Chemical Composition, Antibacterial, and Antioxidant Activities of L. angustifolia Essential Oil Against Human Pathogenic Clinical Bacterial Isolates

Abstract

L. angustifolia is a perennial shrub native to the Mediterranean region with multiple medicinal properties. In this study, we report on the chemical composition of L. angustifolia essential oil (LEO), its antibacterial, antibiofilm, and antioxidant activities against ten clinical isolates. Chemical constituents of LEO were identified using Gas Chromatography-Mass Spectrometry (GC–MS). Its antibacterial activity was evaluated in vitro against Gram-positive and Gram-negative bacteria using disk diffusion and broth microdilution methods. A growth inhibition assay was performed to determine the bacterial growth spectrophotometrically. The antibiofilm activity was assessed using a Crystal Violet assay. Finally, the activities of oxidative stress indicators, including Superoxide dismutase (SOD) and Catalase (CAT), were evaluated. GC–MS findings of the essential oil revealed the predominance of Linalool as the major compound. Antimicrobial tests demonstrated activity against Acetobacter aceti, Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Methicillin-resistant Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumonia, Staphylococcus aureus, Staphylococcus haemolyticus and Stenotrophomonas maltophilia. Furthermore, LEO modulated bacterial growth over time, inhibited biofilm formation and eradicated pre-formed ones. Additionally, LEO significantly decreased the activities of the antioxidant enzymes SOD and CAT. Our findings demonstrated the therapeutic potential of LEO against pathogenic strains and broad antibacterial efficacy.

1. Introduction

Although the discovery of antibiotics, “the magic bullet”, saved millions of lives from bacterial infections, their misuse has led to the emergence of antimicrobial resistance. The World Health Organization (WHO) has classified antimicrobial resistance as one of the three major public health threats [1]. The urgent need to address resistant bacteria has encouraged scientists to search for new antimicrobial agents with potent activity and minimal side effects [2].

In traditional medicine, plants have been used to treat serious diseases. Today, the increased interest in herbal medicine is attributed to its availability, efficiency, and low cost [3]. According to the WHO, approximately 80% of the world’s population relies on herbal remedies for medical care [4]. Their therapeutic efficacy is due to the presence of primary metabolites (PMs) involved in growth and cellular functions and secondary metabolites (SMs), including tannins, terpenoids, alkaloids, and flavonoids that protect the plant against pathogenic infections [4,5,6]. SMs are responsible for antimicrobial, antioxidative, anti-inflammatory, and antiviral activities [5].

The Lavandula genus, comprising 39 species, is a well-known medicinal plant belonging to the plant family Lamiaceae that is rich in essential oils [6]. Different extracts of Lavandula species were used worldwide in traditional medicine for treating common ailments [7]. L. angustifolia exhibits several biological activities, including antifungal, antibacterial, anti-inflammatory, antiseptic, analgesic and cytotoxic properties [8,9].

The biological activities of L. angustifolia are attributed to its chemical profile. Recent studies have expanded the antibacterial activity of its extracts and essential oils. Several bacteria (E. coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella enteritidis, Yersinia enterocolitica) and fungi (Candida albicans, Candida glabrata, Candida tropicalis, Cryptococcus neoformans, Saprochaete capitate) were inhibited by LEO cultivated in Croatia [10]. The antibacterial potential of LEO against Staphylococcus species in a hospital environment was also investigated [11]. In combination with conventional antibiotics, LEO demonstrated synergistic activity against multidrug-resistant E. coli [9]. Another study highlighted the potential of LEO combined with meropenem to reduce the growth of carbapenemase-producing K. pneumoniae [12]. The antibacterial activity of LEO is driven by its ability to destabilize bacterial cell membranes, increase reactive oxygen species (ROS), elevate lipid peroxidation, and alter protein expression [13,14].

In this context, this study aims to investigate the chemical composition of LEO harvested in Lebanon and to evaluate its antibacterial and antioxidant activities against pathogenic bacteria.

2. Materials and Methods

2.1. Chemicals

The following chemicals and reagents were purchased as indicated: (a) Sigma-Aldrich (St. Louis, MO, USA): bovine serum albumin, phosphoric acid (85%), anhydrous sodium phosphate dibasic (Na₂HPO₄), and sodium phosphate monobasic (NaH₂PO₄); (b) Fisher Scientific (Ottawa, ON, Canada): Coomassie brilliant blueG-250, dimethyl sulfoxide (DMSO), hydrogen peroxide (H₂O₂), potassium iodide, and Tris base; (c) HiMedia (Mumbai, India): Mueller-Hinton agar and tryptic soy agar (TSA); (d) Super Chem (Kishangarh, India): crystal violet; (e) Oxoid (Basingstoke, UK): nutrient broth; (f) Amresco (Solon, OH, USA): riboflavin; and (g) Roche Diagnostics (Rotkreuz, Switzerland): phenylmethylsulfonyl fluoride (PMSF).

2.2. Essential Oil

LEO extracted by hydro-distillation of lavender buds and stems was purchased from Lavender Mine, located in Al-Shouf Biosphere Reserve in the Barouk Mountain, Lebanon (longitude 33.7430° N East, and latitude 35.7269° North).

2.3. Essential Oil Analysis

The chemical analysis of LEO was conducted using GC-MS equipped with an Agilent DB-5MS column (30 m × 0.25 mm, film thickness of 0.2 μm). Helium was used as carrier gas at a flow rate of 1 mL/min. Samples were run in dichloromethane with a dilution ratio of 1:100. A 1 µL sample was injected in split mode (split ratio: 1/50). Oven temperature was programmed as follows: 40 °C (held for 3 min), increased at a rate of 5 °C/min to 100 °C, then increased to 200 °C at 8 °C/min. MS scan conditions were a transfer line interface temperature of 270 °C and an ion source temperature of 230 °C. Analytes were identified using the LabSolutions library (GCMS solution Version 4.50 SP1) with a similarity index greater than 90%. In the absence of experimental reference standards and n-alkane series for retention index (RI) calculations, component identities were further validated by cross-referencing their mass spectra and relative elution sequences with published Kovats indices from the NIST Chemistry WebBook and established phytochemical profiles for Lavandula angustifolia (ISO 3515) [15]. This dual-verification approach ensured the biological and chemical plausibility of the identified volatile constituents.

2.4. Antibacterial Activity

2.4.1. Target Organisms

The antibacterial effect of LEO was tested against the following Gram-positive bacterial strains: Enterococcus faecium, Methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus haemolyticus; and the Gram-negative bacteria: Acetobacter aceti, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and Stenotrophomonas maltophilia. These clinical bacterial isolates were obtained from Mount Lebanon Hospital, Lebanon. All tested bacterial strains were maintained on agar Petri dishes at 4 °C and sub-cultured monthly. Inoculates were prepared with fresh cultures by suspending the bacteria in a suitable medium and adjusting the density to 0.5 McFarland standard (1.5 × 10⁸ CFU/mL).

2.4.2. Agar Disk Diffusion Test

A standard agar disk diffusion method was performed for the antibacterial testing. The methodology was based on EUCAST with minor modifications [16].

Fresh bacterial suspensions were prepared and standardized to 0.5 McFarland turbidity. The essential oil was diluted with 1% DMSO to achieve final concentrations of 12.5%, 25% and 50%.

100 μL of the standardized inoculum was pipetted onto Petri dishes containing Mueller-Hinton agar spread evenly using a sterile swab. A sterile filter paper disk (6 mm diameter) was filled with 20 μL of LEO component solution at the specified concentration and applied to the plates (6 mm) that were incubated at 37 °C for 24 h. Finally, the diameters of the zones of inhibition were measured. The tests were performed in triplicate and included a growth, vehicle, and positive control.

Commercial antibiotic disks were used as positive controls: ciprofloxacin (5 µg) for the evaluation of antibacterial activity of Escherichia coli, doxycycline (30 µg) for Klebsiella pneumoniae and Acinetobacter baumannii, Tigecycline (15 µg) for Enterococcus faecium, Stenotrophomonas maltophilia and Proteus vulgaris, vancomycin (30 µg) for Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, and Staphylococcus haemolyticus.

2.4.3. Determination of Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) of LEO was determined using the micro broth dilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines with minor modifications [17]. The assay was performed in sterile, untreated, round-bottom 96-well microtiter plates (Corning, NY, USA) to follow consistent experimental conditions. To ensure a standardized inoculum, bacterial suspensions were prepared by suspending fresh colonies in nutrient broth, and the turbidity was adjusted spectrophotometrically to match a 0.5 McFarland standard (~1.5 × 10⁸ CFU/mL). Then, a plate of 5 µL of bacterial suspension was added to 95 µL of nutrient broth in a 96-well microtiter, resulting in an intermediate concentration of 7.5 × 10⁶ CFU/mL. Subsequently, 100 µL of LEO, serially diluted in 1% DMSO as schematically illustrated in Figure 1, was added to reach a final bacterial concentration of 3.75 × 10⁶ CFU/mL. This specific concentration, while higher than the standard CLSI recommendation, was utilized to ensure a robust and reproducible response against the volatile and hydrophobic nature of LEO components, ensuring that the reported MIC values reflect a high degree of potency against a dense bacterial population.

Plates were incubated at 37 °C for 24 h, and MIC was defined as the lowest concentration of LEO that showed no visible bacterial growth, evidenced by an OD value significantly lower than the positive control (endpoint criterion). Wells containing only nutrient broth served as a negative control, while wells containing 1% DMSO without LEO served as a vehicle control.

To determine the minimum bactericidal concentration (MBC), 10 µL from each well of the MIC assay was transferred onto Tryptic Soy Agar (TSA) plates and incubated at 37 °C for 24 h. MBC is defined as the lowest concentration of LEO that resulted in a reduction of bacterial growth by ≥99.9%. All assays were performed in technical triplicate and repeated across three independent biological replicates to ensure reproducibility.

2.5. Growth Inhibition Assay

Bacterial proliferation was monitored with the treatment of LEO at different concentrations (0.5× MIC, 1× MIC, and 2× MIC) for 24 h with respect to control groups, as previously described [18]. In each well of a 96-well plate, 90 µL of nutrient broth, 10 µL of bacterial suspension (adjusted to 0.5 McFarland standard), and 100 µL of LEO were added. The plate was then incubated at 37 °C, and bacterial growth was monitored by measuring the optical density at 600 nm using a microplate reader at designated time points: 0, 1, 3, 6, 18, and 24 h. To eliminate background interference from LEO turbidity, blank controls containing nutrient broth and LEO were used. Additionally, a negative control comprising the bacterial inoculum for every strain was included. The OD₆₀₀ values of these blanks were subtracted from the experimental readings at each time point. All assays were performed in triplicate across three independent replicates.

2.6. Determination of Antibiofilm Activity

2.6.1. Inhibition of Biofilm Formation

Inhibition of biofilm formation was assessed in 96-well microtiter plates, following a previously described method by Ben Abdallah et al., with minor modifications [19]. Briefly, 100 μL of LEO at different concentrations (0.5× MIC, 1× MIC, and 2× MIC) was mixed with bacterial suspension (100 μL) in nutrient broth (adjusted to 0.5 McFarland standard) in each well. Plates were incubated at 37 °C for 24 h. Following incubation, wells were rinsed several times with distilled water to ensure the removal of planktonic cells, air-dried for 10 min, and incubated at 60 °C for 45 min. A 1% (w/v) aqueous solution of crystal violet (100 μL) was added to each well and incubated for another 15 min at room temperature. After staining, the wells were rinsed thoroughly to remove unbound dye. To solubilize the bound dye, 100 μL of 95% ethanol was added to each well. The absorbance was measured at 600 nm using a microplate reader.

The percentage of biofilm inhibition was calculated using the following equation:

% Inhibition = [(OD control − OD treated)/OD control] × 100

2.6.2. Eradication of Pre-Formed Biofilms

To evaluate the ability of LEO to eradicate pre-formed biofilms, the method described by Ben Abdallah et al. was followed, with minor modifications [19]. 100 μL of bacterial suspension (adjusted to 0.5 McFarland standard) in nutrient broth was added to each well of a 96-well round-bottom microtiter plate. Plates were incubated at 37 °C for 24 h to allow biofilm formation. After incubation, planktonic cells were gently removed, and 100 μL of LEO (at 0.5× MIC, 1× MIC, and 2× MIC concentrations) was added to each well, followed by another incubation at 37 °C for 24 h. Biofilm was quantified using the crystal violet staining method as described previously. This assay was carried out in three independent experiments.

2.7. Determination of Antioxidant Activities

Following tissue homogenization, the total protein concentration was quantified using the Bradford assay [20]. The cellular levels of SOD and CAT were determined according to the method described by Buege & Aust [21]. This test was carried out in three independent experiments.

2.8. Membrane Integrity

Bacterial membrane integrity was assessed spectrophotometrically, as previously described, with some modifications [22]. Bacterial cultures (adjusted to 0.5 McFarland standard) were treated with LEO at their corresponding MIC for 24 h at 37 °C. After incubation, treated bacterial cultures were pelleted by centrifugation (6000 rpm, for 10 min), and the supernatant was filtered through a 0.22 μm filter. Membrane leakage was quantified by measuring the optical density at 260 and 280 nm. This test was carried out in three independent experiments.

2.9. Statistical Analysis

To ensure reproducibility, all assays were conducted as three independent biological replicates, with each measurement performed in technical triplicates. Statistical analyses were carried out using SPSS version 25. All data were presented as mean ± standard deviation (SD). In addition, one-way Analysis of Variance (ANOVA) was performed to determine the significant difference between treatment groups and the control group; p < 0.05 was considered statistically significant.

3. Results

3.1. GC-MS Analysis

GC-MS analysis of LEO identified 46 different compounds listed in

Table 1. Chemical composition (%) of Lavandula angustifolia essential oil.

No.	Compound Name	RT	%	No.	Compound Name	RT	%
1	α-cis-bergamotene	23.36	0.06	24	Juniene	11.18	0.06
2	(E)-α-Bergamotene	22.99	0.04	25	(R)-Lavandulol	20.26	0.8
3	Borneol	17.65	4.64	26	(S)-Lavandulol	17.39	1.23
4	Bornyl formate	19.08	0.05	27	D-Limonene	13.15	0.96
5	Butanoic acid	19.19	0.53	28	Linalool	15.6	50.79
6	Butyric acid	12	0.07	29	Linalyl acetate	19.52	4.3
7	Camphene	10.37	0.28	30	β-myrcene	7.69	0.24
8	Camphor	16.95	11.5	31	(Z)-β-ocimene	13.74	0.16
9	3-Carene	12.42	0.06	32	3-Octanol	12.03	0.42
10	Caryophyllene oxide	26.06	0.06	33	3-Octanone	11.63	0.16
11	Cuminaldehyde	19.33	0.2	34	1-octen-3-ol	11.46	0.17
12	m-Cymene	12.8	0.007	35	1-Octen-3-yl-acetate	15.66	0.24
13	o-Cymene	12.98	0.53	36	1,2-Oxolinalool	14.55	0.49
14	Episesquithujene	22.48	0.23	37	α-pinene	9.8	0.9
15	Eucalyptol	13.24	3.63	38	β-Pinene	13.39	0.14
16	(E)-β-Farnesene	23.81	2.11	39	α -Pinene dimer	13.39	0.9
17	Furanoid	15.08	0.39	40	α-Phellandrene	12.34	0.01
18	Germacrene D	24.36	0.18	41	α-Santalene	23.11	0.1
19	1-Hexanol	7.69	0.06	42	γ-Terpinene	14.14	0.1
20	Hexyl acetate	12.56	0.25	43	4-Terpineol	17.88	9.9
21	n-Hexyl butyrate	18.11	1.96	44	α-Terpinolene	12.72	0.02
22	Hexyl hexanoate	22.37	0.23	45	α-Terpinyl propionate	12.03	0.27
23	Isocaryophyllene	23.19	0.35	46	α-Thujene	9.57	0.11

RT: Retention time in minutes.

. Our results showed the predominance of Linalool (50.79%) as the main compound, followed by Camphor (11.59%), Terpineol (9.9%), Borneol (4.64%), linalyl acetate (4.3%), and eucalyptol (3.63%).

3.2. Antibacterial Activity of L. angustifolia Essential Oil

The antibacterial activity of LEO against both Gram-positive and Gram-negative bacterial strains was evaluated by determining the zones of inhibition and minimum inhibitory and minimum bactericidal concentrations. LEO at varying concentrations demonstrated potent antibacterial effects against all tested strains in a dose-dependent manner. Inhibition zones in Gram-positive strains ranged from 13 ± 3.61 mm to 32.5 ± 3.54 mm, indicating a high sensitivity to LEO as shown in

Table 2. Inhibition zones diameters (mm) by different concentrations of LEO against (a) Gram-positive bacteria and (b) Gram-negative bacteria. Each value is the average of three trials ± standard deviation.

(a)
Gram-positive bacteria	Zone of inhibition (mm)
	25%	50%	100%	Positive control
E. faecium	13 ± 3.61	29.33 ± 5.51	33.33 ± 3.15	35.54 ± 3.41
MRSA	17.5 ± 3.54	25.42 ± 4.25	-	21.00 ± 1.73
S. aureus	10 ± 1.15	23.33 ± 2.89	28.33 ± 2.89	21.67 ± 2.89
S. haemolyticus	15 ± 1.23	22.33 ± 2.52	-	12.00 ± 2.02
(b)
Gram-negative bacteria	Zone of inhibition (mm)
	25%	50%	100%	Positive control
A. aceti	14 ± 1.45	22.67 ± 2.52	32.5 ± 3.54	Resistant to antibiotics
A. baumannii	13.02 ± 1.41	18.33 ± 1.52	-	10.03 ± 1.05
E. coli	NA	NA	NA	37.667 ± 3.21
P. vulgaris	NA	NA	NA	37.67 ± 2.08
K. pneumoniae	NA	15.67 ± 1.15	21.67 ± 0.58	18 ± 1.73
S. maltophilia	24.67 ± 1.53	29 ± 3.61	NA	12.33 ± 0.58

NA: No activity; (-): Large zones to be measured

(a). Similarly, LEO exhibited antibacterial activity against Gram-negative strains, with inhibition zones ranging from 13.02 ± 1.41 mm to 32.5 ± 3.54 mm, which are presented in Table 2(b).

MIC and MBC values of LEO were determined using the broth dilution method for each of the 10 bacterial strains. As shown in

Table 3. MIC and MBC for antibacterial activities of LEO against (a) Gram-positive strains and (b) Gram-negative strains. Values represent the specific concentration (% v/v) at which visible growth was completely inhibited. All experiments were performed in triplicate; the values shown represent the consistent endpoint reached across all three independent replicates. MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration.

(a)
Gram-positive bacteria	MIC % (v/v)	MBC % (v/v)	MBC/MIC
E. faecium	3.12	6.25	2
MRSA	1.56	12.5	8
S. aureus	1.56	3.12	2
S. haemolyticus	3.12	3.12	1
(b)
Gram-negative bacteria	MIC % (v/v)	MBC % (v/v)	MBC/MIC
A. aceti	3.12	3.12	1
A. baumannii	3.12	3.12	1
E. coli	3.12	25	8
P. vulgaris	1.56	3.12	2
K. pneumoniae	3.12	3.12	1
S. maltophilia	3.12	3.12	1

, LEO demonstrated significant bacteriostatic and bactericidal effects. MIC values ranged from 0.78% to 6.25%, while MBC values ranged from 3.12% to 25%. The MBC/MIC ratio was used to determine the mode of antibacterial action, where a ratio ≤ 4 indicates bactericidal activity and a ratio > 4 suggests bacteriostatic activity.

3.3. Growth Inhibition Assay of L. angustifolia Essential Oil

Figure 2 demonstrates the growth curves for all bacterial species. The lag phase was extended across the investigated bacteria within 3 to 6 h. Between 6 and 24 h, the rate of increase in absorbance increased in the control groups and at a lower concentration (0.5 MIC) of LEO. However, bacteria treated with higher concentrations (MIC, 2 MIC) exhibited a significant decrease in growth, suggesting the potential of LEO to inhibit bacterial growth.

3.4. Biofilm Inhibition and Eradication Activity

The ability of LEO to inhibit biofilm formation and to eradicate the preformed ones is shown in Figure 3 and Figure 4, respectively. The greatest inhibition of biofilm formation was observed against MRSA (71.3%) and K. pneumoniae (68.6%), and the lowest inhibition was observed on biofilm formation of S. haemolyticus (27.0%). For the dissolution of mature biofilms, S. aureus was the most sensitive (61.7%), and biofilms of E. coli recorded the lowest sensitivity (13.7%).

3.5. Effect of L. angustifolia on Bacterial Antioxidant Enzymes

To evaluate the effect of LEO on oxidative stress, the activities of SOD and CAT were determined. As illustrated in Figure 5, a decrease in SOD activity was observed across the tested strains. S. aureus was the most sensitive, exhibiting a decrease of 4.1-fold. Among Gram-negative bacteria, S. maltophilia showed the highest sensitivity, with a 3.8-fold reduction in SOD activity.

Figure 6 presents the decrease in CAT activity of all the tested bacteria except for K. pneumoniae. The most sensitive Gram-positive bacteria were S. aureus and MRSA, showing a 4.2-fold decrease in enzyme activity. Similarly, P. vulgaris and S. maltophilia were the most sensitive in Gram-negative strains, exhibiting a decrease of 2.95- fold.

3.6. Effect of LEO on Membrane Integrity

Figure 7 represents the leakage of nucleic acid and protein determined by OD₂₆₀ and OD₂₈₀, respectively. Following LEO treatment, the absorbance values of all treated groups were significantly higher than those of the control groups. The most substantial increase in OD₂₆₀ was recorded for S. haemolyticus (2.9-fold), A. baumannii, and E. coli (both 2.8-fold). Similarly, the increase in OD₂₈₀ was the highest among E. faecium (2.3-fold) and A. aceti (2.2-fold).

4. Discussion

Natural remedies have been used from ancient times to cure severe illnesses and relieve physical symptoms. Plants have been widely used and investigated for their anti-pathogenic activities against a wide variety of bacteria [23]. In the present study, we investigated the chemical composition of Lebanese LEO. The bioactivity of this essential oil was examined through antibacterial screening, growth inhibition, antibiofilm, and antioxidant enzyme activities.

Results of LEO chemical composition revealed a similar composition to other Mediterranean L. angustifolia species. GC-MS analysis of Lebanese Lavender essential oil species grown at different geographic regions revealed the dominance of acyclic monoterpene linalool [24]. Terpenoids show anti-virulence strategies, which include activity against toxin production, bacterial surfactants, biofilm formation, and quorum sensing [25]. In particular, linalool exhibited antibacterial activity against various pathogens, including Staphylococcus, Salmonella, Pseudomonas, Campylobacter, Porphyromonas, and Fusobacterium [26]. Additionally, linalool disrupts bacterial energy pathways and induces metabolic dysfunction. By inhibiting essential enzymes including succinate dehydrogenase, pyruvate kinase, ATPase, and respiratory change dehydrogenases, the membrane integrity of Shewanella putrefaciens was permeabilized, resulting in growth inhibition [27].

Interestingly, our results show that LEO exhibited antibacterial activity based on strain specificity rather than Gram classification. Although Gram-negative strains exhibit higher resistance due to the presence of the outer membrane, our results indicate that these strains were highly susceptible to LEO, with MIC values of 1.5 and 3.12%. The presence of a wide variety of molecules in LEO explains that its antibacterial activity cannot be attributed to a single mechanism. Instead, essential oils exert their activity through distinct biochemical and structural pathways. Substantial chemical alterations of the plasma membrane, cytoplasm, and essential macromolecules can induce a total loss in cellular integrity. Additionally, exposure to EO triggers a sustained ion loss, which alters microbial metabolism and leads to cell death [14]. Our findings are in agreement with a previous study that demonstrated the activity of LEO against novel pathogenic bacteria, including E. coli, K. pneumoniae and S. aureus [10]. Another in vitro study proved that LEO exhibited antibacterial activity against Shigella flexneri, S. aureus, and E. coli with zones of inhibition ranging from 10.06 ± 0.16 to 20.35 ± 0.20 [28]. LEO also showed protective results against antibiotic misuse and the development of bacterial resistance. MRSA is an organism of concern that usually leads to serious clinical infections [29]. Aligned with our findings, the essential oil and hydrosol of L. angustifolia significantly recorded antibacterial activity against MRSA [30]. Similarly, it was reported that LEO had antibacterial activity against S. aureus, S. epidermidis, Enterococcus faecalis, E. coli, and Serratia marcescens [24]. Moreover, LEO exhibited antibacterial activity against E. coli, S. aureus, P. aeruginosa, and B. cereus, along with antifungal activity against C. albicans.” [31].

Biofilm is a natural strategy adopted by bacteria to provide protection against various stressors. Currently, the increase in the misuse of antibiotics leads to drug resistance and tolerance against various biofilm communities [32]. Therefore, the formation of biofilm in organ systems or on medical devices represents a serious health threat [33]. K. pneumoniae is a Gram-negative pathogen that is able to form biofilms on medical devices and on host tissues, leading to a variety of infections, including pneumonia, urinary tract infections and other health issues [34]. In the present study, LEO inhibited biofilm formation of all the tested bacteria, including K. pneumoniae. To our knowledge, this is the first study that demonstrates the antibiofilm activity of the examined essential oil against K. pneumoniae.

Additionally, we observed alterations in SOD and CAT activities coupled with a significant increase in membrane leakage, suggesting that LEO may disrupt bacterial homeostasis. It was found that LEO increased oxidative stress in K. pneumoniae, leading to outer membrane oxidation and ROS efflux, causing cellular damage and death [23]. Another study revealed that LEO reversed E. coli resistance to piperacillin by altering outer membrane integrity and inhibiting quorum sensing [24]. Taken together, these results suggest that LEO induces membrane disruption associated with a decline in the bacterial antioxidant defense system.

While the results of this study provide significant insights into the antibacterial potential of LEO, certain limitations must be acknowledged. First, the current study relies on clinical isolates without the inclusion of ATCC reference strains. Although ATCC strains provide an authenticated and fully characterized baseline that ensures reproducibility, they may not represent the phenotypic profiles and real clinical challenges. Future research should include both ATCC and clinical strains to further validate these findings against globally recognized benchmarks and to explore the efficacy of LEO across broader genetic and resistant pathogens. Second, this study focused on the enzymatic activities of SOD and CAT rather than their quantification. Further investigations employing quantitative techniques would be beneficial to determine whether the observed changes in activity correlate with altered protein levels.

5. Conclusions

In summary, the chemical profile of Lebanese L. angustifolia is defined by high concentrations of Linalool, Camphor, and Terpineol. This study confirms the antibacterial activity of LEO against a diverse range of clinical isolates, supported by the obtained results of antibacterial screening, growth inhibition assay, antibiofilm assays, and antioxidant enzyme activities. Such findings contribute to the development of natural therapeutic alternatives, serving as a foundation for future studies against antibiotic resistance.