Previous Article | Next Article 
Applied and Environmental Microbiology, September 1999, p. 4248-4251, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Variation in Resistance to Hydrostatic Pressure
among Strains of Food-Borne Pathogens
H.
Alpas,1,2
N.
Kalchayanand,1
F.
Bozoglu,2
A.
Sikes,3
C. P.
Dunne,3 and
B.
Ray1,*
Department of Animal Science, University of
Wyoming, Laramie, Wyoming 820711; Food
Engineering Department, Middle East Technical University, Ankara 06531, Turkey2; and Sustainable Directorate,
U.S. Army Natick Research Development and Engineering Center,
Natick, Massachusetts 017603
Received 11 February 1999/Accepted 9 June 1999
 |
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.
| |
TEXT |
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.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Viability loss of strains of two gram-positive food-borne
pathogens following pressurization at 345 MPa for 5 min at 25°C
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Viability loss of strains of two gram-negative food-borne
pathogens following pressurization at 345 MPa for 5 min at 25°C
|
|
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.
View this table:
[in this window]
[in a new window]
|
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
|
|
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
log
10 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.
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.
| |
ACKNOWLEDGMENTS |
This study was funded by NATO Science Fellowships Program by The
Scientific and Technical Research Council of Turkey (TUBITAK), the
North Atlantic Treaty Organization (project no. CRG 960386), and the
U.S. Army Natick Research Development and Engineering Center,
Sustainability Directorate (contract DAAK 60-93-K-0003). H. Alpas was a
graduate student trainee at the University of Wyoming.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Animal Science, University of Wyoming, Laramie, WY 82071. Phone: (307) 766-3140. Fax: (307) 766-2355. E-mail: LABCIN{at}UWYO.EDU.
| |
REFERENCES |
| 1.
|
Benito, A.,
G. Ventoura,
M. Casadei,
T. Robinson, and B. Mackey.
1999.
Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses.
Appl. Environ. Microbiol.
65:1564-1569[Abstract/Free Full Text].
|
| 2.
|
Bradshaw, J. G.,
J. T. Peeler,
J. J. Corrin,
J. R. Barnett, and M. R. Twedt.
1987.
Thermal resistance of disease associated Salmonella typhimurium in milk.
J. Food Prot.
50:95-96.
|
| 3.
|
Cheftel, J.-C.
1992.
Effect of high hydrostatic pressure on food constituents: an overview, p. 195-209.
In
C. Balny, R. Hayashi, K. Heremans, and P. Masson (ed.), High pressure and biotechnology. Colloque INSERM. John Libbey and Co., Ltd., London, England.
|
| 4.
|
Hauben, K. J. A.,
E. Y. Wuytac,
C. F. Soontjens, and C. W. Michiels.
1996.
High pressure transient sensitization of Escherichia coli to lysozyme and nisin by disruption of outer membrane permeability.
J. Food Prot.
59:350-359.
|
| 5.
|
Hoover, D. G.,
C. Metrick,
A. M. Papineau,
D. F. Farkas, and D. Knorr.
1989.
Biological effects of high hydrostatic pressure on food microorganisms.
Food Technol.
43:99-107.
|
| 6.
|
Kalchayanand, N.,
B. Ray,
A. Sikes, and C. P. Dunne.
1998.
Enhancement of safety of processed meat by hydrostatic pressure in combination with temperature and bacteriocin, abstr. B35.
In
Meat consumption and culture, 44th International congress of meat science and technology, Congress proceedings, vol. II. .
|
| 7.
|
Kalchayanand, N.,
T. Sikes,
C. P. Dunne, and B. Ray.
1994.
Hydrostatic pressure and electroporation have increased bactericidal efficiency in combination with bacteriocins.
Appl. Environ. Microbiol.
60:4174-4177[Abstract/Free Full Text].
|
| 8.
|
Kalchayanand, N.,
A. Sikes,
C. P. Dunne, and B. Ray.
1995.
Bacteriocin based biopreservatives add an extra dimension in food preservation by hydrostatic pressure, p. 280-286.
In
Activities report of the R&D associates. Research and Development Associates for Military Food and Packaging Systems, Inc., San Antonio, Tex.
|
| 9.
|
Kalchayanand, N.,
A. Sikes,
C. P. Dunne, and B. Ray.
1998.
Factors influencing death and injury of foodborne pathogens by hydrostatic pressure-pasteurization.
Food Microbiol.
15:207-214.
|
| 10.
|
Kalchayanand, N.,
A. Sikes,
C. P. Dunne, and B. Ray.
1998.
Interaction of hydrostatic pressure, time and temperature of pressurization and pediocin AcH on inactivation of foodborne bacteria.
J. Food Prot.
61:425-431.
[Medline] |
| 11.
|
Knorr, D.
1993.
Effect of high hydrostatic pressure processes on food safety and quality.
Food Technol.
47:156-161.
|
| 12.
|
Ludwig, H.,
C. Bieler,
K. Hallbauer, and W. Scigalla.
1992.
Inactivation of microorganisms by hydrostatic pressure, p. 25-32.
In
C. Balny, R. Hayashi, K. Heremans, and P. Masson (ed.), High pressure and biotechnology. Colloque INSERM. John Libbey and Co., Ltd., London, England.
|
| 13.
|
Metrick, C.,
D. G. Hoover, and D. F. Farkas.
1989.
Effects of high hydrostatic pressure on heat resistant and heat sensitive strains of Salmonella J.
Food Sci.
54:1547-1549, 1564.
|
| 14.
|
Patterson, M. F., and D. J. Kilpatrick.
1998.
The combined effect of high hydrostatic pressure and mild heat on inactivation of pathogens in milk and poultry.
J. Food Prot.
61:432-436.
[Medline] |
| 15.
|
Patterson, M. F.,
M. Quinn,
R. Simpson, and A. Gilmore.
1995.
Sensitivity of vegetative pathogens to high hydrostatic pressure treatment in phosphate-buffered saline and foods.
J. Food Prot.
58:524-529.
|
| 16.
|
Sale, A. J. H.,
G. W. Gould, and W. A. Hamilton.
1970.
Inactivation of bacterial spores by hydrostatic pressure.
J. Gen. Microbiol.
60:323-334[Abstract/Free Full Text].
|
| 17.
|
Shigehisa, T.,
T. Ohmori,
A. Saito,
S. Taji, and R. Hayashi.
1991.
Effect of high pressure on characteristics of pork slurries and inactivation of microorganisms associated with meat and meat products.
Int. J. Food Microbiol.
12:207-216[Medline].
|
| 18.
|
Styles, M. F.,
D. G. Hoover, and D. F. Farkas.
1991.
Response of Listeria monocytogenes and Vibrio parahaemolyticus to high hydrostatic pressure.
J. Food Sci.
56:1404-1407.
|
Applied and Environmental Microbiology, September 1999, p. 4248-4251, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Bowman, J. P., Bittencourt, C. R., Ross, T.
(2008). Differential gene expression of Listeria monocytogenes during high hydrostatic pressure processing. Microbiology
154: 462-475
[Abstract]
[Full Text]
-
Lopez-Pedemonte, T., Sevilla, I., Garrido, J. M., Aduriz, G., Guamis, B., Juste, R. A., Roig-Sagues, A. X.
(2006). Inactivation of Mycobacterium avium subsp. paratuberculosis in Cow's Milk by Means of High Hydrostatic Pressure at Mild Temperatures.. Appl. Environ. Microbiol.
72: 4446-4449
[Abstract]
[Full Text]
-
Moussa, M., Perrier-Cornet, J.-M., Gervais, P.
(2006). Synergistic and Antagonistic Effects of Combined Subzero Temperature and High Pressure on Inactivation of Escherichia coli. Appl. Environ. Microbiol.
72: 150-156
[Abstract]
[Full Text]
-
Rodriguez, E., Arques, J. L., Nunez, M., Gaya, P., Medina, M.
(2005). Combined Effect of High-Pressure Treatments and Bacteriocin-Producing Lactic Acid Bacteria on Inactivation of Escherichia coli O157:H7 in Raw-Milk Cheese. Appl. Environ. Microbiol.
71: 3399-3404
[Abstract]
[Full Text]
-
Aertsen, A., Faster, D., Michiels, C. W.
(2005). Induction of Shiga Toxin-Converting Prophage in Escherichia coli by High Hydrostatic Pressure. Appl. Environ. Microbiol.
71: 1155-1162
[Abstract]
[Full Text]
-
Aertsen, A., Van Houdt, R., Vanoirbeek, K., Michiels, C. W.
(2004). An SOS Response Induced by High Pressure in Escherichia coli. J. Bacteriol.
186: 6133-6141
[Abstract]
[Full Text]
-
Aertsen, A., Vanoirbeek, K., De Spiegeleer, P., Sermon, J., Hauben, K., Farewell, A., Nystrom, T., Michiels, C. W.
(2004). Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli. Appl. Environ. Microbiol.
70: 2660-2666
[Abstract]
[Full Text]
-
Manas, P., Mackey, B. M.
(2004). Morphological and Physiological Changes Induced by High Hydrostatic Pressure in Exponential- and Stationary-Phase Cells of Escherichia coli: Relationship with Cell Death. Appl. Environ. Microbiol.
70: 1545-1554
[Abstract]
[Full Text]
-
Dogan, C., Erkmen, O.
(2003). Note: Ultra High Hydrostatic Pressure Inactivation of Escherichia Coli in Milk, and Orange and Peach Juices. Food Science and Technology International
9: 403-407
[Abstract]
-
Malone, A. S., Shellhammer, T. H., Courtney, P. D.
(2002). Effects of High Pressure on the Viability, Morphology, Lysis, and Cell Wall Hydrolase Activity of Lactococcus lactis subsp. cremoris. Appl. Environ. Microbiol.
68: 4357-4363
[Abstract]
[Full Text]
-
Karatzas, K. A. G., Bennik, M. H. J.
(2002). Characterization of a Listeria monocytogenes Scott A Isolate with High Tolerance towards High Hydrostatic Pressure. Appl. Environ. Microbiol.
68: 3183-3189
[Abstract]
[Full Text]
-
García-Graells, C., Valckx, C., Michiels, C. W.
(2000). Inactivation of Escherichia coli and Listeria innocua in Milk by Combined Treatment with High Hydrostatic Pressure and the Lactoperoxidase System. Appl. Environ. Microbiol.
66: 4173-4179
[Abstract]
[Full Text]
Top
Abstract
Text
