Kaempferol Inhibits the Primary Attachment Phase of Biofilm Formation in Staphylococcus aureus

Abstract

The ability to form biofilms on surfaces makes Staphylococcus aureus the main pathogenic factor in implanted medical device infections. The aim of this study was to discover a biofilm inhibitor distinct from the antibiotics used to prevent infections resulting from S. aureus biofilms. Here, we describe kaempferol, a small molecule with anti-biofilm activity that specifically inhibited the formation of S. aureus biofilms. Crystal violet (CV) staining and fluorescence microscopy clearly showed that 64 μg/ml kaempferol inhibited biofilm formation by 80%. Meanwhile, the minimum inhibitory concentration (MIC) and growth curve results indicated that kaempferol had no antibacterial activity against the tested bacterial strain. Kaempferol inhibited the primary attachment phase of biofilm formation, as determined by a fibrinogen-binding assay. Moreover, a fluorescence resonance energy transfer (FRET) assay and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analyses revealed that kaempferol reduced the activity of S. aureus sortaseA (SrtA) and the expression of adhesion-related genes. Based on these results, kaempferol provides a starting point for the development of novel anti-biofilm drugs, which may decrease the risk of bacterial drug resistance, to prevent S. aureus biofilm-related infections.

Keywords: Staphylococcus aureus; adhesion; biofilm; inhibitor; kaempferol; primary.

Figure 1

Anti-biofilm and antibacterial activities of kaempferol against S. aureus ATCC® 29213™. (A) Chemical structure of kaempferol. (B) S. aureus was incubated with various concentrations of kaempferol for 12 h. CV was used to stain the biofilms. The bound CV was released from the stained cells with 95% ethanol, and the OD595 was measured. Data are represented as the mean ± standard deviation. ^*P < 0.05, and ^***P < 0.001 compared to the control. (C) Fluorescence microscopy. Scale bars represent 200 μm. (D) Growth curve of S. aureus ATCC® 29213™ with or without kaempferol. Kaempferol at 64, 128, and 256 μg/ml had no effect on bacterial growth compared with that of the control group.

Figure 2

(A) Kaempferol specifically inhibited the attachment of phase biofilm formation. Kaempferol was added at various time points during biofilm formation, and the effects were measured after a total of 20 h of incubation. CV was used to stain the biofilms. The bound CV was released from the stained cells with 95% ethanol, and the OD595 was measured. Data are represented as the mean ± standard deviation. ^*P < 0.05, and ^***P < 0.001 compared to the control. (B) Relative % adhesion of S. aureus to fibrinogen. Bacteria treated with kaempferol were cultured in 96-well plates coated with 20 μg/ml fibrinogen at 37°C for 1 h. The OD595 was measured as described above. The relative % adhesion was calculated. ^**P < 0.05, and ^**P < 0.01.

Figure 3

(A) Wild-type and ΔSrtA S. aureus biofilms. (B) Inhibitory effect of kaempferol on the activity of S. aureus SrtA in Vitro. ^*P < 0.05.

Figure 4

TEM. (A,E) Wild-type group. (B,F) Proteinase K group. (C,G) ΔSrtA group. (D,H) Kaempferol 64 μg/ml group. Confocal microscopy images of S. aureus biofilms grown with or without kaempferol. (I) Wild-type group. (J) Kaempferol 64 μg/ml group.

Figure 5

Transcription profiles of S. aureus cells treated with or without kaempferol. S. aureus ATCC® 29213™ was cultivated to an A600 of 1 and incubated with or without kaempferol (64 μg/ml) for 5 h with shaking at 220 rpm. Transcriptional profiles were measured by qRT-PCR. The expression level of 16S rRNA was used to normalize the expression of the genes of interest. The experiment was performed in triplicate (3 qRT-PCR replicates were performed per gene). ^*P < 0.05 vs. non-treated controls (none).