Aeromonas hydrophila (PRJNA808687) used in this study was from our lab, which was isolated from grass carp (Ctenopharyngodon Idella) in 2018. A. hydrophil (CICC25017) was purchased from the China Center of Industrial Culture Collection. A. hydrophila was streaked on Tryptone Soy Agar (TSA, Guangdong Huankai Microbial Sci. & Tech. Co., Ltd., China) and incubated at 28°C for 24 h, and then 5 colonies were transferred to 5 ml of Tryptone Soy Broth (TSB, Guangdong Huankai Microbial Sci. & Tech. Co., Ltd., China) and incubated for 8 h at 28°C with shaking.

Satsuma mandarin was collected from Neijiang city, Sichuan Province, China. In the Clevenger apparatus, 500 g of satsuma mandarin peels were suspended in 2,000 ml of distilled water and subjected to steam distillation. The extraction was carried out for 3 h, and the obtained EO was collected, dried by anhydrous sodium sulfate (Na₂SO₄) for 24 h, and then stored at 4°C in brown glass vials.

The GC-MS analysis was carried out by using an Agilent 7890A GC equipped with a Gerstel MPS autosampler, coupled with an Agilent 5975C MSD detector. The chromatographic separation was performed on an HP-5MS capillary column 30 m × 0.25 mm i.d., 0.25 μm, the GC oven was operated at 40°C held for 1 min, increased to 220°C at a rate of 3°C/min, held at 220°C for 25 min, increased to 250°C at a rate of 5°C/min, and finally held for 10 min. Helium was used as the carrier gas at a flow rate of 1 ml/min. The injector and detector temperatures were set at 250 and 280°C, respectively. The mass spectrometer was operated in the 70 eV EI mode with scanning from 35 to 350 amu/s, and the ion source was set at 230°C. The EO components were identified by matching their recorded mass spectra with the data bank NIST 08 (National Institute of Standards and Technology).

A volume of 100 μl bacterial dilution (PRJNA808687 and CICC25017, 1 × 10⁶ CFU/ml) was evenly smeared on Mueller-Hinton Agar (MHA). Following that, filter paper disks (6 mm in diameter) containing 6 μl SMEO were placed on the surface of the agar plates. After standing for 10 min, the plates were incubated at 28°C for 12 h. Meanwhile, antibiotics (florfenicol and amoxicillin) and water + 1% Tween were used as positive and negative controls, respectively. Finally, the diameters of the inhibition zone (DIZ) were measured (Oonmetta-aree et al., 2006).

Serial dilutions of SMEO ranging from 0.0625 to 16% were prepared in TSB (with 1% Tween 20). A volume of 100 μl of each SMEO serial dilution was dispensed into 96 microtiter plates, respectively. Afterward, 100 μl of the bacterial suspension (PRJNA808687, 2 × 10⁶ CFU/ml) was treated with the dilutions of SMEO. Then, the microtiter plates were cultured at 28°C for 120 rpm. After 24 h incubation of the bacterial suspension, the lowest concentration of SMEO with no bacterial growth was determined as MIC. Furthermore, to determine MBC, 10 μl of the above solutions (from the concentration of MIC to 16%) were placed on TSA plates and incubated at 28°C for 24 h. MBC was the lowest concentration of SMEO without any visible colonies on the plates.

Bacterial growth kinetics was measured by the effect of SMEO on bacterial growth (Sureshkumar et al., 2010). According to the above experimental data, 0 × MIC, 0.0625 × MIC, 0.125 × MIC, 0.25 × MIC, 0.5 × MIC, and 1 × MIC of SMEO were diluted with 40 ml bacterial suspension (PRJNA808687, 1 × 10⁶ CFU/ml). The suspensions were incubated at 28°C for shaking (120 rpm), and the absorbance was measured by a spectrophotometer (UV759S, INESA Analytical Instrument Co., Ltd., China) at an optical density of 600 nm at the following time points (t/h): 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 36, and 48 h.

A total of 3–4 colonies of A. hydrophila were transferred into 10 ml TSB and incubated at 28°C overnight. After incubation, cells were collected by centrifugation at 8,000 rpm for 10 min and adjusted to the concentration of 1 × 10⁹ CFU/ml (PRJNA808687) in 10 ml of phosphate-buffered solution (PBS). Then, SMEO at the concentration of 1 × MIC was added to the abovementioned solutions and treated at 28°C for 4, 8, and 12 h. Finally, the supernatants were separated from bacterial cells by centrifugation (8,000 rpm) and filtration (with 0.22 μm filter, Sigma-Aldrich, United States), which will be used for the nucleic acid and protein assay as described. At the same time, SMEO treatment for 0 h was used as the zeroing sample for UV spectroscopic detection.

For the nucleic acid assay, the absorbance of the supernatant samples was measured by a spectrophotometer (UV759S, INESA Analytical Instrument Co., Ltd., China) at the optical density of 260 nm.

For the protein assay, a Total Protein Kit, Micro (Sigma-Aldrich, United States) was used to measure the concentration of protein. According to the operating instructions, the protein assay solution was mixed with the supernatant samples. After approximately 2 min, the absorbance of the sample was also measured by a spectrophotometer at the optical density of 595 nm (UV759S, INESA Analytical Instrument Co., Ltd., China).

Aeromonas hydrophila (PRJNA808687) was incubated to log phase as the conditions mentioned in the “Microorganism and culture conditions” section; after incubation, bacterial suspensions were adjusted to 1 × 10⁸ CFU/ml. Cells (2 ml suspensions) were collected by centrifugation at 8,000 rpm for 10 min, and the bacterial pellet was carefully washed with PBS and resuspended. Later, 1 × MIC SMEO (with 1% Tween 20) was added to the resuspension and treated for 4, 8, and 12 h. Finally, the samples were collected by centrifugation, washed three times with PBS, and fixed with 2.5% glutaraldehyde at 4°C for 4 h or overnight. Next, the samples were prepared for SEM and TEM assay as described.

For SEM, the glutaraldehyde solution was removed and the bacteria samples were washed with PBS for three times, and then dehydrated with 10, 30, 50, 70, and 90% ethanol; in turn, 15 min each time, and dehydrated two times with 100% ethanol, 20 min each time. After dehydration, ethanol was replaced with tertiary butanol two times for 20 min each, then the samples were vacuum freeze-dried, sprayed with gold, and observed by scanning electron microscope (Hitachi S-4800, Hitachi, Japan).

For TEM, the fixed solution was removed, and the samples were washed three times with PBS, then the bacteria samples were placed on the copper mesh, air-dried for 5 min, and negatively stained with 1% phosphotungstic acid. After that, the samples were observed by a transmission electron microscope (Hitachi HT-7700, Hitachi, Japan).

Flow cytometry analysis was carried out to investigate the effects of SMEO on A. hydrophila (PRJNA808687). The method for SMEO treatment was the same as mentioned in the “Leakage of Cellular Components Assay” section, but the treatment time is 8 h. Meanwhile, PBS and ethanol treatment were used as negative and positive controls, respectively. After treatment, bacterial cells were collected by centrifugation and adjusted to the concentration of 1 × 10⁶ CFU/ml; then different staining procedures proceeded as follows.

Thiazole orange (TO, Sigma-Aldrich, United States) and propidium iodide (PI, Sigma-Aldrich, United States) were used to evaluate the membrane permeability of cells. For TO staining, 1-μl TO solution was added to 1-ml bacterial suspensions (final concentration of TO: 10 μg/ml in DMSO), and then incubated at room temperature for 15 min. For PI staining, 1-μg PI was added to 1 ml bacterial suspensions (final concentration of PI: 1 μg/ml in PBS), and then incubated at 37°C for 15 min.

Bis-1,3-dibutylbarbutiric acid (BOX, Sigma-Aldrich, United States) and PI were used to evaluate the membrane potential of cells. For BOX staining, 2.5-μg BOX was added to 1-ml bacterial suspensions (final concentration of BOX: 2.5 μg/ml in PBS with 4 mM EDTA), and then incubated at 37°C for 15 min. For PI staining, the procedure was the same as mentioned in the “Membrane Permeability (TO and PI Staining)” section.

Ethidium bromide (EB, Sigma-Aldrich, United States) was used to evaluate the efflux activity of cells. For EB staining, 10-μg EB was added to 1-ml bacterial suspensions (final concentration of EB: 10 μg/ml in DMSO) and then incubated at 37°C for 15 min.

After finishing these staining procedures, samples were washed three times with PBS, and the concentration of bacterial suspensions was adjusted to about OD₆₀₀ = 0.1. Then, the samples were placed on ice for flow cytometry analysis by a flow cytometer (BD Accuri C6 plus, BD, United States), green fluorescence was collected in the FL1 channel (533 nm), and red fluorescence in the FL3 channel (>670 nm). Fluorescence signals were collected by FL1 (TO and BOX) and FL3 (PI, EB) bandpass filters. Bacterial cells were gated per the FSC/SSC parameters and a total of 5,000 events were acquired for each sample.

All the experiments were performed in triplicate. Statistical analysis was carried out using SPSS and GraphPad Prism 7 for the t-tests. All asterisks indicate significant differences (p < 0.05).

The chemical composition of SMEO is summarized in Table 1. A total of 79 components were identified, which accounted for the percentage of 99.9997%. Meanwhile, 96.3238% of SEMO constituents were monoterpenes. Limonene was the most abundant component with a percentage of 70.2252%, followed by γ-terpinene (7.8955%), β-myrcene (5.0086%), l-α-pinene (3.8957%), β-terpinene (1.7055%), linalool (1.5054%), and α-terpineol (1.0265%); these seven components account for 91.2624% of all the components. The percentage of the other remaining 72 components was below 1% (0.0097–0.8700%).

As shown in Figure 1A and Table 2, SMEO florfenicol exhibited strong antibacterial activity against A. hydrophila in vitro (PRJNA808687 and CICC25017). Among them, the inhibition diameter of SEMO against A. hydrophila was 31.22 ± 0.46 mm. The bacteria were completely killed when the SEMO concentration was 1%. During bacterial growth period, A. hydrophila was exposed to SEMO to confirm whether the environmental adaptations of cells were affected. The antibacterial kinetic curves of SMEO were demonstrated in Figure 1B, which reflected the kinetic character of SMEO at different concentrations (0.0625 × MIC, 0.125 × MIC, 0.25MIC, 0.5 × MIC, and 1 × MIC) and treated times. In the 0.0625 × MIC, 0.125 × MIC, 0.25 × MIC, and 0.5 × MIC groups, the increasing lag phase time and decreasing concentration of bacteria during the stationary phase indicated that the cells were more sensitive to the environmental stress in which SMEO was located. Meantime, bacterial growth was not observed at the concentration of 1 × MIC.

The release of the bacterial intracellular material was observed by measuring changes in the composition of cell supernatant. Figure 2A shows the changes in nucleic acids. When the cells were exposed to SMEO at the concentrations of 2 × MIC for 4, 8, and 12 h, the values of A₂₆₀ were 0.1950 ± 0.0324, 0.8163 ± 0.0573, and 1.0992 ± 0.0529, respectively. The results demonstrated that the concentration of nucleic acids in the supernatant increased with the increase of exposure time. Figure 2B shows the protein changes by Coomassie blue staining, which indicated that the concentration of proteins increased significantly (p < 0.05) with the increase of exposure time, with values of 0.0237 ± 0.0015, 0.0513 ± 0.0032, and 0.1157 ± 0.0061 in A₅₉₅, respectively. Meanwhile, the changes in protein and nucleic acid concentrations in the extracellular supernatant demonstrate that SMEO was able to rupture bacterial cell membranes.

Scanning electron microscopy was used to reveal the changes in themorphology of A. hydrophila with and without the treatment of SMEO. As shown in Figure 3, cells in the control group were rod-shaped with smooth surfaces and intact structures, about 1–2 μm in length. Compared with the control group, the morphology of cells in those three experimental groups showed significant changes, wrinkled surfaces (pointed by the red arrow, Figure 3, 4h), and collapsed cells (pointed by the blue circle, Figure 3, 4,8h), indicating the huge damage caused by SMEO. The degree of SMEO-induced morphological changes and the quantity of affected cells enhanced with the increase in treatment time. First, after a 6-h treatment (Figure 3, 4 h), bacterial cells began to collapse, and some of the folded cells remained rod-shaped. Further, after an 8-h treatment (Figure 3), 8 h the surfaces of the cells were more densely collapsed and wrinkled, and the cell morphology changed significantly. Moreover, after a 12-h treatment, no intact cells existed and only a fraction of cells could be observed, which indicated the whole lysis of cells.

Transmission electron microscopy was also used to observe more details. As shown in Figure 4, in the control group, the cell membrane and cell wall were intact, and the cytoplasm was evenly dispersed. Compared with the control group, the cell morphology of the other three experimental groups changed significantly, and wrinkled surfaces (pointed by the red arrow, Figure 4), 4 h vacuoles inside the cytoplasm (pointed by the purple arrow, Figure 4), 4, 8, 12 h and the leakage of cytoplasm (pointed by the blue arrow, Figure 4), 8 and 12 h were clearly observed. Meanwhile, the boundaries of the cell membrane and cell wall became vague, and the distribution of cytoplasm became heterogeneous. Moreover, some extreme phenomena were also observed after the treatment of SMEO, such as the bare cell wall without cytoplasm inside it (pointed by the green arrow, Figure 4), 12 h and the individual cytoplasm without the protection of the cell wall (pointed by the blue arrow, Figure 4, 12 h).

Four fluorescent dyes (TO, PI, BOX, and EB) were used to evaluate several vital biological functions in A. hydrophila cells by flow cytometry analysis.

Membrane integrity was evaluated by double staining of TO and PI as shown in Figure 5 (the first row). In the control group, 73.2% of the cells were located in plot Q1 (TO + and PI–), which represented cells with intact cell membranes. Approximately 99.1% of the cells in the positive control group were located in plot Q2 (TO + and PI +), which represented cells with permeabilized cell membranes. In the SMEO-treated group, 75.6% of the cells were located in plot Q2 (TO + and PI +). Moreover, 11.9% of the cells were located in plot Q4 (TO- and PI-), which represented cells with damaged DNA or RNA.

Membrane potential was evaluated by double staining of BOX and PI as shown in Figure 5 (the second row). In the control group, 98.7% of the cells were located in plot Q4 (BOX- and PI-), which represented cells with polarized membranes. By contrast, in the SMEO-treated group and ethanol-treated group, 21.2% of the cells and 57.0% of the cells were located in plot Q1 (BOX + and PI-), respectively, which represented cells with depolarized membrane. Efflux activity was evaluated by EB staining as shown in Figure 5 (the third row). EB represented that the efflux pump functioned properly, while EB + meant the malfunction of the efflux pump. The percentage of EB + cells in the control and treated groups was 4.33 and 84.6%, and the percentage of EB- cells in these two groups was 60.2 and 36.3%.