Antibacterial and Antibiofilm Effects of Different Samples of Five Commercially Available Essential Oils

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—concerning their chemical composition and bioactivities—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 two to four samples of five commercially available essential oils (Oregano, Eucalyptus, Rosemary, Clove, and Peppermint oils) produced by autochthonous companies. The manufacturers provided all EOs’ chemical compositions determined through GC-MS. The EOs’ bioactivities were investigated in vitro against Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). The antibacterial and antibiofilm effects (ABE% and, respectively, ABfE%) were evaluated 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, Gentamycin, and Streptomycin. The results showed that at the first dilution (D1 = 25 mg/mL), all EOs exhibited antibacterial and antibiofilm activity against all Gram-positive and Gram-negative bacteria tested, and MIC value > 25 mg/mL. Generally, both effects progressively decreased from D1 to D3. Only EOs with a considerable content of highly active metabolites revealed insignificant differences. E. coli showed the lowest susceptibility to all commercially available essential oils—15 EO samples had undetected antibacterial and antibiofilm effects at D2 and D3. Peppermint and Clove oils recorded the most significant differences regarding chemical composition and antibacterial/antibiofilm activities. All registered differences could be due to different places for harvesting the raw plant material, various technological processes through which these essential oils were obtained, the preservation conditions, and complex interactions between constituents.


Introduction
Essential oils (EOs) are highly concentrated plant derivatives defined based on their physicochemical properties [1]. The Eos' chemical composition includes phenolic com-

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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 [31][32][33].
The EOs from doTERRA (Pleasant Grove, UT, USA) [34] are commercialized with the label CPTG (Certified Pure Therapeutic Grade). However, the FDA did not regulate essential oils as foods or dietary supplements. Any EO product cannot be marketed with the following mention: "It is intended to treat, prevent, cure or mitigate any disease or other health condition"-even when scientific research supports the claims' validity.
In Europe, the EOs industry growth is promoted by the European Federation of Essential Oils (EFEO) [35]. Currently, EFEO is discussing with the European Commission and the EU Parliament to amend or introduce legislation concerning essential oils. In contrast, due to numerous bioactivities, the European Medicinal Agency (EMA) considers EOs as herbal preparations and as active pharmaceutical ingredients (API) in two groups of herbal products [36]: • 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 [37,38]. 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" [39]. When essential oils are used as APIs of HMPs, the quality guidelines require an analytical characterization of the raw material. Moreover, according to the monograph "Herbal Drugs" from Ph. Eur., other tests must be performed on the essential oils [36]. 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's limits. Compliance with the ISO standards and the Ph. Eur. limits is critical for revealing adulterated EOs, evaluating correct plant material, chemotypes, and provenance, and recognizing changes during fabrication and storage [40].
All manufacturers must have suitable quality documentation for EOs, following EMA regulations. However, it is difficult to achieve all documents [36] when farmers or very Antibiotics 2023, 12,1191 3 of 29 small companies are implied in the manufacturing processes.e 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, measuring the metal content of 34 EOs from various manufacturers, Iordache et al. [41] identified Peppermint oil with Hg levels over six times higher than Ph. Eur. permissible limits. Vargas Jentzsch et al. [42] investigated commercial Clove essential oil samples. They found three adulterated samples containing benzyl alcohol and vegetable oil [42]. Recently, Pierson et al. [43] tested 31 EO samples purchased online by evaluating their compliance with ISO standards; they found that more than 45% did not pass the test, and more than 19% were diluted with various solvents (propylene and dipropylene glycol, triethyl citrate, or vegetable oil) [43]. In a previous study, Brun et al. [44] investigated 10 commercially available Tea Tree essential oils, finding that only 5 samples had significant antimicrobial activity [44].
The present study aims to explore the antibacterial and antibiofilm effects of five commercially available EOs-well-known for their phytotherapeutic applications-against Gram-positive and Gram-negative bacteria. Four essential oils (Eucalyptus oil, Rosemary oil, Clove oil, and Peppermint oil) are registered by EMA as HMPs for human and veterinary use [45], having a periodically updated monography (Table 1) [46]. Moreover, they have individual monographs in Ph. Eur., indicating the bioactive constituents' concentration limits, which are the basis of EMA regulation.
Only Oregano oil is authorized as a feed additive for animal species [47], and these data follow ISO 13171:2016 from International Organization for Standardization, Geneva, Switzerland [48]. Phytogenic feed additives (phytobiotics) are currently used in traditional European animal healthcare [49]. The effect of Oregano oil dietary supplementation in poultry on production parameters, intestinal villi height, and broiler breast's antioxidant capacity is well studied [50,51].
The antibacterial effects of essential oils investigated in our study are implied in all their therapeutical benefits, as mentioned in Table 1.

Essential Oil Name
correlating the data obtained with the bioactive compounds. The results were compared with the most known data from the scientific literature. In addition, a complex statistical analysis was performed to support our results.

Antibacterial and Antibiofilm Activity on S. aureus
The percentage values of antibacterial and antibiofilm efficacy of essential oils against Gram-positive bacteria (S. aureus) tested, compared to standard antibiotics, are displayed in Table 2.  Table 2 shows that, at 50 µg/mL, AMC had remarkable antibacterial efficacy (ABE = 96.83%), while both aminoglycosides exhibited a good one (ABE > 85%). All EOs recorded good antibacterial activity (ABE > 75%) against S. aureus at D1 = 25 mg/mL. OEOs and CEO1 proved to have considerable antibacterial potentials (ABE = 92. 76, 90.40, 91.26%) like the AMC one and higher than GEN and STR. The CEO3, PEO3, and PEO4 had the lowest ABE (78.80, 79.73, 79.83%). S. aureus sensitivity commonly decreases directly proportional to EOs concentration. Only a few EOs recorded good antibacterial effects at all D1, D2, and D3 dilutions: EEOs and REO1 (ABE% = 75-89%). The anti-staphylococcal effect slowly diminished at progressive dilutions (as in the case of REO2) or intensely decreased (OEOs, CEOs, PEOs 1 and 2). PEOs 3 and 4 exhibited antibacterial effects only at 25 mg/mL. As an overview, we could appreciate that the MIC value for all EOs tested is higher than 25 mg/mL. In addition, no significant differences (p > 0.05) between the antibacterial efficacy values of the tested samples at D1 were recorded in the case of OEOs, EEOs, and REOs ( 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 (CEOs 2 and 3 and PEO2), moderate (EEO1), and satisfactory (EEO2, REOs, PEOs 3 and 4). Excepting OEOs, the samples of each EO recorded significant differences (p < 0.05, Table 2). The highest differences were recorded in the case of PEOs 1 and 2 vs. PEOs 3 and 4 (91.13 and 89.23% vs. 32.17 and 38.73%, Table 2). Moderate differences in ABfE values at D1 were also registered in CEOs (95.40, 77.13, 81.77%, Table 2).

Antibacterial and Antibiofilm Activity on E. coli
The percentage values of antibacterial and antibiofilm efficacy of essential oils against Gram-negative bacteria E. coli, compared to standard antibiotics, are displayed in Table 3. Table 3 shows that at D1 = 50 µg/mL, AMC exhibits a very good ABE, while both aminoglycosides display a good one (ABE < 90%).
The antibiofilm activity of standard antibiotics and EOs differs significantly at D1 (  Table 3).
Insignificant differences between D1 and D2 were observed at CEO1, STR, and AMC (Table 3). In the case of other EOs and GEN, the ABfE values significantly decreased (p < 0.05, Table 3). On CEOs 2 and 3 and all PEOs, the antibiofilm activity was undetected at D2 and D3 (Table 3). At D3, the other five EOs (OEO2, EEOs, and REOs) reported undetected antibiofilm activity on E. coli; in addition, the ABfE values of OEO1 and CEO1 substantially decreased (2.93 and 11.20%, respectively, Table 3).

Antibacterial and Antibiofilm Activity on P. aeruginosa
The percentage values of antibacterial and antibiofilm efficacy of essential oils against P. aeruginosa, compared to standard antibiotics, are displayed in Table 4. Table 4. Antibacterial and antibiofilm efficacy of essential oils and antibacterial drugs against P. aeruginosa.
Principal Component Analysis (PCA) was used to evaluate the correlation between the bioactive compounds and antibacterial and antibiofilm efficacy of EOs on Gram-positive and Gram-negative bacteria.
Finally, considering all discussed variable parameters-extensively described in Supplementary Material- Figure 3 shows the place of each PEO sample, thus explaining all differences between them and supporting the results. Finally, considering all discussed variable parameters-extensively described in Supplementary Material- Figure 3 shows the place of each PEO sample, thus explaining all differences between them and supporting the results.
The correlation matrix from Supplementary Material and Figure 4A highlights a strong correlation between antibacterial and antibiofilm effects against both Gram-negative bacteria, P. aeruginosa (r = 0.912, p > 0.05) and E. coli (r = 0.760, p > 0.05). On S. aureus, both effects are poorly correlated (r = 0.425, p > 0.05) when we evaluate this effect on the entire EOs group. 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 that against P. aeruginosa (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. As an overview, on the first dilution (D1 = 25 mg/mL), only OEOs show similar percentage values of antibacterial and antibiofilm effects.

Correlations between EOs Chemical Constituents and Their Antibacterial and Antibiofilm Effects
Knowing each EOʹs chemical composition, the correlations between bioactive constituents and antibacterial and antibiofilm effects were analyzed to explain the differences between the corresponding samples. Figure 5A indicates that only p-cymene's contents in both OEO samples are included The registered data from Results are summarized in Figure 4A, evidencing the place of essential oils reported to both ABE and ABfE against all bacteria tested.
In a simplified manner, the dendrogram obtained by Agglomerative Hierarchical Clustering (AHC) from Figure 4B and Supplementary Material shows how EO samples act similarly. Figure 4B 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. At the same time, OEO2 acts closely to CEO1. The same observation is available on EEOs 1 and 2 and REOs 1 and 2.
The antibacterial activity of CEO and PEO samples is different. CEO1, due to substantial eugenol content, acts like OEOs (Figure 4). The antibacterial and antibiofilm effects decrease in order of CEO1, CEO2, and CEO3. In addition, eugenyl acetate and β-caryophyllene synergistically act with eugenol. CEO2 and CEO3 display similar antibacterial and antibiofilm potential, significantly lower than CEO1. Figures 4 and 7D show that PEO1 and PEO2 and, respectively, PEO3 and PEO4, act similarly. The primary metabolites implied in antibacterial and antibiofilm effects are menthol, menthone, menthyl acetate, and eucalyptol. They synergistically act with the others in lower content.
All previously mentioned observations are available for D1 = 25 mg/mL. At the following two dilutions (D2 = 2.5 mg/mL and D3 = 0.25 mg/mL), the antibacterial and antibiofilm effects could remain similar, slowly decrease, substantially diminish, or be undetected.
On S. aureus and P. aeruginosa, the antibacterial activity of both EEOs did not report significant differences (Tables 2 and 4), while on E. coli, only EEO1 reported similar ABE values (Table 3). Regarding antibiofilm activity, EEO1 and both REOs recorded similar effects (p > 0.05) at all three dilutions against P. aeruginosa.
On S. aureus, the following EOs exhibited no significant differences in antibiofilm activity between D1 and D2: PEOs 1 and 2, CEOs 1 and 3, and OEO1; on E. coli, EEO2, and REOs, regarding antibacterial activity; finally, on P. aeruginosa, REOs and PEOs (ABE) and PEO1, CEO1, EEO2, OEOs (ABfE). In contrast, at D2 and D3, the antibacterial activity of PEOs 3 and 4 against S. aureus was undetected. Similar observations are available on E. coli: PEO3 has no antibacterial effects, and CEO2, CEO3, and all PEOs did not record antibiofilm activities (Table 3). On P. aeruginosa, PEOs 3 and 4 did not show antibiofilm activity, and only PEO3 had no antibacterial efficacy (Table 4).   two constituents mentioned in Ph. Eur. 10 (carvone and isopulegol) were not found in PEOs samples. At the same time, neomenthol, contained by PEO1, PEO2, and PEO4, did not appear in Menthae piperitae aetheroleum monograph from Ph. Eur. 10. The antibacterial activity of CEO and PEO samples is different. CEO1, due to substantial eugenol content, acts like OEOs (Figure 4). The antibacterial and antibiofilm effects decrease in order of CEO1, CEO2, and CEO3. In addition, eugenyl acetate and βcaryophyllene synergistically act with eugenol. CEO2 and CEO3 display similar antibacterial and antibiofilm potential, significantly lower than CEO1.

Figures 4 and 7D
show that PEO1 and PEO2 and, respectively, PEO3 and PEO4, act similarly. The primary metabolites implied in antibacterial and antibiofilm effects are menthol, menthone, menthyl acetate, and eucalyptol. They synergistically act with the others in lower content.
All previously mentioned observations are available for D1 = 25 mg/mL. At the following two dilutions (D2 = 2.5 mg/mL and D3 = 0.25 mg/mL), the antibacterial and antibiofilm effects could remain similar, slowly decrease, substantially diminish, or be unde-

Discussion
According to European Pharmacopoeia, an EO is an odorous product with complex composition obtained by steam distillation, dry distillation, or other mechanical processes without heating from a botanically defined plant raw material. The Eos' separation from the aqueous phase uses a physical process without significantly affecting their composition. This definition reveals that other extraction procedures involving different solvents lead to extracts, not EOs. Due to increased demands and insufficient regulation, the adulteration of essential oils became a common practice along supply chains, generating safety concerns in the EOs industry [56]. The EOs adulteration could be performed using various methods: a cheaper oil addition [57] to the original one (e.g., corn mint to Peppermint oil), EOs' dilution with vegetable oils, and synthetic phytochemicals' inclusion [51] in the original EO [58][59][60]. Thus, supplementary quality control measures should be taken to ensure safety for human use [61,62]. Because aromatherapy is the most crucial application of essential oils, the best way to test the properties of various samples of the same EO is by using biological systems [63]. Pierson et al. [38] recently signaled the potential consumer vulnerability to neroli, mandarin, and bergamot essential oils purchased online.
Many patients request pharmacists' counseling, aiming to know how to select from numerous types of commercially available essential oils the most suitable ones for therapeutic purposes. In Romania, the most known manufacturers mention the following data on each EO packaging and leaflet [64,65].

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The scientific name of the raw plant. • 100% Pure-It is not mixed with other essential oils, synthetic components, plant oils, or mineral oils and has no chemical solvents. • 100% Natural-It is obtained by steam distillation. • 100% Verified-It is biochemically and botanically defined. On some manufacturers' websites, the GC-MS analysis reports are available, indicating the chemical composition of the EOs [68,69]. The others provide GC-MS reports upon request. Therefore, putting ourselves in the patient's place, we have chosen well-known, most studied, and widely used EOs in therapy due to their antibacterial properties precisely so that we can relate our results to the data from the literature. We purchased and analyzed several samples of the same EO from different suppliers. All investigated EOs samples were manufactured by four autochthonous companies and regularly commercialized in pharmacies, pharma markets [70], and online markets. We requested the GS-MS reports with chemical composition to compare them with the one from the Ph. Eur. 10 monograph, or ISO standard, as stipulated by the EMA regulation. The manufacturers provided each EO's chemical composition, and all statistical analyses were performed with available data [71]. Next, we analyzed their influence on antibacterial and antibiofilm effects against Gram-positive and Gram-negative bacteria tested using the microdilution method. Three decimal dilutions, 25, 2.5, and 0.25 mg/mL, were used for EOs; the values were established based on the data used in previously published studies from the scientific literature. We selected the other three (D1 = 50 µg/mL, D2 = 5 µg/mL, and D3 = 0.5 µg/mL) for standard antibiotics, considering their MIC values on bacterial strains according to CLSI and EUCAST [71].
Regarding the OEOs' chemical composition, it was observed that OEO1 contains all four constituents (carvacrol, p-cymene, γ-terpinene, and thymol) in the suitable Ph. Eur. limits ( Figure 5A). OEO2 has a lower carvacrol content, but other metabolites in augmented concentrations than OEO1: thymol content-seven times higher, γ-terpinene-three-pointfive times higher, and p-cymene-two times higher. A similar thymol concentration was quantified by Salehi et al. in OEO from Greece [72]. Moreover, another two compounds, unmentioned in Ph. Eur., were found in the GC-MS report: linalool and β-caryophyllene, in up to 2% concentration. They could contribute to the antibacterial effects due to complex interaction with the other bioactive metabolites [44]. Our results confirm these aspects ( Figure 5B).
The main constituents of OEO are carvacrol and thymol, which have solid pharmacological potential, including antibacterial, anti-inflammatory, and antioxidant activities. At the same time, carvacrol and thymol, obtained by chemical synthesis, could be adulterants of Oregano oil [48]. Gavaric et al. [73] reported the additive antibacterial effect of thymol and carvacrol against tested bacterial strains (S. aureus, E. coli, Salmonella infantis, and Bacillus cereus). p-Cymene is the biological precursor of carvacrol; when used alone, it exhibits a lower antibacterial effect than carvacrol; however, it synergistically acts with carvacrol against E. coli [74] and B. cereus [75]. Furthermore, carvacrol can form a chimera with DNA [76], while thymol reduces enterotoxins A, B, and α-hemolysin secreted by S. aureus isolates [77].
Thus, OEO could be considered a broad-spectrum natural antibiotic [78,79]. Investigating the antibacterial mechanism against MRSA, Cui et al. proved that OEO affects bacterial wall permeability, leading to an irreversible depletion [76]. It can inhibit bacterial respiratory metabolism (perturbing the tricarboxylic acids cycle) and the expression of MRSA's crucial pathogenic factor PVL.
In the present study, both OEOs display similar antibacterial and antibiofilm activities, reporting the highest effects compared to all EOs investigated. Their inhibitory activity against all bacteria tested is similar to Amoxicillin-Clavulanic acid. Our results were similar to those obtained in other studies, which confirm both activities (antibacterial and antibiofilm) on S. aureus [80], E. coli [81], and P. aeruginosa [82,83].
Bachir et al. [99,100], reported the antibacterial efficacy of Eucalypti aetheroleum against S. aureus due to its phytoconstituents (eucalyptol, linalool, β-pinene). Moreover, its bactericidal effect against E. coli and P. aeruginosa was reported [101]. The EEO's bioactive constituent responsible for the antibiofilm activity is eucalyptol [102][103][104]. 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 [105]. Furthermore, eucalyptol obtained by chemical synthesis could be mixed with Eucalyptus oil for adulteration [106].
Our results show that Rosmarini aetheroleum had significant antibacterial and antibiofilm efficacy against P. aeruginosa. The antibacterial activity is similar for all Grampositive and Gram-negative bacteria tested (ABE > 80.00%, MIC > 25 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 [135]. REO1 was slightly more active than REO2, but no significant differences were recorded between the two samples' effects.
Moreover, the synthetic equivalents of their main components, eucalyptol and camphor, could be used for Rosemary oil adulteration [136].
Xu et al. [144] highlighted the antibacterial efficacy of Caryophylli aetheroleum against S. aureus (with a MIC value = 0.625 mg/mL). They hypothesized that the volatile oil destroys the cell wall and membranes, causing loss of vital intracellular materials, resulting in bacterial death. The volatile oil also penetrates the cytoplasmic membrane and inhibits the normal synthesis of DNA and proteins necessary for bacterial growth. Yadav et al. [145] 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 [42].
The MIC value in the present study for all bacteria tested is >25 mg/mL. Generally, the MIC values of CEO against S. aureus vary in the range of 0.52-1.04 mg/mL [146].
Burt et al. [147] 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 [146]. Another study by Kim et al. [148] 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 [149], with a MIC of 4.9 mg/mL [150]. Moreover, the antibiofilm activity of Clove oil is due to its main bioactive compounds, eugenol and eugenyl acetate [151].
The present study reports a few differences between the three CEO samples. Thus, CEO1 showed substantial antibacterial and antibiofilm efficacy against all Grampositive 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, in Peppermint oil, the association of menthol, menthone, limonene, neomenthol, carvone, and eucalyptol with other minor constituents appears to induce a synergistic antibacterial activity. A recent study [153] 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. [154] 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 [155]. All tested samples of Peppermint oil showed appreciable anti-staphylococcal efficacy. Kang et al. [156] showed that PEO inhibits the biofilm of S. aureus by altering the permeability and integrity of bacterial cell membranes. Peppermint oil significantly inhibits biofilm formation and inactivates the mature biofilm [157].
Alamoti et al. [158] proved the antibacterial efficacy of Menthae aetheroleum against E. coli due to pulegone content.
All four PEO samples investigated in the present study had remarkable antibacterial effects against Gram-positive and Gram-negative bacteria, with no significant differences (MIC > 25 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 [153].
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%). Some significant observations are available to corroborate all obtained results and compare them with the literature data. As an overview, the samples with different manufacturers of the same essential oil showed similar activities; only Clove and Peppermint oils showed higher differences. Compared to other studies' results, the low differences between EEO and REO samples appear not to influence the antibacterial effects. Oregano oils showed a substantially lower antibacterial activity against S. aureus and P. aeruginosa; the MIC values (>25 mg/mL) are significantly higher than those mentioned in literature data: 0.16-0.32 mg/mL against MRSA and 0.16-0.64 mg/mL against P. aeruginosa. The same observation is available for Clove oils against S. aureus and E. coli; the MIC values from literature data are substantially lower (0.52-1.04 mg/mL, respectively, 0.64-1.28 mg/mL) than those obtained in the present study. Moreover, appreciable differences were recorded in the chemical composition of CEO samples. Notable differences in bioactive constituents' content were registered in PEOs samples, resulting in high antibacterial and antibiofilm effects variations. However, the literature data indicates large ranges of MIC variation against all bacteria tested.
All these differences could be explained by the significant variation of EOs' chemical composition, bacterial strains selected, and technical aspects implied in microbiological assays used.
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 [163,164]. Sanimed International Impex SRL (Calugareni, Romania) was the Muller-Hinton culture media supplier.

Antibacterial Activity
The current method was adapted from [165,166]. It involved the cultivation of bacteria in 96-well microplates with Muller-Hinton medium with EOs samples and incubation at 37 • C for 24 h.

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 10 8 CFU/mL (CFU = colony-forming unit).

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 [167].
Each emulsion was diluted with double distilled water to achieve the final concentration of each EO stock solution (25 mg/mL).

Standard Antibiotic Solutions Preparation
All antibiotic drug solutions were prepared with double distilled water, the final stock solution concentration being 0.5 mg/mL.

Microdilution 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. [166] and detailed in our recently published study [163]. 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. [163].

Antibiofilm Activity
The method was adapted from [168,169] and detailed in our recently published article [163]. 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 min. The dye incorporated in bacterial cells that formed the biofilm was solubilized with 95% ethanol for 10 min under continuous stirring at 450 rpm.

Quantification and Interpreting of Antibacterial and Antibiofilm Activities
The antibacterial and antibiofilm effect of essential oils was determined by reading the absorbances separately on each experimental variant using the EnSight Multimode Plate Reader. Depending on the measurement needs, the operator selected and modified the wavelengths (562 nm for antibacterial effect and 570 nm for antibiofilm activity). The final absorbance value is the arithmetic mean of the instrument software's 100 readings per second/well-made automatically [122].
The calculation formula is presented in the following equation (Equation (1)) and detailed in the Supplementary Material.

Data Analysis
The analyses were performed in triplicate; the results are expressed as a mean ± Standard Deviation (SD).
The statistically significant differences were determined using Anova single factor from Microsoft 365 Excel ® v.2023 (Microsoft Corporation, Redmond, WA, USA). The statistically significant values were marked in Tables 1-3 with superscripts [122].
The correlations between variable parameters [169] were examined through principal component analysis [170] performed with XLSTAT 2023.1.4. by Lumivero (Denver, CO, USA) using Pearson correlation.
The statistical significance was established at p < 0.05 [170].

Conclusions
All essential oils exhibited antibacterial and antibiofilm activities on the first decimal dilution against all Gram-positive and Gram-negative bacteria tested, and MIC value > 25 mg/mL. The obtained results could be explained by the significant variation of EOs chemical composition, bacterial strains selected, and technical aspects implied in microbiological assays used.
Generally, both effects significantly decreased proportionally with serial dilutions when the concentration of the bioactive compounds recorded a progressive diminution. Only EOs with a considerable content of highly active metabolites revealed insignificant differences. E. coli showed the lowest susceptibility to all commercially available essential oils-15 EO samples had undetected antibacterial and antibiofilm effects at the following two dilutions. Only EOs with a considerable content of highly active metabolites revealed insignificant differences at all decimal dilutions. The essential oils with many bioactive compounds in moderate contents recorded a substantial diminution of antibacterial potential.
Samples with different provenance of the same essential oil showed similar activities; thus, both OEOs and CEO1, EEOs and REOs, CEO2 and CEO3, PEO1 and PEO2, and PEO3 and PEO4 acted similarly. Clove and Peppermint oils showed higher variations due to the bioactive compounds' different contents. The most substantial differences in bioactive constituents' contents were registered in PEO samples, leading to high antibacterial and antibiofilm effects variations.
All these differences could be due to different places for harvesting the raw plant material, various technological processes through which these essential oils were obtained, the preservation conditions, and complex interactions between constituents.
Further research will quantify the bioactive constituents of each EO sample, extending, at the same time, the therapeutical properties investigation.