Acid-Sensitive Enteric Pathogens Are Protected from Killing under ...

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bers of the normal fecal ffora, are very acid resistant (15). S. typhimurium .... pathogens tested were able to survive at pH 2.5 when inocu- lated onto ground beef ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1998, p. 3882–3886 0099-2240/98/$04.0010 Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Vol. 64, No. 10

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

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. 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 ss independent and one that is unresponsive to pH but ss 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

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, ss, encoded by rpoS (7, 31, 34, 35). ss 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 g-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, * 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@nih .gov. 3882

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SURVIVAL OF ACID-SENSITIVE ENTERIC PATHOGENS ON FOOD

TABLE 1. Bacterial strains used in this study Strain

Relevant genotype

S. flexneri M25-8A S. flexneri W422 S. typhimurium SL1344 S. typhi ISP 1820 E. coli O157:H7 PS2 (rpoS) E. coli O157:H7 PS2 (rpoS1)

C. jejuni 81116 GRK1 V. cholerae 14035 a

Cured of large plasmid rpoS deletion mutant of M25-8A Wild type Wild type Natural isolate with rpoS mutation rpoS1, contains pPS4.4 (pACYC184 with 4.4-kb ClaI fragment with rpoS, Cmr) flaA::Kmr Wild type

Source or reference

29 35 17 19 34 34

Chris Grant ATCCa

ATCC, American Type Culture Collection.

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 mg/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 mg/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 ml of an overnight (o/n) culture of the appropriate bacteria diluted 1021 (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 mg/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

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TABLE 2. Survival of pathogens inoculated on ground beef and challenged in acidified LBa % Acid % Survivalb resistancec

Pathogen

S. flexneri M25-8A(pACYC184)d S. flexneri W422(pACYC184) S. typhimurium SL1344(pACYC184) S. typhi ISP 1820(pACYC184) E. coli O157:H7 PS2 (rpoS)(pACYC184) E. coli O157:H7 PS2 (rpoS1)(pPS4.4)e C. jejuni 81116 GRK1 (flaA::Kmr) V. cholerae 14035(pACYC184)

pH of LB after treatment

95.25 41.40 62.86 10.63 43.55

28.03 ,0.001 ,0.001 ,0.001 ,0.009

2.50 2.50 2.50 2.50 2.50

79.06 1.99 0.20

103.70 ,0.001 ,0.001

2.50 2.50 2.50

a Experiments were performed in duplicate, and numbers are means. The inoculum for each experiment was 9 3 105 to 2 3 106 CFU. LB was originally pH 2.5. Ground beef was examined for the presence of contaminating antibioticresistant bacteria, and no Cmr or Kmr bacteria were found. b Percent survival is expressed as 100 3 CFU of bacteria recovered from ground beef after incubation in LB (pH 2.5) for 2 h/CFU of inoculum. c Percent acid resistance is expressed as 100 3 CFU of bacteria recovered from acidified LB (pH 2.5) at 2 h/CFU of inoculum. d pACYC184, Cmr. e pPS4.4 harbors the rpoS gene and Cmr.

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-

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APPL. ENVIRON. MICROBIOL. TABLE 5. Survival of S. typhimurium SL1344(pACYC184) inoculated onto the surface of preacidified ground beef

TABLE 3. Survival of acid-sensitive pathogens at pH 4.0, 5.0, and 6.0 in acidified LB % Acid resistancea at: Pathogen

S. typhimurium SL1344 S. flexneri W422 DrpoS S. typhi ISP 1820 E. coli O157:H7 PS2 C. jejuni 81116 GRK1 V. cholerae 14305

pH 4.0

pH 5.0

91.52 38.06 59.60 25.76 0.12 ,0.002

b

ND ND ND ND 0.19 ,0.002

pH 6.0

ND ND ND ND 52.76 25.67

a Percent acid resistance is expressed as 100 3 CFU recovered after a 2-h exposure to acidified LB/CFU of inoculum. b ND, not done.

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. 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 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 Amt (g) of ground beef

pH of LB after treatmenta

% Survivalb

0.1 0.5 1.0 2.0

2.50 2.70 2.83 2.98

32.07 83.47 86.37 82.93

a

The pH of the medium was examined after a 2-h incubation at 37°C with increasing amounts of ground beef incubated in 10 ml of LB (original pH 2.5). b Ground beef was inoculated with approximately 106 CFU of S. typhimurium, and the number of survivors was determined as a percentage of the original inoculum exposed.

Ground beef acidificationa

pH on surface of beefb

% Survivalc

HCl LB pH 2.5 (1 h) LB pH 2.5 (16 h)

1.0 3.27 2.61

,0.01 103.7 ,0.01

a Ground beef (0.1 g) was acidified by the direct addition of HCl or by being soaked in 10 ml of LB (pH 2.5) for the time indicated; it was then placed in 10 ml of fresh LB (pH 2.5) for 10 min. b The pH on the surface of the ground beef was determined immediately after the acidification treatment and before bacterial inoculation. c Acidified ground beef was inoculated with approximately 106 CFU of S. typhimurium and incubated for 2 h at room temperature. Surviving bacteria were enumerated by vigorous vortexing in 10 ml of PBS and are expressed as a percentage of the original inoculum exposed.

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. 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 ef-

TABLE 6. Survival of S. typhimurium SL1344(pACYC184) inoculated onto ground beef in various inocula and challenged in acidified LBa Inoculum

No. of surviving bacteria (CFU)

% Survival

1.77 3 105 1.77 3 104 1.77 3 103 1.77 3 102

5.00 3 104 3.70 3 103 3.67 3 102 3.33 3 101

28.18 20.86 20.67 18.77

a

Experiments were performed in duplicate, and the results are means.

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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 Inoculum (CFU)

No. of surviving bacteria (CFU)

% Survival

pH of LB after treatment

LB (pH 2.5) Rice Egg white

2.97 3 106 9.40 3 105

0 3.07 3 105

,0.01b 32.66b

2.50 2.50

PBS (pH 7.4)d Rice Egg white

1.72 3 106 9.40 3 105

9.40 3 105 1.72 3 106

54.57c 100.00c

Challenge substance and food source

a

Experiments were performed in duplicate, and numbers are means. Percent survival of S. typhimurium inoculated onto the surface of 0.1 g of boiled rice on egg white and challenged with LB (pH 2.5). c Percent survival of S. typhimurium inoculated onto the surface of 0.1 g of boiled rice or egg white and challenged with PBS (pH 7.4). d PBS was used as a control. b

fect 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. 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 acidsensitive 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

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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 acidsensitive 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 inocu-

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lated 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. Foodborne 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. 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. 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. 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. 4. Benjamin, M. M., and A. R. Datta. 1995. Acid tolerance of enterohemorrhagic Escherichia coli. Appl. Environ. Microbiol. 61:1669–1672. 5. Blaser, M. J., and L. S. Newman. 1982. A review of human Salmonellosis. I. Infective dose. Rev. Infect. Dis. 4:1096–1106. 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. 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. 8. D’Aoust, J. Y. 1985. Infective dose of Salmonella typhimurium in cheddar cheese. Am. J. Epidemiol. 122:717–719. 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

APPL. ENVIRON. MICROBIOL.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21.

22. 23. 24. 25.

26. 27. 28.

29. 30.

31. 32. 33.

34. 35. 36.

urease to acid tolerance in Yersinia enterocolitica. Infect. Immun. 63:3790– 3795. 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. 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. 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. 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. Gordon, J., and P. L. C. Small. 1993. Acid resistance in enteric bacteria. Infect. Immun. 61:364–367. Greenwood, M. H., and W. L. Hooper. 1983. Chocolate bars contaminated with Salmonella napoli: an infectivity study. Br. Med. J. 286:1394. 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. 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. 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. 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. Lee, I. S., J. Lin, H. K. Hall, B. Bearson, and J. W. Foster. 1995. The stationary-phase sigma factor ss (RpoS) is required for sustained acid tolerance response in virulent Salmonella typhimurium. Mol. Microbiol. 17:155– 167. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Waterman, S. R., and P. L. C. Small. 1996. Identification of ss-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri. Mol. Microbiol. 21:925–940. 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.