covR Mediated Antibiofilm Activity of 3-Furancarboxaldehyde Increases the Virulence of Group A Streptococcus

Abstract

Background: Group A streptococcus (GAS, Streptococcus pyogenes), a multi-virulent, exclusive human pathogen responsible for various invasive and non-invasive diseases possesses biofilm forming phenomenon as one of its pathogenic armaments. Recently, antibiofilm agents have gained prime importance, since inhibiting the biofilm formation is expected to reduce development of antibiotic resistance and increase their susceptibility to the host immune cells.

Principal findings: The current study demonstrates the antibiofilm activity of 3Furancarboxaldehyde (3FCA), a floral honey derived compound, against GAS biofilm, which was divulged using crystal violet assay, light microscopy, and confocal laser scanning microscopy. The report is extended to study its effect on various aspects of GAS (morphology, virulence, aggregation) at its minimal biofilm inhibitory concentration (132μg/ml). 3FCA was found to alter the growth pattern of GAS in solid and liquid medium and increased the rate of auto-aggregation. Electron microscopy unveiled the increase in extra polymeric substances around cell. Gene expression studies showed down-regulation of covR gene, which is speculated to be the prime target for the antibiofilm activity. Increased hyaluronic acid production and down regulation of srtB gene is attributed to the enhanced rate of auto-aggregation. The virulence genes (srv, mga, luxS and hasA) were also found to be over expressed, which was manifested with the increased susceptibility of the model organism Caenorhabditis elegans to 3FCA treated GAS. The toxicity of 3FCA was ruled out with no adverse effect on C. elegans.

Significance: Though 3FCA possess antibiofilm activity against GAS, it was also found to increase the virulence of GAS. This study demonstrates that, covR mediated antibiofilm activity may increase the virulence of GAS. This also emphasizes the importance to analyse the acclimatization response and virulence of the pathogen in the presence of antibiofilm compounds prior to their clinical trials.

Fig 1. Quantification of biofilm formation by crystal violet staining.

Biofilms were grown in 24 well micro titre plates for 24 h in 1 ml THYG, with increasing concentrations of 3FCA or acetone (vehicle control). (A) Percentage inhibition of GAS biofilm grown in presence of 3FCA in comparison with that grown in presence of vehicle control (acetone). Significant increase in biofilm inhibition was observed with increasing concentrations of 3FCA. Error bars indicate standard deviations. The presence of 3FCA at 132 μg/ml was found to reduce 90% of group A streptococcal biofilm formation. (B) Photograph showing biofilms grown with (bottom panel) and without (top panel) 3FCA at MBIC (132μg/ml) after crystal violet staining.

Fig 2. Growth of GAS in the presence and absence of 3FCA at MBIC.

(A) A representative graph depicting the OD 600 of cultures of GAS grown in the presence and absence of 3FCA at MBIC and 1/2MBIC. The doubling time of GAS in the presence and absence of 3FCA were found to be around 2 h and the cells reached stationary phase at the eleventh hour. (B) The viability of control and 3FCA (132 μg/ml) treated biofilm and planktonic GAS were quantified after 24 h using XTT reduction assay. An insignificant difference was found in the viability of GAS in control and treated samples.

Fig 3. Microscopic analysis of GAS.

Microscopic images of GAS grown over glass slides (1 x 1 cm) in THYG in the presence and absence of 3FCA at 132μg/ml concentration. (A, B) Light microscopic images (at 400X) of crystal violet stained GAS biofilm grown in the presence of 3FCA (B) and in presence of vehicle control (A). (C, D) CLSM images of acridine orange stained group A streptococcal biofilm grown in the presence (D) and absence (C) of 3FCA. Both the light microscopic and CLSM images clearly display the antibiofilm activity of 3FCA. (E, F, G, H) SEM images of GAS biofilm. (E and F) showing the breach in streptococcal biofilm grown in the presence of 3FCA. Scale-10 μm. (G and H) Displaying the difference in the morphology of GAS grown in the presence (H) and absence of 3FCA (G). Scale-2 μm. (I, J, K, L) TEM images displaying the increased extracellular hyaluronic acid secretion around 3FCA treated GAS (J, L) compared to corresponding control (I, K). [I, J Scale-1 μm, K, L scale-250 nm]. Significant differences in control and treated samples are highlighted with arrows.

Fig 4. Morphology of GAS grown in tryptose agar.

(a, b) Growth of GAS in Tryptose agar plates. GAS growth pattern were found to be mucoid in the presence of 3FCA (b) compared to its control (a). (c, d) Growth of GAS in liquid medium (THYG). GAS grown in the presence of 3FCA (d) were found to be clumped at the centre, whereas their corresponding control (c) were uniformly distributed throughout the well and found to be adhered to the surface.

Fig 5. Auto-aggregation pattern of GAS.

Photograph showing auto-aggregation patterns of GAS suspensions in the absence of 3FCA (Control), presence of 3FCA at 1/2 MBIC (66 μg/ml-T1) and MBIC (132 μg/ml-T2) after 30 min in test tube (A) and in micro titre plate (B). An increased rate of auto-aggregation was observed among treated samples in both the cases.

Fig 6. Hyaluronic acid quantification of GAS.

Percentage increase in hyaluronic acid production by GAS grown in the presence of compound with respect to control. Error bars indicate standard deviations. A concentration dependent increase in hyaluronic acid production was observed.

Fig 7. Differential Gene expression pattern.

Effect of 132 μg/ml of 3FCA on relative gene expression of specific genes involved in virulence, biofilm formation and aggregation in GAS. Data are expressed as mean ± standard deviations. The expression was normalized to the house keeping gene gyrA. * indicates p <0.005 and ** p<0.001 compared to control.

Fig 8. Effect of 3FCA and 3FCA treated GAS on the survival of C. elegans.

C. elegans were exposed to Acetone (vehicle control) + OP50 (♦), 3FCA + OP50 (■), Acetone (vehicle control) + GAS (▲), 3FCA + GAS (×). The survival rate of C. elegans in presence of OP50 was found to be insignificantly affected by 3FCA, whereas significant differences were observed among the lifespan of the nematodes grown in the presence of GAS alone when compared to those grown in 3FCA treated GAS. * denotes p<0.001 compared to control.