Towards Green Strategies of Food Security: Antibacterial Synergy of Essential Oils from Thymus vulgaris and Syzygium aromaticum to Inhibit Escherichia coli and Staphylococcus aureus Pathogenic Food Isolates

Abstract

Foodborne diseases continue to represent an important public health issue. The control of food spoilage and pathogenic microorganisms is achieved mainly by synthetic chemicals, unfortunately associated to several undesirable aspects. The growing requirement for new and safe alternative strategies has resulted in the research of agents from natural sources with antimicrobial properties, such as essential oils (EOs). This study's purpose was to define the antibacterial profile of thyme (Thymus vulgaris) and cloves (Syzygium aromaticum) essential oils against both Gram-positive and Gram-negative important foodborne pathogenic bacteria. Gas chromatography mass spectrometry analysis was performed for EOs' chemical composition. Qualitative in vitro antimicrobial assays (i.e., agar well diffusion method and disk-volatilization method) allowed for verification of the efficacy of EOs, used individually and in binary combination and both in liquid and vapor phase, against Staphylococcus aureus and Escherichia coli food isolates. Minimal inhibitory concentrations and minimal bactericidal concentration values have been used to quantitatively measure the antibacterial activity of EOs, while the fractional inhibitory concentration index has been considered as a predictor of in vitro antibacterial synergistic effects. The microbiological tests suggest that thyme and cloves EOs, rich in bioactive compounds, are able to inhibit the growth of tested foodborne bacteria, especially in vapor phase, also with synergistic effects. Results provide evidence to consider the tested essential oils as promising sources for development of new, broad-spectrum, green food preservatives.

Keywords: antibacterial agents; antibacterial synergy; essential oils; food pathogens; natural food preservatives.

Figure 1

In vitro antibacterial activity of thyme and cloves essential oils, individually used and in binary combination (1:1 ratio), evaluated by the agar well diffusion method, against Escherichia coli (A) and Staphylococcus aureus (B) food isolates. Graphical representation of the results; the mean diameter of inhibition zone (in mm) is reported as the mean of values obtained from assays in triplicate ± standard deviation. Statistical significance was examined by the one-way ANOVA test, with Dunnett’s correction (p < 0.05) for bars comparison with positive control bar, and with Tukey’s correction (p < 0.05) for multiple comparisons between bars. Asterisks indicate the statistical significance respect to the positive control (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05); the absence of asterisks indicates absence of significance. Letters (a, b, c, d) are used for multiple comparisons. Different letters indicate significant differences between bars; bars with no significant differences receive the same letter. MDIZ, mean diameter of the inhibition zone; thy-EO, thyme essential oil; cl-EO, cloves essential oil; GNT, gentamicin; VNC, vancomycin.

Figure 2

In vitro antibacterial activity of the volatile components of thyme and cloves essential oils, individually used and in binary combination (1:1 ratio), evaluated by the disk volatilization method, against Escherichia coli (A) and Staphylococcus aureus (B) food isolates. Graphical representation of the results; the mean diameter of inhibition zone (in mm) is reported as the mean of values obtained from assays in triplicate ± standard deviation. Statistical significance was examined by the one-way ANOVA test, with Dunnett’s correction (p < 0.05), for bars comparison with positive control bar, and with Tukey’s correction (p < 0.05), for multiple comparisons between bars. Asterisks indicate the statistical significance with respect to the positive control (**** p < 0.0001; ** p < 0.01; * p < 0.05); the absence of asterisks indicates absence of significance. Letters (a, b, c, d, e) are used for multiple comparisons. Different letters indicate significant differences between bars; bars with no significant differences receive the same letter. MDIZ, mean diameter of the inhibition zone; thy-EO, thyme essential oil; cl-EO, cloves essential oil; NaClO, sodium hypochlorite 5%.

Figure 3

Comparative analysis of in vitro antimicrobial effects of thyme and cloves essential oils, individually used and in binary combination (1:1 ratio), evaluated with agar well diffusion method and with disk volatilization method against Escherichia coli (A) and Staphylococcus aureus (B) food isolates. The inhibition zone (in mm) is reported as mean value from triplicate assays ± standard deviation, performed using the same volume (20 µL) of each tested antibacterial agent. Statistical significance was examined by two-way ANOVA test, with Tukey’s correction (p < 0.05). Asterisks indicate the statistical significance between selected groups (**** p < 0.0001). Letters (a, b, c, d, e) are used for multiple comparisons between bars. Different letters indicate significant differences; bars with no significant differences receive the same letter. MDIZ, mean diameter of the inhibition zone. MDIZ, mean diameter of the inhibition zone; thy-EO, thyme essential oil; cl-EO, cloves essential oil.