Antibacterial and antibiofilm efficacy of Cymbopogon citratus (DC.) Stapf essential oil and its bioactives against methicillin-resistant Staphylococcus aureus

2.1 Plant material and chemicals

The aerial parts of Cymbopogon citratus were collected from Chiang Khong District, Chiang Rai Province, Thailand. Taxonomic identification was conducted by the Department of Botany, Faculty of Science, Chulalongkorn University (voucher specimen; BCU No.016439). CCEO was extracted by hydrodistillation and stored at 4^°C (avoiding light). The yield of CCEO was 2.5% (v/v) (density 0.9 g/mL). The working solution of CCEO was diluted in dimethyl sulfoxide (DMSO) to obtain a concentration of 400 mg/mL.

2.2 Chemicals

Chemicals: citral (99.3% purity, density 0.888 g/mL), geraniol (≥97.5% purity, density 0.879 g/mL) (Sigma-Aldrich, USA), DMSO (Merck, Germany), crystal violet (88% dye content; Loba Chemie, India), glacial acetic acid (QRec, New Zealand), glucose (Fluka Chemie, Switzerland), potassium dihydrogen phosphate (Merck, Germany), sodium phosphate dibasic anhydrous (Mallinckrodt Baker, USA), phosphate buffer saline (PBS) containing sodium chloride, and potassium chloride (Thermo Fisher Scientific, Australia).

Media: Mueller Hinton broth (MHB), yeast extract (Oxoid, England), Trypticase soy broth (TSB; HiMedia, India), glycerol (Thermo Fisher Scientific, UK), Mueller Hinton agar (MHA), and Mueller Hinton agar with sheep blood (MHS) plates (Clinag Co., Ltd., Thailand).

2.3 Gas chromatography-mass spectrometry analysis

The chemical components of CCEO were identified by gas chromatography-mass spectrometry (GC-MS) using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C inert XL EI/CI mass selective detector with triple-axis detector (Agilent Technologies, USA). The capillary column Mega-5MS (30 m × 0.25 mm, 0.25 µm film thick) with 5% phenyl methylpolysiloxane was applied. The GC-MS conditions were set up according to a previous study (Sreepian et al., 2019). The results were compared with the NIST (National Institute of Standards and Technology) library in the GC-MS database.

2.4 Bacterial strains and culture conditions

Two American Type Culture Collection (ATCC) of S. aureus containing ATCC 43300 (methicillin-resistant; MRSA) and ATCC 25923 (methicillin-susceptible; MSSA), and 10 MRSA isolates (strains 001−010) were included in this study. All tested bacteria were derived from stock cultures in Microbiology Laboratory, Faculty of Medical Technology, Rangsit University. The bacteria were sub-cultivated on blood agar at 37^°C for 18−24 h. The identification of S. aureus was performed by microscopic examination and biochemical testing (Gram-positive cocci in clusters, positive for catalase, coagulase, DNase, and glucose oxidation/fermentation, and susceptible to novobiocin). Methicillin resistance was identified by cefoxitin disk diffusion on MHA agar, following the guideline of the Clinical and Laboratory Standards Institute (CLSI, 2020). The isolates were identified as being methicillin resistant when the IZD of the cefoxitin diskwas <22 mm. The characteristics of the tested bacteria had been revealed in a previous study (Sreepian et al., 2020). MRSA isolates 001−003, 005, and 007 were MDR MRSA, while the other five MRSA isolates were non-MDR MRSA. In addition, all MRSA isolates had a erythromycin-inducible clindamycin-resistant phenotype.

2.5 Screening antibacterial activity of CCEO

Antibacterial activities of CCEO and its main components (citral and geraniol) against MRSA were screened by the disk diffusion method described by Sreepian et al. (2022). In brief, the bacterial suspension was prepared in sterile normal saline by adjusting the turbidity equally to 0.5 McFarland standard using a densitometer (DEN-1; Biosan, England). Then, the bacterial suspension was spread on an MHA plate. Sterilized 6 mm-disks (Whatman^™; Cytiva, UK) impregnated with either 10 µL of undiluted CCEO, 5 µL of citral, or 10 µL of geraniol, equal to 0.888, 0.879, and 0.9 g/mL, respectively, were placed on the MHA and incubated at 37^°C for 18−24 h. DMSO control disk (10 µL, 2.2%) was also included. The IZD was measured, and the antibacterial activity was interpreted according to Lv et al. (2011).

2.6 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The MICs of CCEO and its major components; citral and geraniol, against MRSA were evaluated by broth macrodilution described by Sreepian et al. (2019). Briefly, 1 mL of bacterial suspension (10⁶ CFU/mL) was resuspended with an equal volume of CCEO, citral, or geraniol to prepare the final concentrations from 0.125−16.0 mg/mL. Cationic-adjusted Mueller-Hinton broth (CAMHB) with either CCEO, citral, or geraniol (oil controls), CAMHB with bacterial suspension (bacterial control), and 2.2% DMSO with bacterial suspension (solvent control) were included. After incubating at 37^°C for 18−24 h, bacterial growth based on the turbidity was visually observed. The MIC was defined as the lowest concentration that was visually clear.

Subsequently, the MIC suspension and all controls were inoculated on the MHA plate and subsequently incubated at 37^°C for 18−24 h. The MBC was defined by the lowest concentration that no bacterial colony was observed. To classify antibacterial effect, the MIC index was evaluated by the MBC/MIC ratio and interpreted according to Gatsing et al. (2009).

2.7 Time-killing analysis

The classical time-killing assay was investigated according to the method described by Chimnoi et al. (2018) with some modifications. Briefly, 1 mL of bacterial suspension in sterile normal saline (10⁶ CFU/mL) was treated with an equal volume of either CCEO, citral, or geraniol at the final concentrations 0, 1×, and 2×MICs, and incubated at 37^°C for 0−30 h intervals. DMSO (2.2%) was included as a solvent control. At the end of each timepoint, the optical density (OD) at a wavelength of 600 nm was measured by spectrophotometer (Genesys 20; Thermo Fisher Scientific, USA). The growth curve was constructed by plotting a sigmoidal curve (the x-axis is a various interval timepoint vs. the y-axis is OD at 600 nm) using GraphPad Prism v.10.2.3 (GraphPad Software Inc., USA). Subsequently, one loop of the suspension was inoculated on the MHA and incubated at 37^°C for 18−24 h. After that, the bactericidal endpoint was determined based on the initial time at which no bacterial colony was observed.

2.8 Determination of biofilm-producing ability

The biofilm-producing ability was determined by the conventional crystal violet (CV) staining technique as described by Pedonese et al. (2022). In brief, 100 µL of the bacterial suspension (1.5×10⁸ CFU/mL) was resuspended with an equal volume of TSB supplemented with 0.6% yeast extract, 0.75% glucose, and 1.5% sodium chloride (TSBYEgluc+NaCl) in a sterile flat bottomed 96-well plate (SPL life sciences, Korea). The plate was incubated at 37^°C for 24 h without any agitation to allow biofilm formation. The forming biofilm was fixed by incubating at 60^°C for 1 h and then stained by 100 µL of 2% CV solution at room temperature for 20 min. The plate was soaked three times with sterile PBS and air-dried at room temperature for 30 min. After that, 200 µL of 33% glacial acetic acid was added and incubated at room temperature for 20 min to dissolve the bound dye. The OD at 492 nm was measured by a microplate reader (Infinite^®; F50 TECAN, Singapore). The medium alone (negative control) was included and used as a blank.

The tested OD (ODt) was obtained by subtracting the sample OD from the blank OD. The cutoff OD (ODc) was calculated by adding the lowest mean OD of the tested strain and 2SD.

The biofilm formation was classified into 4 categories by the following criteria: no biofilm (N), the tested OD (ODt) ≤1×ODc; weak producer (W), 1×ODc < ODt < 2×ODc; medium producer (M), 2×ODc < ODt < 3×ODc; and strong producer (S), ODt ≥3×ODc (Pedonese et al., 2022).

2.9 Determination of minimum biofilm inhibitory concentration (MBIC)

The activity on biofilm inhibition was determined by using a method as described by Adukwu et al. (2012) with some modifications. In this study, MRSA ATCC 43300 and five MRSA isolates were selected for investigation due to their strong biofilm-producing properties. Firstly, bacterial biofilm formation was allowed under the above conditions. In brief, 100 µL of the bacterial suspension and an equal volume of various concentrations of either CCEO, citral, or geraniol were added. The final concentrations were 0.25−3.0 mg/mL for CCEO and geraniol and 0.0625−1.0 mg/mL for citral. The bacterial suspension in the medium without any treatment (biofilm control) and bacterial suspension in DMSO (solvent control) were included as controls. The biofilms were fixed, stained, and measured according to the procedures described above. The MBIC was defined as the lowest concentration at which the tested OD (ODt) ≤ the OD of biofilm control (ODb). The biofilm inhibition percentage was calculated as per the following formula:

$\text{Biofilm}\text{inhibition}\left(\%\right)=\frac{\text{ODb}-\text{ODt}}{\text{ODb}}\times 100$

2.10 Determination of minimum biofilm eradication concentration (MBEC)

The activity on biofilm eradication was determined after 24-h biofilm formation. The biofilm-forming plates were gently rinsed three times with sterile PBS, exposed to various concentrations of CCEO, citral, and geraniol (as described above), and incubated at 37^°C for 24 h without any agitation. The biofilm control and solvent control were included. The biofilms were fixed, stained, and measured according to the procedures described above. The MBEC was defined by the lowest concentration at which the biofilm was removed from the bottom of the wells. The biofilm eradication percentage was calculated as per the following formula:

$\text{Biofilm}\text{eradication}\left(\%\right)=\frac{\text{ODb}-\text{ODt}}{\text{ODb}}\times 100$

2.11 Statistical analysis

All experiments were performed in three independent measurements. The quantitative data was expressed as the mean±standard deviation (SD). The differences among different groups were analyzed by the Kruskal-Wallis test and the Dunnett’s multiple comparison test using SPSS version 21.0 (IBM, USA).

3.1 Chemical compositions of CCEO

The identification of chemical components in CCEO provides insights into its potential bioactivities. The chemical compositions analyzed by GC-MS have been presented in Fig. 1 and Table 1. Fourteen compounds were identified, accounting for 92.99% of the total CCEO. It mainly consisted of oxygenated monoterpenes (88.69%) and sesquiterpene hydrocarbons (2.73%). The oxygenated monoterpenes were geraniol (29.09%) and citral (28.53%), which was a combination of two isomeric aldehydes, α-citral (trans-citral or geranial) (16.49%) and β-citral (cis-citral or neral) (12.04%), followed by citronellal (12.16%), citronellol (10.85%), and geranyl acetate (3.66%).

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Fig. 1.

GC-MS chromatogram of CCEO. Major compound peaks marked: (1) citronellal, (2) citronellol, (3) β-citral, (4) geraniol, and (5) α-citral.

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3.2 Antibacterial activity of CCEO and its major components

The antibacterial activity was screened using disk diffusion to indicate antibacterial susceptibility. Almost all MRSA isolates were susceptible to gentamicin (60%, 6/10) and clindamycin (100%, 10/10). There were two gentamicin-resistant (strains 001 and 002) and two gentamicin-intermediated MRSA isolates (strains 003 and 005). The antibacterial activity of CCEO and its major components against two reference strains and 10 MRSA isolates has been shown in Table 2. The IZDs of CCEO, citral, and geraniol against MSSA ATCC 25923 were 21.7, 25.0, and 20.7 mm, while those of MRSA ATCC 43300 were 20.7, 24.0, and 20.3 mm, respectively.

The CCEO displayed strong antibacterial activity against most MRSA isolates (60%, 6/10) with IZDs ranging from 20.0−26.3 mm. Moderate activity was found in 40% (4/10) of those isolates with IZDs ranging from 18.0−18.7 mm. Citral exhibited the highest IZDs across most MRSA isolates, ranging from 17.0−23.7 mm, with the strong and moderate activities observed in 70% (7/10) and 30% (3/10) MRSA isolates, respectively. Geraniol displayed strong antibacterial activity against most MRSA isolates (80%, 8/10) with IZDs ranging from 20.0−23.7 mm. The moderate antibacterial activity of geraniol was observed in 20% (2/10) of those isolates with IZDs ranging from 18.7−19.7 mm. The IZD of CCEO against MRSA isolates was significantly different from that of citral (p <0.01) but not observed in geraniol (p >0.05). It revealed that CCEO and its bioactive compounds had moderate to strong activity, with citral showing a significant difference. These results indicated that citral seems to be a major bioactive compound in CCEO that acts on the inhibitory effect to MRSA isolates.

The MICs and MBCs of CCEO and its major components against two reference strains and 10 MRSA isolates have been shown in Table 2. DMSO and bacterial controls showed ordinary bacterial viability, while that of oil controls were not observed. These results indicated the solvent itself did not affect bacterial growth and all tested bacteria were viable throughout the experiment. The relatively high susceptibility in MRSA ATCC 43300 and MSSA ATCC 25923 was observed against both CCEO and citral (MICs: 2.0−2.7 and 0.8−1.7 mg/mL), while a lower susceptibility was observed in geraniol (MIC: 8.0 mg/mL). The MICs of CCEO, citral, and geraniol against MRSA isolates were significantly different with the ranged from 2.0−4.0, 0.5−1.7, and 4.0−8.0 mg/mL, respectively (p <0.01). In addition, the MIC indexes of CCEO, citral, and geraniol were 1.0−3.4 toward all MRSA isolates (100%, 10/10), suggesting the bactericidal effect on MRSA.

3.3 Growth- curve and time-killing analysis

The results of the time-killing kinetics of CCEO and its major components have been shown in Fig. 2. The bacterial control, and solvent control showed sigmoidal (S-shaped) growth curves, indicating normal viability throughout the 30-h experiment. The growth curves of these controls remained unchanged until 4 h. Following that, there was a rapid increase in turbidity, indicating the onset of the log phases, followed by the observation of stationary phases at 24 h of the experiment (Figs. 2(a) and 2(c)). On the contrary, the bacterial growth curve of MSSA ATCC 25923 after treatment with 1× and 2×MICs CCEO showed no increase in turbidity over 30 h, indicating complete bacterial inhibition. The bactericidal endpoints against MSSA ATCC 25923 were observed after exposure to 1× and 2×MICs CCEO at 4 and 2 h, respectively (Fig. 2(b)).

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Fig. 2.

The bacterial growth curves of (a) MSSA ATCC 25923 after 30-h exposure to 1× and 2× MICs CCEO, citral, and geraniol, and the bactericidal endpoints of 1× and 2× MICs CCEO against (b) MSSA ATCC 25923. The bacterial growth curves of (c) MRSA ATCC 43300 after 30-h exposure to 1× and 2× MICs CCEO, citral, and geraniol, and the bactericidal endpoints of 1× and 2× MICs CCEO against (d) MRSA ATCC 43300. Solvent control (2.2% DMSO) and bacterial control (without any treatment) were included.

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In addition, the bacterial growth curve of MRSA ATCC 43300 was observed only after treatment with 2×MIC CCEO (Fig. 2(c)). The results indicated that only 2×MIC CCEO inhibited the growth of MRSA ATCC 43300. The bactericidal endpoint against MRSA ATCC 43300 was observed after exposure to only 2×MIC CCEO at 12 h, but the bactericidal endpoint was not observed for 1×MIC CCEO. This indicated that 1×MIC was observed to only temporarily suppress the growth of MRSA ATCC 43300. As shown in Figs. 2(b) and 2(d), the bactericidal effect of MSSA was reached when exposed to 1× and 2×MICs at 4 and 2 h, respectively, whereas MRSA were reached when exposed to 2×MIC CCEO at 12 h. These findings highlight the potential of CCEO, particularly at higher concentrations, for eradicating MRSA.

The bacterial growth curves of MSSA ATCC 25923 and MRSA ATCC 43300 after exposure to 1× and 2×MICs citral and geraniol demonstrated horizontal trended lines, which indicated no increasing in the turbidity throughout the 30-h experiment. These results indicated that both 1× and 2×MICs citral and geraniol inhibited the growth of both MSSA and MRSA (Figs. 2a and 2c). The bactericidal endpoints against MSSA ATCC 25923 were observed after exposure to 1× and 2×MICs citral at 8 and 2 h, while those of 1× and 2×MICs geraniol were observed at 12 and 4 h, respectively. For MRSA ATCC 43300, the bactericidal endpoints were also observed after exposure to both 1× and 2×MICs citral at 12 h, while that of 1× and 2×MICs geraniol were observed at 8 and 4 h, respectively. This rapid bactericidal action underscores CCEO’s therapeutic potential, especially in acute or life-threatening infections where rapid bacterial clearance is critical.

3.4 Simple assessment of biofilm formation

Following the assessment of antibacterial activity, we investigated CCEO’s effect on a critical virulence factor of MRSA; biofilm formation. The results of qualitative and quantitative of biofilm formation of two reference strains and 10 MRSA isolates have been shown in Fig. 3. The medium alone (negative control) showed no biofilm formation. MRSA ATCC 43300 and five MRSA isolates (50%); such as strains 004, 006, 008−010, showed strong biofilm production by reaching the optical values of 0.67−1.05. The statistical analysis revealed significant differences in biofilm formation between non-biofilm and strong biofilm producing MRSA isolates (p <0.05). Moreover, the highest biofilm production was observed in MRSA ATCC 43300, followed by MRSA isolate 010, without significant difference (p >0.05).

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Fig. 3.

(a-b) Qualitative and quantitative biofilm formations of MRSA ATCC 43300 and MRSA isolates (a). Each column represents the mean value, and the error bar represents the SD. The biofilm-staining appearances of tested bacterial strains (b). A−J, MRSA isolates 001−010; K, MSSA ATCC 25923; and L, MRSA ATCC 43300. N, non-biofilm producer; S, strong biofilm producer. Different lowercase letters (a, b, and c) between columns indicated significant differences (p <0.05)

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3.5 Antibiofilm activity of CCEO

3.5.1 Biofilm inhibition

Based on biofilm formation, the strong biofilm producers (n =6) were selected to be investigated. The overall results on the activities of CCEO, citral, and geraniol on biofilm inhibition have been shown in Fig. 4. The solvent control produced firm biofilm, demonstrating that DMSO did not affect the biofilm formation. Whereas CCEO, citral, and geraniol inhibited biofilm formation in a dose-dependent manner. The results demonstrated that CCEO at concentrations of 1.0−3.0 mg/mL efficiently inhibited biofilm formation with the inhibition rates 97.0−99.3%, without significant difference (p >0.05). The inhibition rate of CCEO at 3.0 mg/mL (99.3%) was significantly different from those of the lower concentrations of 0.25−0.5 mg/mL (74.7−94.2%) (p <0.05) (Fig. 4a). The results of citral and geraniol were also revealed. Citral at concentrations of 0.125−1.0 mg/mL efficiently inhibited biofilm formation with the inhibition rates of 72.4−93.9%, without significant difference (p >0.05). A significant difference was however observed at 1.0 mg/mL (93.9%) compared to that lower concentration of 0.625 mg/mL (34.2%) (p <0.05) (Fig. 4c). Geraniol at concentrations of 0.5−3.0 mg/mL efficiently inhibited biofilm formation with the inhibition rate of 85.5−96.9%, without significant difference (p >0.05). A significant difference was observed in geraniol at 3.0 mg/mL (96.9%) compared to those lower concentrations of 0.25 mg/mL (67.6%) (p <0.05) (Fig. 4e).

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Fig. 4.

The overall biofilm inhibition by various concentrations of (a) CCEO, and the biofilm-staining appearances after exposure to (b) CCEO; the overall biofilm inhibition by various concentrations of (c) citral, and the biofilm-staining appearances after exposure to (d) citral; and the overall biofilm inhibition by various concentrations of (e) geraniol, and the biofilm-staining appearances after exposure to (f) geraniol. Each column represents the mean value, and the error bar represents the SD, among MRSA ATCC 43300 and MRSA isolates (n = 6). Different lowercase letters (a, b, c, and d) between columns indicated significant differences (p < 0.05)

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The biofilm formation of MRSA ATCC 43300 and MRSA isolates after exposure to CCEO, citral, and geraniol were decreased when compared with the unexposed controls (Figs. 4b, 4d, and f). The study concluded that CCEO and its major compounds influenced biomass-, and therefore biofilm production.

The MBIC of CCEO and its major components have been shown in Table 3. CCEO exhibited inhibitory effects on biofilm formation at MBIC >3.0 mg/mL, while citral demonstrated inhibitory activity against biofilm at MBIC >1.0 mg/mL, except for MRSA isolate 006. Geraniol also demonstrated biofilm inhibition with MBIC >3.0 mg/mL, except MRSA isolate 010 with MBIC 3.0 mg/mL. The MBIC was not determined for CCEO and geraniol, and citral at concentrations >3.0 and >1.0 mg/mL, respectively, due to their inhibitory effects on bacterial viability at these concentrations.

Table 3. Antibiofilm activities of CCEO and its major components.

	MBIC (mg/mL)			MBEC (mg/mL)
Bacterial strains	CCEO	Citral	Geraniol	CCEO	Citral	Geraniol
MRSA ATCC 43300	>3.0	>1.0	>3.0	>3.0	>1.0	>3.0
MRSA004	>3.0	>1.0	>3.0	>3.0	>1.0	>3.0
MRSA006	>3.0	1.0±0.0	>3.0	>3.0	>1.0	>3.0
MRSA008	>3.0	>1.0	>3.0	>3.0	>1.0	>3.0
MRSA009	>3.0	>1.0	>3.0	3.0±0.0	>1.0	>3.0
MRSA010	>3.0	>1.0	3.0±0.0	3.0±0.0	>1.0	>3.0

Values were expressed as mean±SD.

3.5.2 Biofilm eradication

Results about the activities of CCEO, citral, and geraniol on biofilm eradication have been shown in Fig. 5. DMSO (2.2%) had no effect on biofilm et all. CCEO at 2.0−3.0 mg/mL efficiently removed the biofilm with the eradication rates of 85.3−99.1%, without significant difference (p >0.05). However, significant differences were observed in CCEO at 3.0 mg/mL (99.1%) compared to those of the lower concentrations; 0.25−1.0 mg/mL (21.7−56.9%) (p <0.05) (Fig. 5a).

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Fig. 5.

The overall biofilm eradication by various concentrations of (a) CCEO, and the biofilm-staining appearances after exposure to (b) CCEO; the overall biofilm eradication by various concentrations of (c) citral, and the biofilm-staining appearances after exposure to (d) citral; and the overall biofilm eradication by various concentrations of (e) geraniol, and the biofilm-staining appearances after exposure to (f) geraniol. Each column represents the mean value, and the error bar represents the SD among MRSA ATCC 43300 and MRSA isolates (n = 6). Different lowercase letters (a, b, c, and d) between columns indicated significant differences (p < 0.05). Sreepian and SreepianSreepian and Sreepian

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The investigation of biofilm eradication also encompassed major components such as citral and geraniol. Citral at 0.5−1.0 mg/mL efficiently removed biofilm with the eradication rates of 70.9−89.5%, without significant difference (p >0.05). Significant differences were observed in citral at 1.0 mg/mL (89.5%) compared to those of the lower concentrations; 0.0625−0.25 mg/mL (25.2−59.4%) (p <0.05) (Fig. 5c). Geraniol at 1.0−3.0 mg/mL efficiently removed biofilm with the eradication rate of 93.4−96.1%, but no significant differences were observed (p >0.05). Significant differences were observed in geraniol at 1.0−3.0 mg/mL (93.4−96.1%) compared to that of the lower concentrations of 0.25−0.5 mg/mL (63.3−76.6%) (p <0.05) (Fig. 5e).

These findings revealed that CCEO and its principal constituent effectively reduced biofilm. Moreover, the biofilms of MRSA ATCC 43300 and MRSA isolates exposed to CCEO, citral, and geraniol were decreased when compared with the unexposed controls (Figs. 5b, 5d, and 5f). The findings indicated that both CCEO and its primary constituents exhibited a dose-dependent biofilm eradication effect. Consequently, this research suggests that CCEO may serve as a potential agent for inhibiting and removing bacterial biofilms, which often hinder antibiotic penetration.

The biofilm eradication of CCEO in terms of MBEC was observed at 3.0 mg/mL for MRSA isolates 009 and 010, while those of MRSA ATCC 43300 and other isolates were observed at the concentration >3.0 mg/mL (Table 3). Both citral and geraniol exhibited the activity on biofilm eradication of MRSA ATCC 43300 and all MRSA isolates with MBEC >1.0 and >3.0 mg/mL, respectively. Similar to MBIC, MBECs for CCEO and geraniol, and citral could not be determined at concentrations >3.0 and >1.0 mg/mL, respectively, because of their inhibitory effects on bacterial viability at these concentrations.

Results demonstrated that CCEO, citral, and geraniol exhibited antibiofilm activities in a dose-dependent manner through both inhibition of biofilm formation and eradication was affected.