Escherichia coli and Salmonella enterica Are Protected against Acetic Acid, but Not Hydrochloric Acid, by Hypertonicity^▿

Cultures and culture conditions.

Five nonpathogenic strains of E. coli were used in these studies, namely, FRRB (Food Research Ryde Bacterial culture collection) 2697, 2698, 2699, 2700, and 2701. In addition, four strains of Salmonella were used: FRRB 2742 (S. enterica serovar Montevideo), 2743 (S. enterica serovar Poona), 2746 (S. enterica serovar Typhimurium), and 2747 (S. Typhimurium). Cultures were maintained as glycerol stocks at −80°C and activated by transferring a loopful from the stock into 10 ml of nutrient broth (NB) (CM0001; Oxoid, United Kingdom) prepared according to the manufacturer's instructions. NB cultures were then incubated at 37°C (±1°C) for 22 h. For the preparation of experimental inocula, 10 μl of 22-h NB cultures was transferred to 10 ml tryptone soy broth (CM0129; Oxoid) with 1% total glucose (TSB1%G) and incubated with shaking at 200 rpm for 22 h at 37°C (±1°C). At the conclusion of incubation, the pH of TSB1%G cultures was determined using pH indicator papers (type CS, pH 3.8 to 5.5; Whatman International Ltd., United Kingdom) to ensure that it was approximately 4.2, indicating that acid conditioning of the cells was promoted during growth.

Experimental matrix design.

For E. coli, the effect of increasing NaCl concentration on cell survival in NB was assessed for each of five acid treatments: in the presence of acetic acid at pH 3.6, 3.8, and 4.0 and in the presence of HCl at pH 3.6 and 3.8. For Salmonella isolates, the effect of increasing NaCl concentration on cell survival in NB was assessed for each of four acid treatments: in the presence of acetic acid at pH 3.6, 3.8, and 4.0 and in the presence of HCl at pH 3.6 and 3.8. No assessment of inactivation in the presence of HCl at pH 4.0 was attempted because preliminary experiments (data not shown) showed that the selected E. coli and Salmonella isolates were able to grow in NB under this condition. Similarly, no assessment of inactivation of Salmonella in the presence of acetic acid at pH 3.6 was undertaken because preliminary experiments (data not shown) indicated that Salmonella was too rapidly inactivated to enable reliable enumeration.

The survival response of E. coli and Salmonella in acidified NB was assessed at eight concentrations of NaCl: 0.5, 1, 2, 3, 4, 5, 6, and 7% (wt/wt of water) (i.e., 0.5 to 7 g NaCl in 100 g of water used to prepare the broth, or equivalent to 0.5, 1.0, 1.9, 2.9, 3.8, 4.7, 5.6, and 6.5% [wt/wt] of prepared NB, taking into account other broth ingredients). In addition, for all isolates, the effect of sucrose on survival was assessed in NB containing 0.5% (wt/wt of water) NaCl and 10% (wt/wt of water) sucrose (equivalent to 9.0% [wt/wt] of NB) and in NB containing 3% (wt/wt of water) NaCl and 10% (wt/wt of water) sucrose (equivalent to 8.8% [wt/wt] of NB).

Survival experiments.

Twenty grams of NB was dispensed aseptically into sterile 28-ml screw-cap polypropylene containers. Twenty-microliter volumes of TSB1%G cultures were used to inoculate each container to achieve an initial concentration of cells of ∼10⁶ CFU/g. Inoculated NB was incubated statically at 23°C (±1°C). Within 30 min of inoculation, a 1-ml sample of each inoculated NB culture was withdrawn and decimally diluted twice in buffered peptone water (CM0509; Oxoid). Dilutions were surface plated (0.1 ml) onto duplicate tryptone soy agar (TSA) (CM0131; Oxoid) plates. Plates were incubated aerobically at 37°C (±1°) for 48 h prior to counting. Inoculated containers continued to be incubated at 23°C (±1°C) and were sampled at predetermined time intervals for up to 79 h, with a maximum of nine time points (0.5, 1, 7, 24, 31, 48, 55, 72, and 79 h [±0.5 h]). Survivors at each sampling time were enumerated as described above. Each treatment was duplicated using individually grown inocula.

Curve fitting.

Viable cells counts were expressed as log₁₀ of the survival fraction, log [N(t)/N₀], where N(t) and N₀ are the momentary and initial concentrations of cells, respectively. The detection threshold of the experiment was approximately 50 CFU/g. Using the Solver function in Microsoft Excel (Microsoft Corporation), data were fitted to the log logistic model, log [N(t)/N₀] = −ln {1 + exp[k₁ × (t − t_c)]}, where t_c (h) is a measure of the shoulder period (“lag”) or delay time before inactivation was detected, and k₁ is a constant representing the semilogarithmic inactivation rate when t ≫ t_c.

Assessment of relative severity of acid conditions.

Survival in the presence of 0.5% (wt/wt of water) NaCl was used to assess the relative severity of acid treatment conditions against E. coli and Salmonella. The effects of acid treatments were compared by single-factor analysis of variance (ANOVA) (P < 0.05) of average t_c and k₁ (At_c and Ak₁) values using the single-factor ANOVA tool of Microsoft Excel. For E. coli there were generally 10 estimates, comprised of five E. coli isolates tested in duplicate, while for Salmonella there were generally 8 values, derived from four Salmonella isolates tested in duplicate.

Effect of NaCl at different concentrations.

The effect of NaCl concentration was assessed by statistical analysis of average relative t_c (At_cR) values for E. coli and Salmonella as follows. Based on previous observations of the response of E. coli SERL 2 (4), it was assumed that the greatest protection would occur in formulations with 3% (wt/wt of water) NaCl. Thus, for each acid treatment and each E. coli or Salmonella isolate, and for each duplicate, t_cR values were calculated by dividing the t_c at each NaCl concentration by the t_c at 3% (wt/wt of water) NaCl. At_cR values for E. coli and Salmonella for each acid treatment at each NaCl level were then determined by averaging the t_cR values. The effect of NaCl concentration was then evaluated by single-factor ANOVA (P < 0.05) of At_cR using the single-factor ANOVA tool of Microsoft Excel.

Effect of sucrose versus NaCl.

At_cR values were statistically analyzed to compare protection against acid by sucrose, and by sucrose in combination with NaCl, with protection by NaCl alone. For each acid treatment and each E. coli or Salmonella isolate, and separately for each duplicate, t_cR values were calculated as follows. For comparison of t_c values in formulations containing 0.5% NaCl without sucrose (formulation J) and formulations containing 0.5% NaCl and 10% (wt/wt of water) sucrose (formulation I), t_cR values were calculated relative to those in formulations containing 3% NaCl without sucrose (formulation C). For comparison of formulation I and formulation C, t_cR¹ values were calculated relative to t_c values in formulations containing 3% NaCl and 10% (wt/wt of water) sucrose (formulation H). For comparison of formulation C and formulation H, t_cR² values were calculated relative to t_c values in formulations I. At_cR values for E. coli and Salmonella for each treatment were determined by averaging the relevant estimates The effect of NaCl and sucrose treatments on At_cR were compared by single-factor ANOVA (P < 0.05) using the single-factor ANOVA tool of Microsoft Excel.

Assessment of cell wall damage.

Cell wall/cell membrane damage was investigated using E. coli FRRB 2701 inoculated into NB at pH 3.6, acidified with either HCl or glacial acetic acid and containing 0.5 to 7% NaCl as previously described. At 24, 48, and 72 h, samples were withdrawn from the inoculated NB and plated onto both TSA and TSA plus 0.15% bile salts. Plates were incubated as described above. The percentage of sublethally injured cells after different treatment times was calculated by comparing numbers of CFU on TSA to those on TSA plus 0.15% bile salts (13).

Relative severity of acid conditions.

For the five strains of E. coli in NB containing 0.5% NaCl, the At_c was not significantly different (α = 0.05) among broths acidified with acetic acid to pH 3.6, 3.8, or 4.0, and the At_c was not significantly different between NB acidified with HCl to pH 3.6 or 3.8 (Fig. 1A). However, the At_c was significantly different (α = 0.05) between NB acidified with acetic acid and NB acidified with HCl. The Ak₁ for E. coli was significantly different (α = 0.05) between NB acidified with acetic acid at a pH of <4.0 and pH 4.0 and between NB acidified with acetic acid and broths acidified with HCl, but not between NB acidified with HCl to pH 3.6 or 3.8 (Fig. 1B). In general, for E. coli the trend in relative severity of acid conditions as assessed by At_c or Ak₁ was acetic acid at pH 3.6 > acetic acid at pH 3.8 > acetic acid at pH 4.0 > HCl at pH 3.6 > HCl at pH 3.8 (Fig. 1). In NB acidified with HCl to pH 3.8, growth of E. coli FRRB 2701 (only) occurred at 0.5% NaCl in one of the duplicate samples and in the presence of 1% NaCl in both samples (data not included in analyses).

The same trend in the relative severity of the acid treatments for E. coli was also observed for Salmonella, but the effect was more extreme. Much more rapid inactivation of Salmonella than of E. coli occurred when broths were acidified with acetic acid, and growth of some Salmonella strains occurred at pH 3.8 when NB was acidified with HCl, at up to 1% (wt/wt of water) NaCl.

Effect of NaCl at different concentrations.

The ANOVAs of the At_cR for E. coli and Salmonella are shown in Tables 1 and 2, respectively. A summary of the statistically significant (α = 0.05) trends in the At_cR responses of E. coli and Salmonella to increasing NaCl concentration, as a function of acidulent type and pH, is shown in Table 3.

When NB was acidified with HCl, irrespective of pH, a monotonic (consistent and systematic) decrease in the At_cR of both E. coli and Salmonella occurred in response to increasing NaCl concentration (Table 3). In contrast, in the presence of acetic acid at pH 3.8 and 4.0, for both E. coli and Salmonella, a nonmonotonic (i.e., initially increasing and then decreasing) response of the At_cR to the increasing NaCl concentration was observed. The inflection point in the nonmonotonic At_cR response was dependent on pH and was different for E. coli and Salmonella. For E. coli, the maximum At_cR occurred in the range of 4 to 6% NaCl at pH 3.8 and at 2 to 4% NaCl at pH 4.0 (Tables 1 and 3), with no significant differences (α = 0.05) in the At_cR at different NaCl concentrations in those ranges. For Salmonella, the maximum At_cR in the presence of acetic acid occurred at 1 to 2% NaCl at pH 3.8, and at 1 to 4% at pH 4.0 (Tables 2 and 3). For E. coli in the presence of acetic acid at pH 3.6, the response of the At_cR to the increasing NaCl concentration was unexpectedly antitonic; i.e., as the NaCl concentration increased, the At_cR systematically increased for all NaCl concentrations tested (Table 3).

Effect of sucrose versus NaCl.

Comparison of At_cR values for formulation J (0.5% NaCl without sucrose) and formulation I (0.5% NaCl and 10% sucrose) showed that the addition of sucrose to NB acidified with acetic acid significantly (α = 0.05) extended survival of E. coli and Salmonella (i.e., increased the At_cR) at all pH values (Table 4). The addition of sucrose, though, had no effect on survival of E. coli in the presence of HCl (Table 4). Salmonella sometimes grew in NB acidified with HCl and containing sucrose.

In the presence of either acetic acid or HCl, the At_cR¹ in formulation I (containing 0.5% NaCl and 10% sucrose) was not significantly different from the At_cR¹ in formulation C (containing 3% NaCl only). These results suggest that 10% sucrose (with 0.5% NaCl) and 3% NaCl are equally effective at protecting E. coli and Salmonella from inactivation by acetic acid. For E. coli in the presence of HCl, the lack of protection by 10% sucrose (with 0.5% NaCl) is consistent with the lack of protection observed for 3% NaCl alone.

The addition of 10% sucrose (formulation H) to formulations containing 3% NaCl (formulation C) did not significantly affect the At_cR² in the presence of either HCl or acetic acid (Table 4).

Assessment of cell wall damage.

The proportion of sublethally injured E. coli FRRB 2701 as a function of time of exposure to lethal pH (3.6) due to HCl or acetic acid and in the presence of various concentrations of NaCl is shown in Fig. 2. Sublethal injury was observed earlier (i.e., 24 h) at higher concentrations of NaCl, suggesting that damage to the outer membrane/cell wall by NaCl (in the presence of acid) occurred at an early stage of exposure. As the exposure time increased, injury generally increased at all concentrations of NaCl in the presence of either acid. However, for cells exposed to acetic acid, a higher proportion of sublethally injured cells was observed in the presence of “low” (<2%) and “high” (>5%) concentrations of NaCl than at intermediate levels (2 to 5%) as exposure time increased to 72 h.