Willow Bark-Derived Material with Antibacterial and Antibiofilm Properties for Potential Wound Dressing Applications

Tree stems contain wood in addition to 10–20% bark, which remains one of the largest underutilized biomasses on earth. Unique macromolecules (like lignin, suberin, pectin, and tannin), extractives, and sclerenchyma fibers form the main part of the bark. Here, we perform detailed investigation of antibacterial and antibiofilm properties of bark-derived fiber bundles and discuss their potential application as wound dressing for treatment of infected chronic wounds. We show that the yarns containing at least 50% of willow bark fiber bundles significantly inhibit biofilm formation by wound-isolated Staphylococcus aureus strains. We then correlate antibacterial effects of the material to its chemical composition. Lignin plays the major role in antibacterial activity against planktonic bacteria [i.e., minimum inhibitory concentration (MIC) 1.25 mg/mL]. Acetone extract (unsaturated fatty acid-enriched) and tannin-like (dicarboxylic acid-enriched) substances inhibit both bacterial planktonic growth [MIC 1 and 3 mg/mL, respectively] and biofilm formation. The yarn lost its antibacterial activity once its surface lignin reached 20.1%, based on X-ray photoelectron spectroscopy. The proportion of fiber bundles at the fabricated yarn correlates positively with its surface lignin. Overall, this study paves the way to the use of bark-derived fiber bundles as a natural-based material for active (antibacterial and antibiofilm) wound dressings, upgrading this underappreciated bark residue from an energy source into high-value pharmaceutical use.

Briefly, the formed biofilm was washed once with phosphate-buffered saline (1x PBS;137 mM NaCl, 2.7 mM KCl, 1 mM Na 2 HPO 4 ; and 2 mM KH 2 PO 4 ; pH 7.4).For CFU assays, biofilm cells were suspended in 1x PBS by vigorous pipetting, followed by appropriate 10fold dilutions.Then, aliquots of 10 µL were drop-plated on TSA plates and CFU enumerated after 24 h of incubation at 37 °C.For biomass, the washed biofilm was fixed with 100% ethanol for 5 min, air-dried for 10 min and stained with 0.01% of crystal violet (CV, Sigma-Aldrich).After 30 min of incubation biofilm was washed with sterile water and air-dried for 30 min.Finally, 96% w/v of ethanol was added, and stained biofilm was resuspended by vigorous pipetting.CV absorbance measurements at 570 nm were performed using Multiskan GO (Thermo Fisher Scientific).For metabolic activity assessment, resazurin solution (4 µg/mL, Sigma-Aldrich) was added into washed biofilms.Plates were then incubated at 25 °C for 20 min in the dark.A multimode microplate reader (Varioskan LUX, Thermo Fisher Scientific) was used to measure the relative fluorescence units (RFU, λex=530 nm and λem=590 nm).S3.

Fig. S1
Fig. S1 The photographs of the fractionated components and different forms of willow bark fiber bundles (WBFB).a Purified component.b Purified components in their dissolved form for bioactivity experiments.c Fiber bundles that have been used in this study.

Fig. S2
Fig. S2 Biofilm formation assessment of S. aureus DSM 28763 and DSM 25691.a Cell numbers measured by CFU/ml.b Metabolically active cells measured by resazurin fluorescence (λ ex =530 nm and λ em =590 nm).c Biofilm mass measured by crystal violet (CV) absorbance (OD 570nm ) and illustration of stained bacterial biofilm formed on polystyrene wells.Mean of four independent biological experiments (n: 6) for resazurin and CV assays.Mean of three independent experiments for cell numbers determination (n: 4).* Denotes for significant differences (p<0.05).

Fig.
Fig. S10 GC-MS total-ion chromatogram of the major peaks of tannin-like components obtained from WBFB (Fig. 1).Their characteristic m/z are summarized at TableS3.

Table S1 . Chemical composition of detectable components (10.6 ± 0.6 wt % detected) as weight percentage (wt %) of fractionated acetone extracts from WBFB. MS
spectra were referenced with the NIST chemistry book [N].

Table S3 . Chemical composition of tannin-like substances (47.8 wt % detected) that is obtained from WBFB by 0.1M NaOH treatment as weight percentage (wt %) of the analysed tannin-like fraction
. MS spectra were reference with the NIST chemistry book [N].