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Keywords:

  • Campylobacter jejuni;
  • cold storage;
  • cross-resistance responses;
  • high hydrostatic pressure;
  • stressful conditions

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To study the development of resistance responses in Campylobacter jejuni to high hydrostatic pressure (HHP) treatments after the exposure to different stressful conditions that may be encountered in food-processing environments, such as acid pH, elevated temperatures and cold storage.

Methods and Results: Campylobacter jejuni cells in exponential and stationary growth phase were exposed to different sublethal stresses (acid, heat and cold shocks) prior to evaluate the development of resistance responses to HHP. For exponential-phase cells, neither of the conditions tested increased nor decreased HHP resistance of C. jejuni. For stationary-phase cells, acid and heat adaptation-sensitized C. jejuni cells to the subsequent pressure treatment. On the contrary, cold-adapted stationary-phase cells developed resistance to HHP.

Conclusions:  Whereas C. jejuni can be classified as a stress sensitive micro-organism, our findings have demonstrated that it can develop resistance responses under different stressing conditions. The resistance of stationary phase C. jejuni to HHP was increased after cells were exposed to cold temperatures.

Significance and Impact of the Study:  The results of this study contribute to a better knowledge of the physiology of C. jejuni and its survival to food preservation agents. Results here presented may help in the design of combined processes for food preservation based on HHP technology.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial survival under adverse conditions may be greatly increased by the preceding expression of a stress response (Broadbent and Lin 1999). Stress responses may induce the acquisition of tolerance to subsequent severe stress from the same (homologous resistance) or different nature (cross-resistance or cross-protection) (Komatsu et al. 1990). Exploration of bacterial stress responses is motivated by a desire to understand the mechanisms leading to enhanced survival ability in bacteria, but also by industrial and safety concerns in the food industry (Scheyhing et al. 2004).

In recent years, there has been increased research into the use of nonthermal alternative methods for microbial inactivation, such as high hydrostatic pressure (HHP) or pulsed electric fields (PEF) (Raso and Barbosa-Cánovas 2003). The attraction of these technologies lies in the production of microbiologically safe foods with minimal changes in their sensory and nutritional attributes. HHP is now being increasingly applied commercially for the processing of foodstuffs such as seafoods sauces, fruit, oysters and meat products. Also, under study are combinations of high pressure with other methods, such as heat, low pH or antimicrobial peptides (García-Graells et al. 1999; Pagán et al. 2001). To exploit these new approaches fully, we need to better understand the effects of high pressure on different microbes, and in this direction, a considerable body of relevant data has accumulated in recent years (Farkas and Hoover 2000; Patterson 2005). However, although Campylobacter jejuni is now recognized as the leading cause of bacterial foodborne gastroenteritis throughout the developed world (Friedman et al. 2000; Oberhelman and Taylor 2000), we currently know very little about the response of these organisms to high pressure. Poultry products, the most common vehicle of transmission of C. jejuni to human beings, possess a relatively short shelf life and a high consumer demand for freshness, such that application of HHP may be beneficial.

Despite the importance of C. jejuni as a foodborne pathogen, its physiology is poorly understood. Campylobacter spp. are generally considered to be delicate organisms, sensitive to environmental stress and with strict growth requirements (Butzler and Oosterom 1991; Altekruse et al. 1999; Murphy et al. 2006). Campylobacter jejuni is a microaerophile, growing optimally at oxygen concentrations of 3–5% and carbon dioxide concentrations of 5–10%. It is unable to grow at temperatures below 29°C (Solomon and Hoover 1999) and is sensitive to stresses such as heating, freezing, acidification and HHP (Solomon and Hoover 1999; Martínez-Rodriguez and Mackey 2005). In addition, C. jejuni also lacks many of the genetic regulatory networks found in other bacteria that allow them to cope with adverse conditions (Parkhill et al. 2000; Park 2002), such as the general stress response, regulated by σS in Gram-negative cells (Hengge-Aronis 2000). Physiological studies have shown that resistance of C. jejuni to heat, acid or oxidative stress does not increase appreciably on entry to stationary phase, consistent with the lack of σS (Kelly et al. 2001; Murphy et al. 2003a); although resistance does increase somewhat in the case of HHP (Martínez-Rodriguez and Mackey 2005). Despite its apparent sensitivity to stress, recent work has shown that Campylobacter is able to mount novel types of adaptive responses leading to enhanced resistance to mild heat and acid stresses (Murphy et al. 2006); and there is recognition that the organism could be more resistant to adverse conditions than had initially been thought (Food Standards Agency 2005). Moreover, it has been recently demonstrated that it is able to grow in the presence of air if pyruvate is present in the medium (Verhoeff-Bakkenes et al. 2007). Therefore, more studies are needed to understand the physiology of this micro-organism, to fully assure its control especially with the new technologies available for food preservation.

The aim of this work was to study the development of resistance responses in C. jejuni to HHP treatments after the exposure to different stressful conditions encountered in food-processing environments, such as acid pH, elevated temperatures and cold storage. The ability to acquire barotolerance by cross-protection would have implications on high pressure treatments.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains and growth media

Campylobacter jejuni NCTC 11351 was maintained frozen in cryovials. The liquid growth medium consisted of Brucella Broth (Becton, Dickinson and Company, Le Pont de Claix, France) supplemented with ferrous sulfate, sodium metabisulfite and sodium pyruvate, each at 0·25 g l−1 (FBP Campylobacter growth supplement SR0232, Oxoid, Basingstoke, UK), and was referred as BBFBP. The agar-plating medium was tryptone soy agar supplemented with 0·6% yeast extract (Biolife, Milan, Italy) and 0·1% sodium pyruvate (TSA-YEP) (Panreac, Barcelona, Spain). These media were chosen in preliminary studies (data not shown), because they provided the highest numbers of C. jejuni cells, in comparison with various other media (Brain–Heart Infusion, Mueller–Hinton Agar and Columbia Agar with different supplements) used by other authors.

Campylobacter blood-free selective agar base (charcol cefoperazone deoxycholate agar, CCDA) with selective supplement (Oxoid SR0155) was used for plating from the frozen cryovial to assure the purity of the bacterial culture before growth of broth cultures for stress experiments.

Culture conditions

Cultures were prepared by inoculating a flask containing 10 ml of BBFBP with one single colony from a selective agar plate (CCDA). This culture was incubated at 37°C on a shaking platform at 150 rpm for 24 h under microaerobic conditions [5% (v/v) oxygen, 10% (v/v) carbon dioxide and 85% (v/v) nitrogen] maintained using a variable atmosphere workstation (MACS VA 500 Don Whitley, Otley, UK). A volume of 100 μl of this culture were inoculated into 50 ml of fresh BBFBP and incubated at 37°C for 5 h to produce exponential-phase cells (108 UFC ml−1) or for 24 h for stationary-phase cells (2 × 109 UFC ml−1).

Viable counts

Samples were appropriately diluted in 0·1% sterile Maximum Recovery Diluent (MRD; Oxoid CM0733) and surface-plated in TSA-YEP. Plates were incubated for a minimum of 96 h at 37°C under microaerobic conditions. Previous experiments showed that longer incubation times did not influence the profile of survival curves (data not given). After incubation, colony-forming units (CFU) were counted.

Determination of heat resistance

Heat treatments were carried out in a specially designed resistometer as already described (Condón et al. 1993). Briefly, this instrument consists of a 350-ml vessel provided with an electrical heater and thermostat controller for thermoregulation, an agitation device to ensure inoculum distribution and temperature homogeneity, and ports for injecting the microbial suspension and for the extraction of samples. Once treatment temperature had attained stability (±0·1°C), 0·2 ml of an appropriately diluted cell suspension was injected into the main vessel containing the treatment medium which was tryptone soy broth (TSB) of pH 7·0 or 4·5 (adjusted with HCl). After inoculation, 0·2-ml samples were collected at different treatment times and immediately pour-plated in TSA-YEP.

HHP treatments

Cultures were centrifuged at 3000 g for 15 min at 4°C, and the pellets of cells were resuspended in TSB-YE of pH 7·0 or pH 4·5. Cell suspensions (2 ml) were distributed in sterile plastic pouches that were heat-sealed before pressurization at 200 or 300 MPa for different times in a high pressure apparatus (model S-FL-085-9-W; Stanstead Fluid power, Stanstead, UK). The pressure transmitting fluid was monopropylene glycol–water (30 : 70). The temperature in the fluid during pressurization did not exceed 30°C. After decompression, samples were diluted and immediately surface-plated in TSA-YEP.

Determination of acid resistance

The acid resistance was determined by inoculating 500 μl of cell suspension into 9·5 ml of TSB-YE adjusted to pH 3·75 with HCl. Aliquots were taken at different treatment times, diluted and surface-plated in TSA-YEP for survival counts.

Acid, heat and cold-shock treatments

Cultures were exposed to acid shocks (pH 4·5, 5·0 and 5·5), heat shocks (40, 42 and 45°C) or cold shocks (0°C) for different times by inoculating 500 μl of cell suspension into 9·5 ml of TSB-YE acidified with HCl to the desired value, preheated to 40, 42 or 45°C in a thermostated water bath or submerged in an ice-chilled bath, respectively. After the desired adaptation time, samples were collected and challenged for acid resistance, heat resistance or HHP resistance.

Measurement of fluorescence anisotropy

Fluorescence anisotropy of the probe DPH (1,6-diphenyl 1,3,5-hexatriene) (Sigma, St Louis, MO, USA) was used to monitor changes in membrane fluidity as previously described (Zaritsky et al. 1985). Anisotropy values are inversely related to membrane fluidity (Shinitzky 1984). Briefly, samples of bacterial cultures in stationary growth phase were washed twice with PBS containing 0·25% formaldehyde (pH 7·4) for fixation, and then incubated for 45 min at 37°C with 5 × 10−6 mol l−1 DPH (added as a 10−4 mol l−1 solution in tetrahydrofuran) for probe insertion in the membrane. Steady-state fluorescence anisotropy was measured at 37°C with a Cary Eclipse spectrofluorometer provided with a manual polarizer accessory (Varian Inc., Mulgrave, Vic., Australia) with excitation at 355 nm and emission at 425 nm, 5-nm and 5-nm slitwidth values, respectively, and a 3-s integration time.

Anisotropy values (r) were calculated according to Shinitzky (1984):

  • image

V and H stand for polarization direction (vertical and horizontal directions) and I for fluorescence intensity. Fluorescence anisotropy determinations were performed at least three times on independent working days.

Resistance parameters

Survival curves were obtained by plotting the logarithm of the survivor fraction vs treatment time. The profile of the survival curves was different for each agent studied. Therefore, because the traditional first-order exponential kinetics were not adequate, a mathematical model based on the Weibull distribution was used to fit the survival curves as previously recommended by Álvarez et al. (2003). This model is described by the following equation (Mafart et al. 2002):

  • image

where S(t) in the survival fraction; t, the treatment time; and δ and ρ are the scale and the shape parameter, respectively. The δ value represents the treatment time needed to reduce viable numbers of the population by the first log10 cycle; while the ρ parameter indicates the shape of the survival curve, which is related to the distribution of individual resistance of cells in the whole population. ρ values equal to 1 correspond to straight survival curves, ρ values >1 to convex profiles and ρ values <1 to concave profiles. To fit the model to the experimental data and to calculate the δ and ρ parameters, the GraphPad prism® (Graphpad Software, Inc., San Diego, CA, USA) was used. R2 values were always higher than 0·96. All experiments with C. jejuni were repeated at least three times on independent working days, and data in graphs correspond to mean values and standard error (error bars). Statistical significance of differences (P < 0·05) was also calculated using the statistical software package GraphPad prism®.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Adaptation of Campylobacter jejuni to thermal stress

Figure 1 shows the survival curves of native and heat-adapted exponential-phase C. jejuni cells heated at 55°C. For heat adaptation, cells were subjected to different sublethal temperatures, 40, 42 and 45°C for 120 min, before exposure to heat treatment at 55°C cells. Heat shocks carried out at higher temperatures inactivated a substantial part of the original population and were discarded. Campylobacter jejuni developed an adaptation response after the exposure to sublethal temperatures, which was maximum at 45°C within the range studied. Adapted cells were more heat resistant than native cells. For example, after 3 min at 55°C, 3 log10 cycles of inactivation were attained for native cells, whereas no significant inactivation was observed for adapted cells at 45°C for 120 min (P < 0·05). The ratio between the δ values of nonadapted and heat-adapted cells was between 1·8 and 2·4 depending on the adaptation temperature. This protection ratio is similar to that described for other vegetative cells in analogous experimental conditions (Hassani et al. 2007). In all cases, graphs showed an initial portion where bacterial counts remained unchanged, also known as ‘shoulder’, followed by a near-linear portion. The occurrence of a shoulder was more pronounced for heat-adapted cells, although it is remarkable that it was also present in survival curves corresponding to native cells.

image

Figure 1.  Heat resistance of exponential growth-phase cells of Campylobacter jejuni NCTC 11351 at 55°C: survival curves of native cells (•) and cells previously adapted by incubation at 40°C (bsl00001), 42°C (bsl00066) or 45°C (bsl00072) for 120 min.

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Adaptation of Campylobacter jejuni to acid stress

Figure 2 shows the survival curves of native and acid-adapted exponential-phase C. jejuni cells to an acid treatment at pH 3·75. For acid adaptation, cells were incubated at pH 4·5, pH 5·0 or pH 5·5 for 120 min before treatment at pH 3·75. Campylobacter jejuni developed an adaptation response after the exposure to sublethal pH, which was maximum at pH 5·5 within the range here investigated. Adapted cells were more acid resistant than native cells. For example, after 90 min of exposure pH 3·75, 3 log10 cycles of inactivation of native cells were reached compared with <1 log10 cycles of inactivation for adapted cells (pH 5·5/120 min). The ratio between the δ values of nonadapted and acid-adapted cells was between 2·0 and 2·8 depending on the adaptation pH.

image

Figure 2.  Acid resistance of exponential growth-phase cells of Campylobacter jejuni NCTC 11351 at pH 3·75: survival curves of native cells (•) and cells previously adapted by incubation at pH 4·5 (bsl00001), pH 5·0 (bsl00066) or pH 5·5 (bsl00072) during 120 min.

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It has been reported that heat and acid resistance of exponentially growing C. jejuni cells is generally higher than that reported for stationary-phase cells (Kelly et al. 2001; Martínez-Rodriguez and Mackey 2004). For this reason, exponential-phase cells were used to determine the experimental conditions leading to an adaptive response to heat and to acid. However, stationary phase is probably the most common state of bacterial cells in nature, and therefore were also included in this study. Stationary-phase cells were also capable of developing an adaptive response to acid pH and to elevated temperatures, similar to that observed for exponential growth-phase cells (Table 1).

Table 1.   Resistance values to heat and acid treatments of exponential and stationary phase native (Control) and adapted Campylobacter jejuni cells (HS, heat-shocked; AS, acid-shocked)
Treatment conditionsExponential phaseStationary phase
δ (min)ρδ (min)ρ
Heat treatment (55°C)Control2·02 ± 0·172·62 ± 0·571·26 ± 0·812·01 ± 0·52
HS (45°C/2 h)4·79 ± 0·474·07 ± 0·794·43 ± 2·101·49 ± 0·50
Acid treatment (pH 3·75)Control38·4 ± 11·11·20 ± 0·6046·2 ± 7·891·74 ± 0·43
AS (pH 5·5/2 h)107 ± 2·472·24 ± 0·3379·6 ± 5·211·43 ± 0·05

HHP resistance of exponential and stationary-phase cells

Table 2 presents the log cycles of inactivation for exponential and stationary-phase cells of C. jejuni subjected to different HHP treatments. Pressure resistance of C. jejuni depended on the stage of growth such that exponentially growing cells were notably more sensitive than stationary-phase cells confirming the observations of Martínez-Rodriguez and Mackey (2004). The influence of the pH of the pressurizing medium was also examined (Table 2). For cells in both stages of growth, acidification of the treatment medium from pH 7·0 to pH 4·5 increased the lethality of the HHP treatments.

Table 2.   High hydrostatic pressure resistance (log10 cycles of inactivation) of Campylobacter jejuni NCTC 11351 cells in exponential and stationary phase of growth
Treatment conditionsTreatment time (min)Log reduction in viable numbers
Exponential phaseStationary phase
  1. NS, no survivors detected.

200 MPa/pH 7·051·2 ± 0·68no reduction
101·7 ± 0·420·19 ± 0·01
152·5 ± 0·500·46 ± 0·23
203·3 ± 0·680·96 ± 0·20
200 MPa/pH 4·551·5 ± 0·62no reduction
102·5 ± 0·470·30 ± 0·01
153·6 ± 0·311·5 ± 0·23
203·9 ± 0·132·7 ± 0·19
300 MPa/pH 7·055·8 ± 0·352·6 ± 0·55
10NS5·1 ± 0·08

Resistance to HHP in exponential and stationary-phase Campylobacter jejuni cells after the exposure to acid, heat and cold shocks

Figure 3a shows the log10 cycles of inactivation of exponential-phase cells of C. jejuni after pressure treatment of native cells and cells that had been previously exposed to acid (pH 5·5/120 min), heat (45°C/120 min) or cold shocks (0°C/240 min). Results for stationary-phase cells are shown in Fig. 3b. HHP treatments were carried out at neutral pH except in the case of acid-adapted cells and their corresponding nonadapted controls. These HHP treatments were carried out at acidic pH to prevent any reversion of the possible protection exerted by the incubation under acidic conditions prior to HHP treatments.

image

Figure 3.  Log10 cycles of inactivation of Campylobacter jejuni NCTC 11351 cells after a high hydrostatic pressure treatment of native (black bars) and adapted cells (white bars). Cells were adapted by prior exposure to an acid shock (pH 5·5/120 min), to a heat shock (45°C/120 min) or to a cold shock (0°C/4 h). Cells were in exponential (a) or stationary growth phase (b).

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For exponential-phase cells, neither of the adaptation conditions tested increased nor decreased HHP resistance of C. jejuni. However, with stationary-phase cells, acid and heat adaptation sensitized C. jejuni cells to the subsequent pressure treatment. For example, after 15 min at 200 MPa, nearly 6 log10 cycles of inactivation were observed in cells that had been previously exposed to sublethal acid conditions, compared to less than 2 log10 cycles for native cells. These results demonstrate that under our experimental conditions, C. jejuni cells did not show a cross-resistance adaptation response to HHP after the exposure to acid nor to heat shocks. On the contrary, sequential combination of these stressing conditions plus HHP treatments would exert an additional lethal effect on this micro-organism. However, in the case of cold-adapted stationary-phase cells, there was a development of cross-resistance to HHP (Fig. 3b).

A more detailed examination of the effect of cold shock on the development pressure resistance of stationary-phase cells is shown in Fig. 4. The magnitude of the adaptation increased gradually with the duration of the cold storage, and after 4 h at 0°C, there was a difference in the inactivation of native and adapted cells of nearly 3 log10 cycles.

image

Figure 4.  Influence of a cold storage (0°C) in the high hydrostatic pressure resistance of Campylobacter jejuni NCTC in stationary growth phase (bsl00066).Survival fraction after 300 MPa/5 min at pH 7·0.

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Fluorescence anisotropy determinations were performed in native and cold-adapted cells to monitor changes in membrane fluidity after cold storage. No statistically significant changes (P < 0·05) in DPH anisotropy values were observed between native and cold-adapted cells, indicating the absence of significant changes in the fluidity of the membranes, under our experimental conditions. Anisotropy values of 0·197 ± 0·002 and 0·194 ± 0·003 were obtained for native and cold-adapted cells, respectively.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Pressure inactivation of micro-organisms is affected by process and product parameters such as temperature, type of food and pH. In this study, we demonstrated that the physiological status of C. jejuni cells as well as their previous history can influence pressure tolerance. Campylobacter is considered to be sensitive to several agents used for food preservation, including heat, cold, drying and acid stress (Stern and Line 2000). Moreover, recent studies have also shown its sensitivity to HHP (Solomon and Hoover 2004; Martínez-Rodriguez and Mackey 2005). Nevertheless, because of its low infectious dose and its ubiquity, its survival in foods represents an important safety concern. In this investigation, we examined the development of resistance to heat and to acid in C. jejuni NTCT 11351. This micro-organism was able to develop resistance to both agents after being exposed to sublethal temperature and pH, respectively.

To the best of our knowledge, the development of resistance of C. jejuni to heat treatments after being exposed to sublethal elevated temperatures has not been reported previously. It is remarkable that despite being considered as a heat labile organism, its capacity to develop thermotolerance was similar in magnitude to that described for other vegetative cells (Mackey and Derrick 1986; Hassani et al. 2007). Survival curves to heat treatments showed prolonged shoulders, which were more noticeable with heat-adapted cells. The occurrence of shoulders in heat survival curves of heat-shocked cells has been commonly reported (Pagán et al. 1997; Hassani et al. 2007), and it has been attributed to the accumulation of repairable sublethal damages during the first moments of the treatment, multitarget inactivation, cell clumping or activation phenomena in the case of spores (Palop et al. 1999).

According to our results, the maximum thermotolerance induced by heat shock of C. jejuni cells depended on the temperature of the heat shock. In Fig. 1, it is shown that to achieve 3 log10 cycles of inactivation in native cells, 3 min of heat treatment at 55°C were necessary. However, heat-adapted cells had an increased survival capacity, and between 5 and 6 min of heat treatment were necessary to achieve the same lethality. Therefore, C. jejuni heat-adapted cells would have a higher probability to survive mild heat treatments applied to foods. The mechanism responsible for a heat-adaptive response is not fully known. In other organisms, heat-shock induced thermotolerance is associated with synthesis of heat shock proteins (HSPs) (Lindquist 1986). The occurrence of a protective response to heat in C. jejuni was thus not entirely unexpected because, despite the lack of RpoS, RpoE and RpoH sigma factors (Park 2002), the synthesis of at least 24 HSPs has been described, including GroEL, DnaK and Lon (Konkel et al. 1998). Apart from the synthesis of new proteins, other mechanisms of protection may also take place, for instance, the stabilization of the outer membrane via divalent cation binding described for Salmonella (Mañas et al. 2001), or the presence of two-component regulatory systems that appear to be involved in sensing and counteracting stress in Campylobacter (Park 2002).

Despite its importance in pathogenesis, the acid stress response in C. jejuni has been understudied. To cause disease in humans, C. jejuni must survive passage through the stomach, where it is exposed to low pH as well as to reactive oxygen and nitrogen species. While little is known about how C. jejuni survives transit through the stomach, its low infectious dose suggests that it is well equipped to sense and respond to acid shock. It has been shown that the presence of an extracellular component secreted during growth induces acid and heat stress tolerance in C. jejuni (Murphy et al. 2003a), whilst an adaptive acid tolerance response (ATR) that is induced by acid and/or aerobic conditions permits increased survival at lethal pH values (Murphy et al. 2003b). In this work, we have also observed the presence of an adaptation response after exposure to sublethal acid conditions that would increase the possibilities of C. jejuni surviving gastric transit and also acid conditions in food. This adaptation response occurred in both exponential and stationary-phase cells, and should be avoided in food-processing environments. The ATR in C. jejuni involves the upregulation of general stress proteins involved in protein protection or degradation, such as the universal chaperones DnaK and GroEL, which are also involved in the heat-shock response (Reid et al. 2008). In addition, C. jejuni drastically remodels its outer membrane proteins (OMP) composition in response to growth in acid conditions (Reid et al. 2008).

Contrary to what has been described for most stressing agents and in accordance with data reported by Martínez-Rodriguez and Mackey (2004), C. jejuni resistance to HHP was lower in the exponential phase of growth than in the stationary phase. This increase in resistance may be related to changes in cellular fatty acid composition and to an increase in membrane physical stability to pressure that are not dependent on the expression of general stress sigma factors, which are lacking in this species (Martínez-Rodriguez and Mackey 2004). We also observed a higher sensitivity to HHP treatment in media of acidic pH, both for exponential and for stationary-phase cells, as commonly reported for other vegetative micro-organisms (Mackey and Mañas 2008). The levels of inactivation attained either for exponential or for stationary-phase cells, and at both treatment media, pH suggests that this micro-organism would be inactivated by this technology after the treatments normally applied in the industry. Moreover, for most experimental conditions, no protective responses to HHP were observed, indicating that this technology could be combined with other stressing agents without the possible risk of inducing resistance if incorrectly applied. A protective response of practical relevance was seen only with stationary-phase cells that had been cold shocked before the HHP treatment. By contrast, stationary-phase cells that had been previously exposed to mild acid conditions or elevated temperatures showed a greater sensitivity to HHP. This sensitizing effect was particularly important for acid-adapted cells, which were inactivated by more than 5 log cycles after 15 min at 200 MPa and pH 4·5, when compared to only 1·5 log cycles for the nonadapted cells. Therefore, for this micro-organism, the sequential combination of elevated temperatures or acid conditions with subsequent HHP treatments exerted a higher lethal effect than HHP alone.

The reasons for this particular behaviour are not known. Cross-resistances have frequently been demonstrated with different combinations of successive stresses (Broadbent and Lin 1999; Xu et al. 2008). Increased barotolerance resulting from sublethal heat shock has been reported in the yeast Saccharomyces cerevisiae (Iwahashi et al. 1991), in the Gram-negative bacterium Escherichia coli (Aertsen et al. 2004) and in the Gram-positive bacterium Listeria monocytogenes (Hayman et al. 2007; Skandamis et al. 2008). An increase in pressure resistance following cold shock has been reported in L. monocytogenes (Wemekamp-Kamphuis et al. 2002), Staphylococcus aureus (Noma and Hayakawa 2002) and E. coli (Casadei and Mackey 1997). Moreover, in Lactobacillus sanfranciscensis, barotolerance has also been found to be induced by preincubation under a range of other stresses including osmotic and acid stress conditions (Scheyhing et al. 2004). In this work, however, we found no increase in barotolerance of exponential-phase cells, and a different effect on stationary-phase cells depending on the stress applied. This behaviour could be related to the particular physiology of C. jejuni.

Exposure to mild pressure results in the synthesis of cold-shock proteins in E. coli (Welch et al. 1993) but their role, if any, in tolerance to pressure is not clear. In C. jejuni, a possible role for a cold-shock response in pressure resistance is difficult to explain because the major cold-shock protein, CspA, which acts as an RNA chaperone, is not present in the sequenced strain of C. jejuni (Parkhill et al. 2000). A possible alternative explanation would be that changes in the conformation of cell envelopes after exposure to cold temperature lead to enhanced resistance. Bacterial cells can control the fluidity of their membranes by modulation of their composition, to maintain an optimal level of fluidity within the lipid matrix (Beney and Gervais 2001). Casadei et al. (2002) showed that cells of E. coli with more fluid membranes were more resistant to pressure. C. jejuni can regulate its membrane composition in response to changes in temperature even at temperatures below the limit for growth (Hazeleger et al. 1995; Leach et al. 1997; Höller et al. 1998). It has been reported that during heat or acid shock, the membrane initially becomes fluidized (Denich et al. 2002; Mykytczuk et al. 2007) but cells then compensate for the perturbing effect of heat or acid by decreasing membrane fluidity. This decreased fluidity could account for a higher sensitivity to HHP. The contrary effect would take place during a cold shock, when membrane fluidity would increase leading to increased barotolerance of the cell. We tried to demonstrate this hypothesis by studying changes in membrane fluidity (measurement of fluorescence anisotropy), but no significant changes in anisotropy were apparent after the exposure to a cold shock (P < 0·05) for 4 h. Consequently, with the data obtained in this investigation, we cannot confirm whether changes in resistance to HHP of C. jejuni are linked to changes in membrane fluidity.

The absence of changes in barotolerance in exponentially growing cells leads to the conclusion that cellular modifications involved either in the sensitization or in the acquisition of resistance may rely in cellular changes or responses that are active in stationary-phase cells but not in exponentially growing cells. Despite the lack of RpoS, these results confirm that C. jejuni cells in the different phase of growth are different in their physiological responses. This introduces significant uncertainty in the determination of adaptive responses in this micro-organism, and more studies are needed for the design of effective food preservation processes.

Most of the available information with regard to stress adaptation relates to specific adaptive responses. However, from a realistic viewpoint of food processing, foodborne pathogens are likely to be exposed to multiple stresses concurrently or sequentially. Although C. jejuni is considered to be sensitive to different technologies including high pressure processes, as a result of the increase in pressure resistance that we have observed after a cold shock, its barotolerance would be similar to that reported for other Gram-positive and Gram-negative bacteria such as S. aureus, L. monocytogenes and E. coli (Alpas et al. 2000). These results may have important consequences for the use of high-pressure treatment for food preservation. Cold storage is commonly used in all stages of food processing, and it would increase the pressure resistance of stationary-phase C. jejuni cells. However, heat or acid combined sequentially with high pressure would increase the lethality of the treatment with regard to C. jejuni.

This investigation contributes to a better knowledge of the physiology of C. jejuni and its survival to food preservation agents. Whilst C. jejuni can be classified as a stress-sensitive micro-organism, our findings have demonstrated that it can develop resistance responses under different stressing conditions. Results here presented may have important consequences for the use of high-pressure treatment in combination with previous cold storage for food preservation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

N. Sagarzazu is the recipient of a predoctoral fellowship from the Spanish Ministry for Education and Science (FPU scheme).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References