Relationship between membrane permeabilization and sensitization of S. aureus to sodium chloride upon exposure to Pulsed Electric Fields

Innovative Food Science and Emerging Technologies 32 (2015) 91–100

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Relationship between membrane permeabilization and sensitization of S. aureus to sodium chloride upon exposure to Pulsed Electric Fields G. Cebrián ⁎, P. Mañas, S. Condón Tecnología de los Alimentos, Facultad de Veterinaria de Zaragoza, Universidad de Zaragoza, C/ Miguel Servet, 177, 50013 Zaragoza, Spain

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Article history: Received 16 July 2015 Received in revised form 22 September 2015 Accepted 23 September 2015 Available online 8 October 2015 Keywords: Pulsed Electric Fields Bacteria Electroporation Fluorescence microscopy K+ release NaCl

a b s t r a c t The suitability of NaCl-added selective media for the quantification of electroporated bacterial cells was evaluated using Staphylococcus aureus as a model microorganism. A very good correspondence was found between the percentage of cells unable to outgrow in media added with the Maximum Non Inhibitory Concentration of NaCl and the percentage of electropermeabilized cells, as determined by propidium iodide staining and K+ release measurements. The relationship between the percentage of electropermeabilized cells and cells sensitized to NaCl was confirmed for different experimental conditions, such as cells in different growth phase, grown at different temperatures, or treated at different pHs and aw. Among all the factors studied, only when cells were alkalineshocked or treated at 10 °C, lack of correspondence between the percentage of electropermeabilized cells and those unable to outgrow in NaCl added media was found. Finally, the good correspondence between the number of electropermeabilized cells and those inhibited in NaCl-added media was also confirmed for other three bacterial species. Industrial relevance: Recovery in NaCl added media can be used for determining the number of electroporated bacterial cells generated by PEF treatments. This will help in Pulsed Electric Fields process optimization and PEF-based combined processes design. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Pulsed Electric Fields (PEF) technology, which consists of the application of short duration (1–100 μs) high electric field pulses (10– 50 kV/cm) to a food placed between two electrodes (Heinz, Álvarez, Angersbach, & Knorr, 2001), is one of the most promising new technologies for food preservation since it is capable of inactivating microorganisms while causing little changes in food sensory and nutritional quality (Raso & Barbosa-Cánovas, 2003). It is generally acknowledged that the main targets of PEF are the bacterial envelopes (Mañas & Pagán, 2005). Thus, application of PEF would cause temporary or permanent permeabilization of cell membranes, a phenomenon called “electropermeabilization” or “electroporation”. Membrane electroporation has been widely investigated and several theories (Barbosa-Cánovas, Góngora, Pothakamury, & Swanson, 1999; Ho & Mittal, 1996; Kinosita et al., 1992; Pavlin, Leben, & Miklavcic, 2007; Tsong, 1991; Weaver & Chizmadzhev, 1996; Zimmermann, 1986) have been proposed to explain this complex phenomenon. Nevertheless, so far there is no clear evidence on the underlying mechanism of bacterial membrane permeabilization at the molecular level (Pagán & Mañas, 2006). Moreover, it is not clear whether permeabilization of the ⁎ Corresponding author at: Tecnología de los Alimentos, Facultad de Veterinaria, C/ Miguel Servet, 177, 50013 Zaragoza, Spain. Tel.: +34 976761581; fax: +34 976761590. E-mail address: [email protected] (G. Cebrián).

http://dx.doi.org/10.1016/j.ifset.2015.09.017 1466-8564/© 2015 Elsevier Ltd. All rights reserved.

membrane leads to permanent damage. This is an aspect of the most relevance, since to obtain the maximum benefit of PEF technology its mode of action on bacterial cells should be better understood. Recent studies have demonstrated that, at least under some conditions, permeabilization of bacterial envelopes does not always result in microbial inactivation, in other words, PEF inactivation would not be an “all-or-nothing” event. After PEF treatments a certain proportion of cells with permeabilized and/or damaged membranes would be capable of resealing their envelopes and outgrow, given the appropriate recovery conditions are provided (García, Gómez, Condón, Raso, & Pagán, 2003). Conversely, if the environmental conditions are not optimal, cells with damaged membranes could not trigger effective repair processes, and would not survive (García, Mañas, Gómez, Raso, & Pagán, 2006). From the practical side, these cells with damages in their membranes, which are in turn sublethally injured cells, can be efficiently inactivated if an adequately selected additional preservation barrier is used in combination (García, Hassani, Mañas, Condón, & Pagán, 2005). One of the most frequently used methods for the detection of sublethally injured bacterial cells is the so-called differential plating technique. Basically, this technique consists in recovering the stressed cells in two media, one of them is the non-selective medium, in which cells are capable of repairing the sublethal injuries suffered and the second one is the selective medium, which prevents the repair of these damages (Mackey, 2000). Although there are many agents that can be

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used to formulate a selective media, probably sodium chloride is the more frequently used agent for this purpose. Besides its simplicity, the main reason for the widespread use of sodium chloride added media for the detection of sublethally damaged cells is its high sensitivity since this technique allows detecting these cells within populations in which various log cycles of the cellular population have been inactivated (Pagán & Mañas, 2006). This is fairly important because the last surviving cells are of the most relevance to food preservation since a few surviving cells, proven the environmental conditions are adequate, can multiply. However, this technique has also some disadvantages. On the one hand, it is an indirect way of evaluating cellular damage, i.e. it is not possible to directly visualize the damages, and, on the other hand, the mechanism of action of sodium chloride still remains to be elucidated, which hampers the interpretation of the results obtained. Although, as pointed out above, the mechanism of action of sodium chloride is still unknown, it is believed that cells with damaged envelopes, i.e. with impaired homeostatic and/or barrier functions associated with cell envelopes, would not be able to repair their injuries being this the cause for their inability to outgrow in sodium chloride added media (Mackey, 2000). Therefore, this technique would allow identifying and quantifying cells with damaged envelopes, which are regarded as the main targets of PEF treatments (Mañas & Pagán, 2005; Pagán & Mañas, 2006). Various investigators have already used this technique in order to estimate the percentage of sublethally injured cells after PEF treatments (Cebrián et al., 2009; García et al., 2003) but, to date, the mechanisms leading to sodium chloride sensitization after microbial exposure to PEF are far from being completely understood. The aim of this work was to evaluate if selective media based on the addition of NaCl could be used as a tool for determining the number of electroporated bacterial cells. For this purpose Staphylococcus aureus was used as a model microorganism. Thus, the percentage of electroporated cells (determined by propidium iodide staining and K+ release measurements) was compared to the percentage of cells sensitized to NaCl. The influence of factors acting before and during the treatment on this relationship was also evaluated. The usefulness of the method was also checked for other three bacterial species (Cronobacter sakazakii, Escherichia coli and Listeria monocytogenes). Finally, the relationship between the occurrence of electroporation, sensitization to NaCl and cell survival and recovery was analyzed in order to gain insight into the physiology of cell inactivation by PEF. 2. Materials and methods 2.1. Strains and growth conditions The strains used in this study were S. aureus (CECT 4459), C. sakazakii (CECT 858), and L. monocytogenes (CECT 932), and E. coli strain W3110 (ATCC 27325). Bacterial cultures were maintained frozen at −80 °C in cryovials. Pre-cultures were prepared by inoculating 10 mL of tryptone soya broth (Biolife, Milan, Italy) supplemented with 0.6% (w/v) yeast extract (Biolife) (TSBYE) with a loopful of growth from a tryptone soy agar supplemented with 0.6% (w/v) yeast extract (Biolife) (TSAYE) plate. This pre-culture was incubated at 37 °C for 12 h, in a shaking incubator. 50 μL of this pre-culture was inoculated into 50 mL of TSB and incubated until cultures reached stationary-phase. Unless specifically stated, stationary-phase cells were obtained after growth in TSBYE at 37 °C. Before PEF treatments cells were centrifuged and resuspended in McIlvaine citrate phosphate buffer of pH 7.0 (Dawson, Elliot, Elliot, & Jones, 1974) whose conductivity was adjusted with distilled water to 2 mS cm−1. 2.2. Cells treated and/or obtained under non-standard conditions The influence of the following environmental factors on the relationship between the number of cells recovered in NaCl and the number of

electroporated cells was studied: a) growth phase (exponential vs stationary), b) growth temperature (10 vs 37 °C), c) previous exposure to sublethal shocks (heat, oxidative, alkaline, acid, cold and osmotic shock), d) treatment medium pH (pH 4.0 vs pH 7.0), e) treatment medium aw (N0.99 vs 0.96) and, f) treatment temperature (10 vs 30 °C). For these experiments cells were also cultivated in TSBYE at 37 °C until they reached a concentration of approximately 108 cells mL−1 in order to obtain exponentially growing cells (a); grown at 10 °C until they reached stationary growth phase (b); exposed to different sublethal shocks: acid shock at pH 4.5 (adjusted with hydrochloric acid, Panreac S. A., Barcelona, Spain); alkaline shock at pH 9.5 (adjusted with sodium hydroxide, Panreac); oxidative shock with 50 μM hydrogen peroxide (Sigma, S. Louis, USA); osmotic shock with 10% (w/v) NaCl (Panreac) (aw = 0.94); heat shock at 45 °C and cold shock at 4 °C. During acid, alkaline, osmotic and hydrogen peroxide shocks the temperature of the cell suspension was kept at 25 °C. S. aureus cultures were centrifuged (6000 ×g for 5 min) resuspended in the same volume of TSBYE in the presence of the stress factor and then kept for 2 h. (Cebrián, Raso, Condón, & Mañas, 2012) (c); treated in McIlvaine buffer of pH 4.0 (aw N 0.99) or of aw = 0.96 (pH 7.0) (Arroyo, Cebrián, Pagán, & Condón, 2010 (d and e, respectively); or treated at different temperatures as described in Saldaña et al. (2010) (f). 2.3. PEF treatments PEF treatments were carried out in an exponential waveform pulse equipment (Cebrián et al., 2009). High electric field pulses (Fig. 1) were produced by discharging a set of 10 capacitors via a thyristor switch (Behlke, Kronberg, Germany) in a batch treatment chamber. The capacitors were charged using a high voltage dc power supply (FUG, Rosenhein, Germany), and a function generator (Tektronix, Wilsonville, OR, USA) delivered the on-time signal to the switch. Treatments were carried out in a tempered batch parallel-electrode treatment chamber (described in Saldaña et al., 2010). The gap between electrodes was 0.25 cm and the electrode area was 2.01 cm2. The actual electric field strength and electrical intensity applied were measured in the treatment chamber with a high voltage probe and a current probe respectively connected to an oscilloscope (Tektronix). The energy associated with pulses at electric field strengths of 18, 22, 26 and 30 kV cm−1 was 2.24, 3.47, 4.25, and 5.83 kJ kg−1, respectively. Treatments were carried out at 30 °C unless specifically indicated. The microbial suspension, at a concentration of approximately 108 microorganisms mL−1, was placed into the treatment chamber with a sterile syringe and treatments were carried out. After treatment, appropriate serial dilutions were prepared in sterile TSBYE and pour plated. 2.4. Incubation of treated samples and survival counting The recovery medium used was TSAYE with or without different concentrations of NaCl added. After adequately diluting in TSBYE, 0.1 mL samples were pour-plated and then incubated for 24 h at 37 °C, unless NaCl was added. In such case incubation was extended to 3– 4 days and up to 21 days for plates with the highest concentrations. After incubation, colony forming units (CFU) were counted. All resistance determinations were performed at least three times in independent working days. Error bars in figures correspond to the mean standard deviation. 2.5. Assessment of the percentage of cells with permeabilized membranes by PI staining The fluorescent dye propidium iodide (PI; Sigma-Aldrich, 287075) was used to evaluate cell membrane permeabilization by PEF treatments. PI is a fluorescent probe that is commonly used as a marker for membrane permeabilization since membranes of healthy cells prevent its entry inside the cell, where it bounds to nucleic acids rendering a

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strong red fluorescence. A stock solution of 1 mg PI in 1 mL of water was prepared. Samples of cell suspensions were centrifuged (6000 × g for 5 min), resuspended in McIlvaine citrate-phosphate buffer of pH 7.0 at a concentration of approximately 108 microorganisms mL− 1 and mixed with PI solution to a final concentration of 1.5 μM before PEF treatments. After the treatments, cells were incubated for 10 min at room temperature. Previous experiments showed that the presence of PI in the treatment medium did not modify treatment conditions or microbial PEF resistance (data not shown). The percentage of permeabilized cells was determined by microscopic examination using a Nikon Eclipse E4000 microscope (Nippon Kogaku KK, Japan) equipped with phase-contrast optics and an epifluorescence unit. In all cases, a × 100 objective was used with immersion oil, giving a total magnification of × 1000. Cell counts were made on at least five microscopic fields with high cellular concentration (more than 50 cells). The percentage of permeabilized cells was calculated by comparing the total number of cells, determined by using phase-contrast optics, with the number of cells showing fluorescence. The data were normalized by subtracting the percentage of untreated cells showing fluorescence, which was always lower than 2%. The normalized data were plotted as percentages of PI stained cells after the different PEF treatments. It has to be noted that in this investigation PI was added to the bacterial suspension before the PEF treatment was carried out, thus, the percentage of fluorescent cells corresponds to the percentage of cells permeabilized during the treatment. Membrane permeabilization determinations were performed at least three times in independent working days. Error bars in figures correspond to the mean standard deviation. 2.6. Quantification of K+ release from treated cells Samples of cell suspensions were centrifuged (6000 ×g for 5 min), resuspended in McIlvaine citrate-phosphate buffer of pH 7.0 at a concentration of approximately 109 microorganisms mL−1 and PEF treated. After PEF treatments, suspensions were incubated for 5 min at room temperature and then centrifuged. It was checked that further incubation times did not increase the amount of K+ released (data not shown). Cell pellets were discarded and supernatants were filtered through a 0.22-μm syringe filter unit (Millex, USA). After digestion with HNO3 (final concentration of 2% v/v) (Panreac) for 24 h, the amount of K+ released was measured with an ICP (inductively coupled plasma) mass spectrometer (ELAN 6000, Perkin-Elmer, Germany)

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(Laborda, Medrano, & Castillo, 2004). Quantification was performed by specific calibration with K+ standards injected through the chromatographic system, in the same way that samples were measured. The percentage of K+ release was determined as described in Saulis, Satkauskas, and Praneviciute (2007) and expressed as % of cells permeable to K+. Permeabilization to K+ of 100% of cells was achieved by treating the cell suspension with 50 pulses at 26 kV cm−1, a treatment of higher intensity than that leading to electropermeabilization of 100% of cells to PI. 2.7. Recovery of PEF treated cells in liquid media (TSBYE and TSBYE + NaCl) After PEF treatments, cells were diluted (1/10) in TSBYE or TSBYE + 2.39 M NaCl and incubated at room temperature. Samples were collected at preset times and plated onto TSAYE and TSAYE +2.39 M NaCl in order to determine the rate of recovery of tolerance to NaCl. At the same preset times, cells were centrifuged (6000 ×g for 5 min), resuspended in the same volume of citrate-phosphate buffer of pH 7.0 and the percentage of permeabilized cells to PI was evaluated as described above. Experiments were performed at least in triplicate on independent working days. 2.8. Statistical analysis Pearson's correlation coefficient (r), and the slope (s) and intersection (Sy/x) of the regression equations were calculated with an appropriate statistical package (Prism; GraphPad Software, Inc., San Diego, CA). Standard deviations and Bland–Altman plots -scatter plots in which the difference between the paired measurements (A–B) is plotted against their mean value ([A + B] / 2)- were also calculated/obtained with the same package. 3. Results 3.1. Relationship between the number of electroporated cells and the number of cells recovered in NaCl-added media Fig. 2 shows the influence of the addition of different concentrations of NaCl to the recovery medium on the percentage of S. aureus cells recovered after a treatment of 5 pulses at 26 kV cm−1. This treatment was chosen because it led to the permeabilization to PI of ≈ 90% of cells, while resulting in less than a 25% of cell inactivation, measured as the

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percentage of cells unable to grow in non-selective medium (TSAYE). For comparative purposes the influence of the addition of NaCl to the recovery media on the percentage of non-treated S. aureus cells is also shown in the graph. As it can be observed in the figure, addition of NaCl concentrations above 0.85 M resulted in a progressive decrease in the percentage of cells recovered as compared to those recovered in TSAYE without the addition of NaCl. At concentrations of NaCl close to the MNIC (Maximum Non-Inhibitory Concentration; which is defined as the concentration leading to less than a 20% decrease in the number of native cells recovered as compared to those recovered in TSAYE), the percentage of PEF treated cells unable to grow in NaCl added medium was very similar to the percentage of cells permeabilized to propidium iodide (≈90%). Above this concentration (2.39 M), the percentage of cells recovered continued decreasing, but it decreased for the two types of cells: PEF treated and untreated. Similar results were obtained for other treatments assayed, which were also designed to permeabilize ≈90% of cells (data not shown). Thus, 2.39 M was chosen as the adequate concentration of NaCl for studying the relationship between the number of cells recovered in NaCl-added media and the number of electroporated cells. Fig. 3A shows the relationship between the percentage of electroporated cells, determined by PI staining, and the percentage of cells unable to outgrow in media with NaCl added (2.39 M) after PEF treatment. The figure includes data obtained under a wide range of treatment intensities (18–30 kV cm− 1 and 5–100 pulses; more than 80 different experiments). As it can be observed, a very good correlation was found between the percentage of cells permeabilized to PI by PEF and the percentage of cells unable to outgrow in media with NaCl added. A r value of 0.981, a slope of 0.970 and an intersection point of 0.390 were calculated. Furthermore, the Bland–Altman plot shown in Fig. 3B, demonstrates that recovery of S. aureus PEF cells in media with the MNIC of NaCl added is a method comparable to PI staining for evaluating the number of electroporated cells. Recently it has also been proposed that the measurement of K+ exit might be used as a way to determine the extent of cellular electroporation (Saulis et al., 2007). The relationship between the percentage of cells permeabilized to K+ and the percentage of cells unable to outgrow in NaCl added media is illustrated in Fig. 4A. As described for PI staining, results obtained (r = 0.967; slope = 0.968; intersection = 0.483; more than 60 different experiments) also demonstrate that a very good

correspondence exists between the percentage of K+ exit during PEF treatments and the percentage of cells unable to outgrow in media with NaCl added (2.39 M). Therefore it can be concluded that recovery of S. aureus PEF cells in media with the MNIC of NaCl added is also comparable to K+ release measurement as a method for evaluating the number of electroporated cells (Fig. 4B). The relationship between the number of electroporated and sensitized to NaCl cells was also studied after obtaining and/or treating S. aureus cells under non-standard conditions (see Section 2). Results obtained showed that, for S. aureus cells, recovery in NaCl added media would be an accurate way for enumerating the number of electropermeabilized cells regardless of the pH (7.0 vs. 4.0) and aw (N0.99 vs. 0.96) of the treatment media, the growth phase and temperature at which cells were obtained (exponential vs. stationary and 10 vs. 37 °C), or whether they had been previously exposed to heat, acid, osmotic, oxidative or cold shocks or not (Fig. 5A). Only for cells that had been previously exposed to an alkaline shock and when cells were treated at low temperatures (10 °C), the percentage of cells unable to grow in NaCl added medium was significantly lower than that of cells permeabilized to PI (Fig. 5B). Details about the lethality of PEF treatments on S. aureus shocked cells can be found somewhere else (Cebrián et al., 2012). 3.2. Recovery of PEF treated cells in liquid media with and without NaCl The kinetics of NaCl-tolerance recovery (the ability to outgrow in media with 2.39 M of NaCl added) and membrane recovery (recovery of membrane impermeability to PI) were studied by incubating PEFtreated cells (5 pulses; 26 kV cm− 1) in an optimum liquid medium (TSBYE) (Fig. 6A) and in TSBYE with NaCl added (2.39 M) (Fig. 6B). As it can be observed in Fig. 6A, when cells were placed into TSBYE, they were able to regain their tolerance to the presence of NaCl in the recovery agar in 60 min, approximately. In parallel, S. aureus PEF treated cells recovered their impermeability to PI following a similar kinetics. On the contrary, when PEF treated S. aureus cells were incubated in liquid medium with NaCl 2.39 M added, they progressively died, indicating that sequential exposure to PEF plus unfavorable conditions (NaCl) resulted in cell inactivation. Unexpectedly, cells regained simultaneously the impermeability to PI of their membranes, up to 70–90% of the population. These results would indicate that the presence of NaCl

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would provoke the inactivation of the fraction of the population that had been electroporated during the treatment, but would not hamper the process of membrane resealing to PI in S. aureus cells. 3.3. Relationship between the number of electroporated cells and the number of cells recovered in NaCl-added media for microorganisms other than S. aureus The relationship between the number of PI-permeabilized cells and the number of cells sensitized to NaCl was also studied with L. monocytogenes, C. sakazakii and E. coli. A treatment designed to induce the permeabilization to PI of approximately 90% of the population was applied to the different microbial suspensions, and the percentage of L. monocytogenes, C. sakazakii and E. coli recovered cells, as a function

of the amount of NaCl added to the recovery medium was studied. For the three microorganisms a similar behavior to that described for S. aureus (Fig. 2) was found, i.e., increasing NaCl concentration in the recovery medium over a particular threshold resulted in a decrease in the percentage of recovered cells as compared to those recovered in TSAYE, and at concentrations close to the MNIC this percentage was very similar to that of cells stained with PI (data not shown). Then, the percentage of cells recovered in TSAYE + NaCl (at a concentration corresponding to the MNICs for each microorganism) was compared to the percentage of cell stained with PI determined by fluorescence microscopy. Results obtained are shown in Fig. 7A which includes data obtained for stationary growth phase cells of the three microorganisms treated in McIlvaine buffer of pH 7.0 under a variety of treatment intensities (18–30 kV cm−1; 5–100 pulses; 30 °C).

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As described for S. aureus, the results obtained (r = 0.963; slope = 0.957; intersection = 0.940; Fig. 7B) indicate that for E. coli and C. sakazakii and L. monocytogenes NaCl plating at the MNIC concentration would be a good method for enumerating the number of electroporated cells, at least under our standard treatment conditions. 4. Discussion In this work we have studied the relationship between membrane permeabilization during PEF treatments and sensitization to NaCl, using S. aureus as a model microorganism. The results obtained allow us to propose the use of NaCl added media as a tool to determine the number of electroporated bacterial cells. The results also contribute to

obtain a deeper knowledge about some relevant physiological aspects involved in bacterial survival to PEF treatment and sensitization to NaCl. Various techniques have been proposed in order to quantify the number of electroporated cells including fluorescence microscopy, fluorescence spectrometry, flow cytometry and the measurement of the entry/release of different compounds such as bleomycin or K + ions (Wouters, Alvarez, & Raso, 2001; Ulmer, Heinz, Gänzle, Knorr, & Vogel, 2002; Unal, Yousef, & Dunne, 2002; Pagán and Mañas, 2006; Saulis et al., 2007). As described in Section 1, the use of NaCl-added media has also been suggested to determine the percentage of cells with injured membranes (Cebrián et al., 2009; García et al., 2003). Given the fact that membranes are the main targets of PEF treatments and that the action of NaCl resides on its ability to prevent the outgrowth of

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Time (hours) 50 Fig. 6. Influence of recovery time on the percentage of cells recovered in TSAYE (■) and TSAYE + NaCl (2.39 M) (●) and on the percentage of cells impermeable to PI (△; discontinuous lines) when incubated at 25 °C in TSBYE (A) and TSBYE + NaCl (2.39 M) (B). Values represented as time 0 correspond to the percentages of recovered and nonstained cells immediately after a PEF treatment of 5 pulses at 26 kV cm−1 (30 °C; pH 7.0). Error bars correspond to the standard deviation of the means.

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% cells permeabilized to PI Fig. 5. A) Relationship between the percentage of S. aureus cells unable to grow in NaCl (2.39 M) added medium and the percentage of cells permeabilized to propidium iodide after different PEF treatments (18–30 kV cm−1; 5–100 pulses). The figure includes results obtained under non-standard conditions except for those obtained for alkaline shocked cells and for cells treated at 10 °C. B) Relationship between the percentage of S. aureus cells unable to grow in NaCl (2.39 M) added medium and the percentage of cells permeabilized to propidium iodide after different PEF treatments (18–30 kV cm−1; 5–100 pulses; pH 7.0). The figure includes results obtained for alkaline shocked (pH 9.5; 2 h) stationary growth phase cells (▲) and for stationary growth phase cells treated at 10 °C (□).

cells with injured membranes (Mackey, 2000) this technique appears, at least theoretically, as a feasible approach to detect and quantify the number of electroporated bacterial cells. However, to the knowledge of the authors, no specifically designed study to evaluate its appropriateness for this purpose has been carried out to date. Fluorescence microscopy constitutes one of the more widespread and used methods for enumerating the number of electropermeabilized

cells. It is simple to carry out, not very cost expensive, and results can be obtained within minutes (Davey, 2011). Conversely, this method is quite time-consuming and labor-intensive. The relative accuracy of this technique (measured as the estimated standard deviation for the results of three replicate samples) has been reported to be above 10% (Wang, Hammes, De Roy, Verstraete, & Boon, 2010), in our case 11.4%, and it allows calculating the proportion of electroporated cells in the range between 0 and 99%. Imaging techniques (also including fluorescence spectrometry -which is much less time consuming and labor intensive, but that does not provide information about the precise number of electroporated cells- and flow cytometry -that stands out for its accuracy b5% but that is much more expensive, complex and often requires some method development or protocol adjustment (Davey, 2011; Wang et al., 2010)) involve the use of a fluorescent marker. Propidium iodide is widely used to assess membrane integrity since bacterial membranes are generally impermeable to this die. It suffers a displacement in its absorption and emission spectra, as well as an increase in fluorescent intensity, upon binding to nucleic acids, which makes much more simpler the methodologies to be used, due to the low fluorescence backgrounds (Sträuber & Müller, 2010). However, it is known that in eukaryotic cells the plasma membrane can become leaky for small ions (e.g., K+, Na+, Rb+) but still remain impermeable

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Average electropermeabilized cells (%) by PIstaining and NaCl plating Fig. 7. A) Relationship between the percentage of cells unable to grow in media with NaCl added at their respective MNICs and the percentage of cells permeabilized to propidium iodide after different PEF treatments (18–30 kV cm−1; 5–100 pulses; 30 °C; pH 7.0). B) Bland–Altman plot comparing the number of electroporated bacterial cells determined by propidium iodide staining and recovery in media with the MNIC of NaCl added. The graphs include data of E. coli (▲), C. sakazakii (●) and L. monocytogenes (■) stationary growth phase cells.

to larger molecules such as essential proteins, and/or enzymes within the cell (Saulis et al., 2007). Because of this, using test molecules that are larger than the smallest ions (e.g., K+, Na+, Rb+), such as PI (mw = 660), might result in the underestimation of the percentage of electroporated cells. Thus, according to Saulis and co-workers, electroporation must be considered with respect to the membrane permeability to the smallest substances, that is, ions such as K+, Na+, Rb+, Li+, and Cl−. However, not all of these ions are equally appropriate for this purpose. These authors proposed the use of K+ for quantifying cell membrane electroporation -the reasons are well described in their article and will not discussed here-. Several different techniques are available for determination of the concentration of potassium ions in cell

suspensions, including emission flame photometry, radioisotopic tracers, fluorescent indicators and potentiometric measurements with ion-selective electrodes. Both mass spectrometry and, especially, radioisotopic tracers can be compared to flow cytometry in accuracy and sensitivity for some applications but and they are also expensive and methodologically costly. Furthermore, even considering a relative accuracy (estimated standard deviation for the results of three replicate samples) below 5% (7.6% according to our results) they will not allow to precisely determining the percentage of electroporation above 95– 99% in K+ release measurements. Compared to the techniques mentioned above, selective plating (a microbiological technique) has a lower accuracy (estimated standard

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deviation for the results of three replicate samples N 10%; according to our data 18.1%) and will require longer time to obtain the results (days). Conversely, it is very cheap, simple to perform and allows the detection of sublethally damaged cells in samples containing several cycles –up to 5 or more- of inactivated cells (García et al., 2003). Results here reported demonstrate that, for the four microorganisms tested, NaCl added media could substitute PI staining for enumerating the number of electropermeabilized cells under our standard conditions. Moreover, or results also reveal that, in S. aureus, a good correspondence also exists with the release of K+. It should be noted that the good correspondence between the three techniques indicates that the lesions caused by PEF in the membrane of S. aureus would be of a magnitude enough to allow the release of potassium, but also the entrance of propidium iodide. Thus, the size of the lesions or pores formed would be at least that of propidium iodide, under the experimental conditions here used. Our results also point out that under some particular conditions -after alkaline shock or when treated at low temperatures-, the good correspondence between the number of electropermeabilized S. aureus cells and those unable to grow in NaCl-added media did not exist, and in both cases, not all the cells whose membranes were permeabilized to PI during the treatment, became sensitized to NaCl. It should be noted that this particular behavior cannot be attributed to a different sensitivity of non-treated cells to NaCl since, as previously described, the NaCl MNICs and MICs control and alkaline shocked cells were not statistically different (Cebrián, Arroyo, Mañas, & Condón, 2014). On the other hand, since PI is larger than Na+ and Cl− ions, these differences cannot be attributed either to differences in pore size. Thus, it can be hypothesized that permeabilization of bacterial membranes might not be enough for sensitization to NaCl and/or that pores must remain opened for a certain period of time in order to render cells sensitive to NaCl. In this scenario differences in the ability to reseal pores might explain the particular behavior of these cells, although other possibilities cannot be ruled out. Further work is being carried out in our laboratory to elucidate this point and also to determine if these factors – or other factors acting either before or during the PEF treatments – affect the good correspondence found between the number of electroporated cells and those unable to outgrow in media with the MNIC of NaCl added media for the other three bacterial species here studied. In any case, our results demonstrate that recovery in NaCl added media can be considered as an alternative to PI staining and K+ release measurements for exploration studies and even substitute for some applications such as studies in which a large number of inactivated cells are present. On the other hand, our results also point out what probably are two of the most relevant aspects when setting up this experimental approach. The first one is that the percentage of NaCl to be added to the recovery media should be carefully and precisely determined. Thus, adding a concentration below to the MNIC would lead to an underestimation of the percentage of electroporated cells (and also to an underestimation of the percentage of sublethally injured cells) whereas the opposite will happen in the concentration of NaCl exceeds that value. The second one is recovery time. The high NaCl concentration in this media results in an increase in the lag duration and generation time and thus plates should be incubated for longer times than TSAYE plates. For the four microorganisms studied the maximum number of recovered cells in media with the MNIC added was obtained after 3 days for S. aureus, E. coli and C. sakazakii and after 4 days for L. monocytogenes. Further incubation times did not increase the number of CFU per plate. Longer incubation times, up to 21 days, were only required when NaCl concentrations over the MNIC were tested (data not shown). The results here presented confirm once again the essential role of the electroporation phenomena on bacterial inactivation by PEF (Aronsson, Rönner, & Borch, 2005; García, Gómez, Mañas, Raso, & Pagán, 2007). It is worth noting that the percentage of PIpermeabilized cells during the treatment was related to the number of cells unable to grow in TSAYE plus the MNIC of NaCl, and not to the

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number of cells unable to grow in non-selective medium, which is generally considered as the number of inactivated cells. Thus, the formation of pores during PEF treatment is not necessarily a lethal phenomenon, since its consequences can be reversed by bacterial cells if placed under adequate environmental conditions. However, it is reasonable to think that the number of electroporated cells during the treatment, in other words, the number of cells unable to grow onto TSAYE-NaCl, could be the maximum number of inactivated cells, since electroporated cells which remained alive could be further inactivated by the use of an additional preservation barrier, as demonstrated in the experiment included in Fig. 5. This strategy is the basis for a rational design of combined processes, including PEF technology (Condón, Mañas, & Cebrián, 2011; García et al., 2005). Besides, the experiments about the recovery in liquid media (Fig. 6) demonstrate that, under optimal conditions, cells regained tolerance to NaCl and membrane impermeability to PI simultaneously. However, under adverse conditions, cells resealed their membranes to PI, but they did not recover tolerance to NaCl, ever more, they progressively lost viability. These results could indicate that bacterial cells would be able to reseal mechanically, i.e. without the involvement of important metabolic processes, membrane lesions to a certain extent. It is reasonable to think that the smallest lesions (complete pore closure) would be more difficult to repair, as suggested for eukaryotic cells (Saulis, 1997), and these would be involved in tolerance to NaCl. In our opinion, our results indicate that the addition of NaCl to the recovery media would led to the inactivation of sublethally damaged S. aureus cells by PEF by means others than interfering with the first steps of bacterial membrane pore resealing. Our results also suggest that pore formation by PEF could not be enough and/or that pores must remain opened for a certain period of time in order to render sublethally damaged S. aureus cells, sensitive to NaCl and maybe also to other substances of interest to the food industry. We are currently working on elucidating the exact mechanism by which NaCl inhibits the growth and repair of electroporated cells. This will provide the technique with a mechanistic basis and also will help to understand why, for instance, not all the electroporated alkalineshocked S. aureus cells were inhibited by NaCl. Furthermore, this would also help to elucidate the nature of the damages detected when microorganisms are plated in NaCl added media and would give some insights into the mechanisms of action of NaCl on healthy bacterial cells. 5. Conclusions To sum up, results here reported demonstrate that recovery in media with the MNIC of NaCl added is comparable to PI staining and K+ release measurements for the quantification of the number of electroporated bacterial cells during PEF treatments under most of the conditions assayed, provided that recovery conditions (NaCl concentration and recovery time) are precisely determined. This experimental approach, as here described, might be helpful in Pulsed Electric Fields process optimization and PEF-based combined processes design. Acknowledgments The authors would like to thank the European Regional Development Fund MINECO-CICYT (AGL2012-33522) and the Department of Science, Technology and University of the Aragon Government (229307-UZ) for the support. Authors would also like to acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza for K+ release measurements. References Aronsson, K., Rönner, U., & Borch, E. (2005). Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae in relation to membrane permeabilization and subsequent leakage of intracellular compounds due to pulsed electric field processing. International Journal of Food Microbiology, 99, 19–32.

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