Progress 07/15/01 to 01/14/04
Outputs
The purpose of this project was to develop and compare nucleic acid amplification-based methods for the rapid detection of viable enterohemorrhagic Escherichia coli, including E. coli O157:H7. A quantitative competitive PCR (QC-PCR) assay was developed. Estimated CFU from QC-PCR were compared to viable plate counts. E. coli O157:H7 (10E2 to 10E8 CFU/mL) were detected and accurately quantified in broth and in milk. QC-PCR was applied using multiple timepoint quantitative competitive polymerase chain reaction (QC-PCR) to detect viable E. coli O157:H7 in ground beef enrichments. E. coli O157:H7 inoculated at 0.20 CFU/g cooked ground beef (25 g ground beef +225 ml mEC + novobiocin) was detected and confirmed as viable in less than 15 h. Application of immunomagnetic separation processes enhanced PCR detection limits 10 to 100 fold. An RT-PCR assay was developed using the stxII (Shiga toxin) mRNA as a target. Enrichment conditions were examined for their effect on stx mRNA
expression of E. coli O157:H7 as measured by RT-PCR. Toxin expression was constitutive and was unaffected by media. Message RNA of stx toxin was not amplified from dead cells. E. coli O157:H7 was inoculated into trypticase soy broth or cooked ground beef enrichments. Detection by PCR and RT-PCR from both broth and meat enrichments was identical for both inocula at 1 or 10 CFU/gram. Southern blot hybridization detected amplification products at all time points in broth enrichments and at 8, 12 and 24 h for meat enrichments. The effect of storage time and growth in broth culture and in a food medium on DNA extraction efficiency and PCR detection sensitivity of E. coli O157:H7 were investigated. Detection limits were evaluated using dilution series PCR targeting the stx-II gene. The relationship between cell density and DNA yield was generally log-linear for pure cultures. When the bacteria were suspended in skim milk at a density of 10E6 CFU/ml, held at 4C and sampled at 24 h intervals,
cell density, total DNA yield, and PCR detection limits remained stable throughout the entire 96 h storage period. However, when E. coli O157:H7 was grown in skim milk to a final cell density of 10E6 CFU/ml, PCR amplification efficiency was drastically reduced, although overall DNA yields from these samples were consistent with those for the samples in which E. coli O157:H7 growth was static over 96 h of storage at 4C. This is most likely due to poor DNA purity, which consistently occurred when extracting from food matrices in which the pathogen was grown rather than stored. Future studies should address these issues. The impact of sublethal stress on expression and detection of E. coli O157:H7 stx, hly, and eae were investigated. Virulence factor expression and/or production was impacted (usually negatively) by stress conditions encountered in food indicating that an enrichment process would be necessary for application of these targets in an RT-PCR or ELISA assay. Enhanced virulence
factor production was not observed under moderately acidic conditions or in apple juice.
Impacts
Ensuring food safety is an important goal for the sustainability of U.S. agriculture. The development and validation of rapid molecular-based methods offers quicker alternatives to conventional pathogen detection methods. Understanding factors that influence nucleic acid extraction and amplification from foods is critical to optimization of these rapid methods. The further development and refinement of these emerging technologies will enhance the safety of the food supply.
Publications
- Elhanafi, D., Leenanon, B., Bang, W. and Drake, M.A. 2004. Impact of cold and cold/acid stress on post stress tolerance and virulence factor expression of Escherichia coli O157:H7. J. Food Prot. 67:19-24.
- Campbell, J., Bang, W., Isonhood, J. H., Gerard, P.D., Drake, M.A. 2004. Effects of salt, acid, and MSG on cold storage survival and subsequent acid tolerance of Escherichia coli O157:H7. Food Microbiol. 21:727-735.
- Adhikari, S.D., Elhanafi, D., and Drake, M.A. 2004. Impact of organic acids and pH on virulence factor expression of Escherichia coli O157:H7. July 12-16, IFT Annual Meeting, Las Vegas, NV, poster, Abstract 99D-33.
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Progress 10/01/02 to 09/30/03
Outputs
The purpose of this project is to develop and compare nucleic acid amplification-based methods for the rapid detection of viable enterohemorrhagic Escherichia coli, including E. coli O157:H7. A quantitative competitive PCR (QC-PCR) assay was developed. A 400 bp fragment corresponding to the A subunit of the stx II gene product was chosen as a target sequence. A 275 bp competing DNA sequence for use in the QC-PCR assay was constructed from the stx II sequence using composite primer PCR. Estimated CFU from QC-PCR were compared to viable plate counts. E. coli O157:H7 were detected and accurately quantified in broth and in milk. The procedure was subsequently applied to ground beef enrichments with multiple timepoint quantitative competitive polymerase chain reaction (QC-PCR) to quantify and confirm viability. Immunomagnetic separation (IMS) was used to isolate and concentrate cells from ground beef enrichments. Cell viability was confirmed by the quantitative increase in
signal of target bands from QC-PCR across multiple timepoints. E. coli O157:H7 inoculated at 0.20 CFU/g ground beef (25 g ground beef +225 ml mEC + novobiocin) was detected and confirmed as viable in less than 15 h. An RT-PCR assay was developed using the stx (Shiga -like toxin) mRNA as a target. Enrichment conditions were examined for their effect on stx mRNA expression of E. coli O157:H7 as measured by RT-PCR. Four different amplicons within the stx-II mRNA were also examined for relative abundance. Toxin expression was constitutive and was unaffected by media. Optimum toxin expression occurred in late log/early stationary phase. Message RNA of stx toxin was not amplified from dead cells. Southern blot hybridization detected amplification products at all time points in broth enrichments and at 8, 12 and 24 h for meat enrichments. The effect of storage time and growth in broth culture and in a food medium on DNA extraction efficiency and PCR detection sensitivity of E. coli O157:H7
were investigated. Detection limits were evaluated using dilution series PCR targeting the stx-II gene. The relationship between cell density and DNA yield was generally log-linear for pure cultures. When the bacteria were suspended in skim milk at a density of 106 CFU/ml, held at 4oC and sampled at 24 h intervals, cell density, total DNA yield, and PCR detection limits remained stable throughout the entire 96 h storage period. However, when E. coli O157:H7 was grown in skim milk to a final cell density of 106 CFU/ml, PCR amplification efficiency was drastically reduced, although overall DNA yields from these samples were consistent with those for the samples in which E. coli O157:H7 growth was static over 96 h of storage at 4o C. A subsequent study revealed similar PCR results for cells grown in raw or autoclaved ground beef compared to cells inoculated and then directly extracted. Clearly, there are issues with the ability to concentrate cells and/or the subsequent purity of
extracted DNA when cells are allowed to grow in a food matrix. Current work is focused on completing evaluations on the impact of sublethal stress on expression of EHEC virulence factors.
Impacts
Ensuring food safety is an important goal for the sustainability of U.S. agriculture. The development and validation of rapid molecular-based methods offers quicker alternatives to conventional pathogen detection methods. Understanding factors that influence nucleic acid extraction and amplification from foods is critical to optimization of these rapid methods. The further development and refinement of these emerging technologies will enhance the safety of the food supply.
Publications
- Taylor, T.M., Drake, M.A., Jaykus, L. 2003. Development of a method to concentrate E. coli O157:H7 from ground beef to facilitate enumeration and molecular detection. IFT Annual meeting, July 12-16, Chicago, IL. Abstract 59-6.
- Leenanon, B.R., Elhanafi, D., and Drake, M.A. 2003. Acid adaptation and starvation effects on Shiga toxin production of Escherichia coli O157:H7. J. Food Prot. 66:970-977.
- Li, W. and Drake, M.A. 2003. Detection and quantitation of viable Shiga-toxin-producing Escherichia coli by quantitative competitive polymerase chain reaction. J. Food Prot. 66:1277-1282.
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Progress 10/01/01 to 09/30/02
Outputs
The purpose of this project is to develop and compare nucleic acid amplification-based methods for the rapid detection of viable enterohemorrhagic Escherichia coli, including E. coli O157:H7. A quantitative competitive PCR (QC-PCR) assay was developed. A 400 bp fragment corresponding to the A subunit of the slt II gene product was chosen as a target sequence. A 275 bp competing DNA sequence for use in the QC-PCR assay was constructed from the slt II sequence using composite primer PCR. Estimated CFU from QC-PCR were compared to viable plate counts. E. coli O157:H7 (102 to 108 CFU/mL) were detected and accurately quantified in broth and in milk. An RT-PCR assay was developed using the slt (Shiga -like toxin) mRNA as a target. Enrichment conditions were examined for their effect on slt mRNA expression of E. coli O157:H7 as measured by RT-PCR. Four different amplicons within the slt-II mRNA were also examined for relative abundance. Toxin expression was constitutive and
was unaffected by media. Optimum toxin expression occurred in late log/early stationary phase. Message RNA of slt toxin was not amplified from dead cells. E. coli O157:H7 was inoculated into trypticase soy broth or cooked ground beef enrichments. Cells were harvested, and RNA or DNA extracted at 4, 8, 12, and 24 hours. RT-PCR or PCR was conducted and visualized by gel electrophoresis and by Southern blots. Detection by PCR and RT-PCR from both broth and meat enrichments was identical for both inocula at 1 or 10 CFU/gram. Southern blot hybridization detected amplification products at all time points in broth enrichments and at 8, 12 and 24 h for meat enrichments. The effect of storage time and growth in broth culture and in a food medium on DNA extraction efficiency and PCR detection sensitivity of E. coli O157:H7 were investigated. Detection limits were evaluated using dilution series PCR targeting the slt-II gene. The relationship between cell density and DNA yield was generally
log-linear for pure cultures. When the bacteria were suspended in skim milk at a density of 106 CFU/ml, held at 4oC and sampled at 24 h intervals, cell density, total DNA yield, and PCR detection limits remained stable throughout the entire 96 h storage period. However, when E. coli O157:H7 was grown in skim milk to a final cell density of 106 CFU/ml, PCR amplification efficiency was drastically reduced, although overall DNA yields from these samples were consistent with those for the samples in which E. coli O157:H7 growth was static over 96 h of storage at 4o C. This is most likely due to poor DNA purity, which consistently occurred when extracting from food matrices in which the pathogen was grown rather than stored. Currently, work is underway to determine the impact of sublethal stress on expression and detection of slt, hly, and eae mRNA and the application of QC-PCR across two timepoints in a meat enrichment to detect and confirm viability of E. coli O157:H7.
Impacts
Ensuring food safety is an important goal for the sustainability of U.S. agriculture. The development and validation of rapid molecular-based methods offers quicker alternatives to conventional pathogen detection methods. Understanding factors that influence nucleic acid extraction and amplification from foods is critical to optimization of these rapid methods. These results indicate that QC-PCR can be used to accurately detect and quantify E. coli O157:H7 without the need for traditional time-consuming plate counts. Detection of slt mRNA is a good indicator of cell viability and may be of use for detection of viable Shiga-like-toxin producing E. coli in foods including detection of low numbers of viable EHEC in ground meat. The further development and refinement of these emerging technologies will enhance the safety of the food supply.
Publications
- McKillip, J.L., Jaykus, L., and Drake, M.A. 2002. The Influence of Growth in a Food Medium on the Detection of Escherichia coli O157:H7 by PCR. J. Food Prot. 65:1775-1779.
- McIngvale, S.C., Elhanafi, D., and Drake, M.A. 2002. Optimization of reverse transcriptase polymerase chain reaction (RT-PCR) to detect viable Shiga-toxin producing Escherichia coli. Appl. Environ. Micro. 68:799-806.
- Campbell, J., Isonhood, J.H., and Drake, M.A. 2002. Impact of salt and monosodium glutamate on survival and acid tolerance of Escherichia coli O157:H7. IFT Annual Meeting, Anaheim, CA June 15-19. Abstract 61C-27.
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Progress 10/01/00 to 09/30/01
Outputs
The purpose of this project is to develop and compare nucleic acid amplification-based methods for the rapid detection of viable enterohemorrhagic Escherichia coli, including E. coli O157:H7. A quantitative competitive PCR (QC-PCR) assay was developed to detect and quantify E. coli O157:H7 in broth. Three targets within the slt II gene were evaluated. A 400 bp fragment corresponding to the A subunit of the slt II gene product was chosen as a target sequence. A 275 bp competing DNA sequence for use in the QC-PCR assay was constructed from the slt II sequence using composite primer PCR. The amplification efficiencies of the target and competitor DNA were determined to be equivalent. PCR reaction and cycling conditions were optimized. DNA was extracted from broth containing 102 to 103 CFU/mL E. coli O157:H7 and QC-PCR conducted. Estimated CFU from QC-PCR were compared to viable plate counts. E. coli O157:H7 (102 to 108 CFU/mL) were detected and accurately quantified in
broth and in milk (r2 =0.99 and0.93, respectively). An RT-PCR assay was developed for EHEC using the slt (Shiga -like toxin) mRNA as a target. Enrichment conditions including media, temperature, aeration, and growth phase were examined for their effect on slt mRNA expression of E. coli O157:H7 as measured by RT-PCR. Four different amplicons within the slt-II mRNA were also examined for relative abundance. Toxin expression was constitutive and was unaffected by media. Toxin expression was increased at 37C compared to 32C and was stimulated by aeration. Optimum toxin expression occurred in late log/early stationary phase. Message RNA of slt toxin was not amplified from dead cells. Amplification of the B subunit or the conserved region was the most sensitive target. Studies have been initiated to determine the sensitivity of the QC-PCR and RT-PCR of slt mRNA in broth and artificially contaminated ground beef. Preliminary results indicate that the application of QC-PCR across two
timepoints in a meat enrichment can be used to detect and confirm viability of E. coli O157:H7.
Impacts
Ensuring food safety is an important goal for the sustainability of U.S. agriculture. The development and validation of rapid molecular-based methods offers quicker alternatives to conventional pathogen detection methods. These results indicate that QC-PCR can be used to accurately detect and quantify E. coli O157:H7 without the need for traditional time-consuming plate counts. Detection of slt mRNA is a good indicator of cell viability and may be of use for detection of viable Shiga-like-toxin producing E. coli in foods including detection of low numbers of viable EHEC in ground meat. The further development and refinement of these emerging technologies will enhance the safety of the food supply.
Publications
- W. Li and M.A. Drake. 2001. Development of a quantitative competitive PCR assay (QC-PCR) for the detection and quantification of Escherichia coli O157:H7 in broth and skim milk. Appl. Environ. Micro. 67:3291-3294.
- McIngvale, S.C. and Drake, M.A. 2001. Enrichment conditions affect expression of slt-II mRNA of Escherichia coli O157:H7. IFT Annual Meeting, New Orleans, LA June 23-27. Abstract 70-1.
- Li, W. and Drake, M.A. 2001. Detection of viable Escherichia coli O157:H7 by QC-PCR. IFT Annual Meeting, New Orleans, LA June 23-27. Abstract 59F-10.
- Leenanon, B.L. and Drake, M.A. 2001. Acid adaptation and starvation effects on Shiga-like toxin production of Escherichia coli O157:H7. IFT Annual Meeting, New Orleans, LA June 23-27. Abstract 59F-8.
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