Synergistic and Antibiofilm Effects of the Essential Oil from Croton conduplicatus (Euphorbiaceae) against Methicillin-Resistant Staphylococcus aureus

Bacterial resistance refers to the ability of bacteria to resist the action of some antibiotics due to the development of adaptation and resistance mechanisms. It is a serious public health problem, especially for diseases caused by opportunistic bacteria. In this context, the search for new drugs, used alone or in combination, appears as an alternative for the treatment of microbial infections, and natural products, such as essential oils, are important in this process due to their structural diversity, which increases the probability for antimicrobial action. The objective of this study was to extract and identify the chemical components of the essential oil from Croton conduplicatus (EOCC), to evaluate the antimicrobial activity, to investigate the effect of the interaction between the EOCC and different antibiotics and to evaluate its antibiofilm potential. The EOCC was obtained by hydrodistillation. Based on chemical characterisation, 70 compounds were identified, with 1.8 cineole (13.15%), p-cymene (10.68%), caryophyllene (9.73%) and spathulenol (6.36%) being the major constituents. The minimum inhibitory concentration (MIC) values of EOCC were 256 and 512 µg mL−1 for methicillin-sensitive and -resistant Staphylococcus aureus strains (MSSA and MRSA), respectively. The combinations of EOCC with the antibiotics oxacillin and ampicillin were synergistic (OXA/EOCC and AMP/EOCC combined decreased the OXA MIC and AMP MIC to 0.5 and 0.25 for MSSA, respectively, and OXA/EOCC and AMP/EOCC combined decreased the OXA MIC and the AMP MIC to 1 and 0.5 for MRSA, respectively) and could modify the resistance profile of MSSA and MRSA strains. The results indicated that EOCC was also able to partially inhibit biofilm formation. Our study presents important information about the chemical composition of EOCC and its antimicrobial potential and provides a reference to determine the mechanisms of action of EOCC and its use in pharmaceutical formulations.


Introduction
Staphylococcus aureus is a Gram-positive, opportunistic bacterial species characterised by grouped cocci and clusters of cocci that are found mainly in the nasal microbiota. They can cause an infectious condition when they get into the bloodstream by breaking through mucous membrane or skin tissue [1]. This species is highly pathogenic, virulent and shows considerable resistance to environmental factors. A major concern worldwide is the capacity for multi-resistance to antibacterial agents used to combat Gram-positive bacterial infections, such as beta-lactams, glycopeptides and oxazolidones. One of the bacterial resistance profiles of great concern today are those of methicillin-resistant S. aureus (MRSA)

Chemical Characterisation of C. conduplicatus Essential Oil
Essential oils have a high concentration of bioactive compounds such as terpenes, sesquiterpenes, phenolic compounds, phenylpropanoids, non-terpenic aliphatic compounds and heterocyclic compounds, which are responsible for the biological activity of these oils.
Analysis of the chemical composition of the EOCC was performed by GC-MS. Table 1 shows the chemical composition, the retention indices, the retention time, and the relative percentage of each constituent present in EOCC. We verified the presence of 74 distinct peaks, as shown in the Figure 1, of which 70 were identified (Table 1), accounting for 95.94% of the chemical composition of EOCC. The monoterpenes 1,8-cineole (13.15%) and p-cymene (10.68%) and the sesquiterpenes caryophyllene (9.73%) and spathulenol (6.36%) were the major compounds ( Figure 2). The other compounds with percentages below 5% were considered minor, such as αpinene (4.93%), bicyclogermacrene (3.40%), α-phellandrene (3.08%), β-pinene (2.77%) and linalool (2.39%). We verified the presence of 74 distinct peaks, as shown in the Figure 1, of which 70 were identified (Table 1), accounting for 95.94% of the chemical composition of EOCC. The monoterpenes 1,8-cineole (13.15%) and p-cymene (10.68%) and the sesquiterpenes caryophyllene (9.73%) and spathulenol (6.36%) were the major compounds ( Figure 2). The other compounds with percentages below 5% were considered minor, such as α-pinene (4.93%), bicyclogermacrene (3.40%), α-phellandrene (3.08%), β-pinene (2.77%) and linalool (2.39%).  Among the major compounds 1,8-cineole, p-cymene, caryophyllene and spathulenol were identified by other authors as part of the main constituents of the chemical composition of the EO from C. conduplicatus [19][20][21][22][23]. In their results, these compounds showed similar average concentrations to those found here, with the exception of 1,8-cineole, which showed a concentration of 24.09% [20]. In addition, the compounds bicyclogermacrene and α-phellandrene were identified here as minor compounds, whereas in other studies, they were among the major constituents [19][20][21]. Differences in the number and identity of compounds were also identified in the GC-MS analyzes performed by [14] which revealed the presence of 50 peaks and 42 compounds identified in the EOCC obtained from fresh leaves of C. conduplicatus. The monoterpenes 1,8-cineol (21.42%) and pcymene (12.41%) and the sesquiterpenes spathulenol (15.47%) and caryophyllene oxide (12.15%) were considered the majority constituents of the sample (Table 2). In our study, it was possible to identify and quantify 70 different chemical compounds, of which the monoterpenes 1,8 cineole (13.15%) and p-cymene (10.68%) were the majority, as well as the sesquiterpenes caryophyllene (9.73%) and spathulenol (6.36%). This variation in the chemical composition of the EOCC may be related to factors such as the specific location of leaf collection, growing season, botanical origin, climatic factors [24] and the drying process that was used on the plant material.  We verified the presence of 74 distinct peaks, as shown in the Figure 1, of which 70 were identified (Table 1), accounting for 95.94% of the chemical composition of EOCC. The monoterpenes 1,8-cineole (13.15%) and p-cymene (10.68%) and the sesquiterpenes caryophyllene (9.73%) and spathulenol (6.36%) were the major compounds ( Figure 2). The other compounds with percentages below 5% were considered minor, such as α-pinene (4.93%), bicyclogermacrene (3.40%), α-phellandrene (3.08%), β-pinene (2.77%) and linalool (2.39%).  Among the major compounds 1,8-cineole, p-cymene, caryophyllene and spathulenol were identified by other authors as part of the main constituents of the chemical composition of the EO from C. conduplicatus [19][20][21][22][23]. In their results, these compounds showed similar average concentrations to those found here, with the exception of 1,8-cineole, which showed a concentration of 24.09% [20]. In addition, the compounds bicyclogermacrene and α-phellandrene were identified here as minor compounds, whereas in other studies, they were among the major constituents [19][20][21]. Differences in the number and identity of compounds were also identified in the GC-MS analyzes performed by [14] which revealed the presence of 50 peaks and 42 compounds identified in the EOCC obtained from fresh leaves of C. conduplicatus. The monoterpenes 1,8-cineol (21.42%) and pcymene (12.41%) and the sesquiterpenes spathulenol (15.47%) and caryophyllene oxide (12.15%) were considered the majority constituents of the sample ( Table 2). In our study, it was possible to identify and quantify 70 different chemical compounds, of which the monoterpenes 1,8 cineole (13.15%) and p-cymene (10.68%) were the majority, as well as the sesquiterpenes caryophyllene (9.73%) and spathulenol (6.36%). This variation in the chemical composition of the EOCC may be related to factors such as the specific location of leaf collection, growing season, botanical origin, climatic factors [24] and the drying process that was used on the plant material. Among the major compounds 1,8-cineole, p-cymene, caryophyllene and spathulenol were identified by other authors as part of the main constituents of the chemical composition of the EO from C. conduplicatus [20][21][22][23][24]. In their results, these compounds showed similar average concentrations to those found here, with the exception of 1,8-cineole, which showed a concentration of 24.09% [21]. In addition, the compounds bicyclogermacrene and α-phellandrene were identified here as minor compounds, whereas in other studies, they were among the major constituents [20][21][22]. Differences in the number and identity of compounds were also identified in the GC-MS analyzes performed by [14] which revealed the presence of 50 peaks and 42 compounds identified in the EOCC obtained from fresh leaves of C. conduplicatus. The monoterpenes 1,8-cineol (21.42%) and p-cymene (12.41%) and the sesquiterpenes spathulenol (15.47%) and caryophyllene oxide (12.15%) were considered the majority constituents of the sample ( Table 2). In our study, it was possible to identify and quantify 70 different chemical compounds, of which the monoterpenes 1,8 cineole (13.15%) and p-cymene (10.68%) were the majority, as well as the sesquiterpenes caryophyllene (9.73%) and spathulenol (6.36%). This variation in the chemical composition of the EOCC may be related to factors such as the specific location of leaf collection, growing season, botanical origin, climatic factors [25] and the drying process that was used on the plant material.

Antimicrobial Activity of C. conduplicatus Essential Oil
The EOCC showed antibacterial activity, with an minimum inhibitory concentration (MIC) of 256 µg mL −1 for the MSSA strain and 512 µg mL −1 for the MRSA strain ( Figure 3). The bactericidal effect of EOCC was observed only in the presence of twice the MIC concentration. No antimicrobial activity of EOCC was observed against E. coli, P. aeruginosa and C. albicans strains because the MIC values against these strains were > 1024 µg mL −1 ( Table 3). The MRSA strain used in this study presented a resistance profile to oxacillin (OXA) and ampicillin (AMP), which was detected through the determination of the MIC, highlighting the MIC of OXA of 32 µg mL −1 , which is used for the detection of methicillin resistance, according to the breakpoints defined by the CLSI document M100 [26]. Because EOCC showed activity against MRSA and MSSA strains, these were selected for the in vitro combination step with antibiotics to investigate the synergistic effect and reduction in MIC of both the antibiotic and EOCC.

Antimicrobial Activity of C. conduplicatus Essential Oil
The EOCC showed antibacterial activity, with an minimum inhibitory concentration (MIC) of 256 µ g mL −1 for the MSSA strain and 512 µ g mL −1 for the MRSA strain ( Figure 3). The bactericidal effect of EOCC was observed only in the presence of twice the MIC concentration. No antimicrobial activity of EOCC was observed against E. coli, P. aeruginosa and C. albicans strains because the MIC values against these strains were > 1024 µ g mL −1 ( Table 3). The MRSA strain used in this study presented a resistance profile to oxacillin (OXA) and ampicillin (AMP), which was detected through the determination of the MIC, highlighting the MIC of OXA of 32 µ g mL −1 , which is used for the detection of methicillin resistance, according to the breakpoints defined by the CLSI document M100 [25]. Because EOCC showed activity against MRSA and MSSA strains, these were selected for the in vitro combination step with antibiotics to investigate the synergistic effect and reduction in MIC of both the antibiotic and EOCC.     reduction percentages ranging from 75% to 96.9%; the synergistic effect was determined by fractional inhibitory concentration (FICi) values that ranged from 0.0938 to 0.3125. Percentage reductions in the MIC of EOCC alone were also observed when combined with OXA and AMP. Thus, the EOCC showed potential to reduce OXA and AMP MIC, and these antibiotics showed potential to reduce the MIC of EOCC (Table 4).

Synergistic Activity of C. conduplicatus Essential Oil with Oxacillin and Ampicillin against S. aureus
A synergistic effect of combining subinhibitory concentrations of EOCC (≤ 1/2 MIC) with OXA and AMP was observed against MSSA and MRSA (Figure 4a,b) strains, with MIC reduction percentages ranging from 75% to 96.9%; the synergistic effect was determined by fractional inhibitory concentration (FICi) values that ranged from 0.0938 to 0.3125. Percentage reductions in the MIC of EOCC alone were also observed when combined with OXA and AMP. Thus, the EOCC showed potential to reduce OXA and AMP MIC, and these antibiotics showed potential to reduce the MIC of EOCC (Table 4).  The combinations of EOCC with OXA and AMP were able to reverse resistance to these antibiotics, indicating that at concentrations lower than MIC, OXA and AMP became active against the strains when combined with EOCC.
For the MSSA strains, we verified a reduction in the MIC of both EOCC and OXA and AMP, indicating that EOCC in subinhibitory concentration in combination with these antibiotics has the potential to reduce the MIC for both methicillin-sensitive and methicillin-resistant strains ( Table 4). Because of the in vitro combination tests using EOCC with  The combinations of EOCC with OXA and AMP were able to reverse resistance to these antibiotics, indicating that at concentrations lower than MIC, OXA and AMP became active against the strains when combined with EOCC.
For the MSSA strains, we verified a reduction in the MIC of both EOCC and OXA and AMP, indicating that EOCC in subinhibitory concentration in combination with these antibiotics has the potential to reduce the MIC for both methicillin-sensitive and methicillinresistant strains ( Table 4). Because of the in vitro combination tests using EOCC with the antibiotics OXA and AMP, it can be stated that these interactions had a synergistic effect.
The results for MSSA and MRSA strains in terms of sensitivity and resistance to methicillin, respectively, are shown in Figure 1. For the MSSA strain, the MIC of isolated OXA was 2 µg mL −1 . In combination with EOCC, the MIC of OXA was reduced to 0.5 µg mL −1 , changing its profile from resistant to sensitive to this antibiotic. The reduction in OXA MIC was also observed in the MRSA strain ( Figure 5B). A similar effect was observed in the associations of EOCC with AMP, with a synergistic effect against the MRSA strain, with a reduction in the MIC of AMP from 16 to 0.5 µg mL −1 ( Figure 5D). methicillin, respectively, are shown in Figure 1. For the MSSA strain, the MIC of isolated OXA was 2 µ g mL −1 . In combination with EOCC, the MIC of OXA was reduced to 0.5 µ g mL −1 , changing its profile from resistant to sensitive to this antibiotic. The reduction in OXA MIC was also observed in the MRSA strain (Figure 5b). A similar effect was observed in the associations of EOCC with AMP, with a synergistic effect against the MRSA strain, with a reduction in the MIC of AMP from 16 to 0.5 µ g mL −1 (Figure 5d).

Antibiofilm Activities of C. conduplicatus Essential Oil
The antibiofilm activity of EOCC alone (at an concentration equal to the MIC) and in combination with OXA and AMP was measured against MRSA and MSSA strains. The percentages of biofilm inhibition reducing formed biofilms were evaluated ( Figure 6). Figure 7 shows the antibiofilm activity of OECC and the combinations with OXA and AMP against the MRSA strain.
The isolated EOCC at MIC showed inhibited biofilm formation in MSSA and MRSA strains by 18% and 22%, respectively. In the evaluation of the ability of EOCC to reduce formed (mature) biofilms of these strains, isolated EOCC was able to reduce the biofilm of the MSSA strain by 32% and that of the MRSA strain by 27%, highlighting the inhibition or reduction of biofilms by EOCC.

Antibiofilm Activities of C. conduplicatus Essential Oil
The antibiofilm activity of EOCC alone (at an concentration equal to the MIC) and in combination with OXA and AMP was measured against MRSA and MSSA strains. The percentages of biofilm inhibition reducing formed biofilms were evaluated ( Figure 6). Figure 7 shows the antibiofilm activity of OECC and the combinations with OXA and AMP against the MRSA strain.
The isolated EOCC at MIC showed inhibited biofilm formation in MSSA and MRSA strains by 18% and 22%, respectively. In the evaluation of the ability of EOCC to reduce formed (mature) biofilms of these strains, isolated EOCC was able to reduce the biofilm of the MSSA strain by 32% and that of the MRSA strain by 27%, highlighting the inhibition or reduction of biofilms by EOCC.
The EOCC combined with the antibiotics OXA and AMP, in subinhibitory concentrations, also showed activity, for most of the combinations, against the tested strains, with emphasis on the reduction of mature biofilm by the combination EOCC/OXA that was statistically equal (p > 0.05) the inhibition caused by EOCC (MIC concentration) against the strain MSSA and, the combination EOCC/OXA was also able to reduce the biofilm formed by the strain MRSA.
An inhibition of biofilm formation by EOCC and its combinations with OXA and AMP at concentrations lower than the MIC was also observed against the S. aureus strains tested, which was more pronounced for the combination EOCC/AMP against the MRSA strain.
The treatments applied, both regarding the inhibition of biofilm formation and of mature biofilm, showed differences only to the positive control used, indicating that the treatments were effective against MSSA and MRSA antibiofilm activities. Although there was no significant difference between the treatments, the results indicate that EOCC combined with OXA or AMP at subinhibitory concentrations has an effect similar to that of EOCC alone at a higher concentration (equivalent to the MIC).  The EOCC combined with the antibiotics OXA and AMP, in subinhibitory conce trations, also showed activity, for most of the combinations, against the tested strains, wi emphasis on the reduction of mature biofilm by the combination EOCC/OXA that w statistically equal (p > 0.05) the inhibition caused by EOCC (MIC concentration) again  The EOCC combined with the antibiotics OXA and AMP, in subinhibitory concentrations, also showed activity, for most of the combinations, against the tested strains, with emphasis on the reduction of mature biofilm by the combination EOCC/OXA that was statistically equal (p > 0.05) the inhibition caused by EOCC (MIC concentration) against

Discussion
The chemical composition of the essential oil from C. conduplicatus leaves was obtained via GC-MS. Based on the results, it is rich in bioactive compounds, mainly monoterpenes and sesquiterpenes. Generally, EOs are natural products rich in bioactive compounds, such as monoterpenes and phenylpropanoids. These compounds are used by plants as a defense against predators and microorganisms, including those that are pathogenic to humans [27].
Studies indicate that the EOs from plants belonging to the genus Croton have potential antibacterial activity. For example, Barbosa [28] reported that the antimicrobial activity of C. urticifolius EOs against strains of S. aureus and E. coli, and Rocha [29] observed that EOs obtained from the leaves of C. tetradenius and C. pulegiodorus inhibited the growth of clinical isolates of S. aureus, leading to cell death.
Other EOs from plants of the genus Croton with a chemical composition similar to that of C. conduplicatus also possess antimicrobial activity against clinically important strains, such as the activity of C. heliotropiifolius [21], C. ferrugineus [30], C. adipatus, C. thurifer and C. collinus [31] essential oils against S. aureus, Klebsiella pneunomiae, Enterococcus faecalis, Candida albicans, Mycobacterium tuberculosis, Escherichia coli and Pseudomonas aeruginosa.
The activity of C. cajucara EO against an MRSA strain was related by Azevedo et al. [32], who verified that the EO of this species contained 7-hydroxy-calamenene as the major component, with an MIC value of 4.760 µg mL −1 .
The antimicrobial properties are related to the bioactivity of the major compounds as well as to their synergistic action with the minor EO constituents [16,33]. This study presents the first report of the anti-MRSA activity of C. conduplicatus EO.
The antibacterial activity of 1,8-cineole is associated with oxidative stress and damage to the bacterial cell membrane, causing extravasation of the intracellular contents [36]. Previous studies also reported its synergistic and isolated activity, with a consequent reduction in the MIC of the antibiotic mupirocin and betalactamic antibiotics, such as penicillin, respectively, against MRSA strains [38]. A previous study [3] proved the anti-biofilm and anti-quorum-sensing activity toward MRSA strains, highlighting the importance of combating microbial biofilms to avoid complicated infections and the spread of these strains in hospitals.
Other studies also reported the action of 1,8-cineole, p-cymene, caryophyllene and spathulenol against a broad spectrum of microorganisms, including multidrug-resistant bacteria [17,18,[43][44][45]. One of the proposed mechanisms indicates that these compounds permeate the cell wall of bacteria, reversing their resistance and resensitising them to antibiotics [18,45]. A previous study showed the antibacterial activity of EO containing p-cymene against MRSA strains [46]. This compound presents a greater inhibitory potential when associated with other monoterpenes, such as carvacrol and 4-terpineol [47,48]. The p-cymene can affect the membrane integrity of MSSA and MRSA strains, facilitating the passage of other antimicrobial agents and modifying the resistance of these strains to certain antibacterials [49]. Its antibiofilm activity was reported by Miladi et al. [50], who found that p-cymene alone and in combination with tetracycline was effective in preventing biofilm formation in MRSA and MSSA strains as well as clinical S. aureus strains isolated from the human oral cavity.
However, no studies addressed the isolated action of caryophyllene and spathulenol in inhibiting MRSA and MSSA strains. However, EO that contained caryophyllene or spathulenol as one of its major compounds inhibited the action of these strains and their biofilms [51][52][53][54]. This points to the development of studies investigating the potential activity of these compounds in inhibiting MRSA or MSSA strains.
Thus, the synergistic activity of EOCC in combination with betalactamic antibiotics, such as OXA and AMP used in this study, may be related to the joining of the mechanisms of action. The EOCC, containing 1,8-cineole, p-cymene, caryophyllene and spathulenol as major components, causes damage to the cell membrane, with extravasation of intracellular contents, and betalactamic antibiotics act by inhibiting penicillin-binding proteins (PBP), preventing cell wall formation [55]. The combination of the mechanisms of action potentiates both the action of EOCC and those of OXA and AMP against MSSA and MRSA strains.
The MRSA strains have a genetic mutation that results in the production of an alternative PBP, namely PBP2, with a low affinity for penicillin [55], thus ensuring broad resistance to betalactams, except ceftaroline and ceftobiprole [56]. The presence of PBP2 in the cell wall of S. aureus is an example of a specific microbial resistance. Microbial biofilm formation, on the other hand, is a virulence factor that causes nonspecific resistance, especially when prostheses, catheters and other invasive medical devices are infected, in addition to the relationship with endocarditis and osteomyelitis [57].
Given the evidence regarding the bioactivity of the major compounds present in EOCC, it could be inferred that EO from C. conduplicatus presents a potential antimicrobial effect, especially against MRSA strains. This study considerably contributes to the knowledge about plants of the genus Croton, especially regarding the species C. conduplicatus; we confirm the antibacterial activity, synergistic activity against MRSA and MSSA strains and antibiofilm activity of the EO from the leaves of this plant species.

Obtaining Plant Drug
The leaves from C. conduplicatus were separated from the other aerial parts and then dried in a circulating oven at a temperature of 40 • C for a period of 72 h. The dried leaves were ground in a knife mill to a particle size of approximately 10 mesh, and 704.5 g of the plant drug (PD) was obtained and stored in a hermetically sealed container protected from light.

Essential Oil Extraction
The EO was obtained through the hydrodistillation technique at 100 • C, using the simple Clevenger apparatus and a heating mantle (Warmnest, UK), for 3 h. The entire PD was used, considering a ratio of 700 mL of distilled water for every 100 g of PD, the amount of distilled water needed to cover it completely. This procedure enabled the extraction of 2.2 mL of C. conduplicatus essential oil (EOCC), as shown in Figure 8. The obtained EOCC was stored under refrigeration.

Essential Oil Extraction
The EO was obtained through the hydrodistillation technique at 100 °C, using the simple Clevenger apparatus and a heating mantle (Warmnest, UK), for 3 h. The entire PD was used, considering a ratio of 700 mL of distilled water for every 100 g of PD, the amount of distilled water needed to cover it completely. This procedure enabled the extraction of 2.2 mL of C. conduplicatus essential oil (EOCC), as shown in Figure 8. The obtained EOCC was stored under refrigeration.

Gas Chromatography Coupled to Mass Spectrometry (GC-MS)
Gas chromatography-mass spectrometry (GC-MS) analysis was conducted on a Clarus 680 gas chromatograph equipped with a PALCOMBI-xt automatic injector, an Elite-

Gas Chromatography Coupled to Mass Spectrometry (GC-MS)
Gas chromatography-mass spectrometry (GC-MS) analysis was conducted on a Clarus 680 gas chromatograph equipped with a PALCOMBI-xt automatic injector, an Elite-5MS column (30 m × 0.25 mm i.d., 0.25 µm) and a Clarus SQ8S mass spectrometer (Perkin Elmer, Waltham, MA, USA). Helium gas was used as a carrier gas at a flow rate of 1 mL/min. The injector was heated to 250 • C with a 1:10 split, and 1.0 µL of the sample was injected. The oven was programmed as follows: 1st step: heating gradient at 35 • C (for 2 min) to 90 • C (for 2 min) at a rate of 10 • C/min; 2nd step: 90 to 130 • C (for 4 min) at a rate of 8 The compounds present in the EOCC were identified by comparing their respective mass spectra with those of other previously analysed compounds and with the mass spectra of the NIST database (NIST MS Search Version 2.2), with the retention index (RI) of each compound and by comparing the chemical composition of C. conduplicatus essential oils described in other studies [14,[20][21][22]. These analyses were carried out in triplicate. For the antimicrobial screening assay, the initial inocula were diluted to obtain a concentration of 2.0 to 8.0 × 10 5 CFU/mL for the bacteria and 1.0 to 5.0 × 10 3 CFU/mL for C. albicans.

Antimicrobial Agents
Oxacillin sodium (OXA) and ampicillin sodium (AMP), obtained from Teuto Laboratory, were used as antimicrobial agents. Polymyxin B sulphate was obtained from Eurofarma and Amphotericin B obtained from Cristalia Laboratory.
For sample preparation, the EOCC was solubilised in a mixture of 3 mL of dimethylsulfoxide (Neon), 2 mL of Tween 80 ® (Dinâmica) and 6 mL of sterile deionised water. The antimicrobials were solubilised in sterile distilled water. All solutions were filtered through a 0.22-µm membrane before activity testing to ensure sterility.

Antimicrobial Screening
Antimicrobial screening was performed by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal and fungicidal concentration (MBC/FMC).
The MIC of EOCC and antimicrobials was determined by the broth microdilution method as described in the CLSI documents M07 [58] and M27 [59]. For this, serial dilutions of these compounds were performed to obtain final plaque concentrations ranging from 1024 to 0.03 µg/mL. Subsequently, 100 µL of each of these dilutions was added to a 96-well plate and received 100 µL of the standardised inoculum in each well to obtain a final concentration of 1.0 to 4.0 × 10 5 CFU/mL for the bacterial strains and of 0.5 to 2.5 × 10 3 CFU/mL for C. albicans.
Growth inhibition was analysed by evaluating growth in broth compared to an untreated growth control by adding a 0.01% resazurin (Sigma-Aldrich ® , St. Louis, MO, USA) solution. The MIC was taken as the lowest concentration capable of inhibiting growth after 24 h of incubation at 35 • C. A control well of DMSO/Tween 80 ® /water diluent (3.0/1.0/6.0) was included to rule out diluent activity. Wells showing the MIC had a sample seeded onto MHB or PDB plates, which were incubated for 24 and 48 h at 35 • C, respectively, and surviving colonies were counted. The MBC or MFC was considered the lowest concentration capable of inhibiting 99.9% of microbial growth after the incubation period.

Checkerboard Assay against S. aureus
Dilutions of EOCC, oxacillin (OXA) and ampicillin (AMP) were performed in MHB. From these dilutions, 50-µL aliquots were added into 96-well microplates to obtain a final concentration equal to eight dilutions lower than the MIC of EOCC and nine dilutions lower than that of OXA and AMP. Then, 100 µL of the standardised suspension of the MSSA and MRSA strains (10 5 CFU/well) was added to each well. The plates were incubated for 24 h at 35 • C, and growth inhibition was assessed by comparison with the growth control group. The data were interpreted after calculating the Fractional Inhibition Concentration The combination was considered synergistic when FICi ≤ 0.5, additive when 0.5 < FICi ≤ 1, indifferent when 1 < FICi ≤ 2, antagonistic when FICi ≥ 2 [60]. Tests were performed in a triplicate of independent experiments.

Activity against S. aureus Biofilm Formation
To evaluate the activity of EOCC and its combinations with antifungals at subinhibitory concentrations on the formation of biofilms of S. aureus, the methodology described by Manoharan et al. [61], with modifications, was used. Starting from 24-h cultures of MSSA and MRSA strains in MHB, a 50-µL aliquot was inoculated into MHB and then incubated at 35 • C until turbidity equivalent to 0.5 McFarland scale (1.0 × 10 8 to 2.0 × 10 8 CFU/mL) was reached. From this culture, a 100-µL aliquot was dispensed into the wells of the microdilution plates. Subsequently, 100 µL of the compounds and their combinations was dispensed at final plate concentrations corresponding to the MIC and the FICi determined via the checkerboard method, and the plates were incubated at 35 ± 2 • C for 24 h. A positive control of biofilm formation was included, containing 100 µL MHB and 100 µL of the inoculum, and the negative control consisted of 200 µL MHB.
The biomass of the biofilm formed after the treatments was determined according to Munusamy, Vadivelu and Tay [23] via staining with crystal violet, with modifications.
After the incubation period, the plates were washed three times with sterile saline solution (NaCl 0.85%) to remove any cells that were not adhered to the biofilm and placed in a drying oven at 40 • C for 20 min. Subsequently, 200 µL of a 0.4% crystal violet solution was added to all wells, and the plates were incubated for 45 min. After incubation, the plates were washed with sterile distilled water and oven-dried at 40 • C for 20 min. After drying the wells, 200 µL of 96% ethyl alcohol was added for 45 min to detain the biofilm.
To perform the reading, 100 µL was removed and placed in a new 96-well flat-bottom plate. Reading was performed by optical density (OD) in a microplate reader (xMark™, Bio-Rad, Hercules, CA, USA) at a wavelength of 585 nm. The results were expressed as the percentage of inhibition of biofilm formation compared to the positive control, which represents 100% of biofilm formation. 4.5.6. Activity against S. aureus Formed Biofilm Starting from a 24-h culture of S. aureus strains in MHB, a 200-µL aliquot was inoculated into 20 mL of MHB and incubated at 35 • C until turbidity equivalent to McFarland's 0.5 scale (1.0 × 10 6 to 5.0 × 10 6 CFU/mL) was reached. From this culture, 200 µL was dispensed into all 96 wells of the microdilution plates and incubated at 35 ± 2 • C for 24 h to form the biofilm. After the incubation period, the culture medium was carefully removed, and the wells were aseptically washed three times with sterile saline solution (NaCl) at 0.85% to remove the cells that were not adhered to the wells; subsequently, the plates were sealed and placed on a flat surface for 20 min at room temperature (25 • C) for drying. The dried biofilms were spiked with 200 µL of the compounds and their combinations, and the plates were re-incubated at 35 ± 2 • C for 24 h. A positive control for biofilm formation was included, consisting of 100 µL of MHB and 100 µL of the inoculum, and the negative control consisted of 200 µL of MHB.
To evaluate the activity of EOCC and its combinations with OXA and AMP on biofilms formed by S. aureus strains, the methodology described by Uppuluri et al. [62] was used, with modifications.
After the incubation period, the culture medium was removed, and the plates were washed three times with sterile 0.85% NaCl (Dinâmica) to remove any non-adhered cells; subsequently, the plates were placed in an oven at 40 • C for 20 min for drying. The biofilm biomass was determined according to Munusamy, Vadivelu and Tay [23], with some modifications, as described above. The results were expressed as percentage of inhibition of the biofilm formed compared to the positive control (100% of biofilm formation), indicating action against the biofilm formed by S. aureus.

Statistical Analysis
The experiments were performed in triplicates of independent tests, and results were expressed as the mean and standard deviation of the percentage of biofilm inhibition, calculated in the Office Excel 2019 software. The results were submitted to statistical analysis by applying the t-test, performing analysis of variance and evaluating the statistical difference among the treatments. Statistical significance was set at p < 0.05.

Conclusions
The EOCC showed antibacterial activity against methicillin-susceptible and methicillinresistant Staphylococcus aureus strains. This effect was observed together with a potential synergistic effect when EOCC was associated with OXA and AMP, to the point of modifying the resistance profile of MSSA and MRSA strains. Furthermore, EOCC also showed a potential effect of inhibiting biofilm formation and reducing mature biofilms of MRSA and MSSA strains. These results were associated with the chemical composition of EOCC, which showed 1,8-cineole, p-cymene, caryophyllene and spathulenol as the main constituents, compounds known for their activity against multidrug-resistant bacterial strains. These results provide a solid reference for further studies on EOCC in combination with different antibiotics to evaluate its mechanism of action and propose pharmaceutical formulations, with the aim to broaden the therapeutic resources against infections caused by pathogenic microorganisms.
Author Contributions: Conceptualization, data curation, investigation, methodology, writing-original draft, extraction and treatment of plant material and essential oil, G.D.d.O.; Data curation, investigation, acquisition, visualization, interpretation of antimicrobial data, writing-original draft and maintenance of microbial strains, W.R.V.d.R.; Data curation, investigation, acquisition, visualization, analysis and interpretation of GC-MS data, J.F.B.R.; Project administration, resources, supervision,