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Appl Environ Microbiol, June 1998, p. 2065-2071, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Above-Optimum Growth Temperature and
Cell Morphology on Thermotolerance of Listeria monocytogenes
Cells Suspended in Bovine Milk
Neil J.
Rowan* and
John G.
Anderson
Department of Bioscience and Biotechnology,
University of Strathclyde, Glasgow, Scotland
Received 21 November 1997/Accepted 20 March 1998
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ABSTRACT |
The thermotolerances of two different cell forms of Listeria
monocytogenes (serotype 4b) grown at 37 and 42.8°C in
commercially pasteurized and laboratory-tyndallized whole milk (WM)
were investigated. Test strains, after growth at 37 or 42.8°C, were
suspended in WM at concentrations of approximately 1.5 × 108 to 3.0 × 108 cells/ml and were then
heated at 56, 60, and 63°C for various exposure times. Survival was
determined by enumeration on tryptone-soya-yeast extract agar and
Listeria selective agar, and D values (decimal reduction
times) and Z values (numbers of degrees Celsius required to cause a
10-fold change in the D value) were calculated. Higher average recovery
and higher D values (i.e., seen as a 2.5- to 3-fold increase in
thermotolerance) were obtained when cells were grown at 42.8°C prior
to heat treatment. A relationship was observed between thermotolerance
and cell morphology of L. monocytogenes. Atypical
Listeria cell types (consisting predominantly of long cell
chains measuring up to 60 µm in length) associated with rough (R)
culture variants were shown to be 1.2-fold more thermotolerant than the
typical dispersed cell form associated with normal smooth (S) cultures
(P 
0.001). The thermal death-time (TDT) curves of
R-cell forms contained a tail section in addition to the shoulder section characteristic of TDT curves of normal single to paired cells
(i.e., S form). The factors shown to influence the thermoresistance of
suspended Listeria cells (P 0.001) were
as follows: growth and heating temperatures, type of plating medium,
recovery method, and cell morphology. Regression analysis of nonlinear
data can underestimate survival of L. monocytogenes; the
end point recovery method was shown to be a better method for
determining thermotolerance because it takes both shoulders and tails
into consideration. Despite their enhanced heat resistance, atypical
R-cell forms of L. monocytogenes were unable to survive the
low-temperature, long-time pasteurization process when freely suspended
and heated in WM.
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INTRODUCTION |
Listeria monocytogenes is
a facultative anaerobic bacterium that is distributed ubiquitously in
the environment (19) and has a higher thermotolerance than
many other nonsporeforming food-borne pathogens (14, 21).
Because this potentially lethal pathogen is found occasionally in raw
milk (2, 10, 11, 14, 36) and in other nonprocessed foods
(2, 19, 25, 26) and can grow in foods under refrigerated
storage (22), considerable emphasis has been placed on its
complete destruction during pasteurization and during other minimal
thermal food processes (3, 4, 6-8, 12-14, 31, 32).
Many factors are known to influence microbial thermotolerance in foods
such as the composition of the food and the physiological condition of
vegetative cells and spores (15, 24, 52). Bacterial thermotolerance can also increase after exposure to a variety of
environmental stress conditions including heating at sublethal temperatures, presence of deleterious chemicals in the growth medium
(e.g., hydrogen peroxide, dyes, and antibiotics, etc.), viral
infections, and osmotic and acidic shocks (18, 19, 29).
Any temperature above the optimum growth temperature will exert a
stress effect (24); while the optimum temperature for growth
of L. monocytogenes lies between 30 and 37°C, it can grow between 1 and 45°C (19, 48). For most microbial species
growth at or short-term exposure to temperatures above optimum induces higher thermotolerances (1, 37-40). It is believed that
these temperatures trigger physiological responses that lead to the synthesis of special proteins known as heat shock proteins (HSPs) (31, 33). In Escherichia coli K-12, when cells
were shifted to 42°C from an incubation temperature of 30°C, the
rate of HSP formation increased 5 to 20-fold (cited in reference
48). Gram-positive and -negative bacteria appear to
behave similarly: Streptococcus faecalis grown at 45°C
were more heat resistant than when grown at 27°C (53),
while the D55°C value for Salmonella
senftenberg 775W increased as growth temperature was raised from
15 to 44°C (43).
Many authors have reported an increase in the thermotolerance of
L. monocytogenes as a result of heat shock prior to heating (20, 28, 31, 34, 35, 44). Results from Linton et al. (35) and Pagán et al. (44) are particularly
interesting as they suggest that the magnitude of the effect of heat
shock treatments is highly dependent on the temperature and duration of
the treatment, with higher heating temperatures and longer treatments
favoring an increase in heat resistance. Whereas the former researchers observed that L. monocytogenes attained its greatest
thermotolerance after 20 min of heat shocking at 48°C, Pagán et
al. (44) reported a sevenfold increase in thermotolerance
achieved by extending the duration of heat shock to 180 min at 45°C.
While most thermotolerance studies have utilized L. monocytogenes cells at or below 37°C (3, 4, 6-8, 10, 12,
13, 23, 51), the body temperature of a cow suffering from
listeriosis can reach as high as 42.8°C (10, 14). Doyle et
al. (14) reported the low-level survival of L. monocytogenes in high-temperature short-time (HTST)-pasteurized milk from a cow that had been artificially infected with the organism. Also, Knabel et al. (31) showed that growth of
Listeria at temperatures above 37°C for 18 h (39 to
43°C) resulted in cells that were sixfold more thermotolerant than
cells grown at 37°C. Interestingly, both research groups used
serotype 4b strains, a serotype implicated in a number of fatal
food-borne outbreaks (19, 21, 25, 26) and shown recently by
Sörqvist to be the second-most heat resistant of seven serotypes
examined (49). While production of HSPs are typically a
response to temperature upshifts, extended growth at above-optimum
temperatures has been shown to result in the expression of HSPs
(45).
Microbial cell morphology has been linked with increased
thermotolerance (41, 46). The acquisition of thermotolerance in heat-shocked Aquaspirillum arcticum was shown to be
directly related to the formation of long cells (40).
Jørgensen et al. (29) showed that L. monocytogenes cells grown in medium containing 1.5 mol of NaCl
liter
1 prior to heating were 22-fold more heat tolerant
than similarly treated cells grown with 0.09 mol of NaCl
liter1. Cells grown in medium containing 1.5 mol of NaCl
liter1 became 50 times longer, but no link to
thermotolerance could be made. The change from short rods to long cell
chains also occurs under nonstress conditions; Kuhn and Goebel
(32) showed that spontaneous mutants of L. monocytogenes that form long cell chains occur at a relatively
high frequency (about 1 in 10,000 colonies). These long cell chain
forms exhibited thermal death-time (TDT) curves that were characterized
by both shoulder and tail sections. Other researchers have reported
similar TDT curves in L. monocytogenes as a result of heat
shocking (20, 31, 34, 35); none, however, have mentioned
whether the cell morphology of this organism had altered as a result of
heating.
The present study was undertaken to investigate the effect of growth at
the above-optimum temperature of 42.8°C (i.e., similar to the body
temperature of a cow infected with L. monocytogenes) on the
heat resistance of different cell forms of L. monocytogenes (serotype 4b). Thermotolerance was calculated by determining both D
values (decimal reduction times) and Z values (numbers of degrees Celsius required to cause a 10-fold change in the D value) and by using
the end point recovery method.
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MATERIALS AND METHODS |
Bacterial culture and media.
Two different morphological
culture forms of L. monocytogenes were used, the normal
smooth or S type, consisting of short single and/or paired rods (0.4 to
0.5 µm in diameter and 0.5 to 2 µm in length), and the atypical
rough or R type, predominantly consisting of long cell chains of up to
60 µm in length (Fig. 1). The two
S-type strains used were NCTC 9863 and 11994 (both of these serotype 4b
strains were originally isolated from patients with meningitis and are
referred to as S1 and S2, respectively, throughout the text) and were obtained from the National Collection of
Type Cultures, Public Health Laboratories, Colindale, London, United
Kingdom. The two R-form culture variants, R1 and
R2, were derived previously from the S1 and
S2 strains, respectively (46), via heating
studies as follows: the parent S1 and S2
cultures were grown in tyndallized whole milk (WM) at 42.8°C for
24 h without shaking prior to being heated at 60°C for 7 min and
at 63°C for 3 min. The R1 and R2 culture
variants were obtained on tryptone-soya agar plates supplemented with
0.6% yeast extract (TSYEA). The cultures were plated immediately after
the 7- and 3-min intervals described above and were incubated for
48 h at 37°C. The purity of the strains was confirmed by Gram,
catalase, and oxidase reactions; tumbling motility at 25°C; CAMP test
reaction; and biochemical profiling with the API Listeria
gallery (Biomérieux Ltd.). Stock cultures were grown on TSYEA at
37°C for 18 to 20 h and were maintained at 4°C with monthly
transfer.
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FIG. 1.
Cell chain development in L. monocytogenes
NCTC 11994 (R1). A phase contrast micrograph (Nikon
Optiphot-2 light microscope) is shown. Bar = 5µm.
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Fifty milliliters of tryptone-soya broth supplemented with 0.6% yeast
extract (TSYEB) contained in a 250-ml flask was inoculated
with the
test strain and incubated at 30°C with shaking at 150
rpm on a rotary
incubator (model RFI-125; INFORS AG, Botlmingen,
Switzerland). Growth
was monitored by measuring the optical density
at 625 nm
(OD
625) of the culture with a spectrophotometer (model
UV-120-02; Shimadzu Corp., Kyoto, Japan). Cells from the
late-exponential
phase (an absorbance of 0.2 at OD
625,
yielding approximately 10
9 Listeria cells/ml)
were harvested by centrifugation at 3,000
×
g in a
refrigerated (4°C) centrifuge, washed twice, and resuspended
in 5 ml
of precooled phosphate-buffered water (0.01 M, pH 7 at
4°C). Two
duplicate 250-ml flasks each containing 50 ml of commercially
pasteurized WM were tyndallized to sterility and inoculated to
give
initial cell densities of approximately 10
3 cells/ml.
Cultures were grown without shaking for 24 h at 37
or 42.8°C to
the respective maximum stationary stage.
Thermal resistance studies and enumeration.
Heat treatments
were performed using screw-cap 28-ml dilution bottles containing 10 ml
of WM. The bottles were equilibrated at 56, 60, or 63°C utilizing a
circulating constant temperature water bath (model HE30; Grant
Instruments Ltd., Cambridge, United Kingdom) equipped with a
thermoregulator capable of maintaining temperature to within ± 0.05°C (model TE-8A; Techne Ltd., Cambridge, United Kingdom); the
level of the water in the water bath was maintained ca. 5 cm above the
submerged bottles. A mercury thermometer was inserted into an
uninoculated bottle and was checked periodically during the
experimental runs to ascertain that the heating temperatures were
maintained. One milliliter of the overnight-grown cells was then added
to give 1.5 × 108 to 3 × 108 CFU/ml
of WM. At predetermined heating intervals (over 2 h at 56°C,
1 h at 60°C, and 30 min at 63°C), a 1-ml sample was removed from each of the bottles and added to 9 ml of WM. The bottles containing heat-treated samples were transferred quickly into a beaker
containing tap water at 22°C and held for 1 min (which ensured
near-instantaneous cooling of the samples) and then placed into a
beaker containing an ice-water mixture. Preliminary experiments showed
no growth during this period of time (data not shown) (46). Heat treatments at each temperature were repeated at least twice.
Recovery of surviving cell populations was determined at each heating
interval in the heat-treated suspensions, and dilutions
thereof, by
spread, pour, and spiral plating samples (model B;
Spiral Systems Inc.,
Shipley, United Kingdom) onto TSYEA and
Listeria selective
agar (LSA) (Oxford formulation; Oxoid). Successive dilutions
were
performed with 9 ml of WM, and all counts were done with
triplicate
plates. WM was used as the diluent to provide greater
protection
against the deleterious effects of heat treatment when
L. monocytogenes cells were grown, heat treated, and enriched
in the
same nutrient medium compared with that provided by changing
the
nutritional composition of the heating menstruum and/or the
diluent
(data not shown) (
46). Plates were incubated aerobically
at
37°C for 48 h, and colonies were counted. The cell morphologies
of three randomly selected colonies were examined per plate. All
bottles containing treated cultures from each sample heating interval
were then incubated and/or resuscitated without shaking at 30°C
for 2 days, and total aerobic counts were carried out by using
both plating
media as described above. This latter recovery method
is referred to
herein as the end point recovery method, where
the end point is defined
as the last sample interval resulting
in growth of heat-injured cells
(i.e., the last sample interval
just before reaching the interval
yielding total inactivation
of suspended cells) at each treatment
temperature. Near the end
point, samples were removed at 2-, 1-, and
1-min intervals at
56, 60, and 63°C, respectively.
Statistical methods.
All experiments in this study were
performed in triplicate, and results are reported as averages.
Estimates of thermal resistance at each heating temperature were
expressed as D values calculated as the absolute value of the inverse
slope of the least square regression line fitted to log10
reduction in viable cell numbers versus heating time. A linear
regression was computed from log10 D value versus heating
temperature, and the Z value was computed as the absolute value of the
inverse of the slope. Temperatures used to determine Z values were 56, 60, and 63°C, and thermotolerance was computed by using both
selective and nonselective media. Effects of growth and heat treatment
temperatures, plating media, type of recovery method, and cell
morphology on thermal resistance were calculated at the 95 and 99.9%
confidence intervals by analysis of variance (balanced model) with
Minitab software Release 11 (Minitab Inc., State College, Pa.).
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RESULTS AND DISCUSSION |
Effects of growth temperature and other factors on the
thermotolerance of L. monocytogenes.
Growth and heating
temperatures and types of plating media and recovery methods had
significant effects (P < 0.001) on the survival of
heated L. monocytogenes (Tables
1 and 2).
Growth of Listeria cells in bovine WM at the above-optimum
temperature of 42.8°C resulted in a 2.5- to 3-fold increase in
thermotolerance (P 0.001) compared to that for
growth at 37°C (Table 1). This enhanced thermotolerance was seen at
each heating temperature (Table 1). Table 2 lists the factors that
significantly influenced the heat resistance of Listeria
cells shown in Table 1. While treatment temperature was shown to have
the most significant effect on thermotolerance (i.e., the higher
heating temperatures resulting in greater reductions in cell numbers),
other factors which provided greater levels of cell protection were
growth at 42.8°C prior to heat treatment and recovery of thermally
injured cells on the nonselective TSYEA medium (Tables 1 and 2).
Greater recovery of heat-injured Listeria cells was obtained
after a 2-day enrichment period in WM (Table
3). Results from total viable counts
(data not shown) showed that growth occurred in the enriched bottles at
each sample interval up to and including the end point.
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TABLE 1.
Heat resistance of S- and R-culture forms of L. monocytogenes grown in WM at 37 or 42.8°C prior to heating at
56, 60, and 63°C as determined by the interval sample plating method
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TABLE 2.
Factors shown to influence the thermotolerance
of L. monocytogenes suspended in bovine WM as determined
by analysis of variance (balance design)
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TABLE 3.
Effects of growth temperature on the thermotolerances of
S- and R-cell forms of L. monocytogenes using the end point
recovery methoda
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The results of the present study are in agreement with the findings of
other researchers (
14,
31), who showed that growth
of
L. monocytogenes cells at an above-optimum temperature
(i.e.,
39 to 43°C) resulted in an increase in thermotolerance. Growth
of
L. monocytogenes F5069 under anaerobic conditions at
43°C prior
to heat treatment and enumeration of survivors resulted in
D
62 · 8°C values that were at least sixfold
greater than those previously
obtained by using cells grown at 37°C
and with aerobic plating
(
31). Whereas Knabel et al.
(
31) reported that under their
test conditions, high levels
of
L. monocytogenes survived the
minimum low-temperature,
long-time (LTLT) pasteurization process,
we showed that
L. monocytogenes cells grown at 42.8°C were less
heat tolerant,
surviving approximately 27 min of heating at 63°C
when enumerated
after a 2-day enrichment period. Knabel and coworkers
attributed the
greater recovery of severely heat-injured
L. monocytogenes cells to the absence of O
2 in the enrichment medium (i.e.,
the
O
2 sensitivity of heat-injured
Listeria
cells has been attributed
in part to the inactivation of catalase and
superoxide dismutase).
Dallmier and Martin (
9) reported that
catalase and sodium dismutase
were rapidly inactivated when
Listeria cells were heated at temperatures
of 55 to 60°C.
Inactivation of these two enzymes was thought to
result in the
accumulation of toxic levels of O
2 products, such
as
O
2 and H
2O
2.
The production and action of a specific set of HSPs, synthesized during
the growth of
L. monocytogenes cells at 42.8°C, may
also
account for the acquired thermotolerance in the present study
(
27,
31,
45). While the precise role of HSPs in acquired
heat
resistance remains controversial, HSPs might help cells cope
with
stress-induced damage by promoting the degradation of abnormal
proteins
(e.g., lon and Clp proteases) and/or the reactivation
of stress-damaged
proteins by functioning as molecular chaperones,
preventing the
aggregation and promoting the proper refolding
of denatured proteins
(
45). Rapid degradation of damaged proteins
reduces the
possibility of deleterious interactions between polypeptides
and
functional proteins, prevents accumulation of insoluble aggregates,
and
releases the amino acids contained in nonfunctional polypeptides
for
synthesis of new proteins (
45). As is the case for all
organisms
studied so far,
L. monocytogenes responds to
sudden increases
in temperature by synthesizing a particular set of
HSPS (
28).
Some stress-induced proteins are also produced in
organisms in
order to sustain long-term survival at above-optimum
temperatures
(
45). For instance, in
Saccharomyces
cerevisiae, some HSPs are
required for growth at temperatures near
the upper end of the
normal growth range (e.g., HSP70), others are
required for long-term
survival at moderately high temperatures (e.g.,
ubiquitin), and
still others are required for tolerance to extreme
temperatures
(e.g., HSP104) (
45). In some cases, expression
of genes encoding
for the production of proteins associated with one
stimulus (e.g.,
heat shock) can be induced during other stresses; for
example,
various HSPs in
E. coli cells are also synthesized
when the cells
are exposed to hydrogen peroxide, ethanol, UV,
puromycin, and
nutrient or amino acid deprivation (
27).
Therefore, the large
increase in heat resistance observed when
L. monocytogenes cells
were grown at 42.8°C, compared with that of
cells grown at 37°C,
may have been due to the accumulation of large
amounts of postexponential
HSPs that were induced by elevated growth
temperature, nutrient
deprivation, and/or other stresses (
27,
45). Jenkins et al.
(
27) concluded that the increased
heat resistance of stationary-phase
E. coli cells could have
been a result of synthesis of postexponential
HSPs that were induced
during glucose starvation. The induction
of HSPs, therefore, may
prepare cells for growth at elevated sublethal
temperatures while
playing only a minor role in acquired thermotolerance
at lethal
temperatures.
Under the present test conditions, heat-injured
Listeria
cells suspended in the heating menstruum were subjected to minimal
mixing before enumeration and then enriched for 2-days without
shaking.
Due to the differences in cell recovery between immediate
aerobic
plating and 2-day enrichment methods (Table
3), it is
probable that the
enhanced thermotolerance seen with the latter
method was due in part to
the provision of a reduced O
2 environment
in the enrichment
bottles. Nonetheless, R-form cells cultivated
at 42.8°C prior to
heating at 63°C were recovered from the 27-min
sample interval, i.e.,
an additional heat tolerance of 16 min
compared with that of the
immediate plating method resulted (Table
3). As heat-injured cells grew
in WM over the 2-day enrichment
period, it was not possible to convert
end point survivor data
to D values (Table
3). The enhanced recovery of
cells grown at
42.8°C seen after enrichment in WM at 30°C is
important in terms
of milk safety, because HTST pasteurization (which
is used commercially)
has been shown to result in approximately 10-fold
fewer log reductions
of
L. monocytogenes than the LTLT
pasteurization process used
in this study (
6). Therefore,
under similar growth and recovery
conditions (enrichment in WM at
30°C), HTST pasteurization might
yield survivors that can grow in
milk.
The significance of
L. monocytogenes in relation to food
safety is mainly due to its ability to grow in foods under refrigerated
storage (
2,
10,
11,
14,
28). Many authors have reported
that
in non-heat-shocked cells of different bacterial species,
higher growth
temperatures lead to higher thermotolerance (
31,
37-39).
There is little knowledge, however, about the influence
of growth at
above-optimal temperatures in relation to the acquisition
and
maintenance of thermotolerance in foods during refrigeration.
Whereas
the thermotolerance developed by
Salmonella typhimurium after heat shocking was lost during storage in just 1 h
(
37),
Farber and Brown (
17) reported that
L. monocytogenes still maintained,
after a 24-h period at
6°C, the heat resistance developed after
a 2-h heat shock at 48°C.
Smith and Marmer (
47) showed that
L. monocytogenes grown at 10°C did not attain the heat resistance
of non-heat-shocked cells grown at 37°C. Pagán and coworkers
(
44), however, showed that storage of heat-shocked
L. monocytogenes (42.5°C for 180 min) for 24 h at 4°C before
heating did not affect
the D values seen for heat-shocked cells. These
authors also reported
that for non-heat-shocked
Listeria
cells stored for 7 and 14 days
at 4°C, the proportion of cells
responding to heat shock not only
depended on the duration of heat
shock but also on the duration
of previous storage. The longer the
duration of heat shock and
the shorter the duration of storage, the
greater the proportion
of cells responding to heat shock. For instance,
after a 14-day
storage at 4°C, Pagán et al. (
44)
reported that only 10% of
the cells given the longest heat shock of
120 min responded, while
only 1.5% of those given the mildest heat
shock of 15 min did
so.
Factors affecting the thermotolerance of L. monocytogenes R- and S-cell forms.
The thermotolerance of
the R- and S-cell forms varied as a result of the various growth,
heating, and recovery conditions used in the present study. Atypical
R-type culture forms (predominantly consisting of long cell chains)
exhibited higher D and Z values (Table 1) than those of typical S forms
(consisting of single and paired cells). R-cell forms were 1.2-fold
more heat tolerant as measured by D value ratios at each heating
temperature (Table 1). R cultures obtained from the two L. monocytogenes strains (NCTC 11994 and 9863) did not differ in
thermotolerance nor did the parent S forms (P 0.001). Both cell forms of L. monocytogenes grown at 37 and
42.8°C showed nonlinear TDT curves (Fig.
2). Whereas the R-form TDT curves
contained both a shoulder and tail section, S-form survivor curves
exhibited a shoulder section only (Fig. 2).
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FIG. 2.
Thermal resistance of S- and R-cell forms of L. monocytogenes NCTC 11994 (A) and NCTC 9863 (B) grown at 37 and
42.8°C prior to heating at 60°C.
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Atypical R-form cells consistently survived longer at 62.8°C than
S-form cells as shown by both recovery methods (Table
3).
Both cell
forms were recovered after a longer duration of heating
when enumerated
on TSYEA immediately after heating and after a
2-day enrichment period
(Table
3). Other researchers also reported
inferior recovery of
heat-injured
L. monocytogenes as a result
of enumeration on
selective plating media (
31,
34,
35,
47,
48). Despite their
enhanced thermotolerance, atypical cell forms
of
L. monocytogenes were unable to survive the LTLT pasteurization
process (Table
3). Jørgensen et al. (
29) also reported the
existence of long cell chains in
L. monocytogenes which
exhibited
TDT curves that contained both a shoulder and tail section
post
heating. These atypical
Listeria cells, however, were
obtained
by severe osmotic shock due to growth in medium containing 1.5
mol of NaCl/liter, and these cells became up to 50 times longer
than
cells grown in medium containing 0.09 mol of NaCl/liter.
Cells which
had adapted to a high salinity before heat treatment
showed a 10-fold
increase in thermotolerance in minced beef compared
to a 22-fold
increase in tryptic phosphate broth. However, no
link between the
acquisition of thermotolerance and cell morphology
was made. While TDT
curves obtained during the present study,
as a result of growth at an
above-optimum temperature, were similar
to the type of survivor curves
obtained by Jørgensen et al. (
29),
atypical
Listeria cells were shown to be 1.2-fold more heat tolerant
than single-celled cultures at each heating temperature (Table
1).
McCallum and Inniss (
41) reported a direct link between
thermotolerance and cell morphology, where the acquisition of
thermotolerance in
A. arcticum was related to the formation
of
filamentous cells.
It is particularly interesting that the majority of researchers
reporting greater thermotolerance in
L. monocytogenes, as
a
result of growth at above-optimal temperatures and/or heat shock,
showed nonlinear TDT curves that contained both a shoulder and
tail
section (
16,
20,
37,
44) or just an initial shoulder
section
(
30,
35,
50). In addition, the majority of researchers
reporting survivor curves in
L. monocytogenes used the same
serotype,
4b (
16,
20,
29,
31,
35,
37,
50); this serotype has
been isolated from patients with meningitis and from foods implicated
in a number of food-related illnesses (
19,
21,
25,
26).
Irrespective of the shape of the thermal death kinetic data,
calculations on the level of thermotolerance in
Listeria
cells
have been based on logarithmic death kinetics (i.e., D values
that were calculated as the absolute value of the inverse slope
of the
least square regression line fitted to log reduction in
viable cell
numbers versus heating time) (
16,
20,
31,
35,
37,
50). Very
often no allowance has been made in these calculations
for the shoulder
and/or tail section in survivor curves. King
et al. (
30)
showed that the function
(log
N0-log
N)
a = k
t+c could be successfully used to linearize the survivor
curves
obtained from
B. fulva, a mold that produced
heat-resistant ascospores.
N0 and
N
are the initial and surviving number of organisms, respectively,
at
time
t, the death rate constant is given by k, and c is a
constant
for a set of data. The
a value is derived from the
least squares
slope of a plot of
log(log
N0-log
N) versus log time.
King and coworkers showed that the function does not change
significantly as the severity of the lethal treatment is increased.
When comparing the survival of microbial species under different
thermal processes in foods, a large number of log units of kill
should
be used so the final calculations will incorporate the
shoulder and the
rapid death phase of the curve. For instance,
a 1 log unit destruction
of
L. monocytogenes NCTC 11994 (i.e.,
S
1 in Fig.
2) takes 6.1 min at 60°C, but the second log unit takes
3.4 min, the
third takes 3.3 min, the fourth takes 3.1 min, and
the fifth takes 2.6 min. Because of the shoulder of the curve,
a third of the total time
for a 5 log unit reduction is required
for the first log unit of
destruction. While the D
60°C for
S
1 was 3.7 min, the analogous value for a 1-log unit destruction
(using the
linearizing function) increased to 3.9 min. Application
of this
function also showed that the thermotolerance of other
bacterial
pathogens was underestimated. The time taken for a 6-log
unit reduction
in
Salmonella seftenberg 750W was observed to be
119 min but
was calculated to be 132 min using the function (
43).
However, use of this formula did not apply to TDT curves containing
both shoulder and tail sections (data not shown) (
46).
Shoulders
of TDT curves have been postulated to be due to spore
activation,
repair of heat injury, cell disaggregation, or even
methodological
problems (
24,
42). Moats et al.
(
42) reported extensive
tailing in the survivor curves for
E. coli,
S. faecalis,
Salmonella senftenberg 775W, and
S. antum and attributed these
deviations
from the exponential death rates to differences in heat
resistances
in a single bacterial culture and/or clumping (i.e., where
clumps
of two or more cells produce a colony as long as one cell in the
clump is viable). Therefore, while measurement of thermotolerance
by
the TDT curve method (i.e., D value determination) provides
detailed
data on thermal death rate kinetics that cannot be obtained
by the end
point recovery method (
24,
44,
52), regression
analysis of
nonlinear data can underestimate survival of
L. monocytogenes (Table
3). The end point recovery method appears to
be a better
method for determining microbial thermotolerance because it
takes
both shoulders and tails into consideration.
Little knowledge is available on the putative virulence capability of
R-transformed
L. monocytogenes. Kuhn and Goebel
(
32)
reported that long cell chains of this organism result
from an
impairment in the synthesis of a major extracellular protein,
p60 (considered an important housekeeping protein for virulent
strains
of
L. monocytogenes). It was suggested that p60 protein
may
be a murein hydrolase and that its synthesis is not under
the control
of the transcriptional activator, PrfA (which regulates
the synthesis
of many virulence factors in the gene cluster).
The p60 mutants form
long cell chains (also designated R forms),
with unseparated septae
between the individual bacterial cells,
which disaggregate to
normal-sized single bacteria upon treatment
with partially purified
p60. R-mutant forms were reported to be
avirulent as they were unable
to invade phagocytic 3T6 mouse fibroblast
cells (
5,
32).
These researchers showed, however, that these
R-mutant forms were still
capable of adhering to and invading
epithelial human colon carcinoma
cells (CaCo-2), albeit at a reduced
level of invasiveness
(
5). As spontaneously occurring mutants
of
L. monocytogenes with R-form cell characteristics were isolated
previously at a relatively high frequency (1 in 10,000 colonies)
(
32), and were also shown to emerge under conditions of
severe
osmotic (
29) and heat stress (
46), the
potential pathogenicity
of ingested R-form
L. monocytogenes
in vulnerable groups remains
unanswered. In the present study, the
reversion rate from R form
to normal-sized single bacteria was shown to
be approximately
3 in 500 colonies (data not shown) (
46).
In view of the fact that many foods are subject to mild heat treatments
followed by lengthy periods of refrigerated storage,
e.g., sous-vide
(
19), greater research is needed to determine
the
pathogenicity of long cell chain forms of
L. monocytogenes,
their frequency of occurrence in foods, and the relevance of survivor
curves (particularly TDT curves with tail sections) in food systems.
The D value concept, which assumes a linear response between the
log
number of cell survivors and heating time, accurately described
only
some of the data presented. More importantly, D values calculated
from
linear sections of the TDT curve, and not from the entire
curve, could
lead to an underestimation of the time and temperature
required to
achieve the desired level of cell destruction. Therefore,
the end point
method may be the best approach for determining
microbial
thermotolerance as it takes both shoulders and tails
into account.
| |
ACKNOWLEDGMENT |
We thank the Ministry of Agriculture, Fisheries and Food (MAFF)
for funding this research.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Bioscience and Biotechnology, University of Strathclyde, Royal College Bldg., 204 George St., Glasgow G1 1XW, Scotland. Phone: 44 141 548 2531. Fax: 44 141 553 1161. E-mail:
n.j.rowan{at}strath.ac.uk.
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
