Understanding the Role of the Antioxidant Drug Erdosteine and Its Active Metabolite on Staphylococcus aureus Methicillin Resistant Biofilm Formation

Abstract

Increasing numbers of researches have suggested that some drugs with reactive oxygen species (ROS)-mediated mechanisms of action modulate biofilm formation of some pathogenic strains. However, the full contribution of ROS to biofilm development is still an open question. In this paper, the correlations between the antioxidant drug Erdosteine (Er) and its active Metabolite I (Met I), ROS and biofilm development of two strains of methicillin resistant Staphylococcus aureus are presented. Experiments revealed that Er and Met I at 2 and 5 mg/L increased up to three orders of magnitude the number of biofilm-dwelling cells, while the content of ROS within the biofilms was reduced above the 87%, with a major effect of Met I in comparison to Er. Comparative proteomics showed that, 5 mg/L Met I modified the expression of 30% and 65% of total proteins in the two strains respectively. Some proteins involved in cell replication were upregulated, and a nitric oxide-based mechanism is assumed to modulate the biofilm development by changing quorum sensitive pathways. Additionally, several proteins involved in virulence were downregulated in the presence of Met I, suggesting that treated cells, despite being greater in number, might have lost part of their virulence.

Keywords: S. aureus; antioxidant; biofilm; erdosteine; metabolite I; nitric oxide; oxidative stress; proteomics.

Figure 1

Chemical structure of the antioxidant drug Er (N-(carboxymethylthioacetyl)-homocysteine thiolactone) (A) and Met I ((±)-N-(2-carboxymethylthioacetyl)homocysteine) (B).

Figure 2

Adhered cells (CFU/cm²) within MRSA ATCC 43300 (A) and 98825 TX (B) biofilms grown with Er and Met I at 0, 2 and 5 mg/L. Data report the mean ± standard deviation of three independent values. Different superscript letters indicate significant differences (Tukey’s HSD, p ≤ 0.05) between the means of different concentrations whereas a * indicates significative differences between Er and the corresponding counterpart grown with Met I at the same concentration.

Figure 3

Representative views of 3-D CLSM images of biofilm developed without (A,D) and with Er (B,E) and Met I (C,F) at 5 mg/L. Live bacteria were stained green with SYTO 9 fluorescent nucleic acid dye while the biofilm matrix was visualized in red by the lectin Concanavalin A stain. Scale bar = 50 or 100 μm.

Figure 4

Level of oxidative stress within the MRSA ATCC 43300 (A) and 98825 TX (B) biofilms grown with Er and Met I at 0, 2 and 5 mg/L. Data report the mean ± standard deviation of three independent measurements. Different superscript letters indicate significant differences (Tukey’s HSD, p ≤ 0.05) between the means of different concentrations whereas a star indicates significative differences between Er and the corresponding counterpart grown with Met I at the same concentration.

Figure 5

Biofilm dispersion index (%) of MRSA ATCC 43300 (A) and 98825 TX (B) biofilms pre-grown with Er and Met I at 0, 2 and 5 mg/L and soaked in PBS for 1 h. Data report the mean ± standard deviation of three independent measurements. Different superscript letters indicate significant differences (Tukey’s HSD, p ≤ 0.05) between the means of different concentrations whereas a * indicates significant differences between Er and the corresponding counterpart grown with Met I at the same concentration.

Figure 6

Venn diagram and histogram about proteins differentially expressed in ATCC 43300 (A,C) and 98825 TX (B,D) biofilms grown with Met I at 5 mg/L. Panel (A,B): Venn diagram shows proteins not significantly different in abundance between the positive control and treated samples and differentially abundant proteins with a ±1.5-fold change in the treated biofilm compared to the control one. Panel (C,D): histograms displays the number of differentially expressed proteins within a specific range of fold changes.

Figure 7

GO functional classification of proteins differently expressed in both ATCC 43300 and 98825 TX biofilms upon Met I at 5 mg/L treatment. GO classification was performed in terms of molecular function (red), cellular compartment (green) and biological process (blue).