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

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.