Antibacterial Activity and Mechanism of Action of Aspidinol Against Multi-Drug-Resistant Methicillin-Resistant Staphylococcus aureus

Abstract

This study aimed at investigating the antibacterial activity of aspidinol, an extract from Dryopteris fragrans (L.) Schott, against methicillin-resistant Staphylococcus aureus (MRSA). MRSA isolates were treated with aspidinol to determine the differential expression of genes and associated pathways following the drug treatment. Aspidinol displayed significant anti-MRSA activity, both in vivo (minimum inhibitory concentration = 2 μg/mL) and in vitro, and achieved an antibacterial effect comparable to that of vancomycin. In the lethal septicemic mouse study, a dose of 50 mg/kg of either aspidinol or vancomycin provided significant protection from mortality. In the non-lethal septicemic mouse study, aspidinol and vancomycin produced a significant reduction in mean bacterial load in murine organs, including the spleen, lung, and liver. After treatment with aspidinol, we found through RNA-seq and RT-PCR experiments that the inhibition of the formation of ribosomes was the primary S. aureus cell-killing mechanism, and the inhibition of amino acid synthesis and the reduction of virulence factors might play a secondary role.

Keywords: RNA-seq; anti-MRSA activity; antimicrobial mechanism; aspidinol; inhibit ribosomes synthesis.

FIGURE 1

Time-kill analysis and the intracellular bacterial killing activity of aspidinol. (A) Time-kill kinetics of aspidinol against S. aureus ATCC 33591. The error bars show the standard deviations of the results from three independent biological repeats. (B) S. aureus ATCC 33591-infected RAW264.7 cells were treated with aspidinol and control antibiotics (vancomycin or linezolid) for 24 h, and the percent bacterial reduction was calculated compared to that of the untreated control groups. The results are given as the mean ± SD (n = 3). Two-tailed Student’s t-test was employed, and ^∗p-values of ≤0.05 are considered to be significant.

FIGURE 2

Anti-biofilm activity of aspidinol. (A) The effects of aspidinol and antibiotics (linezolid and vancomycin) on established biofilms of S. aureus ATCC 33591 were evaluated. The pre-formed biofilms were treated with linezolid, vancomycin, or aspidinol and then stained with crystal violet. The optical density of the dissolved crystal violet was measured using a spectrophotometer. The values are the mean of the triplicate samples with standard deviation bars. The results are given as the mean ± SD (n = 3). Two-tailed Student’s t-test was employed, and ^∗∗∗ means p-values ≤ 0.005. (B) Scanning electron microscopy images showing the structure of MRSA biofilms treated with aspidinol and antibiotics at 24 h. Magnifications, ×2000.

FIGURE 3

Aspidinol is effective in a mouse model of MRSA septicemic infection. (A) Ten mice per group were infected (i.p.) with a lethal dose of S. aureus ATCC 33591 and intravenously injected with aspidinol, vancomycin (5, 15, or 25 mg/kg), or the vehicle alone for 5 days (one dose per day). Mice were monitored for 5 days and the percentage survival was calculated. The statistical significance was calculated in order to compare treated to control groups. (B) Six mice per group were infected (i.p.) with a non-lethal dose of S. aureus ATCC 33591 and intravenously injected with aspidinol, vancomycin (25 mg/kg) or the vehicle alone for 6 days (one dose per day). Twenty-four hours after the last treatment, the mice were euthanized, and their organs were excised and homogenized in TSB to count viable MRSA colonies. The number of CFUs from each mouse is plotted as individual points. Values are the mean of triplicate samples with standard deviation bars. (C) Histological evaluation of lung and liver of mice infected with S. aureus ATCC 33591 receiving no treatment or a treatment with aspidinol. Both lung and liver in the control group demonstrated acute inflammation; no apparent pathological changes were observed in the treatment group.

FIGURE 4

RNA-Seq gene expression results for S. aureus ATCC 33591 cells treated and not treated with aspidinol. (A) A heatmap generated comparing aspidinol-treated cells to untreated control S. aureus ATCC 33591 cells is shown. Triplicate samples were used for each group. (B) Volcano plot analyses of unigenes in aspidinol and the ATCC 33591 group. (C) Differentially expressed genes enriched in the KEGG pathway.

FIGURE 5

Validation of RNA-seq data for selected genes by real-time PCR. (A) Differentially expressed genes involved in amino acid synthesis; (B) differentially expressed genes involved in iron ABC transporter synthesis; (C) differentially expressed genes involved in ribosome synthesis; (D) differentially expressed genes involved in beta-lactam resistance and virulence factor.