AIMS Microbiology, 2017, 3(4): 872-884. doi: 10.3934/microbiol.2017.4.872.

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Rapid loss of a green fluorescent plasmid in Escherichia coli O157:H7

1 Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, USA
† Current address: School of Veterinary Medicine, Faculty of Medical Sciences, The University of the West Indies, St. Augustine, Trinidad, West Indies
# Current address: Department of Biology and Marine Science, Jacksonville University, Jacksonville, FL 32211, USA

Plasmids encoding green fluorescent protein (GFP) are frequently used to label bacteria, allowing the identification and differentiation from background flora during experimental studies. Because of its common use in survival studies of the foodborne pathogen Escherichia coli O157:H7, it is important to know the extent to which the plasmid is retained in this host system. Herein, the stability of a pGFPuv (Clontech Laboratories Inc) plasmid in six Escherichia coli O157:H7 isolates was assessed in an oligotrophic environment (phosphate buffered saline, PBS) without antibiotic selective pressure. The six test isolates were recovered from a variety of animal and human sources (cattle, sheep, starlings, water buffalo, and human feces). GFP labeling of the bacteria was accomplished via transfer electroporation. The stability of the GFP plasmid in the different E. coli O157:H7 isolates was variable: in one strain, GFP plasmid loss was rapid, as early as one day and complete plasmid loss was exhibited by four of the six strains within 19 days. In one of the two isolates retaining the GFP plasmid beyond 19 days, counts of GFP-labeled E. coli O157:H7 were significantly lower than the total cell population (P < 0.001). In contrast, in the other isolate after 19 days, total E. coli O157:H7 counts and GFP-labeled E. coli counts were equivalent. These results demonstrate strain-to-strain variability in plasmid stability. Consequently the use of GFP-labeled E.coli O157:H7 in prolonged survival studies may result in the underestimation of survival time due to plasmid loss.
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Keywords GFP plasmid; rifampicin; survival studies; marker stability; Escherichia coli O157:H7

Citation: Anil K. Persad, Michele L. Williams, Jeffrey T. LeJeune. Rapid loss of a green fluorescent plasmid in Escherichia coli O157:H7. AIMS Microbiology, 2017, 3(4): 872-884. doi: 10.3934/microbiol.2017.4.872


  • 1. Tarr PI, Gordon CA, Chandler WL (2005) Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 365: 1073–1086.
  • 2. Karch H, Leopold SR, Kossow A, et al. (2015) Enterohemorrhagic E. coli (EHEC): Environmental-Vehicle-Human Interface, In: Zoonoses-Infections Affecting Humans and Animals, Springer, 235–248.
  • 3. Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59: 223–239.    
  • 4. Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339–342.    
  • 5. Ma L, Zhang G, Doyle MP (2011) Green fluorescent protein labeling of Listeria, Salmonella, and Escherichia coli O157:H7 for safety-related studies. PloS One 2011: e18083.
  • 6. Bloemberg GV, O'Toole GA, Lugtenberg BJ, et al. (1997) Green fluorescent protein as a marker for Pseudomonas spp. Appl Environ Microb 63: 4543–4551.
  • 7. Wu VC (2008) A review of microbial injury and recovery methods in food. Food Microbiol 25: 735–744.    
  • 8. Trevors JT (1986) Plasmid curing in bacteria. FEMS Microbiol Lett 32: 149–157.    
  • 9. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2Eds., Cold Spring Harbor: Cold Spring Harbor Laboratory.
  • 10. Dorn CR, Angrick EJ (1991) Serotype O157:H7 Escherichia coli from bovine and meat sources. J Clin Microbiol 29: 1225–1231.
  • 11. Cernicchiaro N, Pearl DL, McEwen SA, et al. (2010) A randomized controlled trial to assess the impact of dietary energy sources, feed supplements, and the presence of super-shedders on the detection of Escherichia coli O157:H7 in feedlot cattle using different diagnostic procedures. Foodborne Pathog Dis 7: 1071–1081.    
  • 12. Food and Drug Administration, FDA and states closer to identifying source of E. coli contamination associated with illnesses at Taco John's restaurants: Food and Drug Administration, 2007. Available from: http://www.fda. gov/bbs/topics/NEWS/2007/NEW015 46.html.
  • 13. Lengacher B, Kline TR, Harpster L, et al. (2010) Low prevalence of Escherichia coli O157:H7 in horses in Ohio, USA. J Food Protect 73: 2089–2092.
  • 14. Williams ML, Pearl DL, Lejeune JT (2011) Multiple-locus variable-nucleotide tandem repeat subtype analysis implicates European starlings as biological vectors for Escherichia coli O157:H7 in Ohio, USA. J Appl Microbiol 111: 982–988.    
  • 15. Kotewicz ML, Mammel MK, LeClerc JE, et al. (2008) Optical mapping and 454 sequencing of Escherichia coli O157 : H7 isolates linked to the US 2006 spinach-associated outbreak. Microbiology 154: 3518–3528.    
  • 16. Zwietering MH, Jongenburger I, Rombouts FM, et al. (1990) Modeling of the bacterial growth curve. Appl Environ Microb 56: 1875–1881.
  • 17. Vialette M, Jandos-Rudnik AM, Guyard C, et al. (2004) Validating the use of green fluorescent-marked Escherichia coli O157:H7 for assessing the organism behaviour in foods. J Appl Microbiol 96: 1097–1104.
  • 18. Fratamico PM, Deng MY, Strobaugh TP (1997) Construction and characterization of Escherichia coli O157:H7 strains expressing firefly luciferase and green fluorescent protein and their use in survival studies. J Food Protect 60: 1167–1173.
  • 19. Fremaux B, Delignette-Muller ML, Prigent-Combaret C, et al. (2007) Growth and survival of non-O157:H7 Shiga-toxin-producing Escherichia coli in cow manure. J Appl Microbiol 102: 89–99.    
  • 20. Jiang X, Morgan J, Doyle MP (2002) Fate of Escherichia coli O157:H7 in manure-amended soil. Appl Environ Microb 68: 2605–2609.
  • 21. Jiang X, Morgan J, Doyle MP (2003) Fate of Escherichia coli O157:H7 during composting of bovine manure in a laboratory-scale bioreactor. J Food Protect 66: 25–30.
  • 22. Himathongkham S, Bahari S, Riemann H, et al. (1999) Survival of Escherichia coli O157:H7 and Salmonella typhimurium in cow manure and cow manure slurry. FEMS Microbiol Lett 178: 251–257.    
  • 23. Summers D (1991) The kinetics of plasmid loss. Trends Biotechnol 9: 273–278.
  • 24. Allison DG, Sattenstall MA (2007) The influence of green fluorescent protein incorporation on bacterial physiology: a note of caution. J Appl Microbiol 103: 318–324.
  • 25. Navarro LJM, Tormo A, Martinez-Garcia E (2010) Stationary phase in gram-negative bacteria. FEMS Microbiol Rev 34: 476–495.
  • 26. Skillman LC, Sutherland IW, Jones MV, et al. (1998) Green fluorescent protein as a novel species-specific marker in enteric dual-species biofilms. Microbiology 144: 2095–2101.    
  • 27. Smith MA, Bidochka MJ (1998) Bacterial fitness and plasmid loss: the importance of culture conditions and plasmid size. Can J Microbiol 44: 351–355.
  • 28. Friehs K (2004) Plasmid copy number and plasmid stability. Adv Biochem Eng Biot 86: 47–82.
  • 29. Wehrli W (1983) Rifampin: Mechanisms of action and resistance. Rev Infect Dis 5: S407–S411.    


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