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Article

Understanding the Salmonella Inactivation Mechanisms of 365, 395 and 455 nm Light Pulses Emitted from Light-Emitting Diodes

by
Amritha Prasad
,
Michael Gänzle
and
M. S. Roopesh
*
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2R3, Canada
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(3), 1501; https://doi.org/10.3390/app13031501
Submission received: 23 December 2022 / Revised: 15 January 2023 / Accepted: 16 January 2023 / Published: 23 January 2023
(This article belongs to the Section Food Science and Technology)
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Abstract

:
Salmonella is a foodborne pathogen responsible for several outbreaks in low-water activity (aw) foods. Treatment using light pulses emitted from light-emitting diodes (LED) is an emerging decontamination method to inactivate foodborne pathogens. The objective of this study was to understand the antibacterial mechanisms of light pulses with 365, 395 and 455 nm wavelengths against Salmonella Typhimurium in low-aw conditions. The 365 nm light pulses showed better inactivation efficacy against low-aw S. Typhimurium than the 395 nm light pulses. For instance, the 365 nm LED treatment with an ~217 J/cm2 dose produced a reduction of 2.94 log (CFU/g) in S. Typhimurium cell counts, as compared with a reduction of 1.08 log (CFU/g) produced by the 395 nm LED treatment with the same dose. We observed a significant generation of intracellular reactive oxygen species (ROS) in S. Typhimurium cells after treatments with the 365, 395 and 455 nm light pulses at low-aw conditions. The LED treatments also showed a significant membrane lipid oxidation of S. Typhimurium cells after treatments with 365, 395 and 455 nm light pulses. Overall, a major role of ROS generation was observed in the inactivation efficacy of the 365, 395 and 455 nm light pulses against S. typhimurium at low-aw conditions.

1. Introduction

Salmonellosis is a disease caused by Salmonella, which infects humans by the consumption of contaminated foods or can be transmitted through pets. The continuing cases of foodborne outbreaks due to Salmonella in low-water activity (aw) foods such as pet food kibble, breakfast cereals, spices, dried coconuts, etc. [1,2,3], is a major concern. Salmonella can contaminate dried food products in any stages of their preparation and can survive in low-aw foods for over a year [4,5,6]. They develop resistance to the conventional decontamination methods such as heat treatments or use of oxidizing chemicals in the food industry by developing certain defense mechanisms such as accumulation of compatible solutes in the cell to maintain their turgor pressure in stress conditions [7,8,9]. This necessitates the need for exploring alternative decontamination technologies for low-aw foods.
Light-emitting diode (LED) technology has gained attention due to several advantages, including cost-effectiveness, ease of incorporation into the existing processing lines, monochromatic light, small size and absence of warm-up time [10,11,12]. This technology can produce antibacterial effects by photodynamic inactivation (PDI), which involves the generation of reactive oxygen species (ROS) upon the absorption of light by chromophores such as porphyrin compounds in the presence of oxygen. This triggers cytotoxic responses in cellular components such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, etc., eventually leading to cell death [13].
Several mechanisms attributed to the inactivation efficacy of LED treatments have been reported in high-aw conditions, especially in cell suspensions [14,15,16,17,18,19,20]. For instance, LEDs emitting light of wavelengths 260 and 280 nm showed inactivation efficacy against E. coli suspension by inducing DNA and RNA damage [15]. In addition, the 365 nm (ultraviolet-A) LED treatment of E. coli DH5α suspension showed DNA damage, DNA oxidation and generation of hydroxyl radical and hydrogen peroxide in the bacterial cell [14]. Similarly, the LEDs emitting blue light (~460 nm) have shown significant oxidative stress and loss of cell membrane integrity in bacteria, when combined with exogenous photosensitizers such as curcumin and riboflavin [21,22].
In low-aw foods, drying can provide stress conditions to the bacteria but could also result in survival of these bacteria [23]. Increases in the accumulation of compatible solutes and in oxidative stress resistance under stress conditions increase the bacterial resistance to lethal intervention processes such as heat and pressure [24,25]. For example, decreasing the aw of the dry bacteria (Salmonella enterica) resulted in improvement in their survival ratio [26]. LED technology has shown its antibacterial efficacy in low-aw food systems, also. For instance, LED emitting light of wavelength 405 nm previously showed its inactivation efficacy against E. coli and Salmonella in shelled almonds [27]. Our previous studies reported the antibacterial effect of 365 (UV-A), 395 (NUV; near ultraviolet-visible) and 455 nm (blue) LEDs against Salmonella in low-aw foods such as wheat flour and pet food kibble [28,29,30,31]. However, studies focusing on understanding the antibacterial mode of action of light pulses emitted from LEDs in low-aw conditions are limited. This study focused on understanding the Salmonella inactivation mechanisms of 365, 395 and 455 nm light pulses emitted from LEDs in the low-aw environment.

2. Materials and Methods

2.1. Preparation of Low-aw Salmonella Typhimurium ATCC13311

Salmonella enterica serovar Typhimurium ATCC13311 strain was used in this study. The low-aw S. Typhimurium for the determination of cell membrane damage was prepared according to our previous studies [29,30]. The frozen culture of the bacteria was streaked on the tryptic soy agar (TSA; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) plates supplemented with 0.6% yeast extract (YE; Fischer Bioreagents, Geel, Belgium). This was followed by two consecutive transfers in tryptic soy broth (TSB; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing 0.6% YE and incubation for 18–24 h in a 37 °C shaker. The overnight cultures were then spread plated on TSAYE plates and incubated for 24 h at 37 °C. The lawn was then washed with 1.5 mL 0.1% peptone water (Fischer Bioreagents, Geel, Belgium) and centrifuged to remove the supernatant. The resultant pellet was dislodged in 1 mL 0.1% peptone water followed by another centrifugation. The pellet was resuspended in 0.1% peptone water to obtain a total volume of 1 mL. Two milliliters of the prepared cell suspension were transferred in each glass vial. The cell count of the inoculum was ~1012 CFU/mL in each glass vial. These glass vials were air-dried in the biosafety cabinet for 4–5 days, followed by drying in a desiccator containing silica gel for 1 day. The cell count of the dried S. Typhimurium was ~1011 CFU/g. These dried S. Typhimurium in the glass vials were incubated in an airtight chamber containing supersaturated sodium chloride solution (prepared by adding additional 5–8 g of NaCl in a saturated solution of NaCl) for 7 days in the room conditions, for equilibration to 0.75 aw. The final cell count of the equilibrated dried bacteria was obtained as ~1010 CFU/g.
To limit the cell membrane damage and oxidation of S. Typhimurium due to the long drying and equilibration period involved above, the low-aw S. Typhimurium was prepared as described by Fang et al. [24] with few modifications. The inoculum was prepared as mentioned above. The cell count of the resultant cell suspension was ~1011 CFU/mL. Two hundred and fifty microliters of this culture was transferred in glass vials and was dried in a vacuum chamber containing silica gel for 18–20 h. The cell count of the vacuum-dried bacteria was ~1011 CFU/g. A previous study has shown that drying in the absence of oxygen eliminates much of its lethal effect in the bacteria [24]. The glass vials were then transferred into an airtight chamber containing supersaturated sodium chloride solution and incubated for 20–24 h for equilibration to 0.75 aw at room conditions. The cell count of the resultant low-aw S. Typhimurium was ~1011 CFU/g.

2.2. Treatment of Low-aw S. Typhimurium with Light Pulses Emitted from the LED

The LED heads of JL3 series (111 × 70 × 128 mm3; six high-intensity LEDs) emitting light of wavelengths 365 nm (ultraviolet-A), 395 nm (near-visible ultraviolet) and 455 nm (blue) compatible with a controller unit (CF3000, Clearstone Technologies Inc., Hopkins, MN, USA) were used in this study. The irradiance of the LEDs was measured using a laser energy meter (7Z01580, Starbright, Ophir Photonics, Har Hotzvim, JRS, Israel) connected to an irradiance and dosage sensor (PD300RM-8 W, Ophir Photonics, A Newport Corporation Brand, Har Hotzvim, JRS, Israel) at a distance of 4 cm from the LED head and at 60 or 80% power level as mentioned in our previous studies [29,30]. The 365, 395 and 455 nm LEDs emitted light pulses with irradiance values of 0.06, 0.30 and 0.35 W/cm2, respectively, at 80% power level, and the irradiance values of the 365 and 395 nm LEDs at 60% power level were 0.05 and 0.23 W/cm2, respectively.
For the determination of cell membrane damage in low-aw S. Typhimurium, the bacteria prepared with longer drying and equilibration periods were used. Ten milligrams of low-aw S. Typhimurium was weighed in a small disc (7.07 cm2) composed of polylactic acid filament and treated with the LED emitting light pulses of wavelengths 365 and 395 nm at 60% power level and at 4 cm from the LED head. The treatment times chosen were 10 and 60 min corresponding to treatment doses of 28.9 and 188.1 J/cm2 for 365 nm and 138.8 and 834.4 J/cm2 for 395 nm LED treatments, respectively. To determine the oxidative stress induced by the treatment with light pulses emitted from the LEDs, low-aw S. Typhimurium prepared with shorter drying and equilibration period was used. Ten milligrams of low-aw S. Typhimurium was treated with the LEDs emitting light pulses at room conditions (~23 °C, 48% relative humidity). The low-aw bacteria were treated with treatment doses of ~217, ~217 and ~250 J/cm2 corresponding to 60, 12 and 12 min treatment times with 365, 395 and 455 nm LEDs, respectively, at 80% power level and at 4 cm distance from the LED heads. Ten milligrams of low-aw S. Typhimurium with no LED treatment was considered as the control. Enumeration of the viable cell counts was performed by 10-fold serial dilution in 0.1% peptone water and spread plating in TSAYE plates, followed by incubation at 37 °C for 24 h.

2.3. Determination of Cell Membrane Damage in Low-aw S. Typhimurium

A LIVE/DEAD [propidium iodide (PI) exclusion] Baclight kit (L7012, Molecular Probes Inc., Eugene, OR, USA) consisting of SYTO9 (labels all cells) and propidium iodide (PI; labels only cells with permeabilized cytoplasmic membrane) was used to determine the cell membrane damage in low-aw S. Typhimurium. The LED-treated low-aw S. Typhimurium was washed with 2 mL 0.85% sodium chloride (NaCl) solution, and the resultant pellet was resuspended in 2 mL 0.85% NaCl solution and was mixed with 20 mL of the same salt solution and incubated at room conditions for 30 min, while vortexing every 10 min. After the incubation, the cell suspensions were centrifuged and washed with 20 mL 0.85% NaCl solution, and the pellet was resuspended again in 10 mL of the same salt solution. Ten milligrams of low-aw S. Typhimurium served as the control, while 10 mg of bacteria heat-treated at 85 °C for 1 h followed by incubation in 70% isopropanol for 30 min was considered as the positive control. The cell suspensions were diluted with sterile deionized water to maintain the cell numbers per second in the range of 300 to 3000 events per second. For staining, equal volumes of the dyes were mixed, and 3 µL of the dye mixture was mixed with 1 mL of the cell suspension followed by incubation in room conditions for 15 min in dark. Flow cytometry was performed using a BD LSR Fotessa X-20 (BD Biosciences, San Jose, CA, USA) with a 488 nm excitation from a blue air laser at 50 mW and a 561 nm excitation from a yellow air laser at 50 mW to excite green (530 ± 30 nm) and red fluorescence (586 ± 15 nm). Sample injection and acquisition were performed simultaneously, and the data were recorded until 10,000 events. The data were analyzed using the FlowJo software (version 10.7.1, Becton Dickinson & Company (BD), San Jose, CA, USA).

2.4. Determination of Intracellular ROS in Low-aw S. Typhimurium

The production of intracellular ROS in the bacterial cell was evaluated using 5-(and-6)-carboxy-2′7′-dichlorodihydroflourescein diacetate (carboxy-H2DCFDA) dye (Invitrogen, Eugene, OR, USA) by following the manufacturer’s instructions with a little modification. The 365, 395 and 455 nm LED-treated low-aw S. Typhimurium were washed with phosphate buffered saline, pH 7.4 (PBS; Gibco, Life Technologies, Waltham, MA, USA), and the pellet was resuspended in PBS containing carboxy-H2DCFDA dye with concentration of 10 µM in 1 mL of cell suspension. The solution was incubated at 37 °C for 30 min in the dark to facilitate the staining of the bacteria. The stained cell suspension was centrifuged, and the resultant pellet was resuspended in PBS, resulting in the final volume of 1 mL. Two hundred microliters of the cell suspension were loaded in each well in a 96-well microtiter plate (Costar, Corning, NY, USA), and four wells were loaded for each sample. Ten milligrams of untreated low-aw S. Typhimurium was considered as the control. Ten milligrams of low-aw S. Typhimurium cells treated with 200 mM H2O2 for 30 min at room temperature served as the positive control. In addition, ten milligrams of vacuum-dried S. Typhimurium were also analyzed for intracellular ROS production due to drying. Fluorescence values were measured using a spectrophotometer (Variskon flash, Thermo Electron Corporation, Nepean, ON, Canada) at an excitation wavelength of 495 nm and an emission wavelength of 525 nm. Here the intracellular ROS generation was reported as an arbitrary value I, calculated as I = (It − Io)/Io, where It and Io are the mean fluorescence values of the treated and untreated low-aw S. Typhimurium as mentioned in [32].

2.5. Determination of Membrane Lipid Oxidation of Low-aw S. Typhimurium

To determine the membrane lipid oxidation of low-aw S. Typhimurium due to treatments with light pulses emitted from the LEDs, C11-BODIPY581/591 (fluorinated boron-dipyrromethene) dye, which detects the peroxide radical (Invitrogen, Eugene, OR, USA), was used in this study. The assay was performed according to the procedure reported previously with few modifications [24,33]. The 365, 395 and 455 nm LED-treated low-aw S. Typhimurium was washed twice with 1 mL of 50 mM Tris × HCl (pH 8.0) containing 20% (wt/vol) sucrose, and the resultant pellet was resuspended in 1 mL of Tris × HCl buffer. The cell suspension was mixed with 200 µL lysozyme (5 mg/mL in 0.25 M Tris × HCl, pH 8.0) and 400 µL of ethylenediaminetetraacetic acid (EDTA; 0.25 M, pH 8.0) to facilitate the disruption of the outer cell membrane and was incubated in 37 °C shaker (200 rpm) for 30 min. This was followed by centrifugation, resuspension of the pellet in 1 mL of 10 mM citrate buffer (pH 7) and 10 µM C11-BODIPY581/591 dye in dimethyl sulfoxide (DMSO) and incubation in 37 °C shaker (200 rpm) for 30 min in the dark for staining. Low-aw S. Typhimurium treated with 200 mM H2O2 for 30 min served as the positive control, while 10 mg of low-aw S. Typhimurium with no treatment (H2O2 and LED) served as the untreated control. Ten milligrams of vacuum-dried S. Typhimurium were also analyzed for membrane lipid oxidation caused due to drying. The cell suspensions were diluted to maintain the cell numbers per second in the range of 300 to 3000 events per second. Flow cytometry was performed using a BD LSR Fotessa X-20, and data were analyzed using the FlowJo software as mentioned in Section 2.3.

2.6. Weight Loss and Surface Temperature Increase Due to LED Treatments

The LED treatments could produce weight loss and an increase in the surface temperature of the samples. Therefore, the weight of the low-aw S. Typhimurium was monitored before and after the LED treatments with 365, 395 and 455 nm light pulses at 80% power level and at 4 cm from the LED head with treatment doses of ~217, ~217 and ~250 J/cm2, respectively, using a weighing balance. In addition, the surface temperature of the bacteria was monitored using a thermocouple connected to a digital thermometer (1507726, Fischer Scientific, Hampton, NH, USA) during the LED treatments.

2.7. Statistical Analysis

All the experiments were conducted with triplicate independent cultures (n = 3). SAS University edition (SAS studio 9.4) was used for the statistical analysis. The significant differences between means were performed by Tukey’s LSD test with p < 0.05. The evaluation of the cell membrane damage and the membrane lipid oxidation due to the LED treatments were analyzed by two-way ANOVA. The determination of log reduction, intracellular ROS production, surface temperature increase and weight loss were analyzed by one-way ANOVA.

3. Results

3.1. Inactivation Effect of 365 and 395 nm LED Treatment

In low-aw S. Typhimurium m prepared with a longer drying and equilibration period, the viable cell counts were reduced from 12.55 ± 0.614 log (CFU/mL) to 10.99 ± 0.473 log (CFU/g) after the drying and equilibration steps used in Section 2.1. The treatment with 365 nm light pulses with 28.9 (10 min) and 188.1 (60 min) J/cm2 doses showed a reduction of 0.37 ± 0.119 and 1.44 ± 0.039 log (CFU/g) in S. Typhimurium cell counts in low-aw conditions, respectively. In addition, the treatment with 395 nm light pulses with 138.8 (10 min) and 834.4 (60 min) J/cm2 doses resulted in reductions of 1.88 ± 0.069 and 2.88 ± 0.397 log (CFU/g) in the low-aw S. Typhimurium cell counts, respectively (Table 1).
In the case of low-aw S. Typhimurium prepared with a shorter drying and equilibration period, the drying and equilibration steps produced no effect on the viable cell counts of the S. Typhimurium cells (Figure 1). The inactivation efficacy of the 365 and 395 nm light pulses was evaluated by treatment of 10 mg of low-aw S. Typhimurium with ~217 J/cm2 dose at 4 cm from the LED head and at 80% power level. Overall, the antibacterial effect of the 365 nm LED treatments against low-aw S. Typhimurium was higher than 395 nm LED treatment with same dose (p < 0.0001). For example, 365 nm light pulses produced a reduction of 2.94 ± 0.339 log (CFU/g) as compared with 1.08 ± 0.073 log (CFU/g) by 395 nm light pulses in low-aw S. Typhimurium cell counts with the ~217 J/cm2 dose treatment (Figure 1).
The high-intensity light pulses increased the surface temperature of the low-aw bacteria (p = 0.0008). The 395 nm LED produced a higher increase in the surface temperature of the low-aw S. Typhimurium as compared with the 365 nm LED treatments with the same dose (p = 0.0036) (Table 2). Although the surface temperature was increased to only ~43 °C, this might play a role in the inactivation efficacy of the 395 nm LED. The LED treatments also reduced the weight of the treated low-aw S. Typhimurium (p < 0.0001), and the weight loss could be attributed to the drying by the 365 and 395 nm LED treatments. Here the 365 nm LED treatment produced a higher reduction in the weight as compared with the 395 nm LED treatments with same dose (p = 0.005) (Table 2), indicating that drying was more pronounced in the case of 365 nm LED treatment, which might influence its inactivation effect against S. Typhimurium in dried form.

3.2. Cell Membrane Damage Due to LED Treatments

High-intensity light pulses emitted from LEDs can induce cell membrane damage in the bacterial cells. Therefore, a PI exclusion assay was performed in low-aw S. Typhimurium in this study. Since the preparation of the low-aw S. Typhimurium involved a total drying period of 5–6 days followed by an equilibration period of 7 days, the PI exclusion analysis was performed after the drying and equilibration steps during the preparation of the dry bacteria. In addition, the cell membrane damage in the overnight culture of S. Typhimurium was performed, which showed damage in 38.9% of the cells (Figure 2). The drying step (air-drying and silica gel drying) produced a higher (p = 0.0008) percentage of damaged cells labelled with PI. However, the percentage of cell population labelled with PI was reduced (p = 0.0391) after equilibration (Figure 2). This could be due to the lysis of dead cells as the flow cytometer measures only intact cells and does not detect the lysed cells.
The PI exclusion assay of the LED-treated low-aw S. Typhimurium did not show additional damage to the cell membrane (Figure 2). Since a majority of the bacterial cells already suffered damage after drying and equilibration, the PI exclusion assay could not detect any significant cellular damage after the LED treatments. There was no effect of treatment time (or dose) on the cell membrane damage in S. Typhimurium cells due to the LED treatments. Moreover, there was no effect of varying the wavelength of the light pulses on the cellular membrane damage in bacterial cells treated by 365 and 395 nm LEDs (Figure 2). Overall, the PI exclusion study was not suitable to understand the effects of the 365 and 395 nm LED treatments on the cell membrane of the low-aw S. Typhimurium due to long drying and equilibration periods involved in the preparation of dry bacteria in this study. In further experiments, the low-aw S. Typhimurium prepared with shorter drying and equilibration periods were used to minimize the cell death by the exclusion of oxygen and faster equilibration [24].

3.3. Intracellular ROS Generation in Low-aw S. Typhimurium

The LED technology produces an antibacterial effect by photodynamic inactivation (PDI), which involves the generation of ROS in the presence of oxygen in the bacteria. Therefore, the formation of intracellular ROS due to the LED treatments was analyzed in this study. Both 365 (p = 0.0123) and 395 (p = 0.0288) nm LED treatments with same dose (~217 J/cm2) resulted in significant intracellular ROS production in the low-aw S. Typhimurium. However, there was no effect of changing the wavelength of the light pulses from 365 to 395 nm (p = 0.5922) on the intracellular ROS generation in the bacterial cells (Table 2). Therefore, the 365 and 395 nm light pulses emitted from the LEDs induced oxidative stress in the bacteria.

3.4. Membrane Lipid Oxidation in Low-aw S. Typhimurium

After observing the generation of intracellular ROS in low-aw S. Typhimurium due to the 365 and 395 nm LED treatments, their influence on the membrane lipid oxidation in S. Typhimurium was explored in this study. The drying step of 18–20 h oxidized membrane lipids in 10.3% of the cell population, while ~85% of the cell population remained unoxidized (Figure 3). A significant reduction in the percentage of unoxidized cells was observed after the LED treatments, with no significant difference among the 365 and 395 nm LED-treated low-aw S. Typhimurium with same dose (Figure 3). The LED treatments with both 365 and 395 nm of low-aw S. Typhimurium increased the percentage of the cell population with membrane lipid oxidation (p < 0.0001) (Figure 3). There was no effect of varying the wavelength of the light pulses from the LEDs used for the treatment of low-aw S. Typhimurium on the membrane lipid oxidation of the bacteria (Figure 3). Therefore, membrane lipid oxidation played an important role in the antibacterial efficacy of both the 365 and 395 nm LED treatments against low-aw S. Typhimurium.

3.5. Effect of 455 nm LED Treatments on Low-aw S. Typhimurium

To understand the inactivation effect and the underlying antibacterial mechanism of 455 nm light pulses against low-aw S. Typhimurium, a treatment dose of ~250 J/cm2 (12 min) at 80% power level and at 4 cm distance from the LED head was selected in this study. We observed that the 455 nm light pulses reduced (p = 0.0014) the cell counts of low-aw S. Typhimurium by 0.859 ± 0.201 log (CFU/g) (Table 3). This inactivation efficacy could be attributed to the generation of intracellular ROS as we observed an increase (p = 0.0072) in the fluorescence values after treatment with the 455 nm LED (Table 3). An increase (p < 0.0001) in the percentage of the cell population of S. Typhimurium oxidized due to the 455 nm LED treatment by C11 BODIPY581/591 assay shows that the membrane lipid oxidation plays a role in the antibacterial effect of the 455 nm LED in this study. In addition, the 455 nm LED treatment reduced (p < 0.0001) the weight of the treated low-aw S. Typhimurium with a weight loss of 9.68% and increased (p < 0.0001) the surface temperature of the bacteria during the LED treatment (Table 3). This indicates that the drying and surface temperature increase might play a role in the antibacterial efficacy of the 455 nm LED treatments against low-aw S. Typhimurium.

4. Discussion

The LED technology is an emerging decontamination method which has shown promising inactivation efficacy against foodborne pathogens in high- and low-moisture foods [19,20,28,29,30,34]. This study focused on further understanding the underlying mode of action of the LEDs emitting light pulses of different wavelengths against S. Typhimurium in low-aw conditions.
In this study, the long drying (4–5 days) and equilibration (7 days) steps involved during the preparation of low-aw S. Typhimurium for the PI exclusion assay resulted in the majority of the cell membrane damage in the bacteria (Figure 2). Hence, the percentage of undamaged S. Typhimurium cells were not sufficient to evaluate the cell membrane damage produced by the treatment with 365 and 395 nm light pulses. The results are confounded by the presence of dead but PI-excluding cells [35], the presence of viable and PI-excluding cells that did not grow on TSBYE after 24 h of [36] and lysis of PI permeant dead cells during storage and treatment. The reduction in the cell counts after the 365 and 395 nm LED treatments observed in this study during this preparation method (Table 1) are similar to the reductions reported in our previous study [30]. Overall, the LED treatments probably do not cause the cell membrane damage in low-aw bacteria. Therefore, the preparation of low-aw S. Typhimurium was modified to limit the effect of drying and equilibration periods in the subsequent studies. Low-aw S. Typhimurium showed susceptibility to the treatments with the 365 and 395 nm light pulses with an ~217 J/cm2 dose at 80% power level and 4 cm distance from the LED head (Figure 2). In addition, low-aw S. Typhimurium showed more sensitivity toward the 365 nm LED treatment as compared with the 395 nm LED treatment with the same dose, power level and distance from the LED head, which was also reported in Prasad et al. (2019) [30], and this observation needs further research.
Understanding the underlying mechanism in the antibacterial effect of the 365 and 395 nm LED treatments against low-aw S. Typhimurium would help in developing these light pulses emitted from the LEDs as a potential decontamination method against foodborne pathogens in low-aw food systems. In this study, carboxy-H2DCFDA was used as a fluorescent indicator of oxidative stress (hydroxyl, peroxyl and other ROS species) to understand the role of ROS production in the inactivation efficacy of the LED treatments. The intracellular esterases in the live cells cleave the diacetate group of the agent, and in the presence of ROS, the non-fluorescent compound converts to the fluorescent form and emits fluorescence [17,32], which was detected by a spectrophotometer in this study. Previously, carboxy-H2DCFDA has been used to study the intracellular oxidative stress in S. Typhimurium ATCC13311 by Yadav and Roopesh (2022) [37]. The LED treatments with the 365 and 395 nm light pulses produced a significant intracellular ROS generation in low-aw S. Typhimurium cells (Table 2). Previously, the generation of ROS due to the 365 nm LED treatment in high-moisture conditions was reported [14,38,39]. Similarly, 405 nm LED treatment showed a significant increase in the intracellular ROS generation in Staphylococcus epidermis by using carboxy-H2DCFDA dye as the fluorescent indicator [40]. In high-aw foods such as fresh-cut papaya inoculated with Salmonella, the 405 nm LED treatments showed oxidation of the bacterial cells [20]. An increase in the oxidative stress might affect the antioxidant defense system of the cells. The generation of intracellular ROS can also lead to the oxidation of cellular components such as lipids and proteins and might inhibit enzyme activity inside the cell, eventually leading to cell death [40,41,42,43].
Since we observed a significant oxidative stress of bacterial cells due to the 365 and 395 nm LED treatments, further evaluation of the membrane lipid oxidation was performed. C11-BODIPY581/591 dye was used to detect the membrane lipid oxidation in low-aw S. Typhimurium that is oxidized by peroxide radicals [24,44]. Flow cytometry analysis facilitated in the quantification of oxidized and unoxidized cells [14] showed that the inactivation efficacy of the 365 nm LED treatment against E. coli DH5α was suppressed by the presence of catalase (a scavenger of hydrogen peroxide), indicating the presence of the peroxide radical in the ROS produced due to the 365 nm LED treatments. Similarly, the 365 nm LED treatment produced a significant membrane lipid oxidation in low-aw S. Typhimurium in this study. In addition, we observed that the percentage of the cell population indicating membrane lipid oxidation due to the treatment with 365 and 395 nm light pulses emitted from the LED with the ~217 J/cm2 dose were not significantly different as opposed to the log reductions observed in this study (Figure 1 and Figure 3). This would indicate that there might be some additional mode of action involved in the inactivation efficacy of 365 nm LED treatments against S. Typhimurium in low-aw conditions.
This study showed a significant inactivation efficacy of the 455 nm LED treatments with the 250 J/cm2 dose against low-aw S. Typhimurium, which is also supported by our previous studies [29,31]. Exogenous photosensitizers such as curcumin, when combined with blue (462 ± 3 nm) LED, enhance their inactivation efficacy against E. coli and S. aureus suspensions and the generation of intracellular ROS in the cells [21]. In this study, we observed a significant generation of intracellular ROS in low-aw S. typhimurium with the 455 nm LED treatment alone (Table 3). This could be attributed to the photosensitization of endogenous photosensitizers such as porphyrin compounds in S. Typhimurium cells by the 455 nm LED treatment, which leads to the generation of ROS in the presence of oxygen [13,45]. On the contrary, the 462 nm LED treatment of E. coli and S. aureus suspensions alone did not produce any significant intracellular ROS [21], indicating the generation of intracellular ROS in a bacterial cell by the blue LED treatment was influenced by the wavelength, sample type and strain used. A significant membrane lipid oxidation of low-aw S. Typhimurium cells was observed with the 455 nm LED treatment in this study, indicating the presence of the peroxide radical in the ROS generated by the LED light pulses. This was supported by Orlandi, Martegani and Bolognese (2018) [46] in their study, where the overexpression of the catalase A enzyme reduced the sensitivity of Pseudomonas aeruginosa to the 464 nm LED treatments, indicating that the blue LED produced the peroxide radical as a major ROS during the treatments.
Previously, 405 ± 5 nm LED treatment of S. Typhimurium suspension did not show significant membrane lipid peroxidation with the thiobarbituric acid reaction substance (TBARS) assay [20]. On the contrary, the treatment of low-aw pet food pellets with the 455 nm light pulses emitted from the LED showed significant lipid oxidation in low-aw pet foods with the TBARs assay [29,47]. Therefore, the generation of membrane lipid oxidation due to the LED treatments would depend upon the sample type, strain used, wavelength of the light used, treatment dose, etc. Additionally, the drying step involved in the preparation of low-aw S. Typhimurium in this study could also contribute to the oxidative stress. This was supported by previous studies by Fang et al. (2022) [24] and Wang et al. (2021) [25], where drying induced oxidative stress in E. coli and Salmonella enterica, respectively. Sanitizing solutions such as peracetic acid were found to be active in dry cells as they have produced promising antimicrobial activity against Salmonella in low-moisture foods such as seeds, dried fruit, nuts, chocolate crumbs and cornflakes [48,49]. The drying and the oxidative stress conditions trigger the bacteria to enter the viable but nonculturable (VBNC) state and increase their resistance to the subsequent intervention methods. The addition of catalase or pyruvate in the recovery media after the drying step would resuscitate the VBNC cells [24,25,36,50] and would aid in evaluating the effect of the LED treatments in bacteria.
Song et al. [51] reported improvement in the inactivation of UV-C-treated E. coli ATCC 11229 by 365 nm (UV-A) LED pre-treatment. Their inactivation mechanism experiments showed that hydroxyl radicals had a major role on the E. coli inactivation during the UVA pre-treatment. Our study showed significant generation of intracellular ROS and membrane lipid oxidation of S. Typhimurium after 365, 395 and 455 nm LED treatments. During the 365 nm LED treatment of S. Typhimurium, the major contributor to oxidative stress and membrane lipid peroxidation could be hydroxyl radicals. Studies focusing on the contribution of individual ROS (e.g., hydroxyl, singlet oxygen, superoxide, and hydrogen peroxide) on bacterial inactivation during 395 and 455 nm LED treatments are limited or not available in the literature. Future research can provide more information on the contribution of the main ROS on the microbial inactivation potential of 395 and 455 nm LED treatments.
Since high doses of light pulses emitted from the LEDs were required to produce antibacterial effects in low-aw conditions in this study, combining this technology with other non-thermal technologies such as high-pressure CO2 treatment or mild heat treatment could result in better inactivation effectiveness in low-aw conditions. The high doses of light pulses from LEDs can also result in photochemical reactions such as oxidation of food ingredients. For instance, previous studies reported significant lipid oxidation in pet foods by 395 and 455 nm LED treatments [29,47]. However, an improvement in functional properties of UV-LED-treated wheat flour was reported in another study [31]. For instance, the wheat flour treated by 365, 395 and 455 nm LEDs showed a reduction in yellowness and water-holding capacity [31]. The changes in food qualities and functional properties by UV-LED treatment will depend on the type of product and the treatment conditions. Future research is needed to optimize the LED treatment conditions, considering food safety and food quality aspects, based on the product of interest.
In conclusion, the LEDs emitting light pulses of wavelengths 365, 395 and 455 nm showed antibacterial efficacy against S. Typhimurium in low-aw conditions. The 365 nm LED treatments showed a better inactivation effect against low-aw S. Typhimurium than the 395 nm LED treatment. Cell membrane damage due to the high-intensity light pulses could not be evaluated due to the drying and equilibration periods involved in the preparation of dried S. Typhimurium equilibrated to 0.75 aw. Significant generation of intracellular ROS was observed with the LED treatments. In addition, the membrane lipid oxidation of the S. Typhimurium cells was observed in the cases of all three LED treatments. Significant weight loss and increase in the surface temperature of the low-aw S. typhimurium was observed with the 365, 395 and 455 nm LED treatments. Overall, this study presented the probable antimicrobial mode of action of the 365, 395 and 455 nm LEDs in the low-aw conditions, which would help in further research for the development of the treatment with light pulses emitted from the LEDs as a potential decontamination method for low-aw food systems.

Author Contributions

Conceptualization, A.P., M.G. and M.S.R.; methodology, A.P., M.G. and M.S.R.; software, A.P.; validation, A.P.; formal analysis, A.P.; investigation, A.P.; resources, M.G. and M.S.R.; data curation, A.P.; writing—original draft preparation, A.P.; writing—review and editing, M.G. and M.S.R.; supervision, M.S.R.; project administration, M.S.R.; funding acquisition, M.G. and M.S.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Alberta Agriculture and Forestry (Grant no. 2018F040R) and the Natural Sciences and Engineering Research Council (Grant no. RGPIN-2017-05051).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is available on request.

Acknowledgments

We acknowledge the Alberta Agriculture and Forestry (Grant no. 2018F040R) and the Natural Sciences and Engineering Research Council (Grant no. RGPIN-2017-05051) for funding this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 13 01501 g001 550
Figure 1. Effects of vacuum drying for 18–20 h (A), followed by equilibration to 0.75 aw for 20–24 h (B) and treatment with 365 nm (C) and 395 nm (D) light pulses emitted from the LEDs of dried and equilibrated S. Typhimurium ATCC13311, on the survival of S. Typhimurium cells. The 365 and 395 nm LED treatments of low-aw S. Typhimurium ATCC13311 were performed at 80% power level and at 4 cm from the LED head with the ~217 J/cm2 dose corresponding to treatment times of 60 and 12 min, respectively. The results are represented as means ± standard deviation of three independent replicates. Values with different letters differ significantly (p < 0.05).
Figure 1. Effects of vacuum drying for 18–20 h (A), followed by equilibration to 0.75 aw for 20–24 h (B) and treatment with 365 nm (C) and 395 nm (D) light pulses emitted from the LEDs of dried and equilibrated S. Typhimurium ATCC13311, on the survival of S. Typhimurium cells. The 365 and 395 nm LED treatments of low-aw S. Typhimurium ATCC13311 were performed at 80% power level and at 4 cm from the LED head with the ~217 J/cm2 dose corresponding to treatment times of 60 and 12 min, respectively. The results are represented as means ± standard deviation of three independent replicates. Values with different letters differ significantly (p < 0.05).
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Figure 2. Effect of vacuum drying and equilibration to 0.75 aw and 365 and 395 nm LED treatments on the cell membrane of the S. Typhimurium ATCC13311 cells by PI (propidium iodide) exclusion assay. Here the Y-axis in the graph represents the percentage of PI-permeable cells of the total population when analyzed using a flow cytometer. Treatments with the 365 and 395 nm light pulses of low-aw S. Typhimurium cells were performed for 10 and 60 min, corresponding to treatment doses of 28.9 and 188.1 J/cm2 for 365 nm and 138.8 and 834.4 J/cm2 for 395 nm, respectively, at 60% power level and at 4 cm from the LED heads. Results are represented as means ± standard deviation of three independent replicates. Values with different letters differ significantly (p < 0.05).
Figure 2. Effect of vacuum drying and equilibration to 0.75 aw and 365 and 395 nm LED treatments on the cell membrane of the S. Typhimurium ATCC13311 cells by PI (propidium iodide) exclusion assay. Here the Y-axis in the graph represents the percentage of PI-permeable cells of the total population when analyzed using a flow cytometer. Treatments with the 365 and 395 nm light pulses of low-aw S. Typhimurium cells were performed for 10 and 60 min, corresponding to treatment doses of 28.9 and 188.1 J/cm2 for 365 nm and 138.8 and 834.4 J/cm2 for 395 nm, respectively, at 60% power level and at 4 cm from the LED heads. Results are represented as means ± standard deviation of three independent replicates. Values with different letters differ significantly (p < 0.05).
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Figure 3. Flow cytometric quantification of membrane lipid oxidation in S. Typhimurium ATCC13311 due to vacuum drying for 18–20 h (A), followed by equilibration to 0.75 aw for 20–24 h (B); and treatment of dried and equilibrated S. Typhimurium with 365 nm (C) and 395 nm (D) LEDs using C11-BODIPY581/591 dye as a fluorescent indicator. The stained and oxidized (black bars) and stained and unoxidized cells (gray bars) are shown as the percentage of cell populations. Treatments with 365 and 395 nm light pulses of low-aw S. Typhimurium ATCC13311 were performed at 80% power level and at 4 cm from the LED head with ~217 J/cm2 dose corresponding to treatment times of 60 and 12 min, respectively. Results are represented as means ± standard deviation of three independent replicates. Values with different letters differ significantly (p < 0.05).
Figure 3. Flow cytometric quantification of membrane lipid oxidation in S. Typhimurium ATCC13311 due to vacuum drying for 18–20 h (A), followed by equilibration to 0.75 aw for 20–24 h (B); and treatment of dried and equilibrated S. Typhimurium with 365 nm (C) and 395 nm (D) LEDs using C11-BODIPY581/591 dye as a fluorescent indicator. The stained and oxidized (black bars) and stained and unoxidized cells (gray bars) are shown as the percentage of cell populations. Treatments with 365 and 395 nm light pulses of low-aw S. Typhimurium ATCC13311 were performed at 80% power level and at 4 cm from the LED head with ~217 J/cm2 dose corresponding to treatment times of 60 and 12 min, respectively. Results are represented as means ± standard deviation of three independent replicates. Values with different letters differ significantly (p < 0.05).
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Table
Table 1. Effect of 365 and 395 nm LED treatment on the survival of S. Typhimurium ATCC13311 cells, dried and equilibrated to 0.75 aw. Treatments with the 365 and 395 nm light pulses of low-aw S. Typhimurium cells were performed for 10 and 60 min corresponding to treatment doses of 28.9 and 188.1 J/cm2 for 365 nm and 138.8 and 834.4 J/cm2 for 395 nm, respectively, at 60% power level and at 4 cm from the LED heads.
Table 1. Effect of 365 and 395 nm LED treatment on the survival of S. Typhimurium ATCC13311 cells, dried and equilibrated to 0.75 aw. Treatments with the 365 and 395 nm light pulses of low-aw S. Typhimurium cells were performed for 10 and 60 min corresponding to treatment doses of 28.9 and 188.1 J/cm2 for 365 nm and 138.8 and 834.4 J/cm2 for 395 nm, respectively, at 60% power level and at 4 cm from the LED heads.
Treatment Time (min)Treatment Dose (J/cm2)Log (CFU/g)
Inoculum before drying--12.55 ± 0.614 a (in Log CFU/mL)
After drying--10.99 ± 0.473 b
Control (after equilibration to 0.75 aw)00.00010.73 ± 0.483 b
365 nm1028.910.36 ± 0.054 b
60188.19.29 ± 0.127 c
395 nm10138.88.85 ± 0.479 c
60834.47.85 ± 0.462 d
The results are represented as means ± standard deviation of three independent replicates. Values with different superscripts in each column differ significantly (p < 0.05).
Table
Table 2. Effect of 365 and 395 nm LED treatment on the weight and surface temperature of low-aw S. Typhimurium ATCC13311. Intracellular ROS generation using carboxy-H2DCFDA dye in low-aw S. Typhimurium due to the 365 and 395 nm LED treatments is also shown. The 365 and 395 nm LED treatments were performed at 80% power level and at 4 cm from the LED head with the ~217 J/cm2 dose corresponding to treatment times of 60 and 12 min, respectively. The low-aw S. Typhimurium cells treated with 200 mM H2O2 for 30 min served as the positive control. Here the arbitrary value I = (It − Io)/Io, where It and Io are the mean fluorescence values of the treated and untreated low-aw S. Typhimurium, represents the intracellular ROS generation.
Table 2. Effect of 365 and 395 nm LED treatment on the weight and surface temperature of low-aw S. Typhimurium ATCC13311. Intracellular ROS generation using carboxy-H2DCFDA dye in low-aw S. Typhimurium due to the 365 and 395 nm LED treatments is also shown. The 365 and 395 nm LED treatments were performed at 80% power level and at 4 cm from the LED head with the ~217 J/cm2 dose corresponding to treatment times of 60 and 12 min, respectively. The low-aw S. Typhimurium cells treated with 200 mM H2O2 for 30 min served as the positive control. Here the arbitrary value I = (It − Io)/Io, where It and Io are the mean fluorescence values of the treated and untreated low-aw S. Typhimurium, represents the intracellular ROS generation.
Weight Loss (%)Surface Temperature (°C)Arbitrary Value (I) Representing the Intracellular ROS Formation
Control0.00 ± 0.00 c23.78 ± 0.69 c0.00 ± 0.00 a
H2O2 treatment--1.22 ± 0.52 b
365 nm15.51 ± 5.42 a31.33 ± 2.31 b0.97 ± 0.53 b
395 nm10.33 ± 3.69 b43.33 ± 4.93 a0.81 ± 0.51 b
Results are represented as means ± standard deviation of three independent replicates. Values with different superscripts in each column differ significantly (p < 0.05).
Table
Table 3. Effect of the 455 nm LED treatment on the survival, weight, and surface temperature of low-aw S. Typhimurium ATCC13311. Flow cytometric quantification of membrane lipid oxidation using C11-BODIPY581/591 dye and intracellular ROS generation using carboxy-H2DCFDA dye analyzed using spectrophotometer in low-aw S. Typhimurium due to the 455 nm LED treatments are also shown. The 455 nm LED treatment was performed at 80% power level and at 4 cm from the LED head with an ~250 J/cm2 dose corresponding to treatment time of 12 min. The low-aw S. Typhimurium cells treated with 200 mM H2O2 for 30 min served as the positive control. Here the arbitrary value I = (It − Io)/Io, where It and Io are the mean fluorescence values of the treated and untreated low-aw S. typhimurium, represents the intracellular ROS generation.
Table 3. Effect of the 455 nm LED treatment on the survival, weight, and surface temperature of low-aw S. Typhimurium ATCC13311. Flow cytometric quantification of membrane lipid oxidation using C11-BODIPY581/591 dye and intracellular ROS generation using carboxy-H2DCFDA dye analyzed using spectrophotometer in low-aw S. Typhimurium due to the 455 nm LED treatments are also shown. The 455 nm LED treatment was performed at 80% power level and at 4 cm from the LED head with an ~250 J/cm2 dose corresponding to treatment time of 12 min. The low-aw S. Typhimurium cells treated with 200 mM H2O2 for 30 min served as the positive control. Here the arbitrary value I = (It − Io)/Io, where It and Io are the mean fluorescence values of the treated and untreated low-aw S. typhimurium, represents the intracellular ROS generation.
Log (CFU/g)Weight Loss (%)Surface Temperature (°C)
Control12.43 ± 0.06 a0.00 ± 0.00 b23.78 ± 0.69 b
455 nm treated11.57 ± 0.18 b9.66 ± 1.57 a52.33 ± 2.52 a
Unoxidized percentage of cell population as per C11 BODIPY assay (%)Oxidized percentage of cell population as per C11 BODIPY assay (%)Arbitrary value (I) representing the intracellular ROS formation
Control86.09 ± 12.93 a15.03 ± 3.28 b0.00 ± 0.00 a
H2O2 treatment19.09 ± 09.89 b76.82 ± 23.96 a1.22 ± 0.52 b
455 nm treated20.29 ± 14.72 b75.94 ± 12.91 a1.19 ± 0.45 b
Results are represented as means ± standard deviation of three independent replicates. Values with different superscripts in each column corresponding to a specific assay differ significantly (p < 0.05).
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Prasad, A.; Gänzle, M.; Roopesh, M.S. Understanding the Salmonella Inactivation Mechanisms of 365, 395 and 455 nm Light Pulses Emitted from Light-Emitting Diodes. Appl. Sci. 2023, 13, 1501. https://doi.org/10.3390/app13031501

AMA Style

Prasad A, Gänzle M, Roopesh MS. Understanding the Salmonella Inactivation Mechanisms of 365, 395 and 455 nm Light Pulses Emitted from Light-Emitting Diodes. Applied Sciences. 2023; 13(3):1501. https://doi.org/10.3390/app13031501

Chicago/Turabian Style

Prasad, Amritha, Michael Gänzle, and M. S. Roopesh. 2023. "Understanding the Salmonella Inactivation Mechanisms of 365, 395 and 455 nm Light Pulses Emitted from Light-Emitting Diodes" Applied Sciences 13, no. 3: 1501. https://doi.org/10.3390/app13031501

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