Progress 01/01/01 to 09/30/04

Outputs
A model system was developed using E. coli phage M13, a well-characterized bacteriophage, for pathogen detection through cell-to-cell communication via a LuxR/3-oxo-C6-HSL system utilizing the a bioluminescent reporter strain designated ROLux. Simulated temperate phage assays tested functionality of the ROLux reporter and production of 3-oxo-C6-HSL by various test strains was performed utilizing a portable luminometer commonly used in food safety hygiene tests. These assays showed detection limits of 100 cells after 24 hours in a varietry of detection formats. Assays incorporating the bacteriophage M13-luxI with the ROLux reporter and a known population of target cells were subsequently developed and have shown consistent detection limits of 105 target organisms. Measurable light response from high concentrations of target cells was almost immediate, suggesting an enrichment step to further improve detection limits and reduce assay time. This approach was subsequently expanded for the detection of Salmonella spp. utilizing a P22 bacteriophage. A recombinant luxI P22 bacteriophage was constructed using genetic recombination and evaluated using the handheld luminometer. The assay utilizing a P22 luxI / ROlux phage system for detection of S. enteriditis showed detection limits of 1000 bacteria and an assay time of approximately 6 hours using a portable luminometer. We further developed this platform for foodborne pathogens utilizing bacteriophage specificity and the Vibrio fischeri AinS synthesized quorum sensing molecule C8-HSL. Once again Phage host specificity is exploited to program a specific target cell to express ainS and produce C8-HSL. The C8-HSL diffuses out of the target cell resulting in autoinduction of V. fischeri ATCC 49387 similar to the model system however with a positive feedback for intense luminescence. A detection assay for E. coli O157:H7 using phage phiV10 was performed by mixing V. fischeri and ainS phiV10 phage with decreasing concentrations of E. coli O157:H7 in a 96 well microtiter plate. Bioluminescence was monitored every 30 min using a Wallac MicroBeta Luminometer. Data was plotted (bioluminescence photons sec -1 versus time). The graph was almost identical to those obtained in real time PCR. Autoinduction, bioluminescence was directly correlated with increasing concentrations of cells producing C8-HSL. Increases in bioluminescence were observed in higher target cell concentrations prior to autoinduction of the control. Previous assays utilizing a luxI / ROlux phage system for detection of S. enteriditis showed similar detection limits of 1000 bacteria and similar assay time of approximately 6 hours using a portable luminometer. The ainS system presented here for O157:H7 showed similar results but had a 1000 fold increase in luminescence allowing visualization of of the luminescent signal. The increased luminescence should allow the use of inexpensive light detection methods (i.e. diodes) facilitating the number of end users of this methodology.

Impacts
The assay developed has the potential of immediate impact in the food safety arena as it can be used with equipment used routinely for the ATP hygiene test. The technology is currently being investigated by a private company to commercialize and additional funding was obtained with these results. The major constraint of the assay was the use of antibiotic resistance markers in the components of the assay. This was remphasized by the interested corporate scientists and should not be a problem with the incorporation of the V. fischeri as the bioluminescent reporter strain.

Publications

• Bright N. G., R. J. Carroll, and B. M. Applegate. 2004. A Model System for Pathogen Detection Using a Two-Component Bacteriophage/Bioluminescent Signal Amplification Assay. In Monitoring Food Safety, Agriculture, and Plant Health. Proceedings of SPIE Vol. 5271 p13-19.
• U. Minocha, N. Bright, L. Perry, B. Applegate. 2004 Detection of the Foodborne Pathogen Escherchia coli O157:H7 Using an ainS Recombinant phiV10 Bacteriophage Based Bioluminescent Reporter System. ASM Biodefense Research meeting: "Future Directions for Biodefense Research: Development of Countermeasures," Baltimore, MD.
• Minocha, U., L.L. Perry, R. Jennings, A. Bhunia, B. Applegate. 2004. Genome Sequence Analysis and Evaluation of Strain Specificity of E. coli O157:H7 Bacteriophage phi V10. 2004. American Society for Microbiology, New Orleans, LA.
• Shroyer, M., U. Minocha, F. Lino, N. Bright, L. Perry, and B. Applegate. 2004. Development of a Recombination System for Rapid Construction of E. coli O157:H7 Reporter Bacteriophage. American Society for Microbiology, New Orleans, LA.

Progress 10/01/02 to 09/30/03

Outputs
To facilitate the construction of a modified P22 bacteriophage for Salmonella spp. it was necessary to construct a recombination vector for insertion of a modified tail spike protein. The vector was modified from a previously constructed vector pTP369 (Casjens et al). Plasmid pTP369 contains a 2558 bp region of the P22 genome corresponding to the region containing gene 23 (antitermination protein), gene 13 (lysis protein), gene 19 (lysozyme) gene 15 (lysis control), orf 201 (unknown protein) and orf 80 (unknown protein). The recombination vector was constructed by removing approximately 1 kb of the P22 genome in the region of orf 201 and orf 80 to allow the insertion of DNA for recombination. A multicloning site and a TA cloning site were inserted to facilitate rapid insertion of modified DNA to construct the luxI phage. Phage based detection of E. coli O157:H7 will be accomplished using phage phiV10. Bacteriophage phiV10 was originally isolated by R. Khakhria and has been shown to specically infect many strains of E.coli O157:H7. It has a genome of approximately 42 kb and classified as a temperate phage (can form lysogens). The phage was obtained from Dr. Rafiq Ahmed at the National Laboratory for Enteric Pathogens in Winnipeg Canada. Total DNA has been isolated from the phage and currently we are generating a genomic library for sequencing the entire phage genome. The sequence data will allow a more deliberate construction of the recombinant phage avoiding the use of transposon mutagenesis. To further evaluate the specificity of phiV10 a previously characterized library from pathogenic E. coli outbreaks responsible for disease was screened for phiV10 susceptability. Environmental isolates were also screened to provide evaluation of false positives. The assay consists of a simple plaque assay using a previously prepared phage solution with a titer of approximately 2 x 10 -3 plaque forming units per mL. Approximately .5ml portions of top agar in capped test tubes were prepared and kept in an isotemp water bath at 50 degrees C. Approximately 100 microliters of the phage was added to the tube immediately followed by the addition of 200 microliters of bacterial test suspension, mixed then the contents of the tube were poured on LB agar plates. Plates were incubated overnight at 37 degrees C and examined for plaque formation. Currently we have screened 440 isolates containing O157:H7 isolates and non O157:H7 isolates with 289 testing positive and 151 negative. Results are currently being analyzed to determine specificity. Dr. Applegate in collaboration with Dr. Rafiq Ahmed at the National Laboratory for Enteric Pathogens (Winnipeg, CA) are currently sequencing the genome of the E. coli O157:H7 bacteriophage phiV10. Project should be completed by the end of 2003. Dr. Applegate is also collaborating with Dr. Len Peruski at the Indiana Medical School in Gary Indiana on development of a Bacillus anthracis bacteriophage using this detection format.

Impacts
The assay being developed has the potential of immediate impact in the food safety arena as it can be incorporated with equipment already deployed and used routinely for the ATP hygiene test. The technology is currently being investigated by a private company to commercialize. The technology could potentially be field ready for Salmonells sp. by late 2004. The major constraint until recent developments was the use of antibiotic resistance markers in the components of the assay. This was emphasized by the interested corporate scientists and should not be a problem as shown in the recent accomplishments above.

Publications

• Gary S. Sayler, Steven A. Ripp, Bruce Applegate. Bioluminescent biosensor device. Patent No. 6,544,729 (February, 2003 ).

Progress 10/01/01 to 09/30/02

Outputs
In 2001-2002 substantial progress was made on objectives 1 and 2. Objective 1. Genetically construct pathogen specific bacteriophage capable of stimulating and enhancing bioluminescence from bioluminescent bioreporter cell populations for the detection, monitoring, and quantitative sensing of microbial pathogenic species. A chromosomally based bioluminescent reporter strain for acyl-homoserine lactone detection was constructed and evaluated. The detection limit was determined to be approximately 10nM with a linear response over 4 orders of magnitude. Stability assays (batch format) were also conducted on the reporter strain to evaluate the maintenance of the reporter construct. The assays showed the reporter construct was maintained after 20 successive transfers. A genetically modified M13 bacteriophage containing the luxI gene in a lacZ-luxI fusion was constructed as a model system to evaluate the fundamental approach of the two-component system. A method for the rapid insertion of luxI into phage genomes, which will allow rapid construction of Bacteriophage/Bioluminescent Reporter Systems was developed using a TNEZ transposome format. The system is currently being utilized to generate temperate bacteriophage/pathogen pairs including: a P22 luxI bacteriophage to detect Salmonella spp, a phiV10 bacteriophage to detect E. coli O157, a phiC bacteriophage to detect Campylobacter jejuni, and an A118 bacteriophage for Listeria monocytogenes. Objective 2. Apply bacteriophage/bioluminescent bioreporter systems with portable field-based photomultiplier units. A simulated temperate phage assay was utilized to evaluate the portable luminometer format for detection limits and overall performance. Using the luminometer format the assay demonstrated detection limits of 100 colony forming units which had bioluminescence levels 12 fold over the negative control. However due to the aqueous nature of the assay oxygen limitation depressed luminescence readings with the higher concentrations of the target organisms. Therefore an alternate approach was examined utilizing a filter format in which cells and phage are concentrated on a membrane and placed on solid media (LB agar) and placed in a light tight enclosure to detect bioluminescence using a Hamamatsu PMT sensor module. The model system using M13 showed an increase in bioluminescence versus phage concentration. This format offers a unique approach using filters to concentrate the phage, bioreporter, and bacterial sample in a close proximity to facilitate detection using the bacteriophage/bioreporter approach.

Impacts
This technology platform once developed will provide rapid detection and monitoring of viable pathogenic bacterial species in food from on-farm production through processing and ultimately consumption by consumers. Field use of this technology will prevent cross contamination of agricultural products by both detecting pathogens in raw product and finding the source of contamination. Early detection could eliminate costly recalls by food processors and also provide intervention strategies

Publications

• No publications reported this period

Progress 10/01/00 to 09/30/01

Outputs
This project has three main objectives. 1) Genetically construct pathogen specific bacteriophage capable of stimulating and enhancing bioluminescence from bioluminescent bioreporter cell populations for the detection, monitoring, and quantitative sensing of microbial pathogenic species. 2) Apply bacteriophage/bioluminescent bioreporter systems with portable field-based photomultiplier units. 3) Develop quantitative RTQ-PCR protocols for mRNA from the recombinant bacteriophage to validate bioluminescent reporter results. To fulfill objective (1) proof in principle experiments have been accomplished validating the approach of biological light amplification to allow the use of low sensitivity light detection. The use of low sensitivity light detection is significant as these technologies are inexpensive and will allow broader use of the technology platform. Genetically modified luxI cassettes for the suite of bacteriophages and the bioreporter gene cassettes have been constructed. Progress has been made in objective (2) by determining the initial experimental parameters associated with a Zylux portable luminometer for field applications of this technology. The portable luminometer results suggest field implementation of this technology will be a simple user friendly protocol. To accomplish objective (3) unique DNA sequences have determined to be utilized in the RT-PCR protocols. The sequences were selected to facilitate the use of the TaqMan protocol for quantitative PCR with the Applied Biosystems 7700 sequence analyzer. This quantitative PCR approach integrated with the reporter technology will allow a second test to detect pathogens using the format in objective one to reduce false positives and negatives.

Impacts
This technology platform once developed will provide rapid detection and monitoring of viable pathogenic bacterial species in food from on-farm production through processing and ultimately consumption by consumers. Field use of this technology will prevent cross contamination of agricultural products by both detecting pathogens in raw product and finding the source of contamination. Early detection could eliminate costly recalls by food processors and also provide intervention strategies.

Publications

• No publications reported this period