Antibacterial Effect of Eugenol on Shigella flexneri and Its Mechanism

Shigella flexneri (Sh. flexneri), which can be found in food and the environment, is a widespread food-borne pathogen that causes human diarrhea termed “shigellosis”. In this study, eugenol, a natural active substance, was investigated for its antibacterial activity against Sh. flexneri. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of eugenol against Sh. flexneri ATCC 12022 was 0.5 and 0.8 mg/mL. The growth curves and inhibitory effect in LB broth, PBS, vegetable juice, and minced pork showed that eugenol had a good activity against Sh. flexneri. Research findings indicated the superoxide dismutase activity of Sh. flexneri was inhibited after eugenol treatment, resulting in concentrations of intracellular reactive oxygen species and an increase in malondialdehyde. The flow cytometry analysis and field emission scanning electron microscopy results revealed obvious damage to cell membrane integrity and changes in the morphology of Sh. flexneri. In addition, the intracellular ATP concentration leaked from 0.5 μM to below 0.05 μM and the membrane potential showed a concentration-dependent depolarization after eugenol treatment. In summary, eugenol exerted strong antibacterial activity and has the potential to control Sh. flexneri in the food industry.


Introduction
Shigella flexneri (Sh. flexneri), which is a rod-shaped, non-spore forming, non-flagella, facultative anaerobic Gram-negative bacterium [1], has been confirmed to be widespread in vegetable salads, meat, raw milk, sea food and other products [2]. In addition, as it tends to spread rapidly in closed and concentrated groups, Sh. flexneri has been detected in schools, restaurants, and other environments [3]. Sh. flexneri can cause bacillary dysentery with clinical symptoms as painful abdominal cramps, nausea, fever, and even colitis [4]. Dysentery caused by Sh. flexneri is considered to be the most common explosive disease in developing countries [5], which has led to 1.7 million episodes of bacillary dysentery in China per year. Additionally, it has become one of the major causes of morbidity and mortality in children with diarrhea in developing countries [6]. Furthermore, the high survival rate and strong tolerance of Sh. flexneri makes it able to survive for extended periods under adverse conditions, such as high acidity or high temperatures. What is more, it can even remain viable following several hours of exposure at pH 2-3 [7]. Thus, the tolerance of Sh. flexneri to low-pH conditions may enhance survival in acidic foods and also in the acidic environment of the human stomach, thereby increasing the potential for causing infection [8].
The control of Sh. flexneri in food has usually adopted sterilization methods, such as heat sterilization, ultra-high pressure, UV, or adding antibacterial substances to achieve

Determinations of MIC and MBC
These assays were determined using the broth microdilution method as recommended by the Clinical and Laboratory Standards Institute [22], with minor modifications. The bacterial suspension, which was prepared as described in Section 2.2, was diluted to approximately 5 × 10 5 CFU/mL in LB broth. Eugenol was diluted in LB broth and was then mixed with an equal volume of bacterial cultures to obtain the final eugenol concentrations of 0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, and 2 mg/mL. Two-hundred microliters of the mixed liquor were added into each well of the 96-well microtiter plate, after which samples were incubated at 37 • C for 24 h. The MIC was defined as the lowest eugenol concentration that bacteria after incubation were tested without producing any visible growth. To determine the MBC, 100 µL of bacterial solution from each well was plated on an LB agar plate and cultured for 48 h. MBC was defined as the lowest eugenol concentration that completely inhibited all bacteria.

Growth Curve Analysis
The determination of the effect of eugenol on the growth curve of Sh. flexneri was done according to the method of Bsbii et al. [23], with minor modifications. The activated Sh. flexneri was cultured as described in Section 2.2 above, and the bacterial suspension was diluted to 2 × 10 6 CFU/mL by LB broth. LB broth dissolved in 0.5% DMSO and different concentrations of eugenol were mixed with equal volumes of bacterial suspension and vortexed, which made the final concentration of eugenol 0 (Control), 1/16MIC, 1/8MIC, 1/4MIC, 1/2MIC, MIC, or 2MIC, respectively. At the same time, the LB broth containing 0.5% DMSO was used as the blank background control. After 200 µL of the mixed solution was added to each well of the honeycomb plate, the growth rate was measured by an automated microbiology growth curve analyzer (Bioscreen C; Labsystems, Helsinki, Finland) at 600 nm at 2 h intervals, which was cultured at 37 • C for 24 h.

Inactivation Effect of Eugenol on Sh. flexneri in LB and PBS
The inactivation effect of eugenol on Sh. flexneri was performed according to the procedure describe by Gao et al. [24], with slight modifications. The bacterial suspension that was prepared according to the method from Section 2.2 was diluted to approximately 1 × 10 6 CFU/mL in LB broth or PBS. Then, equal volumes of LB broth with eugenol dissolved in it were mixed with the individual samples to obtain final eugenol concentrations of 0 (Control), MIC, 3/2MIC, and 2MIC. Similarly, bacteria were treated with 0 (Control), 1/2MIC, MIC, 3/2MIC, and 2MIC eugenol in PBS. After being treated for 0, 0.5, 1, 2, 4, 6, and 8 h at 37 • C, the mixed bacterial suspension was spread on LB agar plates and incubated at 37 • C for 12 h. The total number of colonies of Sh. flexneri were expressed as log CFU/mL. The method for the inactivation effect of eugenol against Sh. flexneri in vegetable juice was carried out according to the method described by Yuan et al. [25], with modifications. The lettuce juice, which was sterilized by irradiation in advance, was incubated with Sh. flexneri to give a final concentration of 1 × 10 6 CFU/mL. Then, eugenol solution was added to the individual samples to achieve the final eugenol concentrations of 0 (Control), MIC, 3/2MIC, and 2MIC, and samples were further placed at 25 • C. The number of Sh. flexneri ATCC 12022 treated by eugenol for 0, 0.5, 1, 2, 4, 6, and 8 h was enumerated by spreading the bacterial suspension on LB agar plates after a 12-h incubation at 37 • C.

Inactivation Effect of Eugenol on Sh. flexneri in Minced Pork
In view of the method used by Sun et al. [26], the inhibitory effect of eugenol on Sh. flexneri in minced pork was explored. The pure lean pork was irradiated and sterilized, and then the cleaned pork was broken into minced meat and packed in homogeneous bags. The Sh. flexneri suspensions prepared in Section 2.2 were grown to an OD 600 value of 0.5 in 0.1% (v/v) BPW. Next, the bacterial suspension (1 × 10 6 CFU/mL) was added to the above homogeneous bags and the attachment of bacteria was completed. Then, the eugenol was added to the individual bacterial suspension bags so that the final concentration was 0 (Control), 5MIC, 10MIC, and 15MIC, respectively. After being mixed uniformly, homogeneous bags with mixed pork were placed in a 10 • C constant temperature incubator to simulate processing and storage. A small portion of the sample was removed from each mixture at 0, 1, 2, 4, 6, and 8 h, was weighed, and then was eluted with 1 mL of BPW to spread on LB agar plates, and finally, was incubated at 37 • C for 12 h. The viable bacterial cells of Sh. flexneri were counted after incubation, and colonies were enumerated and calculated as log CFU/g.

Assays of ATP Concentrations
The method for determining the intracellular ATP concentration change described by Guo et al. [27] was followed. The bacterial suspension prepared according to the method in Section 2.2 was mixed with eugenol solution with different concentrations to make the final concentration of eugenol in the sample 0 (Control), MIC, 2MIC, and 4MIC, respectively. The negative control group was determined by adding different concentrations of eugenol to PBS. Both sets of samples were incubated at 37 • C for 30 min, then transferred to an ice bath and subjected to ultrasonic cell lysis one by one. After lysis, samples were placed at 100 • C for 3 min immediately, which deactivated the ATPase in the sample. The bacterial solution was centrifuged (10,000× g, 4 • C, 5 min) and the supernatant was stored in an ice bath. The supernatant (100 µL) was inoculated into a white 96-well microplate before prepared ATP detection reagent (100 µL) was added to each well and then the luminescence intensity of the mixture was detected by a multimode microplate reader platform (Spark ® ; Tecan, Männedorf, Switzerland). The ATP working curve was drawn by measuring the luminescence intensities of the ATP standard solutions with final concentrations of 0.01, 0.1, 1, and 10 µmol/L, so that the intracellular ATP concentration can be calculated.

Measurement of Membrane Potential
With reference to the method of Guffey et al. [28], the effect of eugenol on the membrane potential of Sh. flexneri was determined with the help of DiBAC 4 (3) anionic fluorescent dye. The bacterial suspension (125 µL) was cultured according to method in Section 2.2 and was added into the black 96-well microplate and incubated at 37 • C for 30 min, after which the anionic fluorescent dye DiBAC 4 (3) was diluted to a final concentration of 1 µmol/L in each well. The different concentrations of eugenol diluent (125 µL) were added to the black microplate to make the final concentration of original bacterial suspension 0, MIC, 2MIC, and 4MIC, respectively. The fluorescence intensity was measured every 5 min at the excitation and emission wavelengths of 492 and 515 nm for 30 min. The background fluorescence resulting from the eugenol added to the PBS was determined and the results were corrected. The membrane potential was expressed as the relative fluorescence intensity of eugenol-treated cells compared to that of the control cells.

Flow Cytometry Investigation
The bacterial cell membrane permeability was detected by staining with propidium iodide (PI) and SYTO 9, followed by analysis using a flow cytometer (CytoFLEX; Beckman, Brea, CA, USA) as described by Wang et al. [29], with some modifications. Briefly, the cell suspension (approximately 1 × 10 6 CFU/mL) was cultured with eugenol (0, MIC, 2MIC, 4MIC) at 37 • C for 1 h. After treatment, the bacterial cells were collected by centrifugation Subsequently, the cells were incubated with 1 µL of an equal volume of SYTO 9 and PI mixed dye for 15 min in the dark. Then, the extent of cell membrane permeability was detected using a flow cytometer.

Effect of Eugenol on Intracellular ROS Production
The level of intracellular ROS generation was estimated using 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology, Shanghai, China) by the method of Li et al. [30]. Bacteria cultured in Section 2.2 were collected by centrifugation at 8000× g for 5 min at 4 • C and washed twice with PBS. After eugenol treatment (0, 1/2MIC, MIC, 2MIC) for 20 min, samples were incubated with DCFH-DA at a final concentration of 5 µmol/L for 15 min at 37 • C in the dark. The blank background control was established by a combination of PBS and DCFH-DA without any pathogens. The strained cells were collected by centrifugation at 12,000× g for 10 min, then washed with PBS twice. The fluorescence was measured with a multimode microplate reader platform (Spark ® ; Tecan, Männedorf, Switzerland) at excitation and emission wavelengths of 488 and 525 nm, respectively. Aliquots taken from each mixture were placed onto LB agar plates to determine the viable bacterial number. The fluorescence intensity of the blank background control was subtracted from that of the treated cells and the data were normalized against the viable bacterial number.

SOD Activity
The effects of eugenol on SOD in Sh. flexneri were studied according to Shi et al. [31], with minor modifications. The cells were harvested after being cultured according to Section 2.2 and were treated with PBS (Control) or eugenol (1/2MIC, MIC, 2MIC) at 37 • C for 20 min. Before the lysozyme (Solarbio, Beijing, China) was lysed at 37 • C for 6 min, bacteria were harvested. Then, the supernatant was collected by centrifugation (10,000× g, 3 min, 4 • C). The NBT/enzyme and reaction start working solution were configured under the guidance of the manual. The NBT/enzyme working solution, the extracted supernatant, and the reaction initiation working solution were sequentially added to the wells, and the 96-well plate was incubated for 30 min at 37 • C, and then the absorbance was read at 560 nm using a microplate reader (Spark ® ; Tecan, Männedorf, Switzerland).

Determination of Lipid Peroxidation
The micro-MDA assay kit (Beijing Solarbio Science & Technology, Beijing, China) was used for measuring the formation of MDA as previously described, with some modifications [32]. In brief, the bacteria were exposed to eugenol (0 (Control), 1/2MIC, MIC, 2MIC) for 20 min at 37 • C, were then harvested by centrifugation at 8000× g for 10 min, and then the supernatant was discarded. After the extracting solution (1 mL) was added to the individual samples, which were placed in an ice bath, the samples were subjected to ultrasound for 6 min under 3/6 s pulse on/off conditions one by one. After lysis, the supernatant and MDA detection working solution were mixed and heated at 100 • C for 10 min and then cooled, followed by centrifugation at 4 • C and 10,000× g for 10 min. The blank control group was established by a combination of the MDA detection working solution and ultra-pure water. The absorbance of the supernatant at 450 nm, 532 nm, and 600 nm was measured by a multimode microplate reader platform (Spark ® ; Tecan, Männedorf, Switzerland), and the result was calculated according to the formula below: where ∆OD 532 , ∆OD 600 , and ∆OD 450 refer to the difference between the absorbance of the positive control group and blank control ground at 532, 600, and 450 nm, respectively.

Statistical Analysis
All assays were performed in triplicate and all experimental data were analyzed using the SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). A difference analysis was performed using a least significant difference test and the results were expressed as the mean ± standard deviation. Significance was expressed as: *, p < 0.05; **, p < 0.01.

MIC and MBC of Eugenol
As shown in Figure 1A, when Sh. flexneri was treated with eugenol at 0.5 mg/mL, the ∆OD 600nm was less than 0.05. Therefore, the MIC value of eugenol was 0.5 mg/mL for Sh. flexneri ATCC 12022. As shown in Figure 1B, the samples at 0.5 mg/mL had bacterial growth on LB agar, while the samples at 0.8, 1.0, and 1.6 mg/mL had no colonies growing on LB agar, indicating that the bacteria were inactivated by eugenol. Therefore, the MBC was 0.8 mg/mL. overnight at 4 °C to fix the cells. After centrifugation, cells were eluted with a w anol gradient (30%, 50%, 70%, 80%, 90%, 100%, v/v) for 10 min before being fixed slides. Following gold spray treatment under vacuum, the cells were observe field-emission scanning electron microscope (S-4800; Hitachi, Tokyo, Japan).

Statistical Analysis
All assays were performed in triplicate and all experimental data were ana ing the SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). A difference analysis formed using a least significant difference test and the results were expressed as ± standard deviation. Significance was expressed as: *, p < 0.05; **, p < 0.01.

MIC and MBC of Eugenol
As shown in Figure 1A, when Sh. flexneri was treated with eugenol at 0.5 m ∆OD600nm was less than 0.05. Therefore, the MIC value of eugenol was 0.5 mg/m flexneri ATCC 12022. As shown in Figure 1B, the samples at 0.5 mg/mL had growth on LB agar, while the samples at 0.8, 1.0, and 1.6 mg/mL had no colonies on LB agar, indicating that the bacteria were inactivated by eugenol. Therefore, was 0.8 mg/mL.

Effects of Eugenol on Sh. flexneri Growth Curve
The results showed that the growth of Sh. flexneri was completely inhibited nol at MIC and 2MIC (Figure 2). Sh. flexneri treated with eugenol at 1/2MIC sh obvious reduction in growth when compared with the control, and the populati

Effects of Eugenol on Sh. flexneri Growth Curve
The results showed that the growth of Sh. flexneri was completely inhibited by eugenol at MIC and 2MIC (Figure 2). Sh. flexneri treated with eugenol at 1/2MIC showed an obvious reduction in growth when compared with the control, and the populations of Sh. flexneri treated with eugenol at 1/4MIC, 1/8MIC, and 1/16MIC were reduced compared with the control group.

Inactivation Effect of Eugenol on Sh. flexneri in LB and PBS
The inhibitory effects of eugenol on Sh. flexneri in LB and PBS were shown in 3. The initial concentration of Sh. flexneri in the bacterial solution was about CFU/mL. The population of Sh. flexneri without eugenol treatment increased to log CFU/mL after 8 h. In the nutrient medium LB broth ( Figure 3A), after 0.5 h, th lation of Sh. flexneri decreased to below the detection limit (1 CFU/mL) after tr with eugenol at 2MIC. In the non-nutrient medium PBS, the population of Sh. treated with eugenol at 2MIC, 3/2MIC, and MIC fell below the detection limit at and 8 h, respectively ( Figure 3B).

Inactivation Effect of Eugenol on Sh. flexneri in Vegetable Juice
The survival of Sh. flexneri treated with eugenol in lettuce juice ( Figure 4) wa ined. The initial concentration of the bacterial solution was about 6.0 log CFU/m population of Sh. flexneri without eugenol treatment increased to 6.6 ± 0.1 log C after 8 h. After 8 h, the population of Sh. flexneri treated with eugenol at MIC and decreased by 5.52 ± 0.21 and 5.10 ± 0.26 log CFU/mL, which was close to the un control. However, the number of Sh. flexneri treated with eugenol at 2MIC dro below the limit of detection (1 CFU/mL) after 0.5 h.

Inactivation Effect of Eugenol on Sh. flexneri in LB and PBS
The inhibitory effects of eugenol on Sh. flexneri in LB and PBS were shown in Figure 3. The initial concentration of Sh. flexneri in the bacterial solution was about 6.0 log CFU/mL. The population of Sh. flexneri without eugenol treatment increased to 8.3 ± 0.1 log CFU/mL after 8 h. In the nutrient medium LB broth ( Figure 3A), after 0.5 h, the population of Sh. flexneri decreased to below the detection limit (1 CFU/mL) after treatment with eugenol at 2MIC. In the non-nutrient medium PBS, the population of Sh. flexneri treated with eugenol at 2MIC, 3/2MIC, and MIC fell below the detection limit at 1 h, 4 h, and 8 h, respectively ( Figure 3B).

Inactivation Effect of Eugenol on Sh. flexneri in LB and PBS
The inhibitory effects of eugenol on Sh. flexneri in LB and PBS were shown in Fig  3. The initial concentration of Sh. flexneri in the bacterial solution was about 6.0 CFU/mL. The population of Sh. flexneri without eugenol treatment increased to 8.3 ± log CFU/mL after 8 h. In the nutrient medium LB broth ( Figure 3A), after 0.5 h, the po lation of Sh. flexneri decreased to below the detection limit (1 CFU/mL) after treatm with eugenol at 2MIC. In the non-nutrient medium PBS, the population of Sh. flex treated with eugenol at 2MIC, 3/2MIC, and MIC fell below the detection limit at 1 h, and 8 h, respectively ( Figure 3B).    The survival of Sh. flexneri treated with eugenol in lettuce juice (Figure 4) was examined. The initial concentration of the bacterial solution was about 6.0 log CFU/mL. The population of Sh. flexneri without eugenol treatment increased to 6.6 ± 0.1 log CFU/mL after 8 h. After 8 h, the population of Sh. flexneri treated with eugenol at MIC and 3/2MIC decreased by 5.52 ± 0.21 and 5.10 ± 0.26 log CFU/mL, which was close to the untreated control. However, the number of Sh. flexneri treated with eugenol at 2MIC dropped to below the limit of detection (1 CFU/mL) after 0.5 h.

Inactivation Effect of Eugenol on Sh. flexneri in Minced Pork
The antibacterial effects of eugenol on Sh. flexneri in ground pork were shown ure 5. The initial concentration of the bacterial solution was about 7.2 ± 0.2 log CFU population of Sh. flexneri without eugenol treatment increased to 7.3 ± 0.1 log CFU 8 h. The samples at 5MIC decreased the concentrations of bacteria to about 6.6 ± CFU/g after being treated for 6 h. However, the population of Sh. flexneri treate eugenol at 10MIC and 15MIC fell below the detection limit at 2 h and 6 h, respecti

Effects of Eugenol on Intracellular ATP Concentration of Sh. flexneri
The effects of eugenol on intracellular ATP concentrations of Sh. flexneri ATCC were shown in Figure 6. There was a good linearity between the relative lumine units and ATP concentration (y = 408,791x + 3732.4, R 2 = 0.9999). Compared with t trol, the intracellular ATP concentrations of the bacteria were significantly reduce being treated with eugenol. The intracellular ATP concentration of bacteria in the was 0.451 ± 0.004 μM, while the intracellular ATP concentrations of the eugenolbacteria (MIC, 2MIC and 4MIC) were significantly reduced (p < 0.01) to below 0.05

Inactivation Effect of Eugenol on Sh. flexneri in Minced Pork
The antibacterial effects of eugenol on Sh. flexneri in ground pork were shown in Figure 5. The initial concentration of the bacterial solution was about 7.2 ± 0.2 log CFU/g. The population of Sh. flexneri without eugenol treatment increased to 7.3 ± 0.1 log CFU/g after 8 h. The samples at 5MIC decreased the concentrations of bacteria to about 6.6 ± 0.1 log CFU/g after being treated for 6 h. However, the population of Sh. flexneri treated with eugenol at 10MIC and 15MIC fell below the detection limit at 2 h and 6 h, respectively.

Inactivation Effect of Eugenol on Sh. flexneri in Minced Pork
The antibacterial effects of eugenol on Sh. flexneri in ground pork were shown ure 5. The initial concentration of the bacterial solution was about 7.2 ± 0.2 log CFU population of Sh. flexneri without eugenol treatment increased to 7.3 ± 0.1 log CFU 8 h. The samples at 5MIC decreased the concentrations of bacteria to about 6.6 ± CFU/g after being treated for 6 h. However, the population of Sh. flexneri treate eugenol at 10MIC and 15MIC fell below the detection limit at 2 h and 6 h, respecti

Effects of Eugenol on Intracellular ATP Concentration of Sh. flexneri
The effects of eugenol on intracellular ATP concentrations of Sh. flexneri ATCC were shown in Figure 6. There was a good linearity between the relative lumin units and ATP concentration (y = 408,791x + 3732.4, R 2 = 0.9999). Compared with t trol, the intracellular ATP concentrations of the bacteria were significantly reduc being treated with eugenol. The intracellular ATP concentration of bacteria in the was 0.451 ± 0.004 μM, while the intracellular ATP concentrations of the eugenolbacteria (MIC, 2MIC and 4MIC) were significantly reduced (p < 0.01) to below 0.05

Effects of Eugenol on Intracellular ATP Concentration of Sh. flexneri
The effects of eugenol on intracellular ATP concentrations of Sh. flexneri ATCC 12022 were shown in Figure 6. There was a good linearity between the relative luminescence units and ATP concentration (y = 408,791x + 3732.4, R 2 = 0.9999). Compared with the control, the intracellular ATP concentrations of the bacteria were significantly reduced after being treated with eugenol. The intracellular ATP concentration of bacteria in the control was 0.451 ± 0.004 µM, while the intracellular ATP concentrations of the eugenol-treated bacteria (MIC, 2MIC and 4MIC) were significantly reduced (p < 0.01) to below 0.05 µM.

Effects of Eugenol on Cell Membrane Potential of Sh. flexneri
As shown in Figure 7, the membrane potential of Sh. flexneri cells treated wit nol indicated membrane depolarization as a result of an increase in membrane p compared with the control, and the degree of depolarization increased with the i in eugenol concentrations and the incubation time during those 5-10 min.

Effects of Eugenol on Cell Membrane Integrity of Sh. flexneri
The changes in the cell membrane integrity of Sh. flexneri were detected by f tometry, which is often used for the qualitive and quantitative detection of cel quadrants were used to represent different cell states, with H1-LL, H1-LR, H1-U H1-UL representing unstained bacteria, stained by SYTO 9 only, stained by SYT PI, and stained by PI only, respectively. As shown in Figure 8, compared with the eugenol could decrease the SYTO 9-stained (H1-LR) rate of cells while increasing t of cells strained by SYTO 9 and PI simultaneously (H1-UR). The PI-labeled pop (H1-UL) in the 0 (Control), MIC, 2MIC, and 4MIC eugenol-treated groups were

Effects of Eugenol on Cell Membrane Potential of Sh. flexneri
As shown in Figure 7, the membrane potential of Sh. flexneri cells treated with eugenol indicated membrane depolarization as a result of an increase in membrane potential compared with the control, and the degree of depolarization increased with the increase in eugenol concentrations and the incubation time during those 5-10 min.

Effects of Eugenol on Cell Membrane Potential of Sh. flexneri
As shown in Figure 7, the membrane potential of Sh. flexneri cells treated wit nol indicated membrane depolarization as a result of an increase in membrane p compared with the control, and the degree of depolarization increased with the i in eugenol concentrations and the incubation time during those 5-10 min.

Effects of Eugenol on Cell Membrane Integrity of Sh. flexneri
The changes in the cell membrane integrity of Sh. flexneri were detected by f tometry, which is often used for the qualitive and quantitative detection of cel quadrants were used to represent different cell states, with H1-LL, H1-LR, H1-U H1-UL representing unstained bacteria, stained by SYTO 9 only, stained by SYTO PI, and stained by PI only, respectively. As shown in Figure 8, compared with the eugenol could decrease the SYTO 9-stained (H1-LR) rate of cells while increasing t of cells strained by SYTO 9 and PI simultaneously (H1-UR). The PI-labeled popu (H1-UL) in the 0 (Control), MIC, 2MIC, and 4MIC eugenol-treated groups were 0.42%, 1.94%, and 16.17%, respectively, which were recognized to increase with crease in eugenol concentration.

Effects of Eugenol on Cell Membrane Integrity of Sh. flexneri
The changes in the cell membrane integrity of Sh. flexneri were detected by flow cytometry, which is often used for the qualitive and quantitative detection of cells. Four quadrants were used to represent different cell states, with H1-LL, H1-LR, H1-UR, and H1-UL representing unstained bacteria, stained by SYTO 9 only, stained by SYTO 9 and PI, and stained by PI only, respectively. As shown in Figure 8, compared with the control, eugenol could decrease the SYTO 9-stained (H1-LR) rate of cells while increasing the rates of cells strained by SYTO 9 and PI simultaneously (H1-UR). The PI-labeled populations (H1-UL) in the 0 (Control), MIC, 2MIC, and 4MIC eugenol-treated groups were 0.01%, 0.42%, 1.94%, and 16.17%, respectively, which were recognized to increase with an increase in eugenol concentration. The effects of eugenol on ROS levels of Sh. flexneri ATCC 9A. The intracellular ROS levels of eugenol-treated samples w while the ROS level of the samples at 2MIC, which was also control, was decreased compared with MIC. After treatmen at 1/2MIC, MIC, and 2MIC, the intracellular ROS levels incr to 7.8 ± 3.0, 11.1 ± 1.4, and 6.8 ± 1.8, respectively.

Effects of Eugenol on ROS Level in Sh. flexneri
The effects of eugenol on ROS levels of Sh. flexneri ATCC 12022 were shown in Figure 9A. The intracellular ROS levels of eugenol-treated samples were higher than the control, while the ROS level of the samples at 2MIC, which was also significantly higher than the control, was decreased compared with MIC. After treatment of Sh. flexneri with eugenol at 1/2MIC, MIC, and 2MIC, the intracellular ROS levels increased significantly (p < 0.01) to 7.8 ± 3.0, 11.1 ± 1.4, and 6.8 ± 1.8, respectively.

Effects of Eugenol on ROS Level in Sh. flexneri
The effects of eugenol on ROS levels of Sh. flexneri ATCC 12022 were shown in Figure  9A. The intracellular ROS levels of eugenol-treated samples were higher than the control, while the ROS level of the samples at 2MIC, which was also significantly higher than the control, was decreased compared with MIC. After treatment of Sh. flexneri with eugenol at 1/2MIC, MIC, and 2MIC, the intracellular ROS levels increased significantly (p < 0.01) to 7.8 ± 3.0, 11.1 ± 1.4, and 6.8 ± 1.8, respectively. The effect of eugenol on SOD of Sh. flexneri was shown in Figure 9B. The activity of

Effect of Eugenol on the Activity of SOD of Sh. flexneri
The effect of eugenol on SOD of Sh. flexneri was shown in Figure 9B. The activity of SOD in Sh. flexneri decreased when the Sh. flexneri was treated with different concentrations of eugenol. The activity of SOD in Sh. flexneri that was treated with eugenol concentrations of 1/2MIC, MIC, and 2MIC was reduced to 1.23 ± 0.09, 0.89 ± 0.09, and 0.25 ± 0.02 units, respectively ( Figure 9B).
3.5.6. Effect of Eugenol on the Intracellular MDA of Sh. flexneri The effects of eugenol on MDA formation in Sh. flexneri cells were determined and shown in Figure 9C. Twenty minutes after the Sh. flexneri were treated by different concentrations of eugenol, the MDA content of the eugenol-treated bacteria increased and were significantly higher (p < 0.01) than that of the control, but there was no significant difference within the three groups.
3.5.7. Effect of Eugenol on the Cell Morphology of Sh. flexneri FESEM was carried out to observe the changes in cell morphology. As shown in Figure 10, the untreated cells ( Figure 10A) were smooth rod-shaped, while the eugenoltreated Sh. flexneri cells ( Figure 10B-D) were wrinkled with rough surfaces. As the concentration of eugenol increased, the degree of shrinkage gradually increased from MIC to 4MIC.  Figure 10A) were smooth rod-shaped, while the eugenoltreated Sh. flexneri cells ( Figure 10B-D) were wrinkled with rough surfaces. As the concentration of eugenol increased, the degree of shrinkage gradually increased from MIC to 4MIC.

Discussion
The results of the study indicated that eugenol shows an inhibitory effect against Sh. flexneri, and its MIC and MBC for Sh. flexneri are 0.5 mg/mL and 0.8 mg/mL, respectively (Figure 1). Similarly, Kang et al. studied the antibacterial activity of gallic acid against Sh. flexneri, and its MIC and MBC for Sh. flexneri were 2 mg/mL and 8 mg/mL, respectively [33]. Another study reported that the MIC and MBC of ferulic acid against Sh. flexneri were 2 mg/mL and 4 mg/mL [34]. Therefore, compared with the other natural products that have been reported so far, eugenol exerted stronger antibacterial activity against Sh. flexneri.
Outbreaks of Sh. flexneri have been linked to unpasteurized vegetable juices and meat in recent years [35,36]. Eugenol had a bactericidal effect on Sh. flexneri in LB broth and PBS (Figure 3) in this research. In order to explore its application in controlling Sh. flexneri in food media, minced pork and lettuce juice were selected for the experiments. In this study, regardless of whether it was the LB nutrient medium ( Figure 3A), PBS non-nutrient medium ( Figure 3B), lettuce juice fluid food medium (Figure 4), or pork minced solid food medium ( Figure 5), it was demonstrated that eugenol greatly inactivated Sh. flexneri. Similarly, Ashrafudoulla et al. [37] indicated that Vibrio parahaemolyticus in LB broth decreased to below the detection limit after being treated with 0.4% eugenol for 10 min. The study

Discussion
The results of the study indicated that eugenol shows an inhibitory effect against Sh. flexneri, and its MIC and MBC for Sh. flexneri are 0.5 mg/mL and 0.8 mg/mL, respectively ( Figure 1). Similarly, Kang et al. studied the antibacterial activity of gallic acid against Sh. flexneri, and its MIC and MBC for Sh. flexneri were 2 mg/mL and 8 mg/mL, respectively [33]. Another study reported that the MIC and MBC of ferulic acid against Sh. flexneri were 2 mg/mL and 4 mg/mL [34]. Therefore, compared with the other natural products that have been reported so far, eugenol exerted stronger antibacterial activity against Sh. flexneri.
Outbreaks of Sh. flexneri have been linked to unpasteurized vegetable juices and meat in recent years [35,36]. Eugenol had a bactericidal effect on Sh. flexneri in LB broth and PBS ( Figure 3) in this research. In order to explore its application in controlling Sh. flexneri in food media, minced pork and lettuce juice were selected for the experiments. In this study, regardless of whether it was the LB nutrient medium ( Figure 3A), PBS non-nutrient medium ( Figure 3B), lettuce juice fluid food medium (Figure 4), or pork minced solid food medium ( Figure 5), it was demonstrated that eugenol greatly inactivated Sh. flexneri. Similarly, Ashrafudoulla et al. [37] indicated that Vibrio parahaemolyticus in LB broth decreased to below the detection limit after being treated with 0.4% eugenol for 10 min. The study by Cui et al. [38] showed that the number of Listeria monocytogenes treated by clove oil at MIC concentrations was reduced by 95.82% and 99.99% after 4 h and 8 h in the peptone yeast glucose solution. In addition, the antibacterial effects of eugenol on Sh. flexneri in PBS, LB, lettuce juice, and minced pork were inconsistent in this study. The strength of the inhibitory effect on Sh. flexneri in PBS, LB, lettuce juice, and minced pork was PBS > LB > vegetable juice > minced pork. The study by Fei et al. [39] reported that treatment with MIC olive oil polyphenol extract in normal saline non-nutrient medium and LB nutrient medium for 3 h and 10 h, respectively, reduced Enterobacter sakazakii concentrations below the detection limit. Differences in growth media led to different inhibitory effects. On the one hand, the difference may be related to the growth state of the bacteria, while on the other hand, this was probably due to the complex chemical composition, texture, and pH of different media affecting the antibacterial effect of eugenol against Sh. flexneri.
The results of our study indicated that the intracellular ROS level of Sh. flexneri significantly increased after eugenol treatment ( Figure 9A). The study by Khan et al. [40] reported that carvacrol increased the level of ROS in Escherichia coli (E. coli) cells. Studies have shown that bacteria owned ROS removal systems, such as SOD enzyme, catalase, etc., so that ROS can maintain harmless levels in the body [41,42]. Studies have shown that the decreased liveness of SOD enzyme [30] leads to the increase in the level of intracellular ROS, which in turn leads to the inhibition of bacterial life activities. When Li et al. [30] assessed the inhibitory effect of epigallocatechin gallate on Vibrio mimicus, they found that epigallocatechin gallate would reduce the activity of SOD enzyme and lead to an increase in the intracellular ROS level of Vibrio mimicus. The intracellular SOD enzyme activity of Sh. flexneri treated with eugenol decreased ( Figure 9B) in our study. Therefore, as an effective antibacterial method, eugenol inactivates the ROS scavenging enzymes of Sh. flexneri, thereby accumulating ROS in the bacteria and inducing the damage and death of Sh. flexneri.
The results also showed that the intracellular ROS levels of Sh. flexneri treated with a eugenol concentration of 2MIC were decreased compared with the intracellular ROS levels of bacteria treated with MIC eugenol ( Figure 9A). This may be due to the leakage of intracellular ROS due to the damage to cell membrane integrity, resulting in the decrease of ROS at the detection level, which was similar to the findings of Liao et al. [43].
Excessive ROS in cells under oxidative stress can cause damage to important biomolecules such as lipids and proteins, thereby posing a threat to bacterial cells [44]. Polyunsaturated fatty acids in cell lipid membranes are particularly peroxidized by ROS to generate the lipid peroxidation product, MDA [45]. At 37 • C, MDA levels of Sh. flexneri significantly increased after eugenol treatment (p < 0.01) ( Figure 9C). The study by Shi et al. [31] showed that the MDA content in Escherichia coli treated with octyl gallate was significantly increased by 1.45 times compared with the control group. Liang et al. [46] pointed out that 6% (w/v) buckwheat honey can induce Pseudomonas aeruginosa cells to produce malondialdehyde, indicating that membrane lipid peroxidation caused oxidative stress and led to bacterial oxidative damage. Combined with the results of the ROS experiment, as an effective antibacterial method, eugenol significantly increased the intracellular ROS level of Sh. flexneri (p < 0.01), oxidative stress occurred, and the content of malondialdehyde increased significantly (p < 0.01), which led to oxidative damage to the bacteria, thereby inducing the damage and death of Sh. flexneri.
In this study, a quantitative analysis through flow cytometry showed that eugenol treatment reduced the cell membrane integrity of Sh. flexneri (Figure 8). A similar study showed that 4.0 mg/mL of polysaccharides damaged the cell membrane integrity of E. coli [47]. It was found that the cell membranes of Pseudomonas fluorescens were damaged by the action of lactobionic acid at a concentration of 2MIC for 2 h [48]. Studies have shown that the excessive accumulation of ROS promotes oxidative damage to cell membranes and destroys cell membrane integrity [49]. This was the same as the results of this study (Figures 8 and 9A,C), which indicated that the impaired cell membrane integrity was caused by the elevation of intracellular ROS in bacteria caused by eugenol.
The membrane potential of the bacterial cell membrane reflects the difference in potential between the inner and outer membranes of the cell and is an important indicator for measuring the permeability of the cell membrane [50]. After eugenol treatment, the membrane potential of the cell membrane was depolarized, which showed the same trend as eugenol concentration and action time within the first 15 min of processing (Figure 7). Research by Khan et al. [40] demonstrated that in carvacrol-treated E. coli, membrane depolarization increased, oxidation rupture increased, and bacterial cell death increased. It shows that carvacrol has a significant antibacterial effect on E. coli. Silverman et al. [51] reported that daptomycin caused Staphylococcus aureus K + to be released, which led to the depolarization of the membrane potential of the cell membrane, thereby causing cell death. Therefore, it was believed that cell membrane depolarization was caused by K + released to maintain membrane homeostasis, further illustrating that the cell membrane integrity of Sh. flexneri was damaged by eugenol.
ATP plays a vital role in life activities such as bacterial growth, replication, and survival. Intracellular ATP is necessary for functions such as the storage and supply of metabolic energy and enzyme reactions [52]. Compared with the control, the eugenol treatment significantly reduced the intracellular ATP concentration of Sh. flexneri (p < 0.01) in this study ( Figure 6). Turgis et al. [53] showed that the intracellular ATP concentrations of E. coli O157:H7 and Salmonella typhi when treated with mustard essential oil were significantly decreased. The study by Guo et al. [27] showed that the intracellular ATP concentration of Cronobacter sakazakii treated with coenzyme Q 0 was significantly reduced. Studies have shown that cells can change the permeability of the cell membrane through the production of plasma membrane carriers or conductance channels, thereby causing the release of ATP [52], and resulting in a decrease in the concentration of intracellular ATP. The study by Khan et al. [40] showed that carvacrol destroys the cell membrane of E. coli, leading to the release of ATP in the cell, and, as the concentration of carvacrol increases, the release of ATP increases. On the other hand, adverse external conditions will increase the activity of ATPase and increase the rate of ATP hydrolysis, leading to the rapid consumption of ATP in cells [52,54]. It is speculated that eugenol reduces the ATP level of Sh. flexneri, on the one hand, because eugenol changes the integrity of bacterial cell membranes, leading to ATP leakage, and on the other hand, because eugenol increases bacterial ATPase activity and increases ATP consumption.
In this study, the FESEM was used to observe the morphological changes of Sh. flexneri cells treated with eugenol ( Figure 10). Based on the study by Niu et al. [55], after treatment with 0.80 mg/mL of eugenol, E. coli cells deformed and became irregularly shaped, with large areas of deformation and wrinkles. The study by Kang et al. [1] showed that the MIC concentration of gallic acid ruptured the cell membrane of Sh. flexneri, and the bacteria treated with an MBC concentration of gallic acid almost did not show normal morphology. In this study, Sh. flexneri, after eugenol treatment, showed atrophy in a concentrationdependent manner ( Figure 10). By combining the results from the study of cell membrane integrity, severe membrane damage caused by changes in cell permeability led to the leakage of contents, thereby causing Sh. flexneri to shrink and become non-directional cells.

Conclusions
In this study, eugenol was confirmed to exhibit an antibacterial effect against Sh. flexneri. The activity of the SOD enzyme of Sh. flexneri was reduced by eugenol, resulting in increased intracellular ROS levels, which further led to oxidative damage to the cell membrane. The permeability and integrity of the cell membrane were disrupted by eugenol, accompanied by the leakage of intracellular ATP and the depolarization of the membrane potential. FESEM observation further confirmed the disruption of the cell membrane; all of these result in Sh. flexneri cell death. Eugenol also effectively inactivated Sh. flexneri in LB broth, PBS, minced pork, and vegetable juice. In summary, this study provided a theoretical possibility that eugenol might be used as an attractive antibacterial agent to reduce the food safety issues caused by Sh. flexneri.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.