Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses

Abstract

Strains of Escherichia coli O157 isolated from patients with clinical cases of food-borne illness and other sources exhibited wide differences in resistance to high hydrostatic pressure. The most pressure-resistant strains were also more resistant to mild heat than other strains. Strain C9490, a representative pressure-resistant strain, was also more resistant to acid, oxidative, and osmotic stresses than the pressure-sensitive strain NCTC 12079. Most of these differences in resistance were observed only in stationary-phase cells, the only exception being acid resistance, where differences were also apparent in the exponential phase. Membrane damage in pressure-treated cells was revealed by increased uptake of the fluorescent dyes ethidium bromide and propidium iodide. When strains were exposed to the same pressure for different lengths of time, the pressure-sensitive strains took up stain sooner than the more resistant strain, which suggested that the differences in resistance may be related to susceptibility to membrane damage. Our results emphasize the importance of including stress-resistant strains of E. coli O157 when the efficacy of a novel or mild food preservation treatment is tested.

FIG. 1

Pressure resistance of E. coli strains. Cells were grown to the stationary phase in TSB, harvested by centrifugation, resuspended in PBS (pH 7) (10⁹ CFU · ml⁻¹), and then treated at a pressure of 500 MPa. Each curve shows the average values obtained from at least two independent experiments. The mean standard deviation, shown for each curve, varied between 0.14 and 0.6. The strains used were E. coli O157 C9490 (●), 30-2C4 (□), NCTC 12079 (▴), W2-2 (○), and H1071 (■) and E. coli O124 NCTC 8003 (▵).

FIG. 2

Heat resistance of E. coli strains. Cells grown to the stationary phase in TSB were diluted 1:100 in fresh TSB (10⁷ CFU · ml⁻¹) and heated at 52°C (A) or 57°C (B). Each curve shows the average values obtained from at least two independent experiments. The mean standard deviation, shown for each curve, varied between 0.15 and 0.7. The strains used were E. coli O157 C9490 (●), 30-2C4 (□), NCTC 12079 (▴), W2-2 (○), and H1071 (■) and E. coli O124 NCTC 8003 (▵).

FIG. 3

Acid resistance of E. coli O157 at 37°C. Strains C9490 (● and ○) and NCTC 12079 (▴ and ▵) were grown to the stationary phase in TSB and diluted 1:100 in TSB acidified to pH 2.5 with HCl without an additive (solid symbols) or supplemented with 50 mM undissociated acetic acid (open symbols). The curves show the data obtained in single experiments.

FIG. 4

Resistance of E. coli O157 to high osmotic pressure. Strains C9490 (●) and NCTC 12079 (▴) were grown to the stationary phase in TSB and then diluted in TSB containing 20% sodium chloride at 37°C (10⁷ CFU · ml⁻¹). Each curve shows the average values obtained from at least two independent experiments. The mean standard deviations shown for each curve were around 0.4 in both cases.

FIG. 5

Pressure resistance of exponential-phase E. coli strains. Cells from an overnight culture were diluted 1:1,000 in fresh TSB, grown to a density of 10⁸ CFU · ml⁻¹, harvested by centrifugation, resuspended in PBS (pH 7.0), and treated at a pressure of 200 MPa. Each curve shows the average values obtained from at least two independent experiments. The strains used were E. coli O157 C9490 (●), 30-2C4 (□), NCTC 12079 (▴), and H1071 (■).

FIG. 6

Acid resistance of exponential-phase E. coli O157 strains at 37°C. Strains C9490 (● and ○) and NCTC 12079 (▴ and ▵) were grown to the stationary phase, diluted in fresh TSB (10⁶ CFU · ml⁻¹), grown to a concentration of 10⁸ CFU · ml⁻¹, and then inoculated (1:10) into TSB acidified to pH 3 with HCl without an additive (solid symbols) or supplemented with 50 mM undissociated acetic acid (open symbols). Each curve shows the average values obtained from at least two independent experiments. The mean standard deviation shown for each curve varied between 0.3 and 0.5.

FIG. 7

Uptake of fluorescent dyes by pressure-treated E. coli cells. EB (●) and PI (■) were added to stationary-phase cells of strains C9490 (A), NCTC 12079 (B), and H1071 (C) treated at a pressure of 500 MPa in PBS for different lengths of time. Viable counts (▵) were determined at each measurement time. Fluorescence was expressed as a percentage of the value obtained with cells heated at 80°C for 5 min, which was assumed to be 100%. The curves show the data obtained in single experiments.