Variation in Resistance to Hydrostatic Pressure among Strains of Food-Borne Pathogens

Abstract

Among food-borne pathogens, some strains could be resistant to hydrostatic pressure treatment. This information is necessary to establish processing parameters to ensure safety of pressure-pasteurized foods (N. Kalchayanand, A. Sikes, C. P. Dunne, and B. Ray, J. Food Prot. 61:425–431, 1998). We studied variation in pressure resistance among strains of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella species at two temperatures of pressurization. Early-stationary-phase cells in 1% peptone solution were pressurized at 345 MPa either for 5 min at 25°C or for 5, 10, or 15 min at 50°C. The viability loss (in log cycles) following pressurization at 25°C ranged from 0.9 to 3.5 among nine L. monocytogenes strains, 0.7 to 7.8 among seven S. aureus strains, 2.8 to 5.6 among six E. coli O157:H7 strains, and 5.5 to 8.3 among six Salmonella strains. The results show that at 25°C some strains of each species are more resistant to pressure than the others. However, when one resistant and one sensitive strain from each species were pressurized at 345 MPa and 50°C, the population of all except the resistant S. aureus strain was reduced by more than 8 log cycles within 5 min. Viability loss of the resistant S. aureus strain was 6.3 log cycles even after 15 min of pressurization. This shows that strains of food-borne pathogens differ in resistance to hydrostatic pressure (345 MPa) at 25°C, but this difference is greatly reduced at 50°C. Pressurization at 50°C, in place of 25°C, will ensure greater safety of foods.

Hydrostatic pressure is being investigated as a nonthermal processing technique to destroy food-borne microorganisms and enhance safety and shelf life of food (5, 11). Bacterial cells, yeasts, and molds are relatively sensitive to pressurization below 700 MPa, but bacterial spores, especially of Clostridium species, are quite resistant to it (5, 9–11, 16). The effectiveness of several variables in hydrostatic pressure-induced death of microbial cells has been reported. These include magnitude of pressure, pressurization time and temperature, microbial types, cell growth phase, suspending media, and the presence of antimicrobial substances (1, 9, 10, 13–15, 17, 18). In general, cell destruction increased with increases in pressure, pressurization time, and temperature; in suspending media with low solid content; and in the presence of antimicrobial substances. Gram-negative bacteria and cells in exponential growth phase were, respectively, more sensitive than gram-positive bacteria and stationary-phase cells. Microbial cells surviving pressurization also sustained sublethal injury in the wall and the membrane (4, 6–8, 10).

Limited studies have indicated that among the food-borne pathogenic bacterial species, some strains could be resistant to hydrostatic pressure than other strains. This could be the reason for variation in results obtained by researchers using different strains of the same species. For example, Listeria monocytogenes CA was more pressure resistant than L. monocytogenes ScottA (18). Differences in pressure resistance was also observed among three strains of L. monocytogenes, two strains of Salmonella spp., and three strains of Escherichia coli O157:H7 (15). In both studies, cells in phosphate buffer were pressurized at 20 to 25°C. It has been shown that bacterial cells were relatively less sensitive to hydrostatic pressure at 20 to 25°C but that above 35°C, they became highly sensitive to pressurization due to phase transition of membrane lipids (9, 10, 12). Recently we reported that a 7- to 8-log-cycle viability loss for eight bacterial species could be achieved by pressurization at 345 MPa for 5 min at 50°C (6, 10). However, in that study we did not consider the strain variation in pressure-resistant food-borne bacteria. We undertook this study to examine the variation in pressure resistance among six to nine strains of food-borne pathogens from four genera at 25 and 50°C. The results showed that variation in pressure resistance among strains exists mainly at lower temperature and not at 50°C.

Bacterial strains.

The following 28 strains were used: L. monocytogenes CA, Ohio₂, ScottA, 35091, 103, V7, Camp⁺/Beta⁺, 117, and SLR1; Staphylococcus aureus 315, 485, 565, 743, 765, 778, and 582; E. coli O157:H7 strains 932, 933, C7927, EDL 931, 35748-88, and SLR 503; Salmonella typhimurium ATCC 14028 and E 21274; Salmonella enteritidis VL and FDA; Salmonella choleraesuis subsp. choleraesuis ATCC 10708; and Salmonella choleraesuis subsp. choleraesuis serotype typhi ATCC 6539.

Pressurization of cell suspensions.

Cells from the early stationary phase of growth in tryptic soy broth (Difco, Detroit, Mich.) supplemented with 0.6% yeast extract at 37°C were used for pressurization. A culture broth was diluted with sterile 1% peptone solution to obtain about 10⁸ CFU/ml. The cell suspensions were dispensed in 2-ml portions in sterile plastic cryovials (Simport Plastics, Quebec, Canada). The vials were individually vacuum sealed in sterile plastic bags (Fisher Scientific, Pittsburgh, Pa.) and kept at 4°C prior to pressurization, which did not exceed 1 h. A hydrostatic pressurization unit (Engineered Pressure Systems, Wilmington, Mass.), capable of operating up to 690 MPa and between 22 and 95°C, was used. The pressure chamber (10-cm internal diameter by 45-cm length) was filled with a mixture of deionized water with 5% soluble oil (Hydrolubric 2; Houghton International, Valley Forge, Pa.). The liquid was warmed prior to pressurization to the desired temperature by an electric heating system around the chamber. The rate of pressure increase was about 140 MPa/min, and pressure come-down time was less than 2 min. Pressurization time reported in this study did not include the come-up and come-down times. The pressure level and time and temperature of pressurization were set by an automatic device, which recorded all the parameters during a pressurization cycle.

The cryovials containing cell suspensions were placed in a wire basket in duplicate and submerged in the liquid at 25 or 50°C in the pressure chamber. After the chamber was closed, the cell suspensions were kept for 5 min at 25°C and for 6 min at 50°C for temperature equilibration. These temperature and time relations for equilibration, especially for 50°C, were determined earlier. The cell suspensions were pressurized at 345 MPa either at 25°C for 5 min or at 50°C for 5, 10, and 15 min. Immediately after pressurization, the vials were removed, cooled in an ice bath, and stored at 4°C prior to enumeration of CFU per milliliter (within 2 h). Unpressurized cell suspensions were enumerated as controls.

Thermal inactivation of bacterial cell suspensions.

Cells from the early stationary phase of growth, diluted in 1% peptone solution to 10⁸ to 10⁹ CFU/ml, were used. Duplicate cryovials in vacuum-sealed plastic bags containing the cell suspensions were placed in a water bath that was set to 52°C for inactivation at 50°C and to 62°C for inactivation at 60°C for 5 and 6 min, respectively, for temperature equilibration. This temperature and time relation for equilibration was determined earlier. Duplicate vials were removed at 5, 10, and 15 min from the water bath and then cooled in an ice bath; the CFU per milliliter were enumerated within 30 min. The cells in unheated suspensions were enumerated as controls.

Enumeration of viable CFU.

Pressurized, thermally-inactivated, and control cell suspensions were serially diluted in 0.1% peptone solution. From the selected dilutions, 0.1-ml portions were surface plated in duplicate on prepoured tryptic soy agar plates (Difco) supplemented with 0.6% yeast extract. With samples containing less than 30 CFU/ml in a 1:10 dilution, 1 ml of undiluted cell suspension was plated in three plates (0.3, 0.3, and 0.4 ml). The plates were incubated at 37°C for 2 days before enumeration. Each experiment, with duplicate vials for each strain, was performed twice, and the average results are presented.

Variation in pressure resistance among strains of food-borne pathogens at 25°C.

Among the nine L. monocytogenes strains, the viability loss ranged from 0.92 to 3.53 log cycles (Table 1). The most resistant (strain CA) and most sensitive (strain SLR1) differed in viability loss by a factor of 4. Strain ScottA, which has been used in many L. monocytogenes studies, was also quite resistant to pressure (7, 9, 10, 15, 18). Viability loss among seven S. aureus strains ranged from 0.7 to 7.8 log cycles, about an 11-fold difference between the most resistant and most sensitive strains. In the six E. coli O157:H7 strains, viability loss ranged from 2.8 to 5.64 log cycles, which is about a twofold difference between the most resistant and most sensitive strains (Table 2). Among the six Salmonella strains, the smallest viability loss, of 5.45 log cycles, was in strain S. enteritidis FDA, and the highest, of 8.34 log cycles, was in strain S. choleraesuis subsp. choleraesuis ATCC 10708; this is about a 1.5-fold difference. For the two strains of either S. typhimurium or S. enteritidis, viability loss differed by about 1.5 to 2.0 log cycles. Under the conditions of these studies, the strains of pathogens differed widely in resistance to hydrostatic pressure.

TABLE 1.

Viability loss of strains of two gram-positive food-borne pathogens following pressurization at 345 MPa for 5 min at 25°C

Bacterial strain	Log10 CFU/mla		Viability loss (log10)
	Control	Pressurized
L. monocytogenes
CA	8.00	7.08	0.92
ScottA	8.00	7.04	0.96
Camp+ Beta+	8.00	6.26	1.74
V7	8.04	6.11	1.93
35091	8.00	5.90	2.10
117	7.78	5.30	2.48
103	7.90	5.41	2.49
Ohio2	7.95	5.15	2.80
SLR1	8.00	4.47	3.53
S. aureus
778	8.18	7.48	0.70
485	8.11	7.41	0.70
743	8.14	7.28	0.86
315	8.18	7.28	0.90
565	8.26	7.14	1.12
765	8.11	6.60	1.51
582	8.80	1.00	7.80

^an = 8. 

TABLE 2.

Viability loss of strains of two gram-negative food-borne pathogens following pressurization at 345 MPa for 5 min at 25°C

Bacterial strain	Log10 CFU/mla		Viability loss (log10)
	Control	Pressurized
E. coli O157:H7 strains
933	8.28	5.48	2.80
C7927	8.25	5.39	2.86
931	8.34	5.08	3.26
SLR 503	8.32	5.04	3.28
35748-88	8.30	3.95	4.35
932	8.26	2.62	5.64
Salmonella spp.
S. enteritidis FDA	8.14	2.69	5.45
S. typhimurium E21274	8.60	2.89	5.71
S. typhimurium ATCC 14028	8.30	1.00	7.30
S. enteritidis VL	8.78	1.30	7.48
S. choleraesuis subsp. choleraesuis typhi ATCC 6539	7.70	NDb	7.70
S. choleraesuis subsp. choleraesuis ATCC 10708	8.34	ND	8.34

^an = 8. 

^bND, no CFU were detected in 1 ml of cell suspension from each of the samples tested. 

Sensitivities of pressure-resistant and pressure-sensitive strains to pressure at 50°C.

Two strains, one resistant and one sensitive, from each species were used in this study. The cell suspensions were subjected to 345 MPa at 50°C for 5, 10, or 15 min, and survivors were enumerated as before (Table 3). No survivors were detected for seven of eight strains after 5 min of pressurization at 50°C; a viability loss of more than 8 log cycles occurred in all seven strains. The viability losses in pressure-resistant S. aureus 485 were 5.38, 6.08, and 6.30 log cycles, respectively, after pressurization for 5, 10, and 15 min. In comparison, viability loss due to thermal inactivation alone at 50°C was less than 2 log cycles after 15 min in all eight strains. However, in all four genera, the pressure-resistant strains were also comparably resistant to thermal treatment.

TABLE 3.

Viability loss of pressure-resistant and pressure-sensitive strains of food-borne pathogens caused by a combination of 345 MPa and 50°C or by thermal inactivation at 50°C

Bacterial strain	Log10 CFU/mla after pressurization for:				Log10 CFU/mla after thermal inactivation for:
	0 min (control)	5 min	10 min	15 min	0 min (control)	5 min	10 min	15 min
L. monocytogenes
CA	8.11	NDb	ND	ND	8.30	7.60	7.23	7.00
Ohio2	8.00	ND	ND	ND	8.14	7.04	6.90	6.70
S. aureus
485	8.08	2.70c	2.00c	1.78c	8.11	8.08	7.60	7.48
765	8.11	ND	ND	ND	8.08	7.46	7.15	7.04
E. coli O157:H7
933	8.30	ND	ND	ND	8.04	7.30	6.95	6.48
931	8.18	ND	ND	ND	8.11	6.84	6.60	6.30
Salmonella spp.
S. enteritidis FDA	8.18	ND	ND	ND	8.32	7.26	7.08	6.70
S. typhimurium E 21274	8.39	ND	ND	ND	8.08	6.60	6.48	6.00

^an = 8. 

^bND, no CFU were detected in 1 ml of cell suspension from each of the samples tested. 

^cThe survivors of S. aureus 485 following pressurization after 5, 10, and 15 min failed to form colonies in tryptic soy agar–0.6% yeast extract supplemented with 5% NaCl due to sublethal injury (9). 

From the viability loss data (Table 3), decimal reduction times (D values) were calculated (2, 10). Where no survivors were detected after 5 min, D values were estimated by dividing the initial log₁₀ CFU/ml by 5. The D value for pressurization for seven strains was ≤0.60; for S. aureus 485, it was 2.55 (Table 4). The D values for thermal treatment were much higher, at both 50 and 60°C. These results indicate that the D value of a bacterial species or strain due to thermal treatment at a lower temperature range (50 or 60°C) can be greatly reduced by combining thermal treatment with pressurization even at a moderate pressure range of 345 MPa.

TABLE 4.

Estimated D values of strains following pressurization and thermal inactivation

Bacterial strain	D valuea after:
	Pressurization at 345 MPa and 50°C	Thermal inactivation at temp
		50°C	60°C
L. monocytogenes
CA	≤0.62	11.71	7.12
Ohio2	≤0.62	11.21	6.56
S. aureus
485	2.55	21.10	6.91
765	≤0.62	14.58	6.33
E. coli O157:H7
933	≤0.60	9.94	4.96
EDL 931	≤0.61	8.82	4.78
Salmonella spp.
S. enteritidis FDA	≤0.61	9.92	3.68
S. typhimurium E 21274	≤0.60	7.86	1.96

^aCalculated from the absolute value of the inverse of the slope from linear regression between logarithm of survivors and times (10). In the absence of any survivors, D value was estimated by dividing the initial population (log₁₀/ml) by time (in minutes). Each value is the mean of eight counts. 

Parameters for hydrostatic pressure pasteurization of food have to be developed to reduce populations of vegetative cells of food-borne pathogens probably by more than 6 log cycles. At ambient temperature (25 to 30°C), viability loss of this magnitude may be achieved either at a high pressure range of 600 to 700 MPa, in 15 min (15) or over 40 min at 350 MPa (13). The quality of many protein-rich foods could be adversely affected by processing at such ultrahigh pressure or for a prolonged period (3, 17).

Ultrahigh pressure and log pressurization time are also not economical and may not be commercially acceptable (5, 10). Elevated temperature, lower pH, and antibacterial compounds, such as bacteriocins, lysozyme, and chitosan, were found to considerably enhance bactericidal efficiency of hydrostatic pressure (4, 8, 10, 11). In view of the limited available information on differences in pressure resistance among strains of food-borne pathogens (13, 15, 17), we undertook the present study to determine how widely strains of food-borne pathogens differ in pressure resistance and how this is modified by moderate pressurization at 50°C.

The results of this study confirmed the results of other researchers (1, 14, 15, 18) showing that at a lower temperature of pressurization, the strains of a species varied in pressure resistance. However, this difference in pressure resistance was greatly eliminated by pressurizing the cells at 50°C, even for 5 min. Thus, a combination of moderate hydrostatic pressure (such as 345 MPa) and a temperature of 50°C can be used to obtain a viability loss of pathogens of more than 6 log cycles. Incorporation of other parameters, such as the presence of a bacteriocin, during pressurization will increase the viability loss of pathogens further (7, 9). This combination treatment could be used for pressure pasteurization of foods. Also, in developing pressurization parameters, pressure-resistant strains of bacterial species should be used to ensure greater safety of foods.