Phage-based detection of Escherichia coli O157:H7 in leafy greens
Escherichia coli O157:H7, due to its low infectious dose and severe symptoms such as hemolytic uremic syndrome, is particularly dangerous. Detecting pathogens in foods like lettuce, a common vehicle for contamination, requires a faster and more sensitive method than conventional techniques. This pro...
- Autores:
-
Mora Pérez, Luisa Valentina
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2025
- Institución:
- Universidad de los Andes
- Repositorio:
- Séneca: repositorio Uniandes
- Idioma:
- eng
- OAI Identifier:
- oai:repositorio.uniandes.edu.co:1992/75605
- Acceso en línea:
- https://hdl.handle.net/1992/75605
- Palabra clave:
- Bacteriophage
Bioluminescence
Detection
E. coli O157:H7
Foodborne
Microbiología
- Rights
- openAccess
- License
- Attribution-NonCommercial-NoDerivatives 4.0 International
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Phage-based detection of Escherichia coli O157:H7 in leafy greens |
| title |
Phage-based detection of Escherichia coli O157:H7 in leafy greens |
| spellingShingle |
Phage-based detection of Escherichia coli O157:H7 in leafy greens Bacteriophage Bioluminescence Detection E. coli O157:H7 Foodborne Microbiología |
| title_short |
Phage-based detection of Escherichia coli O157:H7 in leafy greens |
| title_full |
Phage-based detection of Escherichia coli O157:H7 in leafy greens |
| title_fullStr |
Phage-based detection of Escherichia coli O157:H7 in leafy greens |
| title_full_unstemmed |
Phage-based detection of Escherichia coli O157:H7 in leafy greens |
| title_sort |
Phage-based detection of Escherichia coli O157:H7 in leafy greens |
| dc.creator.fl_str_mv |
Mora Pérez, Luisa Valentina |
| dc.contributor.advisor.none.fl_str_mv |
Reyes Muñoz, Alejandro |
| dc.contributor.author.none.fl_str_mv |
Mora Pérez, Luisa Valentina |
| dc.subject.keyword.eng.fl_str_mv |
Bacteriophage Bioluminescence Detection E. coli O157:H7 Foodborne |
| topic |
Bacteriophage Bioluminescence Detection E. coli O157:H7 Foodborne Microbiología |
| dc.subject.themes.spa.fl_str_mv |
Microbiología |
| description |
Escherichia coli O157:H7, due to its low infectious dose and severe symptoms such as hemolytic uremic syndrome, is particularly dangerous. Detecting pathogens in foods like lettuce, a common vehicle for contamination, requires a faster and more sensitive method than conventional techniques. This project proposes a detection method based on bioluminescence using a modified phage specific for E. coli O157:H7, фV10, by inserting the luxCDABE operon and the kanamycin resistance gene. The infection and genome insertion of фV10lux into O157:H7 generate luciferase and its substrate through the complete luxCDABE operon, producing light proportional to the number of viable E. coli cells. The kanamycin gene allows the selection of successfully modified cells. Concentration techniques and food-related conditions were applied to evaluate phage performance, determining the minimum number of bacteria required to produce detectable light and the minimum amount of phage necessary to ensure E. coli detection. Laboratory assays are expected to achieve detection limits below 10^3 CFU/ml for bacterial cells and 10^5 CFU/ml for phage particles. Various temperatures, concentrations, and incubation times were assessed before adding kanamycin, with no significant differences expected in bioluminescence detection. In food matrices such as parsley and cilantro, detection was anticipated to be comparable to laboratory conditions. This method aims to be easily implemented at critical points in the food supply chain to improve pathogen detection and food safety. |
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2025 |
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2025-01-23T14:49:50Z |
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2025-01-23T14:49:50Z |
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2025-01-22 |
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Trabajo de grado - Pregrado |
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Lim, J. Y., Yoon, J. W., & Hovde, C. J. (2010). A Brief Overview of Escherichia coli O157:H7 and Its Plasmid O157. PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645889/ Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson M, Roy SL, Jones JL, and Griffin PM. (2011). Foodborne illness acquired in the United States- Major Patohens. Emerging Infectious Diseases. 17(1), 7-15. Chauret, C. (2011). Survival and control of Escherichia coli O157:H7 in foods, beverages, soil and water. Virulence, 2(6), 593-601. https://doi.org/10.4161/viru.2.6.18423 [USDA-FSIS], United States Department of Agriculture. (2011). Report on the food safety and inspection service’s microbiological and residue sampling programs. United States. Food Safety and Inspection Service [Accessed 2020 February 20] Report on FSISs Microbiological and Residue Sampling Programs (usda.gov) Rohde, A., Hammerl, J. A., Boone, I., Jansen, W., Fohler, S., Klein, G., Dieckmann, R., & Dahouk, S. A. (2017). Overview of validated alternative methods for the detection of foodborne bacterial pathogens. Trends In Food Science & Technology, 62, 113-118. https://doi.org/10.1016/j.tifs.2017.02.006 Zhang, D., Coronel-Aguilera, C. P., Romero, P. L., Perry, L., Minocha, U., Rosenfield, C., Gehring, A. G., Paoli, G. C., Bhunia, A. K., & Applegate, B. (2016). The Use of a Novel NanoLuc -Based Reporter Phage for the Detection of Escherichia coli O157:H7. Scientific Reports, 6(1). https://doi.org/10.1038/srep33235 Priyanka, B., Patil, R. K., & Dwarakanath, S. (2016). A review on detection methods used for foodborne pathogens. The Indian Journal Of Medical Research, 144(3), 327-338. https://doi.org/10.4103/0971-5916.198677 Johnson, L., Rose, B., Sharar, A., Ransom, G., Lattuada, C., & Mcnamara, A. (1995). Methods Used for Detection and Recovery of Escherichia coli O157:H7 Associated with a Food-Borne Disease Outbreak. Journal Of Food Protection, 58(6), 597-603. https://doi.org/10.4315/0362-028x-58.6.597 Richter, Ł., Janczuk-Richter, M., Niedziółka-Jönsson, J., Paczesny, J., & Hołyst, R. (2018). Recent advances in bacteriophage-based methods for bacteria detection. Drug Discovery Today, 23(2), 448-455. https://doi.org/10.1016/j.drudis.2017.11.007 Gu, M. B., Min, J., & LaRossa, R. A. (2000). Bacterial bioluminescent emission from recombinant Escherichia coli harboring a recA::luxCDABE fusion. Journal Of Biochemical And Biophysical Methods, 45(1), 45-56. https://doi.org/10.1016/s0165-022x(00)00100-7 Kim, J., Kim, M., Kim, S., & Ryu, S. (2017). Sensitive detection of viable Escherichia coli O157:H7 from foods using a luciferase-reporter phage phiV10lux. International Journal Of Food Microbiology, 254, 11-17. https://doi.org/10.1016/j.ijfoodmicro.2017.05.002 Oats, J.,Michael F. (2018). Determinants of Specificity of the Escherichia coli O157:H7 Bacteriophage PHIV10 (Order No. 10808940). Available from Dissertations & Theses @ Big Ten Academic Alliance; ProQuest Dissertations & Theses Global Closed Collection; ProQuest One Academic. (2267059863). https://www.proquest.com/dissertations-theses/determinants-specificity-em-escherichia-coli-o157/docview/2267059863/se-2 Feng, P., Weagant, S. D., & Jinneman, K. (2020). BAM Chapter 4A: Diarrheagenic Escherichia coli. FDA. Recuperado 29 de julio de 2024, de https://www.fda.gov/food/laboratory-methods-food/bam-chapter-4a-diarrheagenic-escherichia-coli |
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Reyes Muñoz, Alejandrovirtual::22445-1Mora Pérez, Luisa Valentina2025-01-23T14:49:50Z2025-01-23T14:49:50Z2025-01-22https://hdl.handle.net/1992/75605instname:Universidad de los Andesreponame:Repositorio Institucional Sénecarepourl:https://repositorio.uniandes.edu.co/Escherichia coli O157:H7, due to its low infectious dose and severe symptoms such as hemolytic uremic syndrome, is particularly dangerous. Detecting pathogens in foods like lettuce, a common vehicle for contamination, requires a faster and more sensitive method than conventional techniques. This project proposes a detection method based on bioluminescence using a modified phage specific for E. coli O157:H7, фV10, by inserting the luxCDABE operon and the kanamycin resistance gene. The infection and genome insertion of фV10lux into O157:H7 generate luciferase and its substrate through the complete luxCDABE operon, producing light proportional to the number of viable E. coli cells. The kanamycin gene allows the selection of successfully modified cells. Concentration techniques and food-related conditions were applied to evaluate phage performance, determining the minimum number of bacteria required to produce detectable light and the minimum amount of phage necessary to ensure E. coli detection. Laboratory assays are expected to achieve detection limits below 10^3 CFU/ml for bacterial cells and 10^5 CFU/ml for phage particles. Various temperatures, concentrations, and incubation times were assessed before adding kanamycin, with no significant differences expected in bioluminescence detection. In food matrices such as parsley and cilantro, detection was anticipated to be comparable to laboratory conditions. This method aims to be easily implemented at critical points in the food supply chain to improve pathogen detection and food safety.Pregrado11 páginasapplication/pdfengUniversidad de los AndesMicrobiologíaFacultad de CienciasDepartamento de Ciencias BiológicasAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Phage-based detection of Escherichia coli O157:H7 in leafy greensTrabajo de grado - Pregradoinfo:eu-repo/semantics/bachelorThesisinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_7a1fTexthttp://purl.org/redcol/resource_type/TPBacteriophageBioluminescenceDetectionE. coli O157:H7FoodborneMicrobiologíaLim, J. Y., Yoon, J. W., & Hovde, C. J. (2010). A Brief Overview of Escherichia coli O157:H7 and Its Plasmid O157. PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645889/Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson M, Roy SL, Jones JL, and Griffin PM. (2011). Foodborne illness acquired in the United States- Major Patohens. Emerging Infectious Diseases. 17(1), 7-15.Chauret, C. (2011). Survival and control of Escherichia coli O157:H7 in foods, beverages, soil and water. Virulence, 2(6), 593-601. https://doi.org/10.4161/viru.2.6.18423[USDA-FSIS], United States Department of Agriculture. (2011). Report on the food safety and inspection service’s microbiological and residue sampling programs. United States. Food Safety and Inspection Service [Accessed 2020 February 20] Report on FSISs Microbiological and Residue Sampling Programs (usda.gov)Rohde, A., Hammerl, J. A., Boone, I., Jansen, W., Fohler, S., Klein, G., Dieckmann, R., & Dahouk, S. A. (2017). Overview of validated alternative methods for the detection of foodborne bacterial pathogens. Trends In Food Science & Technology, 62, 113-118. https://doi.org/10.1016/j.tifs.2017.02.006Zhang, D., Coronel-Aguilera, C. P., Romero, P. L., Perry, L., Minocha, U., Rosenfield, C., Gehring, A. G., Paoli, G. C., Bhunia, A. K., & Applegate, B. (2016). The Use of a Novel NanoLuc -Based Reporter Phage for the Detection of Escherichia coli O157:H7. Scientific Reports, 6(1). https://doi.org/10.1038/srep33235Priyanka, B., Patil, R. K., & Dwarakanath, S. (2016). A review on detection methods used for foodborne pathogens. The Indian Journal Of Medical Research, 144(3), 327-338. https://doi.org/10.4103/0971-5916.198677Johnson, L., Rose, B., Sharar, A., Ransom, G., Lattuada, C., & Mcnamara, A. (1995). Methods Used for Detection and Recovery of Escherichia coli O157:H7 Associated with a Food-Borne Disease Outbreak. Journal Of Food Protection, 58(6), 597-603. https://doi.org/10.4315/0362-028x-58.6.597Richter, Ł., Janczuk-Richter, M., Niedziółka-Jönsson, J., Paczesny, J., & Hołyst, R. (2018). Recent advances in bacteriophage-based methods for bacteria detection. Drug Discovery Today, 23(2), 448-455. https://doi.org/10.1016/j.drudis.2017.11.007Gu, M. B., Min, J., & LaRossa, R. A. (2000). Bacterial bioluminescent emission from recombinant Escherichia coli harboring a recA::luxCDABE fusion. Journal Of Biochemical And Biophysical Methods, 45(1), 45-56. https://doi.org/10.1016/s0165-022x(00)00100-7Kim, J., Kim, M., Kim, S., & Ryu, S. (2017). Sensitive detection of viable Escherichia coli O157:H7 from foods using a luciferase-reporter phage phiV10lux. International Journal Of Food Microbiology, 254, 11-17. https://doi.org/10.1016/j.ijfoodmicro.2017.05.002Oats, J.,Michael F. (2018). Determinants of Specificity of the Escherichia coli O157:H7 Bacteriophage PHIV10 (Order No. 10808940). Available from Dissertations & Theses @ Big Ten Academic Alliance; ProQuest Dissertations & Theses Global Closed Collection; ProQuest One Academic. (2267059863). https://www.proquest.com/dissertations-theses/determinants-specificity-em-escherichia-coli-o157/docview/2267059863/se-2Feng, P., Weagant, S. D., & Jinneman, K. (2020). BAM Chapter 4A: Diarrheagenic Escherichia coli. FDA. 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