Chemical Composition, Antioxidant, Antibacterial, and Antibiofilm Activities of Backhousia citriodora Essential Oil

The essential oil of Backhousia citriodora, commonly known as lemon myrtle oil, possesses various beneficial properties due to its richness in bioactive compounds. This study aimed to characterize the chemical profile of the essential oil isolated from leaves of Backhousia citriodora (BCEO) and its biological properties, including antioxidant, antibacterial, and antibiofilm activities. Using gas chromatography–mass spectrometry, 21 compounds were identified in BCEO, representing 98.50% of the total oil content. The isomers of citral, geranial (52.13%), and neral (37.65%) were detected as the main constituents. The evaluation of DPPH radical scavenging activity and ferric reducing antioxidant power showed that BCEO exhibited strong antioxidant activity at IC50 of 42.57 μg/mL and EC50 of 20.03 μg/mL, respectively. The antibacterial activity results showed that BCEO exhibited stronger antibacterial activity against Gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis) than against Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae). For the agar disk diffusion method, S. epidermidis was the most sensitive to BCEO with an inhibition zone diameter of 50.17 mm, followed by S. aureus (31.13 mm), E. coli (20.33 mm), and K. pneumoniae (12.67 mm). The results from the microdilution method showed that BCEO exhibited the highest activity against S. epidermidis and S. aureus, with the minimal inhibitory concentration (MIC) value of 6.25 μL/mL. BCEO acts as a potent antibiofilm agent with dual actions, inhibiting (85.10% to 96.44%) and eradicating (70.92% to 90.73%) of the biofilms formed by the four tested bacteria strains, compared with streptomycin (biofilm inhibition, 67.65% to 94.29% and biofilm eradication, 49.97% to 89.73%). This study highlights that BCEO can potentially be a natural antioxidant agent, antibacterial agent, and antibiofilm agent that could be applied in the pharmaceutical and food industries. To the best of the authors’ knowledge, this is the first report, on the antibiofilm activity of BCEO against four common nosocomial pathogens.


Introduction
Antibiotics, which are also called antibacterial or antimicrobial drugs, have significantly improved human health and life expectancy by preventing or treating various and ethanolic leaf extracts of BC have been reported, in several studies, to possess good antioxidant, antimicrobial, and anti-inflammatory activities [33,34]. It has been shown that BCEO is a rich source of citral, which possesses versatile biological activities, such as antimicrobial, antifungal, antiviral, and antioxidant activities [35].
However, the chemical composition of EOs can vary remarkably with extrinsic conditions, such as geographic origin and climate conditions. Additionally, few studies have been carried out on the antibiofilm activity of BC, and only two studies reported on the antibiofilm activity of aqueous and ethanolic leaf extracts of BC against Pseudomonas aeruginosa and Streptococcus mutans [36,37]. Therefore, this study aimed to evaluate the chemical composition and antioxidant activity of BCEO from Malaysia, as well as its antibacterial and antibiofilm activities against four human pathogenic bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Klebsiella pneumoniae. To the best of our knowledge, this is the first report on the antibiofilm activity of BCEO against four common nosocomial pathogens.

Chemical Composition
BCEO was analyzed by GC-MS and presented in Figure 1. The chemical compositions of BCEO are listed in detail in Table 1, along with their retention times (Rt), molecular formulae, compound groups, Kovats indices (KI), and percentages (trace components below 0.1% are not listed). Twenty-one compounds, corresponding to 98.50% of the total oil, were identified and quantified in BCEO, which contains a complex mixture of compounds. GC-FID and GC-MS analysis showed that BCEO obtained from the leaves were separated into four compound classes: oxygenated monoterpenes (91.95%), monoterpene hydrocarbons (0.22%), sesquiterpene hydrocarbons (0.33%), and others (6.00%) ( Table 1). The oxygenated monoterpenes were the most abundant phytocompounds in BCEO, dominated by geranial phytocompounds (52.13%) and neral phytocompounds (37.65%). The monoterpene hydrocarbon in BCEO was dominated by β-Myrcene (0.22%), while the predominant sesquiterpene hydrocarbons were Germacrene B (0.18%) and α-Gurjunene (0.15%) ( Table 1). widely used as ingredients in food flavorings, perfumes, and personal care products in Australia [32]. This Australian native plant has recently received increasing attention in Malaysia, due to its unique flavor, fragrance, and biological properties. Methanolic and ethanolic leaf extracts of BC have been reported, in several studies, to possess good antioxidant, antimicrobial, and anti-inflammatory activities [33,34]. It has been shown that BCEO is a rich source of citral, which possesses versatile biological activities, such as antimicrobial, antifungal, antiviral, and antioxidant activities [35]. However, the chemical composition of EOs can vary remarkably with extrinsic conditions, such as geographic origin and climate conditions. Additionally, few studies have been carried out on the antibiofilm activity of BC, and only two studies reported on the antibiofilm activity of aqueous and ethanolic leaf extracts of BC against Pseudomonas aeruginosa and Streptococcus mutans [36,37]. Therefore, this study aimed to evaluate the chemical composition and antioxidant activity of BCEO from Malaysia, as well as its antibacterial and antibiofilm activities against four human pathogenic bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Klebsiella pneumoniae. To the best of our knowledge, this is the first report on the antibiofilm activity of BCEO against four common nosocomial pathogens.

Antioxidant Activity of BCEO
In this study, BCEO exhibited considerable antioxidant activity, based on both the DPPH method and the FRAP method, compared with that of standard antioxidant ascorbic acid. BCEO had an IC 50 value of 42.57 µg/mL for DPPH radical scavenging activity, while the IC 50 value of ascorbic acid was 7.23 µg/mL. Using the FRAP method, BCEO had an EC 50 value of 20.03 µg/mL, which was approximately 1.49 times the value obtained for ascorbic acid (13.48 µg/mL).

Antibacterial Activity Analyzed by the Disk Diffusion Method
In this study, BCEO had an inhibition effect on the four tested bacteria using the agar disk diffusion method, as shown in Table 2. BCEO exhibited significantly (p < 0.05) higher antibacterial activity, as indicated by the larger inhibition zone, against Gram-positive bacteria (S. aureus, 31.13 ± 0.29 mm; S. epidermidis, 50.17 ± 0.29 mm) than that of gentamicin (S. aureus, 22.30 ± 0.60 mm; S. epidermidis, 24.30 ± 0.60 mm). No significant difference was found between BCEO and gentamicin in the antibacterial activity against E. coli. However, the results revealed that the antibacterial effect of BCEO against K. pneumoniae was significantly (p < 0.05) lower than that of gentamicin. The negative control, 10% DMSO, showed no inhibitory effect against any of the four tested bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of BCEO against the four bacterial strains were determined, as shown in Table 3. It was observed that BCEO exhibited bacteriostatic and bactericidal activities against all four bacterial strains. The lowest MIC of BCEO was found against S. aureus and S. epidermidis (6.25 µL/mL), followed by E. coli and K. pneumoniae (12.5 µL/mL). The MIC values of BCEO against S. aureus and S. epidermidis were lower than those of the standard antibiotic streptomycin. However, the MIC values of E. coli and K. pneumoniae were higher than that of streptomycin. The BCEO concentration of 50 µL/mL was determined as the MBC for all four tested bacteria. Overall, the results indicated that the MBC values of both BCEO and streptomycin against the four pathogenic bacteria strains were higher than their MIC values. Bacterial growth was observed in all assays for both the negative control (10% DMSO) and the growth control, confirming the non-bacterial inhibition of 10% DMSO and the viability of the bacteria strains used, and demonstrating the antibacterial effect of BCEO. However, the biofilm inhibitory ability of BCEO (86.98% to 88.79%) against K. pneumoniae was comparable to that of streptomycin (93.36% to 94.29%) at 0.5×, 1×, and 2×MICs.

Biofilm Eradication Activity
A modified microdilution assay was used to investigate the ability of BCEO in eradicating the established biofilms of S. aureus, S. epidermidis, E. coli, and K. pneumoniae at 0.5×, 1×, and 2× MICs. The effect of BCEO on the eradication of established biofilms of tested bacterial strains at 0.5×, 1×, and 2× MICs, expressed as biofilm eradication (%), are illustrated in Figures 3A, 3B  bacterial strains at 0.5×, 1×, and 2×MICs, expressed as biofilm eradication (%), are illustrated in Figure 3A, Figure 3B and Figure 3C, respectively.

Discussion
In the present study, the chemical composition of BCEO was rich in citral, a mixture of two geometric isomers: an E-isomer, known as citral A or geranial (52.13%), and a Z-isomer, known as citral B or neral (37.65%), which constituted 89.78% of the total composition. Various studies have identified the chemical composition of BCEO from different countries. The BCEO composition results of this study are approximately consistent with those of de Andrade Santiago et al. [38], who reported that the basic compounds were citral (91.19%), made from a combination of the isomeric aldehydes geranial (39.82%) and neral (51.37%).
The method for measuring antioxidant activity is still evolving. In this study, the antioxidant activity of BCEO was determined by two different methods: DPPH and FRAP. Although the expression of antioxidant results has not been standardized, the DPPH and FRAP methods can be used to determine the antioxidant capacity of EOs [45,46]. The DPPH method is one of the most simple, acceptable, and widely used antioxidant methods for evaluating the radical scavenging activity of organic compounds. It has typically been used for plant extracts, EOs, and isolated organic substances [47]. Additionally, DPPH is a stable free radical that is used to simulate the antioxidant activity of chemical constituents of EOs, extracts, and other substances from natural products [48]. The purple DPPH free radical transforms into a yellow stable molecule when the odd electron of the DPPH radical is paired with hydrogen from a free radical scavenging antioxidant. Hence, the degree of DPPH discoloration may indicate the scavenging potential of the antioxidant extract.
The FRAP assay was applied in this study to evaluate the reducing power of EOs. For the FRAP method, the presence of reducing agents in the plant extract leads to the reduction of a colored pale yellow ferric-tripyridyltriazine (Fe 3+ -TPTZ) complex to a blue-colored ferrous tripyridyltriazine (Fe 2+ -TPTZ) [49]. The reducing capacity is related to the degree of hydroxylation and the degree of conjugation of the bonds that are present in the phenolic compounds of EOs and extracts [50]. At low cost, this method was useful for screening the reducing capacities and comparing the efficiencies of different compounds, such as those in EOs [51]. The results of the study showed that BCEO possesses the activity to reduce the free radicals of DPPH and Fe 3+ -TPTZ. The great antioxidant activity of BCEO could be attributed to the effect of its key compounds, geranial and neral (two isomers of citral), as they were the significant oxygenated monoterpenes identified in the current study. Furthermore, minor compounds, such as monoterpene hydrocarbons and sesquiterpene hydrocarbons in BCEO, can enhance antioxidant activity. These chemical compounds of EO might act individually or synergistically as antioxidants. Several studies have indicated that enhanced antioxidant activity was found in EOs which were rich in oxygenated monoterpenes [52,53]. It has been reported that the antioxidant capacity of oxygenated compounds could be ascribed to the free electrons, due to high oxygenation [54,55]. The major compounds, geranial and neral, have been reported in high levels in EO of Cymbopogon citratus (lemongrass) (70% to 85%) [56] and Lippia alba (70.6% to 79.0%) [57], exhibiting strong antioxidant activity [58][59][60]. Recent studies have associated antioxidant activity to geranial and neral by demonstrating that these compounds are active in scavenging ROS [61][62][63]. Furthermore, citral is known to exhibit various medicinal properties, including inhibiting oxidant activity, cyclooxygenase-2 (COX-2) expression, and nuclear factor kappa B (NF-kB) activation [64]. A study carried out by Bouzenna et al. [65] demonstrated the antioxidant effects of citral in rat small intestine epithelial cells (IEC-6 cells), indicating that citral can protect against aspirin-induced oxidative stress. Therefore, BCEO can be a promising source of natural antioxidant with its high level of oxygenated monoterpenes (citral).
In this study, BCEO displayed considerable antibacterial activity against Gram-positive and Gram-negative bacterial isolates. Four bacterial strains, S. aureus, S. epidermidis, E.coli, and K. pneumoniae, were used in this study, as they are common nosocomial pathogens and often form biofilms on medical devices [66]. BCEO showed varying inhibitory activity on the four tested bacterial strains, in the following order: S. epidermidis > S. aureus > E. coli > K. pneumoniae. The high antibacterial activity of BCEO against S. aureus is strongly correlated with the results of Wilkinson et al. [67], which demonstrated a significant antibacterial effect of two different BCEO samples against S. aureus with inhibition zones ranging from 11.50 mm to 32.00 mm. Wilkinson et al. also stated that the two BCEO samples exhibited antibacterial activity against E. coli in the range of 8.00 mm to 16.50 mm. These inhibition zones of BCEO against E. coli were lower than those of this study.
Another previous study investigated 91 essential oils and found that BCEO exhibited the largest inhibition zone (65.00 mm) against methicillin-resistant Staphylococcus aureus (MRSA), compared with the other EOs that were tested [68]. Recently, da Silva Júnior et al. [69] found that a citral-chemotype from Lippia alba EO displayed strong antibacterial activity against S. epidermidis with an inhibition zone of >40 mm and MIC at 2.50 µL/mL. A MIC study carried out by Hayes and Markovic [70] in Australia revealed that BCEO and 100% citral displayed compatible activity against S. aureus, MRSA, E. coli, K. pneumoniae, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger in the range of 0.05% to 2.00% v/v. Their study observed that S. aureus was the only organism that showed greater susceptibility to citral. They also showed that the MIC results of BCEO in their study were considerably lower than those of tea tree oil against all of the microorganisms that were tested. Similarly, a recent study indicated that BCEO exerted significant antimicrobial activity against two foodborne pathogenic bacteria, S. aureus DSM 1104 and E. coli DSM 1103, with MIC values of 50 µg/mL and 200 µg/mL, respectively [71]. Recently, Beikzadeh et al. [72] revealed that BCEO exhibited high antimicrobial activity in inhibiting the growth of pathogenic and spoilage bacteria at MICs ≤ 0.125 and 0.06 µL/mL for E. coli and S. aureus, respectively. However, their findings were lower than the MICs found in this study, where BCEO showed strong antimicrobial activity at MICs ≤ 12.5 and 6.25 µL/mL for E. coli and S. aureus, respectively. These variations may be attributed to several factors, such as extraction methods, time, temperature, plant parts, and agroclimatic conditions. Furthermore, although the types of expression of MIC values are variable in the studies, all authors identified great inhibitory activity of BCEO, which corresponds with our findings.
Previous studies showed that the antibacterial activities of EOs from different plant species were mainly associated with their chemical compositions, especially oxygenated monoterpenes, which usually occur in high amounts [73][74][75]. In the present study, the results obtained for antibacterial activity correspond with the great antioxidant activity of BCEO. Based on this observation, it can be speculated that the strong antibacterial activity exerted by BCEO can be attributed to the high amounts of oxygenated monoterpenes, especially geranial and neral. Citral, or one of its isomers (geranial or neral), has been reported to be a molecule that inhibits the growth of several pathogenic and food-spoilage bacteria, such as S. aureus, E. coli, Salmonella enterica, and Bacillus cereus [76,77]. Chueca et al. [78] demonstrated that oxygenated monoterpenes of citral (a mixture of geranial and neral) exhibited antimicrobial activity by causing oxidative stress in E. coli. Similarly, a recent study highlighting the antimicrobial activity of BCEO corresponded directly to the high citral content, which was an isomeric mixture of neral (Z-isomer) and geranial (E-isomer) [79]. Meanwhile, the contribution of other minor chemical components in BCEO in intensifying antimicrobial activity, such as β-myrcene and linalool, in combination with citral, has also been reported [80][81][82]. Several studies have revealed that the antimicrobial activity of oxygenated monoterpenes acts by disrupting the microbial cytoplasmic wall, which improves cell permeability and allows the passage of large protons and ions, leading to cell death [83,84]. Shi et al. [85] confirmed that citral exhibited antimicrobial activity against Cronobacter sakazakii, an opportunistic Gram-negative bacterium, by causing changes in ATP concentration, cell membrane hyperpolarisation, and a reduction in the cytoplasmic pH. Recently, Zhang et al. [84] reported that citral could change a cell's shape by destroying the cell wall and the membrane structure, leading to the loss of cytoplasm and the intracellular leakage of protein, nuclei acid, and other cell substances, as well as influencing the pH balance inside and outside the cell. Therefore, the strong antibacterial activity of BCEO, as indicated in this study, could be due to the high amounts of oxygenated monoterpenes of citral, which contains a mixture of geranial and neral.
Microorganisms can attach to surfaces and produce extracellular polysaccharides, resulting in biofilm formation as a microbial survival strategy, especially in adverse conditions. The National Institutes of Health (NIH) estimated that biofilm formation is responsible for 65% of microbial diseases and more than 80% of chronic infections [86]. Biofilms have great significance for public health because of the increased resistance of biofilm-associated microorganisms to antimicrobial agents. Biofilm formation is strongly associated with indwelling or implanted medical devices, such as urinary catheters, endotracheal tubes, enteral feeding tubes, ventilators, orthopaedic implants, and prosthetic joints, causing medical treatments to become increasingly difficult. Therefore, the use of a new natural compound to inhibit or eradicate biofilms is of great importance. In the present study, BCEO has been shown to exhibit dual actions in preventing and eradicating biofilm formation in all of the tested bacterial strains. The biofilm inhibition and eradication abilities of BCEO on the four tested bacterial strains in this study are classified in the following decreasing order: S. epidermidis > S. aureus > E. coli > K. pneumoniae. This observation corresponds with this study's results on antibacterial activity. It was found that BCEO demonstrated significant biofilm inhibition activity (85.10% to 96.44%) and eradication activity (70.92% to 96.44%) against S. aureus, S. epidermidis, and E. coli, compared with those of the standard antibiotic (streptomycin), at 0.5× to 2× MICs. These results agree with those of Duarte et al. [87], who revealed that coriander EO inhibited at least 85% of biofilm formation and eradicated up to 97.14% of 48 h pre-formed biofilms of Acinetobacter baumannii. However, the biofilms of K. pneumoniae appeared to be less sensitive to being inhibited or eradicated by BCEO than by streptomycin in this study. Several recent reports revealed that the bacteria biofilm could be inhibited or eradicated effectively by EOs, including clove basil oil [88], tea tree oil [89], cedar oil [90], and garlic and thyme oil [91]. Recently, Cáceres et al. [92] reported that EO from Lippia origanoides showed detrimental effects against biofilm formation in E. coli O33, E. coli O157:H7, and S. epidermidis ATCC12228, with percentages of biofilm inhibition of 75%, 73%, and 74%, respectively. These biofilm inhibition results were generally lower than our results, which may be due to different plant chemotypes, as L. origanoides EO is rich in thymol-carvacrol, while BCEO is rich in citral. Another recent study found that Origanum majorana, Rosmarinus officinalis and Thymus zygis EOs demonstrated high biofilm inhibition and eradication activities against MRSA clinical isolates, with the percentages of inhibition in the range of 10.20% to 95.91%, and the percentages of eradication ranging from 12.65% to 98.01% [93].
The antibiofilm activity of EOs have been investigated in the past two decades; however, there is limited information about the antibiofilm effects of BCEO on pathogenic bacteria. The biofilm inhibition effects of BCEO found in the present study suggest that the addition of EOs prior to biofilm formation eliminates planktonic cells, rendering the abiotic (polystyrene) surface less susceptible to cell adhesion. Furthermore, the biofilm inhibitory effects of BCEO identified in this study can be explained by the modification of bacterial surface proteins, due to the effect of their interactions with EOs in reducing the adhesion of planktonic cells to surfaces, which is the initial attachment phase [94,95]. Several studies have demonstrated that EOs could penetrate the exopolysaccharide matrix of established biofilms and destabilize them, due to their strong intrinsic antimicrobial actions [95,96]. The significant biofilm inhibition and eradication activities of BCEO can also be explained by the fact that the major constituent in this oil, citral, has an effect on the biofilm formation process.
The biofilm inhibition ability of citral and citral-rich EO from three Lippia alba (LAEO) specimens was demonstrated by Porfírio et al. [97], who found a 100% inhibition of S. aureus biofilm formation at the concentration of 0.5 mg/mL of citral and all three LAEOs (LA1EO-LA3EO). They also reported that the biofilm elimination ability of citral, LA1EO, LA2EO, and LA3EO were confirmed at concentrations of 0.5 mg/mL, 1 mg/mL, 2 mg/mL, and 2 mg/mL, respectively. A recent study reported that citral applied individually or in combination with linalool, eugenol, and thymol, could exert growth inhibition in planktonic cells and a bacteriostatic effect on the biofilm cells of Shigella flexneri [98]. A study on the biofilm inhibition activity of an ethanolic extract of B. citriodora by Almousawi et al. [37] showed that this plant extract was effective in inhibiting ten clinical isolates of P. aeruginosa that were isolated from various burn sites. Gao et al. [96] indicated that EO of lemongrass (Cymbopogon flexuosus) (LGEO) and its major component citral exhibited strong antibiofilm action against mono-and dual-species biofilms formed by S. aureus and Candida species at low concentrations ranging from 0.0156% to 0.1563%. Gao et al. stated that the biofilm biomass and the cell viability of S. aureus and Candida spp. (C. albicans and C. tropicalis) were reduced after exposure to LGEO and citral in a biofilm staining and viability test. The microscopic examinations of these pathogenic bacteria and yeast found that LGEO and citral interfered with the adhesive characteristics of each species and disrupted the exopolysaccharide of biofilm matrix by counteracting carbohydrates, proteins, and nuclei acids in the biofilm. Additionally, the transcriptional analyses revealed that citral reduced the expression of genes involved in quorum sensing, fatty acids, and peptidoglycan biosynthesis in S. aureus, and downregulated the virulence factors and hyphal of C. albicans. In another study, C. citratus EO, citral, and geraniol were found to inhibit the planktonic growth of E. coli O157:H7 (MIC = 2.2 mg/mL, 1.0 mg/mL, and 3.0 mg/mL, respectively), the bacterial adhesion (2.0, 2.0, and 4.0 mg/mL, respectively), and the glucan production on stainless steel surfaces. They suggested that this biofilm inhibition effect could be associated with an uncompetitive inhibition of glucosyltransferase activity that was caused by citral and geraniol [99].
The present study also found that Gram-negative bacteria are more resistant to BCEO than Gram-positive bacteria. These results were correlated with a study conducted by Man et al. [100], who showed that the six studied EOs were more susceptible to Gram-positive cocci, such as MRSA, while being more resistant to Gram-negative bacilli including Pseudomonas aeruginosa. Several recent studies also indicated that Gram-positive bacteria are more sensitive than Gram-negative bacteria to EOs [83,101]. It has been documented that different compositions of the cell wall in Gram-positive and Gram-negative bacteria may be the key contributors to various inhibition zones, MICs, MBCs, and antibiofilm activities. In fact, the cell wall structure of Gram-negative bacteria is more complex than that of Gram-positive bacteria [102,103]. The outer membrane of Gram-negative bacteria containing hydrophilic lipopolysaccharides could create an effective barrier for hydrophobic compounds, such as those found in EOs [104]. The structure of the Gram-positive bacteria cell wall enables hydrophobic molecules to penetrate the cells and act on both the cell wall and the cytoplasm. Thus, the hydrophobic phenolic compounds present in EOs generally exhibit antimicrobial activity against Gram-positive bacteria [102]. It can be postulated that the strong antimicrobial and antibiofilm effect of BCEO against Gram-positive bacteria in this study may be due to the high amount of hydrophobic citral.

Plant Material Collection and Identification
Backhousia citriodora (BC) leaves were obtained from an organic lemon myrtle plantation area in Kuala Linggi, Malacca, Malaysia. The taxonomic identification of BC was confirmed by the botanist from the Biodiversity Unit, Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM), Malaysia. The voucher specimen (MFI 0145/19) was deposited at the Biodiversity Unit, IBS, UPM.

B. citriodora Essential Oil (BCEO) Extraction
Fresh leaves of BC were air-dried for three days and grounded into powder in a grinding machine. BCEO was extracted by hydrodistillation in a Clavenger-type apparatus for 4 h (2 kg of BC leaves in 5 L of distilled water) to obtain the EO. The moisture in the EO was removed by using anhydrous magnesium sulphate (MgSO 4 ). The EO obtained was then transferred to sealed dark vials and stored at 4 • C for subsequent analysis.

Gas Chromatography-Flame Ionization Detection (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) Analyses of the BCEO
The GC-MS for the analyses of the volatile constituents of BCEO was performed on an Agilent 7890A/5975C series gas chromatography-mass spectrometry (GC-MS) system (Agilent Technologies Inc., Santa Clara, CA, USA). The system was equipped with an HP-5MS fused silica capillary column (30 m long × 0.25 mm i.d. × 0.25 µm film thickness, Agilent Technologies, Santa Clara, CA, USA). The ultra-pure helium was used as a carrier gas at the flow rate of 1 mL/min with a spilt ratio of 50:1. The oven temperature was programmed from 60 • C for 10 min to 230 • C at a rate of 3 • C/min for 1 min. The injector port temperature was 250 • C. The mass spectra of the BCEO compounds were acquired by electron ionisation (EI) at 70 eV. Data acquisition was performed in full scan mode, using a spectral range of m/z 50-550. The temperatures of the auxiliary heating zone, the ion source, and the MS quadrupole were 280 • C, 230 • C, and 150 • C, respectively. The GC-FID was carried out for the quantitative analyses on Shimadzu GC-2010 gas chromatography (Shimadzu, Tokyo, Japan), using a fused silica capillary column Zebron ZB-5MS (30 m long × 0.25 mm i.d. × 0.25 µm film thickness) (Phenomenex, Inc., Torrance, CA, USA). The sample injection volume was 0.5 µL. The oven temperature was programmed at 60 • C for 10 min to 230 • C at a rate of 3 • C/min for 1 min. The detector temperature was 250 • C. The carrier gas was ultra-pure helium at a flow rate of 1.9 mL/min, equipped with FID with a spilt ratio of 50:1. The chemical compounds in BCEO were identified by retention indices, calculated using linear interpolation relative to retention times of C 8 -C 23 of nalkanes standards, and compared to reference spectra (Adams and NIST databases). The percentages composition of BCEO were calculated using the peak area normalization method without correction factors. The measurement was conducted in triplicate. The DPPH free radical scavenging activity of BCEO was determined by following the method described by Semiz et al. [105]. The stock solution (1 mg/mL) of BCEO was prepared in methanol. Dilutions were made to obtain concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90, 1.99, and 0.97 µg/mL. One mL of the diluted solution was mixed with 1 mL of 0.2 mM of DPPH (2,2-diphenyl-1-picrylhydrazyl) methanolic solution. After 30 min of incubation in the dark at room temperature, the absorbance was recorded at 517 nm against a blank (methanol solution). The control samples contained all the reagents except BCEO. All measurement was performed in triplicate. Ascorbic acid was used as the standard. The DPPH radicals scavenging activity was calculated using the following equation: where A control = the absorbance value of the control and A sample = the absorbance values of the DPPH radical in the presence of BCEO. The inhibition percentage was plotted against the sample concentrations, and 50% of the inhibitory concentration (IC 50 ) of the DPPH values were defined by linear regression analysis.

Ferric Reducing Antioxidant Power Assay (FRAP)
The total antioxidant activity was measured by the ferric reducing antioxidant power assay (FRAP), following the method described by Vijayalakshmi et al. [106]. In brief, different concentrations of BCEO (0.97-1000 µg/mL) were added to 2.5 mL of 0.2 M of sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide [K 3 Fe(CN) 6 ] solution. Then, the reaction mixture was vortexed and incubated at 50 • C for 20 min using a vortex shaker. After incubation, 2.5 mL of 10% trichloroacetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min. After that, 2.5 mL of the supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride. Ascorbic acid was used as a reference standard. The colored solution was read at 700 nm against blank with reference to standard using a UV-Spectrophotometer. Blank for each solvent was run using the same procedure but replacing BCEO with an equal volume of solvent. The experiment was conducted in triplicate. The EC 50 value (µg/mL) was BCEO concentration at the absorbance 0.5 (a.u.) for the reducing power and was calculated from the graph of absorbance at 700 nm against extracts and EO concentrations.

Agar Disk Diffusion Method
The antibacterial activity of BCEO was measured using an agar disk diffusion assay, as described in a previous work [107], with minor modifications. Before antimicrobial testing, each bacteria strain was subcultured twice onto nutrient agar (NA) plates and incubated for 24 h at 37 • C to obtain colonies. After overnight incubation, colonies of each bacteria strain were taken from their respective cultures with a sterile inoculating loop and transferred to a sterile physiological saline glass tube and vortexed thoroughly. The turbidity of each bacterial suspension was then compared with that of the 0.5 McFarland standard solution (containing about 1.5 × 10 8 CFU/mL). BCEO was dissolved in 10% dimethyl-sulfoxide (DMSO) to 10 mg/mL. Each adjusted bacterial suspension was inoculated on Muller-Hinton agar (MHA) plates and allowed to dry for 5 min. The blank sterile antibiotic disks (diameter = 6 mm) were impregnated with 30 µL of BCEO. The BCEO-containing disks were then aseptically placed on the inoculated MHA plates. Gentamicin (30 µg) was used as the positive control, while a 10% DMSO-soaked disk was used as the negative control. The plates were incubated for 24 h at 37 • C. The diameters of the inhibition zones were measured and recorded in mm after incubation. All assays were conducted in triplicate in three independent experiments.

Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)
The antibacterial activities of BCEO were investigated using the microdilution method in 96-well plates to determine MIC values, as described in an earlier work [107], with minor modifications. This test was carried out with S. aureus, S. epidermidis, E. coli, and K. pneumoniae. Each bacterium was prepared and adjusted to 0.5 McFarland (containing about 1.5 × 10 8 CFU/mL). BCEO was dissolved in 10% DMSO to adequate concentrations and then syringe-filtered through 0.22 µm nylon membrane filters. Two-fold serial dilutions were conducted to yield volumes of 100 µL per well, with final concentrations ranging from 0.024 µL/mL to 50 µL/mL in the MHB medium. An aliquot of 50 µL of the bacterial dilution was added to each well and the final volume of 200 µL/well was adjusted with the MHB medium. As a negative control, an aqueous solution of 10% DMSO was used. Then, 50 µL of the inoculum solution was added to each of the wells. The wells containing only MHB medium with inoculum and the wells containing MHB medium, inoculum, and streptomycin were used as growth and positive controls, respectively. Microplates were incubated for 24 h at 37 • C. After overnight incubation, bacterial growth was evaluated with resazurin. Resazurin at a concentration of 0.015% was prepared, using sterile distilled water, and filtered. An aliquot of 30 µL of 0.015% resazurin was added to each well. The plates were incubated for 30 min in the dark. The viable bacteria were detected by the change of blue-purple to pink. The MIC was defined as the lowest concentration that completely inhibited any visual growth of the tested bacteria. To determine the MBC values, an aliquot of 10 µL from each well that did not show an apparent growth as confirmed by MIC determination were plated and streaked on an NA agar plate. The plates were incubated at 37 • C for 24 h. The MBC was determined as the lowest concentration where no growth was visually observed. All assays were conducted in triplicate in three independent experiments. 4.6. Assessment of Antibiofilm Activity of BCEO 4.6.

Preparation of Bacterial Cultures
The four bacterial cultures (S. aureus, S. epidermidis, E. coli, and K. pneumoniae) were cultured by streaking onto sterile tryptone soy agar (TSA) and incubated at 37 • C for 24 h. After incubation, the bacteria were inoculated in sterile tryptone soy broth (TSB) and incubated in a shaking incubator at 37 • C for 24 h. The overnight bacterial culture was standardized to a concentration of 1.0 × 10 6 CFU/mL prior antibiofilm assays. This was achieved by diluting the overnight culture with TSB supplemented with 2% glucose to obtain an absorbance of 0.02 at OD 590 nm, using a spectrophotometer [102].

Inhibition of Biofilm Formation, Prevention of Initial Bacteria Cell Attachment
BCEO was evaluated for its potential anti-adhesion properties on a 96-well microplate, as described by Sandasi et al. [108] with minor modifications. One hundred µL of standardised culture dilution (1.0 × 10 6 CFU/mL) was dispensed into each well. A volume of 100 µL of BCEO dissolved in TSB, containing 0.5% (v/v) of Tween 80, was added to each 96-well microplate to reach final concentrations of 0.5×, 1× and 2× MICs. The wells containing TSB supplemented with 0.5% (v/v) of Tween 80 and inoculum without BCEO were used as growth controls. Streptomycin (0.5×, 1× and 2× MIC values) was included as a positive antibiotic control. The microplate was incubated at 37 • C for 24 h without shaking to allow cell attachment and biofilm development. Each experiment was carried out in triplicate in three independent experiments. Following overnight incubation, the modified crystal violet assay was conducted to assess biofilm biomass [106].

Inhibition of Development of Pre-Formed Biofilms-Evaluation of Eradication of Biofilm Mass
The effects of BCEO on established biofilm were evaluated using the procedure as described by Jardak et al. [44], with slight modifications. Briefly, biofilm formation was achieved by aliquoting 100 µL of a standardized bacterial culture (1.0 × 10 6 CFU/mL) into a 96-well microplate and incubated at 37 • C for 4 h. After 4 h incubation, an aliquot of 100 µL of BCEO diluted in TSB supplemented with 0.5 % (v/v) Tween 80 was dispensed into each well to yield final concentrations of 0.5×, 1×, and 2× MICs. The microplates were then incubated at 37 • C for 24 h. Streptomycin (0.5×, 1× and 2× MIC values) was included as a positive control. The wells containing TSB supplemented with 0.5% (v/v) of Tween 80 and inoculum without BCEO served as growth controls. The biofilm biomass was quantified using the modified crystal violet staining method after incubation. Each experiment was performed in triplicate in three independent experiments.

Modified Crystal Violet Assay
Cell attachment and eradication were assessed using the modified crystal violet (CV) assay, as described by Djordjevic et al. [109]. Following incubation, the microplates were washed three times with sterile distilled water to remove the loosely attached cells. The plates were air-dried, then oven-dried at 60 • C for 10 min. After drying, 200 µL of methanol was added for 15 min, then air-dried for 10 min. Then, 100 µL of 1% CV was added to each well and incubated at room temperature for 15 min. Next, the microplates were washed three times with sterile distilled water to remove excess CV. Finally, 125 µL of ethanol was added to destain the wells and 100 µL of the destaining solution was transferred to a new microtiter plate. The optical density (OD) of each well was measured at 595 nm using a microplate reader. The percentages of adherence inhibition and biofilm eradication were calculated using the following formula: [(OD (growth control) − OD (sample))/OD (growth control)] × 100 where OD growth control refers to the absorbance of the bacteria growth without BCEO and OD sample refers to the absorbance of BCEO or streptomycin with bacteria.

Statistical Analysis
The Statistical Package for the Social Sciences (SPSS) Version 25 was used to carry out the independent samples t-test to determine the significant differences between the means. All differences were considered significant at p < 0.05.

Conclusions
This study assessed the chemical composition and the antioxidant, antibacterial, and antibiofilm activities of BCEO. The GC-MS analysis of BCEO revealed the presence of 21 compounds. Oxygenated monoterpenes compounds were predominant, especially geranial and neral (isomers of citral), which constituted 89.78% of the total oil. BCEO exhibited promising antioxidant, antibacterial, and antibiofilm activities. The antibacterial and antibiofilm activities of BCEO against the tested bacteria stains were classified in the following decreasing order: S. epidermidis > S. aureus > E. coli > K. pneumoniae. The findings of this study indicated that BCEO was more effective against Gram-positive bacteria than against Gram-negative bacteria. The dual action of BCEO in preventing and eradicating biofilm formation has made this plant a potential candidate for nosocomial infection therapy. Taken together, these findings indicate the potential of BCEO as a promising agent against human pathogenic bacteria and their biofilms. Therefore, BCEO can be a potential candidate for developing a new therapeutic system or a drug adjuvant in the future. Additional research works should be conducted to evaluate the safety and effectiveness of BCEO as an alternative to synthetic drugs.