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Applied and Environmental Microbiology, October 1998, p. 3882-3886, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Acid-Sensitive Enteric Pathogens Are Protected from
Killing under Extremely Acidic Conditions of pH 2.5 when They Are
Inoculated onto Certain Solid Food Sources
Scott R.
Waterman1 and
P. L. C.
Small2,*
Department of Infectious Diseases, Imperial
College, Hammersmith Hospital, London W12 ONN, United
Kingdom,1 and
Rocky Mountain
Laboratories, National Institute of Allergy and Infectious
Diseases, Hamilton, Montana 598402
Received 2 January 1998/Accepted 6 July 1998
 |
ABSTRACT |
Gastric acidity is recognized as the first line of defense against
food-borne pathogens, and the ability of pathogens to resist this pH
corresponds to their oral infective dose (ID). Naturally occurring and
genetically engineered acid-sensitive enteric pathogens were examined
for their ability to survive under acidic conditions of pH 2.5 for
2 h at 37°C when inoculated onto ground beef. Each of the
strains displayed significantly high survival rates under these
normally lethal conditions. The acid-sensitive pathogens Campylobacter jejuni and Vibrio cholerae, which
were protected at lower levels from acid-induced killing by ground beef
under these conditions, were sensitive to killing in acidified media at
pH 5.0 but survived at pH 6.0. Salmonella inoculated onto
the surface of preacidified ground beef could not survive if the pH on
the surface of the beef was 2.61 or lower but was viable if the surface
pH was 3.27. This implies that the pH of the microenvironment occupied
by the bacteria on the surface of the food source is critical for their
survival. Salmonella was also shown to be protected from
killing when inoculated onto boiled egg white, a food source high in
protein and low in fat. These results may explain why Salmonella species have a higher oral ID of approximately
105 cells when administered under defined conditions but
have been observed to cause disease at doses as low as 50 to 100 organisms when consumed as part of a contaminated food source. They may also help explain why some pathogens are associated primarily with
food-borne modes of transmission rather than fecal-oral transmission.
| |
INTRODUCTION |
The low pH of gastric secretions has
long been recognized as the first line of defense against food-borne
enteric pathogens (13, 28). The ability of enteric bacteria
to resist killing by acid during transit through the stomach increases
their likelihood of colonizing the intestines and causing an infection.
The infective dose (ID) of different enteric pathogens corresponds to
their relative abilities to resist killing by acid (23). The
ID of Vibrio cholerae, nontyphi Salmonella
species, and Shigella flexneri are approximately
109, 105, and 102, respectively
(5). These doses correspond with the relative levels of acid
resistance of these pathogens, with V. cholerae being the
least resistant and S. flexneri being the most resistant (23).
Enteric microorganisms have evolved several mechanisms for handling
acid stress. Escherichia coli O157:H7 and S. flexneri, which both have a low ID, can survive extreme acid
conditions of pH 2.5 or less for a number of hours in vitro (2, 4, 15, 23, 34, 35). This acid resistance is induced in stationary phase or under starvation conditions and is dependent upon the alternate sigma factor, 
s, encoded by rpoS
(7, 31, 34, 35). s regulates a set of genes
which may enable the cell to transport glutamate from the acidified
media to the interior of the cell, where it is converted by glutamate
decarboxylase to 
-amino butyric acid. This basic amine presumably
provides a buffering effect and maintains the internal pH homeostasis
of the cell (35). S. flexneri is commonly
transmitted by person-to-person spread via the fecal-oral route and by
waterborne infections, but it can also be transmitted via a
contaminated food source. Although E. coli O157:H7 is
usually a food-borne pathogen, person-to-person spread can also occur
and is a significant cause of secondary infections (3). The
acid resistance of E. coli O157:H7 is not surprising, since
most E. coli strains, as members of the normal fecal flora,
are very acid resistant (15).
S. typhimurium possess two stationary-phase acid tolerance
response systems, one that is acid induced and s
independent and one that is unresponsive to pH but s
dependent (21). This inducible acid tolerance system is
important to the virulence of the organism (12) but does not
allow Salmonella to survive at the extremes of pH (1.5 to
2.5) that are tolerated by the stationary-phase acid resistance
phenotype of S. flexneri and E. coli
(15). Yersinia enterocolitica can also tolerate a
pH less than 1.5 in vitro. This tolerance is dependent upon a
cytoplasmic urease induced in the stationary phase and during cytoplasmic acidification (10, 36).
Despite the amount of information derived from studies using in vitro
acid resistance assays, the minimum number of ingested Salmonella organisms necessary to produce clinical symptoms
in humans remains a controversial issue. Earlier work on experimental human salmonellosis involving volunteers from a penal institution showed that ingestion of greater than or equal to 105 CFU
of Salmonella meleagridis or Salmonella anatum
was required to produce illness (24). Results with other
Salmonella species found similar requirements for infection
(18, 25, 26). In contrast, the infectivity of
Salmonella in pharmaceutical preparations and foods
including pancreatin, oral vaccine, water, hamburger, milk chocolate,
and cheddar cheese was found to be less than 103 CFU
(5). There have been other reports of ID of less than 100 CFU of Salmonella eastbourne (9) or 50 CFU of
Salmonella napoli (16) in chocolate and of 100 to
500 CFU of Salmonella heidelberg (11) or 1 to 6 CFU of S. typhimurium (8) in cheddar cheese
(8). These results suggest that Salmonella
species can cause infection at a much lower ID if they are ingested
with a food source, which implies that food may provide protection
against the acidity of the stomach for this pathogen.
The acidity of the human stomach is dependent on physiological
variables that include previous food intake. Under fasting conditions,
the median of the luminal pH in healthy volunteers is around 2.0, ranging from 1.5 to 5.5 (33). Ingestion of a meal
characteristic of a Western diet produces an immediate rise in the
median gastric pH to about 6.0 (20, 32). Reduction of
gastric acidity has been associated with an increase in the survival
rates of some food-borne pathogens (28) and with a lowering
of the ID (6, 30).
In this study we have shown that pathogens classified as acid sensitive
in an in vitro acid resistance assay can survive under the same acidic
conditions if inoculated onto certain food sources. To further test
this hypothesis, we used a genetically engineered acid-sensitive
rpoS deletion mutant of Shigella flexneri as an acid-sensitive control.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The strains of
pathogenic enteric bacteria and their isogenic derivatives used in this
study are described in Table 1. The acid-resistant S. flexneri M25-8A and its acid-sensitive
isogenic rpoS mutant W422 were from our laboratory
collection and have been described previously (35). E. coli O157:H7 strain PS2 is a naturally occurring rpoS
mutant which is restored to its acid-resistant state when
complemented by pPS4.4 (containing rpoS) (34).
All strains used in this study were transformed with pACYC184
(Cmr) as a selectable marker unless stated otherwise. This
has no effect on the viability of the organisms or their ability to
resist killing by acid. Campylobacter jejuni 81116 GRK1
contains a kanamycin cartridge inserted in the flaA gene and
was obtained from C. Grant. Strains were grown in 5 ml of Luria-Bertani
broth (LB) with chloramphenicol (30 µg/ml) at 37°C for 24 h
with shaking. C. jejuni was grown for 48 h at 37°C in
5% CO2 on LB plates containing 5% sheep erythrocytes and
kanamycin (30 µg/ml).
Acid resistance assays.
The ability of enteric bacteria to
survive killing by acid when inoculated onto a food source was tested
by using ground beef, boiled egg white, and boiled rice. Ground beef
(9% fat) was purchased at a local supermarket. Samples (0.1 g) of the
ground beef were weighed and inoculated with 10 µl of an overnight
(o/n) culture of the appropriate bacteria diluted 10
1
(approximately 106 CFU) in phosphate-buffered saline (PBS),
and the bacteria were allowed to dry on the surface of the beef for 10 min at room temperature. For inoculations with C. jejuni,
the bacteria were harvested from a blood plate and resuspended in 1 ml
of PBS (pH 7.4) prior to dilution. The inoculated beef was then placed
into 10 ml of LB acidified to pH 2.5 with HCl and incubated at 37°C
with gentle shaking (100 rpm) for 2 h in a 20-ml plastic tube
(Sterilin). Following incubation, the acidified medium was decanted
from the beef fragments and its pH was measured. Surviving bacteria
were recovered by extracting the ground beef with 10 ml of PBS followed by vigorous vortexing. Appropriate aliquots were taken from the resuspended samples, plated onto MacConkey lactose agar containing chloramphenicol (30 µg/ml), and incubated o/n at 37°C unless stated otherwise. Surviving bacteria were enumerated and expressed as a
percentage of the original inoculum exposed to acid challenge. An
uninoculated ground-beef control was placed in acidified LB under
identical conditions and examined for the presence of any contaminating
bacteria. Ground-beef samples inoculated with S. typhimurium
or S. flexneri were placed in 10 ml of PBS under identical conditions to those used for acid challenge as a control to determine the percent cell recovery obtained from untreated ground beef. This
indicates the efficiency of the extraction procedure. Samples taken
from ground beef inoculated with C. jejuni were plated onto LB agar containing 5% sheep erythrocytes and kanamycin and incubated as described above. Samples taken from ground beef inoculated with
V. cholerae were plated onto LB containing chloramphenicol.
For experiments involving the direct acidification of ground beef,
0.1 g of the beef was acidified with HCl to pH 1.0. Ground
beef
was also acidified by being soaked in LB (pH 2.5) for 1 h
or o/n
where stated and then placed in 10 ml of fresh LB (pH 2.5)
for 10 min
before being inoculated with bacteria. The pH at the
surface of the
acidified ground beef was determined after treatment.
Acidified ground
beef was inoculated with bacteria for 2 h at
room temperature, and
the surviving bacteria were recovered by
extraction with 10 ml of PBS
as described above. In some experiments,
0.05 g of boiled rice
grains and 0.1 g of boiled egg white were
used as a food source
instead of ground beef and were inoculated
with bacteria in the same
manner as ground beef. In vitro acid
resistance assays of freely
suspended cells were performed as
described previously (
34)
with LB acidified with HCl to pH 2.5,
4.0, 5.0, and 6.0, and the
results are given as the percentage
of the number of bacteria surviving
compared with the original
inoculum exposed. Results are expressed as
the mean of duplicate
samples from a typical experiment. Larger amounts
of uninoculated
ground beef were used in some experiments under
identical conditions,
and the pH of the acidified media was measured
after incubation,
as described above.
| |
RESULTS |
Survival of enteric pathogens inoculated onto ground beef under
acidic conditions.
Our studies show that all the enteric pathogens
tested were able to survive at pH 2.5 when inoculated onto ground beef
(Table 2) even though many of these
isolates could not survive when assayed in acidified LB at the same pH
(Table 2). Even sensitive rpoS mutants of S. flexneri and E. coli O157:H7, as well as S. typhimurium, S. typhi, V. cholerae, and
C. jejuni, were able to survive when inoculated onto ground
beef (Table 2). Measurement of the pH of the acidified LB used to
challenge each pathogen revealed that the addition of ground beef did
not alter the pH of the medium (Table 2). This is in contrast to
results obtained with larger amounts of ground beef in which the pH of
the medium was raised significantly (see Table 4). A control ground
beef sample which was not inoculated was treated at pH 2.5 under
identical conditions and did not harbor any contaminating
chloramphenicol- or kanamycin-resistant bacteria (Table 2). The percent
survival rates were significant for all the acid-sensitive strains
tested, compared to their ability to survive as freely suspended cells in acidified LB (Table 2). The survival (recovery) rates of control samples where S. typhimurium and S. flexneri were
inoculated onto ground beef but challenged in PBS instead of acidified
LB (Table 2) were measured to gauge the efficiency of the extraction
procedure and were found to be 100.00 and 66.52%, respectively.
Acid-sensitive strains were tested in acidified LB for their ability to
survive at pH 4.0, 5.0, and 6.0. We found that the
acid-sensitive
rpoS mutants of
S. flexneri and
E. coli and all
the
Salmonella species could survive at pH
4.0; however,
C. jejuni survival at this pH 4.0 and 5.0 was
low and
V. cholerae was unable
to survive at either pH 4 or
5 (Table
3). At pH 6.0, both species
showed significant survival rates. These studies suggest that
the pH of
the media to which these enteric pathogens are exposed
is a critical
factor in determining their survival.
Effect on the pH of LB caused by increasing the inoculation size of
ground beef.
To determine if the addition of ground beef was
having a neutralizing effect on the pH of the acidified medium used for
challenge, we examined the pH of the medium after incubation with
different amounts of ground beef under identical conditions of acidity
(Table 4). We also examined the effect of
different amounts of ground beef on the survival of S. typhimurium. We observed that the larger the amount of ground beef
challenged under acidic conditions, the greater the increase in the pH
of the medium. This rise in pH was correlated with an increase in the
survival of S. typhimurium. This demonstrates that ground
beef can raise the pH of acidified media in vitro.
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TABLE 4.
Effect on pH of 10 ml of acidified LB and on S. typhimurium SL1344(pACYC184) survival of the addition of
increasing amounts of ground beef
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|
Survival of S. typhimurium inoculated onto the surface
of preacidified ground beef.
To demonstrate if there was a
protective effect against pH observed at the surface level of the
ground beef, 0.1 g of ground beef was acidified before being
inoculated with S. typhimurium. Ground beef acidified
directly with HCl to a pH of 1.0 was shown to be unable to support the
survival of S. typhimurium (Table 5). In other experiments, ground beef was
also acidified by being soaked in LB (pH 2.5) for either 1 h or
o/n and then placed in fresh LB (pH 2.5) for 10 min. Following this
acidification, the ground beef was removed from the medium, its surface
pH was measured, and was inoculated with S. typhimurium and
incubated at room temperature for 2 h. The ground beef soaked for
1 h had a surface pH of 3.27 and provided an environment that was
demonstrated to support the survival of S. typhimurium
(Table 5). S. typhimurium has been demonstrated previously
to be able to tolerate conditions of this pH (22). In
contrast, the ground beef soaked o/n had a surface pH of 2.61 and did
not support the survival of S. typhimurium. However, the
ground beef had raised the pH of the LB to 2.66 as a result of the o/n
soaking. These results demonstrate that even 0.1 g of ground beef
has an alkalinating effect on the acidified LB over time and that if
the surface pH of the ground beef is below the threshold for S. typhimurium survival, it cannot support this acid-sensitive
pathogen.
Survival of serial dilutions of S. typhimurium
inoculated onto ground beef under acidic conditions.
To determine
if the inoculum size had any effect on the bacterial survival rate,
serial dilutions of S. typhimurium were inoculated onto
ground beef and challenged as described above. The survival rates
obtained for all inoculations ranging from 102 to
105 CFU were similar (Table
6) despite variations in the inoculum size.
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TABLE 6.
Survival of S. typhimurium SL1344(pACYC184)
inoculated onto ground beef in various inocula and challenged in
acidified LBa
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Survival of S. typhimurium inoculated onto boiled rice
and boiled egg white under acidic conditions.
S. typhimurium
was inoculated onto boiled rice under the same conditions as those
described for the ground-beef challenge to examine whether a food
source high in carbohydrate would have a protective effect against an
acidic environment. S. typhimurium inoculated onto boiled
rice was not protected against low-pH challenge, in contrast to the
protective effect observed with ground beef (Table
7). Rice inoculated with S. typhimurium and incubated under identical conditions in PBS (pH
7.4) was used as a control to determine the percent recovery.
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TABLE 7.
Survival of S. typhimurium SL1344(pACYC184)
inoculated onto boiled rice or boiled egg white and challenged in
acidified LBa
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S. typhimurium was also inoculated onto boiled egg white to
determine whether a food source high in protein and low in fat
could
offer protection against extreme acid conditions.
S. typhimurium was protected from killing by low pH at a significant
level when
inoculated on egg white (Table
7), suggesting that this
survival
was not due to a protective effect of fat. The percent
recovery
of
S. typhimurium inoculated on egg white was
determined by using
a similar PBS control.
| |
DISCUSSION |
The studies presented in this paper were undertaken to examine
what effect food might have on the survival of acid-sensitive pathogens
challenged under acidic conditions and perhaps to shed light on the
contradictory evidence concerning the ID of Salmonella species. In clinical trials where defined inocula were fed to human
volunteers, the ID was demonstrated to be at least 105
bacteria (5). However, careful analysis of ID in outbreak studies has shown that individuals ingesting as few as 50 to 100 of
these organisms have become ill (5, 8). Since we had previously shown that S. typhimurium cannot survive under
extreme acidic conditions (below pH 3.0) (15), we examined
whether food could provide a protective effect to acid-sensitive
bacteria by facilitating their survival under extreme acidic
conditions.
In vitro acid resistance assays have been used by a number of
investigators to study the ability of pathogens to resist acidic conditions (10, 15, 22). Likewise, the ability of pathogens to survive in acidic food sources such as yogurt, fermented sausage, and apple cider has received similar attention (1, 14, 27). In the present study, we have brought the two fields together to
determine if a solid-food source can contribute to the survival of a
pathogen under extreme acidic conditions. Information gleaned from
these studies may help explain why pathogens which have a high ID when
studied in clinical trials may require a much lower dose to cause
natural infections.
We have shown previously that the stationary-phase acid resistance
phenotype of S. flexneri and E. coli O157:H7 is
dependent upon the expression of rpoS. rpoS mutants of these
species are completely sensitive to killing under extreme acidic
conditions of pH 2.5 (34, 35). Other pathogens, such as
Salmonella, behave similarly to rpoS mutants of
Shigella in this in vitro assay. In this study, we have
shown that acid-sensitive rpoS mutants of
Shigella and E. coli, as well as naturally
acid-sensitive pathogens such as Salmonella, survive under
these extreme low-pH conditions in vitro when inoculated onto the
surface of certain food sources. This suggests that in some cases the
solid food source provides a protection against low pH.
It has been speculated by D'Aoust (8) that
Salmonella outbreaks with a low ID are often associated with
a food source with a high fat content such as chocolate and cheese.
This led to the hypothesis that the fat content of contaminated foods
may play a significant role in human salmonellosis. The rationale
behind this hypothesis is that organisms trapped in hydrophobic lipid moieties may readily survive the acidic conditions of the stomach and
pass into the intestinal tract. The precise role played by fat in
protecting bacteria from killing by low pH has yet to be determined and
may be important in the protection offered by other food sources not
examined here. If organisms are trapped in hydrophilic lipid moieties,
it would be expected that the protective effect afforded by ground beef
would have been equal for all the pathogens that were tested. This was
not the case, since C. jejuni and V. cholerae
were not as well protected as the other pathogens. We observed,
however, that if S. typhimurium was inoculated onto boiled
egg white, which is low in fat, it was protected from killing by acid.
We have demonstrated that increasing the amount of ground beef
challenged in the assay raised the pH of the medium and increased the
survival rate of S. typhimurium. By raising the pH of the medium in vitro, we confirmed that the pH of the acidified medium is a
critical factor in determining the survival of each enteric pathogen.
The importance of pH was also confirmed by the ability of
Salmonella to survive when inoculated onto the surface of
preacidified ground beef if the surface pH was high enough to be
tolerated by this pathogen. However, if the pH of the ground beef was
acidified so that its pH at the surface remained lethal to S. typhimurium, no survival was observed. These results imply that
the survival of acid-sensitive bacteria on the surface of ground beef
is most probably the result of the ability of ground beef to raise the pH of the acidified medium at the microenvironment occupied by the
bacteria.
The number of bacteria consumed in contaminated food can vary
considerably, and so we looked at the effect of inoculum size on the
survival of S. typhimurium in our ground-beef assay. We found that the inoculum size had no significant effect on the survival
rates of S. typhimurium in ground beef. This experiment also
demonstrated that a very low inoculum of S. typhimurium can survive extreme acidity when present on the surface of certain solid
food sources and may explain the low ID observed in some food-borne
outbreaks involving Salmonella species.
At present it is not clear exactly how food protects bacteria from
acidic conditions. The fat content of food may still provide a
significant barrier against acidic conditions, but other food sources
with lower fat content can still offer significant protection. This
could be tested in the future by examining the protective effect of a
solid food which has an extremely high fat content and is low in
protein. It should be noted, however, that foods high in fat may not
maintain their consistency under these conditions of temperature and pH
(indeed, fatty oils were observed at the surface of the acidified media
following the ground-beef challenge). Carbohydrate does not seem to
offer protection, since Salmonella did not survive when
inoculated onto the surface of rice. It has been demonstrated in vitro
that the acid resistance phenotype of Shigella flexneri and
E. coli is dependent upon the presence of amino acids in the
acidified media (22, 23, 35). Acid resistance is expressed
under these conditions by supplementing the acidified media with
extremely small quantities of amino acids. It is possible that the
protein content of some solid foods may be the essential component
responsible for the protective effect observed in the experiments
described here.
In this study, we have demonstrated that bacteria can survive acidic
conditions in vitro when inoculated onto the surface of certain solid
food sources whereas the same level of acidity is lethal to the
inoculum in an acidified broth environment. The protective effect of
some solid foods may be the result of at least partially raising the pH
of the acidified medium at the microenvironment occupied by the
bacteria on the surface of the food source. This is consistent with
evidence that the ID of some food-borne pathogens is lowered when
gastric acidity is reduced. Studies with the acid-sensitive food-borne
pathogen Listeria monocytogenes have shown that the ID of
this organism in the Sprague-Dawley rat model can be lowered by raising
the pH of the stomach to 5.5 to 6.0 with cimetidine (30).
Rats that were inoculated with a lower ID exhibited the same degree of
severity of bacterial colonization in specific organs and tissue as did
rats that received a higher ID, suggesting that once the pH barrier of
the stomach has been breached, the number of surviving bacteria
reaching the intestines does not affect the severity of the disease.
This implies that the ability to survive the acidic conditions of the
stomach is the principal factor determining the ID of a specific
enteric pathogen.
In summary, these studies may help resolve the controversy surrounding
the ID of pathogenic Salmonella species. In volunteer studies, the inoculum was administered in a liquid form, which may
offer little protection against stomach acidity. Food-borne outbreaks
characterized by a low ID are often associated with consumption of
bacteria on solid food. This environment may protect bacteria from the
lethal effects of stomach acidity.
| |
ACKNOWLEDGMENTS |
We thank Chris Grant for providing us with C. jejuni
81116 GRK1. We also thank Lucia Barker, Katie George, Joe Hinnebusch, and Lisa Pascopella for kindly reviewing the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Rocky Mountain
Laboratories, Microscopy Branch, 903 South 4th St., Hamilton, MT 59840. Phone: (406) 363-9280. Fax: (406) 363-9371. E-mail:
pam_small{at}nih.gov.
| |
REFERENCES |
| 1.
|
Abdul-Raouf, U. M.,
L. R. Beuchat, and M. S. Ammar.
1993.
Survival and growth of Escherichia coli O157:H7 in ground, roasted beef as affected by pH, acidulants, and temperature.
Appl. Environ. Microbiol.
59:2364-2368[Abstract/Free Full Text].
|
| 2.
|
Arnold, K. W., and C. W. Kaspar.
1995.
Starvation-and stationary-phase induced acid tolerance in Escherichia coli O157:H7.
Appl. Environ. Microbiol.
61:2037-2039[Abstract].
|
| 3.
|
Bell, B. P.,
M. Goldoft,
P. M. Griffin,
M. A. Davis,
D. C. Gordon,
P. I. Tarr,
C. A. Bartleson,
J. H. Lewis,
T. J. Barrett,
J. G. Wells,
R. Baron, and J. Kobayashi.
1994.
A multistate outbreak of Escherichia coli O157:H7-associated blood diarrhea and hemolytic uremic syndrome from hamburgers.
JAMA
272:1349-1353[Abstract/Free Full Text].
|
| 4.
|
Benjamin, M. M., and A. R. Datta.
1995.
Acid tolerance of enterohemorrhagic Escherichia coli.
Appl. Environ. Microbiol.
61:1669-1672[Abstract].
|
| 5.
|
Blaser, M. J., and L. S. Newman.
1982.
A review of human Salmonellosis. I. Infective dose.
Rev. Infect. Dis.
4:1096-1106[Medline].
|
| 6.
|
Cash, R. A.,
S. I. Music,
J. P. Libonati,
M. J. Snyder,
R. P. Wenzel, and R. B. Hornick.
1974.
Response of man to infection with Vibrio cholerae. I. Clinical, serologic, and bacteriologic responses to a known inoculum.
J. Infect. Dis.
129:45-52[Medline].
|
| 7.
|
Cheville, A. M.,
K. W. Arnold,
C. Buchrieser,
C.-M. Cheng, and C. W. Kaspar.
1996.
rpoS regulation of acid, heat, and salt tolerance in Escherichia coli O157:H7.
Appl. Environ. Microbiol.
62:1822-1824[Abstract].
|
| 8.
|
D'Aoust, J. Y.
1985.
Infective dose of Salmonella typhimurium in cheddar cheese.
Am. J. Epidemiol.
122:717-719[Free Full Text].
|
| 9.
|
D'Aoust, J. Y.,
B. J. Aris,
P. Thisdele,
A. Durante,
N. Brisson,
D. Dragon,
G. Lachapelle,
M. Johnston, and R. Laidley.
1975.
Salmonella eastbourne outbreak associated with chocolate.
Can. Inst. Food Sci. Technol. J.
8:181-184.
|
| 10.
|
de Koning-Ward, T. E., and R. M. Robins-Browne.
1995.
Contribution of urease to acid tolerance in Yersinia enterocolitica.
Infect. Immun.
63:3790-3795[Abstract].
|
| 11.
|
Fontaine, R. E.,
M. L. Cohen,
W. T. Martin, and T. M. Vernon.
1980.
Epidemic salmonellosis from cheddar cheese: surveillance and prevention.
Am. J. Epidemiol.
111:247-253[Abstract/Free Full Text].
|
| 12.
|
Gardia-del Portillo, F.,
J. W. Foster, and B. B. Finlay.
1993.
Role of acid tolerance response genes in Salmonella typhimurium virulence.
Infect. Immun.
61:4489-4492[Abstract/Free Full Text].
|
| 13.
|
Giannella, R. A.,
S. A. Broitman, and N. Zamcheck.
1972.
Gastric acid barrier to ingested microorganisms in man: studies in vivo and in vitro.
Gut
13:251-256[Abstract/Free Full Text].
|
| 14.
|
Glass, K. A.,
J. M. Loeffelholz,
J. P. Ford, and M. P. Doyle.
1992.
Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented dry sausage.
Appl. Environ. Microbiol.
58:2513-2516[Abstract/Free Full Text].
|
| 15.
|
Gordon, J., and P. L. C. Small.
1993.
Acid resistance in enteric bacteria.
Infect. Immun.
61:364-367.
|
| 16.
|
Greenwood, M. H., and W. L. Hooper.
1983.
Chocolate bars contaminated with Salmonella napoli: an infectivity study.
Br. Med. J.
286:1394.
|
| 17.
|
Hoiseth, S. K., and B. A. Stocker.
1981.
Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines.
Nature (London)
291:238-239[Medline].
|
| 18.
|
Hornick, R. B.,
S. E. Greisman,
T. E. Woodward,
H. L. DuPont,
A. T. Dawkins, and M. J. Snyder.
1970.
Typhoid fever: pathogenesis and immunological control.
N. Engl. J. Med.
283:739-746.
|
| 19.
|
Hone, D. M.,
A. M. Harris,
S. Chatfield,
G. Dougan, and M. M. Levine.
1991.
Construction of genetically defined double aro mutants of Salmonella typhi.
Vaccine
9:810-816[Medline].
|
| 20.
|
Konturek, J. W.,
P. Thor,
M. Maczka,
R. Stoll,
W. Domschke, and S. J. Konturek.
1994.
Role of cholecystokinin in the control of gastric emptying and secretory response to a fatty meal in normal subjects and duodenal ulcer patients.
Scand. J. Gastroenterol.
29:583-590[Medline].
|
| 21.
|
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 sustained acid tolerance response in virulent Salmonella typhimurium.
Mol. Microbiol.
17:155-167[Medline].
|
| 22.
|
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].
|
| 23.
|
Lin, J.,
M. P. Smith,
K. C. Chapin,
H. S. Baik,
G. N. Bennett, and J. W. Foster.
1996.
Mechanisms of acid resistance in enterohemorrhagic Escherichia coli.
Appl. Environ. Microbiol.
62:3094-3100[Abstract].
|
| 24.
|
McCullough, N. B., and C. W. Eisele.
1951.
Experimental human salmonellosis. I. Pathogenicity of strains of Salmonella meleagridis and Salmonella anatum obtained from spray-dried whole egg.
J. Infect. Dis.
88:278-289.
|
| 25.
|
McCullough, N. B., and C. W. Eisele.
1951.
Experimental human salmonellosis. III. Pathogenicity of strains of Salmonella newport, Salmonella derby and Salmonella bareilly obtained from spray-dried whole egg.
J. Infect. Dis.
89:209-213.
|
| 26.
|
McCullough, N. B., and C. W. Eisele.
1951.
Experimental human salmonellosis. IV. Pathogenicity of strains of Salmonella pullorum obtained from spray-dried whole egg.
J. Infect. Dis.
89:259-265.
|
| 27.
|
Miller, L. G., and C. W. Kaspar.
1994.
Escherichia coli O157:H7 acid tolerance and survival in apple cider.
J. Food Prot.
57:460-464.
|
| 28.
|
Peterson, W. L.,
P. A. Mackowiak,
C. C. Barnett,
M. Marling-Cason, and M. L. Haley.
1989.
The human gastric bactericidal barrier: mechanisms of action, relative antibacterial activity, and dietary influences.
J. Infect. Dis.
159:979-983[Medline].
|
| 29.
|
Sansonetti, P. J.,
D. J. Kopecko, and S. B. Formal.
1982.
Involvement of a plasmid in the invasive ability of Shigella flexneri.
Infect. Immun.
35:852-860[Abstract/Free Full Text].
|
| 30.
|
Schlech, W. F.,
D. P. Chase, and A. Badley.
1993.
A model of food-borne Listeria monocytogenes infection in the Sprague-Dawley rat using gastric inoculation: development and effect of gastric acidity on infective dose.
Int. J. Food Microbiol.
18:15-24[Medline].
|
| 31.
|
Small, P.,
D. Blankenhorn,
D. Welty,
E. Zinser, and J. L. Slonczewski.
1994.
Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH.
J. Bacteriol.
176:1729-1737[Abstract/Free Full Text].
|
| 32.
|
Snepar, R.,
G. A. Poporad,
J. M. Romano,
W. B. Kobasa, and D. Kay.
1982.
Effect of cimetidine and antacid on gastric microbial flora.
Infect. Immun.
36:518-524[Abstract/Free Full Text].
|
| 33.
|
Verdu, E.,
F. Viani,
D. Armstrong,
R. Fraser,
H. H. Siegrist,
B. Pignatelli,
J.-P. Idstrom,
C. Lederberg,
A. L. Blum, and M. Fried.
1994.
Effect of omeprazole on intragastric bacterial counts, nitrates, nitrites, and N-nitroso compounds.
Gut
35:455-460[Abstract/Free Full Text].
|
| 34.
|
Waterman, S. R., and P. L. C. Small.
1996.
Characterization of the acid resistance phenotype and rpoS alleles of Shiga-like toxin-producing Escherichia coli.
Infect. Immun.
64:2808-2811[Abstract].
|
| 35.
|
Waterman, S. R., and P. L. C. Small.
1996.
Identification of s-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri.
Mol. Microbiol.
21:925-940[Medline].
|
| 36.
|
Young, G. M.,
D. Amid, and V. L. Miller.
1996.
A bifunctional urease enhances survival of pathogenic Yersinia enterocolitica and Morganella morganii at low pH.
J. Bacteriol.
178:6487-6495[Abstract/Free Full Text].
|
Applied and Environmental Microbiology, October 1998, p. 3882-3886, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
