Arch Microbiol (2006) 186:229–239

DOI 10.1007/s00203-006-0137-1

O RI G I NAL PAPE R

The acid tolerance response of Bacillus cereus ATCC14579

is dependent on culture pH, growth rate and intracellular pH
Séverine Thomassin · Michel P. Jobin ·
Philippe Schmitt

Received: 1 December 2005 / Revised: 6 June 2006 / Accepted: 19 June 2006 / Published online: 12 August 2006
© Springer-Verlag 2006

Abstract The food pathogen Bacillus cereus is likely Abbreviations

to encounter acidic environments (i) in food when ATR Acid tolerance response
organic acids are added for preservation purposes, and cFDASE CarboxyXuorescein diacetate succinimidyl
(ii) during the stomachal transit of aliments. In order to ester
characterise the acid stress response of B. cereus cFSE CarboxyXuorescein succinimidyl ester
ATCC14579, cells were grown in chemostat at diVerent HBL Haemolysin BL
pH values (pHo from 9.0 to 5.5) and diVerent growth JB J broth
rates (
from 0.1 to 0.8 h¡1), and were submitted to NHE Non-haemolytic enterotoxin
acid shock at pH 4.0. Cells grown at low pHo were pHi Internal pH
adapted to acid media and induced a signiWcant acid pHo External pH
tolerance response (ATR). The ATR induced was
pH, Delta pH
modulated by both pHo and
, and the
eVect was GLM General linear model
more marked at pHo 5.5. Intracellular pH (pHi) was HDS Honest signiWcant diVerence
aVected by both pHo and
. At a pHo above 6, the pHi
decreased with the decrease of pHo and the increase of

. At pHo 5.5, pHi was higher compared to pHo 6.0, Introduction
suggesting that mechanisms of pHi homeostasis were
induced. The acid survival of B. cereus required protein Bacillus cereus is a sporulating bacterium which is
neo-synthesis and the capacity of cells to maintain their found in cooked chilled foods and various vegetables
pHi and
pH (pHi - pHo). Haemolysin BL and non- (Roberts et al. 1982; Kramer and Gilbert 1989; Carlin
haemolytic enterotoxin production were both inXu- et al. 2000; Choma et al. 2000; Valero et al. 2003). This
enced by pHo and
. bacterium is responsible for food poisoning with two
relevant symptoms: (i) diarrheic symptoms due to the
Keywords Bacillus cereus · Chemostat · production of enterotoxins [haemolysin BL (HBL),
Acid tolerance response · Stress · Intracellular pH non-haemolytic enterotoxin (NHE) and Cyt K] in the
intestine, and (ii) emetic symptoms due to the produc-
tion of cereulide, an emetic toxin produced in the food
(Agata et al. 1995; Lund and Granum 1997; Lund et al.
S. Thomassin · M. P. Jobin · P. Schmitt
IUT Génie Biologique, Univ Avignon, UMR 408,
2000).
Sécurité et Qualité des Produits d’Origine Végétale, Bacillus cereus responsible for food poisoning has to
Avignon, 84029, France have been able to resist and adapt to environmental
stresses during food processing and conservation and/
S. Thomassin · M. P. Jobin (&) · P. Schmitt
INRA, UMR 408, Site Agroparc,
or during the transit through the gastro-intestinal tract.
Avignon cedex, 9 84914, France B. cereus spores or vegetative cells therefore have to
e-mail: michel.jobin@univ-avignon.fr overcome barriers including heat treatment, low

123
230
temperature preservation, freezing, dehydration, vari-
able compositions of atmospheres and acid environ-
ments. B. cereus frequently encounters these acid
conditions in foods when organic acids are added or
produced during fermentation, and during the passage
of contaminated food through the stomach. B. cereus
vegetative cells are more acid-sensitive than spores,
and this sensitivity depends on the growth pH and on
the type of food (Clavel et al. 2004). However, it has
been shown that B. cereus vegetative cells, like many
other bacteria, are able to induce an acid tolerance
response (ATR) (O’Hara and Glenn 1994; Davis et al.
1996; Jobin et al. 2002; Tiwari et al. 2004). These mech-
anisms of resistance to acid may involve (i) F0F1
ATPase and/or glutamate decarboxylase, which is
implicated in intracellular pH (pHi) homeostasis, (ii)
metabolic modiWcations and (iii) protein synthesis to
protect and/or repair macromolecules (Cotter and Hill
2003). It has been shown that B. cereus TZ415 grown in
regulated batch culture is more tolerant to acid shock
when cells are cultivated at low pH (pHo). The ATR
induced is maximal at pHo 5.0 and is not aVected by
growth phase. Furthermore, at pHo 6.0 and 7.0, the
ATR is induced at a higher level in the late stationary
growth phase than in other growth phases. This estab-
lished the eVect of culture pH and growth phase on
inducible acid resistance (Jobin et al. 2002). Browne
and Dowds also demonstrated such an adaptation of
B. cereus NCIMB11796 in non-regulated batch cultures,
where cells were found to maintain their pHi at a
higher level than the external acid pHo, and cells pre-
adapted by exposure to moderately low pH had a
higher pHi than unadapted cells. This suggested that
pHi may be involved in acid resistance in this bacte-
rium (Browne and Dowds 2002).
These studies observed the eVects of growth phase
and pH on acid resistance. However, according to the
growth phase, the environmental conditions were
either favourable, encouraging fast growth, or unfa-
vourable, thus slowing growth down. Thus, the growth
rate, which varies according to growth phase, may have
an eVect on the behaviour of B. cereus in acid condi-
tions. Chemostat culture enables control of all parame-
ters (pH, temperature, pO2, agitation, growth rate,
nutrient concentration), making it possible to change
the pHo or the growth rate of a culture independently
in order to study the individual roles of each parameter
on acid resistance.
Studies in other micro-organisms have demon-
strated that the ATR of cells growing in chemostat cul-
ture is both growth rate- and pHo-dependent.
Streptococcus mutans, Lactococcus lactis and Strepto-
coccus sobrinus cells growing at low pH are thus acid
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Arch Microbiol (2006) 186:229–239
resistant (Belli and Marquis 1991; O’Sullivan and Con-
don 1999; Nascimento et al. 2004). In S. mutans, quick-
growing steady-state cells are more acid tolerant than
slow-growing cells (Belli and Marquis 1991). However,
diVerent results were obtained in L. lactis because
steady-state cells growing quickly at a pHo from 7.0 to
6.0 ( = 0.5 h¡1) are less resistant to acid shock than
cells growing slowly ( = 0.17 h¡1). At pHo 5.0, the
ATR induced is not growth rate-dependent. Under
these conditions, pHi was found to be aVected by both
dilution rate and pHo (O’Sullivan and Condon 1999).
In the present study, chemostat cultures were car-
ried out at diVerent pH and growth rates in order to
diVerentiate between the eVects of growth medium pH
and growth rate on the acid resistance of B. cereus
ATCC14579. Thus, the eVect of these two parameters
on acid shock resistance and on pHi was analysed. The
study highlighted a pHo- and growth rate-dependent
ATR induced in B. cereus.
Materials and methods
Bacterial strains and growth conditions
Bacillus cereus strain ATCC 14579 was obtained from
the American Type Culture Collection. Growth
medium was J Broth (JB; 5 g l¡1 peptone, 15 g l¡1 yeast
extract, 3 g l¡1 K2HPO4 and 15 g l¡1 agar for plate
count J Agar) (Claus and Berkeley 1986). The pH of
the medium was adjusted to the desired value before
autoclaving for 20 min at 120°C. JB medium was
checked for pH value after sterilisation and supple-
mented with 2 g l¡1 Wlter-sterilised glucose. B. cereus
from stock culture was puriWed on J Agar. One colony
was transferred in an anaerobic Xask containing 100 ml
of JB at pH 7.0. The medium was sparged with oxygen-
free nitrogen gas for 15 min to eliminate oxygen.
Growth was at 34°C for 15 h with agitation at 100 rpm.
Chemostat cultures
Chemostat cultures were performed in a 2-l bioreactor
(Adagio, BiolaWtte et Morizt) using a 1-l working vol-
ume. All experiments were carried out at 34°C with
agitation at 300 rpm. Culture pH was monitored and
maintained at pH 5.5, 6.0, 7.0 or 9.0 § 0.05. During fer-
mentation, the culture was continuously sparged with
oxygen-free nitrogen gas to ensure anaerobiosis.
Medium inXow rates (F, l h¡1) were adjusted to gener-
ate the desired dilution rate (D, h¡1). Since the volume
of the growth vessel (V, in l) is set by the operator, dilu-
tion rate was equal to speciWc growth rate (
). The
Arch Microbiol (2006) 186:229–239
chemostat was operated at steady-state dilution rates
of 0.1, 0.2, 0.4, 0.6 or 0.8 h¡1. To ensure a steady-state,
cells were maintained at each deWned growth rate for
at least Wve generations before sampling for analysis.
Cell counting
Decimal dilutions in 100 mM potassium phosphate
buVer pH 7 were surface spread on J Agar plates using
a spiral plate maker (Spiral système®, Interscience, St
Nom la Bretèche, France). The cell concentration was
calculated according to the manufacturer’s instructions
and expressed as colony forming units per ml
(cfu ml¡1). The detection limit of the method was
200 cfu ml¡1.
Measurement of pHi
pHi was determined based on the Xuorescent probe
method described by Breeuwer et al. (1996) and
adapted for B. cereus by Ultee et al. (1999). Steady-
state cells were harvested at 3,500 g for 5 min at 4°C,
washed three times in 50 mM HEPES buVer (pH 7.0),
and diluted to an OD600 of 1. The cells were then incu-
bated in the presence of 1.5 M carboxyXuorescein
diacetate succinimidyl ester (cFDASE, Fluka, Lyon,
France) for 10 min at 30°C in a 15-ml tube with agita-
tion at 100 rpm. cFDASE was hydrolysed to carboxy-
Xuorescein succinimidyl ester (cFSE) in the cell and
subsequently conjugated to aliphatic amines. After
washing in 50 mM potassium phosphate buVer (pH
5.81), cells were incubated with 10 mM glucose for
30 min at 30°C with agitation at 100 rpm to eliminate
non-conjugated cFSE. In addition, cells were washed
twice and resuspended in 50 mM potassium phosphate
(pH 5.81), then kept on ice until further use. Analysis
started with addition of 30 l of the cell suspension to a
quartz cuvette containing 3 ml of a 50-mM phosphate
buVer (pH 5.81). The suspension was sparged with
nitrogen for 1 min to eliminate any oxygen. Fluores-
cence intensities were determined using a Xx-Xenius
spectroXuorometer (Safas Monaco, Monaco) with an
excitation spectrum of 400–500 nm wavelength range
including wavelengths of 490 nm (pH-sensitive) and
440 nm (pH-insensitive). Emission was determined at
525 nm. The excitation and emission slit widths were
set at 5 nm for the cell suspension and 10 nm for the
corresponding Wltrate. The ratio of the emission result-
ing from excitation at 490 and 440 nm obtained for
both cell suspension (C) and Wltrate (F) was calculated
as R490/440 = (C490 - F490)/(C440 - F440). A calibration
curve was determined as follows: B. cereus cells were
suspended in potassium phosphate buVers adjusted to
231
pH values ranging from 5.8 to 8.5 by mixing 50 mM
solutions of K2HPO4 and KH2PO4. pHi and pHo were
equilibrated by addition of 1 M valinomycin and 1 M
nigericin, and Xuorescence was measured after 2 min.
The calibration curve of treated cells showed a non-lin-
ear relationship between R490/440 and pHi. Sample pHi
values were calculated from the calibration curve by
linear interpolation.
Determination of viability loss
One millilitre of steady-state B. cereus cells was diluted
in 19 ml of JB acidiWed to pH 4.0 with HCl and main-
tained at 34°C with agitation at 100 rpm. Viability loss
was expressed as log(N/N0)t, where N is the viable
count after 10, 20 and 30 min (time t) exposure at pH
4.0 and N0 is the viable count at the beginning of acid
shock. The time to achieve one logarithmic unit reduc-
tion in population (T¡1 log) was estimated graphically.
Acid shift in the fermentor
Steady-state growing cells were submitted to an acid
shift at pH 4.0 for 5 min in the fermentor by rapid addi-
tion of 3 M HCl. The viability loss and pHi of the
B. cereus cells were determined as described above.
EVect of ionophores on cell survival after acid shock
In order to determine the role of pHi in acid tolerance,
acid shock was performed on steady-state cells with a
pHi value equilibrated with the pHo of the initial cul-
ture using ionophores. Control experiments were per-
formed via a pHi equilibration procedure with the
medium pH equal to the pHi value. One millilitre of
cells (grown at pHo 5.5 or 7.0 and at
= 0.2 h¡1) was
centrifuged at 10,000 g for 2 min. The pellet was resus-
pended in 1 ml of JB either adjusted to pHo 5.5 or 6.4
(control) for cells grown at pHo 5.5 or adjusted to pHo
7.0 or 7.9 (control) for cells grown at pHo 7.0. Cells
were incubated for 2 min in the presence of 1 M vali-
nomycin and 1 M nigericin to equilibrate pHi with
pHo. Treated cells were transferred in 19 ml of JB at
pH 4.0 and incubated for 30 min at 34°C and 100 rpm.
Viability loss of B. cereus cells was determined as
described above.
EVect of chloramphenicol on acid shock
One millilitre of steady-state B. cereus cells was incu-
bated in the absence or presence of 70 g ml¡1 of chl-
oramphenicol for 10 min at 34°C and at 100 rpm. The
cells were then diluted in 19 ml of JB at pH 4.0 (acid
123
232
challenge) or at a pH equivalent to the culture pH
(control) and maintained at 34°C with agitation at
100 rpm. Viability loss was determined by viable
counts after 10, 20 and 30 min.
Toxin measurements
Haemolysin BL detection was performed using the
B. cereus enterotoxin reverse passive latex agglutina-
tion assay kit BCET-RPLA TD950 (Oxoid, Darilly,
France), which detects the L2 component of HBL. The
amount of HBL product was determined from the
titre, which was deWned as the reciprocal of the highest
dilution of crude supernatant that gave a positive sig-
nal. NHE detection was performed using the Bacillus
diarrhoeal enterotoxin visual immunoassay (VIA,
TECRA, French Forest, Australia), which detects the
NheA component of NHE. Samples absorbance was
measured at 420 nm and the amount of NHE present
in culture supernatants was expressed in OD420 (g of
dry weight)¡1.
Statistical analysis
Acid resistance, pHi and toxin concentrations were all
determined in triplicate at diVerent times on the same
chemostat for each pH and growth rate combination
tested. Results were submitted to variance analysis
using Systat 9 software (SPSS, Chicago, IL, USA).
General linear models were used to test the eVect of
pH (four levels), growth rate (Wve levels) and their
interactions on the variables pHi, log(N/N0)5, log(N/
N0)10, log(N/N0)20 and T¡1 log. Analysis of variance of
the production of NHE and HBL enterotoxins was
performed for multiple comparisons of means using
Tukey’s honest signiWcant diVerence test at the 5%
level.
Results
EVect of medium pH and growth rate on acid tolerance
Chemostat cultures were carried out to distinguish
between the pH and growth rate eVects on acid resis-
tance. Growth rate varied independently of the pHo
following changes in dilution rate. B. cereus
ATCC14579 was grown at a pHo ranging from 9.0 to
5.5 and at a growth rate ranging from 0.1 to 0.8 h¡1 at
each pH. Steady-state B. cereus cells were harvested
and subjected to acid shock in JB at pH 4.0 for 30 min.
Cell survival was determined before and after 10, 20
and 30 min of acid shock. In a Wrst experiment, in order
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Arch Microbiol (2006) 186:229–239
to distinguish the eVect of culture pHo on cell survival
after acid shock, average of log(N/N0) was calculated
for the 15 sets of experimental data obtained at each
pHo (triplicate of acid shock for the Wve dilution rates
tested) (Fig. 1). Cell survival was greater when the cul-
ture pHo decreased from 9.0 to 5.5. Viability loss after
30 min of acid shock, i.e. log(N/N0)30, was roughly
¡4.5, ¡3.3, ¡2.6 and ¡2.2 log at pHo 9.0, 7.0, 6.0 and
5.5, respectively. Thus, B. cereus cells grown at low pHo
were adapted to acid shock, indicating the induction of
a marked ATR.
In a second experiment, in order to distinguish the
eVect of dilution rate on B. cereus survival to acid
shock, viability loss during acid shock [expressed as
log(N/N0)20] was determined for each culture pHo and
growth rate combination (Table 1). Statistical analysis
allowed us to classify the data into Wve diVerent groups
(P < 0.05). At pHo 9.0, 7.0 and 6.0, there were only
moderate variations in population decrease as a func-
tion of growth rate. At pHo 5.5, the population
decrease was more strongly aVected by dilution rate,
and varied from ¡0.6 § 0.1 at 0.1 h¡1 to ¡2.9 § 0.1 at
0.4 h¡1. However, the acid resistance of B. cereus grow-
ing cells is not only related to growth rate, since cells
growing either quickly or slowly presented similar acid
resistance. This suggests that other parameters aVect
the induction of B. cereus ATR.
EVect of pH and growth rate on intracellular pH (pHi)
In order to investigate whether pHi was implicated in
B. cereus ATR, pHi values of cells grown in chemostat
Time (min) at pH 4.0
0 10 20 30
-1
-2
log (N/N0 )
-3
-4
-5
-6
Fig. 1 Population reduction of steady-state B. cereus ATCC14579
cells grown at a pHo of 9.0 (Wlled circle), 7.0 (Wlled upward triangle),
6.0 (Wlled square) or 5.5 (Wlled diamond) and subjected to acid
shock at pH 4.0. N0 initial population, N population after exposure
to acid shock at pH 4.0, log(N/N0) logarithm of population reduc-
tion during acid shock at pH 4.0. Each point is the mean of 15
experimental data, i.e. three replicates of acid shock for each of the
dilution rates 0.1, 0.2, 0.4, 0.6 and 0.8 h¡1. Bars represent standard
deviation between the 15 experimental data
Arch Microbiol (2006) 186:229–239 233

Table 1 EVect of a growth rate ranging from 0.1 to 0.8 h¡1 on the almost constant, regardless of dilution rate. pHi
population decrease of B. cereus ATCC14579 after 20 min of acid declined when pHo decreased from 9.0 to 6.0
shock: cells were steady-state cells grown at a pHo ranging from
5.5 to 9.0 (P < 0.05), regardless of dilution rate (Fig. 2). How-
ever, pHi values were slightly higher (P < 0.05) at pHo
Growth Log(N/N0)20 5.5, with a mean of 6.49 § 0.16, compared to pHi values
rate (h¡1)
pHo 5.5 pHo 6.0 pHo 7.0 pHo 9.0 obtained at pHo 6.0, with a mean of 6.11 § 0.17. Thus,
pHo and culture dilution rate both had an eVect on the
0.1 -0.6a § 0.1 ¡1.7b § 0.1 ¡3.8c § 0.0 ¡4.9d § 0.2
pHi of B. cereus cells.
0.2 -1.8b § 0.3 ¡2.9e § 0.3 ¡3.6c § 0.1 ¡3.8c § 0.5
0.4 -2.9e § 0.1 ¡2.1be § 0.3 ¡3.8c § 0.2 ¡4.8d § 0.5
0.6 -2.1b § 0.4 ¡1.4b § 0.2 ¡2.8e § 0.1 ¡4.6d § 0.1 EVect of pHo and growth rate on
pH
0.8 -1.8b § 0.0 ¡1.2b § 0.1 ¡3.1ce § 0.0 ¡4.8d § 0.1
Data represent the mean values of three replicate experiments We have shown that pHi varied with pHo, resulting in
N0 initial population, N population after exposure to acid shock further modiWcations in
pH (pHi - pHo). In order to
at pH 4.0, log(N/N0)20 logarithm of population reduction after characterise the evolution of
pH, the
pH values cal-
20 min of acid shock at pH 4.0 culated were plotted against dilution rate for each pHo
a–e
For each pHo column, diVerent superscript letters indicate a tested (Fig. 3).
pH increased linearly with the pHo
statistically signiWcant diVerence (P < 0.05)
decrease.
pH tended to increase with dilution rate
decrease, regardless of pHo, except at pHo 5.5 where
cultures were determined for each dilution rate and pH
pH remained constant. Thus, pHo and dilution rate
tested (Fig. 2). The sensitivity of the cFDASE probe both had an eVect on the
pH of B. cereus cells.
used for pHi measurement was greater at pH values
between 5.87 § 0.1 and 8.28 § 0.23 (data not shown). Relationships between ATR, pHi and
pH
This was in good agreement with the probe sensitivity
obtained for pHi measurements in Lactobacillus del- Both the ATR and pHi of B. cereus cells were aVected
brueckii and Listeria innocua (Siegumfeldt et al. 1999). by pHo and growth rate. In order to characterise the
At pHo 9.0 and 7.0, the pHi increased signiWcantly relationship between acid shock survival and pHi,
(P < 0.05) with the decrease in dilution rate. pHi values log(N/N0)20 values were plotted versus pHi values
varied from 6.96 to 8.21 and from 6.62 to 7.93 at pH 9.0 (Fig. 4). Two patterns could be distinguished. For
and pH 7.0, respectively. At pHo 6.0, pHi increased B. cereus cells with pHi above 7.0, the population
slightly with the decrease in dilution rate (P < 0.05) decrease was maximal and seemed to be independent
from 6.01 to 6.33. Surprisingly, at pH 5.5, pHi was of pHi value. For cells with pHi below 7.0, the popula-
tion decrease was lesser as pHi decreased. Cells show-
ing better resistance to acid shock had a pHi between
6.0 and 6.5. These results indicate that the ATR of
B. cereus is strongly related to cell pHi.

Fig. 2 pHi values of B. cereus ATCC14579 cells grown in chemo-
stat cultures as function of pHo and growth rate. pHo values were
9.0, 7.0, 6.0 or 5.5 and dilution rates were 0.6 h¡1 (open bars),
0.4 h¡1 (striped bars), 0.2 h¡1 (hatched bars) and 0.1 h¡1 (black
bars). Data represent the mean values for at least three replicate
experiments
Fig. 3
pH values of B. cereus ATCC14579 cells grown in che-
mostat cultures as function of pHo and growth rate. pHo values
were 9.0, 7.0, 6.0 or 5.5 and dilution rates were 0.6 h¡1 (open bars),
0.4 h¡1 (striped bars), 0.2 h¡1 (hatched bars) and 0.1 h¡1 (black
bars). Data represent the mean values for at least three replicate
experiments

123
234 Arch Microbiol (2006) 186:229–239

pH i pH drop to 4.0 directly in the fermentor. Cell survival

5.5 6.5 7.5 8.5 and pHi were determined before and 5 min after the
0.0 pH shift (Table 2). Acid-adapted cells maintained their
viability after 5 min of acid shift to pH 4.0, and their
-1.0
pHi decreased by 0.74 units. The eVect of the acid shift
-2.0 was more marked in non-adapted cells, where popula-
log(N/N0 )20

tion reduction was 3.2 log units together with a pHi

-3.0 decrease of 1.15 units after 5 min. Thus, the pHo of
growth has a direct eVect on ATR induction level (as
-4.0
previously described), and may also inXuence the abil-
-5.0 ity of cells to maintain their pHi.

-6.0 Role of pHi in the ATR of B. cereus

Fig. 4 Population decrease after 20 min of acid shock at pH 4.0
of B. cereus ATCC14579 cells from steady-state chemostat cul- We studied the role of pHi in the survival of adapted
tures as a function of pHi values. Cells were grown at pH 9.0 and non-adapted B. cereus cells to acid stress. Cells
(Wlled circle), 7.0 (Wlled upward triangle), 6.0 (Wlled square) and 5.5
(Wlled diamond) at dilution rates ranging from 0.1 to 0.8 h¡1
grown at either pHo 7.0 (unadapted cells) or 5.5
(adapted cells) were transferred (i) at a pH equal to the
initial value (pH 7.0 and 5.5, respectively) as a control
In order to characterise the relationship between
experiment, or (ii) at a pH equal to the initial value or
(
pH and the population decrease following acid
at a pH equal to the measured pHi (pH 7.9 and 6.4,
shock, log(N/N0)20 values were plotted against the cal-
respectively), in the presence of valinomycin and nige-
culated (
pH values (Fig. 5). The reduction in popula-
ricin for pHi with pHo equilibration (
pH = 0). Valino-
tion decrease following acid shock was related to the
mycin renders the plasma membrane permeable to
increase in (
pH. Cells showing better resistance to
potassium ions, while nigericin exchanges potassium
acid shock had a positive
pH. These results indicate
for protons, so that the combined actions of these com-
the relationship between the
pH of B. cereus cells and
pounds result in an equilibration of both potassium
their ATR.
ions and protons across the membrane. Cells were sub-
mitted to acid shock at pH 4.0 and the corresponding
Acid shock aVects cell pHi
population decrease was determined (Fig. 6). Non-
adapted cells pre-incubated at pH 7.0 without iono-
To characterise the physiology of acid-adapted and
phore (pHi = 7.9,
pH = 0.9), and at pH 7.0 (pHi = 7.0,
non-adapted B. cereus cells, we investigated the eVect

pH = 0) or pH 7.9 (pHi = 7.9,
pH = 0) with the two
of acid shock on pHi. Non-adapted (i.e. grown at pHo
ionophores presented identical populations at the
7.0,
= 0.2 h¡1) and adapted (i.e. grown at pHo 5.5,
beginning of the acid shock (Fig. 6a). Thereafter, the

= 0.2 h¡1) steady-state cells were submitted to a rapid
population decrease was 3.2 § 0.1, 4.0 § 0.3 and
3.6 § 0.6 log, respectively, after 10 min. After a 30-min
∆ pH acid shock, the population decrease was similar
-2.5 -1.5 -0.5 0.5 1.5 (P < 0.05) under all three conditions, reaching a value
0 of ¡4 log. On one hand, acid-adapted cells pre-incu-
-1
bated at pH 5.5 without ionophore (pHi = 6.4,
2
R = 0.5
log(N/N0 )20

-2
Table 2 EVect of an acid shift (5 min at pHo 4.0) on population
-3 decrease and pHi of steady-state B. cereus ATCC14579 cells
grown at pHo 5.5 or 7.0 and at a growth rate of 0.2 h¡1
-4
pHo Log(N/N0)5 pHai pHbi
p
-5 Hci
-6
5.5 0.1 6.42 § 0.07 5.68 § 0.18 0.74
Fig. 5 Relationship between population decrease after 20 min of 7.0 -3.2 7.93 § 0.24 6.78 § 0.23 1.15
acid shock at pH 4.0 and the
pH (pHi - pHo) of B. cereus a
pHi before the acid shift in the fermentor
ATCC14579 cells grown at pH 5.5 (Wlled diamond), 6.0 (Wlled b
square), 7.0 (Wlled upward triangle) and 9.0 (Wlled circle) and at pHi after the acid shift in the fermentor
diVerent dilution rates ranging from 0.1 to 0.8 h¡1
c

pHi = pH-ai pHbi

123
Arch Microbiol (2006) 186:229–239 235

Time (min) B. cereus viability. The equilibration of pHi at pH 7.0

0 5 10 15 20 25 30 or 7.9 led to no change in acid resistance of the non-
A 0 adapted cells, suggesting that pHi Xuctuation in a range
from 7.0 to 7.9 and
pH abolition had no signiWcant
-1
eVects on the acid shock resistance of these cells.
pH
abolition in adapted cells (with a constant pHi) did,
log(N/N0 )

-2
however, have an eVect on acid shock resistance. The
-3
equilibration of pHi at 5.5 in addition to
pH abolition
strongly aVected the resistance of adapted cells at the
-4 beginning of the acid shock. These results suggest that
the ATR induced in B. cereus depends on the cells’
-5 ability to maintain their
pH and pHi. The decrease in
the adapted cell populations was less marked than in
0 5 10 15 20 25 30 the non-adapted cell populations, regardless of the
B 0
conditions tested. This suggests that factors other than
-1 pHi and
pH were involved in the ATR of B. cereus.

B. cereus acid shock resistance depends

log(N/N0 )

-2
-3
-4
-5
Fig. 6 Population decrease during acid shock at pH 4.0 of
B. cereus ATCC14579 cells from steady-state chemostat cultures
grown at a dilution rate of 0.2 h¡1 and at pHo 7.0 (a) and pHo 5.5
(b). Prior to acid shock at pH 4.0, the cells were incubated for
2 min without ionophores at pH 7.0 (open diamond) or 5.5 (Wlled
diamond) as controls or with 1 M nigericin and 1 M valinomy-
cin at pH 7.0 (open square) or 7.9 (open upward triangle), and pH
5.5 (Wlled square) or 6.4 (Wlled upward triangle), respectively.
Data represent mean values for at least three replicate experi-
ments

pH = 0.9), and at pH 6.4 with the two ionophores
(pHi = 6.4,
pH = 0) had identical populations at the
beginning of the acid shock (Fig. 6b), while on the
other hand, adapted cells pre-incubated at pH 5.5 with
the two ionophores (pHi = 5.5,
pH = 0) presented an
initial one log population reduction. There was no pop-
ulation decrease in adapted cells pre-incubated at pH
5.5 in the absence of ionophore during the Wrst 10 min
of acid shock; thereafter, the population decrease
peaked after 30 min of acid shock (2.3 § 0.5 log reduc-
tion). The population of adapted cells pre-incubated at
pH 5.5 or 6.4 with the two ionophores decreased by
2.7 log after 10 min of acid shock, and remained similar
after 20 and 30 min (P < 0.05). The fact that identical
initial populations were obtained at the beginning of
the acid shock (for cells pre-incubated at pH 7.0 or 5.5
without ionophore and 7.9 or 6.4 with ionophores)
adds to the evidence that the use of ionophores for
equilibration of pHi with pHo has no direct eVect on
on neo-synthesis of proteins
We investigated the impact of the neo-synthesis of pro-
teins on the acid resistance of B. cereus. Steady-state
cells grown at pH 7.0 (non-adapted cells) or at pH 5.5
(adapted cells) were incubated for 10 min in the
absence or presence of 70 g ml¡1 of chloramphenicol.
The cells were submitted to acid shock at pH 4.0 or
transferred at a pH equivalent to growth pH (as a con-
trol experiment), and the population was measured for
30 min (Table 3). Chloramphenicol had no direct eVect
on B. cereus viability. When cells were subjected to
acid shock, pre-treatment with chloramphenicol
resulted in a population decrease of about one log for
non-adapted cells and about 1.5 log for adapted cells.
Moreover, the time required for a 1 log population
decrease (T¡1 log) was reduced by half with chloram-
phenicol. These results indicate that protein neo-syn-
thesis is required for acid tolerance of B. cereus cells.
The production of NHE and HBL enterotoxins
depends on growth pHo, growth rate and pHi
In order to investigate the eVects of culture pHo and
growth rate on the production of HBL and NHE in
B. cereus, cells were grown at diVerent pHo values and
at various growth rates and enterotoxin production was
determined. The yields of HBL and NHE are pre-
sented in Table 4. At pHo 9.0, B. cereus produced the
maximum level of HBL at 0.4 h¡1 with a value of
25,600 U g¡1 dry weight. At pH 7.0, HBL production
had increased at 0.1 h¡1 with a value of 4,708 U g¡1 dry
weight, and production decreased as dilution rate
increased (P < 0.05). At pHo 6.0 and 5.5, HBL yield
peaked at 0.2 h¡1 with values of 2,560 and 4,947 U g¡1

123
236 Arch Microbiol (2006) 186:229–239

Table 3 EVect of chloramphenicol on B. cereus ATCC14579 acid resistance

Pre-incubation with + - +
chloramphenicol

Acid shock - + +

pHo Log(N/N0)10 T¡1 log (min) Log(N/N0)10 T¡1 log (min) Log(N/N0)10 T¡1 log (min)

7.0 0.1 § 0.2 ND ¡2.2 § 0.3 4 -3.3 § 0.2 2

5.5 -0.1 § 0.1 ND 0.2 § 0.2 15 -1.4 § 0.1 7
Cells from steady-state chemostat cultures grown at a dilution rate of 0.2 h¡1 and at pHo 7.0 or 5.5 were pre-incubated for 10 min in the
presence or absence of 70 g ml¡1 of chloramphenicol prior to an acid challenge at pH 4. Cells pre-incubated with chloramphenicol and
transferred in JB at a pH identical to the culture pH (7.0 or 5.5) instead of the acid challenge were included as controls. Log(N/N0)10
and T¡1 log values are the means of data for at least three replicate experiments
ND not determinable

Table 4 Yields of HBL and NHE production by cells grown at a enterotoxins, HBL and NHE production rates were
pHo ranging from 5.5 to 9.0 and at diVerent growth rates ranging plotted against pHi values. There was no relationship
from 0.1 to 0.6 h¡1
between HBL production rate and pHi (data not
YHBL [Ua(g of dry weight)¡1] shown). However, at a culture pHo of 9.0, 7.0 and 6.0,
YNHE [OD420(g of dry weight)¡1) the speciWc decrease in NHE production rate was cor-
related to the increase in cell pHi (Fig. 7). These results
Growth pHo 5.5 pHo 6.0 pHo 7.0 pHo 9.0
suggest that pHi may be involved in the regulation of
rate (h¡1)
NHE production.
0.1 1,280 2,226 4,708 582
0.7 1.9 1.5 2.8
0.2 4,947 2,560 3,724 217 Discussion
1.1 3.2 1.1 1.9
0.4 1,778 400 316 25,600
0.4 4.3 1.4 2.2 Food-borne bacteria encounter various barriers during
0.6 75 36 298 6,024 food processing, in food conservation and during tran-
1.2 3.9 3.0 3.6 sit through the gastro-intestinal tract, including acidiW-
Values in italic for YNHE
cation, heating, freezing, dehydration, starvation or
a
One unit (U) was deWned as the reciprocal of the highest dilution
low redox potential. However, many bacteria are able
of cell-free supernatant giving a positive response using the to induce an adaptive stress response which could facil-
BCET-RPLA kit itate survival to lethal conditions. Concerning acid
stress, Clavel et al. suggested that the probability of
dry weight, respectively. HBL yield subsequently viable B. cereus cells reaching the small intestine
decreased as dilution rate increased. These results
showed that HBL production (i) peaked at alkaline
pH, and (ii) was growth rate-dependent, i.e. enhanced 5.0
qNHE (toxin. (g dry weight) . (h) )
-1

at high growth rates at alkaline pHo and at slow growth 4.5

-1

rates at acidic pHo. 4.0

At pHo 7.0 and 9.0, NHE yield peaked at 0.6 h¡1, 3.5
reaching 3.0 and 3.6 OD420 g¡1 dry weight, respectively. 3.0
At pHo 6.0, NHE yield was slightly higher than at the 2.5
2.0
other pHo values tested, regardless of dilution rate,
1.5
except at 0.1 h¡1. The highest NHE yield was observed
1.0
at 0.4 h¡1 with 4.3 OD420 g¡1 dry weight. At pHo 5.5,
0.5
NHE yield ranged from 0.4 to 1.2 DO420 g¡1 dry
0.0
weight, peaking at 0.6 h¡1. These results indicate that 5.5 6 6.5 7 7.5 8 8.5
NHE production was aVected by both pHo and growth pHi
rate, and was enhanced at high growth rate.
Fig. 7 Relationship between the speciWc production rate of NHE
Non-haemolytic enterotoxin and HBL production as and the pHi values of B. cereus ATCC14579 cells grown at pHo 9.0
well as pHi were all aVected by both growth rate and (Wlled circle), 7.0 (Wlled upward triangle), 6.0 (Wlled square) or 5.5
pHo. To characterise the relationship between pHi and (Wlled diamond) and at diVerent growth rates

123
Arch Microbiol (2006) 186:229–239
(where HBL enterotoxin is produced) depends (i) on
the form of the cells ingested (vegetative cells or
spores), (ii) on the food they have contaminated and
(iii) on the pH of the stomach (Clavel et al. 2004).
Thus, food intoxication may be due to toxin production
by cells resulting in a germination of spores that have
resisted acid stress, or vegetative cells which have
adapted and survived the acid stress. Therefore, it is
particularly important to characterise the precise
behaviour of B. cereus in a low pH environment. Adap-
tation and survival at low pH are important factors in
the pathogenicity of vegetative cells, and are of great
concern in food safety and health.
We thus investigated the eVect of growth medium
pHo and growth rate on the ability of B. cereus
ATCC14579 to resist acid shock. Anaerobic growths
were carried out to avoid the obvious eVect of variation
in oxygen concentration on B. cereus physiology. Our
results demonstrated that an ATR is inducible at low
pHo in B. cereus ATCC14579, as previously established
in the TZ415 strain (Jobin et al. 2002), as well as in
many other bacteria (Karem et al. 1994; O’Hara and
Glenn 1994; Davis et al. 1996; O’Sullivan and Condon
1999; Jobin et al. 2002; Nascimento et al. 2004).
B. cereus ATCC14579 cells showed a stronger ability to
survive an acid stress when grown at pH 5.5. The
induced ATR of the B. cereus ATCC14579 strain is
more strongly aVected by growth rate at pHo 5.5. This
result is interesting, because B. cereus TZ415 ATR was
more aVected by a growth phase at pH0 ¸ 6.0 (Jobin
et al. 2002). Thus, growth rate and growth phase may
aVect the induced ATR diVerently, and must therefore
be clearly distinguished. This distinction is only
allowed by chemostat cultures. It has been demon-
strated in L. lactis and S. mutans that the acid shock
survival of cells grown in chemostat culture is growth
rate-dependent (Belli and Marquis 1991; O’Sullivan
and Condon 1999). In L. lactis, acid resistance
increased as growth rate decreased at a pHo of (6.0
(O’Sullivan and Condon 1999). At a pHo above 6.0, no
growth rate eVect on acid resistance was observed in
L. lactis, whereas the acid resistance of B. cereus
ATCC14579 was most strongly inXuenced by growth
rate at pHo 5.5. This suggests that B. cereus and L. lac-
tis have diVerent ATR mechanisms. This study high-
lighted that acid shock survival can be similar in either
fast-growing or slow-growing cells, suggesting that
ATR is aVected by other parameters.
Acid resistance in gram-positive bacteria involves
multiple strategies, including mechanisms of pHi
homeostasis (Cotter and Hill 2003). Bacteria could
maintain a neutral pHi despite an acidic external envi-
ronment, as is the case with L. innocua (Siegumfeldt
237
et al. 2000), or else they may enable a moderate
decrease in pHi with pHo while maintaining a pHi com-
patible with the cell’s physiology, as in Streptococcus
thermophilus, L. lactis, L. delbrueckii, Mycobacterium
smegmatis and Mycobacterium bovis BCG (Siegum-
feldt et al. 2000; Rao et al. 2001). We observed that the
pHi of B. cereus decreased from 8.21 to 6.49 when pHo
decreased from 9.0 to 5.5, indicating that this bacte-
rium employs the second strategy. This relatively lim-
ited decrease in pHi together with the corresponding
increase in
pH could prevent an even more dramatic
decline in pHi at lower pHo. The lower the growth pHo,
the less the pHi varied with growth rate. In B. cereus,
pHi and positive
pH were almost constant at pHo 5.5
and were higher than at pHo 6.0. These results suggest
that a mechanism of pHi homeostasis is induced at a
pHo below 6.0 in order to ensure that pHi remains com-
patible with cell physiology. Comparable results were
observed in M. smegmatis and M. bovis BCG, and
lethal pHi for both strains was less than pH 6.0 (Rao
et al. 2001).
We observed that induction of the ATR of B. cereus
ATCC14579 was correlated with a pHi decrease, but
only at a pHi above 7.0. Acid-adapted B. cereus cells
presented lower pHi compared to non-adapted cells,
and the better acid resistance was obtained with pHi
ranging from 6.0 to 6.5. Comparable results were
obtained in B. cereus NCIMB11796 by Brown and
Dowds, that performed non-regulated batch cultures at
pHo = 6.3 for acid-adapted cells (measured pHi = 7.9)
or at pHo = 7 for unadapted cells (measured pHi = 9)
(Brown and Dowds 2002). In L. lactis ssp. cremoris
NCDO712, the more acid-resistant cells have a pHi
below 6.65 (O’Sullivan and Condon 1999). This sug-
gests that reaching a low pHi induces acid resistance in
B. cereus ATCC14579. Thus, pHi is a potential factor
for ATR activation. We observed that B. cereus
ATCC14579 cells with a positive
pH also present a
better acid resistance compared to cells with a negative

pH. A positive
pH favours the entrance of protons
into the cells and the resulting ATP synthesis repre-
sents an energy supply for the cells. Such conditions
may be favourable during stress responses such as
ATR.
An acid shift in the fermentor resulted in a decrease
in B. cereus ATCC14579 pHi values. This pHi drop was
less for the acid-adapted cells compared to unadapted
cells. Similar results were observed in the B. cereus
strain NCIMB11796 (Brown and Dowds 2002). The
ability of acid-adapted B. cereus cells to maintain their
pHi with better eYciency may explain their enhanced
acid resistance. The use of the two ionophores nigeri-
cin and valinomycin allowed pHi equilibration with
123
238
various pHo values, and consequently
pH abolition.
These experiments led us to approach the issue of how
pHi and
pH are implicated in B. cereus acid resis-
tance. We have thus demonstrated that the pHi equili-
bration of unadapted cells at 7.0 or 7.9 may correspond
to physiological values and have no eVect on acid resis-
tance. However, pHi equilibration of adapted cells at
pH 5.5 or 6.4 results in a decrease in their acid resis-
tance. Thus, the eVect of ionophores did not result
from any direct toxic action on B. cereus cells, which is
consistent with observations in Listeria monocytogenes
(Datta and Benjamin 1997). In adapted cells (at
pHi = 6.4), acid resistance was strongly aVected by
pH
abolition. This tends to conWrm that maintenance of a
positive
pH is an important factor in the ATR of
B. cereus ATCC14579. Furthermore, a decrease in pHi
from 6.4 to 5.5 together with
pH abolition resulted in
an initial 1 log population decrease. A pHi value of 5.5
combined with
pH abolition are conditions that may
not allow essential metabolic activities and/or activity
of proteins that are essential for ATR (such as F0F1
ATPase). The permeability of the cytoplasmic mem-
brane to protons and proton extrusion by the F0F1
ATPase have been established as crucial for pHi main-
tenance in mycobacteria (Rao et al. 2001). We
observed that pHi and thus
pH maintenance were
both essential to the ATR of B. cereus ATCC14579.
If survival of B. cereus ATCC14579 depended solely
on pHi, cells with the same pHi—irrespective of how it
was established—should present similar behaviour
during acid shock. However, this was not the case, and
it is unlikely that the ability of cells to maintain their
pHi during an acid challenge is the only reason for their
resistance. Thus, we have demonstrated that resistance
of B. cereus to an acid shock required protein neo-syn-
thesis, as has been established in both L. monocytoge-
nes and L. lactis (O’Driscoll et al. 1996; O’Sullivan and
Condon 1999). Chaperones are proteins speciWcally
and highly synthesised in response to stress, and are
known to be involved in the general stress response
since they protect essential proteins and repair dam-
aged proteins (Minton et al. 1982).
We examined HBL and NHE production in relation
to pHo and growth rate in B. cereus ATCC14579. HBL
enterotoxin was produced at pHo from 9.0 to 5.5. Suth-
erland and Limond obtained similar results in the
HRM44 strain, since they observed that HBL was pro-
duced in a growth pH ranging from 10 to 5.5 (Suther-
land and Limond 1993). However, HBL remained
undetectable at a pHo above 6.0 in the TZ415 strain
(Jobin et al. 2002). We have shown that HBL produc-
tion was maximal at a pHo above 7.0 in the
ATCC14579 strain, at slow growth rate, and decreased
123
Arch Microbiol (2006) 186:229–239
as growth rate increased. At pHo 9.0, a high level of
HBL enterotoxin production was observed at high
growth rate, peaking at 0.4 h¡1. These results suggest
that HBL production is regulated by growth rate, as
previously demonstrated in the F4430/73 strain
(Duport et al. 2004). It appears clear that the regula-
tion of HBL production depends on the B. cereus
strain as well as on the environmental conditions
encountered by the cells.
We also demonstrated that NHE production is pH-
dependent since it was favoured at pH 6.0, except at

= 0.1 h¡1. Furthermore, NHE production was also
growth rate-dependent, and peaked at high growth
rate. However, the eVects of pH and growth rate on
HBL and NHE production are unrelated, indicating
distinct regulation mechanisms for the two toxins. In
support of this hypothesis, the correlation between pHi
and toxin production was observed with NHE but not
with HBL. Indeed, the NHE-speciWc production rate
decrease correlated with an increase in pHi, suggesting
that NHE production is pHi-dependent.
Acknowledgements We are grateful to Claire Dargaignaratz
for her technical assistance. This work was supported by the Min-
istère de l’Education Nationale, de l’Enseignement Supérieur et
de la Recherche (French Ministry for Education and Research).
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