Preprint
Article

Screening of Antibacterial and Antibiofilm Activities of Commercially Available Essential Oils' Different Samples in Comparison to Conventional Antibiotics

Altmetrics

Downloads

208

Views

155

Comments

0

A peer-reviewed article of this preprint also exists.

Submitted:

30 May 2023

Posted:

30 May 2023

You are already at the latest version

Alerts
Abstract
Essential oils (EOs) have gained economic importance due to their biological activities, and increasing amounts are demanded everywhere. However, substantial differences between the same essential oil samples from different suppliers are reported due to numerous companies involved in EOs production and the continuous development of online sales. The present study investigates the antibacterial and antibiofilm activities of 2-4 samples of five commercially available essential oils (Oregano, Eucalyptus, Rosemary, Clove, and Peppermint oils) from different manufacturers. Their effects were evaluated in vitro on Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa). The antibacterial efficacy (ABE%) and antibiofilm efficacy (ABfE%) were determined spectrophotometrically at 562 and 570 nm using microplate cultivation techniques. The essential oils' calculated parameters were compared with those of three standard broad-spectrum antibiotics: Amoxicillin/Clavulanic acid (AMC), Gentamycin (GEN), and Streptomycin (STR), active on tested bacteria. The results showed that at the first dilution (D1 = 2.5 mg/mL), all essential oils (EOs) exhibited antibacterial activity against Gram-positive and Gram-negative bacteria. On S. aureus and E. coli, EOs had considerable antibacterial effects (ABE = 79.70—92.80%, respectively, 71.30 ‒ 94.00%). The highest antibacterial effects of commercially available EOs were against P. aeruginosa because all exhibited a significant antibiofilm activity. Their antibiofilm efficacy intensively decreased on E. coli and S. aureus. Generally, the samples with different manufacturers of the same EO showed similar effects. Only Clove and Peppermint oils samples displayed a higher variability associated with active metabolites' different contents, maybe due to various zones of harvesting raw material, numerous technologies involved in EOs obtaining processes, and complex interactions between components.
Keywords: 
Subject: Medicine and Pharmacology  -   Pharmacy

1. Introduction

Essential oils are highly concentrated plant derivatives defined based on their physicochemical properties [1]. The EOs’ chemical composition includes phenolic compounds, terpenes, terpenoids, phenylpropanoids, and other aliphatic and aromatic constituents. European Pharmacopoeia defines EOs as odorous products, usually of complex composition, obtained from a botanically defined plant raw material by steam distillation, dry distillation, or a suitable mechanical process without heating [2]. Generally, EOs have high contents (20-70%) of two or three major phytoconstituents; other compounds are quantified in trace concentrations [3].
Currently, substantial growth in the EOs’ general use worldwide has been reported in various domains: the food industry, fragrances, aromatherapy, and cosmetics, personal care, spa and relaxation, home care, and healthcare (pharmaceuticals and nutraceuticals) [4]. Especially, aromatherapy (implying professionals – aromatherapists - and individual customers) has gained extensive applications in numerous countries. The pharmacological potential of EOs has been extensively explored: ranging from antioxidant [5][6], anti-inflammatory [7][8], immunomodulatory [9][10], antinociceptive [11][12], antiulcer [13][14], anticancer [15][16], insecticidal [17][18], larvicidal [19][20], anthelmintic [21][22], antiviral [23][24], and antimicrobial [25][26] properties.
Therefore, the global market for essential oils is anticipated to have a continuous expansion, with regional differences. For example, in the USA, its size is expected to be approximately double in 2030 than in 2021. The USA Food and Drug Administration (FDA) divides essential oils into three categories:
  • Cosmetics – Products intended to clean the body (except for soap).
  • Household items/Other - Fragrance products, like scented candles, household cleaners, and air fresheners.
  • Drugs – Products intended for therapeutic use that can treat or prevent various diseases or affect the body structure or function [27] [28][29].”
The essential oils from doTERRA (Pleasant Grove, Utah, USA) [30] are commercialized with the label CPTG (Certified Pure Therapeutic Grade). However, FDA did not regulate essential oils as foods or dietary supplements; it means that any essential oil product cannot be marketed with the following mention: “it intended to treat, prevent, cure or mitigate any disease or other health condition” – even when there is scientific research to back up the validity of the claims.
In Europe, the EOs industry growth is promoted by the European Federation of Essential Oils (EFEO) [31]. Currently, EFEO is discussing with the European Commission and the EU Parliament to amend or introduce legislation concerning essential oils. Contrariwise, due to numerous bioactivities, the European Medicinal Agency (EMA) considers essential oils as herbal preparations and also as active pharmaceutical ingredients (API) in two groups of herbal products [32]:
  • Herbal medicinal products (HMPs), both for human and veterinary use.
  • Traditional herbal medicinal products (THMPs) for human use.
Thus, EMA established rigorous quality documentation for all manufacturers, and competent national authorities can refer to one unique set of information concerning registered EOs as HMPs/THMPs when evaluating marketing applications [33][34]. In 2022, EMA revised the “Guideline on specifications: test procedures and acceptance criteria for herbal substances, herbal preparations, and herbal medicinal products/traditional herbal medicinal products” [35]. The quality guidelines when essential oils are used as APIs of HMPs, an analytical characterization of the raw material is required. Moreover, other tests (according to the Ph. Eur. monograph Herbal Drugs) must be performed on the essential oils [32]. According to GMP standards, the manufacturing process is another point, implying the quality of water used for the EOs distillation from fresh plants. The composition of essential oils should be within the Ph. Eur. monograph limits.
The documentation regarding the quality of EO must contain all manufacturers. It is difficult to achieve all documents [32] when implied farmers or very small companies are in the manufacturing process.
Therefore, substantial differences could be recorded between the same essential oil samples from different suppliers due to a lack of regulation, numerous companies involved in EOs production, and continuous development of online sales. Thus, Iordache et al. measured the metal content of 34 plant essential oils from various manufacturers [36] and identified Hg levels over six times higher than Ph. Eur. permissible limits in Peppermint oil. Vargas Jentzsch et al. investigated 19 samples of commercial clove essential oil. They found that two samples were adulterated by the addition of benzyl alcohol and a third by the addition of vegetable oil [37]. Recently, Pierson et al. tested 31 EO samples purchased online by evaluating their compliance with ISO standards; they found that more than 45% of the samples did not pass the test, and more than 19% were diluted with solvents as propylene and dipropylene glycol, triethyl citrate, or vegetal oil [38].
Numerous authors investigated the pharmacological activities of commercially available EOs, especially antimicrobial effects [39][40][41] [42]. In a previous study, Brun et al. investigated the antimicrobial effects of commercially available Tea Tree essential oils, finding that only five out of ten samples had significant antimicrobial activity [43]. The present study aims to explore the antibacterial and antibiofilm effectiveness of five commercially available essential oils—well-known for their phytotherapeutic applications —against Gram-positive and Gram-negative bacteria. The novelty of the present study consists of a different design. We checked various Romanian markets (including online suppliers) and selected only four that concomitantly provided the essential oils’ chemical composition. Then, 2-4 samples of each essential oil from different manufacturers were evaluated, comparing their antibacterial and antibiofilm effects with conventional antibiotics and correlating the data obtained with the bioactive secondary metabolites content. In addition, a complex statistical analysis supports our results.

2. Results

2.1. Antibacterial and Antibiofilm Activity on S. aureus

The percentual values of antibacterial and antibiofilm efficacy of essential oils against Gram-positive bacteria (S. aureus) tested, compared to standard antibiotics, are displayed in Figure 1.
Figure 1A shows that, at 50 µg/mL, AMC has very good antibacterial efficacy (ABE > 90%), while both aminoglycosides exhibit a good one (ABE > 85%). All EOs have significant antibacterial activity against S. aureus at 2.5 mg/mL. OEO and CEO1 proved very good antibacterial potential, like the AMC one, and higher than GEN and STR. CEO3, PEO3 and PEO4 have the lowest ABE% (< 80%). S. aureus sensitivity commonly decreases directly proportional to EOs concentration. Only a few EOs recorded a good antibacterial effect at all D1, D2, and D3 dilutions: EEOs and REO1. The anti-staphylococcal effect can slowly diminish at progressive dilutions (as in the case of REO2) or intensely decrease (OEOs, CEOs, PEO1-2). PEO3-4 exhibited antibacterial efficacy only at 2.5 mg/mL. As an overview, we could appreciate that the MIC value for all EOs tested is higher than 2.5 mg/mL. In addition, no significant differences between the antibacterial efficacy of the tested samples of each EO at D1 were recorded (Figure 1A).
Figure 1B shows that all standard antibiotics and EOs have antibiofilm activity at D1 and D2. At D1, AMC shows a moderate AbfE, and both aminoglycosides report a satisfactory one. For EOs, it decreases from “very good” (OEOs, CEO1 and PEO1) to good (CEO2-3 and PEO2), moderate (EEO1), and satisfactory (EEO2, REOs, PEO3-4). The highest differences were recorded in the case of PEO’s four samples. Moderate differences in ABfE values at D1 were also registered in CEOs and EEOs (Figure 1B).
Figure 1. Antibacterial (A) and Antibiofilm efficacy (B) of essential oils and antibacterial drugs against S. aureus: 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. For EOs, D1 = 2.5 mg/mL, D2 = 0.25 mg/mL and D3 = 0.025 mg/mL. For standard antibiotics, D1 = 50 µg/mL, D2 = 5 µg/mL, and D3 = 0.5 µg/mL.
Figure 1. Antibacterial (A) and Antibiofilm efficacy (B) of essential oils and antibacterial drugs against S. aureus: 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. For EOs, D1 = 2.5 mg/mL, D2 = 0.25 mg/mL and D3 = 0.025 mg/mL. For standard antibiotics, D1 = 50 µg/mL, D2 = 5 µg/mL, and D3 = 0.5 µg/mL.
Preprints 75124 g001

2.2. Antibacterial and Antibiofilm Activity on E. coli

The percentual values of antibacterial and antibiofilm efficacy of essential oils against Gram-negative bacteria E. coli, compared to standard antibiotics, are displayed in Figure 2. Figure 2A shows that at D1 (= 50 µg/mL), AMC exhibits a very good ABE, while both aminoglycosides display a good one (ABE < 90%). All EOs inhibited E. coli strains growing at 2.5 mg/mL; the antibacterial effect decreased from “very good” (OEO1 and CEO1) to moderate (PEO2-4). Most EOs recorded good inhibitory effects against E. coli (Figure 2A).
Figure 2. Antibacterial (A) and Antibiofilm efficacy (B) of essential oils and antibacterial drugs against E. coli: 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. For EOs, D1 = 2.5 mg/mL, D2 = 0.25 mg/mL, and D3 = 0.025 mg/mL. For standard antibiotics, D1 = 50 µg/mL, D2 = 5 µg/mL, and D3 = 0.5 µg/mL..
Figure 2. Antibacterial (A) and Antibiofilm efficacy (B) of essential oils and antibacterial drugs against E. coli: 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. For EOs, D1 = 2.5 mg/mL, D2 = 0.25 mg/mL, and D3 = 0.025 mg/mL. For standard antibiotics, D1 = 50 µg/mL, D2 = 5 µg/mL, and D3 = 0.5 µg/mL..
Preprints 75124 g002
The antibiofilm activity of standard antibiotics and EOs differs significantly at D1 (Figure 2B). AMC and GEN show a high ABfE, while STR has a moderate one. However, both OEOs and CEO1 show a very good inhibition of E. coli biofilm formation, while CEO2-3 have a good one. Figure 2B also shows that EEOs have a moderate antibiofilm effect, followed by REOs and PEO1 with satisfactory ABfE values. PEO2-4 recorded the lowest inhibitory activity on E. coli biofilm formation.

2.3. Antibacterial and Antibiofilm Activity on P. aeruginosa

The percentual values of antibacterial and antibiofilm efficacy of essential oils against P. aeruginosa, compared to standard antibiotics, are displayed in Figure 3.
Figure 3. Antibacterial (A) and Antibiofilm efficacy (B) of essential oils and antibacterial drugs against P. aeruginosa; 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 3 different manufacturers; PEO1-4—Peppermint essential oil from 4 different manufacturers. For EOs, D1 = 2.5 mg/mL, D2 = 0.25 mg/mL and D3 = 0.025 mg/mL. For standard antibiotics, D1 = 50 µg/mL, D2 = 5 µg/mL, and D3 = 0.5 µg/mL.
Figure 3. Antibacterial (A) and Antibiofilm efficacy (B) of essential oils and antibacterial drugs against P. aeruginosa; 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 3 different manufacturers; PEO1-4—Peppermint essential oil from 4 different manufacturers. For EOs, D1 = 2.5 mg/mL, D2 = 0.25 mg/mL and D3 = 0.025 mg/mL. For standard antibiotics, D1 = 50 µg/mL, D2 = 5 µg/mL, and D3 = 0.5 µg/mL.
Preprints 75124 g003
Figure 3A shows that all standard antibiotics exhibit very good inhibitory activity on P. aeruginosa strains growing at 50 µg/mL (ABE > 90%), AMC having a lower one than both aminoglycosides. At 2.5 mg/mL, both OEOs and CEO1 reveal an antibacterial efficacy similar to standard antibiotics, while CEO2-3 reported a moderate one. All the other EOs displayed good antibacterial efficacy against P. aeruginosa. Only between CEO samples (CEO1 and CEO2-3) were registered significant differences at D1.
Figure 3B evidences the same differences in the antibiofilm activity of CEO1 compared to CEO2-3. At D1, all antibiotics show good antibiofilm activity, while AMC recorded the highest ABfE value. On P. aeruginosa, most EOs reported the most increased antibiofilm effects (compared with E. coli and S. aureus). Therefore, 7 EO samples (OEOs, EEOs, REOs, and CEO1) showed a very good ABfE. On the other hand, minimal differences were registered between PEOs (PEO3-4 have a good antibiofilm effect, while PEO1-2 prove a moderate one). Substantial differences were observed in CEOs, CEO2-3 showing the lowest antibiofilm efficacy on P. aeruginosa.

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.
Preprints 75124 g004
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.
Preprints 75124 g005
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).
Preprints 75124 g006
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.
Preprints 75124 g007
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.
Preprints 75124 g008
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.
Preprints 75124 g009
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 MaterialFigure 9 shows the place of each PEO sample, thus explaining all differences between them and supporting the results.

3. Discussion

The present study investigated 5 common essential oils used in phytotherapy [44] with a well-known phytochemical and pharmacological profile. All samples were manufactured by four autochthonous companies and regularly commercialized in pharmacies, pharma markets [45], and online markets.
Four essential oils are registered by European Medicinal Agency (EMA) as HMPs for human and veterinary use [45][46], having a periodically updated monography (Table 1) [47]. Moreover, they have individual monographs in European Pharmacopoeia, indicating the bioactive constituents’ concentration limits.
Only Oregano oil is authorized as a feed additive for animal species [48], and these data follow ISO 13171:2016 from International Organization for Standardization, Geneva, Switzerland (Table 1) [49]. Phytogenic feed additives (phytobiotics) are currently used in traditional European animal healthcare [50]. The effect of oregano oil dietary supplementation in poultry on production parameters, intestinal villi height, and broiler breast’s antioxidant capacity is well-studied [51].
The antibacterial effects of essential oils investigated in our study are implied in all their therapeutical benefits, as mentioned in Table 1.
Table 1. The main phytoconstituents and applications of essential oils according to Ph. Eur. 10 and the European Medicinal Agency.
Table 1. The main phytoconstituents and applications of essential oils according to Ph. Eur. 10 and the European Medicinal Agency.
Essential oil
name
Botanical
name
Main
phytoconstituents
Therapeutic area /
Applications
1. Caryophylli floris
aetheroleum
(Clove oil)
Syzygium aromaticum (L.) Merr. et L.M. Perry,
syn. Eugenia caryophyllus (Spreng.) Bullock et S.G. Harrison
  • β-caryophyllene: 5 - 14%
  • Eugenol: 75 – 88%
  • Eugenyl-acetate: 4 - 15%
[52]
Mouth and throat disorders [53]
2. Menthae piperitae aetheroleum
(Peppermint oil)
Mentha x piperita L.
  • Menthol: 30 - 55%
  • Menthone: 14 – 32%
  • Isomenthone: 1.5 - 10 %
  • Menthyl acetate: 2.8 - 10.0 %
  • 1,8-cineol: 3.5 - 8.0 %
  • Limonene: 1.0 - 3.5 %
  • Menthofuran: 1.0 - 8.0 %
  • Pulegone: ≤ 3.0 %
  • Carvone: ≤ 1.0 %
  • Isopulegol: ≤ 0.2 %
[52]
Pain and inflammation;
Skin disorders and minor wounds;
Cough and cold;
Gastrointestinal disorders [54]
3. Eucalypti
aetheroleum
(Eucalyptus oil)
Eucalyptus globulus Labill.
Eucalyptus polybractea R.T. Baker.
Eucalyptus smithii R.T. Baker.
  • α-pinene: 1 – 9%
  • β-pinene: < 1.5%
  • α-phellandrene: < 1.5%
  • Limonene: 4 -12%
  • 1,8-cineol: ≥ 70%
  • Camphor: < 0.1%
[52]
[55]
Pain and inflammation
Cough and cold [56]
4. Rosmarini
aetheroleum
(Rosemary oil)
Rosmarinus officinalis L.
  • α-pinene: 18 – 26%
  • Camphene: 8 – 12%
  • β-pinene: 2 – 6%
  • β-myrcene: 1.5 - 5%
  • Limonene: 2.5 – 5%
  • 1,8-cineol:16 – 25%
  • p-cymene: 1 – 13%
  • Camphor: 13 – 21%
  • Bornyl acetate: 0.5 – 2.5%
  • α-terpineol:1– 3.5%
  • Borneol: 2 – 4.5%
  • Verbenone: 0.7 – 2.5%
[52]
Circulatory disorders;
Gastrointestinal disorders
[57]
7. Origani aetheroleum
(Oregano oil)
Origanum vulgare ssp. hirtum (Link) Ietsw.
  • Carvacrol: 60 - 80%
  • p-cymene: 4 - 10%
  • γ-terpinene: 3 - 9%
  • Thymol: 0.5 - 5%
[49]
Feed additive for certain
animal species [48]

3.1. Antibacterial and Antibiofilm Activity of Oregano Oils

The main constituents of OEO are carvacrol and thymol, which have solid pharmacological potential, including antibacterial, anti-inflammatory, and antioxidant activities. OEO could be a broad-spectrum natural antibiotic [58][59]. Both compounds act synergic in combination, having an additive effect [60]. Investigating the antibacterial mechanism against MRSA, Cui et al. proved that OEO affects bacterial wall permeability, leading to an irreversible depletion [61]. It can inhibit bacterial respiratory metabolism (perturbing the tricarboxylic acids cycle) and the expression of MRSA’s crucial pathogenic factor PVL. Furthermore, carvacrol can form a chimera with DNA [61], and thymol reduces enterotoxins A, B, and α-hemolysin secreted by S. aureus isolates [62]. Other studies confirm both activities (antibacterial and antibiofilm) on S. aureus [63], E. coli [64], and P.aeruginosa [65][66]. Carvacrol exhibits a synergic effect with its biological precursor, p-cymene.
Moreover, carvacrol and thymol, obtained by chemical synthesis, could be adulterants of oregano oil [49].
In the present study, both OEOs display similar antibacterial and antibiofilm activities, evidencing the highest effects of all EOs investigated. Their inhibitory activity against all bacteria tested is similar to Amoxicillin&Clavulanic acid. Regarding the chemical composition, OEO1 contains all four constituents in the suitable Ph. Eur. limits (Table 1). OEO2 has a lower carvacrol content, but other metabolites in augmented concentrations than OEO1: thymol content, 7 times higher; γ-terpinene, 3.5 times higher and p-cymene, 2 times higher. A similar thymol concentration was quantified by Salehi et al. in OEO from Greece [67]. Moreover, another 3 compounds, unmentioned in Ph. Eur., were found in the GC-MS report: linalool, myrcene, and β-caryophyllene, in up to 2% concentration. They could contribute to the antibacterial effects due to complex interaction with the other bioactive metabolites [43].
Our PCA analysis, separately performed on OEOs, reported a strong statistically significant correlation between the bioactive constituents and ABE, respectively ABfE, thus justifying OEOs’ records. Thus, carvacrol and linalool were substantially correlated (r = 0.999, p < 0.05) with ABE and ABfE against S. aureus and P. aeruginosa and only ABE against E. coli. In contrast, thymol, linalool, terpinene alpha, and gamma evidenced the same correlation with antibiofilm activity in E. coli. For all bacteria tested, MIC > 2.5 mg/mL. Other studies indicated various MIC values: > 3.2 mg/mL against E. coli [68], [2—4] mg/mL against S. aureus, and 63 mg/mL against P. aeruginosa [69].

3.2. Antibacterial and Antibiofilm Activity of Eucalyptus Oils

Bachir et al. [70][71] reported the antibacterial efficacy of Eucalypti aetheroleum against S. aureus due to its phytoconstituents (1,8–cineol, linalool, β-pinene). Moreover, its bactericidal effect against E. coli and P. aeruginosa was reported [72]. The EEO’s bioactive constituents are responsible for the antibiofilm one due to 1,8-cineol (eucalyptol) [73][74][75]. Therefore, Eucalyptus oil penetrates the biofilm matrix, interfering with the essential constituents’ synthesis and the metabolic processes of the biofilm. EEO has synergistic antibacterial activity against Gram-positive bacteria, while against Gram-negative ones, it is additive [76]. 1, 8 cineol obtained by chemical synthesis could be mixed with Eucalyptus oil for adulteration [77].
The present study proved Eucalypti aetheroleum’s appreciable antibacterial effectiveness against S. aureus, E. coli, and P. aeruginosa (MIC > 2.5 mg/mL). The EEO’s MIC against MRSA varies between 0.032—307 mg/mL [78]. Mulyaningsih et al. [79] evidenced antibacterial activity against E. coli with a MIC > 4 mg/mL, while Van et al. reported a median MIC of 27.26 mg/mL against P. aeruginosa isolates [80]. However, EEOs recorded a moderate antibiofilm efficacy on S. aureus and E. coli strains and a substantial one against P. aeruginosa, like OEOs. Minimal differences were registered between both tested samples, with EEO1 acting higher than EEO2. Of six metabolites mentioned in the EEO monograph from Ph. Eur. (Table 1), only 4 (1,8-cineol, α-pinene, α-phellandrene, and limonene) appear in the suppliers’ GC-MS results. Their concentration in EEO1 is included in regulatory limits. EEO2 has a lower content of eucalyptol (61.45% vs. 79.73%) and a 6 times higher concentration. The samples have small contents of other different compounds: myrcene (in EEO1), trans-pinocarveol, terpinyl acetate (in EEO2), and p-cymene and γ-terpinene (in EEO1 and EEO2). Both common constituents are considerably correlated with antibacterial/antibiofilm activities. Thus, data analysis indicated a strong statistically significant correlation between eucalyptol, limonene, and p-cymene and both antibacterial and antibiofilm activities against S. aureus and E. coli; at the same time, α-pinene is substantially correlated with ABE and ABfE against P. aeruginosa (r = 0.999, p < 0.05).

3.3. Antibacterial and Antibiofilm Activity of Rosemary Oils

Previous studies [68][81][82][83] evidenced the antibacterial effects of Rosmarini aetheroelum against S. aureus and E. coli. Santoyo et al. [84] highlighted the antibacterial efficacy of REO against P. aeruginosa due to the bioactive constituents, camphor, borneol, and verbenone. Rosemary oil also inhibits P. aeruginosa biofilm formation [85].
Our results show that Rosmarini aetheroleum had significant antibacterial and antibiofilm efficacy against P. aeruginosa. The antibacterial activity is similar for all Gram-positive and Gram-negative bacteria tested (ABE > 80.00%, MIC > 2.5 mg/mL). Other studies reported MIC values of [6.2—25] mg/mL against S. aureus, [12.5—25] mg/mL against E. coli, and 50 mg/mL against P. aeruginosa [86]. REO1 was slightly more active than REO2, but no significant differences were recorded between the two samples’ effects. Compared to Ph. Eur. data, REOs have around 2 times higher eucalyptol content (48.10% in REO1 and 42.50% in REO2) and no verbenone. In addition, REO2 contains 4.45% β-caryophyllene. Pinene alpha, beta, camphor, borneol, and camphene were substantially correlated with ABE and ABfE on P. aeruginosa (r = 0.999, p < 0.05). Eucalyptol has a similar correlation with ABE against E coli; camphor and camphene are correlated with ABE and ABfE against S. aureus; pinene beta only correlates with ABfE on S. aureus.
Moreover, the synthetic equivalents of their main components, 1.8 cineole and camphor could be used for Rosemary oil adulteration [87].

3.4. Antibacterial and Antibiofilm Activity of Clove Oils

Xu et al. [88] highlighted the antibacterial efficacy of Caryophylli aetheroleum against S. aureus (with a MIC value = 0.0625 mg/mL). They hypothesized that the volatile oil destroys the cell wall and membranes, causing loss of vital intracellular materials, resulting in bacterial death. Generally, the MIC values of CEO against S. aureus vary in the range of [0.52—1.04] mg/mL [89]. The volatile oil also penetrates the cytoplasmic membrane and inhibits the normal synthesis of DNA and proteins necessary for bacterial growth. Yadav et al. [90] reported the antibiofilm effect of Clove oil on S. aureus attributed to eugenol. It inhibits biofilm formation, interrupts intercellular connections, detaches pre-existing biofilms, and kills bacteria in biofilms. Synthetic eugenol is also used for Clove oil adulteration [37].
Burt et al. [91] evidenced the antibacterial efficacy of Caryophylli aetheroleum against E. coli, The CEO’s MIC value belonged to the range of [0.64—1.28] mg/mL [89]. Another study by Kim et al. [92] reported the antibiofilm efficacy of Clove oil against E. coli due to eugenol inhibitory activity on biofilm formation.
The CEO’s antibacterial efficacy against P. aeruginosa is also demonstrated [93], with a MIC of 4.9 mg/mL [94]. Moreover, the antibiofilm activity of Clove oil is due to its main bioactive compounds, eugenol and eugenyl acetate [95].
The present study reports a few differences between the three CEO samples.
Thus, CEO1 showed substantial antibacterial and antibiofilm efficacy against all Gram-positive and Gram-negative bacteria tested (with ABE and ABfE values > 91.80%)
CEO2 and CEO3 proved good antibacterial and antibiofilm effectiveness against S. aureus and E. coli. However, significant differences were registered in their effects against P. aeruginosa, exhibiting moderate and satisfactory antibacterial and antibiofilm activity.
Generally, their chemical composition corresponds to Ph. Eur.; CEO1 has the highest eugenol content, followed by CEO3 and CEO2. However, CEO3 has the lowest eugenyl acetate concentration and no β-caryophyllene; both constituents are highly and moderately correlated with antibacterial and antibiofilm activities.

3.5. Antibacterial and Antibiofilm Activity of Peppermint Oils

In PEO, the association of menthol, menthone, limonene, neomenthol, carvone, and 1,8-cineol with other minor constituents appears to induce a synergistic antibacterial activity. A recent study [96] evaluated the antibacterial activity of volatile oil obtained from Mentha×piperita L. leaves on MDR strains from hospitalized patients. The authors used bacterial cell lines (ATCC) and isolates of S. aureus, E coli, and P. aeruginosa, proving PEOs’ bactericidal effects against all microorganisms.
Li et al. [97] evidenced that Menthae aetheroleum (with a high content of carvone, menthone, isomenthone, neomenthol, menthol, and menthyl acetate) has a significant antibacterial effect against S. aureus [98]. All tested samples of Peppermint oil showed appreciable anti-staphylococcal efficacy. Kang et al. [99] showed that PEO inhibits the biofilm of S. aureus by altering the permeability and integrity of bacterial cell membranes. Peppermint oil also significantly inhibits biofilm formation and inactivates the mature biofilm [100].
Alamoti et al. [101] proved the antibacterial efficacy of Menthae aetheroleum against E. coli due to pulegone content.
Peppermint oil also inhibits P. aeruginosa [102], showing substantial antibiofilm activity [103][104].
All four Menthae aetheroleum samples investigated in the present study had remarkable antibacterial effects against Gram-positive and Gram-negative bacteria, with no significant differences (MIC > 2.5 mg/mL). They recorded the highest ABE (> 85.00%) on P. aeruginosa and S. aureus (ABE > 79.70%). On E. coli, the PEOs’ antibacterial efficacy was good to moderate, in the range of [71.30 – 79.00] %; PEO1 shows the highest effect. Evaluating the antibacterial effect of EO from Mentha×piperita L. against MDR bacterial strains, Muntean et al. reported the following MIC values range: [5—20] mg/mL on S. aureus, [10—20] mg/mL on E. coli, and [20—40] mg/mL on P. aeruginosa [96].
Regarding the antibiofilm activity, the PEOs displayed considerable effects on P. aeruginosa, ABfE = [73.20 – 85.80] %. On E. coli, PEOs registered the lowest effects: AbfE = [2.90 – 34.20] %. The most significant differences were highlighted in the antibiofilm efficacy evaluation against S. aureus. The obtained data show that PEO1 and PEO2 have a substantial antibiofilm activity AbfE = [89.20 – 91.00] %. Concomitantly, PEO3 and PEO4 exhibited a poor antibiofilm effect [32.10 – 38.70] %. These differences could be explained by their bioactive metabolite content. PEO1 and PEO2 have the highest concentrations of menthol, menthyl acetate, isomenthone, and eucalyptol; these constituents considerably correlate with antibacterial and antibiofilm activities. Moreover, synthetic menthol could substitute peppermint in adulterant oil [105].

4. Materials and Methods

4.1. Materials

All chemicals and reagents were on analytical grade. Poly (ethylene glycol)-block-poly (propylene glycol)-block-poly (ethylene glycol) (Poloxamer 407) and Crystal Violet (Gentian Violet) were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany).
Gentamicin® 80 mg/2 ml (GEN) injectable solution was supplied by KRKA (Novo mesto, Slovenia). Antibiotice SA (Iași, Romania) provided Streptomycin (Strevital®) 1 g (STR) powder for an injectable solution and Amoxicillin/Clavulanic acid (Amoxiplus®) 1.2 g (AMC) [106] powder for an injectable solution.
Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria were obtained from the sub-collection of the Experimental Microbiology Laboratory of the “Cantacuzino” National Military Medical Institute for Research and Development, Bucharest. Other recently published studies used these strains for antibacterial activity screenings [107],[108]. Sanimed International Impex SRL (Calugareni, Romania) was the Muller Hinton culture media supplier.
The laboratory equipment consisted of an EnSight Multimode Plate Reader (PerkinElmer, Waltham, Massachusetts, USA), an adjustable incubator (Memmert GmbH + Co.KG, Büchenbach, Germany), a microplate shaking incubator (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany), microbiological hood class II with laminar flow (Jouan SA, Saint-herblain, Pays de la Loire, France), Evoqua double-water distiller (Evoqua Water Technologies GmbH, Barsbüttel, Germania) and an electronic scale (Ohaus Corporation, Parsippany, NJ, USA). The NUNC™ MaxiSorp™ 96 well plates were supplied from Electron Microscopy Sciences (Hatfield, Pennsylvania, USA).
Five commercially available essential oils were purchased from Romanian markets (2-4 samples for each EO, having different manufacturers, noted with 1, 2, 3, and 4):
Origani aetheroleum 1, 2 (Oregano essential oil, OEO);
Eucalypti aetheroleum 1, 2 (Eucalyptus essential oil, EEO);
Rosmarini aetheroleum 1, 2 (Rosemary essential oil, REO);
Caryophylli aetheroleum 1. 2, 3 (Clove essential oil, CEO);
Menthae aetheroleum 1, 2, 3, 4 (Peppermint essential oil, PEO).

4.2. Antibacterial Activity

The current method was adapted from [109][110]. It involved the cultivation of bacteria in 96-well microplates with Muller Hinton medium with EOs samples and incubation at 37 °C for 24 hours.

4.2.1. Inoculum Preparation

The direct colony suspension method (CLSI) was used for preparing the bacterial inoculum. First, bacterial colonies selected from a 24 h agar plate were suspended in an MHA medium. The bacterial inoculum was accorded to the 0.5 McFarland standard, measured at Densimat Densitometer (Biomerieux, Marcy-l’Étoile, France) with around 108 CFU/mL (CFU = colony-forming unit).

4.2.2. Sample Preparation

The samples were O/W emulsions prepared with an essential oil concentration of 30% w/w; the emulsifier was Poloxamer 407 5% in water, as previously mentioned [111].
Each emulsion was diluted with double distilled water to achieve the final concentration of each EO stock solution (25 mg/mL).

4.2.3. Standard Antibiotic Solutions Preparation

All antibiotic drug solutions were prepared with double distilled water, the final stock solution concentration being 0,5 mg/mL.

4.2.4. Microdillution Method

All successive steps were performed in a laminar flow; In 96-well plates, we performed serial dilutions, adapting the protocol described by Gómez-Sequeda et al. [110] and detailed in our recently published study [107]. All well plates were incubated for 24 h at 37 °C. After incubation, the antibacterial efficacy of essential oils was determined by reading the absorbance values using the EnSight Multimode Plate Reader and calculated according to Sandulovici et al. [107].

4.3. Antibiofilm Activity

The method used was adapted from [112] and detail presented in our recently published article [107]. After incubation, the bacterial biofilm production was evidenced by staining with 0.1% Gentian Violet after removing the culture medium, washing twice with sterile distilled water, and drying at room temperature under airflow. After dye removal, the microplates were dried at 50 °C for 60 minutes. The dye incorporated in bacterial cells that formed the biofilm was solubilized with 95% ethanol for 10 minutes under continuous stirring at 450 rpm.

4.4. Quantification and Interpreting of Antibacterial and Antibiofilm Activities

The antibacterial and antibiofilm effects of essential oils were determined by reading the absorbencies using the EnSight Multimode Plate Reader at 562 and 570 nm, respectively, and calculated according to Sandulovici et al. [107].
The obtained results were compared to standard antibiotics.
Interpretation of antibacterial (ABE%—bacterial growth inhibition%) and antibiofilm (ABfE%—biofilm formation inhibition%) efficacy was quantified on conventional arithmetic intervals: very good efficacy: ≥ 90%, good efficacy: 75—89%, moderate efficacy: 50—74%, satisfactory: 25—49% and unsatisfactory: 0—24%.

4.5. Data Analysis

The analyses were performed in triplicate. The absorbance values, expressed as a mean of three determinations, were included in the calculation formula detailed in our previous publication [107].
The correlations between variable parameters [113] were examined through principal component analysis [114] performed with XLSTAT 2023.1.4. by Lumivero (Denver, CO, USA); their statistical significance was available when p < 0.05 [115].

5. Conclusions

All essential oils and antibacterial drugs exhibited antibacterial efficacy against Gram-positive and Gram-negative bacteria tested.
The most active essential oils were Oregano oils, with a substantial antibacterial and antibiofilm effect on all tested bacteria.
P. aeruginosa was the most susceptible bacteria, all EOs having concomitantly antibacterial and antibiofilm effects. The antibiofilm effectiveness decreases in order: P aeruginosa, E. coli, and S. aureus.
The highest antibacterial effects of commercially available essential oils were against P. aeruginosa because they exhibited a significant antibiofilm activity. Their antibiofilm efficacy intensively decreased on E. coli and S. aureus.
Generally, the samples with different manufacturers of the same essential oil showed similar activities; only Clove and Peppermint oils showed higher differences, maybe due to different places of harvesting the raw plant material, various technological processes through these essential oils were obtained, and complex interactions between constituents.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org, Principal Component Analysis.

Author Contributions

Conceptualization, R.N., V.P., and L.E.I.; methodology, L.E.I., D.M.P., and E.A.O.; software, R.N., and V.P.; validation, L.E.I., and V.O.; formal analysis, V.P. and C.E.G.; investigation, R.N., and V.P.; resources, R.N.; data curation, L.E.I.; writing—original draft preparation, R.N., and V.P.; writing—review and editing, V.P., V.O., and C.E.G.; visualization, D.M.P., and E.A.O.; supervision, C.E.G; project administration, L.E.I.; funding acquisition, R.N., and V.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of National Defense through the Sectoral Research and Development Plan.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available in the present manuscript and Supplementary Material.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Masyita, A.; Mustika Sari, R.; Dwi Astuti, A.; Yasir, B.; Rahma Rumata, N.; Emran, T. Bin; Nainu, F.; Simal-Gandara, J. Terpenes and Terpenoids as Main Bioactive Compounds of Essential Oils, Their Roles in Human Health and Potential Application as Natural Food Preservatives. Food Chem X 2022, 13, 100217. [Google Scholar] [CrossRef] [PubMed]
  2. European Pharmacopoeia Aetherolea. Europeian Pharmacopoeia 2008.
  3. Chouhan, S.; Sharma, K.; Guleria, S. Antimicrobial Activity of Some Essential Oils—Present Status and Future Perspectives. Medicines 2017, 4, 58. [Google Scholar] [CrossRef] [PubMed]
  4. Manion, C.R.; Widder, R.M. Essentials of Essential Oils. American Journal of Health-System Pharmacy 2017, 74, e153–e162. [Google Scholar] [CrossRef] [PubMed]
  5. Valdivieso-Ugarte, M.; Gomez-Llorente, C.; Plaza-Díaz, J.; Gil, Á. Antimicrobial, Antioxidant, and Immunomodulatory Properties of Essential Oils: A Systematic Review. Nutrients 2019, 11, 2876. [Google Scholar] [CrossRef] [PubMed]
  6. Mutlu-Ingok, A.; Devecioglu, D.; Dikmetas, D.N.; Karbancioglu-Guler, F.; Capanoglu, E. Antibacterial, Antifungal, Antimycotoxigenic, and Antioxidant Activities of Essential Oils: An Updated Review. Molecules 2020, 25, 4711. [Google Scholar] [CrossRef]
  7. Xu, N.; Lei, H.; Li, X.; Wang, Q.; Liu, M.; Wang, M. Protective Effects of Ginger Essential Oil (Geo) against Chemically-Induced Cutaneous Inflammation. Food Science and Technology (Brazil) 2019, 39, 371–377. [Google Scholar] [CrossRef]
  8. Pandur, E.; Balatinácz, A.; Micalizzi, G.; Mondello, L.; Horváth, A.; Sipos, K.; Horváth, G. Anti-Inflammatory Effect of Lavender (Lavandula Angustifolia Mill.) Essential Oil Prepared during Different Plant Phenophases on THP-1 Macrophages. BMC Complement Med Ther 2021, 21, s12906. [Google Scholar] [CrossRef]
  9. Fan, H.; Zhang, L.; Li, Y.; Soo Khoo, C.; Han, D.; Liu, Q.; Li, P.; Zhang, X. Antioxidant and Immunomodulatory Activities of Essential Oil Isolated from Anti-Upper Respiratory Tract Infection Formulation and Their Chemical Analysis. Evidence-based Complementary and Alternative Medicine 2022, 2022, 7297499. [Google Scholar] [CrossRef]
  10. Pelvan, E.; Karaoğlu, Ö.; Önder Fırat, E.; Betül Kalyon, K.; Ros, E.; Alasalvar, C. Immunomodulatory Effects of Selected Medicinal Herbs and Their Essential Oils: A Comprehensive Review. J Funct Foods 2022, 94, 105108. [Google Scholar] [CrossRef]
  11. González-Velasco, H.E.; Pérez-Gutiérrez, M.S.; Alonso-Castro, Á.J.; Zapata-Morales, J.R.; Niño-Moreno, P.D.C.; Campos-Xolalpa, N.; González-Chávez, M.M. Anti-Inflammatory and Antinociceptive Activities of the Essential Oil of Tagetes Parryi A. Gray (Asteraceae) and Verbenone. Molecules 2022, 27, 2612. [Google Scholar] [CrossRef]
  12. Gómez-Betancur, I.; Benjumea, D.; Gómez, J.E.; Mejía, N.; León, J.F. Antinociceptive Activity of Essential Oils from Wild Growing and Micropropagated Plants of Renealmia Alpinia (Rottb.) Maas. Records of Natural Products 2019, 13, 10–17. [Google Scholar] [CrossRef]
  13. Eftekhari, M.; Hoseinsalari, A.; Mansourian, M.; Farjadmand, F.; Shams Ardekani, M.R.; Sharifzadeh, M.; Hassanzadeh, G.; Khanavi, M.; Gholami, M. Trachyspermum Ammi (L.) Sprague, Superb Essential Oil and Its Major Components on Peptic Ulcers: In Vivo Combined in Silico Studies. DARU, Journal of Pharmaceutical Sciences 2019, 27, 317–327. [Google Scholar] [CrossRef]
  14. Abu Bakar, N.A.; Hakim Abdullah, M.N.; Lim, V.; Yong, Y.K. Essential Oils Derived from Momordica Charantia Seeds Exhibited Antiulcer Activity against Hydrogen Chloride/Ethanol and Indomethacin. Evidence-based Complementary and Alternative Medicine 2021, 2021, 5525584. [Google Scholar] [CrossRef] [PubMed]
  15. Sharma, M.; Grewal, K.; Jandrotia, R.; Batish, D.R.; Singh, H.P.; Kohli, R.K. Essential Oils as Anticancer Agents: Potential Role in Malignancies, Drug Delivery Mechanisms, and Immune System Enhancement. Biomedicine and Pharmacotherapy 2022, 146, 112514. [Google Scholar] [CrossRef] [PubMed]
  16. Osanloo, M.; Yousefpoor, Y.; Alipanah, H.; Ghanbariasad, A.; Jalilvand, M.; Amani, A. In-Vitro Assessment of Essential Oils as Anticancer Therapeutic Agents: A Systematic Literature Review. Jordan Journal of Pharmaceutical Sciences 2022, 15, 173–203. [Google Scholar] [CrossRef]
  17. Zimmermann, R.C.; Aragão, C.E. de C.; Araújo, P.J.P. de; Benatto, A.; Chaaban, A.; Martins, C.E.N.; Amaral, W. do; Cipriano, R.R.; Zawadneak, M.A.C. Insecticide Activity and Toxicity of Essential Oils against Two Stored-Product Insects. Crop Protection 2021, 144, 105575. [Google Scholar] [CrossRef]
  18. Rants’o, T.A.; Koekemoer, L.L.; Panayides, J.L.; van Zyl, R.L. Potential of Essential Oil-Based Anticholinesterase Insecticides against Anopheles Vectors: A Review. Molecules 2022, 27, 7026. [Google Scholar] [CrossRef]
  19. Huong, L.T.T.; Huong, T.T.T.; Huong, N.T.T.; Hung, N.H.; Dat, P.T.T.; Luong, N.X.; Ogunwande, I.A. Mosquito Larvicidal Activity of the Essential Oil of Zingiber Collinsii against Aedes Albopictus and Culex Quinquefasciatus. J Oleo Sci 2020, 69, 153–160. [Google Scholar] [CrossRef]
  20. Budiman; Ishak, H.; Stang; Ibrahim, E.; Daud, A.; Amiruddin, R. Essential Oil as a New Tool for Larvicidal Aedes Aegypti: A Systematic Review. Gac Sanit 2021, 35, S459–S462. [CrossRef]
  21. Castro, L.M.; Pinto, N.B.; Moura, M.Q.; Villela, M.M.; Capella, G.A.; Freitag, R.A.; Berne, M.E.A. Antihelminthic Action of the Anethum Graveolens Essential Oil on Haemonchus Contortus Eggs and Larvae. Brazilian Journal of Biology 2021, 81, 183–188. [Google Scholar] [CrossRef] [PubMed]
  22. Upadhyay, R. Essential Oils: Antimicrobial, Antihelminthic, Antiviral, Anticancer and Antiinsect Properties. J Appl Biosci 2010, 36, 1–22. [Google Scholar]
  23. Mieres-Castro, D.; Ahmar, S.; Shabbir, R.; Mora-Poblete, F. Antiviral Activities of Eucalyptus Essential Oils: Their Effectiveness as Therapeutic Targets against Human Viruses. Pharmaceuticals 2021, 14, 1210. [Google Scholar] [CrossRef] [PubMed]
  24. Asif, M.; Saleem, M.; Saadullah, M.; Yaseen, H.S.; Al Zarzour, R. COVID-19 and Therapy with Essential Oils Having Antiviral, Anti-Inflammatory, and Immunomodulatory Properties. Inflammopharmacology 2020, 28, 1153–1161. [Google Scholar] [CrossRef] [PubMed]
  25. Tohidi, B.; Rahimmalek, M.; Trindade, H. Review on Essential Oil, Extracts Composition, Molecular and Phytochemical Properties of Thymus Species in Iran. Ind Crops Prod 2019, 134, 89–99. [Google Scholar] [CrossRef]
  26. Garzoli, S. Chemical Composition and Antimicrobial Activity of Essential Oils. Plants 2023, 12, 800. [Google Scholar] [CrossRef]
  27. Bollyky, T.J.; Kesselheim, A.S. Reputation and Authority: The FDA and the Fight over U.S. Prescription Drug Importation. Vanderbilt Law Rev 2020, 73. [Google Scholar]
  28. Darrow, J.J.; Avorn, J.; Kesselheim, A.S. FDA Approval and Regulation of Pharmaceuticals, 1983-2018. JAMA - Journal of the American Medical Association 2020, 323, 164–176. [Google Scholar] [CrossRef]
  29. Farrar, A.J.; Farrar, F.C. Clinical Aromatherapy. Nursing Clinics of North America 2020, 55, 489–504. [Google Scholar] [CrossRef]
  30. doTerra DoTerra.
  31. Stringaro, A.; Colone, M.; Angiolella, L. Antioxidant, Antifungal, Antibiofilm, and Cytotoxic Activities of Mentha Spp. Essential Oils. Medicines 2018, 5, 112. [Google Scholar] [CrossRef]
  32. Committee on Herbal Medicinal Products (HMPC) Reflection Paper on Quality of Essential Oils as Active Substances in Herbal Medicinal Products / Traditional Herbal Medicinal Products. EMA/HMPC/84789/2013 2014, 44.
  33. Peschel, W. The Use of Community Herbal Monographs to Facilitate Registrations and Authorisations of Herbal Medicinal Products in the European Union 2004-2012. J Ethnopharmacol 2014, 158, 471–486. [Google Scholar] [CrossRef] [PubMed]
  34. Petrović, S. Herbal and Traditional Herbal Medicinal Products, EU Herbal Monographs and EU List. Arh Farm (Belgr) 2019, 69, 221–269. [Google Scholar] [CrossRef]
  35. European Medicines Agency Guideline on Similar Biological Medicinal Products. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2014/10/WC500176768.pdf 2014, 44, 1–7.
  36. Iordache, A.M.; Nechita, C.; Voica, C.; Roba, C.; Botoran, O.R.; Ionete, R.E. Assessing the Health Risk and the Metal Content of Thirty-Four Plant Essential Oils Using the ICP-MS Technique. Nutrients 2022, 14, 2363. [Google Scholar] [CrossRef]
  37. Vargas Jentzsch, P.; Gualpa, F.; Ramos, L.A.; Ciobotă, V. Adulteration of Clove Essential Oil: Detection Using a Handheld Raman Spectrometer. Flavour Fragr J 2018, 33, 184–190. [Google Scholar] [CrossRef]
  38. Pierson, M.; Fernandez, X.; Antoniotti, S. Type and Magnitude of Non-Compliance and Adulteration in Neroli, Mandarin and Bergamot Essential Oils Purchased online: Potential Consumer Vulnerability. Sci Rep 2021, 11, 11096. [Google Scholar] [CrossRef]
  39. De Clerck, C.; Maso, S.D.; Parisi, O.; Dresen, F.; Zhiri, A.; Haissam Jijakli, M. Screening of Antifungal and Antibacterial Activity of 90 Commercial Essential Oils against 10 Pathogens of Agronomical Importance. Foods 2020, 9, 1418. [Google Scholar] [CrossRef]
  40. Thomsen, P.S.; Jensen, T.M.; Hammer, K.A.; Carson, C.F.; Mølgaard, P.; Riley, T. V. Survey of the Antimicrobial Activity of Commercially Available Australian Tea Tree (Melaleuca Alternifolia) Essential Oil Products in Vitro. Journal of Alternative and Complementary Medicine 2011, 17, 835–841. [Google Scholar] [CrossRef]
  41. Ogbaini-Emovon, E.; Schuster, H.; Waze, J.; Iche, K. In Vitro Antimicrobial Activity of Commercially Available Melaleuca Alternifolia (Tea Tree) Oil on Some Selected Clinical Pathogens. Br J Pharm Res 2015, 5, 202–208. [Google Scholar] [CrossRef]
  42. Ju, J.; Xie, Y.; Yu, H.; Guo, Y.; Cheng, Y.; Qian, H.; Yao, W. Synergistic Interactions of Plant Essential Oils with Antimicrobial Agents: A New Antimicrobial Therapy. Crit Rev Food Sci Nutr 2022, 62, 1740–1751. [Google Scholar] [CrossRef]
  43. Brun, P.; Bernabè, G.; Filippini, R.; Piovan, A. In Vitro Antimicrobial Activities of Commercially Available Tea Tree (Melaleuca Alternifolia) Essential Oils. Curr Microbiol 2019, 76, 108–116. [Google Scholar] [CrossRef] [PubMed]
  44. Anquez-Traxler, C. The Legal and Regulatory Framework of Herbal Medicinal Products in the European Union: A Focus on the Traditional Herbal Medicines Category. Ther Innov Regul Sci 2011, 45, 15–23. [Google Scholar] [CrossRef]
  45. Peschel, W.; Alvarez, B.M. Harmonised European Standards as a Basis for the Safe Use of Herbal Medicinal Products and Their Marketing Authorisation in European Union Member States. Pharmaceut Med 2018, 32, 275–293. [Google Scholar] [CrossRef]
  46. Steinhoff, B. Harmonised Assessment Criteria for Efficacy and Safety of Herbal Medicinal Products. Revista de Fitoterapia 2002, 2, 47. [Google Scholar]
  47. Yevale, R.; Khan, N.; Kalamkar, P. Overview on “Regulations of Herbal Medicine. ” J Pharmacogn Phytochem 2018, 7, 61–63. [Google Scholar] [CrossRef]
  48. Commission, T.H.E.E. COMMISSION IMPLEMENTING REGULATION (EU) 2022/1248 of 19 July 2022 Concerning the Authorisation of Essential Oil from Origanum Vulgare Ssp. Hirtum (Link) Ietsw. as a Feed Additive for Certain Animal Species. 2022, 1248, 18–20. [Google Scholar]
  49. Bejar, E. Adulteration of Oregano Herb and Essential Oil. Botanical Adulterants Prevention Bulletin 2019, 10, 1–10. [Google Scholar]
  50. Mohammadi Gheisar, M.; Kim, I.H. Phytobiotics in Poultry and Swine Nutrition–a Review. Ital J Anim Sci 2018, 17. [Google Scholar] [CrossRef]
  51. Fonseca-García, I.; Escalera-Valente, F.; Martínez-González, S.; Carmona-Gasca, C.A.; Gutiérrez-Arenas, D.A.; Ramos, F. Effect of Oregano Oil Dietary Supplementation on Production Parameters, Height of Intestinal Villi and the Antioxidant Capacity in the Breast of Broiler. Austral J Vet Sci 2017, 49, 200083. [Google Scholar] [CrossRef]
  52. European Pharmacopoeia (Ph. Eur.) 10th Edition | EDQM - European Directorate for the Quality of Medicines Available Online: Https://Www.Edqm.Eu/En/European-Pharmacopoeia-Ph-Eur-10th-Edition accessed on 17 May 2023. 17 May.
  53. Committee, on H.M.P. Assessment Report on Syzygium Aromaticum (L .) Merill et L . M . Perry, Flos and Syzygium Aromaticum (L .) Merill Et. European Medicines Agency (EMA) 2011, 44, 26.
  54. Medicines Agency, E. Peppermint Oil - Herbal Summary for the Public. EMEA European Medicines Agency 2020, 31, 1–3. [Google Scholar]
  55. Sebei, K.; Sakouhi, F.; Herchi, W.; Khouja, M.L.; Boukhchina, S. Chemical Composition and Antibacterial Activities of Seven Eucalyptus Species Essential Oils Leaves. Biol Res 2015, 48, 7. [Google Scholar] [CrossRef]
  56. EMA. Committee on Herbal Medicinal Products (HMPC) Community Herbal Monograph on Eucalyptus Globulus Labill., Eucalyptus Polybractea R.T. Baker and/or Eucalyptus Smithii R.T. Baker, Aetheroleum. European Medicines Agenc 2014, 44, 2–11. [Google Scholar]
  57. Revision, D. European Union Herbal Monograph on Rosmarinus Officinalis L., Aetheroleum. EMEA European Medicines Agency 2022, 31, 1–6. [Google Scholar]
  58. Luo, K.; Zhao, P.; He, Y.; Kang, S.; Shen, C.; Wang, S.; Guo, M.; Wang, L.; Shi, C. Antibacterial Effect of Oregano Essential Oil against Vibrio Vulnificus and Its Mechanism. Foods 2022, 11, 403. [Google Scholar] [CrossRef]
  59. Yuan, Y.; Sun, J.; Song, Y.; Raka, R.N.; Xiang, J.; Wu, H.; Xiao, J.; Jin, J.; Hui, X.L. Antibacterial Activity of Oregano Essential Oils against Streptococcus Mutans in Vitro and Analysis of Active Components. BMC Complement Med Ther 2023, 23, 61. [Google Scholar] [CrossRef] [PubMed]
  60. Gavaric, N.; Mozina, S.S.; Kladar, N.; Bozin, B. Chemical Profile, Antioxidant and Antibacterial Activity of Thyme and Oregano Essential Oils, Thymol and Carvacrol and Their Possible Synergism. Journal of Essential Oil-Bearing Plants 2015, 18, 1013–1021. [Google Scholar] [CrossRef]
  61. Cui, H.; Zhang, C.; Li, C.; Lin, L. Antibacterial Mechanism of Oregano Essential Oil. Ind Crops Prod 2019, 139, 111498. [Google Scholar] [CrossRef]
  62. Qiu, J.; Wang, D.; Xiang, H.; Feng, H.; Jiang, Y.; Xia, L.; Dong, J.; Lu, J.; Yu, L.; Deng, X. Subinhibitory Concentrations of Thymol Reduce Enterotoxins A and B and α-Hemolysin Production in Staphylococcus Aureus Isolates. PLoS One 2010, 5, 9736. [Google Scholar] [CrossRef]
  63. Kryvtsova, M. V.; Fedkiv, O.K.; Hrytsyna, M.R.; Salamon, I. Anty-Microbial, and Anty-Biofilm-Forming Properties of Origanum Vulgare L. Essential Oils on Staphylococcus Aureus and Its Antioxidant Action. Studia Biologica 2020, 14, 27–38. [Google Scholar] [CrossRef]
  64. Sipahi, N.; Kekeç, A.I.; Halaç, B. In Vitro Effect of Some Essential Oils against Multiple Antibiotic-Resistant Bacteria from Cats and Dogs. Pak Vet J 2022, 42, 561–565. [Google Scholar]
  65. Schillaci, D.; Napoli, E.M.; Cusimano, M.G.; Vitale, M.; Ruberto, G. Origanum Vulgare Subsp. Hirtum Essential Oil Prevented Biofilm Formation and Showed Antibacterial Activity against Planktonic and Sessile Bacterial Cells. J Food Prot 2013, 76, 1747–1752. [Google Scholar] [CrossRef]
  66. Lu, M.; Wong, K.I.; Li, X.; Wang, F.; Wei, L.; Wang, S.; Wu, M.X. Oregano Oil and Harmless Blue Light to Synergistically Inactivate Multidrug-Resistant Pseudomonas Aeruginosa. Front Microbiol 2022, 13, 810746. [Google Scholar] [CrossRef]
  67. Salehi, B.; Mishra, A.P.; Shukla, I.; Sharifi-Rad, M.; Contreras, M. del M.; Segura-Carretero, A.; Fathi, H.; Nasrabadi, N.N.; Kobarfard, F.; Sharifi-Rad, J. Thymol, Thyme, and Other Plant Sources: Health and Potential Uses. Phytotherapy Research 2018, 32. [Google Scholar] [CrossRef] [PubMed]
  68. Lara, V.M.; Carregaro, A.B.; Santurio, D.F.; Sá, M.F. De; Santurio, J.M.; Alves, S.H. Antimicrobial Susceptibility of Escherichia Coli Strains Isolated from Alouatta Spp. Feces to Essential Oils. Evidence-based Complementary and Alternative Medicine 2016, 2016, 1643762. [Google Scholar] [CrossRef] [PubMed]
  69. Man, A.; Santacroce, L.; Jacob, R.; Mare, A.; Man, L. Antimicrobial Activity of Six Essential Oils against a Group of Human Pathogens: A Comparative Study. Pathogens 2019, 8, 15. [Google Scholar] [CrossRef]
  70. Ghalem, B.R.; Mohamed, B. Antibacterial Activity of Leaf Essential Oils of Eucalyptus Globulus and Eucalyptus Camaldulensis. Afr J Pharm Pharmacol 2008, 2, 211–215. [Google Scholar]
  71. Bachir, R.G.; Benali, M. Antibacterial Activity of the Essential Oils from the Leaves of Eucalyptus Globulus against Escherichia Coli and Staphylococcus Aureus. Asian Pac J Trop Biomed 2012, 2, 739–742. [Google Scholar] [CrossRef]
  72. Ameur, E.; Sarra, M.; Yosra, D.; Mariem, K.; Nabil, A.; Lynen, F.; Larbi, K.M. Chemical Composition of Essential Oils of Eight Tunisian Eucalyptus Species and Their Antibacterial Activity against Strains Responsible for Otitis. BMC Complement Med Ther 2021, 21, 209. [Google Scholar]
  73. Merghni, A.; Noumi, E.; Hadded, O.; Dridi, N.; Panwar, H.; Ceylan, O.; Mastouri, M.; Snoussi, M. Assessment of the Antibiofilm and Antiquorum Sensing Activities of Eucalyptus Globulus Essential Oil and Its Main Component 1,8-Cineole against Methicillin-Resistant Staphylococcus Aureus Strains. Microb Pathog 2018, 118, 74–80. [Google Scholar] [CrossRef]
  74. Quatrin, P.M.; Verdi, C.M.; de Souza, M.E.; de Godoi, S.N.; Klein, B.; Gundel, A.; Wagner, R.; de Almeida Vaucher, R.; Ourique, A.F.; Santos, R.C.V. Antimicrobial and Antibiofilm Activities of Nanoemulsions Containing Eucalyptus Globulus Oil against Pseudomonas Aeruginosa and Candida Spp. Microb Pathog 2017, 112, 230–242. [Google Scholar] [CrossRef]
  75. Khedhri, S.; Polito, F.; Caputo, L.; Manna, F.; Khammassi, M.; Hamrouni, L.; Amri, I.; Nazzaro, F.; De Feo, V.; Fratianni, F. Chemical Composition, Phytotoxic and Antibiofilm Activity of Seven Eucalyptus Species from Tunisia. Molecules 2022, 27, 8227. [Google Scholar] [CrossRef] [PubMed]
  76. Azzam, N.F.A.E.M. Antibacterial Effect of Eucalyptus Essential Oil. Indian J Sci Technol 2020, 13, 799–804. [Google Scholar] [CrossRef]
  77. https://oshadhi.co.uk/kb/purity-adulteration-testing/ accessed on 20 May 2023.
  78. Elangovan, S.; Mudgil, P. Antibacterial Properties of Eucalyptus Globulus Essential Oil against MRSA: A Systematic Review. Antibiotics 2023, 12, 474. [Google Scholar] [CrossRef] [PubMed]
  79. Mulyaningsih, S.; Sporer, F.; Reichling, J.; Wink, M. Antibacterial Activity of Essential Oils from Eucalyptus and of Selected Components against Multidrug-Resistant Bacterial Pathogens. Pharm Biol 2011, 49, 553625. [Google Scholar] [CrossRef] [PubMed]
  80. Van, L.T.; Hagiu, I.; Popovici, A.; Marinescu, F.; Gheorghe, I.; Curutiu, C.; Ditu, L.M.; Holban, A.M.; Sesan, T.E.; Lazar, V. Antimicrobial Efficiency of Some Essential Oils in Antibiotic-Resistant Pseudomonas Aeruginosa Isolates. Plants 2022, 11, 2003. [Google Scholar] [CrossRef]
  81. Jafari-Sales, A.; Pashazadeh, M. Study of Chemical Composition and Antimicrobial Properties of Rosemary (Rosmarinus Officinalis) Essential Oil on Staphylococcus Aureus and Escherichia Coli in Vitro. International Journal of Life Sciences and Biotechnology 2020, 3, 62–69. [Google Scholar] [CrossRef]
  82. Liu, T.; Wang, J.; Gong, X.; Wu, X.; Liu, L.; Chi, F. Rosemary and Tea Tree Essential Oils Exert Antibiofilm Activities in Vitro against Staphylococcus Aureus and Escherichia Coli. J Food Prot 2020, 83, 1261–1267. [Google Scholar] [CrossRef]
  83. Stojiljkovic, J. Antibacterial Activities of Rosemary Essential Oils and Their Components against Pathogenic Bacteria. Advances in Cytology & Pathology 2018, 3, 93–96. [Google Scholar]
  84. Santoyo, S.; Cavero, S.; Jaime, L.; Ibañez, E.; Señoráns, F.J.; Reglero, G. Chemical Composition and Antimicrobial Activity of Rosmarinus Officinalis L. Essential Oil Obtained via Supercritical Fluid Extraction. J Food Prot 2005, 68, 790–795. [Google Scholar] [CrossRef] [PubMed]
  85. Ceylan, O.; Uğur, A.; Saraç, N.; Ozcan, F.; Baygar, T. The in Vitro Antibiofilm Activity of Rosmarinus Officinalis L. Essential Oil against Multiple Antibiotic Resistant Pseudomonas Sp. and Staphylococcus Sp. J Food Agric Environ 2014, 12, 82–86. [Google Scholar]
  86. Bogavac, M.A.; Karaman, M.A.; Sudi, J.J.; Radovanović, B.B.; Janjušević, L.N.; Ćetković, N.B.; Tešanović, K.D. Antimicrobial Potential of Rosmarinus Officinalis Commercial Essential Oil in the Treatment of Vaginal Infections in Pregnant Women. Nat Prod Commun 2017, 12, 200136. [Google Scholar] [CrossRef]
  87. https://www.wildherbsofcrete.com/rosemary accessed on 20 May 2023.
  88. Xu, J.G.; Liu, T.; Hu, Q.P.; Cao, X.M. Chemical Composition, Antibacterial Properties and Mechanism of Action of Essential Oil from Clove Buds against Staphylococcus Aureus. Molecules 2016, 21, 1194. [Google Scholar] [CrossRef]
  89. Bai, J.; Li, J.; Chen, Z.; Bai, X.; Yang, Z.; Wang, Z.; Yang, Y. Antibacterial Activity and Mechanism of Clove Essential Oil against Foodborne Pathogens. LWT 2023, 173. [Google Scholar] [CrossRef]
  90. Yadav, M.K.; Chae, S.W.; Im, G.J.; Chung, J.W.; Song, J.J. Eugenol: A Phyto-Compound Effective against Methicillin-Resistant and Methicillin-Sensitive Staphylococcus Aureus Clinical Strain Biofilms. PLoS One 2015, 10, 119564. [Google Scholar] [CrossRef] [PubMed]
  91. Burt, S.A.; Reinders, R.D. Antibacterial Activity of Selected Plant Essential Oils against Escherichia Coil O157:H7. Lett Appl Microbiol 2003, 36, 162–167. [Google Scholar] [CrossRef]
  92. Kim, Y.G.; Lee, J.H.; Gwon, G.; Kim, S. Il; Park, J.G.; Lee, J. Essential Oils and Eugenols Inhibit Biofilm Formation and the Virulence of Escherichia Coli O157:H7. Sci Rep 2016, 6, 36377. [Google Scholar] [CrossRef]
  93. Musthafa, K.S.; Voravuthikunchai, S.P. Anti-Virulence Potential of Eugenyl Acetate against Pathogenic Bacteria of Medical Importance. Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology 2015, 107, 703–710. [Google Scholar] [CrossRef]
  94. Costa, L.V.; Moreira, J.M.A.R.; de Godoy Menezes, I.; Dutra, V.; do Bom Parto Ferreira de Almeida, A. Antibiotic Resistance Profiles and Activity of Clove Essential Oil (Syzygium Aromaticum) against Pseudomonas Aeruginosa Isolated of Canine Otitis. Vet World 2022, 15. [Google Scholar] [CrossRef]
  95. Purwasena, I.A.; Astuti, D.I.; Taufik, I.; Putri, F.Z. The Potential of Clove Essential Oil Microemulsion as an Alternative Biocide against Pseudomonas Aeruginosa Biofilm. J Pure Appl Microbiol 2020, 14, 261–269. [Google Scholar] [CrossRef]
  96. Muntean, D.; Licker, M.; Alexa, E.; Popescu, I.; Jianu, C.; Buda, V.; Dehelean, C.A.; Ghiulai, R.; Horhat, F.; Horhat, D.; et al. Evaluation of Essential Oil Obtained from Mentha×piperita L. against Multidrug-Resistant Strains. Infect Drug Resist 2019, 12, 2905–2914. [Google Scholar] [CrossRef]
  97. Li, J.; Dong, J.; Qui, J.Z.; Wang, J.F.; Luo, M.J.; Li, H.E.; Leng, B.F.; Ren, W.Z.; Deng, X.M. Peppermint Oil Decreases the Production of Virulence- Associated Exoproteins by Staphylococcus Aureus. Molecules 2011, 16, 1642. [Google Scholar] [CrossRef]
  98. Horváth, P.; Koščová, J. In Vitro Antibacterial Activity of Mentha Essential Oils Against Staphylococcus Aureus. Folia Vet 2017, 61, 71–77. [Google Scholar] [CrossRef]
  99. Kang, J.; Jin, W.; Wang, J.; Sun, Y.; Wu, X.; Liu, L. Antibacterial and Anti-Biofilm Activities of Peppermint Essential Oil against Staphylococcus Aureus. LWT 2019, 101, 639–645. [Google Scholar] [CrossRef]
  100. Sarwar, W.; Ali, Q.; Ahmed, S. Microscopic Visualization of the Antibiofilm Potential of Essential Oils against Staphylococcus Aureus and Klebsiella Pneumoniae. Microsc Res Tech 2022, 85, 3921–3931. [Google Scholar] [CrossRef] [PubMed]
  101. Pajohi Alamoti, M.; Bazargani-Gilani, B.; Mahmoudi, R.; Reale, A.; Pakbin, B.; Di Renzo, T.; Kaboudari, A. Essential Oils from Indigenous Iranian Plants: A Natural Weapon vs. Multidrug-Resistant Escherichia Coli. Microorganisms 2022, 10, 109. [Google Scholar] [CrossRef]
  102. Metin, S.; Didinen, B.I.; Telci, I.; Diler, O. Essential Oil of Mentha Suaveolens Ehrh., Composition and Antibacterial Activity against Bacterial Fish Pathogens. An Acad Bras Cienc 2021, 93, 20190478. [Google Scholar] [CrossRef]
  103. Pazarci, O.; Tutar, U.; Kilinc, S. Investigation of the Antibiofilm Effects of Mentha Longifolia Essential Oil on Titanium and Stainless Steel Orthopedic Implant Surfaces. Eurasian Journal of Medicine 2019, 51, 18432. [Google Scholar] [CrossRef]
  104. Iseppi, R.; Di Cerbo, A.; Aloisi, P.; Manelli, M.; Pellesi, V.; Provenzano, C.; Camellini, S.; Messi, P.; Sabia, C. In Vitro Activity of Essential Oils against Planktonic and Biofilm Cells of Extended-Spectrum β-Lactamase (ESBL)/Carbapenamase-Producing Gram-Negative Bacteria Involved in Human Nosocomial Infections. Antibiotics 2020, 9, 272. [Google Scholar] [CrossRef]
  105. https://www.perfumerflavorist.com/flavor/ingredients/article/21856932/authentication-of-natural-peppermint-oil accessed on 20 May 2023.
  106. Singh, N.S.; Singhal, N.; Kumar, M.; Virdi, J.S. Exploring the Genetic Mechanisms Underlying Amoxicillin-Clavulanate Resistance in Waterborne Escherichia Coli. Infection, Genetics and Evolution 2021, 90, 104767. [Google Scholar] [CrossRef] [PubMed]
  107. Sandulovici, R.C.; Carmen-Marinela, M.; Grigoroiu, A.; Moldovan, C.A.; Savin, M.; Ordeanu, V.; Voicu, S.N.; Cord, D.; Costache, G.M.; Galatanu, M.L.; et al. The Physicochemical and Antimicrobial Properties of Silver/Gold Nanoparticles Obtained by “Green Synthesis” from Willow Bark and Their Formulations as Potential Innovative Pharmaceutical Substances. Pharmaceuticals 2023, 16, 10048. [Google Scholar] [CrossRef] [PubMed]
  108. Stefan, D.S.; Popescu, M.; Luntraru, C.M.; Suciu, A.; Belcu, M.; Ionescu, L.E.; Popescu, M.; Iancu, P.; Stefan, M. Comparative Study of Useful Compounds Extracted from Lophanthus Anisatus by Green Extraction. Molecules 2022, 27, 7737. [Google Scholar] [CrossRef] [PubMed]
  109. Martínez, A.; Manrique-Moreno, M.; Klaiss-Luna, M.C.; Stashenko, E.; Zafra, G.; Ortiz, C. Effect of Essential Oils on Growth Inhibition, Biofilm Formation and Membrane Integrity of Escherichia Coli and Staphylococcus Aureus. Antibiotics 2021, 10, 1474. [Google Scholar] [CrossRef] [PubMed]
  110. Gómez-Sequeda, N.; Cáceres, M.; Stashenko, E.E.; Hidalgo, W.; Ortiz, C. Antimicrobial and Antibiofilm Activities of Essential Oils against Escherichia Coli O157:H7 and Methicillin-Resistant Staphylococcus Aureus (MRSA). Antibiotics 2020, 9, 730. [Google Scholar] [CrossRef] [PubMed]
  111. Popovici, V.; Bucur, L.; Gîrd, C.E.; Rambu, D.; Calcan, S.I.; Cucolea, E.I.; Costache, T.; Ungureanu-Iuga, M.; Oroian, M.; Mironeasa, S.; et al. Antioxidant, Cytotoxic, and Rheological Properties of Canola Oil Extract of Usnea Barbata ( L.) Weber Ex F. H. Wigg from Călimani Mountains, Romania. Plants 2022, 11, 854. [Google Scholar] [CrossRef]
  112. Guillín, Y.; Cáceres, M.; Torres, R.; Stashenko, E.; Ortiz, C. Effect of Essential Oils on the Inhibition of Biofilm and Quorum Sensing in Salmonella Enteritidis 13076 and Salmonella Typhimurium 14028. Antibiotics 2021, 10, 1191. [Google Scholar] [CrossRef] [PubMed]
  113. Popovici, V.; Bucur, L.; Gîrd, C.E.; Popescu, A.; Matei, E.; Caraiane, A.; Botnarciuc, M. Phenolic Secondary Metabolites and Antiradical and Antibacterial Activities of Different Extracts of Usnea Barbata ( L.) Weber Ex F. H. Wigg from Călimani Mountains, Romania. Pharmaceuticals 2022, 15, 829. [Google Scholar] [CrossRef]
  114. Popovici, V.; Matei, E.; Cozaru, G.C.; Bucur, L.; Gîrd, C.E.; Schröder, V.; Ozon, E.A.; Musuc, A.M.; Mitu, M.A.; Atkinson, I.; et al. In Vitro Anticancer Activity of Mucoadhesive Oral Films Loaded with Usnea Barbata ( L. ) F. H. Wigg Dry Acetone Extract, with Potential Applications in Oral Squamous Cell Carcinoma Complementary Therapy. Antioxidants 2022, 11, 1934. [Google Scholar] [CrossRef]
  115. Popovici, V.; Matei, E.; Cozaru, G.; Bucur, L.; Gîrd, C.E.; Schröder, V.; Ozon, E.A.; Sarbu, I.; Musuc, A.M.; Atkinson, I.; et al. Formulation and Development of Bioadhesive Oral Films Containing Usnea Barbata ( L.) F. H. Wigg Dry Ethanol Extract ( F-UBE-HPC ) with Antimicrobial and Anticancer Properties for Potential Use in Oral Cancer Complementary Therapy. Pharmaceutics 2022, 14, 1808. [Google Scholar] [CrossRef]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
Prerpints.org logo

Preprints.org is a free preprint server supported by MDPI in Basel, Switzerland.

Subscribe

© 2024 MDPI (Basel, Switzerland) unless otherwise stated