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Keywords:

  • food preservation;
  • food safety;
  • heat;
  • nonthermal processes;
  • stress response;
  • sublethal injury

Abstract

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

Aims:  The objective was to study the response of Cronobacter sakazakii ATCC 29544 cells to heat, pulsed electric fields (PEF), ultrasound under pressure (Manosonication, MS) and ultraviolet light (UV-C) treatments after exposure to different sublethal stresses that may be encountered in food-processing environments.

Methods and Results: Cronobacter sakazakii stationary growth-phase cells (30°C, 24 h) were exposed to acid (pH 4·5, 1 h), alkaline (pH 9·0, 1 h), osmotic (5% NaCl, 1 h), oxidative (0·5 mmol l−1 H2O2, 1 h), heat (47·5°C, 1 h) and cold (4°C, 4 h) stress conditions and subjected to the subsequent challenges: heat (60°C), PEF (25 kV cm−1, 35°C), MS (117 μm, 200 kPa, 35°C) and UV-C light (88·55 mW cm−2, 25°C) treatments. The inactivation kinetics of Csakazakii by the different technologies did not change after exposure to any of the stresses. The combinations of sublethal stress and lethal treatment that were protective were: heat shock–heat, heat shock–PEF and acid pH–PEF. Conversely, the alkaline shock sensitized the cells to heat and UV-C treatments, the osmotic shock to heat treatments and the oxidative shock to UV-C treatments. The maximum adaptive response was observed when heat-shocked cells were subjected to a heat treatment, increasing the time to inactivate 99·9% of the population by 1·6 times.

Conclusions: Cronobacter sakazakii resistance to thermal and nonthermal preservation technologies can increase or decrease as a consequence of previous exposure to stressing conditions.

Significance and Impact of the Study:  The results help in understanding the physiology of the resistance of this emerging pathogen to traditional and novel preservation technologies.


Introduction

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

During food processing, foodborne pathogens may undergo a series of stresses, such as heating and freezing, acid pH, salt stress or exposure to cleaners and disinfectants. Food preservation and safety assurance are associated with these stresses, which are considered hurdles to foodborne pathogens (Leistner 1995). However, micro-organisms can develop adaptive responses when exposed to sublethal stresses (Abee and Wouters 1999). According to Hill et al. (2002), the general principle of stress adaptation can be simply stated in that a bacterium that is exposed to a sublethal stress may become more resistant to subsequent applications of the same stress (homologous resistance). Nevertheless, stress responses may sometimes induce the acquisition of tolerance to subsequent severe stress of a different nature in a phenomenon called cross-resistance response or cross-protection (Abee and Wouters 1999; Hill et al. 2002). These phenomena may counteract the effectiveness of food preservation hurdles and compromise food safety (Lou and Yousef 1997), and its magnitude should not be left out: for instance, the exposure of Staphylococcus aureus cells to a heat shock can increase up to 49 times its resistance to a subsequent heat treatment (Cebrián et al. 2009). The relevance of the increased resistance to lethal factors after adaptation to environmental stresses might now be even greater as increasing consumer demand for fresh-like foods is leading to a decrease in the intensity of traditional preservation technologies or their substitution with new ones.

Stress adaptation and protection has been studied more extensively in model micro-organisms such as Gram-negative Escherichia coli and Salmonella or Gram-positive Bacillus subtilis and Listeria monocytogenes, and little is known about the ability of other foodborne pathogens such as Cronobacter sakazakii to develop adaptive responses under these new circumstances. Cronobacter spp. is often found contaminating a great variety of food and food ingredients of animal and vegetable origin (Friedemann 2007), and it has increasingly gained the interest and concern of regulatory agencies, health care providers, the scientific community and the food industry because of its potential impact on human health (Chang et al. 2009). The evaluation of its capability to adapt to stressing environmental conditions and their impact on its resistance either to traditional or novel technologies is, therefore, required. Recent published reports have evaluated the effect of previous heat (Chang et al. 2009; Arku et al. 2011) or cold (Shaker et al. 2008) shocks on Cronobacter spp. resistance to subsequent heat treatments. To the best of our knowledge, the ability of Cronobacter spp. to acquire tolerance to food preservation technologies other than thermal treatments after exposure to shocks other than heat or cold shocks has not been previously described. Furthermore, the comparison of the ability of a micro-organism to develop resistance to the different technologies after being exposed to the same stresses and applied under the same experimental conditions offers a great opportunity to obtain some clues about the physiology of its resistance and the mechanisms of inactivation of the different technologies.

This study was undertaken to explore the ability of Csakazakii ATCC 29544 to increase its resistance to thermal, pulsed electric fields (PEF), ultrasound under pressure (Manosonication, MS) or ultraviolet light (UV-C) treatments after exposure to certain environmental stresses encountered in food-processing environments (i.e. acid, alkali, NaCl, H2O2, heat and cold).

Material and methods

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

Micro-organism and growth conditions

Cronobacter sakazakii ATCC 29544 used in this study was supplied by the Spanish Type Culture Collection (CECT Type strain 858). The bacterial culture was kept frozen at −80°C in cryovials. Micro-organisms were recovered from the cryovial by surface spreading onto Oh and Kang (OK) agar plates (Vitaltech Ibérica S.L., Madrid, Spain) and incubated for 24 h at 37°C. A broth subculture was prepared by inoculating a flask containing 10 ml of tryptone soya broth (Biolife, Milan, Italy) supplemented with 0·6% (w/v) yeast extract (Biolife) (TSBYE) with one of the colonies isolated as described above. After inoculation, the flask was incubated at 30°C overnight in a rotary shaker. Flasks containing 50 ml of TSBYE were inoculated with the overnight subculture to a concentration of 5 × 104 cells ml−1 and then incubated under agitation at 30°C for 24 h, which resulted in stationary-phase cultures with a final concentration of approx. 5 × 109 CFU ml−1.

Acid, alkaline, osmotic, oxidative, heat and cold shocks

Acid-, alkaline-, osmotic- and oxidative-stressed cells were prepared by pelleting 1 ml of a 24-h culture at 8000 g for 5 min and then resuspending it in 1 ml of TSBYE acidified to pH 4·5 with hydrogen chloride, 1 ml of TSBYE alkalinized to pH 9·0 with sodium hydroxide, 1 ml of TSBYE with 5% sodium chloride (w/v) or 1 ml of pH 7·0 100 mmol l−1 Tris–HCl buffer with the appropriate amount of 30% (v/v) H2O2 to obtain a concentration 0·5 mmol l−1, respectively, followed by incubation for 1 h at 30°C. Heat- and cold-stressed cells were prepared in a similar way, but the pelleted cells were resuspended in 1 ml of TSBYE and incubated for 1 h at 47·5°C or 4 h at 4°C, respectively. The preliminary data indicated that the number of cells remained constant after the adaptation times. After the shocks, cell suspensions were immediately used to study their resistance to heat, PEF, MS and UV-C treatments in pH 7·0 McIlvaine citrate-phosphate buffer.

Determination of heat resistance

Heat treatments were carried out in a specially designed resistometer described by 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 sample injection and extraction. Once the treatment temperature had attained stability (60 ± 0·2°C), 0·2 ml of an appropriately diluted microbial suspension was injected into the vessel containing the buffer. After inoculation, 0·1-ml samples were collected at different treatment times and immediately pour-plated.

Determination of PEF resistance

Cultures were centrifuged at 8000 g for 5 min and resuspended in the buffer whose concentration was adjusted to an electrical conductivity of 2 mS cm−1 with sterile distilled water. Then, 0·5 ml of the microbial suspension was placed into the treatment chamber with a sterile syringe and subjected to 5, 10, 25, 50 and 100 pulses. The treatment chamber consisted of a cylindrical plastic tube closed with two polished stainless steel electrodes. The distance between electrodes was 0·25 cm and the electrode area was 2·01 cm2. Exponential waveform pulses of 4 μs of duration at an electrical field strength of 25 kV cm−1 and a pulse repetition rate of 1 Hz were used in an apparatus previously described (García et al. 2005). The specific energy input of each pulse was 4·25 kJ kg−1. During treatments, the temperature inside the treatment chamber never exceeded 35°C.

Determination of MS resistance

The MS treatments were carried out in a specially designed resistometer (Raso et al. 1998). A 450 W Digital Sonifier® ultrasonic generator (Branson Ultrasonics Corp., Danbury, CT, USA) with a constant frequency of 20 kHz was used. The amplitude of the ultrasonic waves was 117 μm and the pressure applied during the treatment was 200 kPa. All treatments were carried out at a constant temperature (35 ± 0·2°C). Once the temperature, pressure and amplitude of the ultrasonic waves were stabilized, 0·2 ml of an appropriate dilution of the microbial suspension was injected into the 23-ml treatment vessel containing the buffer. After inoculation and at preset intervals, 0·1-ml samples were collected and directly pour-plated.

Determination of UV-C resistance

UV-C treatments were carried out in equipment previously described (Gayán et al. 2011). In brief, the equipment consists of eight individual annular thin-film flow-through reactors emitting at 254 nm (low-pressure UV lamp, TUV8WT5; Philips, Andover, MA, USA) and connected in series. In the annular gap (2·5 mm), a stainless steel coil spring was installed to improve the flow turbulence. The equipment includes a feed tank, a peristaltic pump, a heating/cooling coil exchanger and eight sampling valves placed at the outlet of each reactor. Ten millilitres of the microbial suspension was inoculated into 1 l of the buffer with 0·25 g l−1 of tartrazine (Sigma-Aldrich, St Louis, MO, USA) (absorption coefficient: α = 10·5 cm−1) and then pumped through the reactors. One-millilitre samples were withdrawn through the sampling valves and immediately pour-plated. The temperature during the treatment never exceeded 25°C.

Incubation of treated samples and survival counting

Samples were pour-plated into a nonselective media: tryptone soya agar (Biolife), supplemented with 0·6% (w/v) yeast extract (TSAYE), and incubated for 24 h at 35°C. To study the presence of sublethally injured cells, samples were also plated onto TSAYE with 5% (w/v) sodium chloride added (TSAYE-SC). This level of sodium chloride was previously determined to be the maximum noninhibitory concentration for untreated cells (data not shown). Samples recovered in selective media were incubated for 48 h. After incubation, CFUs were counted. The number of sublethally injured cells was estimated by subtracting the counts obtained in the nonselective (TSAYE) from the counts in the selective medium (TSAYE-SC).

Curve fitting and resistance parameters

Survival curves were obtained by plotting the log number of survivors vs the treatment time expressed in minutes for heat and MS, as the number of pulses for PEF, and as the dose (J ml−1) for UV-C treatments. A mathematical model based on the Weibull distribution was used to fit the curves (Mafart et al. 2002):

  • image

where S(t) is the survival fraction, t is the treatment time, and δ and ρ are the scale and shape parameters, respectively. The δ value represents the treatment time needed to inactivate the first log cycle of the population, and the ρ value reveals the profile of the curve (ρ < 1: concave upward curve; ρ = 1: linear curve; ρ > 1: concave downward curve). To fit the model to the experimental data and to calculate the δ and ρ parameters, the GraphPad prism® software (GraphPad Software, Inc., SanDiego, CA, USA) was used. Statistical analyses: t-tests were carried out with the same software and differences were considered significant for  0·05. All resistance determinations were performed at least in triplicate on independent working days. Error bars in the figures correspond to the standard deviation of the mean values.

Results

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

Prior to studying the possible development of cross-resistance to heat, PEF, MS and UV-C treatments of Csakazakii ATCC 29544 stationary growth-phase cells, the development of homologous resistance to heat, extreme pH, H2O2 and salt was explored to establish shock conditions. These cells were able to develop homologous resistance to heat and acid pH (data not shown), and therefore, the combination of temperature/time and pH/time inducing the maximum homologous resistance was used as shock conditions. For the remaining stresses that did not afford homologous resistance, the shock conditions used were the most intense not causing significant microbial inactivation (data not shown) and were in agreement with previous reports on other Gram-negative bacteria such as E. coli and Salmonella (Humphrey et al. 1991; Evrendilek and Zhang 2003; Bae and Lee 2010). Subsequent technological treatment conditions were selected from previous works (Arroyo et al. 2009, 2010, 2011a; Gayán et al. 2011) to attain at least 99·9% inactivation under suitable experimental conditions.

Figure 1 shows the profiles of the survival curves of C. sakazakii cells exposed to thermal (Fig. 1a), PEF (Fig. 1b), MS (Fig. 1c) and UV-C (Fig. 1d) treatments in pH 7·0 citrate-phosphate buffer. As the figure shows, the inactivation of C. sakazakii followed different profiles depending on the technology assayed: whereas the survival curves to heat and MS were log-linear, the PEF and UV-C survival curves showed deviations from linearity. The PEF survival curves displayed a tailing region and the UV-C curves showed a shoulder region. The estimation of the resistance parameters for the different survival curve profiles required the application of a flexible model capable of describing both nonlinear and linear survival curves. Table 1 comprises the mean values of the scale (δ) and shape (ρ) parameters from the fitting of the Mafart equation to the experimental data and, for comparison purposes, the time for a 3 log cycle reduction. Table 1 also includes the kinetic parameters for survival curves for heat, PEF, MS and UV-C treatments obtained after the exposure of C. sakazakii cells to acid, alkaline, osmotic, oxidative, heat and cold shocks. As shown in the table, depending on the combination of sublethal stress and technological treatment applied, an increase, decrease or no change in Csakazakii resistance was observed. Accordingly, the heat resistance of C. sakazakii increased significantly after exposure to a heat shock, and the same heat shock or an acid shock afforded cross-protection against PEF ( 0·05). By contrast, alkaline and osmotic shocks sensitized Csakazakii cells against a further heat challenge, and the alkaline and oxidative shocks decreased its tolerance to a UV-C treatment ( 0·05). The maximum adaptive response was observed when heat-shocked cells were subjected to a heat treatment (homologous response), increasing by 64% the time to inactivate 3 log cell cycles. The sensitization phenomena to heat and UV-C treatments implied reductions of 18–50% on the time to inactivate 3 log cell cycles. Interestingly, only the cold shock caused neither significant adaptation nor sensitization to any of the subsequent challenges (> 0·05).

image

Figure 1.  Different profiles of the survival curves of Cronobacter sakazakii ATCC 29544 stationary growth-phase cells subjected to (a) a thermal treatment at 60°C, (b) a PEF treatment (25 kV cm−1, 35°C), (c) a MS treatment (200 kPa, 117 μm, 35°C) and (d) a UV-C treatment (88·55 mW cm−2, 25°C) in pH 7·0 citrate-phosphate buffer. Data points represent the mean values of at least three independent replicates, and the error bars show the standard deviations. PEF, pulsed electric field; MS, Manosonication.

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Table 1. δ and ρ values and time for a 3-log cell reduction estimated from the fitting of the Mafart equation to the experimental data of Cronobacter sakazakii ATCC 29544 cells subjected to different sublethal stresses and then treated by heat, PEF, MS and UV-C treatments
ChallengeShockδ Value† mean (SD)ρ Value mean (SD)3-Log† mean (SD)RMSER2
  1. δ Value, scale parameter; ρ value, shape parameter (dimensionless); SD, standard deviation; RMSE, root mean square error; R2, determination coefficient; PEF, pulsed electric field; MS, Manosonication.

  2. *Values significantly different from their control value (t-test;  0·05).

  3. †Units: minutes for heat and MS, number of pulses for PEF, J ml−1 for UV-C treatments.

HeatControl0·75 (0·09)0·85 (0·09)2·74 (0·03)0·0820·99
Acid0·68 (0·28)0·73 (0·18)3·04 (0·74)0·2050·95
Alkaline0·35 (0·17)0·82 (0·22)1·36 (0·28)*0·1200·99
Osmotic0·42 (0·26)0·82 (0·25)1·57 (0·36)*0·1510·98
Oxidative0·73 (0·09)0·82 (0·09)2·84 (0·54)0·1810·96
Heat1·56 (0·46)1·03 (0·17)4·49 (0·52)*0·1580·97
Cold0·85 (0·17)1·06 (0·11)2·40 (0·21)0·1030·99
PEFControl4·57 (1·38)0·51 (0·08)39·02 (4·33)0·1630·99
Acid4·20 (2·50)0·41 (0·08)56·88 (6·62)*0·1470·99
Alkaline4·12 (1·28)0·52 (0·03)34·59 (9·18)0·1110·99
Osmotic2·63 (0·47)0·45 (0·001)30·01 (5·53)0·1160·99
Oxidative4·53 (1·28)0·56 (0·06)31·95 (5·34)0·1550·99
Heat4·95 (1·12)0·45 (0·05)56·45 (7·66)*0·2270·98
Cold4·84 (1·56)0·55 (0·11)37·71 (12·8)0·1510·99
MSControl0·41 (0·05)0·99 (0·05)1·24 (0·11)0·0750·99
Acid0·38 (0·05)0·92 (0·07)1·25 (0·06)0·1260·99
Alkaline0·36 (0·05)0·88 (0·05)1·26 (0·09)0·1130·99
Osmotic0·45 (0·002)1·01 (0·002)1·32 (0·01)0·0760·99
Oxidative0·37 (0·06)0·90 (0·13)1·26 (0·02)0·0940·99
Heat0·44 (0·08)1·00 (0·10)1·33 (0·08)0·0860·99
Cold0·39 (0·03)0·91 (0·09)1·32 (0·05)0·0960·99
UV-CControl6·47 (1·07)1·53 (0·23)13·3 (0·78)0·2140·99
Acid6·58 (0·96)1·70 (0·003)12·6 (1·83)0·1880·99
Alkaline5·55 (0·22)1·63 (0·03)10·9 (0·55)*0·2550·99
Osmotic6·13 (0·04)1·71 (0·14)11·7 (0·67)0·2530·99
Oxidative6·57 (0·13)2·17 (0·24)10·9 (0·40)*0·2730·99
Heat6·53 (0·82)1·64 (0·11)12·7 (1·03)0·1710·99
Cold7·26 (0·03)1·84 (0·18)13·3 (0·74)0·1740·99

When the influence of a previous stress on the resistance of Csakazakii cells to the different technologies is compared, it can be observed that the response of shocked cells to the four subsequent challenges differed. The MS resistance of Csakazakii was not significantly influenced by any of the sublethal shocks assayed (> 0·05). In addition, whereas the resistance to UV-C light of shocked cells was similar to or lower (for the alkaline- and oxidative-shocked cells) than that of nonshocked cells (control cells), the resistance of shocked cells to PEF was similar or higher (1·5-fold increase for the time to inactivate 3 log cell cycles when the cells were exposed to heat and acid shocks). Finally, the tolerance of shocked cells to heat showed the three alternatives: it remained constant after cold, acid and oxidative shocks; it was enhanced after the heat shock (adaptation); and it was worsened after alkaline and osmotic shocks (sensitization). It should be also noted that these different responses did not imply a significant modification to the profile of the survival curves, which in all cases kept their original shape (ρ values, Table 1).

Finally, the occurrence of sublethally injured cells was studied by recovering the treated samples after heat, PEF, MS and UV-C treatments in a selective medium (TSAYE-SC), and the proportion of sublethally damaged cells was estimated by comparing the counts in TSAYE and TSAYE-SC – the differential plating technique – (Mackey 2000). For control and shocked cells, survival counts in the selective medium (TSAYE-SC) after PEF, MS and UV-C treatments were the same as in the nonselective medium (TSAYE) (> 0·05) (data not shown), suggesting that neither the treatments alone nor the sublethal shocks induced the occurrence of sublethally injured cells. On the contrary, the differential plating technique allowed us to observe that the response of C. sakazakii cells to the subsequent heat challenge was much more diverse and complex. Figure 2 shows the survival curves of control and shocked cells after a heat treatment at 60°C and recovered in TSAYE and TSAYE-SC, and Table 2 includes the resistance parameters (δ and ρ values) and the time for 3 log cell reduction when control and shocked cells were heat-treated and recovered in TSAYE-SC. A simple comparison of the survival counts obtained in both recovery media demonstrated the occurrence of sublethal injury in C. sakazakii cells after heat treatments under all the conditions tested.

image

Figure 2.  Survival curves of Cronobacter sakazakii ATCC 29544 stationary growth-phase cells subjected to a heat treatment (60°C) after exposure to different sublethal shocks: (a) acid shock, (b) alkaline shock, (c) osmotic shock, (d) oxidative shock, (e) heat shock and (f) cold shock. Nonshocked or control cells (inline image, □) and shocked cells (bsl00072, inline image) were recovered in the nonselective medium TSAYE (inline image, bsl00072, continuous lines) and in the selective medium TSAYE-SC (□, inline image, discontinuous lines). Data points represent the mean values of at least three independent replicates, and the error bars show the standard deviations.

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Table 2. δ and ρ values and time for a 3-log inactivation estimated from the fitting of the Mafart equation to the experimental data of Cronobacter sakazakii ATCC 29544 cells subjected to different sublethal stresses and then treated by heat (60°C) and recovered in the selective media TSAYE-SC
Shockδ Value (min) mean (SD)ρ Value mean (SD)3-Log (min) mean (SD)RMSER2
  1. δ value, scale parameter; ρ value, shape parameter (dimensionless); SD, standard deviation; RMSE, root mean square error; R2, determination coefficient.

  2. *Values significantly different from their control value (t-test;  0·05).

Control0·48 (0·01)1·32 (0·12)1·10 (0·06)0·0550·99
Acid0·37 (0·01)1·16 (0·07)0·95 (0·07)0·0740·99
Alkaline0·17 (0·02)0·92 (0·14)0·56 (0·05)*0·1400·98
Osmotic0·36 (0·05)1·31 (0·01)0·83 (0·12)*0·1250·98
Oxidative0·23 (0·03)1·37 (0·01)0·52 (0·06)*0·0560·99
Heat0·53 (0·07)0·98 (0·06)1·61 (0·13)*0·1250·98
Cold0·28 (0·02)1·01 (0·21)0·81 (0·20)0·0710·99

As previously pointed out, whereas the heat shock enhanced survival to heat treatments, alkaline and osmotic shocks worsened it (Table 1). Moreover, heat shock also enhanced survival after a heat challenge when cells were recovered in the selective medium (Fig. 2e), but lower survival counts were found when alkaline- (Fig. 2b), osmotic- (Fig. 2c) and oxidative-shocked (Fig. 2d) cells were heated and recovered in the selective medium in comparison with control cells ( 0·05). Consequently, whereas the same trend was found regardless of the recovery medium – selective or nonselective – for heat-shocked (increase) and for alkaline- and osmotic-shocked cells (decrease), an oxidative shock only caused lower counts in the selective recovery medium. Finally, acid- and cold-shocked cells behaved the same as control cells against heat in both recovery media (> 0·05). On the other hand, the proportion of sublethally injured cells after heat treatments was higher for oxidative-shocked cells, lower for osmotic-shocked cells ( 0·05) and similar for the remaining shocks (> 0·05) when compared to the proportion of sublethally injured cells appearing after a heat treatment for control cells.

Discussion

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

In recent years, our research group has carried out an ambitious research project to characterize the resistance of Csakazakii to heat and nonthermal processes of food preservation such as PEF, MS, high hydrostatic pressure and UV-C light, aiming to describe the kinetics of inactivation of each technology and evaluate the resistance of this species under the influence of the main process parameters and environmental factors (Arroyo et al. 2009, 2010, 2011a,b). Nevertheless, it is well known that bacterial survival after these treatments may also be affected by preceding exposure to a sublethal stress. Therefore, the present study was designed to evaluate whether the most resistant Csakazakii ATCC 29544 cells to heat, PEF, MS and UV-C light treatments – those grown at 30°C for 24 h (Arroyo et al. 2009, 2010, 2011a) – were able to increase their thermal, PEF, MS and UV-C tolerance by cross-protection via temperature-, pH-, salt- and H2O2-stress conditions.

The results obtained in this study demonstrate that the influence of a prior stress on the resistance of Csakazakii to the subsequent challenges varies depending on the stressor and the technology studied. This fact, together with the different inactivation kinetics obtained when Csakazakii was treated by heat, PEF, MS and UV-C light (Fig. 1), indicates that there should be great diversity within the mechanisms of inactivation of these technologies.

Concerning heat treatments, the effect of the different prestresses on Csakazakii resistance differed. Whereas Csakazakii thermotolerance remained constant after acid, oxidative and cold shocks, it was enhanced after the heat shock (homologous resistance) and worsened after the alkaline and osmotic shocks. The development of resistance to heat after a heat shock is a widely observed phenomenon (Mackey and Derrick 1987; Linton et al. 1990; Pagán et al. 1997; Jørgensen et al. 1999; Silva-Laport et al. 2003; De Angelis et al. 2004; Cebrián et al. 2009) that has been associated with the synthesis of heat shock proteins (HSPs) (Lindquist 1986). The acquisition of thermotolerance after exposure to heat shock has also been observed for this species: Chang et al. (2009) observed that the highest homologous response for C. sakazakii BCRC 13988 cells was achieved after an exposure of 15 min at 47°C, such that the D51 value was increased 2·5-fold. This increase in thermotolerance is higher than the increase reported here (1·6 times), but it is still lower than other magnitudes reported (Pagán et al. 1997; Cebrián et al. 2009). In addition, Arku et al. (2011) observed greater survival at 52°C in Cronobacter spp. pretreated at 46°C for 30 min, which they attributed to a more rigid membrane in adapted cells owing to a decreased ratio of unsaturated to saturated membrane fatty acids. Conversely, Shaker et al. (2008) commented that a heat stress (55°C, 5 min) reduced by 50% the thermal resistance of Cronobacter spp. in milk. The authors attributed this sensitization to the severity of the shock conditions prior to the heat challenge. In addition, our study indicated that the adaptive response to heat did not involve a prolonged shoulder in the profile of the survival curves, contrary to what has been commonly observed with other bacteria (Mackey and Derrick 1987; Pagán et al. 1997; Juneja et al. 1998; Cebrián et al. 2009). This fact permits the continued use of the linear model, which eases the estimation of microbial inactivation even when the micro-organisms have been subjected to prior stresses. On the other hand, it is generally acknowledged that thermotolerance can also be induced by stresses other than heat, such as starvation and acid shock (Wesche et al. 2009). However, our results indicate that exposure to any of the stressing conditions tested did not result in the development of resistance to heat. It is remarkable that, in spite of the development of homologous resistance to acid pH observed in this study (data not shown), acid shock did not confer cross-protection against a subsequent heat challenge; especially, because this cross-protection has been previously reported for various micro-organisms such as Salmonella Typhimurium, E. coli, L. monocytogenes and Staph. aureus (Farber and Pagotto 1992; Leyer and Johnson 1993; Duffy et al. 2000; Cebrián et al. 2010). The thermal sensitization of Csakazakii caused by the alkaline and osmotic shocks and the absence of response observed after hydrogen peroxide shocks was also somewhat unexpected as these stresses proven to be protective for many species (Flahaut et al. 1997; Taormina and Beuchat 2001; Cebrián et al. 2009; Bae and Lee 2010) in similar conditions. Nevertheless, some exceptions have also been observed (Bae and Lee 2010). Our results demonstrated that cold shock did not afford cross-protection against heat, which is in agreement with observations of Shaker et al. (2008). This seems to indicate a characteristic behaviour of Csakazakii because most authors have found enhanced thermotolerance (Jackson et al. 1996; Evrendilek and Zhang 2003) or sensitization (Miller et al. 2000) of other species after a cold shock.

All these data suggest that the effect of a previous shock in bacterial thermotolerance will depend strongly on the micro-organism studied. As the development of cross-resistance responses in bacteria has been generally associated with the activation/development of common resistance responses such as the induction of rpoS – for Gram-negative bacteria – (Hengge 2011) and the synthesis of HSPs (Taglicht et al. 1987; Foster 1993), further work should be carried out to determine whether the stresses applied can induce similar responses in C. sakazakii or not, which might help to explain the results reported here. On the other hand, it is well known that other factors, such as the physiological state of cells, can also influence these phenomena. Thus, it can be hypothesized that the absence of the development of cross-resistance responses to heat as well as the limited ability to develop homologous resistance responses – only heat and acid stresses – might be owing to the fact that stationary growth-phase cells were used in this study. It is well known that the physiological changes that happen as a consequence of the entry into the stationary growth phase usually overlap – at least partially – with those induced by all the stressing agents studied in this work (Hengge 2011). Further work should also be carried out to verify this hypothesis.

Much less is known about the development of resistance responses to nonthermal technologies. In the present study, PEF was the only technology to which cross-protection was achieved (after pre-exposure to heat and acid shocks). Both heterologous responses increased the time to achieve the 3 log reduction by approx. 1·5 (Table 1). Similarly, higher PEF tolerance was also observed for E. coli after exposure to an acid shock at pH 3·6 – but not at pH 5·2 (Evrendilek and Zhang 2003) – and with Staph. aureus pre-exposed to heat (Cebrián et al. 2011). Contrary to Csakazakii, enhanced PEF tolerance has been also observed after a cold (Evrendilek and Zhang 2003; Somolinos et al. 2008) and alkaline shocks (Cebrián et al. 2011) with other species.

No shock exerted any kind of response in Csakazakii cells subjected to MS treatments. As it is generally acknowledged that MS causes cell death by the mechanical disruption of cell envelopes and that this phenomenon has proven to be mostly independent of any kind of environmental factor (Raso et al. 1998; Arroyo et al. 2010), it is not surprising that the inactivation of Csakazakii by MS was independent of any previous stress condition. Similarly, Pagán et al. (1999) did not find a cross-protective effect on the MS tolerance of Lmonocytogenes after a heat shock. No previous studies were found on the effect of other sublethal stresses on microbial resistance to ultrasound.

Whereas both alkaline and oxidative shocks sensitized Csakazakii cells to UV-C radiation at a similar magnitude (twofold), the remaining shocks did not influence its UV-C tolerance. Several studies indicate that the killing of Ecoli cells exposed to H2O2 is due mainly to damage to DNA (Imlay and Linn 1987), and DNA is also the key target of the UV-C light inactivating effect (Bintsis et al. 2000). It can be hypothesized that the sensitization of Csakazakii cells to UV-C light may be caused by the induction of DNA damage by H2O2. However, Asad et al. (2000) found that a pretreatment with 2·5 mmol l−1 H2O2 protected mid-exponential growth-phase Ecoli cells against UV-C light. Further work should be carried out to determine whether this discrepancy is owing to the existence of relevant physiological differences in the response to hydrogen peroxide between the species studied, to the different methodologies used (mid-exponential vs stationary growth-phase cells) or to the different pretreatment/treatment intensities and equipment. It is also remarkable that, conversely to what it has been observed in this work, acid-stressed Lmonocytogenes cells were reported to be more resistant to UV-C light than unstressed cells were (McKinney et al. 2009). No previous studies were found addressing the effect of alkaline pre-exposure on microbial sensitivity to UV-C light.

The occurrence of sublethally injured cells after the treatments allowed us to study in depth the response of Csakazakii to the different stressing environmental conditions. To the best of our knowledge, this is the first time that the occurrence of sublethally injured cells after heat, PEF, MS and UV treatments has been studied in cells pre-exposed to different stressors. The results obtained indicate that, as for control cells, no sublethally injured cells were detected after PEF, MS and UV-C treatments. This suggests that neither the increase in resistance to PEF observed after heat and acid shocks nor the decrease in resistance to UV-C after exposing Csakazakii cells to H2O2 and alkaline pH was related either with the appearance of sublethally injured cells or with a change in the ability of cells to repair the resulting damage. On the other hand, cells subjected to heat showed sublethal injuries in all the conditions assayed. The results indicate that, although heat shock enhanced survival and alkaline shock diminished it, they did not modify the proportion of sublethally injured cells, suggesting that the different resistance of these cells might be related to an increase or decrease, respectively, in the stability of the structures that determine the heat resistance of C. sakazakii. By contrast, the osmotic shock resulted not only in a decreased resistance when cells were recovered in both media but also in a decreased proportion of sublethally injured cells, suggesting that this shock might affect both the heat stability and the ability to repair themselves. Finally, it is surprising that a higher proportion of sublethally injured cells was found for oxidative-shocked cells, although survival in nonselective media was not modified. This would imply that oxidative shock induced different changes in C. sakazakii physiology with opposite consequences on its heat resistance. As the inhibition of any repair process would also imply an increase in the efficacy of the heat treatments, our results indicate that this strategy might be successfully used to increase the lethality of heat treatments on C. sakazakii cells both for stressed cells, especially oxidative-shocked cells, and control cells.

The results reported here show the great complexity of adaptive responses and variety in the effects – protection, sensitization or no effect – that they can induce depending on the stress applied and the technology studied. Our results indicate that the resistance of the C. sakazakii strain assayed (CECT 858, ATCC type strain 29544) stationary growth-phase cells to MS and UV-C will be affected little or not at all by its previous exposure to sublethal stressing conditions and that heat and acid shocks will lead to an increase in its PEF resistance. Furthermore, its tolerance to heat can be either increased or decreased depending on the shock applied. It is also important to conclude that only those conditions that induced homologous adaptation (heat and acid shocks) caused cross-protection, and only against PEF. From a practical point of view, our results indicate that stressing conditions such as heat and acid pH should be avoided before heat and PEF treatments. Moreover, further work should be carried out to test whether the behaviour reported here is representative for other strains of the same species.

Acknowledgements

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

This study was supported by MICINN-FEDER (project: CIT-020000-2009-40). Authors acknowledge Departamento de Ciencia, Tecnología y Universidad (Gobierno de Aragón, Spain) for providing C. Arroyo with a doctoral grant. Authors also wish to thank Fondo Social Europeo.

References

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