Synergistic Effect of Abietic Acid with Oxacillin against Methicillin-Resistant Staphylococcus pseudintermedius

Abstract

Resin acids are valued in traditional medicine for their antiseptic properties. Among these, abietic acid has been reported to be active against methicillin-resistant Staphylococcus aureus (MRSA) strains. In veterinary healthcare, the methicillin-resistant Staphylococcus pseudintermedius (MRSP) strain is an important reservoir of antibiotic resistance genes including mecA. The incidence of MRSP has been increasing, and treatment options in veterinary medicine are partial. Here, we investigated the antimicrobial and antibiofilm properties of abietic acid against three MRSP and two methicillin-susceptible Staphylococcus pseudintermedius (MSSP) strains, isolated from diseased pet animals and human wound samples. Abietic acid showed a significant minimal inhibitory concentration (MIC) value ranging from 32 to 64 μg/mL (MRSPs) and 8 μg/mL (MSSP). By checkerboard method we demonstrated that abietic acid increased oxacillin susceptibility of MRSP strains, thus showing a synergistic interaction with oxacillin. Abietic acid was also able to contrast the vitality of treated MSSP and MRSP1 biofilms at 20 μg/mL and 40 μg/mL, respectively. Finally, the compound moderately reduced mecA, mecR1 and mec1 gene expression. In conclusion, the results here reported demonstrate the antimicrobial activity of abietic acid against MRSP and support the use of this compound as a potential therapeutic agent to be used in combinatorial antibiotic therapy.

Keywords: Staphylococcus pseudintermedius; abietic acid; antimicrobial activity; methicillin-resistant Staphylococcus pseudintermedius; methicillin-susceptible Staphylococcus pseudintermedius; synergistic interaction.

Figure 1

Time-kill assay of abietic acid against Ctrl, MRSP1-3 and MSSP strains. Each experiment is the result of three independent experiments performed in triplicate. Ctrl: human antibiotics susceptible S. pseudintermedius strain; MSSP: veterinary methicillin-susceptible strain; MRSP1: veterinary methicillin-resistant S. pseudintermedius strain; MRSP2 and 3: human methicillin-resistant S. pseudintermedius strains. * represents p  <  0.05.

Figure 2

Relative expression of genes of mec operon in MRSP strains treated with subinhibitory concentration of abietic acid (16 mg/mL for MRSP1 and MRSP3, and 32 mg/mL for MRSP2) and 1 mg/mL oxacillin. Values represent the mean ± SD for three independent experiments. MRSP1: veterinary methicillin-resistant S. pseudintermedius strain; MRSP2 and 3: human methicillin-resistant S. pseudintermedius strains. Aa: abietic acid; Oxa: oxacillin.

Figure 3

Activity of the abietic acid on S. pseudintermedius biofilm formation. Values represent the mean ± SD for three independent experiments. * indicates statistically significant difference (p < 0.05).

Figure 4

Effect of abietic acid on one-day-old biofilms of S. pseudintermedius. Upper panel: biofilm biomass obtained by crystal violet staining; bottom panel: biofilm vitality obtained by XTT assay. Bars represent the average values ± SD from three independent experiments. * represents p < 0.05.

Figure 5

Confocal laser scanner microscopy micrographs of MSSP and MRSP1 biofilms. The panels contain three-dimensional images, green and red fluorescence is associated with live and dead cells, respectively. (A) MSSP untreated biofilm (B) MSSP biofilm treated with abietic acid at 20 µg/mL (C) MRSP1 untreated biofilm (D) MRSP1 biofilm treated with abietic acid at 40 µg/mL.