The Anti-Biofilm Potential of Linalool, a Major Compound from Hedychium larsenii, against Streptococcus pyogenes and Its Toxicity Assessment in Danio rerio

Abstract

The anti-biofilm and anti-virulence potential of the essential oil (E.O.) extracted from Hedychium larsenii M. Dan & Sathish was determined against Streptococcus pyogenes. A crystal violet assay was employed to quantify the biofilm. Linalool, a monoterpene alcohol from the E.O., showed concentration-dependent biofilm inhibition, with a maximum of 91% at a concentration of 0.004% (v/v). The AlamarBlue^TM assay also confirmed Linalool's non-bactericidal anti-biofilm efficacy (0.004%). Linalool treatment impeded micro-colony formation, mature biofilm architecture, surface coverage, and biofilm thickness and impaired cell surface hydrophobicity and EPS production. Cysteine protease synthesis was quantified using the Azocasein assay, and Linalool treatment augmented its production. This suggests that Linalool destabilizes the biofilm matrix. It altered the expression of core regulons covRS, mga, srv, and ropB, and genes associated with virulence and biofilm formation, such as speB, dltA, slo, hasA, and ciaH, as revealed by qPCR analysis. Cytotoxicity analysis using human kidney cells (HEK) and the histopathological analysis in Danio rerio proved Linalool to be a druggable molecule against the biofilms formed by S. pyogenes. This is the first report on Linalool's anti-biofilm and anti-virulence potential against S. pyogenes.

Keywords: AlamarBlueTM assay; Danio rerio; Hedychium larsenii; Streptococcus pyogenes; biofilm; linalool.

Figure 1

Structure of Linalool.

Figure 2

Effect of increasing the concentration of Linalool on the growth and biofilm development in S. pyogenes. Graph displaying anti-biofilm activity of Linalool at different concentrations (0.0002% to 0.006%) against S. pyogenes SF370. A concentration of 0.004% was considered the MBIC exhibiting 91% inhibition of biofilm without affecting growth. Error bars represent the S.D. Asterisks indicate statistical significance (p < 0.05).

Figure 3

(a) AlamarBlue^TM assay showing the metabolic viability of the control and Linalool-treated S. pyogenes at its MBIC (0.004%). (b) CFU analysis of the control and Linalool-treated S. pyogenes exhibiting the non-antibacterial nature of Linalool at its MBIC (0.004%). Error bars represent the S.D. Asterisks indicate statistical significance (p < 0.05).

Figure 4

Microscopic visualization of Streptococcus pyogenes biofilm. (A) Light microscopic images (400×) representing Linalool’s inhibitory effect on biofilm microcolony formation. (B) CLSM images (200×) of the acridine orange-stained biofilm of S. pyogenes, showing a decrease in biofilm architecture formation on Linalool treatment. Scale bar 250 μm. (C) SEM images (25,000×) comparing the cell surface features of S. pyogenes biofilms produced in the presence and absence of Linalool (at its MBIC of 0.004%). Scale bars 5 μm.

Figure 5

Effect of Linalool on EPS production, cell surface hydrophobicity, and extracellular protease production. (A) Percentage inhibition of EPS grown in the presence of Linalool (0.004%). (B) Graph showing the cell surface hydrophobicity (CSH) of Streptococcus pyogenes SF370 in the absence and presence of Linalool (0.004%). (C) The graph displays extracellular cysteine protease production by S. pyogenes in the absence and presence of Linalool at the MBIC concentration. Data are represented as mean ± S.D. Asterisks denote statistical significance (p < 0.05).

Figure 6

Linalool modulates virulence gene expression differentially. qPCR analysis of S. pyogenes’ expression of biofilm- and virulence-related genes following a 24-h Linalool treatment. Data are represented as S.D. Asterisks denote statistical significance (p < 0.05).

Figure 7

(A) Effect of Linalool on HEK cells. HEK cells treated with different concentrations of Linalool showed 100% viability at the tested concentrations. Data are represented as S.D. (B) Effect of Linalool on HEK (Human Embryonic Kidney) cells. (a) Control HEK cells (b) HEK cells treated with Linalool (0.004%) were healthy and normal compared to the control.

Figure 8

(A) Experimental setup for establishing an in vivo toxicity analysis on Danio rerio at different concentrations of Linalool. S.C. represents the solvent control. (B) In vivo study in Danio rerio. Survival percentage of Zebrafish at different concentrations of Linalool (%). Data are expressed as the mean ± S.D., p < 0.05. Blank is the control group, and S.C. is the solvent control. Linalool-treated histopathological samples of vital organs after the 4th day of treatment. (C) Light micrographs of gill morphology of adult zebrafish exposed to Linalool for 96 h (100×). (a) Control group; (b) solvent control exhibiting normal structure, including a gill arch formed by filaments (F) and lamellae (S.L.); (c–e) gills exposed to MBIC/2, MBIC, and MBIC × 2 of Linalool exhibited normal histology. (D) Light micrographs of the kidneys of adult zebrafish exposed to Linalool for 96 h (100×). (a) Control group; (b) solvent control exhibiting normal histology with regular renal tubules (R.T.); (c–e) kidney from fish exposed to MBIC/2, MBIC, MBIC × 2 exhibiting normal histology. (E) Light micrographs of liver of adult zebrafish exposed to Linalool for 96 h (100×). (a) Control group (b) solvent control exhibiting normal structure with normal hepatocytes (H); (c–e) liver from fish exposed to MBIC/2, MBIC, and MBIC × 2 exhibited normal histology.