Phytochemical Screening and Antibacterial Activity of Commercially Available Essential Oils Combinations with Conventional Antibiotics against Gram-Positive and Gram-Negative Bacteria

Abstract

The present study aims to evaluate the antibacterial activity of five commercially available essential oils (EOs), Lavender (LEO), Clove (CEO), Oregano (OEO), Eucalyptus (EEO), and Peppermint (PEO), against the most-known MDR Gram-positive and Gram-negative bacteria-Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853)-alone and in various combinations. Gas Chromatography-Mass Spectrometry (GC-MS) analysis established their complex compositions. Then, their antibacterial activity-expressed as the inhibition zone diameter (IZD) value (mm)-was investigated in vitro by the diffusimetric antibiogram method, using sterile cellulose discs with Ø 6 mm impregnated with 10 µL of sample and sterile borosilicate glass cylinders loaded with 100 µL; the minimum inhibitory concentration (MIC) value (µg/mL) for each EO was calculated from the IZD values (mm) measured after 24 h. The following EO combinations were evaluated: OEO+CEO, CEO+EEO, CEO+PEO, LEO+EEO, and EEO+PEO. Then, the influence of each dual combination on the activity of three conventional antibacterial drugs-Neomycin (NEO), Tetracycline (TET), and Bacitracin (BAC)-was investigated. The most active EOs against S. aureus and E. coli were LEO and OEO (IZD = 40 mm). They were followed by CEO and EEO (IZD = 20-27 mm); PEO exhibited the lowest antibacterial activity (IZD = 15-20 mm). EEO alone showed the highest inhibitory activity on P. aeruginosa (IZD = 25-35 mm). It was followed by CEO, LEO, and EEO (IZD = 7-11 mm), while PEO proved no antibacterial action against it (IZD = 0 mm). Only one synergic action was recorded (OEO+CEO against P. aeruginosa); EEO+PEO revealed partial synergism against S. aureus and CEO+PEO showed additive behavior against E. coli. Two triple associations with TET showed partial synergism against E. coli, and the other two (with NEO and TET) evidenced the same behavior against S. aureus; all contained EEO+PEO or CEO+PEO. Most combinations reported indifference. However, numerous cases involved antagonism between the constituents included in the double and triple combinations, and the EOs with the strongest antibacterial activities belonged to the highest antagonistic combinations. A consistent statistical analysis supported our results, showing that the EOs with moderate antibacterial activities could generate combinations with higher inhibitory effects based on synergistic or additive interactions.

Keywords: GC-MS; Gram-positive and Gram-negative bacteria; antibacterial activity; antibacterial drugs; combinations; diffusimetric antibiogram; essential oils; interactions.

Figure 1

GC-MS chromatograms of EO samples: (A). Clove Essential Oil, (B). Eucalyptus Essential Oil, (C). Lavender Essential Oil, (D). Peppermint Essential Oil, and (E). Oregano Essential Oil.

Figure 1

GC-MS chromatograms of EO samples: (A). Clove Essential Oil, (B). Eucalyptus Essential Oil, (C). Lavender Essential Oil, (D). Peppermint Essential Oil, and (E). Oregano Essential Oil.

Figure 2

Stacked columns chart, as an overview of antibacterial activity against Gram-positive and Gram-negative bacteria, expressed as IZD (mm). LEO—Lavender Essential Oil; CEO—Clove Essential Oil; OEO—Oregano Essential Oil; EEO—Eucalyptus Essential Oil; PEO—Peppermint Essential Oil; TET—Tetracycline; NEO—Neomycin; BAC—Bacitracin; and IZD—inhibition zone diameter (mm), the scale of measurement was as follows: powerful inhibitory effect at IZD ≥ 35 mm, strong inhibitory effects at 35 > IZD ≥ 25 moderate inhibitory effect at 25 > IZD ≥ 15 mm, mild inhibitory effect when 15 > IZD ≥ 10 mm, and no inhibitory effect at IZD < 10 mm. Sa—S. aureus, Ec—E. coli, and Pa—P. aeruginosa.

Figure 3

Bland–Altman comparison between both techniques of diffusimetric antibiogram for all bacteria tested: S. aureus (A,B), E. coli (C,D), and P. aeruginosa (E,F). The differences are statistically significant at p < 0.05.

Figure 3

Bland–Altman comparison between both techniques of diffusimetric antibiogram for all bacteria tested: S. aureus (A,B), E. coli (C,D), and P. aeruginosa (E,F). The differences are statistically significant at p < 0.05.

Figure 4

Heat map of FICI values for double and triple combinations. LEO—Lavender Essential Oil; CEO—Clove Essential Oil; OEO—Oregano Essential Oil; EEO—Eucalyptus Essential Oil; PEO—Peppermint Essential Oil; NEO—Neomycin; TET—Tetracycline; and BAC—Bacitracin. FICI—fractional inhibitory concentration index. If IZD = 0 mm, it has no effect; no MIC exists. FICI ≤ 0.5 indicates synergism (S). 0.5 < FICI < 1—partial synergism (PS); FICI = 1—additive effects (Add.); 1 < FICI ≤ 4—indifference (I); FICI > 4—antagonism (Ant.) [39]; 4 < FICI ≤ 10—low antagonism; 10 < FICI ≤ 15—moderate antagonism; 15 20—very strong antagonism; FICI1—determined by DDM (disc diffusion method); and FICI2—determined by cylinder diffusion technique. Sa—S. aureus, Ec—E. coli, and Pa—P. aeruginosa. The FICI values ≤ 4 are marked with green and the color intensity decreases from dark green (synergism) to light green (indifference) the FICI values > 4 are marked with brown and the color intensity increases from light brown (low antagonism) to dark brown (very strong antagonism).

Figure 5

Correlations between EOs’ antibacterial activity (expressed as IZD) and the main phytoconstituents: (A) EEO-PEO-CEO group; (B) CEO-OEO-EEO-LEO group; and (C) all EOs. IZD—inhibition zone diameter (mm), Sa—S. aureus, Ec—E. coli, Pa—P. aeruginosa, LEO—Lavender Essential Oil; CEO—Clove Essential Oil; OEO—Oregano Essential Oil; EEO—Eucalyptus Essential Oil; PEO—Peppermint Essential Oil; IZD1—determined by DDM (disc diffusion method); and IZD2—determined by cylinder technique.

Figure 5

Correlations between EOs’ antibacterial activity (expressed as IZD) and the main phytoconstituents: (A) EEO-PEO-CEO group; (B) CEO-OEO-EEO-LEO group; and (C) all EOs. IZD—inhibition zone diameter (mm), Sa—S. aureus, Ec—E. coli, Pa—P. aeruginosa, LEO—Lavender Essential Oil; CEO—Clove Essential Oil; OEO—Oregano Essential Oil; EEO—Eucalyptus Essential Oil; PEO—Peppermint Essential Oil; IZD1—determined by DDM (disc diffusion method); and IZD2—determined by cylinder technique.

Figure 6

An overview of interactions between EOs and conventional antibiotics against Gram-positive and Gram-negative bacteria, assessed by both techniques of diffusimetric antibiogram and expressed as FICI. LEO—Lavender Essential Oil; CEO—Clove Essential Oil; OEO—Oregano Essential Oil; EEO—Eucalyptus Essential Oil; PEO—Peppermint Essential Oil; NEO—Neomycin; TET—Tetracycline; and BAC—Bacitracin. FICI—fractional inhibitory concentration index. If IZD = 0 mm, it has no effect; no MIC exists. FICI ≤ 0.5 indicates synergism (S). 0.5 < FICI < 1—partial synergism (PS); FICI = 1—additive effects (Add.); 1 < FICI ≤ 4—indifference (I); FICI > 4—antagonism (Ant.) [39]; 4 < FICI ≤ 10—low antagonism; 10 < FICI ≤ 15—moderate antagonism; 15 < FICI ≤ 20—strong antagonism; FICI > 20—very strong antagonism; FICI1—determined by DDM (disc diffusion method); and FICI2—determined by cylinder diffusion technique. Sa—S. aureus, Ec—E. coli, and Pa—P. aeruginosa.

Figure 7

(A) Correlations between all tested double and triple combinations and FICI values. (B) AHC Dendrogram, with 5 clusters: C1 = LEO+EEO+NEO; C2 = CEO+EEO+NEO; C3 = CEO+EEO+TET; C4 = CEO+PEO; and C5 = LEO+EEO+BAC and heat map. LEO—Lavender Essential Oil; CEO—Clove Essential Oil; OEO—Oregano Essential Oil; EEO—Eucalyptus Essential Oil; PEO—Peppermint Essential Oil; NEO—Neomycin; TET—Tetracycline; and BAC—Bacitracin. FICI—Fractional inhibitory concentration index. FICI1 is determined by DDM (disc diffusion method), and the cylinder diffusion technique determines FICI2. Sa—S. aureus, Ec—E. coli, and Pa—P. aeruginosa.

Figure 8

Correlation of MIC values of EOs and those of conventional antibiotics in double and triple combinations and FICI value. (A–C) CEO+PEO+EEO group in triple combinations with various antibiotics: (A) TET, (B) NEO, (C) BAC, and (D) CEO+OEO+EEO+LEO in double and triple combinations, with 3 antibiotics. LEO—Lavender Essential Oil; CEO—Clove Essential Oil; OEO—Oregano Essential Oil; EEO—Eucalyptus Essential Oil; PEO—Peppermint Essential Oil; NEO—Neomycin; TET—Tetracycline; BAC—Bacitracin; MIC—minimum inhibitory concentration (µg/mL), and FICI—fractional inhibitory concentration index. FICI1 was determined by DDM (disc diffusion method), and the cylinder diffusion technique provided FICI2. Sa—S. aureus, Ec—E. coli, and Pa—P. aeruginosa.

Figure 8

Correlation of MIC values of EOs and those of conventional antibiotics in double and triple combinations and FICI value. (A–C) CEO+PEO+EEO group in triple combinations with various antibiotics: (A) TET, (B) NEO, (C) BAC, and (D) CEO+OEO+EEO+LEO in double and triple combinations, with 3 antibiotics. LEO—Lavender Essential Oil; CEO—Clove Essential Oil; OEO—Oregano Essential Oil; EEO—Eucalyptus Essential Oil; PEO—Peppermint Essential Oil; NEO—Neomycin; TET—Tetracycline; BAC—Bacitracin; MIC—minimum inhibitory concentration (µg/mL), and FICI—fractional inhibitory concentration index. FICI1 was determined by DDM (disc diffusion method), and the cylinder diffusion technique provided FICI2. Sa—S. aureus, Ec—E. coli, and Pa—P. aeruginosa.

Figure 8

Correlation of MIC values of EOs and those of conventional antibiotics in double and triple combinations and FICI value. (A–C) CEO+PEO+EEO group in triple combinations with various antibiotics: (A) TET, (B) NEO, (C) BAC, and (D) CEO+OEO+EEO+LEO in double and triple combinations, with 3 antibiotics. LEO—Lavender Essential Oil; CEO—Clove Essential Oil; OEO—Oregano Essential Oil; EEO—Eucalyptus Essential Oil; PEO—Peppermint Essential Oil; NEO—Neomycin; TET—Tetracycline; BAC—Bacitracin; MIC—minimum inhibitory concentration (µg/mL), and FICI—fractional inhibitory concentration index. FICI1 was determined by DDM (disc diffusion method), and the cylinder diffusion technique provided FICI2. Sa—S. aureus, Ec—E. coli, and Pa—P. aeruginosa.