INTRODUCTION

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Members of the genus Salmonella are major international food-borne pathogens and reportedly cause approximately 30,000 cases of food-borne illness each year in England and Wales (4). In the United States, approximately 40,000 Salmonella infections are confirmed by culturing each year, and the true figure is estimated to be between 800,000 and 4 million infections, with approximately 500 fatalities (2, 13). Salmonella enterica serovar Enteritidis phage type 4 (PT4) and S. enterica serovar Typhimurium definitive type 104 (DT104) are the two most prevalent serotypes isolated from infected humans and together accounted for approximately 80% of all isolates obtained from 1990 to 1994 in England and Wales (PHLS Communicable Disease Surveillance Centre). It has been known for 10 years that S. enterica serovar Enteritidis PT4 is a key cause of infections associated with foods of animal origin, especially shelled eggs and poultry (2). More recently, S. enterica serovar Typhimurium DT104 has emerged as an important Salmonella strain in Europe and the United States, and the percentage of antibiotic-resistant S. enterica serovar Typhimurium isolates increased from 1% in 1979 and 1980 to 34% in 1996 in the United States (13). S. enterica serovar Typhimurium DT104 is of particular concern to the food industry due to the severity of the human disease which it causes, its multiple drug resistance, and the extensive animal reservoirs of the organism (40).

Reducing the available water in food is a long-established method for controlling bacterial growth. Desiccation or increasing the humectant content of a food results in a reduced water activity (a_w) (5). Optimal growth of Salmonella strains occurs at an a_w of 0.99, but these bacteria tolerate many stressful conditions and can survive in low-a_w foods for long periods (36). Various solutes are incorporated into food in order to reduce the a_w and maintain a reasonable safety margin before growth of microorganisms can occur. However, consumer pressure to reduce the levels of salt and sugar in food products has resulted in increases in the a_w in intermediate-moisture-level foods (a_w, 0.9 to 0.6 [24]). This affects primarily the spoilage stability of the products at the lower end of this a_w range, but pathogens, including Salmonella spp., have caused illness following survival in foods at the higher end. For example, S. enterica serovar Napoli and S. enterica serovar Agona have caused international food poisoning outbreaks associated with low-a_w chocolate (12, 15) and a low a_w-crisp type of snack, respectively (3, 21, 35). In addition, it has been reported that the infectious dose of Salmonella cells is reduced (e.g., 10 to 100 cells) when the organism is present in low-a_w foods (15, 32), possibly due to acid tolerance that occurs after sublethal osmotic stress, which has increased concern over the survival of the bacteria under these conditions. For these reasons it is important to continue to assess safety when either food composition or processing changes and to assess the ability of emerging pathogens, such as S. enterica serovar Typhimurium DT104, to survive at low a_w.

Recent research has revealed that the virulence of S. enterica serotype Enteriditis PT4 in animal models infected orally is linked to greater tolerance of a range of stressful conditions (18, 19). In Escherichia coli, the major stationary-phase sigma factor (RpoS) encoded by the rpoS gene is important for survival during an osmotic upshift (16). Understanding the survival of Salmonella cells in low-a_w foods should facilitate the design of safe preservation procedures. Since RpoS expression is important for survival under certain stressful conditions, it may influence the ability of Salmonella cells to survive at low a_w.

In this paper we describe the long-term survival of isolates of S. enterica serovar Enteriditis PT4 and S. enterica serovar Typhimurium DT104 at reduced a_w values (achieved by using various humectants), the associated morphological changes (as determined by microscopy and flow cytometry), and the involvement of the rpoS gene. To the best of our knowledge, this is the first report of filamentation in S. enterica serovar Enteriditis PT4 and S. enterica serovar Typhimurium DT104 in response to low-a_w conditions achieved by using NaCl, glycerol, and sucrose.

	MATERIALS AND METHODS

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Salmonella strains. S. enterica serovar Typhimurium DT104 strain 30 (41) was isolated from cattle feces. S. enterica serovar Typhimurium DT104 strain 10 (41) and S. enterica serovar Enteriditis PT4 strain E (18, 19) are human isolates. S. enterica serovar Enteriditis PT4 strain I (18, 19) was obtained from a chicken carcass, and strain LA5 was obtained from a natural chicken infection (1). Strain EAV54 is an otherwise isogenic rpoS mutant of LA5 (1) which was kindly provided by M. J. Woodward, Veterinary Laboratory Agency, Weybridge, United Kingdom. Strains 10 and I are naturally occurring rpoS mutants (F. Jørgensen, S. J. Wilde, G. S. A. B. Stewart, and T. J. Humphrey, submitted for publication). Strain I exhibits reduced virulence in animals infected orally and lower tolerance to certain environmental stresses (18, 19).

Preparation of log- and stationary-phase cultures. Salmonella strains were recovered from storage at 20°C on Protect beads (Mast Diagnostics, Merseyside, United Kingdom). A single bead was streaked onto 5% horse blood agar (Columbia agar base [Oxoid, Basingstoke, United Kingdom] containing horse blood [E & O Laboratories, Bonnybridge, Scotland]) prior to incubation at 37°C for 24 h. Log-phase cultures were prepared by inoculating 9 ml of tryptone soya broth (TSB) (Oxoid) which contained 0.25% glucose or nutrient broth (NB) (Oxoid) which contained no glucose with a single typical colony. The broth was incubated at 37°C for 3 h before it was used. One microliter of a log-phase culture was used to inoculate 9 ml of TSB before incubation at 37°C for 15 h to produce a stationary-phase culture. Before cultures were used, their turbidities at 600 nm were measured with a spectrophotometer (Cecil model CE1010) and standardized to values of 0.2 for NB and 0.4 for TSB, which gave a final cell density of 10⁸ CFU ml¹ in each case.

Preparation of low-a_w broth media. AnalaR grade NaCl, glycerol, and sucrose (BDH, Poole, Dorset, United Kingdom) were used as humectants to produce a range of low-a_w TSB or NB preparations (2, 4, 8, and 12% [wt/vol] NaCl, corresponding to a_w values of 0.99, 0.98, 0.95, and 0.92, respectively; 16, 32, 64, and 96% [wt/vol] sucrose, corresponding to a_w values of 0.99, 0.98, 0.96, and 0.94, respectively; 16, 32, 64, and 96% [vol/vol] glycerol, corresponding to a_w values of 0.96, 0.92, 0.86, and 0.79, respectively).

Survival of Salmonella cells at low a_w values (achieved by using sucrose, glycerol, or NaCl) over a 144-h period. Ten-microliter portions of log- or stationary-phase cultures of strains E, I, 30, and 10 were added to 190-µl portions of NB or TSB with the a_w reduced to between 0.79 and 0.99 by NaCl, glycerol, or sucrose in a microtiter plate. Cells inoculated into reduced-a_w NB were previously grown in NB, and cells inoculated into reduced-a_w TSB were previously grown in TSB. The microtiter plate was incubated statically at 21 or 37°C, and the number of CFU per milliliter was determined after 24, 72, and 144 h of incubation by using the method of Miles and Misra (28) and blood agar plates, which were incubated at 37°C for 24 h. The limit of detection was 40 CFU ml¹.

Long-term survival of Salmonella cells at low a_w values (achieved by using NaCl) over a 5-month period. Ten-milliliter aliquots of TSB preparations with the a_w reduced to between 0.92 and 0.99 by NaCl were prepared in duplicate, and 100-µl portions of stationary-phase cultures of strains E, I, 30, and 10 were inoculated. The preparations were incubated statically at 21 and 37°C for up to 5 months. Counts were determined every 14 days by using the method described above.

Filamentation of Salmonella cells at low a_w values and recovery from filamentation. Ten-microliter portions of log- or stationary-phase cultures of strains E, I, 30, and 10 were added to 190-µl portions of NB or TSB with the a_w reduced to between 0.79 and 0.99 by NaCl, glycerol, or sucrose in a microtiter plate, and the preparations were incubated statically at 21 or 37°C. Changes in cell morphology were assessed by examining wet preparations and by Gram staining; the preparations were viewed by using an oil immersion lens (magnification, ×100) and model CH-2 light microscope (Olympus Instruments, London, United Kingdom). Images of Gram-stained cells were passed via the microscope through a high-performance charge-coupled device video camera to a Mac 7200/90 computer. Image capture and editing were accomplished by using Scion Image LG5 software (available from zippy.nimh.nih.gov).

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Data analysis. Data analysis was performed with Microsoft Excel 97.

	RESULTS

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Survival of Salmonella cells at low a_w values over a 144-h period. All of the Salmonella strains tested could survive for long periods at reduced a_w values, although survival was greatest at an a_w of 0.99 (in TSB or NB containing no added humectant). Concentrations at or above 8% NaCl (a_w, 0.95), 96% sucrose (a_w, 0.94), and 32% glycerol (a_w, 0.92), were usually bactericidal for Salmonella cells (Tables 1 and 2), although this depended on the broth base and the incubation temperature. For example, after 72 h of incubation, 8% NaCl was always bactericidal when NB was the broth base but was not always bactericidal when TSB was the broth base, and 32% glycerol was always bactericidal at 37°C but was not always bactericidal at 21°C. The minimum a_w for growth of Salmonella cells was between 0.95 (when NaCl was bactericidal) and 0.92 (when glycerol was bactericidal). Incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C at lethal a_w values. The variable minimum a_w values for growth when different humectants were used supported the hypothesis that there was a specific solute effect (NaCl > sucrose > glycerol).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Survival of log-phase S. enterica serovar Enteriditis PT4 strain E (open symbols) and strain I (solid symbols) (A) S. enterica serovar Typhimurium DT104 strain 30 (open symbols) and strain 10 (solid symbols) (B), and S. enterica serovar Enteriditis PT4 strain LA5 (open symbols) and strain EAV54 (solid symbols) (C) at 37°C in TSB with the aw reduced by no NaCl (circles), 8% NaCl (triangles), or 12% NaCl (squares). The detection limits (40 CFU ml1) are indicated by dashed lines. The error bars indicate the standard errors of the means.

Survival of Salmonella cells at low a_w values over a 5-month period. During incubation in TSB supplemented with 8% NaCl, the levels of all rpoS mutants became undetectable sooner than the levels of the strains that expressed RpoS became undetectable (Table 3). For example, strain E survived for 43 days in the presence of 8% NaCl before it became undetectable, whereas strain I survived for only 15 days. As with short-term survival, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C.

View this table: [in this window] [in a new window]   TABLE 3.   Time taken for the concentration of Salmonella cells to decrease from an initial value of 106 CFU ml1 to an undetectable level (<40 CFU ml1) when preparations were incubated at 21 or 37°C in TSB supplemented with NaCl

Filamentation of Salmonella cells at low a_w values over a 144-h period. In response to an a_w that was suboptimal for growth but not bactericidal (approximately 0.93 to 0.98, depending on the solute), all of the Salmonella strains tested formed filaments at 21 or 37°C, and some of these filaments were at least 200 µm long and had regularly spaced nucleoids visible by Gram staining (Fig. 2). Filaments were observed after approximately 24 h of incubation at 37°C in TSB supplemented with 8% NaCl. Filaments were observed in both NB and TSB but were longer and more numerous in the latter medium. Filaments formed whether sucrose, NaCl, or glycerol was used as the humectant, but with NaCl at a_w values of 0.95 to 0.98 the filaments appeared to be longer and more numerous. TSB supplemented with NaCl at an a_w of 0.95 to 0.98 was optimal for filament formation. The filaments had straight sides and no indentations visible by light microscopy.

View larger version (55K): [in this window] [in a new window]   FIG. 2.   Image analysis of S. enterica serovar Typhimurium DT104 strain 30, showing a filamentous cell formed during incubation at 21°C in TSB supplemented with 8% NaCl for 144 h (A) and subsequent recovery following 3 h (B) and 8 h (C) of rehydration in fresh TSB containing no added humectant.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Light scattering of S. enterica serovar Enteriditis strain I, showing the mean particle sizes (bars) and the coefficients of variation (line) after incubation at 21°C in TSB supplemented with various concentrations of NaCl for 144 h.

	DISCUSSION

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In this study, S. enterica serovar Enteriditis PT4 and S. enterica serovar Typhimurium DT104 survived at reduced a_w values for long periods. Under low-a_w conditions, strains of S. enterica serovar Enteriditis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 µm long.

Previous work by Scott on the survival of microorganisms at low a_w values revealed that the response to a_w was solute independent (33). More recently, specific solute effects were described (6), and NaCl was found to be more inhibitory than glycerol for Salmonella cells at the same a_w (27). Our estimates of the minimum a_w for growth confirmed the findings of other researchers (10). The levels of survival at low a_w values were greater at 21°C than at 37°C, which is consistent with research describing improved survival in the presence of high salt concentrations at a lower temperature (22). Our study revealed that optimal survival at a low a_w requires RpoS expression. The survival data presented here highlight the importance of choosing the right strain to obtain mathematical modelling data to simulate worst-case scenarios. The results obtained for inactivation of PT4 and DT104 in this study were compared to the results predicted for Salmonella cells by the USDA Pathogen Modelling Program. Our results generally revealed less reduction in the number of Salmonella cells than would be predicted by the model. The discrepancies may have been due to differences in the conditions and strains used.

The filamentous cells observed in this study presumably formed as a result of a continued increase in biomass in the absence of cell septation during the low-a_w stress. The filaments formed in this study had regularly spaced nucleoids and no indentations visible by light microscopy, which is consistent with an early block in the cell division genes involved in septation, the fts genes (26). Filamentous phenotypes have been found for cell division gene mutants (9) and, more recently, for wild-type organisms in response to a number of stresses. The latter organisms include Escherichia coli at a low temperature (34), S. enterica serovar Enteriditis at a low temperature (29), and Listeria monocytogenes under high osmotic stress conditions (20). Filamentation of S. enterica serovar Oranienburg in response to a low a_w has been observed (34), and Yoshida et al. observed filamentation in S. enterica serovar Paratyphi in response to salts in solid media (42).

It is thought that low-a_w stress results in inhibition of the production of (or action of) cell division proteins in Salmonella cells, which blocks septation but allows the proteins involved in biomass formation to function to some extent. The specific mechanism involved in filamentation remains unclear, but we believe that there are four main possibilities. First, a low a_w affects the extent of DNA supercoiling, which in turn affects the regulation of osmotically controlled genes (14, 17) and may interfere with the regulation of cell division genes, thus causing filamentation. Second, the SOS response is a repair system induced by DNA damage (30) which upregulates SulA, an inhibitor of the initial protein involved in septation (FtsZ). If osmotic stress causes DNA damage, a filament with an early block in septation would be formed. Third, the heat shock response involves production of cell division inhibitors (8, 38). Cross-protection of osmotic shock and heat shock has been reported in non-Salmonella species (20, 37), and it has been demonstrated that common stress proteins occur in response to heat shock and osmotic shock in Bacillus subtilis (39). Therefore, it seems reasonable to suppose that the osmotic shock response could produce inhibitors of cell division. Finally, cell division is driven in part by turgor (11). If turgor is disturbed by osmotic stress, then there may be a "lack of signal" for cell division to proceed.

The longer, more numerous filaments and improved survival in reduced-a_w TSB compared with NB could have been the result of acid habituation due to the presence of fermentable glucose in TSB. During overnight culture of Salmonella cells in TSB (containing 0.25% glucose), the pH of the culture decreased from around neutral to 4.5 (7). In any case, the presence of glucose can also lead to acid habituation at neutral pH by inducing a novel tolerance response (31). Acid habituation has been found to confer resistance to osmotic stress (23), so growth in TSB could cross-protect against osmotic stress.

The presence of live filaments after 144 h of incubation in TSB supplemented with 8% NaCl indicated that filamentation may improve survival under low-a_w conditions which would kill cells that do not elongate. Further work is required to establish whether filamentation actually aids survival or if the normal (small-phenotype) cells that do survive go on to form filaments.

The filaments formed in response to a low a_w are not dependent on rpoS expression since strains 10, I, and EAV54 (rpoS mutants) elongate; this is unlike the response to chilling, which is rpoS dependent (29). Thus, chilling appears to be more inhibitory to rpoS mutants than low a_w is.

If filamentation is found to occur in foods, there are clear implications for low-a_w food products. The DNA of a single contaminating Salmonella cell could continue to replicate, the biomass could increase, and long filaments could form in the food. Following an increase in the a_w of the food (e.g., after rehydration with water), septation could resume and rapidly result in a large number of viable Salmonella cells, which could cause infection after consumption. The possible presence of filamentous Salmonella cells should also be considered when workers design methods for microbiological monitoring. The increase in Salmonella biomass (without a concomitant increase in the microbial count) would not be detected by traditional microbiological enumeration tests if a food product was tested by direct plating immediately after it was removed from low-a_w storage conditions. The bacterial count would appear to be low, but under favorable conditions a rapid increase in the number of cells could occur in as little as 2 h. Note that testing for the presence of Salmonella cells by an enrichment method would probably not be negatively affected by the presence of filaments.

The survival and filamentation of Salmonella strains at low a_w values clearly require further research to determine if the filamentation of Salmonella cells in food products (a_w, 0.92 to 0.98) constitutes a risk to public health and to determine whether methods for microbiological monitoring are affected by the presence of filamentous Salmonella cells.