Effect of Temperature on Antimicrobial Activity and Mode of Action of Thymol against Staphylococcus aureus

Objective: Staphylococcus aureus is one of the important food borne pathogens. The use of chemical food preservations to prevent the contamination of such bacterial strain in foods has potential to pose harm to consumers. Therefore, currently there have been attempts to find safe natural products such as thymol to use as alternatives. Methods: This study was to examine the effect of temperature on antimicrobial activity and mode of action of thymol against S. aureus. As studied at 370C and 200C, thymol had ability to inhibit the bacterium with the Minimal Inhibitory Concentration (MIC) of 1.25 mM and had bacteriocidal mode of action. Findings: These results suggest that temperature has no effect on antimicrobial activity and mode of action of thymol against S. aureus that is a gram positive bacterium. The findings are different from those of previous research works reporting that temperature had an effect on antimicrobial activity of thymol against gram negative bacteria. Application: Thus, this report is the first to propose the possible explanation on why temperature affects antimicrobial activity of thymol against gram positive and gram negative bacteria differently. *Author for correspondence


Introduction
TFood borne pathogens are groups of disease causing microorganisms that are associated with foods. They can be bacteria, fungi and viruses. Symptoms resulting from these pathogens range from mild to severe depending on infected persons' health and amount of pathogens that they receive. Among food borne pathogens, Staphylococcus aureus is considered to be the most common causative agents of food borne diseases in humans. Therefore, it is often used as representatives of bacterial food borne pathogens in several experiments. S. Indian Journal of Science and Technology, Vol 12(15)   aureus when grow in foods make toxins that cause nausea, stomach cramps, vomiting, and diarrhea 1 . Currently, chemical food preservatives such as benzoic acid, sorbic acid, sulfur dioxide, nitrites and nitrates have been the most common used agents to prevent the growth of pathogens in foods. However, their potential harms to consumers' health have been continuously reported 2 and recently driven consumer demand for natural and safe alternatives.
Essential oils and their components are natural products obtained from many parts of plants including Keywords: Antimicrobial Activity, Mode of Action, Staphylococcus aureus, Thymol flowers, seeds, leaves, twigs and roots. They have been known for their antimicrobial activity since ancient times. The safe use of essential oils and their components has led to their current status of generally recognized as safe (GRAS) food ingredients. Thymol is a major component of several plants' essential oils including thyme and oregano 3,4 . It has been found to exhibit antimicrobial activity against a variety of food borne pathogenic bacteria, including S. aureus [5][6][7][8] . However, effect of temperature on antimicrobial activity and mode of action of thymol against the bacterium has not been performed. Therefore, in the present study, we focused on how temperature affects the MIC and mode of action of thymol against S. aureus.

Bacterial Strain and Culture Conditions
The bacterial strain used in this study was S. aureus ATCC 25923 that was obtained from the American Type Culture Collection (ATCC). Its identity was confirmed using the API Stap-Ident system (bioMerieux Industry, Hazelwood, MO, USA), respectively. The bacteria strain was grown at 37°C in BHI (Brain Heart Infusion) broth. Its stock culture was stored as a frozen culture at -80°C in BHI broth containing 20% glycerol (v/v).

Chemicals
Thymol was obtained from Himedia, Mumbai, India. The stock solution of thymol (100 mM) was made in 95% ethanol and kept at 4°C in a tightly sealed container in the dark.

Antimicrobial Activity of Thymol against S. aureus
In this study, the swab-paper disc technique was used to detect the antimicrobial activity of thymol against S.
aureus. An exponential phase culture of the tested bacterial strain was spread with a sterile swab on a BHI agar plate. A sterile 6-mm-diameter filter paper disc (Schleicher & Schuell, Inc., Keene, N.H.) was placed on the agar plate containing the tested bacterial strain. Ten mL of thymol were spotted onto the paper disc. For a control, 95% ethanol was used instead of thymol. The plates were incubated overnight at 37°C and then checked for an inhibition zone around the paper disc. This experiment was performed in triplicate.

Determination of Minimal Inhibitory Concentration
Thymol stock solution was subjected to twofold serial dilution using 95% ethanol as a diluent. One hundred μL of each dilution were added to 1.

Examination of Mode of Action
To examine whether thymol exhibited bacteriostatic or bactericidal effect on S. aureus, 100 μL of thymol solution (at a final concentration equal to the MIC value) were added to a 1.9 mL culture of the tested bacterial strain (approximately 10 3 CFU/mL). For control, 95% ethanol was used to replace thymol solution. After incubation for 24 h (at 20°C or 37°C), a 100 μL aliquot of the mixture was spread onto a (thymol free) BHI agar plate. The appearance of bacterial colony on the agar plate was observed after incubation the agar plate at 37°C for 24 h. The presence and absence of (thymol treated) bacterial colony on thymol free agar indicated the bacteriostatic and bactericidal effects of thymol, respectively.
By using swab paper disc method, it was found that thymol had antimicrobial activity against S. aureus, indicating by an inhibition zone on the lawn of the bacterial strain. The diameter of the inhibition zone was about 18 mm ( Figure  1). On the other hand, no inhibition zone was observed in the control experiment using 95% ethanol instead of thymol ( Figure 1). Our results support reports of previous research works demonstrating the broad antimicrobial spectrum of thymol against both grams positive and gram negative bacteria 5-8 .

Determination of Minimal Inhibitory Concentration
The MIC value of thymol for S. aureus was determined by observing bacterial growth at 20°C and 37°C in the BHI broth having thymol with concentrations increased twofold ranging from 0.08 mM to 5.00 mM. No difference in Figure 1. Antimicrobial activity of (a) thymol (b) 95% ethanol against S. aureus as studied by the swab-paper disc technique. bacterial growth in BHI broth containing different concentrations of thymol was found at 20°C and 37°C. At both temperatures, no growth of bacteria was observed in BHI broth having thymol with concentrations of 1.25, 2.50 and 5.00 mM (Figure 2). These results suggested that temperature might have no effect on the antimicrobial activity of thymol against S. aureus because at both temperatures the MIC of thymol was 1.25 mM. Temperature has been shown to be a major factor influencing antimicrobial activity of several essential oil components [9][10][11][12] . Reported that thymol had higher antimicrobial activity against gram negative Shigella sonnei at 37°C than at 4°C. Similarly, our previous study reported that thymol had higher antimicrobial activity against gram negative E. coli O157:H7 at 37°C than at 20°C. However, this study found that temperature had no effect on antimicrobial activity of thymol against gram positive S. aureus. This difference might be resulted from high lipid solubility of thymol and high lipid contents of bacterial outer membrane (OM), a structure that exists only in gram negative bacteria. At low temperature (20°C), the fluidity of bacterial OM may lessen and become more rigid, which would lower the number of thymol dissolving in the OM and sensitivity of the bacterium to the compound. This situation may not occur in gram positive bacteria, including S. aureus, because it has no OM and the temperature may not affect the structure of its cell wall ( Figure 3). However, further investigation is required to elucidate the actual cause of this difference.

Examination of Mode of Action
Mode of action of thymol was examined by observing the ability of bacterial cells pre-treated with thymol (at MIC) for 24 h to grow on thymol free BHI agar.
S. aureus cells pre-treated with thymol (at MIC) for 24 h at both 20°C and 37°C could not resume their growth on thymol free BHI agar indicating that thymol had a temperature independent bacteriocidal effect against S. aureus. These results are in agreement with previous reports showing that thymol had bactericidal effect on several strain of bacteria including Bacillus cereus, Campylobacter jejuni, E. coli, Listeria monocytogenes and Salmonella enterica 13,14 . Our previous work also found that thymol had a temperature independent bactericidal effect against E. coli O157:H7. The temperature independent bactericidal mode of action of thymol may broaden its applications in a wide range of temperature.

Conclusion
Thymol has been shown by many research works including this work to have antimicrobial activity against a variety of pathogenic bacteria. However, in order to use it effectively as a biological control substance, information about its properties and factors influencing its properties is required. This study is the first report presenting the difference in effect of temperature on antimicrobial activity of thymol against gram negative and gram positive bacteria and proposing the possible cause of the difference.