Staphylococcus aureus ATCC 29213, obtained from the American Type Culture Collection, was stored in tryptone soybean broth (TSB; Land Bridge Technology Co. Ltd., Beijing, China) containing 25% glycerol at −80°C. It was taken out from the refrigerator and inoculated on tryptone soybean agar (TSA; Land Bridge Technology Co. Ltd., Beijing, China) plate before each experiment. Single colony of the strain was picked out, inoculated into TSB, and cultured at 37°C for about 12 h. The absorbance of the bacterial suspension was measured by a spectrophotometer (Smart Spec™ plus, Bio-Rad Laboratories, Hercules, CA) to obtain the optical density about 0.5 at 600 nm.

The growth curves of S. aureus were detected when treated with or without punicalagin using the Bioscreen C automated microbiology growth curve analysis system (Labsystems, Helsinki, Finland) as described by Zheng et al. (2020). Punicalagin (≥98%, CAS 65995–63-3, Must Bio-Technology Co. Ltd., Chengdu, China) was added into the wells to obtain the final concentrations of 0 (control), 1/64, 1/32, 1/16 and 1/8 MIC, respectively. The above bacterial suspension was inoculated at a ratio of 1% containing about 10⁶ colony-forming units (CFU)/mL. Sterile TSB containing corresponding concentrations of punicalagin was taken as negative control. The microplate was incubated statically at 37°C and the absorbance at 600 nm wavelength was measured in 1 h intervals for 24 h.

The effect of punicalagin on biofilm biomass was conducted by crystal violet staining method referred to Fan et al. (2022b). Briefly, S. aureus suspensions exposure to punicalagin at 0 (control), 1/64, 1/32 and 1/16 MIC were incubated in a 96-well microplate at 37°C for 24 h. After removing the planktonic cells, wells were washed with sterile phosphate buffered saline (PBS) for three times and fixed with methanol for 15 min. Then crystal violet solution (1%) was added to stain biofilms and the wells were rinsed thrice with distilled water, followed by the addition of acetic acid (33%, vol/vol). After shaking at low speed for 5 min, the absorbance at 570 nm was measured using a microplate reader (model 680; Bio-Rad). The relative biofilm formation was calculated by the OD_treatment normalized with OD_control.

The effect of punicalagin on biofilm metabolism was examined by the method of Jadhav et al. (2013). The prepared bacterial suspensions containing different concentrations of punicalagin (0, 1/64, 1/32 and 1/16 MIC) were separately added to a 96-well plate and incubated at 37°C for 24 h. Then the bacterial suspensions were removed. The plate was gently rinsed three times with PBS. A total of 250 μl of 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) solution at the concentration of 0.5 mg/ml was added to each well, and incubated at 37°C for 3 h. The insoluble purple formazan was further dissolved in dimethyl sulfoxide (DMSO) and the absorbance was measured at a wavelength of 570 nm using a microplate spectrophotometer (model 680; Bio-Rad).

The number of viable bacteria in biofilms was counted as previously reported with some modifications (Amalaradjou and Venkitanarayanan, 2011). Briefly, the bacterial suspensions containing different concentrations of punicalagin (0, 1/64, 1/32 and 1/16 MIC) were inoculated into a 24-well polystyrene plate at 2 ml per well and cultured at 37°C for 24 h. Then the wells were carefully washed three times with sterile PBS, followed by the addition of another 2 ml of PBS. The biofilms were wiped off and mixed thoroughly. After a series of 10-fold dilution, the suspensions were spread on TSA, and cultured at 37°C overnight before the colonies were counted.

FESEM was performed as described by Li et al. (2021) with some modifications. The bacterial suspensions containing punicalagin at different concentrations (0, 1/64, 1/32 and 1/16 MIC) were added to a 12-well plate pre-placed with sterile glass sheets. After incubated at 37°C for 24 h, the glass sheets were rinsed gently with 2 ml of PBS for three times to wash off the planktonic bacteria. Then they were placed in 2.5% glutaraldehyde solution (prepared in PBS) at 4°C for 5 h, followed by fixation with 1% osmic acid solution for 5 h. Samples were washed with PBS and dehydrated with different concentrations of ethanol solution (30, 50, 70, 80, 90 and 100%). After naturally air dried in a fume hood overnight, the samples were immobilized on a support, and sprayed with Au-Pd under vacuum. Finally, the morphology of the biofilm was observed and photographed by a scanning electron microscope (S-4800, Hitachi, Tokyo, Japan).

CLSM was carried out according to Fan et al. (2022a) with minor changes. As described above, S. aureus biofilm was formed on glass sheets with or without the treatment of punicalagin (0, 1/64, 1/32 and 1/16 MIC) at 37°C for 24 h. After gently rinsed three times with PBS, the glass sheets were transferred to a new 12-well plate. A total of 1 ml of SYTO 9 and propidium iodide (PI) fluorescent dye mixture was added to the wells, followed by the incubation at room temperature in the dark for 15 min. The biofilms were observed under a confocal laser scanning fluorescence microscope (A1; Nikon, Tokyo, Japan). Live bacteria with intact cell membranes emitted green fluorescence, while dead or damaged bacteria emitted red fluorescence.

The effect of punicalagin on the cell surface hydrophobicity of S. aureus was assessed by the method of microbial adhesion to hydrocarbons (MATH) as reported previously (Tang et al., 2020). In brief, overnight S. aureus culture was collected, washed, and resuspended in PBS. Punicalagin was added to reach the concentrations of 0, 1/64, 1/32 and 1/16 MIC. The absorbance of each sample was detected at 600 nm. Then, 1 ml of hexadecane was added to 2 ml of the bacterial suspension and mixed thoroughly for 1 min. The absorbance of aqueous phase was detected again after incubated for 15 min at 37°C. The hydrophobicity was calculated as follow.

Where Aa is the initial absorbance at 600 nm, and Ab is the absorption value after the treatment of punicalagin. Control groups were those without punicalagin treatment.

Mean values and standard deviations were obtained from three replicate experiments. Statistical analysis was performed by the analysis of variance (ANOVA) with SPSS 20.0 software. Tukey’s multiple range test was used to calculate the significant differences (p < 0.05) between the control and treatment groups.

The effect of punicalagin on the growth of S. aureus was shown in Figure 1. After about 2 h of lag, the strains of control groups quickly entered the logarithmic growth phase and reached a stationary phase within 8 h. The growth of S. aureus was obviously inhibited by punicalagin at 1/8 MIC compared to the control. However, punicalagin has minor effect on the growth of S. aureus at 1/64 MIC to 1/16 MIC. Therefore, the concentrations of punicalagin from 1/64 MIC to 1/16 MIC were considered as sub-inhibitory concentrations (SICs) against S. aureus which were chosen for the following experiments.

As shown in Figure 2, the relative biofilm formation by S. aureus on polystyrene microplate was markedly decreased after punicalagin treatment. The biomass of the biofilm was decreased to 42.0% at the presence of punicalagin at 1/64 MIC compared to the control. And a higher reduction to 8.1% occurred as the concentration of punicalagin increased to 1/32 MIC. There was no significant difference between the 1/32 MIC and 1/16 MIC groups. This result indicated that punicalagin prevented biofilm formation effectively at SICs.

MTT staining method reflects the metabolism of live bacteria in the biofilm and the result was depicted in Figure 3. At 1/64 MIC, punicalagin has a significant inhibitory effect on the metabolism of bacteria in the biofilm. The inhibitory effect increased with the increase of the concentration of punicalagin. At the highest concentration tested (1/16 MIC), the optical density was reduced by 97.3% in comparison with the control.

Figure 4 shows the number of viable bacteria in biofilm. It can be observed that the biofilm-associated population of S. aureus was reduced by the treatment of punicalagin and higher concentrations of punicalagin lead to more reduction in viable bacteria. Specifically, the viable cell counts were decreased by 0.94 and 1.75 log CFU/mL in comparison with the control after incubated with punicalagin at the 1/32 MIC and 1/16 MIC, respectively.

FESEM was used to observe the effect of punicalagin on the structure of biofilm. The results are shown in Figure 5. In Figure 5A, the cells are tightly adhered to each other on the surface of the glass slide and stacked on top of each other, suggesting the strong biofilm formation ability of the strain. The cells in Figures 5B–D are under the action of SICs of punicalagin. The adhesion between the cells became looser and the distribution became sparser than the control. This experiment visually illustrates the inhibitory effect of punicalagin on the biofilm formation of S. aureus. It also can be clearly seen that the surface of the control cells was smooth, while the surface of the punicalagin-treated cells was rough, indicating that punicalagin disrupted the cell surface morphology.

The inhibitory effect of punicalagin on the biofilm formation of S. aureus was also observed by CLSM. As can be seen from Figure 6, the fluorescence intensity of the control group was significantly higher than that of the experimental group after the action of punicalagin. A large number of S. aureus aggregates can be seen in the control group. After the treatment of punicalagin, the ability of S. aureus to form biofilm was significantly reduced. The amount of biofilm formation decreased as the concentration of punicalagin increased. The proportion of viable bacteria in all groups accounted for the majority (green). Only a few bacteria emitted red fluorescence, suggesting impaired cell membranes.

The result of bacterial surface hydrophobicity was shown in Figure 7. No significant difference was seen on the hydrophobic rates between the control and 1/64 MIC groups with the result of 70.9 and 69.6%, respectively. But the hydrophobic rates were significantly reduced to 55.5 and 37.2% after treated with 1/32 MIC and 1/16 MIC of punicalagin.