence if they can be utilized. In a key experiment the effects of carbon-deprivation on luminescence of the construct was evaluated and demonstrated loss of luminescence over time as carbon became liminting. As luminescence is dependent on the presence of high energy intermediates and a fatty acid substrate, an absence of either could account for the decrease in luminescence observed.. After initial light readings, n-decyl aldehyde was added to aliquots of carbon deprived culture. The addition
of aldehyde did not result in a significant increase in bioluminescence suggesting that the decrease in luminescence is not a result of fatty-acid substrate limitation. This result suggests the loss of luminescence can be solely attributed to lack of energy in the cell. The luminescent response of carbon deprived E. coli O157:H7-lux to the addition of glucose showed that the culture regardless of the length of carbon deprivation, increased in bioluminescent intensity with the addition of glucose. However, as the time of carbon deprivation increased, there was a delayed and attenuated response to the glucose. The delay is most likely due to the cells initially possess pools of biomolecules that are available for metabolism, and energy derived from added glucose results in a rapid increase in bioluminescence and as the period of carbon-deprivation lengthens, biomolecule pools are depleted. Dose dependency with regards to glucose concentration and bioluminescence was also observed. It
was also determined that carbon-deprived cells were able to metabolize mannose, fructose, glucose, N-acetyl-glucosamine, galactose, galactouronic acid, and xylose. Each sugar yielded distinct bioluminescence profiles suggesting different utilization stratagies To determine the usefulness of bioluminescence in the detection of metabolic available carbon from produce assays were conducted with extracts of fresh lettuce, chosen due to its relevance to food safety as a vehicle for E. coli O157:H7 infections. When carbon deprived cultures were incubated with lettuce extracts a strong bioluminescent response was observed and was approximately 7 times the luminescence generated by a glucose control and was achieved in half the time. This result is probably due to the presence of preformed biosynthetic precursors in the whole lettuce extract alleviating the biosynthetic burden on the carbon-deprived cells and less energy is required for the synthesis of necessary compounds.
Impacts
The developed assay utilizing bioluminescence and carbon deprivation showed its applicability to detect the presence of metabolically available carbon in both simple and complex carbon sources using increases in bioluminescence. Previous applications of luminescence for the detection of available carbon simply used bioluminescence as an indicator of cell number (i.e., a more rapid analysis of growth). The assay developed in this study is focused on increased metabolic activity, as measured by luminescence, not an increase in cell numbers, although that may occur. The kinetics of luminescence also provide insight into the one of the key issues in the contamination of lettuce. The rapid increase in luminescence as compared to glucose shows the potential of cells to dramatically increase their numbers on produce, so that they would be metabolically active when consumed. The active cells could be potentially more virulent than those which are not as active putting the
consumer at higher risk. This bioluminescence assay will allow investigation of produce that pose food safety risks, due to high levels of metabolically available complex carbon.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
The complete geneome of phiV10 was sequenced to provide data which will allow a more deliberate construction of the recombinant phage avoiding the use of transposon mutagenesis. Sequence analysis determined the phiV10 genome to be 39,104 bp long with a G+C content of 49.0%. It contains 56 proposed open reading frames (ORFs). Functions were proposed for 19 of the 56 predicted proteins. The genomic organization and hypothetical proteomic composition of phiV10 reveal a probable common ancestry with the Salmonella enterica Group E1 phage epsilon 15. Forty one hypothetical phiV10 proteins have homologs in epsilon15, thirty of which are more similar to their epsilon15 homologs than to any other protein. The serotype conversion genes of epsilon15 are absent from phiV10. A putative acyltransferase gene occupies a position in the phiV10 genome where serotype conversion genes are located in epsilon15. A 2425 bp cluster of seven ORFs located between the putative DNA replication
and terminase genes has considerable sequence identity with DNA from E. coli O157:H7phage VT2 Sa. No known bacterial virulence genes were found in the phiV10 genome. The apparent host specificity of phiV10, its ability to lysogenically convert without transducing host genes, and its lack of identifiable virulence determinants suggest that phiV10 should be useful in biotechnology and that its use in biotechnology should not constitute any undue hazard, making it a good candidate for detection assays. A recombination system for insertion of foreign DNA into the phage genome was developed for the modification of the E. coli O157:H7 bacteriophage phiV10. This was accomplished by isolating a phiV10 lysogen of E. coli O157:H7. The lysogen was further characterized and shown to be inducible for the lytic phenotype. The strain was subsequently transformed with pKD46,which contains an arabinose inducible recombinase. This system allows the electroporation of linear DNA containing foreign genes
flanked by DNA sequences from regions of the phage genome facilitating insertion of reporter genes into the phage genome. Initial work has identified regions for modification, allowing insertion of reporter genes which do not affect the phage life cycle. We are also utilizing pKD46 for analysis of the unknown open reading frames found in the genome of bacteriophage phiV10. Key parameters were determined for optimization of the recombination system for reporter gene insertion and gene inactivation. Primers were designed to begin the systematic evaluation of the unknown open reading frames. Amplicons were constructed for the systematic disruption of identified open reading frames in phiV10. The recombination and lysogen rescue experiments are continuing. We have begun collaboration with Embedded Concepts, which has patented technology which allows the embedding of antibodies into plastics. This innovation would greatly enhance the potential for integration of the phage based assay into
food packaging material without the need for lateral flow simplifying development of stand alone kits involving sample preparation and detection all in one.
Impacts
This technology platform can be integrated with antibody-based assays coupled with impedance based spectroscopy, biochips, fluorescence microscopy, and enzyme-linked immuno-assays(ELISA)which address the diverse needs of detection methods for food and waterborne pathogens. However these technologies can require significant equipment expense. Therefore the proposed technology could lead to the development of a test strip format which will provide an inexpensive and multiplexed detection assay for a variety of pathogens. The test strip could then be utilized for direct testing of potable water and food for both coliforms and pathogens. The test strip will be utilized as a stand-alone kit or in an integrated food packaging format, which will allow the testing of foods prior to their removal from the package.
Publications
- No publications reported this period
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