The S. aureus strain ATCC® 29213™ (National Center For Medical Culture Collections) was used in this study for its high ability to form biofilms in vitro (Abouelhassan et al., 2014). The ΔSrtA strain was constructed using a method described previously (Chen et al., 2014). The SrtA_ΔN59 protein was constructed in the previous study by our team (Wang L. et al., 2015). The fluorescent peptide Dabcyl-QALPETGEE-Edans was synthesized by GL Biochem (Shanghai, China). The S. aureus ATCC® 29213™ and ΔSrtA strains were cultured in brain–heart infusion (BHI) solution (Oxoid, Basingstoke, UK) supplemented with 0.5% glucose and 3% NaCl at 37°C with shaking at 220 rpm.

Kaempferol was purchased from the Chengdu Ruifensi Biotech Company (Chengdu, China) and dissolved in dimethyl sulfoxide (DMSO) (Changchun Baotaike Biotech Company, China) to a storage concentration of 100 mg/ml.

The S. aureus ATCC® 29213™ strain was cultured overnight and then diluted 1:100 in sterile BHI broth supplemented with 0.5% glucose and 3% NaCl. Then, 200 μl of diluted bacteria was placed in a polystyrene Costar 96-well plate (Tiangen, Changchun Baotaike Biotech Company, China) coated with plasma (20% rabbit freeze-dried plasma, incubated overnight at 4°C). Compounds were added to the assay plates at a final concentration of 512 μg/ml, and the plate was incubated at 37°C for 12 h without shaking. Then, the supernatant was completely removed. After rinsing in phosphate buffered saline (PBS), the biofilms were stained with 12.5 g/l crystal violet (CV) for 10 min, washed again with PBS and dried. Images were obtained using an AMT 2 k charge-coupled device (CCD) camera. Finally, 0.2 ml of ethanol (95%) was added to the CV-stained wells, which were then incubated for 30 min to quantify biofilm growth, and 100 μl of each final dissolved CV solution was transferred into new 96-well plates and measured at an OD of 595 nm using a microplate reader (Infinite® F500, Tecan, Shanghai, China). Each data point consisted of three independent samples. The compounds that showed 90% inhibition compared with the negative control (no compound added) were selected as validated hits.

The minimum inhibitory concentration (MIC) of kaempferol were determined in accordance with the Clinical and Laboratory Standards Institute (CLSI) Approved Standard M7-A8, (CLSI, Wayne, PA, 2009, pp. 19–91). To obtain growth curves, S. aureus was cultured overnight and diluted 1:100 into sterile BHI (supplemented with 0.5% glucose and 3% NaCl) broth with or without kaempferol at different concentrations. The absorbance at 600 nm was measured at different time intervals.

Sytox green was obtained from Invitrogen-Molecular Probes (Oregon). After treatment with 128, 64, and 32 μg/ml kaempferol for 3, 6 and 12 h, the biofilms produced by each group were washed with PBS. To permeabilize the bacterial membranes, the cell growth substrates were soaked in 0.1% (vol/vol) Triton X-100 (VWR) in PBS (PBST) for 15 min. The cells were labeled by replacing the PBST with 0.5 μM Sytox green nucleic acid stain in PBST for 30 min. The cells were then washed with PBS to remove the excess stains. Afterwards, fluorescent images were obtained using a fluorescence microscope (Leica DMRX).

The S. aureus ATCC® 29213™ strain was cultured overnight and then diluted 1:100 in sterile BHI (supplemented with 0.5% glucose and 3% NaCl) broth containing different concentrations of kaempferol and cultured with shaking at 37°C. All cells were collected by centrifugation (5,000 × g for 5 min) when the OD600 reached 0.5. The cells were suspended in PBS to obtain an OD600 of 1.0. The resuspended cells were placed to polystyrene Costar 96-well plates coated with fibrinogen (incubated overnight with 20 μg/ml bovine fibrinogen at 4°C) and incubated for 1 h at 37°C. The supernatant was removed, and the cells were washed with PBS and fixed with 25% (v/v) formaldehyde. After 30 min, the adherent bacteria were washed again with PBS and stained with 12.5 g/l CV for 10 min. The wells were then washed with PBS and dried. Subsequently, different samples were measured at 595 nm. The percentage of the tested group compared to the control group was used to report results. To ensure reproducibility, each experiment was repeated at least three times.

The influence of kaempferol on SrtA was examined by a fluorescence resonance energy transfer (FRET) method (Ton-That et al., 1999; Mazmanian et al., 2002). Sortase A (SrtA), an enzyme that anchors surface proteins to the cell wall of Gram-positive bacteria, cleaves sorting signals between the threonine (T) and glycine (G) of the LPXTG motif (Ton-That et al., 1999). During SrtA catalysis, the change in fluorescence was monitored using the fluorescent peptide substrate Dabcyl-QALPETGEE-Edans of SrtA. The inhibitory effect of kaempferol on the activity of SrtA was measured based on the fluorescence changes. The sortase A (SrtA) activity inhibition assay was conducted according to a method described previously (Bi et al., 2016). The experiment was performed in black 96-well plates. The reaction consisted of 4 μM SrtA_ΔN59 protein, 10 μM peptide substrate and the assay buffer (150 mM NaCl, 5 mM CaCl₂, 0.1% Triton X-100 and 50 mM Tris-HCl, pH 7.5) in a final volume of 300 μl. The SrtA_ΔN59 protein and different concentrations of kaempferol were added to the plate and incubated at 37°C for 1 h. Then, the peptide substrate was added, and the reaction continued for another 1 h at 37°C. The change in fluorescence intensity was detected using a microplate reader (Infinite®F500, Tecan, Shanghai, China) with 495 nm as the emission wavelength and 350 nm as the excitation wavelength. To ensure the reproducibility of this assay, each reaction was repeated three times.

Transmission electron microscopy (TEM) was performed using a JEM-2100 transmission microscope (JEOL, Tokyo, Japan). Biofilm samples were prepared according to the method described above. For the proteinase K group, proteinase K (2 μg/ml) was added to the pre-established biofilms and incubated for 2 h at 37°C. Samples prepared for TEM imaging were spotted onto formvar-coated copper grids, incubated for 5 min, washed with sterile distilled H₂O, and negatively stained with 2% uranyl acetate for 60 s (Chu et al., 2016).

The confocal microscopy experiment was performed using confocal laser-scanning microscopy (Olympus, Shanghai, China). Syto 63 and fluorescein isothiocyanate (FITC) were purchased from Invitrogen Molecular Probes (Oregon). The biofilms were cultured according to the above method on glass cover slides with or without kaempferol (64 μg/ml). The biofilms produced by each group were washed with PBS after 12 h. Syto 63 (100 μM) was added to each well, and the plate was incubated with shaking for 5 min. FITC (0.001%) was then added, and the plate was incubated for another 30 min without shaking. The cells were washed with PBS to remove excess stain. Confocal microscopy images were obtained from NIS-Elements C version 3.2 (Nikon Eclipse).

For quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) experiments, RNA from S. aureus cells was isolated using the following procedure. The S. aureus ATCC® 29213™ strain was cultured overnight and diluted 1:100 into sterile BHI broth supplemented with 0.5% glucose and 3% NaCl. After culturing at 37°C for 3 h with shaking at 220 rpm, kaempferol was added to a concentration of 64 μg/ml—at which it showed significant anti-biofilm activity—and the culture was incubated again for 5 h. Subsequently, RNA was isolated using the TRIzol (Tiangen, Changchun Baotaike Biotech Company, China) RNA extraction method. The concentration of RNA was assessed using a NanoVue Plus (Biochrom Ltd., Cambridge, UK).

qRT-PCR was used to assess the transcription levels of adhesion-related genes (fnbA, fnbB, clfA, clfB, sarA) in S. aureus ATCC® 29213™. Gene-specific primers (listed in Table 1) were used for these genes, and appropriate primers were used for 16S rRNA as a housekeeping control to normalize the expression of genes of interest. The isolated RNAs were reverse-transcribed into cDNA using the TransScript® All-in-One First-Strand cDNA Synthesis SuperMix (Quanshijin, Changchun Weierkete Biotech Company, China). Then, qRT-PCR was performed using the TransStart Top Green qPCR SuperMix (Quanshijin, Changchun Weierkete Biotech Company, China) under the following conditions: initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 10 s and 72°C for 10 s. As a negative control, qRT-PCR was performed without cDNA. The experiments were performed three times in parallel, and the data were analyzed using a previously described relative quantitative (2^−ΔΔCt) method (Livak and Schmittgen, 2001).

According to the screening assay, kaempferol at 512 μg/ml showed 90% inhibition on the biofilm formation representing that it had obvious anti-biofilm activity. To detect the influence of different concentrations of kaempferol on S. aureus biofilms, S. aureus was co-cultured with kaempferol (1–512 μg/ml) using the microdilution method, which was similar to the MIC assay for planktonic cells in 96-well plates coated with 20% rabbit freeze-dried plasma at 37°C for 12 h without shaking. After 12 h, we stained the biofilm with crystal violet and measured the absorbance at 595 nm. We observed that kaempferol was effective at lower concentrations, and it could inhibit biofilm formation by 80% at 64 μg/ml (Figure 1B).

The nucleic acid dye Sytox Green is a fluorescent indicator that can efficiently label the entire cytoplasm (Hochbaum et al., 2011). Therefore, the effect of kaempferol on biofilms was observed by the fluorescence microscopy. Fluorescent images indicated that biofilm of the control group increased gradually with time, whereas kaempferol dose-dependently inhibited the biofilm formation (Figure 1C).

To test whether the effect of kaempferol on biofilms was dependent on the inhibition of the growth of planktonic bacteria, MIC of kaempferol was determined by MIC experiment. The results showed that the MIC of kaempferol on S. aureus was greater than 1,024 μg/ml. In addition, the results of the growth curve were consistent with the MIC results: bacteria treated with different concentrations of kaempferol (64–256 μg/ml) showed the same growth trend as the control group (Figure 1D). These results confirmed that kaempferol did not affect the growth of S. aureus at concentrations of 64–256 μg/ml but could significantly inhibit the formation of S. aureus biofilms.

The development of bacterial biofilm can be divided into the following three phases, which involve specific molecular factors: attachment, maturation, and detachment (Otto, 2013). To explore which phases of biofilm development kaempferol influenced, we added kaempferol (64 μg/ml) at different time points during biofilm formation and measured the effects after a total of 20 h of incubation. There was no inhibitor in the control group. As shown in Figure 2A, compared to the control group, kaempferol significantly inhibited the formation of biofilms only when added immediately after inoculation (0 h). After bacteria were incubated for 1, 2, 3, 4, 8, and 12 h, the addition of kaempferol had no effect and the biofilms were completely resistant to kaempferol. These results demonstrated that kaempferol specifically inhibited the attachment phase of biofilm formation.

The above results showed that kaempferol only affected the attachment phase of biofilm formation, and this phase was primarily mediated by the binding of S. aureus surface-anchored proteins and host matrix proteins (Otto, 2013). Fibrinogen, as a plasma protein, can be used as a substrate for staphylococcal adhesion (Patti et al., 1994). To examine the influence of kaempferol on S. aureus adhesion, we therefore employed a fibrinogen-bing assay in which cell adhesion to fibrinogen-coated plates was stained with crystal violet and quantified by measuring the absorbance at 595 nm. The relative % adhesion was reduced after treatment with kaempferol at 32 μg/ml compared to the control group. When treating with 64 μg/ml of kaempferol, the relative % adhesion decreased by approximately 75% (Figure 2B). These results indicated that kaempferol inhibited the formation of biofilm by reducing S. aureus adhesion.

S. aureus surface proteins include clumping factors (ClfA and ClfB), which are essential for the adhesion of S. aureus to fibrinogen (Bi et al., 2016). These proteins are mainly anchored by sortase A to the cell wall and play significant roles in the formation of biofilms (Cascioferro et al., 2014). Thus, we cultured the biofilm of a sortase A-null mutant of S. aureus (ΔSrtA). The result shown in Figure 3A proved that the ΔSrtA strain had no ability to form biofilms, further indicating that SrtA-mediated surface proteins were essential for this strain (S. aureus ATCC® 29213™) to form biofilms under these conditions. Based on this result, to test the effect of kaempferol on the activity of SrtA, we performed a FRET (Ton-That et al., 1999; Mazmanian et al., 2002) assay in vitro in which a fluorescent peptide substrate Dabcyl-QALPETGEE-Edans of SrtA was used to monitor the fluorescence changes during SrtA catalysis. The result showed that SrtA activity was blocked by 47% (Figure 3B) after treatment with 64 μg/ml kaempferol, suggesting that kaempferol weakened the adhesion of S. aureus by blocking the activity of SrtA, resulting in the inhibition of biofilm formation.

In addition, under a transmission electron microscope (TEM), we observed many fibrous protrusions around the wild-type strain (Figures 4A,E). Biofilms treated with proteinase K were dispersed (data not shown) and the fibrous protrusions disappeared (Figures 4B,F). As shown in Figures 4C,G, the surface of ΔSrtA strain was also smooth. A significant reduction of fibrous protrusions on the surface of bacteria was observed after treatment with 64 μg/ml kaempferol (Figures 4D,H). Moreover, we chose two fluorescent dyes, Syto63 and FITC. The intracellular DNA were stained red with Syto63 dye and the extracellular proteins were stained green with FITC dye. Under confocal laser microscopy, there was obvious green fluorescence around the wild-type strain (Figure 4I) and the green fluorescence decreased after treatment with kaempferol at 64 μg/ml (Figure 4J).

Based on these data, we inferred that kaempferol reduced the anchoring of surface proteins by inhibiting the activity of SrtA, which led to biofilm formation inhibited.

According to the preceding conclusions, kaempferol blocked the activity of SrtA by only 47% at 64 μg/ml, whereas inhibited the biofilm formation of S. aureus by 80% at this concentration. So, there may be other targets for kaempferol to affect the biofilm formation. Above results illustrated that kaempferol only acted on the adhesion stage of biofilm formation. To further investigate the molecular mechanism of adhesion inhibition, qRT-PCR was conducted to detect the transcription level of several adhesion-related genes. As shown in Figure 5, the expression of several genes was altered. The genes clfA and clfB, which encode Clumping factor A (ClfA) and ClfB were repressed by 45 and 88%. In addition, kaempferol also down-regulated fnbA and fnbB which encode Fibronectin-binding proteins (FnbpA and FnbpB). The inhibition rates were 56 and 72%. The global regulatory gene sarA was studied here as well and it was inhibited by 77%. The expression level of the 16S rRNA gene served as the internal control. These results suggested that kaempferol down-regulated the expression of adhesion-related genes, which was responsible to explain its inhibitory effect on the biofilm formation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.