78

Journal of Food Protection, Vol. 74, No. 1, 2011, Pages 78–85

doi:10.4315/0362-028X.JFP-10-117
Copyright G, International Association for Food Protection

Adaptive Growth Responses of Listeria monocytogenes to Acid

and Osmotic Shifts above and across the Growth Boundaries
C.-I. A. BELESSI,1 Y. LE MARC,2 S. I. MERKOURI,1 A. S. GOUNADAKI,1 S. SCHVARTZMAN,3 K. JORDAN,3
E. H. DROSINOS,1 AND P. N. SKANDAMIS1*

1Laboratory of Food Quality Control and Hygiene, Department of Food Science and Technology, Agricultural University of Athens, 11852, Athens, Greece;
2Institute of Food Research, Norwich, NR4 7UA, UK; and 3Teagasc, Moorepark Food Research Centre, Moorepark, Fermoy, County Cork, Ireland

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MS 10-117: Received 17 March 2010/Accepted 10 September 2010

ABSTRACT
The effect of acid and osmotic shifts on the growth of Listeria monocytogenes was evaluated at 10uC. Two types of shifts
were tested: (i) within the range of pH and water activity (aw) levels that allow growth of L. monocytogenes and (ii) after
habituation at no-growth conditions back to growth-permitting conditions. A L. monocytogenes cheese isolate, with high survival
capacity during cheesemaking, was inoculated (102 CFU/ml) in tryptic soy broth supplemented with 0.6% yeast extract at six pH
levels (5.1 to 7.2; adjusted with lactic acid) and 0.5% NaCl (aw 0.995), or four aw levels (0.995 to 0.93, adjusted with 0.5 to
10.5% NaCl) at pH 7.2 and grown to early stationary phase. L. monocytogenes was then shifted (at 102 CFU/ml) to each of the
aforementioned growth-permitting pH and aw levels and incubated at 10uC. Shifts from no-growth to growth-permitting
conditions were carried out by transferring L. monocytogenes habituated at pH 4.9 or aw 0.90 (12.5% NaCl) for 1, 5, and 10 days
to all pH and aw levels permitting growth. Reducing aw or pH at different levels in the range of 0.995 to 0.93 and 7.2 to 5.1,
respectively, decreased the maximum specific growth rate of L. monocytogenes. The lag time of the organism increased with all
osmotic downshifts, as well as by the reduction of pH to 5.1. Conversely, any type of shift within pH 5.5 to 7.2 did not markedly
affect the lag times of L. monocytogenes. The longer the cells were incubated at no-growth aw (0.90), the faster they initiated
growth subsequently, suggesting adaptation to osmotic stress. Conversely, extended habituation at pH 4.9 had the opposite effect
on subsequent growth of L. monocytogenes, possibly due to cell injury. These results suggest that there is an adaptation or injury
rate induced at conditions inhibiting the growth of the pathogen. Thus, quantifying adaptation phenomena under growth-limiting
environments, such as in fermented dairy and meat products or products preserved in brine, is essential for reliable growth
simulations of L monocytogenes during transportation and storage of foods.

In dynamic growth modeling, it is assumed that the
growth rate of cells is instantaneously adapted to momen-
tary temperature changes, whereas there is contrasting
evidence for induction of short lag times before growth is
resumed (1, 3, 20, 21). Studies also suggest that preincu-
bation of Listeria monocytogenes at 4uC results in a faster
growth rate at 7uC than preculture at 7uC (19). Therefore,
there seems to be marked uncertainty about the role of
previous environmental conditions or the effect of inoculum
history on subsequent microbial growth.
The lag phase of a microorganism in a new environ-
ment is affected by the intrinsic (e.g., pH and water activity
[aw]) and/or extrinsic (e.g., temperature) properties of the
environment and the physiological state of cells, as
determined by the previous environment. Subsequent
changes of environmental factors may induce an ‘‘interme-
diate’’ lag phase (27), the duration of which depends on the
magnitude and direction of the environmental shift. Quanti-
fication of the effect of temperature shifts on the lag time of
bacteria has received more attention (2, 18, 27, 28, 30, 31)
* Author for correspondence. Tel/Fax: z30-210-5294684;
E-mail: pskan@aua.gr.
than the corresponding effect of osmotic shifts (15–17),
whereas relevant information for shifts in pH is currently
lacking.
The most widely used approach for quantifying the
effect of sudden changes in the environment on bacterial lag
time is based on the amount of work that cells need to
undertake and the rate at which this work is accomplished
(work to be done, h0) (15, 23). According to the latter
approach, if changes in the environment do not pose
additional adaptation work, then the inverse of lag time (the
so-called lag rate) (18) and growth rate should follow the
same trend as a function of the shift and they should be
linearly correlated. This may be true for shifts of low
magnitude (e.g., 3 to 5uC) that are far from the growth
boundaries. However, linearity is lost when the intensity of
the stress imposed is so high that the bacteria cannot adapt
to the new conditions with only the available homeostatic
mechanisms and require additional effort to overcome the
imposed stress. This is the case with osmotic or acid shifts,
which are considered more severe stresses than temperature
(23, 24), or with shifts close to the growth boundaries.
Regarding the latter, even though the lag time of
Lactobacillus plantarum was consistently well predicted
J. Food Prot., Vol. 74, No. 1
after temperature upshifts, marked variations in lag time
were evident in temperature downshifts close to the growth
boundaries, which rendered the prediction of growth
difficult (31).
The response of bacteria to dynamic environmental
conditions varies with the Gram type, genus, etc. For
instance, the lag time of gram-negative Escherichia coli and
Salmonella increased nonlinearly as osmotic conditions
became less favorable for growth, suggesting the need for
additional adaptation work after the shift. Conversely, it has
been suggested that gram-positive bacteria have a higher
ability to withstand osmotic stress than gram-negative
bacteria, due to inherent constitutive turgor pressure
mechanisms available to the former (15). Nonetheless, the
findings of the aforementioned studies are restricted to
observations following shifts within the growth domain of
microorganisms. Another key hypothesis in growth model-
ing addresses whether shifts in the environment across the
growth boundaries induce additional adaptation work to
microorganisms compared with shifts in the growth region,
or allow cells to adapt to adverse conditions and, hence,
enhance their subsequent growth. Mitchell et al. (21)
stressed that extended exposure to temperatures that do
not support growth of Salmonella may affect subsequent
growth of the bacterium at growth-permitting temperatures.
However, although aw shifts to harsher conditions are
known to induce adaptation work (15–18), growth under
dynamic pH or for osmotic and pH shifts across the growth
boundaries is not clearly understood. Such knowledge is
crucial for development or optimization of dynamic growth
models for L. monocytogenes, because it is ubiquitous in
nature and may be sequentially exposed to growth-limiting
or growth-permitting conditions in various products of low
pH and/or low aw, as well as during fermentation and
ripening of meat and dairy foods.
Based on the above, the objectives of this work were to
evaluate the growth responses of L. monocytogenes under
(i) environmental shifts within the range of pH and aw levels
that allow growth of the bacterium and (ii) shifts from
optimum aw (0.995) or pH (7.2) conditions to no-growth
conditions, habituation for 1, 5, or 10 days, and then back to
growth-permitting conditions.
MATERIALS AND METHODS
Bacterial strain and culture preparation. L. monocyto-
genes C5 (serotype 4b), shown previously to survive the stressful
environment of cheesemaking better than other strains, was used in
this study. The strain was selected among other isolates from dairy
products, factory equipment, and human outbreaks (including Scott
A), after a preliminary experiment where all the strains were grown
at the pH and NaCl levels tested in the present study. The particular
strain demonstrated the highest growth rate and the shortest lag
time among all the tested strains (data not shown). The strain was
maintained at 222uC in tryptic soy broth supplemented with 0.6%
yeast extract (TSBYE; Biolife Italiana Srl, Milan, Italy) and 20%
glycerol (Glycerine 4810, Oleon, Ertvelde, Belgium). Cultures
were activated by transferring 0.1 ml of stock culture to 10 ml of
TSBYE followed by incubation at 30uC for 24 h. Then working
cultures were obtained by subculturing 0.1 ml of the previous
culture to 10 ml of TSBYE followed by incubation at 30uC for
L. MONOCYTOGENES RESPONSE TO PH AND AW SHIFTS 79
16 h, in order to obtain late-exponential-phase to early-stationary-
phase cells. The culture was harvested by centrifugation (3,600 |
g for 15 min at 4uC; Megafuge 1.0R, Heraeus Instruments, Hanau,
Germany). The cell pellet was washed twice and resuspended in
10 ml of maximum recovery diluent (MRD; Biolife). The bacterial
population of the latter suspension, which was constantly at 109
CFU/ml (confirmed by four independent cultures, duplicate plates),
was serially diluted in MRD to the level of 104 CFU/ml in order to
be used as the initial inoculum for the experiments.
Media preparation. A total of seven pH values (7.2, 6.0, 5.8,
5.5, 5.3, 5.1, and 4.9) and five aw levels (0.995, 0.97, 0.95, 0.93,
and 0.90) by adding NaCl were evaluated in TSBYE. Of these
conditions, one pH and one aw level, namely pH 4.9 and aw 0.90,
respectively, did not allow growth of L. monocytogenes at 10uC;
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these were selected for the experiments involving habituation of
the bacterium at no-growth conditions, as will be further described.
We did not use TSBYE without glucose, because none of the
experimental cases was expected to cause inactivation. Thus,
potential acid adaptation due to glucose levels as low as 0.25%,
contained in commercially available TSBYE formulation, was
assumed to have no influence on the growth of L. monocytogenes.
In order to achieve the target pH values of the tested media by
using the smallest possible volume of lactic acid, a concentrated
lactic acid solution of 85% (wt/wt) was used (Sigma-Aldrich Ltd.,
Taufkirchen, Germany), along with 10 N NaOH (when necessary;
sodium hydroxide pellets, Panreac, Castellar del Valles, Spain). To
achieve the lowest tested pH (4.9) in 100 ml of commercial
TSBYE (initial pH 7.3) prepared according to manufacturer’s
recommendations in 250-ml Duran bottles (Duran Group GmbH,
Mainz, Germany), the maximum volume required was 0.6 ml of
the concentrated lactic acid solution (85% [wt/wt]). Therefore, the
total volume of the lactic acid added was not considered to change
the total volume of the broth. The target aw levels were achieved by
adding 0, 4.5, 7.5, 10, and 12 g of NaCl in 100 ml of TSBYE, also
in 250-ml Duran bottles. Given that the commercial composition of
TSB contains 0.5% NaCl, the final NaCl concentrations of the
tested media were 0.5, 5, 8, 10.5, and 12.5%. The pH and aw of all
media were measured after autoclaving with a pH electrode
(pH 691, Metrohm, Zofingen, Switzerland) and aw meter
(HygroLab, Rotronic, Bassersdorf, Switzerland), respectively.
Two pairs of growth media adjusted at each of the aforementioned
pH and aw levels were prepared for two independent experiments.
Experimental procedure. To evaluate the growth of L.
monocytogenes in response to environmental shifts within the
range of pH or aw levels permitting growth, the following
procedure was applied: pairs of sterile broths (100 ml) adjusted
to all the growth-permitting pH values (7.2, 6.0, 5.8, 5.5, 5.3, and
5.1) or aw levels (0.995, 0.97, 0.95, and 0.93) were inoculated with
1 ml of the L. monocytogenes C5 suspension in MRD (104 CFU/
ml) in order to obtain an initial level of 100 CFU/ml. Inoculated
media were placed in high-precision (¡0.5uC) incubation
chambers (MIR-153, Sanyo Electric Co., Osaka, Japan) and stored
at 10uC. Changes in viable counts were monitored at regular
intervals, ranging from 6 h to 3 days, depending on the growth rate
and lag time of the pathogen at each tested condition, until late
exponential to early stationary phase. Enumeration of viable cells
was performed by serial dilutions in MRD, surface plating using a
spiral plater (Autoplate 400, Automated Spiral Plater, Spiral
Biotech, Inc., Norwood, MA) onto duplicate tryptic soy agar
supplemented with 0.6% yeast extract (Biolife) and incubation at
30uC for 24 to 48 h. After the L. monocytogenes culture had
reached the late exponential to early stationary phase (ca. 8 to 9 log
80 BELESSI ET AL.
FIGURE 1. Schematic representation of
the experimental design (A) and of the
experimental procedure (B). Arrows in
upper graph indicate shifts to all six pH
or four aw levels reported in the vertical
rectangles, followed by incubation at 10uC
as shown in the lower graph. Cases on the
left of the vertical rectangles represent
shifts within growth-permitting conditions,
whereas cases on the right of the vertical
rectangles represent habituation at growth-
inhibiting conditions of pH 4.9 or aw 0.90
for 1, 5, and 10 days and then back to
growth-permitting pH and aw levels, as
shown in the lower graph.
CFU/ml), aliquots (1 ml) were serially diluted and then shifted to
duplicate media adjusted at all the growth-permitting levels of the
same factor, as illustrated in Figure 1, at an initial population of
100 CFU/ml. In particular, six pH shifts were performed from each
of the six initial pH levels, resulting in a total of 36 shifts and four
transfers from each of the four initial aw levels, resulting in 16 total
NaCl shifts. We performed both downshifts (toward lower pH or
aw levels) and upshifts (toward optimal pH and aw levels); we also
tested transfers of L. monocytogenes to noninoculated medium of
the same pH or aw level as those applied before shift (Fig. 1).
The application of shifts from optimum aw (0.995) or pH (7.2)
conditions to no-growth conditions for 1, 5, or 10 days and then
back to growth-permitting conditions (Fig. 1) took place as
follows: pairs of adjusted TSBYE at pH 4.9 (aw 0.995; 0.5%
NaCl) or aw 0.90 (12.5% NaCl at pH 7.2) were inoculated with L.
monocytogenes C5 at 104 CFU/ml. The tested pH and aw levels
allowed survival of L. monocytogenes at the initial inoculation
level for more than 10 days, without any marked reduction (i.e.,
.0.5 log CFU/ml; data not shown). Thus, the initial inoculation
level of L. monocytogenes after shifting to growth-permitting
conditions was also approximately 100 CFU/ml, similar to shifts
within growth-permitting levels of pH and aw. After 1, 5, and
10 days of habituation at growth-inhibiting pH or aw levels at
10uC, aliquots (1 ml) were transferred to double bottles of TSBYE
(100 ml) adjusted at all growth-permitting levels of the same factor
(Fig. 1). The number of days of habituation prior to the shift to
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growth-permitting conditions was chosen to examine the effect of
the length of incubation at no-growth conditions on the ability of L.
monocytogenes to adapt to subsequent environments. These shifts
were also considered as a simulation of cross-contamination
between those products with physicochemical properties that do
not allow proliferation of the pathogen and those products with
optimal or suboptimal properties. Incubation of tested media at
growth-permitting or growth-inhibiting pH and aw levels was
carried out at 10uC in high-precision (¡0.5uC) incubation
chambers, in four replicates (two replicates per condition in two
separate experiments). The incubation temperature (10uC) was
chosen to ensure a broader range of pH and aw conditions that
permit growth of L. monocytogenes compared with conditions at a
refrigeration temperature of 4uC, thus yielding more data about the
effect of acid and osmotic shifts on the growth of the bacterium.
Also, 10uC is a common temperature at dairy processing sites or in
cheese ripening chambers.
Fitting of growth curves. Changes in viable counts during
incubation at 10uC were transformed to log values. Log-transformed
data were fitted to the Baranyi model (3) in order to determine the
maximum specific growth rate mmax (h21), the lag time (h), and the
dimensionless parameter h0 representing the ‘‘work-to-be-done’’
before entering the exponential phase of growth (23), also analogous
to the ‘‘relative lag time’’ (15, 18). The estimated mmax, lag times,
and h0 values were subjected to one-way analysis of variance, and
J. Food Prot., Vol. 74, No. 1
FIGURE 2. Effect of aw (a) and pH (b) shifts on maximum
specific growth rate (mmax) of L. monocytogenes, following growth
at different initial aw and pH conditions. Daw at horizontal axis is
equal to the difference awfinal 2 awinitial, where awfinal is the aw
after the shifts and awinitial is aw before the shifts. Likewise, DpH is
equal to pHfinal 2 pHinitial, where pHfinal is the pH after the shifts
and pHinitial is the pH before the shifts. Zero value represents shifts
to the same aw or pH, positive sign values represent upshifts, and
negative sign values represent downshifts.
differences in least-squares means were evaluated with the Tukey
test at a significance level of a ~ 0.05.
RESULTS AND DISCUSSION
Effect of shifts between growth-permitting levels of
pH and aw . The mmax of L. monocytogenes was influenced
by the direction, but not by the magnitude, of aw shifts (Daw),
as shown by the almost parallel position of mmax curves in
response to shifts for different starting aw values (i.e.,
conditions before the shift; Fig. 2a). More specifically,
upshifts in aw increased mmax, and the opposite occurred
when aw decreased (Fig. 2a). The change of mmax in response
to pH shifts indicated a similar trend to that caused by aw
changes, independently of the pH before shift (Fig. 2b).
Nevertheless, as expected, mmax was influenced by the pH and
aw levels after each shift (P , 0.05; Fig. 2). Therefore, our
results suggest that in growth simulations under dynamic
acidic or osmotic conditions, mmax is instantaneously adapted
to the new environment, similarly to the bacterial growth
responses to fluctuating temperatures.
L. MONOCYTOGENES RESPONSE TO PH AND AW SHIFTS 81
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FIGURE 3. Effect of aw (a) and pH (b) shifts on lag times of L.
monocytogenes following growth at different initial aw and pH
conditions. Daw at horizontal axis is equal to the difference awfinal
2 awinitial, where awfinal is the aw after the shifts and awinitial is aw
before the shifts. Likewise, DpH is equal to pHfinal 2 pHinitial,
where pHfinal is the pH after the shifts and pHinitial is the pH before
the shifts. Zero value represents shifts to the same aw or pH,
positive sign values represent upshifts, and negative sign values
represent downshifts.
Lag times were influenced by both the direction and the
magnitude of aw shifts (Fig. 3a). Specifically, lag times of
L. monocytogenes sharply increased (P , 0.05) as the
conditions of aw became harsher (i.e., in the case of negative
shifts; Fig. 3a). This is in accordance with Mellefont et al.
(15), who observed increased relative lag times at low aws for
Salmonella, although this effect was less evident for L.
monocytogenes. Lebert et al. (13) also demonstrated an
exponential increase in the lag time of Listeria innocua
grown under optimum aw conditions (0.995) and subsequently
exposed to various aw levels in broth and gelatine, from 0.995
down to the growth-inhibiting aw level of 0.91. However, in
our study we also showed that lag times are markedly reduced
(P , 0.05) when shifts occurred toward more favorable
conditions than the initial aw (Fig. 3a). This suggests that any
possible injury due to low aw is quickly repaired when the
conditions become more favorable for growth. Similar
observations regarding the effect of environmental shifts on
the intermediate lag time of microorganisms have been
reported for temperature downshifts in studies predicting
growth of Salmonella and Brochothrix thermosphacta (4, 20)
82 BELESSI ET AL.
FIGURE 4. Additional work needed for growth initiation (addi-
tional h0*) of L. monocytogenes at growth-permitting conditions,
N
due to shifts from different initial aw levels: (%) 0.995, ( ) 0.97,
(n) 0.95, (e) 0.93. For the calculation of additional h0, lag times
for no-shift conditions were subtracted from lag times at each
shifted condition and multiplied with maximum specific growth
rate according to equation 1.
and for temperature upshifts in studies involving E. coli
growth (27, 28). Therefore, changes in the aw of the food have
a marked influence on the lag time of L. monocytogenes,
similar to the effect posed by temperature shifts on the growth
of the bacterium.
In contrast with the observations of aw shift effects, lag
times were not influenced by pH shifts within the range of 5.3
to 7.2 (P $ 0.05; Fig. 3b), and the magnitude of h0 was
relatively lower (0 to 0.4; data not shown) compared with that
caused by aw shifts (Daw in the range 0 to 1.5). However,
remarkable increases in lag times were caused by shifts close
to the growth-limiting pH value of 5.1 (P , 0.05; Fig. 4b), in
agreement with Robinson et al. (23). It is suggested that cells
are capable of restoring their homeostasis rapidly when mild
pH stress is imposed, whereas the combination of low
temperature (10uC) with growth-limiting pH values may
have posed a marked energetic burden that necessitated the
expenditure of additional energy by the cells in order to
initiate growth. Therefore, these data indicate that the
intermediate lag times induced by pH shifts should be
considered only when they occur near the growth boundaries
(i.e., pH 4.9 to 5.2). This is notable in regard to the risk of L.
monocytogenes growth during the shelf life of some dairy
products (commonly semihard or hard cheeses) that may
marginally support growth of the bacterium due to their low
pH values (5.0 to 5.5).
Notably, the hypothesis that the adaptation work of the
bacterium to a new environment is independent of the
environmental factor controlling its growth is not valid for
aw. To reach this conclusion, first, we calculated h0 as a
product of lag time and mmax for each shift, and no
meaningful trend of h0 with aw was evident. Then we
applied the following transformation:
h0 ~ mmax |ðlagnew { lagsame Þ ð1Þ
where lagnew is the lag time of L. monocytogenes after shift
to a different aw from that in which the organism was
J. Food Prot., Vol. 74, No. 1
precultured and lagsame is the lag after shifting to fresh
medium with the same aw as in preculture. These
calculations revealed a trend: habituation at low aw
enhanced subsequent adaptation to aw in the range of 0.93
to 0.995 (Fig. 4). No such trend was obtained under the pH
conditions tested in the present study (results not shown).
Consequently, models for the effects of osmotic shifts on the
intermediate lag time of L. monocytogenes should consider
not only the aw after the shift but also the direction and the
magnitude of shift. Cheroutre-Vialette and Lebert (5)
presented a dynamic growth model for L. monocytogenes
in response to shifts in NaCl and pH based on recurrent
neural networks. Even though good agreement was obtained
between predictions and data, the model was complex; its
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learning base was oversimplified and perhaps inadequate to
cover all potential shifts from different initial NaCl and pH
levels. Our findings will assist in the development of
simpler growth models based on biologically meaningful
assumptions related to potential adaptation phenomena of L.
monocytogenes. Such phenomena may occur during transfer
of L. monocytogenes from environments of low aw (e.g.,
brines) or pH (fermented product) to environments of higher
aw or pH (e.g., milk or other raw materials) and vice versa.
Effect of shifts from growth-inhibiting to growth-
permitting levels of pH and aw . The duration of
habituation at no-growth conditions did not influence the
mmax of L. monocytogenes at subsequent shifts to growth-
permitting levels (data not shown). However, an apparent
effect on lag time and, hence, on h0 estimates was evident
(Fig. 5). Specifically, for the same level of aw, the h0 tended
to decrease (P , 0.05) proportionally to the time the cells
were held at 0.90 (Fig. 5a). Furthermore, lag times and h0
estimates at aw 0.93 and 0.95 after 10 days at 0.90 were
lower (P , 0.05) than the corresponding values after growth
at 0.995 (Fig. 6); however, no differences (P $ 0.05) in lag
times were observed at aw . 0.95 in cultures previously
maintained at aw 0.90 compared with those grown at 0.995
(Fig. 6). These results suggest that the cells are able to carry
out some of the ‘‘work to be done’’ required for growth in
aw 0.93 to 0.995, during habituation at 0.90. Nonetheless,
the lag times and h0 values of L. monocytogenes after
preadaptation to aw 0.90 decreased with increasing aw
(Fig. 5a). This in turn means that the adaptation work
represented by the parameter h0 decreased as the magnitude
of the shift from no-growth to growth conditions increased.
The observation that lag time is proportional to the
magnitude of the shift toward the growth limits (i.e., in
negative shifts) is established for bacterial growth under
fluctuating temperatures and suggests a positive temperature
history effect on lag time on bacteria (8, 30). More
specifically, although lag was induced upon shifts from
high temperature (25 to 37uC) to 3 to 4uC, preincubation at
4uC markedly reduced such a lag time (7). The putative
mechanisms associated with resistance of L. monocytogenes
to osmotic stress involve the uptake of compatible solutes
such as glycine, betaine, and carnitine (11). Our results
suggest that during incubation at aw 0.90, the metabolic
activity of cells continues and probably assists in accumu-
J. Food Prot., Vol. 74, No. 1
FIGURE 5. Work needed (h0) for growth initiation of L.
monocytogenes at growth-permitting aw (a) or pH (b), following
N
habituation for (%) 1, (n) 5, or ( ) 10 days at aw 0.90 or pH 4.9.
lation of the necessary resources that will enable cells to
grow once they encounter favorable conditions. Further-
more, although the growth rate of exponentially growing
cells is thought to adapt instantaneously to changes in aw,
our results suggest that the bacterial lag time depends on
the duration of preadaptation at no-growth conditions.
Not taking this adaptation into account may lead to an
underestimation of the actual growth of L. monocytogenes,
thereby compromising the safety of foods of low aw (e.g.,
0.90 to 0.94), especially close to the growth boundaries of
this bacterium. These findings are valuable in quantitative
microbial risk assessment; they provide quantitative data
to confirm implications on the adaptive responses of L.
monocytogenes, an important area of uncertainty that is not
sufficiently addressed by the existing predictive models.
However, based on these implications, further research is
required to quantify the rate at which cells adapt while they
are in no-growth status, especially various L. monocyto-
genes strains that are involved in outbreaks or that persist in
processing and retail environments.
In contrast with the observations with aw, the longer the
cells were held at no-growth pH (4.9), the greater was the
work needed for growth initiation in a more favorable
environment (Fig. 5b). Specifically, the value of h0 at pH
levels decreasing from 7.2 to 5.1, increased from 0 (no lag)
to 0.7, 0.6 to 2.5, and 1.1 to 4.2, after 1, 5, and 10 days of
L. MONOCYTOGENES RESPONSE TO PH AND AW SHIFTS 83
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FIGURE 6. Lag times of L. monocytogenes at growth-permitting
N
aw following growth at aw 0.995 (e), 0.97 (n), or habituation ( )
for 10 days at aw 0.90.
incubation at pH 4.9, respectively (Fig. 5b). Furthermore,
lag times and h0 estimates at pH 5.1 (near-growth and no-
growth interface), after 10 days at pH 4.9 were higher (P ,
0.05) than the corresponding values at pH 7.2 and 5.5,
following the same habituation scenario (Fig. 5b). This may
be explained on the basis of the higher energetic burden
posed by pH on cells compared with aw under no-growth
conditions (24, 29). The present experimental approach
consisted of three consecutive steps: (i) growth at optimum
pH; (ii) shift to no-growth pH for 1, 5, and 10 days; and (iii)
regrowth at different pH above the growth boundaries.
According to Goodson and Rowbury (9), prior growth of E.
coli at pH 7.0 reduced the ability of cells to grow at the same
pH after short exposure (26 min) to pH 3.0 (adjusted with
HCl). It is likely that habituation at pH 4.9, in the present
study, induced physiological changes to cells which
rendered them more resistant to subsequent lethal acid
challenges (6, 26) or reduced their minimum pH for growth
initiation. For instance, Skandamis et al. (25) found that
habituation of E. coli O157:H7 in acidic meat decontam-
ination run-off fluids (pH 4.9) enabled growth of cells at
lower pH than nonhabituated cells. Note, however, that the
pH-dependent acid resistance mechanisms of E. coli
O157:H7 are different from those of L. monocytogenes
(10, 14). Moreover, such mechanisms presumably differ
from those needed to resume growth under subsequent
favorable conditions. Kroll and Patchett (12) found that
growth of L. monocytogenes at pH 5.0 before acid shock at
pH 3.5 delayed regrowth at pH 7.0, in comparison with
growth at pH 7.0 before the shock. Conversely, preadap-
tation at pH 5.0 enhanced subsequent survival at pH 3.0, as
opposed to cells initially grown at pH 7.0. It may thus be
postulated that the conditions that increase acid resistance
(e.g., gradual drop of pH during fermentation) or even
reduce the minimum pH allowing growth may not impact
the adaptation work required for entrance into the
exponential phase of growth under favorable conditions.
Additional research involving the transcriptional or proteo-
mic changes mediated by habituation at growth-inhibiting
pH would offer a better insight to the above-speculated
mechanisms. A potential area of application of such findings
84 BELESSI ET AL.
could also be associated with the passage of L. monocyto-
genes from the stomach (low pH) to the intestine (high pH).
In the present study, shifts were applied on bacterial
cells in late exponential to early stationary phase. Therefore,
our results may not be directly comparable to those of
relevant studies, in which cells in the midexponential or late
stationary phase were used. According to previous reports
(16, 17, 22), cells in the exponential phase are more
sensitive to osmotic shifts than cells in stationary phase. Our
findings represent the response of cells in a state between
exponential and stationary phase. Furthermore, the present
results may provide inferences for the effects of aw and pH
shifts on the growth of L. monocytogenes and, hence, reduce
this area of uncertainty in dynamic growth modeling and
quantitative microbial risk assessment. In particular, we
indicated that habituation of L. monocytogenes under
growth-inhibiting conditions, (e.g., in a product with pH
,4.4 or aw ,0.92 or a combination of pH 5.0 and aw 0.94)
is not only a period when the pathogen simply does not
grow. Instead, these situations may trigger adaptation
phenomena, which could enhance growth of the bacterium
upon subsequent transfer to a growth-supporting environ-
ment (e.g., due to cross-contamination). Therefore, the
predictions of models for microbial responses near the
growth boundaries of pathogens should be viewed with
caution, and perhaps proper adjustments of existing models
might be necessary in order to take into account the above
adaptation phenomena. However, the effects of aw and pH
shifts were studied independently, and further work is
needed to study the effect of simultaneous shifts on the
intermediate lag time. In addition, the effects of such shifts
(pH and aw) on the growth of L. monocytogenes need to be
studied not only on liquid cultures but also on cultures
growing on solid surfaces or immobilized in emulsified (oil-
in-water or water-in-oil) foods. Even though the present
findings refer to a single strain, they could be considered
representative of the potential behavior of the bacterium in
dairy processing environments, since the selected strain was
a persistent dairy isolate. However, our findings should be
evaluated for multiple strains in order to account for
potential strain variability in growth and adaptive responses
(24).
ACKNOWLEDGMENT
This work was funded by the European Union Integrated 6th
Framework Programme under the project ‘‘BIOTRACER: Improved bio-
traceability of unintended microorganisms and their substances in food and
feed chains’’ (proposal/contract: 036272).
REFERENCES
1. Alavi, S. H., V. M. Puri, S. J. Knabel, R. H. Mohtar, and R. H.
Whiting. 1999. Development and validation of a dynamic growth
model for Listeria monocytogenes in fluid whole milk. J. Food Prot.
62:170–176.
2. Augustin, J. C., L. Rosso, and V. Carlier. 2000. A model describing
the effect of temperature history on lag time for Listeria
monocytogenes. Int. J. Food Microbiol. 57:169–181.
3. Baranyi, J., and T. A. Roberts. 1994. A dynamic approach to
predicting bacterial growth in food. Int. J. Food Microbiol. 23:277–
294.
J. Food Prot., Vol. 74, No. 1
4. Baranyi, J., T. P. Robinson, A. Kaloti, and B. M. Mackey. 1995.
Predicting growth of Brochothrix thermosphacta at changing
temperature. Int. J. Food Microbiol. 27:61–75.
5. Cheroutre-Vialette, M., and A. Lebert. 2000. Modelling the growth of
Listeria monocytogenes in dynamic conditions. Int. J. Food Microbiol.
55:201–207.
6. Davis, M. J., P. J. Coote, and P. O’Byrne. 1996. Acid tolerance in
Listeria monocytogenes: the adaptive acid tolerance response (ATR)
and growth-phase-dependent acid resistance. Microbiology 142:
2975–2982.
7. Delignette-Muller, M. L., F. Baty, M. Cornu, and H. Bergis. 2005.
Modelling the effect of a temperature shift on the lag phase duration
of Listeria monocytogenes. Int. J. Food Microbiol. 100:77–84.
8. Gay, M., O. Cerf, and K. R. Davey. 1996. Significance of pre-
incubation temperature and inoculum concentration on subsequent
growth of Listeria monocytogenes at 14uC. J. Appl. Microbiol. 81:
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/1/78/1681479/0362-028x_jfp-10-117.pdf by guest on 13 October 2020
433–438.
9. Goodson, M., and R. J. Rowbury. 1989. Habituation to normally
lethal acidity by prior growth of Escherichia coli at a sub-lethal acid
pH value. Lett. Appl. Microbiol. 8:77–79.
10. Hill, C., P. D. Cotter, R. D. Sleator, and C. G. M. Gahan. 2002.
Bacterial stress response in Listeria monocytogenes: jumping the
hurdles imposed by minimal processing. Int. Dairy J. 12:273–283.
11. Ko, R., L. T. Smith, and G. M. Smith. 1994. Glycine betaine confers
enhanced osmotolerance and cryotolerance on Listeria monocyto-
genes. J. Bacteriol. 176:426–431.
12. Kroll, R. G., and R. A. Patchett. 1992. Induced acid tolerance in
Listeria monocytogenes. Lett. Appl. Microbiol. 14:224–227.
13. Lebert, I., C. G. Dussap, and A. Lebert. 2004. Effect of aw, controlled
by the addition of solutes or by water content, on the growth of
Listeria innocua in broth and in a gelatine model. Int. J. Food
Microbiol. 94:67–78.
14. Lin, J., M. P. Smith, K. C. Chapin, H. B. Baik, G. N. Bennet, and
J. W. Foster. 1996. Mechanisms of acid resistance in Enterohemor-
rhagic Escherichia coli. Appl. Environ. Microbiol. 62:3094–3100.
15. Mellefont, L. A., T. A. McMeekin, and T. Ross. 2003. The effect of
abrupt osmotic shifts on the lag phase duration of foodborne bacteria.
Int. J. Food Microbiol. 83:281–293.
16. Mellefont, L. A., T. A. McMeekin, and T. Ross. 2004. The effect of
abrupt osmotic shifts on the lag phase duration of physiologically
distinct populations of Salmonella typhimurium. Int. J. Food
Microbiol. 99:111–120.
17. Mellefont, L. A., T. A. McMeekin, and T. Ross. 2005. Viable count
estimates of lag time responses for Salmonella typhimurium M48
subjected to abrupt osmotic shifts. Int. J. Food Microbiol. 105:399–
410.
18. Mellefont, L. A., and T. Ross. 2003. The effect of abrupt shifts in
temperature on the lag phase duration of Escherichia coli and
Klebsiella oxytoca. Int. J. Food Microbiol. 83:295–305.
19. Membré, J.-M., T. Ross, and T. A. McMeekin. 1998. Behaviour of
Listeria monocytogenes under combined chilling processes. Lett.
Appl. Microbiol. 28:216–220.
20. Mitchell, G. A., T. F. Brocklehurst, R. Parker, and A. C. Smith. 1994.
The effect of transient temperatures on the growth of Salmonella
typhimurium LT2. I: cycling within the growth region. J. Appl.
Microbiol. 77:113–119.
21. Mitchell, G. A., T. F. Brocklehurst, R. Parker, and A. C. Smith. 1995.
The effect of transient temperatures on the growth of Salmonella
typhimurium LT2. II: excursions outside the growth region. J. Appl.
Microbiol. 79:128–134.
22. Robinson, T. P., O. O. Aboaba, A. Kaloti, M. J. Ocio, J. Baranyi, and
B. N. Mackey. 2001. The effect of inoculum size on the lag phase of
Listeria monocytogenes. Int. J. Food Microbiol. 70:163–173.
23. Robinson, T. P., M. J. Ocio, A. Kaloti, and B. M. Mackey. 1998. The
effect of the growth environment on the lag phase of Listeria
monocytogenes. Int. J. Food Microbiol. 44:83–92.
24. Shabala, L., S. H. Lee, P. Cannesson, and T. Ross. 2008. Acid and
NaCl limits of growth of Listeria monocytogenes and influence of
sequence of inimical acid and NaCl levels on inactivation kinetics. J.
Food Prot. 71:1169–1177.
J. Food Prot., Vol. 74, No. 1
25. Skandamis, P. N., J. D. Stopforth, P. A. Kendall, K. E. Belk, J. A.
Scanga, G. C. Smith, and J. N. Sofos. 2007. Modeling the effect of
inoculum size and acid adaptation on growth/no growth interface of
Escherichia coli O157:H7. Int. J. Food Microbiol. 120:237–249.
26. Skandamis, P. N., Y. Yoon, J. D. Stopforth, P. A. Kendall, and J. N.
Sofos. 2008. Heat and acid tolerance of Listeria monocytogenes after
exposure to single and multiple sublethal stresses. Food Microbiol.
25:294–303.
27. Swinnen, I. A. M., K. Bernaerts, K. Gysemans, and J. F. Van Impe.
2005. Quantifying microbial lag phenomena due to a sudden rise in
temperature: a systematic macroscopic study. Int. J. Food Microbiol.
100:85–96.
L. MONOCYTOGENES RESPONSE TO PH AND AW SHIFTS 85
28. Swinnen, I. A. M., K. Bernaerts, and J. F. Van Impe. 2006. Modelling
the work to be done by Escherichia coli to adapt to sudden
temperature upshifts. Lett. Appl. Microbiol. 42:507–513.
29. Tiganitas, T., N. Zeaki, A. S. Gounadaki, E. H. Drosinos, and P. N.
Skandamis. 2009. Study of the effect of lethal and sublethal pH and
aw stresses on the inactivation or growth of Listeria monocytogenes
and Salmonella Typhimurium. Int. J. Food Microbiol. 134:104–112.
30. Whiting, R. C., and L. G. Bagi. 2002. Modelling the lag phase of
Listeria monocytogenes. Int. J. Food Microbiol. 73:291–295.
31. Zwietering, M. H., J. C. De Wit, H. G. A. M. Cuppers, and K. Van’t
Riet. 1994. Modeling of bacterial growth with shifts in temperature.
Appl. Environ. Microbiol. 60:204–213.
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/1/78/1681479/0362-028x_jfp-10-117.pdf by guest on 13 October 2020