Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Letters in Applied Microbiology 2001, 32 (5), 312-315

Sodium chloride enhances recovery  and growth of acid-stressed  E. coli  O157:H7

Jordan KN and Davies KW

ABSTRACT

Aims: Combinations of sodium chloride and acid are frequently used to inhibit growth of spoilage and pathogenic bacteria in food. The influence of differing sodium chloride, lactate and pH values on the growth of stressed and unstressed cells of a non-toxigenic strain of Escherichia coli O157:H7 was studied.

Methods and Results: At pH 5·5 or 6·0, there was little or no effect on the growth rate in the presence of lactate and/or sodium chloride, but the lag times were longer as the lactate concentration increased. At pH 5·0, in the absence of sodium chloride, increasing the lactate concentration increased the growth rate and the lag time; no growth occurred in the presence of 1·5 g 100 g ¹ lactate. In the presence of 4-6 g 100 g ¹ sodium chloride, growth occurred at 1·5 g 100 g ¹ lactate. The growth rate was similar at all lactate concentrations.

Conclusions: The results demonstrate that the presence of sodium chloride promoted growth of E. coli O157:H7, especially under stressful conditions of low pH.

Significance and Impact of the Study: These findings could have implications for the use of acid and sodium chloride as a preservation treatment for the inhibition of E. coli O157:H7 in food.

INTRODUCTION

Since its first appearance as a food pathogen in 1983 (Riley et al. 1983), Escherichia coli O157:H7 has graduated from being an emerging pathogen to becoming a significant threat to consumer safety. A large number of food-poisoning outbreaks have been reported and a wide variety of foods have been implicated, including meat, dairy products and vegetables (Buchanan and Doyle 1997). The number of reported incidents is increasing. In 1996 there were 660 confirmed cases in England, compared with 1102 cases in 1999.

A considerable amount of research has been directed at attempting to understand the factors that contribute to the ability of this organism to become a successful food pathogen. Its low infective dose, possibly as few as 10 organisms ingested with food (Willshaw et al. 1994), certainly contributes to this. However, its ability to resist acid is also important. Acid tolerance in E. coli strains has been shown to be growth phase-dependent. Stationary phase cultures are considerably more acid-tolerant than mid-exponential phase cultures (Arnold and Kasper 1995), although this resistance is lost rapidly on subsequent growth (Jordan et al. 1999a). A more stable acid tolerance can be induced in mid-exponential phase cultures by exposure of the cells to mild acid prior to exposure to low pH. This inducible adaptive tolerance response (ATR) requires protein synthesis and confers a considerable degree of acid tolerance to the cells (Jordan et al. 1999a). Induction of ATR increases the survival of E. coli O157:H7 (Leyer et al. 1995) or Listeria monocytogenes (Gahan et al. 1996) in acidic foods.

Studies on E. coli O157:H7 have concentrated on developing better methodologies for its detection, or on understanding its survival during stress. Few studies have examined growth of E. coli O157:H7 in food. This is an important aspect since survival of the organism in food could result in growth if the storage conditions become slightly more favourable. Studies have showngrowth of E. coli O157:H7 in milk at 8°C (Massa et al. 1999), during the manufacture of Feta cheese (Ramsaran et al. 1998) and in the presence of different acids (Connor and Kotrola 1995). Sodium chloride is a commonly-used preservative in foods and will contribute to the prevention of growth of undesirable organisms. However, studies have shown that sodium chloride can have a stimulatory effect on the growth of L. monocytogenes (Cole et al. 1990), Salmonella enteritidis PT4 (Radford and Board 1995), or Salmonella heidelberg (Larson et al. 1993), at low pH.

In dairy products there are many inhibitory factors that contribute to the prevention of food pathogen growth. In this study, the focus was on sodium chloride, lactate concentrations and pH to determine the relative importance of these inhibitors in preventing the growth of E. coli O157:H7. Acid-stressed cells, in addition to unstressed and ATR-induced cells, were studied, since the physiological state of cells may contribute to their ability to grow under sub-optimal conditions.

MATERIALS AND METHODS

Strains, growth and media used

The strain used in this study was the non-toxigenic E. coli 0157:H7 isolate P1432 (obtained from P. Chapman, PHLS). This strain was isolated from a patient showing symptoms of gastro-enteritis. It does not carry the toxin genes, as shown by PCR and using a cell culture toxin assay. For routine use it was maintained on Brain Heart Infusion (BHI) agar plates at 4°C. For growth, one colony was transferred to 9 ml Tryptone Phosphate Broth (TPB) and incubated at 30°C for 16 h.

Acid treatment and induction of the adaptive tolerance response (ATR)

Mid-exponential phase cultures (O.D.₆₀₀ of 0·3-0·4), inoculated at 0·5% (v/v) from a freshly-grown culture and incubated at 30°C, with agitation (150 rev min ¹), were grown. After reduction of the pH to 3·0 with HCl, they were incubated for 90 min at 30°C. In one of these cultures, an ATR had been induced by adjusting the pH to 5·0 (with HCl) and incubating at 30°C for 60 min, prior to reduction of the pH to 3·0. This protocol resulted in mid-log phase stressed cells, with and without the induction of ATR. The number of viable cells in each culture and in a stationary phase culture were determined by diluting in Maximum Recovery Diluent (MRD) and spread-plating on BHI agar plates, which were incubated at 30°C for 18 h.

Growth of cells in media containing different salt and lactic acid concentrations at different pH values

Cells were inoculated at 10% (v/v) into TPB at the required pH, sodium chloride concentration and/or lactic acid concentration. The stationary phase and ATR-induced cultures were diluted in TPB at the required pH so that the inoculum resulted in the same number of viable cells (10³ cfu ml ¹) as were present in the acid-treated culture. All cultures were incubated in a Bioscreen instrument (Labsystems, Helsinki, Finland) with a 96-well plate, and O.D. (at 600 nm) was recorded hourly for 72 h.

Calculation of lag time and specific growth rate

A calibration curve was constructed relating O.D. to cell numbers. Due to variability of O.D. from various sources, this necessitated using a complex alogrithm written in SAS. Using this calibration curve, O.D. values from the Bioscreen data were converted to cell numbers. A graph of log cell numbers as a function of time was drawn. Lag times (i.e. the length of time during which no increase in O.D. was observed) and growth rates were then calculated from this graph. The specific growth rates may not be the maximum specific growth rates, since these may have occurred at O.D. values below the threshold of the Bioscreen.

Reproducibility of results

All growth experiments were repeated at least four times. Values shown are the mean of these experiments with the standard deviation.

RESULTS

Table 1 shows the effect of pH and lactate on the growth rate and lag times of stressed cells of strain P1432 in the absence of sodium chloride. At pH 5·5 or 6·0, there was little or no effect of increasing the lactate concentration on the growth rate, but the lag time was increased. At pH 5·0, increasing the lactate concentration reduced the growth rate and extended the lag time. The presence of 1·5 g 100 g ¹ lactate at this pH resulted in growth inhibition. Similar results were obtained with unstressed cells, or cells with an induced ATR (data not shown). Decreasing the pH from 6·0 to 5·0 extended the lag time from 0·45 to 3·5 h (Table 1).

Addition of sodium chloride to the medium at pH 5·5 or 6·0 had little influence on the growth rate, but the lag times were decreased (data not shown). Addition of sodium chloride to the medium at pH 5·0 resulted in a dramatic increase in the growth rate of stressed cells, which was dependent on the lactate concentration (Table 2). In the absence of lactate, the addition of 4% sodium chloride resulted in a >30% increase in growth rate (from 0·79 to 1·21 h ¹). In the presence of 1·5 g 100 g ¹ lactate, the growth rate increased from 0 to 1·22 h ¹ as the sodium chloride concentration increased from 0 to 4%. Similar results were obtained with unstressed cells and cells with an induced ATR. At concentrations of 4-6 g 100 g ¹ sodium chloride, the increase in growth rate was not affected by the lactate concentration (Table 2). If the sodium chloride concentration was increased to 7, 8 or 9 g 100 g ¹, the growth rate decreased dramatically. In this case, the physiological state of the cells did affect the growth rate. Acid-stressed cells showed some growth at all sodium chloride concentrations, whereas ATR-induced and unstressed cells showed very little or no growth at 7, 8 or 9 g 100 g ¹ sodium chloride at pH 6·5 (Fig. 1). Even at pH 5·0, stressed cells could grow in the presence of 9 g 100 g ¹ sodium chloride (lag time was 42 h), whereas unstressed cells were inhibited and showed no growth in 72 h.

FIGURES

Table 2 The effect of different sodium chloride and lactate concentrations on the growth rate (h ¹) of ...

Fig. 1 Effect of sodium chloride concentration on the growth rate of Escherichia coli O157:H7 at pH 6·5....

DISCUSSION

Decreasing the pH and increasing the lactate concentration had the effect of inhibiting growth of E. coli O157:H7. However, rather than contributing to the inhibition, the addition of sodium chloride had the effect of reversing the inhibitory effect and enabling growth under conditions of low pH and high lactate. Similar results have been obtainedby Casey and Condon (2001), who showed that E. coli O157:H45 and other strains of E. coli showed greater survival of acid stress in the presence of sodium chloride. Radford and Board (1995) have also reported a similar phenomenon in Salm. enteritidis. In that case, the type of acidulant was important as the effect was only observed with acetic acid and not citric, propionic or hydrochloric acids. Cole et al. (1990) also observed that low concentrations (2%) of sodium chloride can have a protective effect on L. monocytogenes at low pH, or can stimulate the recovery of acid-injured cells, with citric acid as the acidulant. Obviously, the acidulant used has a different effect with different species. It would be interesting to determine the effect of different acidulants on the growth of E. coli O157:H7 in the presence of sodium chloride.

The addition of sodium chloride (3%) to the diluent used to count bacteria has been shown to result in the improved recovery of stressed cells of strain P1432 by reducing osmotic stress (Jordan et al. 1999b). The osmolarity of TPB would be similar to that of a cell, and addition of sodium chloride would increase the osmotic pressure, thereby increasing the osmotic stress on the cell. Therefore, the improved growth rate and decreased lag times in the presence of sodium chloride are not the result of reduced osmotic stress. An improved ability of stressed cells to grow in the presence of Ca⁺⁺ has been reported previously for Rhizobium spp. (Watkin et al. 1997). Better recovery in the presence of Na⁺⁺ may be analogous to this. The mechanism by which this works has not been elucidated, but it may be due to an improved ability to regulate the internal pH of the cell. In addition to improving growth of E. coli O157:H7, sodium chloride can also increase the tolerance of the organism to low pH in the presence of lactate. Cheng and Kasper (1998) reported that the time taken for a 90% reduction in cell numbers at pH 1·5 was 65·3 min in the presence of 1 g 100 g ¹ sodium chloride, compared with 38·1 min in the absence of sodium chloride.

The induction of ATR has been shown to enhance survival of E. coli O157:H7 during stress (Leyer et al. 1995). Furthermore, induction of an ATR (to one stress) can confer cross-protection to other stress factors (Wang and Doyle 1998). However, the present results show that the ability to grow under adverse conditions of pH, sodium chloride and/or lactate was not enhanced by ATR induction. This indicates that survival and growth mechanisms are independently controlled within the cell.

While these results have been obtained with a non-toxigenic strain of E. coli O157:H7, they could be applicable to toxigenic strains. A model for the growth of non-toxigenic strains was shown to be applicable to toxigenic strains (Salter et al. 1998), while the gene encoding universal stress protein from a toxigenic strain showed 97% homology with that of E. coli K-12 (Chen and Griffiths 1999). In addition, the unpublished results that we have obtained for acid and heat tolerance in this strain are similar to those obtained for toxigenic strains.

These results have implications for dairy products if they are contaminated with E. coli O157:H7. Many dairy products contain sodium chloride which can promote growth of E. coli O157:H7 if conditions within the food become favourable. Growth of this organism can occur under apparently stressful conditions of pH 5·0 in the presence of 6 g 100 g ¹ sodium chloride and 1·5 g 100 g ¹ lactate. In addition, stressed cells have a competitive advantage if the sodium chloride concentration is between 6 and 9 g 100 g ¹.

ACKNOWLEDGEMENTS

This research has been part-funded by grant aid under the Food Sub-Programme of the Operational Programme for Industrial Development which is administered by the Department of Agriculture, Food and Forestry and supported by national and EU funds (97/R & D/T/173).

REFERENCES

1 Arnold, K.W. & Kasper, C.W. (1995) Starvation- and stationary-phase-induced acid tolerance in Escherichia coli O157:H7. Applied and Environmental Microbiology 61, 2037-2039.

2 Buchanan, R.L. & Doyle, M.P. (1997) Foodborne disease significance of E. coli O157:H7 and other enterohemorrhagic E. coli. Food Technology 51, 69-76.

3 Casey, P. & Condon, S. (2001) Sodium chloride reduces the effect of organic acid on Escherichia coli O157:H45. Irish Journal of Agricultural and Food Research in press.

4 Chen, J. & Griffiths, M.W. (1999) Cloning and sequencing of the gene encoding universal stress protein from Escherichia coli O157:H7 isolated from Jack-in-a-Box outbreak. Letters in Applied Microbiology 29, 103-107.

5 Cheng, C.-M. & Kasper, C.W. (1998) Growth and processing conditions affecting acid tolerance in Escherichia coli O157:H7. Food Microbiology 15, 157-166.

6 Cole, M.B., Jones, M.V., Holyoak, C. (1990) The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes. Journal of Applied Bacteriology 69, 63-72.

7 Connor, D.E. & Kotrola, J.S. (1995) Growth and survival of Escherichia coli O157:H7 under acidic conditions. Applied and Environmental Microbiology 61, 382-385.

8 Gahan, C.G.M., O'Driscoll, B., Hill, C. (1996) Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation. Applied and Environmental Microbiology 62, 3128-3132.

9 Jordan, K.N., Hall, S., McClure, P.J. (1999b) Osmotic stress on dilution of acid injured Escherichia coli O157:H7. Letters in Applied Microbiology 28, 389-393.

10 Jordan, K.N., Oxford, L., O'Byrne, C.P. (1999a) Survival of low pH stress by Escherichia coli O157:H7: a correlation between alterations in the cell envelope and increased acid tolerance. Applied and Environmental Microbiology 65, 3048-3055.

11 Larson, A., Johnson, E.A., Nelson, J.H. (1993) Behaviour of Listeria monocytogenes and Salmonella heidelberg in rennet whey containing added sodium or potassium chloride. Journal of Food Protection 56, 385-389.

12 Leyer, G.J., Wang, L.-L., Johnson, E.A. (1995) Acid adaptation of Escherichia coli O157:H7 increases survival in acid foods. Applied and Environmental Microbiology 61, 3752-3755.

13 Massa, S., Goffredo, E., Altieri, C., Natola, K. (1999) Fate of Escherichia coli O157:H7 in unpasteurised milk stored at 8°C. Letters in Applied Microbiology 28, 89-92.

14 Radford, S.A. & Board, R.G. (1995) The influence of sodium chloride and pH on the growth of Salmonella enteriditis PT4. Letters in Applied Microbiology 20, 11-13.

15 Ramsaran, H., Chen, J., Brunke, B., Hill, A., Griffiths, M.W. (1998) Survival of bioluminescent Listeria monocytogenes and Escherichia coli O157:H7 in soft cheeses. Journal of Dairy Science 81, 1810-1817.

16 Riley, L.W., Remis, R.S., Helgerson, S.D.et al. (1983) Hemorrhagic colitis associated with a rare Escherichia coli serotype O157:H7. New England Journal of Medicine 308, 681-685.

17 Salter, M.A., Ross, T., McMeekin, T.A. (1998) Applicability of a model for non-pathogenic Escherichia coli for predicting the growth of pathogenic Escherichia coli. Journal of Applied Microbiology 85, 357-364.

18 Wang, G. & Doyle, M.P. (1998) Heat shock response enhances acid tolerance of Escherichia coli O157:H7. Letters in Applied Microbiology 26, 31-34.

19 Watkin, E.L.J., O'Hara, G.W., Glenn, A.R. (1997) Calcium and acid stress interact to affect the growth of Rhizobium leguminosarum bv. trifolii. Soil Biology and Biochemistry 29, 1427-1432.

20 Willshaw, G.A., Thirlwell, J., Jones, A.P., Parry, S., Salmon, R.L., Hickey, M. (1994) Vero cytotoxin-producing Escherichia coli O157 in beefburgers linked to an outbreak of diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome in Britain. Letters in Applied Microbiology 19, 304-307.
 

(order Full Text from publisher)

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005