The natural antimicrobial carvacrol inhibits quorum sensing in Chromobacterium violaceum and reduces bacterial biofilm formation at sub-lethal concentrations

Abstract

The formation of biofilm by bacteria confers resistance to biocides and presents problems in medical and veterinary clinical settings. Here we report the effect of carvacrol, one of the major antimicrobial components of oregano oil, on the formation of biofilms and its activity on existing biofilms. Assays were carried out in polystyrene microplates to observe (a) the effect of 0-0.8 mM carvacrol on the formation of biofilms by selected bacterial pathogens over 24 h and (b) the effect of 0-8 mM carvacrol on the stability of pre-formed biofilms. Carvacrol was able to inhibit the formation of biofilms of Chromobacterium violaceum ATCC 12472, Salmonella enterica subsp. Typhimurium DT104, and Staphylococcus aureus 0074, while it showed no effect on formation of Pseudomonas aeruginosa (field isolate) biofilms. This inhibitory effect of carvacrol was observed at sub-lethal concentrations (<0.5 mM) where no effect was seen on total bacterial numbers, indicating that carvacrol's bactericidal effect was not causing the observed inhibition of biofilm formation. In contrast, carvacrol had (up to 8 mM) very little or no activity against existing biofilms of the bacteria described, showing that formation of the biofilm also confers protection against this compound. Since quorum sensing is an essential part of biofilm formation, the effect of carvacrol on quorum sensing of C. violaceum was also studied. Sub-MIC concentrations of carvacrol reduced expression of cviI (a gene coding for the N-acyl-L-homoserine lactone synthase), production of violacein (pigmentation) and chitinase activity (both regulated by quorum sensing) at concentrations coinciding with carvacrol's inhibiting effect on biofilm formation. These results indicate that carvacrol's activity in inhibition of biofilm formation may be related to the disruption of quorum sensing.

Figure 1. (A–D). Carvacrol reduces biofilm formation by C. violaceum, S. Typhimurium and S. aureus but not P. aeruginosa.

Percentage biofilm formation measured as OD (590 nm) after crystal violet staining after 24 h incubation in the presence or absence of carvacrol (0–0.8 mM) (bars, left axis) compared to colony counts (log cfu/ml) from the same samples (connected dots, right axis). C. violaceum (A), S. Typhimurium (B), S. aureus (C), P. aeruginosa (D). Data points which are significantly different from each other are denoted by different letters (p<0.05). Bars indicate standard error of the mean.

Figure 2. (A–C). Carvacrol has no significant effect on pre-formed biofilms.

Percentage remaining of pre-formed biofilms after 24 h incubation in presence or absence of carvacrol (0–8 mM). Expressed as percentage original biofilm measured as OD at 590 nm. C. violaceum (A), S. Typhimurium (B), S. aureus (C). Data points marked with asterisks are just significantly lower than the 24 h control (p = 0.05). Bars indicate standard error of the mean.

Figure 3. Carvacrol inhibits gene expression of cviI at concentrations too low to influence bacterial growth or survival.

Expression of cviI by C. violaceum after incubation with carvacrol for 24, 48 and 72 h. Mean fold increases compared to t = 24 h, 0 mM carvacrol for three experiments are shown. Data points which are significantly different from each other are denoted by different letters (p<0.05). Bars indicate standard error of the mean.

Figure 4. Carvacrol inhibits production of violacein by C. violaceum.

The amount of violacein pigment present in a butanol extraction after 24(0–0.8 mM) measured as OD 590 nm (bars, left axis), compared to the colony count of bacteria present in the suspension (log cfu/ml) (connected dots, right axis). Data points which are significantly different from each other are denoted by different letters (p<0.05). Bars indicate standard error of the mean.

Figure 5. Carvacrol inhibits chitinolytic activity of C. violaceum.

Diameter of clear chitinolytic zone around C. violaceum centrally inoculated onto low-nutrient agar plates containing chitin after 14 d incubation (mean of three experiments). Data points which are significantly different from each other are denoted by different letters (p<0.05). Bars indicate standard error of the mean.