After treating V. parahaemolyticus with PAW 20 and PAW 30 for varying durations (0, 3, 10, and 30 s), bacterial cells were collected by centrifugation (3-30 KS, Sigma, Germany) at 4°C for 10 min at 8000 r/min. The cells were then fixed overnight at 4°C in 1 mL of 2.5% (v/v) glutaraldehyde. After washing twice with PBS, a series of ethanol concentrations (30, 50, 70, 90, and 100%) were employed to dehydrate the cells. Subsequently, they were freeze-dried overnight using a freeze-dryer (FDU-1100, Shanghai, China). After being glued onto a sticky stage and gold sprayed, the bacterial powder was observed under a scanning electron microscope (S3400, Hitachi, Tokyo, Japan) at a magnification of 20,000×.

The nucleic acid and protein leakage rates of bacterial solutions were determined after PAW treatments (0, 3, 5, 10, 20, and 30 s) (18). The samples were centrifuged at 4°C for 10 min with a speed of 8,000 r/min, and pure water was used as the reference. The leakage of nucleic acids and proteins were measured using a UV-2550 UV–Vis spectrophotometer (Shimadzu, Kyoto, Japan), and the absorbance values were measured at 260 nm and 280 nm, respectively. The following Equations (1) and (2) were used to determine the leakage ratio,

The permeability of V. parahaemolyticus outer membrane was assessed by investigating the fluorescence intensity of NPN. After PAW treatment for varying durations (0, 3, 5, 10, 20, and 30 s), the bacterial solution was centrifuged (4°C, 2 min with a speed of 10,000 r/min) and resuspended in 1 mL of PBS buffer (0.1 mol/L, pH 7.2). Then, 20 μL of NPN solution was added into 1 mL of the bacterial suspension to make a final concentration of 10 μmol/L. The mixture was incubated at 25°C for 10 min in darkness. To measure the fluorescence intensity, a multifunctional enzyme marker (Molecular Devices, California, United States) with an excitation wavelength of 350 nm and an emission wavelength of 401 nm was applied. The following Equation (3) was used to calculate the relative fluorescence intensity of NPN.

where, F₀ represents the fluorescence intensity of the untreated PAW bacterial suspension, and F₁ represents the fluorescence intensity of the PAW-treated bacterial suspension.

PI fluorescence intensity was used to evaluate V. parahaemolyticus cell membrane permeability. After PAW treatment for varying durations (0, 3, 5, 10, 20, and 30 s), the bacterial solution was centrifuged (4°C, 2 min with a speed of 10,000 r/min) and then resuspended in 1 mL of PBS buffer (0.1 mol/L, pH 7.2). Then, 20 μL of PI solution was added into 1 mL of the bacterial suspension to make a final concentration 3 μmol/L. The mixture was incubated at room temperature for 30 min in darkness. After centrifuging at 4°C for 2 min at 10000 r/min, the mixture was washed twice with PBS, and resuspended. A multifunctional enzyme marker with an excitation wavelength of 482 nm and an emission wavelength of 635 nm was used to measure fluorescence intensity. The results were expressed as the relative fluorescence intensity using the following Equation (4),

where, F₀ represents the fluorescence intensity of the untreated PAW bacterial suspension, and F₁ represents the fluorescence intensity of the PAW-treated bacterial suspension.

After PAW treatment for varying durations (0, 3, 5, 10, 20, and 30 s), the bacterial solution was centrifuged at 4°C for 10 min with a speed of 8,000 r/min and then resuspended in 1 mL of PBS buffer (0.1 mol/L, pH 7.2). The EC was measured immediately using an electric conductivity meter (DDB-303A, Shanghai, China).

The intracellular levels of ROS were evaluated using 2′,7′-dichlorofluorescent yellow diacetate (DCFH-DA). After PAW treatment for varying durations (0, 3, 5, 10, 20, and 30 s), the bacterial solution was centrifuged at 4°C for 10 min with a speed of 8,000 r/min. Then, the pellet was resuspended using 2 mL of DCFH-DA (10 μmol/L) and the solution was incubated in darkness for 30 min. Following the reaction, the solution was centrifugated at 4°C for 2 min with a speed of 10,000 r/min. The pellet was resuspended using an equal volume of PBS. A fluorescence spectrophotometer was used to detect fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

The enzyme activity of SOD was measured according to the kit instructions (Solarbio, Beijing, China). After treatment, the bacterial solution was centrifuged at 4°C for 10 min with a speed of 8,000 r/min. Then, the pellet was resuspended using 1 mL of extract solution in the kit. The mixture was sonicated and centrifuged at 4°C for 10 min with a speed of 8,000 r/min. The supernatant was used to measure the absorbance at 560 nm.

Quantitative Real-Time Polymerase Chain Reactions (qPCR) were used to determine the expression levels of target genes within related biofilms. To verify changes in the expression of biofilm-related genes in the treatment groups compared with the control group, target genes (emrD, luxS, ompW) were selected (19).

The RNA extraction kit (Sangon Biotech, Shanghai, China) was used to extract total RNA from the sample cells, which was then quantified using an ELISA (Molecular Devices, California, United States). RNA yield and integrity were assessed through absorbance and agarose gel electrophoresis, respectively. To generate complementary DNA (cDNA), cDNA Synthesis Kit Ver 2 (Tsingke, Beijing, China) was used for random reverse transcription. For each qCR reaction, the mixture comprised 25 μL of SYBR Green Dye Method Mix (2×), 2.5 μL of each primer (100 μM), 2.5 μL of cDNA, and 17.5 μL of ddH₂O. The cycling parameters were set as follows: an initial denaturation step at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s. Subsequently, a melting curve analysis of 95°C for 5 s and 60°C for 1 min was performed, followed by a gradual increase in temperature from 60°C to 95°C at a rate of 0.15°C per second while continuously collecting fluorescence data. The primers were synthesized by Bioengineering Co, China and the sequences were presented in Table 1. The changes in relative gene expression were calculated with the 2^−ΔΔCT method using the following Equations (5), (6), and (7):

Changes in the cell morphology of V. parahaemolyticus after PAW treatment are shown in Figure 3. Scanning electron microscopy (SEM) images revealed that the surface of the untreated group of V. parahaemolyticus cells displayed an intact, well-defined, rod-shaped structure with clear boundaries. However, after PAW treatment, V. parahaemolyticus cells underwent severe deformation, resulting in a shriveled, irregular appearance. The cell surfaces displayed marked wrinkling, and immersion for 30 s resulted in noteworthy leakage of intracellular solutes. These findings were similar with studies by Liu et al. (26), who reported cell surface damage and disruptions following PAW treatment. The cell surface appeared to be obviously wavy and deep “holes.” These results demonstrate that PAW effectively disrupts cell membrane structures, leading to the leakage of intracellular components and bacterial inactivation.

The leakage of nucleic acids and proteins from cells are general indicators of microbial damage. The extravasation of intracellular substances is a good criterion for studying the integrity of cell membranes. When the bacterial cells are damaged, the internal solution leaked out of the cells. Therefore, the extent of cellular damage can be determined by measuring the leakage rate of nucleic acids and proteins in the supernatant. To assess cell membrane integrity, the leakage of nucleic acids and proteins of V. parahaemolyticus exposed to PAW treatment was measured (Figures 4A,B). The leakage rates of proteins and nucleic acids increased with longer plasma discharge time and immersion time compared with the control group (p < 0.05). With longer plasma discharge time and immersion time, the leakage ratio of DNA/RNA significantly increased from initial value 1.06 to 1.78 for PAW 30 (p < 0.05), and the leakage ratio of proteins significantly increased from initial value 1.05 to 1.91 for PAW 30 (p < 0.05). Studies have shown that PAW contains many active substances, such as H₂O₂, O₃, NO₂⁻, and NO₃⁻, among others (27). These active substances penetrate bacterial cells, damaging cell membranes, and inducing oxidative damage to nucleic acids, proteins, and other cellular components (28, 29). Xiang et al. (18) also reported a significant increase in intracellular leakage of nucleic acids and proteins with prolonged PAW treatment times. Thus, PAW destroyed the cellular membrane of V. parahaemolyticus and caused the leakage of intracellular solutes that becomes more pronounced with longer immersion times.

The outer membrane of Gram-negative bacteria is crucial when it comes to adapting to various ambient pressure such as heat, acid, and antibiotics. To assess the impact of PAW on outer membrane permeability, the intracellular fluorescence intensity of NPN was measured (Table 3). NPN is a fluorescent probe that fluoresces strongly in non-polar or hydrophobic environments, making it suitable for evaluating Gram-negative bacterial outer membrane damage (26). The results demonstrated that the fluorescence intensity of NPN increased with longer plasma discharge time and PAW immersion time. After treatment of PAW 30 for 3, 5, 10, 20, and 30 s, the fluorescence intensity of NPN increased by 110.81, 130.04, 135.01, 136.35, and 148.82%, respectively. These findings suggest that PAW treatment destroyed the outer membrane of V. parahaemolyticus cells as compared with the control group.

The permeability of the inner membrane was assessed using PI, which can penetrate damaged cell membranes and bind to DNA, emitting red fluorescence when excited at 540 nm (30). As shown in Table 3, the relative fluorescence intensity of PI in the PAW-treated group was generally higher than that in the control group. With the increase of plasma discharge time, the relative fluorescence intensity of PI in the PAW treatment group increased gradually (p < 0.05), indicating a time-dependent increase in cell membrane penetration. This observation is consistent with the study by Liu et al. (16), who indicated that the interaction between ROS and RNS in PAW may destroy the membrane. However, further investigation is needed to identify the specific active substances and interactions for membrane disruption.

The cell membrane is an important protective barrier for bacterial cells. When bacterial cells are in an unfavorable environment, the cell membrane becomes damaged, resulting in the change in cell membrane permeability. This is followed by the leakage of internal electrolytes and increased EC (31). Therefore, the change in cell membrane permeability can be reflected by measuring the change in EC of the bacterial suspension. As illustrated in Table 3, EC increased with longer plasma discharge time and immersion time (p < 0.05). The highest conductivity was observed after 30 s of PAW immersion. The increase in EC is attributed to the damage to the cell membrane, which caused leakage of intracellular electrolytes into the solution, ultimately increasing conductivity.

The DCFH-DA probe was utilized to measure intracellular ROS levels in V. parahaemolyticus after PAW treatment (Figure 5A). The relative expression level of intracellular ROS increased dramatically with the increasing plasma discharge time and immersion time (p < 0.05). The highest ROS accumulation was observed in V. parahaemolyticus cells immersed in PAW 20 for 30 s, with a relative expression level reaching 1.97-fold. These results demonstrate that PAW stimulates high ROS generation in V. parahaemolyticus cells, resulting in substantial oxidative damage to the cell membrane. The intracellular ROS are crucial for maintaining cell function and signal transduction. However, when intracellular ROS exceed the cellular antioxidative threshold, large amount of ROS cause irreversible oxidative damage to biomacromolecules including DNAs, proteins, lipids, and enzymatic systems from inside, thereby disrupting the physiological functions of bacterial (32). Additionally, the active substances in PAW, such as H₂O₂, O₃, ONOO⁻, NO₂⁻, and NO₃⁻, may induce oxidative injury to bacterial cell membrane lipids and intracellular nucleic acids and proteins, leading to oxidative stress and cell dysfunction (33).

SOD is a metalloenzyme widely found in living organisms and is an important oxygen radical scavenger that catalyzes the disproportionation of superoxide anions to produce H₂O₂ and O₂, which has an important role in the antioxidant system. A gradual decrease in SOD activity was observed with longer plasma discharge time and PAW immersion time (Figure 5B). After 30 s of PAW immersion, SOD activity in V. parahaemolyticus treated with PAW 5, PAW 10, PAW 20, and PAW 30 decreased by 61.47, 60.44, 66.50, and 72.74%, respectively. These results indicate that PAW may destroy the antioxidant defense system of V. parahaemolyticus, promoting peroxidation process (16).

To understand the mechanisms of cell membrane damage, the relative expression levels of the ompW, emrD, and luxS genes were evaluated by qRT-PCR (Figure 5C) (6). As an outer membrane protein synthesis gene, ompW has functions in disrupting the regular transport of biofilm formation substances, interfering with the early adhesion of bacterial cells, and inhibiting the growth of biofilms (34). As a drug resistance and metabolism-related gene, emrD can interfere with metabolic function and reduce drug resistance (35). The population-sensing gene luxS is a key gene that affects the normal function of other population-sensing signaling systems (36). As shown in Figure 5C, the relative expression levels of ompW, emrD, and luxS genes of V. parahaemolyticus dramatically decreased after PAW treatment (p < 0.05). The result suggests that PAW destroy the normal physiological balance in V. parahaemolyticus cells by regulating gene expression. This disruption leads to cell membrane damage, impairing normal bacterial function and contributing to enhanced bactericidal efficiency (37).