2.4. Data Analysis
Data regarding antibacterial and antibiofilm effectiveness for all standard antibiotics and essential oils at the first dilution (D1) are displayed in
Figure 4.
Figure 4.
Antibacterial and Antibiofilm efficacy of essential oils and antibacterial drugs against Gram-positive and Gram-negative bacteria; very good efficacy: ≥ 90%, good efficacy: 75—89%, moderate efficacy: 50—74%, satisfactory: 25—49% and unsatisfactory: 0—24%. GEN — Gentamicin; STR —Streptomycin, AMC — Amoxicillin&Clavulanic acid; OEO1-2—Oregano essential oil from two different manufacturers; EEO1-2—Eucalyptus essential oil from two different manufacturers; REO1-2 —Rosemary essential oil from 2 different manufacturers; CEO1-3—Clove essential oil from three different manufacturers; PEO1-4—Peppermint essential oil from 4 different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
Figure 4.
Antibacterial and Antibiofilm efficacy of essential oils and antibacterial drugs against Gram-positive and Gram-negative bacteria; very good efficacy: ≥ 90%, good efficacy: 75—89%, moderate efficacy: 50—74%, satisfactory: 25—49% and unsatisfactory: 0—24%. GEN — Gentamicin; STR —Streptomycin, AMC — Amoxicillin&Clavulanic acid; OEO1-2—Oregano essential oil from two different manufacturers; EEO1-2—Eucalyptus essential oil from two different manufacturers; REO1-2 —Rosemary essential oil from 2 different manufacturers; CEO1-3—Clove essential oil from three different manufacturers; PEO1-4—Peppermint essential oil from 4 different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
Principal Component Analysis (PCA) was used to evaluate the correlation between the antibacterial and antibiofilm efficacy of EOs on Gram-positive and Gram-negative bacteria (
Figure 5A) and compared it to standard antibiotics.
The correlation matrix from
Supplementary Material and
Figure 5A highlight a strong correlation, statistically significant, between antibacterial and antibiofilm effects against both Gram-negative bacteria,
P. aeruginosa (
r = 0.878,
p < 0.05) and
E. coli (r = 0.772,
p < 0.05). On
S. aureus, both effects are poorly correlated (
r = 0.359,
p > 0.05).
However, for all EOs and standard antibiotics, the antibacterial activity against S. aureus is considerably associated with that against E. coli (r = 0.895, p < 0.05) and moderately with an inhibitory effect against P. aeruginosa strains growing (r = 0.628, p < 0.05). Antibacterial effects against Gram-negative bacteria also show a moderate correlation (r = 0.878, p < 0.05). All data are statistically significant (p < 0.05). Generally, antibiofilm activities on all bacteria tested are poorly correlated.
The registered data from Results are summarized in
Figure 5A, evidencing the place of essential oils and standard antibiotic drugs reported to both
ABE and
ABfE against all bacteria tested.
In a simplified manner, the dendrogram obtained by Agglomerative Hierarchical Clustering (AHC) from
Figure 5B and
Supplementary Material shows how standard antibiotics and EO samples act similarly.
Figure 5B shows that PEO1 acts similarly to PEO2, PEO3 to PEO4, and CEO2 to CEO3. On the other hand, both OEOs have similar effects; at D1, they act similarly to AMC. The same observation is available on EEO1-2 and REO1-2, their activities being more closely to Aminoglycosides (STR and GEN).
Figure 5.
A. PCA-Biplot displays the antibacterial and antibiofilm efficacy of essential oils and antibacterial drugs against Gram-positive and Gram-negative bacteria. B. AHC-Dendrogram. GEN — Gentamicin; STR —Streptomycin, AMC — Amoxicillin&Clavulanic acid; OEO1-2—Oregano essential oil from two different manufacturers; EEO1-2—Eucalyptus essential oil from two different manufacturers; REO1-2 —Rosemary essential oil from 2 different manufacturers; CEO1-3—Clove essential oil from three different manufacturers; PEO1-4—Peppermint essential oil from 4 different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
Figure 5.
A. PCA-Biplot displays the antibacterial and antibiofilm efficacy of essential oils and antibacterial drugs against Gram-positive and Gram-negative bacteria. B. AHC-Dendrogram. GEN — Gentamicin; STR —Streptomycin, AMC — Amoxicillin&Clavulanic acid; OEO1-2—Oregano essential oil from two different manufacturers; EEO1-2—Eucalyptus essential oil from two different manufacturers; REO1-2 —Rosemary essential oil from 2 different manufacturers; CEO1-3—Clove essential oil from three different manufacturers; PEO1-4—Peppermint essential oil from 4 different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
Knowing each EO’s chemical composition (
Figure 6), the correlations between bioactive constituents and antibacterial and antibiofilm effects were analyzed to explain the differences between the corresponding samples.
Figure 6.
Chemical constituents of all EOs’ samples: A. Eucalyptus Oil (EEO1 and EEO2), Rosemary Oil (REO1 and REO2), Oregano Oil (OEO1 and OEO2); B. Clove oil (CEO1-3); C. Peppermint Oil (PEO1-4).
Figure 6.
Chemical constituents of all EOs’ samples: A. Eucalyptus Oil (EEO1 and EEO2), Rosemary Oil (REO1 and REO2), Oregano Oil (OEO1 and OEO2); B. Clove oil (CEO1-3); C. Peppermint Oil (PEO1-4).
Therefore, the PCA-Biplot from
Figure 7 shows the correlation between the previously mentioned variable parameters for 3 EOs: EEO, OEO, and REO.
Figure 7.
PCA-Biplot displaying the correlations between bioactive constituents and antibacterial and antibiofilm effects on Gram-positive and Gram-negative bacteria in each OEO, EEO, and REO sample. OEO1-2—Oregano essential oil from two different manufacturers; EEO1-2—Eucalyptus essential oil from two different manufacturers; REO1-2 —Rosemary essential oil from 2 different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
Figure 7.
PCA-Biplot displaying the correlations between bioactive constituents and antibacterial and antibiofilm effects on Gram-positive and Gram-negative bacteria in each OEO, EEO, and REO sample. OEO1-2—Oregano essential oil from two different manufacturers; EEO1-2—Eucalyptus essential oil from two different manufacturers; REO1-2 —Rosemary essential oil from 2 different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
All three EOs contain
p-cymene. EEOs and REOs have eucalyptol (1,8-cineol), pinene alpha, and limonene, while in EEOs and OEOs, terpinene gamma was quantified. In this EOs group, the Correlation Matrix from
Supplementary Material evidence strong and statistically significant correlations between several secondary metabolites and antibacterial and antibiofilm effects.
Antibacterial effects on S. aureus and E. coli are substantially correlated with antibiofilm ones (r = 0.927, r = 0.898, p < 0.05). On P. aeruginosa, both activities are moderately correlated (r = 0.591, p > 0.05).
From EOs constituents, carvacrol and linalool displays the highest correlation with previously mentioned activities, except ABfE on P. aeruginosa (carvacrol: r = 0.985, r = 0.959, r = 0.974, r = 0.887, r = 0.965, p < 0.05; linalool: r = 0.991, r = 0.943, r = 0.980, r = 0.870, r = 0.952, p < 0.05).
P-cymene is significantly correlated with ABE against P. aeruginosa and ABfE against S. aureus and E. coli (r = 0.830, r = 0.886, r = 0.886, p < 0.05) and moderately with S.a. ABE and E.c. ABE (r = 0.680, r = 0.687, p > 0.05). Similarly, terpinene gamma shows good and moderate correlation with all 5 previously mentioned effects (r = 0.788, r = 0.799, r = 0.750, r = 0.539, r = 0.539, p > 0.05).
Eucalyptol and alpha-pinene evidence a high negative correlation with antibacterial and antibiofilm effects on S. aureus and P. aeruginosa (r = ‒0.813, r = ‒0.861, r = ‒0.828, r = ‒0.830, r = ‒0.885, p < 0.05). Camphor, pinene beta, camphene, bornyl-acetate, terpineol alpha, and borneol are significantly negatively correlated only with P.a. ABfE: r = ‒ [0.849 – 0.898], p < 0.05. With the other activities (excepting S.a. ABE), they report a moderate negative correlation: r = ‒ [0.560 – 0.738], p > 0.05.
Thymol and terpinene alpha are moderately correlated with ABfE on S. aureus and E. coli and ABE against P. aeruginosa (r = [0.555 - 0.727], p > 0.05).
Figure 7 also shows the place of each OEO, EEO, and REO correlated to chemical composition and antibacterial and antibiofilm activities, with both samples of each EO having similar properties without significant differences.
Figure 8 reveals a good and moderate correlation between bioactive constituents and CEOs’ antibacterial and antibiofilm effectiveness. Therefore, beta-caryophyllene has a good correlation with
ABE against
S. aureus (r = 0.758
, p > 0.05
) and moderate ones with
ABE against
P. aeruginosa and
ABfE against
E. coli (r = 0.559
, r = 0.543
, p > 0.05). Eugenol is moderately correlated with
S. aureus and
P. aeruginosa biofilm inhibition
(r = 0.638,
r = 0.516
, p > 0.05), and eugenyl acetate with antibacterial activity against
S. aureus (r = 0.609
, p > 0.05
).
Figure 8.
PCA-Biplot displaying the correlations between bioactive constituents’ content and antibacterial and antibiofilm effects on Gram-positive and Gram-negative bacteria in each CEO sample. CEO1-3—Clove essential oil from three different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
Figure 8.
PCA-Biplot displaying the correlations between bioactive constituents’ content and antibacterial and antibiofilm effects on Gram-positive and Gram-negative bacteria in each CEO sample. CEO1-3—Clove essential oil from three different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
Moreover, antibacterial and antibiofilm effects are strongly correlated on Gram-negative bacteria (P. aeruginosa and E. coli, r = 0.976, r = 0.990, p > 0.05), and moderately on S. aureus (r = 0.791, p > 0.05).
Finally, the PCA Biplot shows each CEO sample’s place considering these variable parameters; it evidences that CEO1 is usually the most active sample regarding antibacterial and antibiofilm effects. All detailed data are found in
Supplementary Material.
Figure 9 reveals a strong and statistically significant correlation between antibacterial and antibiofilm activities in
S. aureus and
E. coli (
r = 0.974,
r = 0.975,
p < 0.05) and an appreciable negative correlation in
P. aeruginosa (
r = ‒ 0.856
, p > 0.05).
Figure 9.
PCA-Biplot displaying the correlations between bioactive constituents’ content and antibacterial and antibiofilm effects on Gram-positive and Gram-negative bacteria in each PEO sample; PEO1-4—Peppermint essential oil from 4 different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
Figure 9.
PCA-Biplot displaying the correlations between bioactive constituents’ content and antibacterial and antibiofilm effects on Gram-positive and Gram-negative bacteria in each PEO sample; PEO1-4—Peppermint essential oil from 4 different manufacturers; S.a.—S. aureus, E.c.—E. coli, P.a.—P. aeruginosa, ABE—Antibacterial efficacy, ABfE—Antibiofilm efficacy.
From bioactive constituents, eucalyptol shows a remarkable and significant statistical correlation with antibacterial activity against P. aeruginosa (r = 0.995, p < 0.05). It is also considerably correlated with ABE against E. coli (r = 0.830, p > 0.05) and moderately associated with ABE against S. aureus (r = 0.716, p > 0.05) and ABfE in S. aureus and E. coli (r = 0.593, r = 0.685, p > 0.05). However, eucalyptol negatively correlates with ABfE in P. aeruginosa (r = ‒0.803, p > 0.05).
Concomitantly, menthol is considerably correlated with both ABE and ABfE in S. aureus (r = 0.847, r = 0.826, p > 0.05) and E. coli (r = 0.680, r = 0.754, p > 0.05).
Moderate correlations were evidenced between menthone et pulegone and antibiofilm activity against P. aeruginosa (r = 0.736, r = 0.503, p > 0.05); both previously mentioned constituents and menthofuran are negatively correlated with P.a. ABE (r = ‒0.579, r = ‒0.818, r = ‒0.617, p > 0.05).
Isomenthone and neomenthol with ABfE on S. aureus (r = 0.708, r = 0.550, p > 0.05). Moreover, isomenthone is moderately correlated with antibacterial activity against S. aureus (r = 0.636, p > 0.05).
Menthyl acetate shows a robust negative correlation with ABE and ABfE against E. coli (r = ‒0.809, r = ‒0.855, p > 0.05), while pulegone reports a moderate one, only with E.c. ABE (r = ‒0.516, p > 0.05)
Finally, considering all discussed variable parameters—extensively described in
Supplementary Material—
Figure 9 shows the place of each PEO sample, thus explaining all differences between them and supporting the results.