Effect of Achillea fragrantissima Extract on Excision Wound Biofilms of MRSA and Pseudomonas aeruginosa in Diabetic Mice

Abstract

Achillea fragrantissima, a desert plant commonly known as yarrow, is traditionally used as an antimicrobial agent in folklore medicine in Saudi Arabia. The current study was undertaken to determine its antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA) and multi-drug-resistant Pseudomonas aeruginosa (MDR-P. aeruginosa) using in vitro and in vivo studies. A biofilm model induced through an excision wound in diabetic mice was used to evaluate its effect in vivo. The skin irritation and cytotoxic effects of the extract were determined using mice and HaCaT cell lines, respectively. The Achillea fragrantissima methanolic extract was analyzed with LC-MS to detect different phytoconstituents, which revealed the presence of 47 different phytoconstituents. The extract inhibited the growth of both tested pathogens in vitro. It also increased the healing of biofilm-formed excision wounds, demonstrating its antibiofilm, antimicrobial, and wound-healing action in vivo. The effect of the extract was concentration-dependent, and its activity was stronger against MRSA than MDR-P. aeruginosa. The extract formulation was devoid of a skin irritation effect in vivo and cytotoxic effect on HaCaT cell lines in vitro.

Keywords: HaCaT cell lines; LC-MS; cytotoxicity; diabetic; skin irritation.

Figure 1

Chromatogram of the Achillea fragrantissima methanolic extract showing different peaks in positive mode (A) and negative mode (B) in LC-MS analysis.

Figure 1

Chromatogram of the Achillea fragrantissima methanolic extract showing different peaks in positive mode (A) and negative mode (B) in LC-MS analysis.

Figure 2

Antibiofilm activity of methanolic extract of A. fragrantissima in vitro against MRSA and P. aeruginosa in crystal violet assay. Absorbance is measured for the quantification of crystal violet. Amount of crystal violet is directly proportional to the biofilm formation. Bars represent mean ± SEM, n = 3., * p < 0.05, ** p < 0.01, *** p < 0.001 compared to 0 µg/mL concentration.

Figure 3

Wound contraction and epithelization period after different treatments on excision wound in MRSA-induced biofilm. Wound contraction indicates decrease in the wound area as compared to the initial wound area. Day of epithelization shows complete healing of wounds without any raw wound. Bars represent mean ± SEM, n = 6, * p < 0.05, *** p < 0.001 compared to the MRSA + base treatment. Af—Achillea fragrantissima formulation; MPN—mupirocin.

Figure 4

Wound contraction and the epithelization period after different treatments on excision wound in P. aeruginosa-induced biofilm. Wound contraction indicates a decrease in the wound area as compared to the initial wound area. Day of epithelization shows complete healing of wounds without any raw wound. Bars represent mean ± SEM, n = 6, *** p < 0.001 compared to the base treatment. Af—Achillea fragrantissima formulation; GEN—gentamicin.

Figure 5

Histological changes (400×) in the wounded tissue after different treatments. Arrows indicate regenerated skin epithelium. The thickness of the epithelium is lowest in animals treated with base-treated controls (A,D). The thickness of regenerated epithelium was higher after extract treatment in both MRSA (B) and P. aeruginosa (E) infected wounds and the regenerated epithelial height was maximum in animals treated with respective antibiotics (C,F).

Figure 6

Cell viability of HaCaT cell lines treated with different concentrations of the extract.