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Applied and Environmental Microbiology, September 1998, p. 3458-3463, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Induction of Acid Resistance of Salmonella
typhimurium by Exposure to Short-Chain Fatty Acids
Y. M.
Kwon and
S. C.
Ricke*
Department of Poultry Science, Texas A&M
University, College Station, Texas 77843-2472
Received 19 March 1998/Accepted 19 June 1998
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ABSTRACT |
Exposure to short-chain fatty acids (SCFA) is one of the stress
conditions Salmonella typhimurium encounters during its
life cycle, because SCFA have been widely used as food preservatives and SCFA are also present at high concentrations in the
gastrointestinal tracts of host animals. The effects of SCFA on the
acid resistance of the organism were examined in an attempt to
understand the potential role of SCFA in the pathogenesis of S. typhimurium. The percent survival of S. typhimurium
at pH 3.0 was determined after exposure to SCFA for 1 h at pH 7.0. The percent acid survival, which varied depending on the SCFA species
and the concentration used, was 42 after exposure to 100 mM propionate
at pH 7.0 under aerobic incubation conditions, while less than 1%
could survive without exposure. The SCFA-induced acid resistance was
markedly enhanced by anaerobiosis (64%), lowering pH conditions (138%
at pH 5.0), or increasing incubation time (165% with 4 h) during exposure to propionic acid. When protein synthesis during exposure to
propionate was blocked by chloramphenicol, the percent acid survival
was less than 1, indicating that the protein synthesis induced by
exposure to propionate is required for the induction of the acid
resistance. The percent acid survival determined with the isogenic
mutant strains defective in acid tolerance response revealed that AtrB
protein is necessary for the full induction of acid resistance by
exposure to propionate, while unexpectedly, inactivation of PhoP
significantly increased acid resistance over that of the wild type
(P < 0.05). The results suggest that the virulence of
S. typhimurium may be enhanced by increasing acid resistance upon exposure to SCFA during its life cycle and further enhanced by anaerobiosis, low pH, and prolonged exposure time.
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INTRODUCTION |
Salmonella spp. are among
the major foodborne pathogens which are of public concern with respect
to food safety (22). Since Salmonella is a
facultatively anaerobic bacterium and does not require strict
conditions for its growth, this pathogen is able to proliferate
and survive in diverse environmental niches, including most
environmental ecosystems, food production and processing systems, and
intestinal tracts of the host animals. During its life cycle,
Salmonella can encounter various environmental stress conditions, such as nutrient starvation, pH extremes, oxidative stress,
osmotic shock, and heat shock (12), which may have dramatic effect(s) on its survival and virulence (1).
Depending on the severity and duration of the exposure to the
stressors, either the growth or survival of Salmonella
is inhibited, or the cells lose viability. This organism also has
the capability of sensing the stress conditions as signals for inducing
dramatic changes in gene expression and protein synthesis
(12). Although the mechanisms of how Salmonella
sense the stress conditions are not well understood, the general
function of the stress response is to enable the cells to be more
tolerant or resistant to the stress conditions encountered.
One important adaptation mechanism of Salmonella typhimurium
is the acid tolerance response (ATR), where the acid resistance of
S. typhimurium is greatly enhanced when the cells are
exposed to conditions considered mildly acidic (pH 5.8) (9).
Acid adaptation of S. typhimurium appears to have an
important role in the survival in various stress conditions. Leyer and
Johnson (24) reported that acid adaptation induces
cross-protection against several environmental stresses, and
Wilmes-Riesenberg et al. (33) showed that mutants which were
more sensitive to acid were highly attenuated, suggesting a strong
correlation in S. typhimurium between the ability
to mount an ATR and virulence.
One of the potential stress conditions frequently encountered by
S. typhimurium is the presence of short-chain fatty acids (SCFA), such as acetate, propionate, and butyrate. SCFA are produced as
fermentation products by native intestinal microflora and can be
present at high concentrations in gastrointestinal ecosystems possessing large numbers of highly fermentative anaerobic bacteria. In
humans, for example, the concentrations of SCFA are 35 mmol/kg in the
small intestine and 134 mmol/kg in the large intestine (4).
Salmonella spp. may also encounter the SCFA acetate and propionate in food products, such as meat carcasses, salad dressing, and mayonnaise, where they are widely used as preservatives (100 to 300 mM) due to their antibacterial activities (3, 5, 16, 20).
In this study we report that the acid resistance of S. typhimurium is greatly increased after exposure of this organism
to SCFA, and this SCFA-induced acid resistance is further enhanced by
acid pH, anaerobiosis, and prolonged exposure to SCFA. The results
suggest that the exposure of S. typhimurium to SCFA
increases the virulence of this pathogen by increasing the acid
resistance.
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MATERIALS AND METHODS |
Bacterial strains.
S. typhimurium strains used are
listed in Table 1. Generalized
transduction with P22 HT105/1 int-201 was conducted by the method of Maloy et al. (27). Transductants selected on
Luria-Bertani (LB) agar plates containing relevant antibiotics were
purified on Evans Blue-Uranine (EBU) (27) plates and
subsequently cross-streaked with P22 H5 for identification of and
subsequent elimination of pseudolysogens and lysogens. A spontaneous
mutant of S. typhimurium ATCC 14028s resistant to nalidixic
acid (NA) was used in all of the experiments, except for Fig. 2 and 5,
where wild-type and ATR mutants of S. typhimurium ATCC
14028s and SL1344 were used. Using the antibiotic marker strain allowed
the use of broth and agar media containing the antibiotics, so that the
potential problems by contamination could be avoided. We confirmed that
the results were not affected by the Nar marker of the
mutant strain.
Preparation of media.
Tryptic soy broth (TSB; Difco
Laboratories, Detroit, Mich.) and E medium (0.02%
MgSO4 · 7H2O, 0.2% citric acid
(C6H8O7 · H2O), 1% K2HPO4, 0.35%
NaHNH4PO4 · 2H2O) containing
0.2% glucose (27) were used for the SCFA adaptation (pH
7.0) and acid survival assay (pH 3.0). For the preparation of anaerobic
TSB medium, aerobically prepared TSB medium were dispensed into test
tubes (13 by 100 mm) (4 ml/tube) and placed in an anaerobic chamber
(Coy Laboratories, Ann Arbor, Mich.) with a mixed-gas atmosphere
consisting of 10% H2, 10% CO2, and 80%
N2 gas. After 4 to 5 h of equilibrium, the tubes were
sealed with butyl rubber stoppers and autoclaved for 20 min. The
initial oxidation-reduction (O/R) potential of the medium was measured
with a Corning pH meter 240 (Corning Inc., Corning, N.Y.), which was
kept in an anaerobic chamber. The electrode was stored in a standard
ferrous-ferric solution and calibrated with a reference electrode by
the method of Light (25).
Conditions for SCFA adaptation and acid resistance assay.
The SCFA adaptation assay was conducted as follows, unless described
otherwise. Ten microliters of a fresh culture of S. typhimurium grown overnight was inoculated into each test tube
containing 4 ml of TSB medium (pH 7.0), and grown in a water bath at
37°C until the optical density (A600) reached
0.2 on a Spectronic 20D spectrophotometer (Milton Roy Co., Rochester,
N.Y.). This was followed by addition of filter-sterilized SCFA stock
solution (1.0 M; pH 7.0, adjusted with NaOH) to the specified final
concentration and incubation at 37°C for 1 h. To determine the
acid resistance of the cells, 100 µl of the adapted culture was
transferred to 4 ml of phosphate-buffered saline (PBS) buffer (pH 7.2)
and of TSB medium (pH 3.0, adjusted with HCl). The CFU/milliliter in PBS was determined by plating serial dilutions in PBS buffer (pH 7.2)
on tryptic soy agar (TSA; Difco Laboratories) plates containing NA (25 µg/ml) and used as initial cell populations. The TSB medium (pH 3.0)
inoculated with adapted S. typhimurium was incubated for an
additional hour at 37°C, and the CFU/milliliter in TSB medium (pH
3.0) was determined in the same way and used as final cell populations.
The percent acid survival was then calculated as (initial
population/final population) × 100.
Agar well diffusion assay.
Growth inhibition by SCFA was
measured by an agar well diffusion assay. A fresh culture of S. typhimurium grown overnight in TSB was diluted 100-fold in PBS
buffer (pH 7.2), and approximately 105 cells in 100 µl
were spread on an M9 (0.6% Na2HPO4, 0.3%
KH2PO4, 0.05% NaCl, 0.1% NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2) agar plate containing 0.2% glucose. The well was made using a Pasteur pipette, and 80 µl
of 1.0 M SCFA stock solution (pH 7.0) was added to the well. After
24 h of incubation in a 37°C incubator, the diameter of the
inhibitory zone was measured. For the assay under anaerobic growth
conditions, the plates were transferred and incubated at 37°C in an
anaerobic chamber.
Statistical analysis.
The diameters of inhibition zones and
the percent survivals were analyzed by least-squares mean separations
which were accomplished by the pdiff option of the General Linear
Models (GLM) procedure in SAS statistical analysis software program,
version 6.11 (31). Each mean was the average of three
independent trials, and means were considered significantly different
at P < 0.05.
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RESULTS AND DISCUSSION |
Acid resistance after adaptation to SCFA.
In our preliminary
study we found that adaptation to propionate markedly increased the
acid resistance of an S. typhimurium strain isolated from
poultry (21). Guilfoyle and Hirshfield (14)
also reported that the acid resistance of Escherichia
coli can be induced by exposure to SCFA at neutral pH. In
order to investigate the observations in detail, we determined the acid resistance of S. typhimurium after adaptation to
various SCFA as described in Materials and Methods. The percent
survival of S. typhimurium after 1-h exposure to pH 3.0 was
conveniently used to represent the acid resistance of the cells by the
method of Foster and Hall (11). We also compared their
effects under both aerobic and anaerobic incubation conditions (Fig.
1A). The levels of acid resistance
induced by acetate and propionate were significantly greater than the
levels induced by other SCFA or NaCl under both aerobic and anaerobic
conditions (P < 0.05). NaCl (100 mM, pH 7.0) was used
as a negative control because NaOH was used to adjust the pH of the
SCFA stock solutions (1.0 M, pH 7.0). No survivors were recovered when
acid resistance was determined before adaptation. We also found that
the acid resistance induced by the various SCFA was significantly
enhanced by anaerobiosis (P < 0.05). Greater than 60%
survival was seen after 1 h at pH 3.0 after adaptation to either
acetate or propionate. The measured O/R potentials of the aerobic and
anaerobic TSB media were 378.0 ± 5.0 and 
62.2 ± 16.7 mV,
respectively. This result may have an important implication in the
sense that anaerobic conditions resulting in a negative O/R potential
can be found in the gastrointestinal tracts of host animals and also in
vacuum-packaged meat products (20). The O/R potential in the
cecum of a conventional rat with native microflora was 206 ± 40 mV when it was read 5 min after insertion of the electrode in the cecum
(34), and Broberg (2) reported the O/R of the
ruminal contents was approximately 150 mV. However, it remains to be
determined if there is a correlation between O/R potential and
SCFA-induced acid resistance while the O/R potential reaches the levels
observed in the gastrointestinal tract or rumen.
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FIG. 1.
Percent acid survival of S. typhimurium ATCC
14028s after adaptation to various SCFA (100 mM) in aerobic and
strictly anaerobic TSB media (A) and when TSB and E media were
used for both adaptation and acid challenge (B).
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It was previously shown by Lin et al. (
26) that the
induction of acid resistance by adaptation to mild pH requires
components
of complex medium in
E. coli and
Shigella
flexneri, but not in
S. typhimurium, suggesting
that nutrient composition also plays
an important role in SCFA-induced
acid resistance. Therefore,
we determined the acid resistance of
S. typhimurium in complex
(TSB) and minimal (E) media
at pH 3.0 after exposure to SCFA in
the medium at pH 7.0 (Fig.
1B). We
found that the acid resistance
after adaptation to the same SCFA was
not significantly different
between minimal and complex media, except
when butyrate was used
for adaptation (
P < 0.01). When
butyrate was added, the acid resistance
in minimal medium increased
approximately 2.7-fold over that in
complex medium.
Growth inhibition by SCFA.
S. typhimurium is able
to use SCFA, such as acetate or propionate, as its sole carbon and
energy source under aerobic growth conditions but requires considerable
adaptation time (18 to 20 h) to grow on propionate (7,
19). On the other hand, SCFA have been widely used as food
preservatives because of their antibacterial activity (3, 5,
16) and also have been suggested to be a major factor
inhibiting the colonization by S. typhimurium by competitive exclusion of native microflora in the mouse intestine (17, 28).
Therefore, we reasoned that the acid resistance of
S. typhimurium induced by exposure to SCFA shown in Fig.
1 may be the
result
of growth inhibition by SCFA rather than specific activities of
SCFA. To answer this question, we quantitatively measured the
growth-inhibitory activities of various SCFA species at pH 7.0
and
compared them under both aerobic and anaerobic growth conditions.
We
used an agar well diffusion assay for convenient screening,
and the
growth inhibition of
S. typhimurium was represented by
the diameters (in millimeters) of the inhibitory zones (Fig.
2).
Under both aerobic and anaerobic
conditions, the growth inhibition
by propionate was significantly
higher than those by other SCFA
and NaCl (
P < 0.01).
No inhibition of
S. typhimurium was observed
with
acetate or NaCl. We also found that the growth-inhibitory
effects of
all SCFA species tested were significantly lower under
anaerobic growth
conditions than those under aerobic growth conditions
(
P < 0.05).
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FIG. 2.
Growth inhibition of S. typhimurium ATCC
14028s by SCFA determined by the agar well diffusion assay.
Susceptibility was measured as the zone diameter (in millimeters)
surrounding a SCFA-containing agar well.
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The acid resistance was induced by acetate, although acetate did not
inhibit the growth of
S. typhimurium. Also, the
acid
resistance induced by SCFA was greatly enhanced by
anaerobiosis,
while the growth inhibition by SCFA was suppressed
under anaerobic
conditions. Therefore, the above results indicate
that the induction
of acid resistance of
S. typhimurium by exposure to SCFA was not
mediated by a
growth-inhibitory effect.
Effects of various adaptation conditions on SCFA-induced acid
resistance.
In order to examine the effects of various
adaptation conditions on the induction of acid resistance, we chose to
use propionate for further study, because it appears to be most
effective in terms of both growth inhibition and induction of acid
resistance.
To examine the dose response of SCFA-induced acid resistance, we
determined the acid resistance of
S. typhimurium after
adaptation
to propionate at various concentrations (0 to 100 mM) at pH
7.0
(Fig.
3A). As expected, the percent
survival increased from <0.1
to 54 as the propionate concentration was
increased from 0 to
100 mM in 20 mM increments.
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FIG. 3.
Percent acid survival of S. typhimurium
ATCC 14028s after adaptation to propionate when various concentrations
of propionate (A), various adaptation times (B) and different pHs (C)
were used.
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The effect of adaptation time on the level of acid resistance was
also examined by increasing the time of adaptation to 100
mM
propionate at pH 7.0 from 0 to 4 h (Fig.
3B). Without adaptation,
only <0.1% survived, while the acid resistance was significantly
increased by increasing the adaptation time to longer than
1 h
(
P < 0.01). When the adaptation time was
extended to 4 h, 165%
of the cells survived, which means that
the adapted cells of
S. typhimurium were able
to multiply even at pH 3.0. One possibility
is that the cells present
as clumps were separated upon exposure
to acidic pH, which then
resulted in increased cell numbers, as
determined by CFU. As the
exposure time of
S. typhimurium to SCFA
in
environmental niches is likely to be much longer than 1 h,
the
enhancement of SCFA-induced acid resistance by prolonged exposure
to
SCFA appears to be relevant in its life cycle. However, whether
SCFA-induced acid resistance could be enhanced further by exposure
to
SCFA for much longer times (e.g., several days) remains to
be
determined.
It is known that SCFA enter bacterial cells only in undissociated
forms, and as extracellular pH decreases, the portion of
the
undissociated SCFA increases, increasing their activities
to bacterial
cells (
3). Although the mode of action by which
SCFA
increase the acid resistance of
S. typhimurium is not
known,
we assumed that pH level may be one critical factor
controlling
SCFA-induced acid resistance. Therefore, we determined the
acid
resistance of
S. typhimurium after adaptation to
100 mM propionate
at different pHs (Fig.
3C). For this study, the
adaptation protocol
was partially modified so that the adaptation to
100 mM propionate
could be done exactly at pH 5.0, 6.0, 7.0, and 8.0. The cells
of
S. typhimurium were grown in TSB medium
(pH 7.0) overnight,
and 400 µl of the culture was transferred to TSB
medium at pH
5.0, 6.0, 7.0, and 8.0 containing 0 or 100 mM propionate.
After
incubation for 1 h, the adapted cultures were assayed for
acid
survival as described in the Materials and Methods. When adapted
to propionate at pH 8.0, only 2% survived. However, as the pH
was
decreased, the percent survival greatly increased, leading
to 138%
survival when adapted at pH 5.0, which indicates the capability
of the
adapted cells to multiply at pH 3.0. However, the percent
survival of
the cells without exposure to propionate was significantly
lower than
the cells adapted to propionate at the same pH (
P <
0.05). The enhanced survival in acidic pH conditions might be
explained
either by combined effects of both ATR and SCFA-induced
acid resistance
or increased concentrations of propionate anion
in acidic
conditions. According to Cummings et al. (
4), the
pHs of
different sections of the human intestine are 6.3 ± 0.1
in
the ileum, 5.6 ± 0.2 in the cecum, and 6.2 ± 0.1 in the
colon.
Therefore, it seems reasonable to suppose that the mildly acidic
conditions in the human intestine play a role in enhancing SCFA-induced
acid resistance in the intestinal environment.
Role of protein synthesis in SCFA-induced acid resistance.
We
reasoned that the SCFA-induced acid resistance of S. typhimurium described thus far should be conferred by the
synthesis of a specific set of proteins. Therefore, we determined the
acid resistance of S. typhimurium after adaptation to
100 mM propionate at pH 7.0 while protein synthesis was blocked by
addition of chloramphenicol (50 µg/ml) at different time points
during the adaptation (Fig. 4). This
concentration (50 µg/ml) was previously shown to block the protein
synthesis of S. typhimurium completely (10).
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FIG. 4.
Effect of blocking protein synthesis on the percent acid
survival of S. typhimurium ATCC 14028s after adaptation
to propionate (100 mM). Chloramphenicol (50 µg/ml) was added at
different time points during the adaptation procedure.
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The results show that the addition of chloramphenicol 5 min before the
addition of propionate completely abolished the acid
resistance,
whereas the addition of the antibiotics 30 min after
the addition of
propionate and 5 min before the acid challenge
did not have a
significant effect on the induction of acid resistance
compared to that
of the control (
P < 0.05). The results indicate
that a
set of proteins newly synthesized within 30 min after the
cells are
exposed to propionate was responsible for the induction
of acid
resistance by adaptation to propionate. However, protein
synthesis
after the acid challenge at pH 3.0 did not appear to
have an essential
role in the acid resistance. By using two-dimensional
gel
electrophoresis, Guilfoyle and Hirshfield (
15) identified
six proteins which are specifically synthesized in response to
butyrate
(11 mM) at pH 5.5, including inducible arginine decarboxylase,
lipoamide acetyltransferase, inducible lysyl-tRNA synthetase.
Although it is not known if these proteins are also synthesized
in
S. typhimurium by exposure to other SCFA, they may play
an
important role in expressing the SCFA-induced acid resistance
phenotype.
SCFA-induced acid resistance of isogenic ATR mutant
strains.
The best-characterized mechanism for the induction of
acid resistance in S. typhimurium is the ATR, which
occurs when the acid resistance of this microorganism is greatly
induced after the cells are adapted to mildly acidic pH conditions
(9). Numerous genes of S. typhimurium that
are necessary to express ATR phenotypes have been identified and
characterized, including rpoS, phoPQ, fur, mviA, atp, and atrB
(9). It is reasonable to speculate that there may be an
overlap between the genetic systems for ATR and SCFA-induced acid
resistance. Therefore, we examined the effects of the ATR mutations on
SCFA-induced acid resistance (Fig. 5). To
compare the ATR mutations in different genetic backgrounds, the
mutations linked to antibiotic markers were transferred by P22
transduction from the ATR mutant strains obtained from Foster and Hall
(11) and Lee et al. (23) to the wild-type
S. typhimurium ATCC 14028s and SL1344 strains, and the
resulting transductants were selected on LB plates containing relevant
antibiotics. Among the four ATR mutations tested, only the mutants
harboring a mutation in atrB appeared to be defective in
inducing acid resistance after adaptation to 100 mM propionate at pH
7.0 in both strain backgrounds but not statistically significant
(P > 0.05) compared to that of the wild type. The
mutation in the rpoS and atp gene did not change
the acid resistance after adaptation to propionate significantly (P > 0.05), whereas unexpectedly, the phoP
mutation in ATCC 14028s background significantly increased the acid
survival of S. typhimurium over that of the wild type
(P < 0.05). Wilmes-Reisenberg et al. (33)
reported that the atrB mutation in SL1344 strain just
slightly increased the 50% lethal dose (LD50) in mouse
infection study over that of the wild type. It is possible that the
partial defect of the atrB mutant in SL1344 background in
SCFA-induced acid resistance is the reason for the minor role of the
gene in virulence during mouse infection. If that is correct, it may be
interesting to determine the LD50 of atrB mutant
in ATCC 14028s background, because this mutation almost completely
abolished the SCFA-induced acid resistance (0.8% survival) in this
strain background. Although it was not shown in Fig. 5, we also found
that a mutant strain of S. typhimurium SF1 with a
mutation in the fur gene (JF2391 [23]) could
not mount SCFA-induced acid resistance.
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FIG. 5.
Percent acid survival of wild-type (WT) and
ATR mutant strains of S. typhimurium ATCC 14028s and
SL1344 after adaptation to propionate (100 mM). The percent acid
survival determined before adaptation was <0.0001% for all the
strains tested (not shown on the graph).
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Concluding remarks.
Given that S. typhimurium
is a major foodborne pathogen and also an enteropathogen which can use
an oral route of infection to reach the gastrointestinal tract and
colonize the intestine or invade the epithelial cells of the small
intestine in its host animal (8), S. typhimurium should be frequently exposed to SCFA during its life
cycle. The concentration range of SCFA used as food preservatives is
approximately 100 to 300 mM (3, 5, 16), and the SCFA
concentrations in the human intestine varies, depending on the region.
The concentrations of acetate, propionate, butyrate, and valerate are
57.9, 23.1, 24.4, and 4.2 mmol/kg, respectively, in the human colon but
only 7.9, 1.5, 2.3, and 0.2 mmol/kg, respectively, in the human ileum
(4). The concentrations of SCFA in the chicken cecum, which
is a major site of Salmonella colonization are 56, 29, and
10 mmol/kg for acetate, propionate, and butyrate, respectively
(13). However, little information is available on the
potential role of SCFA on either the survival and virulence of
Salmonella in the host gastrointestinal tract. Recently,
El-Gedaily et al. (6) found that expression of the spv operon, which is located on the virulence plasmid of
Salmonella dublin, is markedly induced specifically by SCFA
with two to six carbons. The rpoS gene is also known to be
induced by SCFA, such as acetate and propionate (29).
Although it is not certain that SCFA is the physiological inducer of in
vivo expression of these virulence genes, SCFA production in the
intestine may be related to them.
The results in this study provide additional evidence indicating that
SCFA in the gastrointestinal tract of a host animal
or in food
materials may contribute to the enhancement of the
virulence of
S. typhimurium by increasing acid resistance. Also
we
report here that various environmental conditions, which can
be found
in the gastrointestinal tracts of host animals, such
as acidic pH and
low redox potential, and prolonged exposure to
SCFA, markedly
enhance the SCFA-induced acid resistance.
There are two stages during the process of infection in which acid
resistance would be required for successful pathogenesis
of
S. typhimurium. One is the gastric acidity (pH 3.0) in
the
stomach that
Salmonella ingested with contaminated food
materials
would pass through (
32), and the other one is the
acidification
(pH 4.0 to 5.0) of the phagolysosome which
Salmonella would encounter
when phagocytosed by
macrophages (
30).
To probe the hypothesis that the SCFA-induced acid resistance reported
in this study is required for
Salmonella to cause systemic
disease in a host animal, we will need to isolate mutant strains
of
S. typhimurium defective in SCFA-induced acid
resistance and
determine the LD
50s of the mutant strains in
an infection study.
In addition to the results in this study, we also found that the
resistance of
S. typhimurium to other stress conditions
including
reactive oxygen and high osmolarity was increased by exposure
to the mixture of SCFA species at the concentrations comparable
to
those in large intestines of animals (data not shown). The
capability
of SCFA to induce cross-protection to various stress
conditions
indicates that SCFA could have a more profound effect
on the survival
or virulence of
S. typhimurium in a host animal.
This
could have biological and practical implications for controlling
Salmonella spp. in food production systems.
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ACKNOWLEDGMENTS |
We thank J. W. Foster (Department of Microbiology and
Immunology, University of South Alabama) and K. E. Sanderson
(Department of Biological Sciences, University of Calgary, Calgary,
Alberta, Canada) for sharing S. typhimurium ATR mutant
strains and wild-type SL1344 strain, respectively. We also thank
D. A. Siegele (Department of Biology, Texas A&M University) for
critical reading of the manuscript and helpful suggestions.
This research was supported in part by Hatch grant 8311 administered by
the Texas Agricultural Experiment Station and by Faculty minigrant (FMG
97-152) administered by the Office of the Vice President for Research
and Associate Provost for Graduate Studies in Texas A&M University.
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FOOTNOTES |
*
Corresponding author. Mailing address: Poultry Science
Department, Room 101, Kleberg Center, Texas A&M University, College Station, TX 77843-2472. Phone: (409) 862-1528. Fax: (409) 845-1921. E-mail: sricke{at}poultry.tamu.edu.
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REFERENCES |
| 1.
|
Archer, D. L.
1996.
Preservation microbiology and safety: evidence that stress enhances virulence and triggers adaptive mutations.
Trends Food Sci. Technol.
7:91-95.
|
| 2.
|
Broberg, G.
1957.
Measurement of the redox potential in rumen contents. I. In vitro measurements on healthy animals.
Nord. Vetmed.
9:918-930.
|
| 3.
|
Cherrington, C. A.,
M. Hinton,
G. C. Mead, and I. Chopra.
1991.
Organic acids: chemistry, antibacterial activity, and practical applications.
Adv. Microb. Physiol.
32:87-108[Medline].
|
| 4.
|
Cummings, J. H.,
E. W. Pomare,
W. J. Branch,
C. P. E. Naylor, and G. T. Macfarlane.
1987.
Short chain fatty acids in human large intestine, portal, hepatic and venous blood.
Gut
28:1221-1227[Abstract/Free Full Text].
|
| 5.
|
Dorsa, W. J.
1997.
New and established carcass decontamination procedures commonly used in the beef-processing industry.
J. Food. Prot.
60:1146-1151.
|
| 6.
|
El-Gedaily, A.,
G. Paesold,
C. Y. Chen,
D. G. Guiney, and M. Krause.
1997.
Plasmid virulence gene expression induced by short-chain fatty acids in Salmonella dublin: identification of rpoS-dependent and rpoS-independent mechanisms.
J. Bacteriol.
179:1409-1412[Abstract/Free Full Text].
|
| 7.
|
Fernandez-Briera, A., and A. Garrido-Pertierra.
1988.
A degradation pathway of propionate in Salmonella typhimurium LT-2.
Biochemie
70:757-768[Medline].
|
| 8.
|
Finlay, B. B.
1994.
Molecular and cellular mechanisms of Salmonella pathogenesis.
Curr. Top. Microbiol. Immunol.
192:163-185[Medline].
|
| 9.
|
Foster, J. W.
1995.
Low pH adaptation and the acid tolerance response of Salmonella typhimurium.
Crit. Rev. Microbiol.
21:215-237[Medline].
|
| 10.
|
Foster, J. W.
1991.
Salmonella acid shock proteins are required for the adaptive acid tolerance response.
J. Bacteriol.
173:6896-6902[Abstract/Free Full Text].
|
| 11.
|
Foster, J. W., and H. K. Hall.
1990.
Adaptive acidification tolerance response of Salmonella typhimurium.
J. Bacteriol.
172:771-778[Abstract/Free Full Text].
|
| 12.
|
Foster, J. W., and M. P. Spector.
1995.
How Salmonella survive against the odds.
Annu. Rev. Microbiol.
49:145-174[Medline].
|
| 13.
| Goldstein, D. L. 1989. Absorption by the cecum
of wild birds: is there interspecific variation? J. Exp. Zool.
3(Suppl.):103-110.
|
| 14.
|
Guilfoyle, D. E., and I. N. Hirshfield.
1996.
The survival benefit of short-chain organic acids and the inducible arginine and lysine decarboxylase genes for Escherichia coli.
Lett. Appl. Microbiol.
22:393-396[Medline].
|
| 15.
|
Guilfoyle, D. E., and I. N. Hirshfield.
1994.
The molecular response of Escherichia coli to the short chain organic acid butyrate.
Ann. N.Y. Acad. Sci.
730:246-248[Medline].
|
| 16.
|
Hardin, M. D.,
G. R. Acuff,
L. M. Lucia,
J. S. Oman, and J. W. Savell.
1995.
Comparison of methods for contamination removal from beef carcass surfaces.
J. Food Prot.
58:368-374.
|
| 17.
|
Hentges, D. J.
1983.
Role of the intestinal microflora in host defense against infection, p. 311-331.
In
D. J. Hentges (ed.), Human intestinal microflora in health and disease. Academic Press, New York, N.Y.
|
| 18.
|
Hoiseth, S. K., and B. A. Stocker.
1981.
Aromatic-dependent Salmonella typhimurium are nonvirulent and effective as live vaccines.
Nature
291:238-239[Medline].
|
| 19.
|
Horswill, A. R., and J. C. Escalante-Semerena.
1997.
Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon.
J. Bacteriol.
179:928-940[Abstract/Free Full Text].
|
| 20.
|
Jay, J. M.
1997.
Modern food microbiology, 5th ed.
Chapman & Hall, New York, N.Y.
|
| 21.
|
Kwon, Y. M., and S. C. Ricke.
1996.
Characterization of propionic acid sensitivity of Salmonella typhimurium under strict anaerobic growth conditions, abstr. P-30, p. 373.
In
Abstracts of the 96th General Meeting of the American Society for Microbiology 1996. American Society for Microbiology, Washington, D.C.
|
| 22.
|
Lax, A. J.,
P. A. Barrow,
P. W. Jones, and T. S. Wallis.
1995.
Current perspectives in salmonellosis.
Br. Vet. J.
151:351-377[Medline].
|
| 23.
|
Lee, I. S.,
J. Lin,
H. K. Hall,
B. Bearson, and J. W. Foster.
1995.
The stationary-phase sigma factor  s (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium.
Mol. Microbiol.
17:155-167[Medline].
|
| 24.
|
Leyer, G. J., and E. A. Johnson.
1993.
Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium.
Appl. Environ. Microbiol.
59:1842-1847[Abstract/Free Full Text].
|
| 25.
|
Light, T. S.
1972.
Standard solution for redox potential measurements.
Anal. Chem.
44:1038-1039.
|
| 26.
|
Lin, J.,
I. S. Lee,
J. Frey,
J. L. Slonczewski, and J. W. Foster.
1995.
Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli.
J. Bacteriol.
177:4097-4104[Abstract/Free Full Text].
|
| 27.
|
Maloy, S. R.,
V. J. Stewart, and R. K. Taylor.
1996.
Genetic analysis of pathogenic bacteria: a laboratory manual.
Cold Spring Harbor Laboratory Press, Plainview, N.Y.
|
| 28.
|
Meynell, G. G.
1963.
Antibacterial mechanisms of the mouse gut. II. The role of Eh and volatile fatty acids in the normal gut.
Br. J. Exp. Pathol.
44:209-219[Medline].
|
| 29.
|
Mukhopadhyay, S., and H. E. Schellhorn.
1994.
Induction of Escherichia coli hydroperoxidase I by acetate and other weak acids.
J. Bacteriol.
176:2300-2307[Abstract/Free Full Text].
|
| 30.
|
Rathman, M.,
M. D. Sjaastad, and S. Falkow.
1996.
Acidification of phagosomes containing Salmonella typhimurium in murine macrophages.
Infect. Immun.
64:2765-2773[Abstract].
|
| 31.
|
SAS Institute.
1988.
SAS user's guide.
SAS Institute Inc, Cary, N.C.
|
| 32.
|
Small, P. L. C.
1994.
How many bacteria does it take to cause diarrhea and why?, p. 479-489.
In
V. L. Miller, J. B. Kaper, D. A. Portnoy, and R. R. Isberg (ed.), Molecular genetics of bacterial pathogenesis. ASM Press, Washington, D.C.
|
| 33.
|
Wilmes-Riesenberg, M. R.,
B. Bearson,
J. W. Foster, and R. Curtiss, III.
1996.
Role of the acid tolerance response in virulence of Salmonella typhimurium.
Infect. Immun.
64:1085-1092[Abstract].
|
| 34.
|
Wostmann, B. S., and E. Bruckner-Kardoss.
1966.
Oxidation-reduction potentials in cecal contents of germfree and conventional rats.
Proc. Soc. Exp. Biol. Med.
121:1111-1114[Medline].
|
Applied and Environmental Microbiology, September 1998, p. 3458-3463, Vol. 64, No. 9
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Abstract
Introduction
