Departamento de Investigación y Posgrado en Alimentos, Facultad de Química, Universidad Autónoma de Querétaro, Querétaro, Querétaro, México

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1 2047 Journal of Food Protection, Vol. 68, No. 10, 2005, Pages Copyright, International Association for Food Protection Effect of Acid Shock with Hydrochloric,, and Acids on the Survival and Growth of Salmonella Typhi and Salmonella Typhimurium in Acidified Media SOFÍA M. ARVIZU-MEDRANO AND EDUARDO F. ESCARTÍN* Departamento de Investigación y Posgrado en Alimentos, Facultad de Química, Universidad Autónoma de Querétaro, Querétaro, Querétaro, México MS : Received 26 August 2004/Accepted 14 March 2005 ABSTRACT The effect of acid shock with hydrochloric, citric, or lactic acid on the survival and growth of Salmonella Typhi and Salmonella Typhimurium in acidified broth was evaluated. Salmonella serovars were acid shocked (1 h at 35 C) in Trypticase soy broth acidified with hydrochloric, citric, or lactic acid at ph 5.5. cells were exposed to the same media that had been neutralized before use to ph 7.0. and unshocked cells were inoculated into broth acidified with hydrochloric acid (ph 3.0), citric acid (ph 3.0), or lactic acid (ph 3.8), and growth and survival ability were evaluated. The acid shock conferred protection to Salmonella against the lethal effects of low ph and organic acids. The adaptive response was not specific to the anion used for adaptation. The biggest difference in reduction of survival between shocked and unshocked strains ( 2 log CFU/ml) was observed when the microorganisms were shocked with lactic acid and then challenged with citric acid. Salmonella Typhi was more tolerant of citric acid than was Salmonella Typhimurium, but Salmonella Typhimurium had higher acid tolerance in response to acid shock than did Salmonella Typhi. The acid shock decreased the extension of the lag phase and enhanced the physiological state values of Salmonella Typhi and Salmonella Typhimurium when the ph of growth was 4.5. This increased ability to tolerate acidity may have an important impact on food safety, especially in the case of Salmonella Typhi, given the very low infectious dose of this pathogen. Highly acid foods have long been considered generally safe for human consumption. Outbreaks related to these foods have rarely been recorded. However, some acidic foods such as mayonnaise (37), yogurt (30), apple cider (8, 29), apple juice (14, 16, 28), and orange juice (11, 12, 15, 36) have been associated with foodborne outbreaks. Salmonella and Escherichia coli O157:H7 were the etiologic agents in 80% of outbreaks associated with acidic foods. These bacteria are notoriously tolerant of acidic conditions (7, 20, 33). Bacterial survival in acidified environments can be enhanced by previous exposure to moderately acidic conditions. This phenomenon, the acid tolerance response, has been found in several microorganisms during the exponential and stationary phases (6, 17, 21, 24, 28) and has been demonstrated in real foods (38). Acid adaptation prolonged the survival of Salmonella in acidic foods (27, 42). Acid adaptation in Salmonella Typhimurium induces cross-protection against heat, high osmolarity, and the lactoperoxidase system (26). Wilmes-Riesenberg et al. (44) observed a close relationship between virulence and the ability to mount an acid tolerance response in some strains of Salmonella Typhimurium. Studies with other pathogenic bacteria such as E. coli O157:H7 (34) and Shigella flexneri (39) have revealed that the acid tolerance response can be influenced by the type of acid used during the adaptation and challenge phases. The ability of bacteria to survive in acid foods is es- * Author for correspondence. Tel: , Ext 5581; Fax: ; efescart@sunserver.uaq.mx. pecially relevant for pathogens with low infective doses, such as Salmonella Typhi. Although Salmonella Typhi causes a severe life-threatening illness, there is not much information regarding its behavior in foods. Typhoid fever is not a major health problem in industrialized countries (13). By contrast, in developing countries 33 million cases per year and 600,000 deaths per year from this disease have been estimated (31, 40). In Mexico, typhoid fever remains an endemic disease. In the last 5 years, an average of 8,200 cases per year were reported (35); however, these figures probably underestimate the actual incidence. Several characteristics of Salmonella Typhi differentiate it from the rest of the Salmonella serovars. These differences include the ability to infect only humans and to colonize the gallbladder. Its infective dose is very low, it is highly invasive, and some biochemical reactions are unique among other Salmonella serovars (e.g., no gas from glucose and ornithine decarboxylase negative) (19). Thus, there is a pending request to the Judicial Commission of the International Committee of Systematic Bacteriology to reclassify Salmonella Typhi as a species (9). Information on acid tolerance response is limited for Salmonella Typhi. Tiwari et al. (41) found that Salmonella Typhi in exponential phase is able to adapt and increase its acid tolerance in culture medium. In the present work, we studied the behavior of Salmonella Typhi in stationary phase, because the response of bacteria to stressing agents such as acidity may be different in this phase. This difference could be important because Salmonella in contaminated foods may be in stationary phase when acid is added.

2 2048 ARVIZU-MEDRANO AND ESCARTÍN J. Food Prot., Vol. 68, No. 10 The objectives of this work were to (i) investigate the capacity of Salmonella Typhi in stationary growth phase to increase its acid tolerance by an acid shock and (ii) compare the effect of acid shock with hydrochloric, citric, or lactic acid on the survival and growth of Salmonella Typhi and Salmonella Typhimurium in broth acidified with each acid. MATERIALS AND METHODS Bacterial strains and preparation of inocula. Four strains of Salmonella were included. Salmonella Typhimurium strain 1 was obtained from the culture collection at the Center of Food Safety and Quality Enhancement (University of Georgia, Griffin), and Salmonella Typhimurium strain 2 was ATCC Salmonella Typhi strain 1 was obtained from the National Institute of Neurology (México City), and Salmonella Typhi strain 2 was from the National Public Health Laboratory (México City). Stock cultures of each strain grown on Trypticase soy agar (TSA; Bioxon, Becton Dickinson México, México City) were stored at 4 C. Each strain was activated by culturing in Trypticase soy broth (TSB; Bioxon) for 24 h at 35 C and then overnight for 18hat35 C before use. Acid shock treatment. Activated Salmonella Typhi and Salmonella Typhimurium cultures were centrifuged and resuspended in TSB previously acidified to ph 5.5 with 10% solutions of hydrochloric, citric, or lactic acid (J. T. Baker, México City). These cell suspensions were incubated for 1 h at 35 C (shocked cells). Controls (unshocked cells) consisted of Salmonella Typhi and Salmonella Typhimurium cells suspended in media that had been acidified with hydrochloric, citric, or lactic acid but then neutralized (ph 7.0) with NaOH (2.5 M; J. T. Baker) before addition of the cells. Controls were also incubated for 1 h at 35 C. and unshocked bacterial suspensions were used to assess the survival of bacteria cells and their ability to grow in acidified media. Survival of Salmonella in acidified media. In this experiment only strain 1 of each serovar was used. Each of the acidshocked or control suspensions was inoculated ( 8 log CFU/ml) into TSB acidified with 10% solutions of hydrochloric acid (ph 3.0), citric acid (ph 3.0), or lactic acid (ph 3.8) and incubated for 1hat35 C (acid challenge). Ten-fold dilutions of the treated samples were prepared in phosphate-buffered saline (0.25 M, ph 7.4). Cell survival was determined by pour plate on TSA and Salmonella-Shigella agar (SSA; Bioxon); plates were incubated for24hat35 C. The differences between TSA and SSA counts were considered an estimate of the number of injured cells: % injured cells (TSA count SSA count) 100/TSA count. Growth of Salmonella in acidified media. The Bioscreen C automated turbidimetric analyzer (Labsystems, Helsinki, Finland) was employed to measure Salmonella growth. Two hundred microliters of TSB acidified with hydrochloric, citric, or lactic acid (J. T. Baker) at ph 4.5, 5.0, 5.5, and 7.0 was dispensed into 100- well honeycomb plates. Twenty microliters ( 5 log CFU) of the shocked or unshocked suspensions of Salmonella Typhi and Salmonella Typhimurium was inoculated into each cuvette of the plates containing acidified broth. These plates, with all combinations, were incubated at 35 C for 24 h. At 15-min intervals, the optical density (OD) was automatically measured at 600 nm; before measurements, the plates were shaken at medium intensity for 5 s. Five treatments were randomly selected and performed in quadruplicate. Detection times and maximum OD were obtained directly from the Bioscreen-Link software program (Labsystems). The relationship between OD and cell concentration was experimentally determined using viable counts on TSA. OD data for all combinations of the studied variables were obtained using Bioscreen-Link software and were transformed to cell concentration (growth kinetics data). Curve fitting and statistical analysis. For evaluation of survival of shocked and unshocked microorganisms in acidified media, two trials with three replicates each were conducted for each treatment. A split plot experimental design was used, with challenge acid as the plot. The combinations of the type of acid used for the acidic shock and the microorganism used were randomized on the plot. Reduction data were subjected to analysis of variance and t test using the R software (22). The Gompertz equation (10) was fitted to the growth kinetics data for each of the variable combinations for shocked and unshocked microorganisms using R software: L(t) A C e e[ B(t M)] where L(t) is the log count of the number of bacteria at time t (in hours), A is the asymptotic log count as t decreases indefinitely, C is the asymptotic amount of growth (log number) that occurs as t increases indefinitely, M is the time (in hours) at which the absolute growth rate is maximum, and B is the relative growth rate at M. The Gompertz parameters (A, C, M, and B) were used to calculate traditional growth parameters (10): exponential growth rate BC/e; lag phase duration M (1/B); generation time log 2e/BC; and maximum population density A C. A physiological state parameter was also calculated from detection times (4): e T(X 0/X det) where is the physiological state, is the exponential growth rate, T is the detection time, X 0 is the bacterial concentration at time 0 (initial count), and X det is the minimal bacterial concentration detected by Bioscreen (detection level). The effects of acid shock, ph, type of acid used for the acid shock, type of acid used for challenge, microorganism, and strain on all parameters were evaluated by analysis of variance and t test using the R software. RESULTS AND DISCUSSION The organic acids used in this study are found as major components of orange juice and fermented milk products. Both acidic food types have been reported as vehicles of foodborne outbreaks (11, 12, 15, 30, 36). Effect of acid shock on the survival of Salmonella Typhi and Salmonella Typhimurium in acidified medium. The acid shock with the three acids conferred protection to Salmonella against the lethal effect of acidified media (P 0.05; Fig. 1). The increase in acid tolerance found in this study was similar to that reported by Tetteh and Beuchat (39) for S. flexneri shocked with lactic acid and then challenged with lactic and acetic acids. Tiwari et al. (41) observed a 1.48-log CFU reduction when Salmonella Typhi hydrochloric acid adapted cells in the exponential phase were challenged at ph 3.3, whereas for unadapted cells the reduction was 4.61 log CFU. This increment in acid tolerance was larger than the one observed in our study, where shocked and unshocked Salmonella Typhi decreased 2.5 and 3.9 log CFU, respectively. This finding is congruent with the generally accepted hypothesis that cells

3 J. Food Prot., Vol. 68, No. 10 BEHAVIOR OF ACID-SHOCKED SALMONELLA TYPHI AND SALMONELLA TYPHIMURIUM 2049 FIGURE 1. Reduction of Salmonella Typhi and Salmonella Typhimurium populations that have not been shocked (open bars) or have been shocked (solid bars) in hydrochloric (A and D), citric (B and E), or lactic (C and F) acids and then challenged with each acid. Plotted values are the means of six replicates. Error bars are standard deviations. in stationary phase are more resistant to adverse environmental conditions. In general, Salmonella Typhimurium had a greater capacity for adaptation than did Salmonella Typhi. The average difference in reduction between shocked and unshocked cells in all treatments was 0.84 log CFU for Salmonella Typhi and 1.59 log CFU for Salmonella Typhimurium. Significant differences between the susceptibility of the two serovars to the challenge acids were observed. Salmonella Typhimurium was more tolerant to lactic acid than was Salmonella Typhi (Fig. 1). Salmonella Typhi cells were more tolerant to citric acid than were counterpart Salmonella Typhimurium cells. However, shocked microorganisms were similarly tolerant of citric acid (Fig. 1A, 1B, 1D, and 1E). The acid used for challenge was the main factor influencing the viability reduction of both serovars (P 0.05). The acid tolerance exhibited by Salmonella Typhi and Salmonella Typhimurium was independent of the acid employed in the shock treatment (P 0.05) (Fig. 1); thus, the adaptive response apparently is not specific to the anion used in adaptation. The biggest difference in reduction between shocked and unshocked cells ( 2 log CFU/ml) was observed when the microorganisms had been shocked with lactic acid and then challenged with citric acid. The acid shock with hydrochloric acid improved the survival of Salmonella in media acidified with hydrochloric acid and in media acidified with the organic acids (Fig. 1A and 1D). Similar results were obtained by Baik et al. (3), who observed that the lethal effect of acidity on Salmonella Typhimurium was minimized with a previous acid shock (hydrochloric acid at ph 4.4). This acid shock protected the pathogen against the antibacterial effect of butyric, acetic, and propionic acids. In our work, shocked and unshocked Salmonella Typhi cells had a similar reduction in viability only when they were challenged with citric acid (Fig. 1A). However, the acid shock improved the physiological condition of survivors of the challenge. The injured proportion of Salmonella Typhi survivors to the challenge with citric acid was 69% for shocked and 99% for unshocked cells (data not shown). Uljas and Ingham (43) observed that E. coli O157:H7 preexposed for 72 h to TSB acidified to ph 3.5 with hydrochloric acid survived better in synthetic gastric fluid (ph 2.5) that did controls preincubated at ph 6.5. Preexposure of pathogen cells to citric or malic acid did not improve survival in synthetic gastric fluid, and preexposure to lactic acid (ph 3.5) resulted in a diminished ability of these cells to survive in the synthetic gastric fluid. In preliminary studies in our laboratory, the protection conferred by the acid shock was ph dependent. The ph values near 5.0 induced acid tolerance; ph 3.5 may be too harmful to the cell to permit the induction of acid tolerance, even for E. coli O157:H7. To determine whether the organic acids anions affected the acid tolerance of Salmonella, the unshocked strains were exposed to the dissociated form of the acids during the shock step. Although all tested acids (ph 5.5) induced acid tolerance in both serovars, only lactate (ph 7.0) influenced the acid tolerance (Fig. 1A, 1C, 1D, and 1F). This effect was observed when hydrochloric acid was used as an acid challenger. Salmonella suspensions exposed to lactate were reduced 0.5 to 1.2 log CFU/ml less than unshocked suspensions exposed to hydrochloric acid. Lactate was effective in inducing tolerance to low ph, but it did not protect the cells against the antibacterial action of citric or lactic acid. Acid tolerance induction resulting from exposure to anions of organic acids has been reported for Salmonella Typhimurium and E. coli (2, 25). When an exponential culture of Salmonella Typhimurium was exposed to acetate, propionate, or butyrate, the cells improved their survival in a culture medium acidified at ph 3.0 (25). Baik et al. (3) identified some genes implicated in the tolerance to organic acids. They reported that fur (ferric uptake regulator) and atp (Mg 2 -dependent ATPase) cell mutants did not have increased tolerance at low ph but were more tolerant of organic acids. The lactic acid effect observed suggests that this anion could be an activator of such genes or of some other genes with similar physiological roles. To obtain a more sensitive response than the viability reduction, we assessed the level of stress among cells that survived acid challenge. The percentage of stressed cells among survivors of acid challenge for both pathogens was affected by the type of acid used for the challenge (data are not shown). Virtually all the cells surviving the challenge with citric or lactic acid had some degree of injury ( 98%).

4 2050 ARVIZU-MEDRANO AND ESCARTÍN J. Food Prot., Vol. 68, No. 10 TABLE 1. Length of lag phase for Salmonella Typhimurium and Salmonella Typhi cells shocked and unshocked in hydrochloric, citric, and lactic acids and challenged in media acidified with each acid a Lag phase values by challenge acid Salmonella Typhi cells Salmonella Typhimurium cells Growth ph Strain Shock acid HCl HCl HCl HCl HCl HCl 5.8 B f 5.2 C d 4.9 D e 6.0 A f 4.4 E d 6.0 A f 1.8 G cd 2.0 F b 1.9 FG bc 1.4 I b 1.6 H b 1.5 HI bc 14.6 C b 19.6 B b 1.9 E bc 2.1 D ab 1.9 E bc 1.4 G b 1.8 E a 1.6 F ab 2.1 B a 2.1 B ab 2.2 B a 1.6 D a 1.9 C a 1.7 D a 6.6 C d 4.3 E f 4.6 D f 7.2 B c 3.5 F e 12.6 A c 1.5 G e 1.6 G d 1.5 G e 1.3 H bc 1.5 H bc 1.3 H d 1.9 B b 1.7 C cd 1.7 C d 1.4 D b 1.4 D cd 2.0 B ab 1.7 C cd 1.7 C d 1.4 D b 5.8 B f 4.5 F e 5.2 D d 5.5 C e 4.8 E c 6.4 A e 1.9 H b 2.2 G a 1.8 H c 1.3Ibc 1.3Id 1.3Id 12 E c 13.2 CE c 20.5 A b 19.3 A b 14.8 BC b 15.0 B b 1.9 E b 2.1 D 1b 2.0 DE b 1.2 H c 1.4 G cd 1.4 G cd 2.0 C ab 2.2 B a 2.0 C b 1.4 D b 6.3 D e 4.3 F f 7.6 C c 5.6 E e 8.1 B d 1.7 G d 1.6 G d 1.7 G d 1.3 H bc 1.4 H cd 1.1Ie 1.9 B b 1.8 B c 1.8 B cd 1.4 C b 1.5 C b 1.5 C bc 2.0 B ab 2.0 B b 2.0 B b 1.4 C b 1.5 C b 1.4 C cd a Lag phase is reported in hours. Values in the same column that are followed by the same capital letter are not significantly different (P 0.05); values in the same row that are followed by the same lowercase letter are not significantly different (P 0.05).

5 J. Food Prot., Vol. 68, No. 10 BEHAVIOR OF ACID-SHOCKED SALMONELLA TYPHI AND SALMONELLA TYPHIMURIUM 2051 TABLE 2. Physiological state of Salmonella Typhimurium and Salmonella Typhi cells shocked and unshocked in hydrochloric, citric, and lactic acids and challenged in media acidified with each acid a Physiological state by challenge acid Salmonella Typhi cells Salmonella Typhimurium cells ph Strain Shock acid HCl HCl HCl HCl HCl HCl 0.28 F b 0.38 E a 0.38 E a 0.23 G a 0.29 F b 0.65 A a 0.63 A a 0.4 E ef 0.47 D d 0.51 C bc 0.47 D d 0.55 B b 0.15 D e 0.06 E d 0.07 E c 0.00 G d F e 0.00 G d 0.51 C c 0.54 BC a 0.64 A a 0.56 B a 0.63 A a 0.57 B b 0.00 F f 0.00 F e 0.00 F d 0.00 F d 0.00 F e 0.00 F d 0.43 D f 0.4 D ef 0.15 E g 0.48 C cd 0.55 B b 0.62 A a 0.23 H c 0.33 G b 0.37 F a 0.12 I c 0.46 D a 0.06 A a 0.5 C cd 0.42 E de 0.52 C bc 0.42 E e 0.56 B b 0.49 CD c 0.00 E f 0.57 B b 0.45 D cd 0.52 C bc 0.54 BC a 0.61 A a 0.63 A a 0.00 D D D C ef 0.45 C cd BC d 0.45 C de 0.55 A b 0.49 B c 0.34 D a 0.36 D ab 0.21 E b 0.16 F b 0.20 E c 0.22 E b 0.42 BC fg 0.40 C ef 0.47 A d 0.44 AB e 0.42 BC e 0.47 A d 0.00 E f 0.03 D de 0.01 E d 0.02 E d 0.04 D d 0.06 D c 0.47 B de 0.38 C f 0.52 A bc 0.53 A ab 0.45 B de 0.51 A c 0.00 D f 0.30 C h 0.28 C g 0.41 B e 0.50 A bc 0.47 A d 0.50 A c 0.19 G d 0.27 F c 0.19 G b 0.25 F a 0.00 I e 0.08 H D g 0.49 B b 0.35 E f 0.55 A a 0.45 C de 0.45 C de 0.40 C g 0.46 B bc 0.53 A b 0.55 A a 0.53 A bc 0.43 BC e 0.00 E f 0.42 C fg 0.37 D f 0.49 B cd 0.56 A a 0.51 B c 0.50 B c a Physiological state was estimated as the proportion of the population with no lag period (arbitrary scale from 0 to 1). Physiological state was recorded as 0.00 when no growth was observed within 24 h. Values in the same column that are followed by the same capital letter are not significantly different (P 0.05); values in the same row that are followed by the same lowercase letter are not significantly different (P 0.05).

6 2052 ARVIZU-MEDRANO AND ESCARTÍN J. Food Prot., Vol. 68, No. 10 In contrast, only 60% were injured when hydrochloric acid was used for challenge. Organic acids cause more cellular damage than do inorganic acids (1). Only for Salmonella Typhi shocked with hydrochloric acid and challenged with citric acid were significant differences in the proportion of the injured population between the shocked and unshocked cells observed. Effect of acid shock on growth of Salmonella Typhi and Salmonella Typhimurium in acidified media. Under certain conditions, some strains of Salmonella cause illness after ingestion of only a few viable cells (23). However, the illness risk increases with higher numbers of cells in the implicated food. Therefore, we studied the growth kinetics of shocked and unshocked Salmonella Typhi and Salmonella Typhimurium in acidified media. One limitation of using OD data to describe the growth of a microorganism is that detectable absorbance changes occur at a minimum bacterial concentration of 10 6 CFU/ ml. Thus, changes in OD occur only after the start of bacterial multiplication. The estimation of the length of the lag phase using absorbance data is dependent of the precision with which has been calculated (18). When bacteria were grown under adverse conditions, max values estimated from absorbance data with the Gompertz equation tended to be lower that those values obtained from viable counts (18). This potential source of variation probably did not affect our conclusions because comparisons of growth parameters between shocked and unshocked cells were made only under the same set of experimental conditions. We also used an initial concentration of 10 5 CFU/ml, which was very close to the detection limit of the Bioscreen turbidimetric analyzer. The growth curve data fitted well to the Gompertz equation; the root mean square error was small (0.0887), and only a few dispersed data points were observed. By using the Gompertz parameters (A, C, M, and B), we calculated lag phase durations, exponential growth rates, generation times, and the maximum population densities. The acid shock affected only the extension of the lag phase of Salmonella Typhi and Salmonella Typhimurium (Table 1). This effect was observed only when the ph of growth media was 4.5. At ph 5.0 and 5.5, shocked and unshocked cells had very similar behavior (only data at ph 4.5 and 5 are shown). At ph 7.0, unshocked cells had shorter lag phases and larger maximum population densities than did shocked cells (data not shown). The effect of acid shock on the duration of the lag phase was dependent on the type of acid in the growth media. Only Salmonella Typhi shocked with hydrochloric or citric acid grew within 24 h at 35 C (Table 1). Only when the cells grew in media containing citric acid were differences in the lag phase extension between shocked and unshocked cells observed. Acid-shocked Salmonella Typhimurium began its growth between 12 and 20.5 h, whereas unshocked cells did not grow in 24 h (Table 1). In contrast with the behavior observed during survival evaluation, Salmonella Typhi was less able than Salmonella Typhimurium to grow on medium acidified with citric acid. When the serovars grew in medium acidified with hydrochloric acid, small differences in the length of the lag phase between shocked and unshocked cells were observed. Our results suggest that exposure to hydrochloric acid does not require the utilization of the additional systems of defense triggered by acid shock. However, lactic acid was injurious, even for shocked cells. No growth was observed at ph 4.5 in the media containing this acid. The shorter duration of the lag phase for shocked cells was expected; when the microorganism is found at neutral ph and then is transferred to an acidic medium, it should activate its homeostatic mechanisms to adapt and grow in the new environment. When a bacterial population has been exposed to mildly acidic conditions before inoculation into a medium at ph 4.5, it should be better prepared to grow and its lag phase should be shorter. Differences in lag phase duration were greater among strains (P ) than between serovars (P 0.014). The type of acid used in the initial shock had less of an effect (P 0.003) on lag phase duration than did the type of challenge (P ). To evaluate the physiological condition of the inoculum, Baranyi and Roberts (5) introduced the parameter. This parameter depends on the maximum rate of growth ( ) and on lag phase duration ( ). The parameter is the fraction of the inoculated population that does not show a lag phase and thus starts its growth at a rate. This value ranges from 0 (no growth) to 1 (immediate growth without a lag phase). Some researchers have referred to the parameter as work to be done by the cell to adjust to the actual environment (32). In agreement with the lag phase duration, the physiological state values for the shocked strains of both Salmonella serovars were generally higher than those for the unshocked strains in a medium at ph 4.5 (Table 2). These results suggest that the acid shock enhanced the physiological state of the cells, and therefore they required less time to adapt to the new environment and start growth. To our knowledge, this work is the first demonstrating that the survival and growth potential of stationary-phase Salmonella Typhi in acidified media are enhanced when the pathogen has been previously exposed to moderately low ph. In Mexico, some food-processing facilities process fruit juices, pasteurized milk, and/or yogurt using the same equipment. If the equipment is not properly sanitized, milk or yogurt residues could induce acid adaptation in contaminating bacteria. If these acid-adapted microorganisms ever contaminate juice, they could survive longer in the beverage. This enhanced ability to tolerate acidity and organic acids may have an important impact on the safety of acidic foods, especially in the case of Salmonella Typhi, given the very low infectious dose of this organism. ACKNOWLEDGMENTS We thank Consejo Nacional de Ciencia y Tecnologia, México, for the scholarship for S. M. Arvizu-Medrano. We especially thank Dr. Dean O. Cliver for grammatical revision of the manuscript. REFERENCES 1. Alakomi, H. L., E. Skyttä, M. Saarela, T. Mattila-Sandholm, K. Lavtva-Kala, and I. M. Helander acid permeabilizes gram-

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