Antibacterial Properties of Origanum vulgare and Eucalyptus staigeriana Essential Oils Single or in Combination With Cloxacillin Against Corynebacterium pseudotuberculosis

ABSTRACT

Caseous lymphadenitis is a chronic infectious disease caused by Corynebacterium pseudotuberculosis that affects small ruminants, resulting in significant economic losses. In response, essential oils (EO) have emerged as promising natural alternatives for disease control. The present study evaluated the chemical composition, antioxidant capacity, and antibacterial activity of EO from Origanum vulgare (OVEO) and Eucalyptus staigeriana (ESEO) against C. pseudotuberculosis isolated from Northeastern Brazil. The antibacterial effect was assessed by disk diffusion, minimum inhibitory concentration (MIC), Minimum Bactericidal Concentration, time–kill assays, and combination tests with cloxacillin. Gas chromatography analysis identified carvacrol as the major compound in OVEO, and D‐limonene in ESEO. OVEO demonstrated strong antibacterial efficacy, exhibiting inhibition zones of up to 74 mm and MICs ranging from 31.2 to 250 µg/mL. Both EOs demonstrated bactericidal properties and exhibited additive interactions with cloxacillin (FICi 0.56–1.03). Furthermore, OVEO exhibited strong antioxidant activity (IC₅₀ = 6.89 ± 0.58 µg/mL). These findings underscore the significance of bioactive compounds with antibacterial potential, although further studies are necessary to evaluate their applicability for managing caseous lymphadenitis.

A Caseous lymphadenitis is an infectious disease that affects small ruminants. In light of this issue, the present study proposes the use of essential oils derived from a species of Eucalyptus and Origanum to combat bacteria associated with this pathology. Both oils demonstrated in vitro activity, with the oregano essential oil showing superior efficacy.

1. Introduction

Caseous lymphadenitis (CL) is a chronic disease of goats and sheep caused by Corynebacterium pseudotuberculosis. The disease is characterized by the formation of encapsulated granulomas. The consequences of CL include significant productivity losses and elevated management costs, which exert a substantial impact on small ruminant production systems [1]. The disease has been shown to result in a variety of economic consequences, including reduced milk and wool production, reproductive failures, carcass condemnation, and decreased skin value. Fertility problems are also related, causing smaller herds and long‐term declines in productivity. It is imperative to recognize the gravity of CL, as underestimating its severity can result in substantial financial losses and detrimental impacts to herd health. This underscores the necessity for the implementation of effective disease control measures [2].

The current vaccines are expensive, and their availability differs throughout the country. The contemporary CL treatment protocol entails the opening, drainage, and cleansing with an iodine solution (10%). However, this procedure is associated with the potential for environmental contamination and lesion reappearance [3]. In this context, the antibiotic cloxacillin has been investigated as a therapeutic alternative for CL [5], including in nanotechnological formulations designed to overcome the limitations of conventional treatments [4]. This semi‐synthetic isoxazolylpenicillin demonstrates in vitro susceptibility against C. pseudotuberculosis, a wide safety margin, and is classified as a priority antimicrobial for veterinary use. Furthermore, benzathine cloxacillin exhibits a greater degree of lipophilicity compared to penicillin, a property that may contribute to its preferential interaction with lipid‐rich abscess environments [5].

Despite these advantages, the therapeutic performance of antibiotics under field conditions remains limited by poor penetration into encapsulated abscesses and difficulty sustaining effective concentrations at the infection site. This can hinder the attainment of optimal pharmacokinetic/pharmacodynamic (PK/PD) targets [5]. In this scenario, combination strategies emerge as a promising approach. Evaluating the interaction between cloxacillin and natural products, such as essential oils, could enhance antimicrobial activity and improve PK/PD target attainment in future in vivo studies (Supporting Information).

Plants synthesize a variety of secondary metabolites, (phytochemicals), that are involved in defense mechanisms. It is recognized that major classes of these molecules have beneficial effects on human and animal health, including antioxidants and antimicrobials. The attractive antioxidant and antibacterial properties of phytochemicals warrant attention because they could replace synthetic antioxidants, which can cause adverse health effects, such as cancer. The plant kingdom is rich in various phytochemicals, including phenolic acids, flavonoids, gingerol, and curcumin [6]. EOs have gained attention as an alternative for combating antibiotic‐resistant bacteria. Among EOs, Eucalyptus staigeriana (ESEO) has been highlighted in scientific literature for its notable antimicrobial, analgesic, antiseptic, insecticidal, wound‐healing, and anti‐inflammatory properties [7, 8, 9, 10]. Another essential oil with antimicrobial potential is Origanum vulgare (OVEO), also known as oregano. This plant from the Lamiaceae family exhibits various properties, including antimicrobial, anti‐inflammatory, sedative, antispasmodic and antioxidant activities [11]. Its antioxidant constituents may reduce oxidative stress, support immune function, and contribute to host defense against infections [12].

The EOs described above are obtained from plant species belonging to different families with distinct chemical compositions and profiles of biological and therapeutic activity. However, their effects on corynebacteria associated with CL cases are not well‐documented in the literature. Thus, this study aimed to evaluate the susceptibility of C. pseudotuberculosis strains isolated from small ruminants with CL to OVEO and ESEO. Additionally, we investigated the potential synergistic effect of these oils in conjunction with the antibiotic cloxacillin.

2. Results and Discussion

2.1. Chemical Characterization

The composition of OVEO varies depending on the plant's chemotype, cultivation conditions, and geographical origin. It generally includes compounds such as carvacrol, thymol, α‐pinene, and β‐caryophyllene. Carvacrol was identified as the major compound (77.12%), which is consistent with previous reports [11, 13, 14], followed by o‐cymene (11.07%). The other three compounds were identified at amounts ranging from 1%–3%, including caryophyllene, gamma‐terpinene, and linalol (Table 1).

TABLE 1.

Chemical analysis of Origanum vulgare essential oil by gas chromatography coupled to mass spectrometry.

Compound	RI	%
Alpha‐pinene	921	0.79
o‐Cymene	1010	11.07
D‐Limonene	1014	0.69
Gamma‐terpinene	1045	3.14
Linalol	1089	1.38
Carvacrol	1295	77.12
Caryophyllene	1403	3.25

Retention indices (RI) of compounds were estimated by linear regression using the Kovat´s index from the NIST library and the retention times of the main compounds.

A GC/MS analysis of the ESEO revealed 26 chemical compounds. The major components were D‐limonene (25.88%), eucalyptol (9.55%), and α‐citral (9.31%). Four compounds were detected at concentrations greater than 4%: geranyl methyl ester (7.04%), β‐citral (5.93%), α‐pinene (6.81%), and β‐pinene (4.40%) (Table 2).

TABLE 2.

Chemical analysis of Eucalyptus staigeriana essential oil by gas chromatography coupled to mass spectrometry.

Compound	RI	%	Compound	RI	%
α‐Thujene	931	0.23	Citronellal	1152	4.20
α‐Pinene	936	6.81	Terpinen‐4‐ol	1175	1.52
β‐Pinene	975	4.40	p‐Cymen‐8‐ol	1184	0.66
β‐Myrcene	991	0.41	α‐Terpineol	1189	1.01
α‐Phellandrene	1002	0.95	Citronellol	1227	1.58
o‐Cymene	1022	3.10	β‐Citral	1239	5.93
D‐Limonene	1026	25.88	cis‐Geraniol	1252	3.69
Eucalyptol	1029	9.55	α‐Citral	1267	9.31
Trans‐beta‐Ocimene	1037	0.22	Geranyl methyl ester	1317	7.04
γ‐Terpinene	1057	1.04	Citronelol acetate	1344	0.44
Terpinolene	1086	5.45	Nerol acetate	1355	1.09
Linalool	1099	0.68	Geraniol acetate	1372	3.69
Mirtenol	1144	0.89	β‐(E)‐cariofileno	1402	0.26

Retention indices (RI) of compounds were estimated by linear regression using the Kovat's index from NIST library and retention times of main compounds.

ESEO was characterized by a high content of unsaturated and oxygenated monoterpenes. D‐limonene (25.88%) was identified as the major compound, which is consistent with previous analyses of Brazilian samples [15]. The chemical composition of ESEO is highly variable and is influenced by chemotype, environmental factors, agronomic factors, genetic background, harvesting conditions, and processing methods [8, 16]. Rodrigues and Faria reported distinct chemical profiles, including three samples rich in limonene, findings consistent with the present study. Other samples showed a predominance of geranial, geraniol, and methyl geranate [17]. Similarly, Pedrotti et al. identified citral (30.51%, consisting of 19.74% geranial and 11.17% neral) as the primary component, alongside 1,8‐cineole (24.59%) and limonene (19.47%) [18]. However, Gilles et al. reported a different profile, with 1,8‐cineole (34.8%) as the predominant compound, followed by neral (10.8%), geranial (10.8%), α‐phellandrene (8.8%), and methyl geranate (5.2%). These findings underscore the presence of various chemotypes in ESEO, demonstrating significant variation in the relative proportions of monoterpenes and oxygenated compounds.

The OVEO chemotypic is a key determinant of its biological activity. Raal et al. described six main chemotypes characterized by the predominance of caryophyllene oxide, sabinene, (E)‐β‐caryophyllene, carvacrol, thymol, and carvacrol [19]. This chemical diversity directly influences the antimicrobial potential of essential oils; chemotypes rich in phenolic compounds, such as carvacrol and thymol, are generally associated with higher efficacy.

Thus, chemotypic variability may account for the differences in antimicrobial efficacy observed across studies, as well as the relative performance of ESEO compared to OVEO in the present work. Additionally, factors such as geographical origin, climatic conditions, and the developmental stage of the plant may significantly impact the final chemical composition of the oil and its biological activity [20].

2.2. Antioxidant Activity

OVEO showed antioxidant activity over 70% at concentrations between 4000 and 125 µg/mL. At 62.5 and 31.25 µg/mL, the activities were 67.26% and 58.99%, respectively. ESEO also demonstrated strong antioxidant activity, with a free radical scavenging ability of 71.50% at the highest concentration tested (4000 µg/mL), as shown in Figure 1.

FIGURE 1.

Antioxidant activity of Origanum vulgare (dark gray) and Eucalyptus staigeriana (light gray) essential oils by the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) method. Control is represented in black. Data are expressed as mean ± SD (n = 5). Statistically significant differences compared to control (*) and between oils (#) with p < 0.01 (Bonferroni test).

OVEO exhibited strong antioxidant activity (IC₅₀ = 6.89 ± 0.58 µg/mL), surpassing values reported in other regions, [21] which can be attributed to its high concentration of phenolic monoterpenes, such as carvacrol and thymol [22]. ESEO also demonstrated potent activity comparable to the standards BHT and Trolox. This effect is primarily due to its high content of unsaturated monoterpenes (e.g., α‐pinene, β‐pinene, limonene, terpinolene, and γ‐terpinene) and oxygenated functional groups (e.g., alcohols, aldehydes, ketones, and esters), which enhance free radical scavenging [23]. 1,8‐cineole stands out among these compounds due to its antioxidant and anti‐inflammatory effects, as well as its ability to interact with other monoterpenes, such as α‐pinene, thereby enhancing the oil's overall antioxidant capacity [24, 25]. OVEO and ESEO exhibited medium inhibitory concentrations (IC₅₀) of 6.89 and 10.36 µg/mL, respectively. Both oils exhibited high DPPH radical‐scavenging capacity, with IC₅₀ values lower than those of standard antioxidants (Table 3).

TABLE 3.

Antioxidant activity 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) of the essential oils Origanum vulgare (OVEO) and Eucalyptus staigeriana (ESEO).

Samples	IC50 DPPH● (µg/mL)
OVEO	6.89 ± 0.58
ESEO	10.36 ± 0.51
BHT (standard)	10.25 ± 0.02
Trolox (standard)	12.35 ± 0.05

CI₅₀, medium inhibitory concentration; BHT, Butylated hydroxytoluene.

Both OVEO and ESEO exhibited strong DPPH radical‐scavenging activity, indicating high chemical antioxidant potential. While the DPPH assay does not directly reflect biological activity under in vivo conditions, antioxidant compounds such as carvacrol, citral, and 1,8‐cineole may contribute to these essential oils' overall bioactivity. Further studies using cellular and animal models are needed to determine if these antioxidant properties have biological effects relevant to C. pseudotuberculosis infections.

2.3. Antibacterial Activity

Table 4 shows the inhibition zone diameter (IZD), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of the essential oils against the five strains of C. pseudotuberculosis tested. The ESEO IZD ranged from 21 to 29 mm, and the MIC was 500 µg/mL after 24 h for all strains. However, after 48 h, the MIC increased to 1000 µg/mL for some strains. The OVEO showed an IZD ranging from 69 to 74 mm. The MIC ranged from 31.2 to 250 µg/mL. The MIC/MBC ratio for both oils ranged from 1 to 2.

TABLE 4.

Inhibition zone diameter (IZD), minimal bacteriostatic concentration (MIC), and minimal bactericidal concentration (MBC) of Origanum vulgare (OVEO) and Eucalyptus staigeriana (ESEO) essential oils, and their respective major constituent, carvacrol and D‐limonene, against Corynebacterium pseudotuberculosis isolates.

Strains	OVEO			Carvacrol			ESEO			D‐Limonene
	IZD (mm)	MIC (µg/mL)	MBC (µg/mL)	IZD (mm)	MIC (µg/mL)	MBC (µg/mL)	IZD (mm)	MIC (µg/mL)	MBC (µg/mL)	IZD (mm)	MIC (µg/mL)	MBC (µg/mL)
BRM 064776	74.0 ± 1.3	250	250	78.3 ± 2.2	62.5	62.5	28.3 ± 2.2	1.000	2.000	NI	>4000
BRM 064777	69.0 ± 1.3	250	250	81.6 ± 2.2	125	250	21.7 ± 2.2	500	2.000
BRM 064784	70.0 ± 0.0	31,25	125	73.3 ± 2.2	62.5	62.5	23.0 ± 2.7	500	2.000
BRM 050764	72.7 ± 5.1	125	125	80.0 ± 0.3	125	125	22.3 ± 1.8	1.000	1.000
BRM 064788	69.3 ± 0.9	125	125	85.0 ± 3.3	125	125	29.3 ± 0.9	1.000	2.000

NI, no inhibition.

Preliminary antibacterial activity testing showed that OVEO exhibited zones of inhibition exceeding 60 mm, indicating strong activity, as reported by Valerio et al. [26]. ESEO showed inhibition zones above 20 mm, which is considered moderate. Previous studies have reported similar antimicrobial activity of ESEO against Staphylococcus epidermidis, S. aureus, Bacillus cereus, and other Gram‐positive strains [8, 15]. The inhibition zones observed for OVEO in this study notably exceed those previously reported against pathogens such as Salmonella spp., Pseudomonas aeruginosa, Escherichia coli, and S. aureus, which range from 9 to 36 mm depending on the oil concentration and composition [14, 25, 27]. These exceptionally large inhibition zones can be primarily attributed to OVEO's high carvacrol content (77.12%). Carvacrol is a phenolic monoterpene widely recognized for its potent antibacterial activity against Gram‐positive bactéria [30]. The remarkable susceptibility of C. pseudotuberculosis to OVEO suggests that this pathogen may be sensitive to phenolic compounds, which affect cell membrane integrity and bacterial homeostasis. Furthermore, the complex mixture of volatile constituents present in the essential oil may contribute to its overall antimicrobial effect through complementary or synergistic interactions.

Additionally, both EOs exhibited bacteriostatic activity against C. pseudotuberculosis, with MIC of ≤1000 µg/mL. OVEO was particularly potent, ranging from 31.2 to 250 µg/mL, whereas ESEO ranged from 500 µg/mL after 24 h to 1000 µg/mL after 48 h. Both oils were classified as bactericidal agents, with MBC/MIC ratios of 1 or 2 [28].

The antimicrobial effects of OVEO resemble those of carvacrol alone, suggesting that OVEO's effectiveness primarily stems from its high carvacrol content. However, the presence of other constituents, such as thymol, γ‐terpinene, and p‐cymene, may enhance its activity through synergistic interactions; for example, p‐cymene facilitates carvacrol's entry into bacterial cells [13, 24]. OVEO activity is often explained by several mechanisms, including the inhibition of bacterial enzymes, interference with motility and adhesion, disruption of quorum sensing, and, most notably, membrane disruption. The phenolic constituents of OVEO, including carvacrol, thymol, and β‐caryophyllene, disrupt membrane integrity, resulting in ion leakage and loss of cytoplasmic contents. Carvacrol causes DNA damage and inhibits DNA gyrase and ATPases. It also depletes intracellular ATP and inhibits efflux pumps, enhancing its overall antimicrobial efficacy [29, 30, 31, 32].

Previous studies have documented how C. pseudotuberculosis isolated from goats responds to EOs. Specifically, Issa reported the antibacterial effects of thyme, lavender, and peppermint EOs against this bacterium. This effect is likely due to its high content of phenolic monoterpenes, such as carvacrol and thymol, which disrupt the bacterial plasma membrane, leading to increased permeability and cell death [33, 34]. These findings are consistent with our observations for OVEO, which exhibited strong antibacterial activity due to its high carvacrol content. However, the lower efficacy of ESEO compared to OVEO against the C. pseudotuberculosis isolates evaluated in this study may be explained by its distinct chemical profile. ESEO is characterized by a high content of D‐limonene and a lack of potent phenolic compounds, such as carvacrol. Similarly, lavender and peppermint essential oils exhibited higher MIC/MBC values, further demonstrating the superior potency of phenolic‐rich oils against this pathogen [33, 35].

In vitro activity of essential oils [36] and their components, including carvacrol [37], against Corynebacterium species isolated from clinical samples further supports the present study's findings regarding the antimicrobial potential of these volatile compounds. However, the activity of OVEO and ESEO observed in this study is noteworthy, as it remains underexplored, particularly against strains isolated from CL.

2.4. Time Kill‐Curve

Time‐kill curves were evaluated using EOs at concentrations corresponding to 1× and 2× MBC over 48 h (Figure 2). Both ESEO and OVEO exhibited a bactericidal effect dependent on concentration and exposure time, with greater activity observed at higher concentrations (2× MBC), particularly after eight hours of incubation for most analyzed strains. For OVEO, however, no statistically significant difference was observed between the 1× and 2× MBC concentrations after eight hours of incubation for strain BRM 050764. The 2× MBC concentration led to bacterial death within 8 h for the other strains, while the 1× MBC concentration achieved bacterial elimination within 24 h.

FIGURE 2.

Time‐kill curves and area under the curve (AUC) analysis for Corynebacterium pseudotuberculosis strains untreated (●) and treated with 1× MBC (■) and 2× MBC (▲) of ESEO (a) and OVEO (b) over 48 h. The inset graphs show bars comparing AUC values between the control and MBC treatments (C: control; 1×: MBC; 2×: 2× MBC). Statistically significant differences compared to the control (*) and differences between EOs (#) with p < 0.01. Strain codes from left to right: BRM 064776, BRM 064777, BRM 064784, BRM 050764, and BRM 064788.

OVEO exhibited a pronounced, time‐dependent bactericidal effect, reducing viable counts even at sub‐MIC levels. Carneiro de Barros et al. reported a significant inhibitory effect of OVEO on S. aureus cell viability and observed that treatment with OVEO at a concentration of 0.596 mg/mL reduced bacterial counts to less than 2 log₁₀ CFU/mL after 2 h, indicating a rapid and consistent bactericidal rate. Similarly, Vasconcelos et al. [38] demonstrated that OVEO induced a reduction of approximately 5 log₁₀ CFU/mL in Gram‐negative bacteria, including Klebsiella pneumoniae, Serratia marcescens, and Acinetobacter baumannii, after four hours of exposure. Building upon these findings, the present study confirms OVEO's potent antimicrobial action [39]. These results, consistent with previous evidence, highlight OVEO's therapeutic potential under various exposure conditions.

Both OVEO and ESEO exhibited time‐dependent bactericidal activity: OVEO rapidly reduced bacterial counts by up to 5 log₁₀ CFU/mL, while ESEO eliminated C. pseudotuberculosis within 8 to 24 h, depending on concentration and strain. Together, these findings reinforce the promise of both essential oils as potential alternatives or adjuncts for controlling C. pseudotuberculosis.

2.5. Combinatorial Effect

Importantly, combining essential oils with antibiotics at subinhibitory concentrations can enhance the antibiotics antimicrobial effects by inhibiting efflux pumps and increasing membrane permeability. This strategy may reduce the required antibiotic dosage, mitigate the development of resistance, and broaden the antimicrobial efficacy [11]. Previous studies have shown that OVEO exhibits synergy with antibiotics, inhibiting efflux pumps and enhancing the activity of fluoroquinolones and tetracyclines against S. pneumoniae and S. aureus, respectively, by downregulating resistance‐related genes [40]. Combining EOs with cloxacillin produced an additive effect against all tested strains except one OVEO strain, which exhibited an indifferent interaction. Table 5 displays the individual and combined MIC, fractional inhibitory concentration indices (FICI), their interpretations, and fold reductions.

TABLE 5.

Fractionated inhibitory concentration index (FICI) of Origanum vulgare (OVEO) and Eucalyptus staigeriana (ESEO) essential oils in combination with cloxacillin (CLOX) against Corynebacterium pseudotuberculosis strains.

Strains	Compounds	MIC (µg/mL)		FICi	Outcome	x‐Fold reduction
		Individual	Combined
BRM 064776	OVEO	250	125	0.56	Additive	2
	CLOX	2	0.13			16
BRM 064777	OVEO	250	125	0.56	Additive	2
	CLOX	2	0.13			16
BRM 064784	OVEO	31.25	15.62	1.00	Additive	2
	CLOX	2	1			2
BRM 050764	OVEO	125	125	1.13	Indifferent	1
	CLOX	2	0.25			8
BRM 064788	OVEO	125	62.50	0.63	Additive	2
	CLOX	1	0.13			8
BRM 064776	ESEO	1,000	500	0.63	Additive	2
	CLOX	1	0.13			8
BRM 064777	ESEO	500	15.62	1.03	Additive	32
	CLOX	2	2			1
BRM 064784	ESEO	500	15.62	1,03	Additive	4
	CLOX	2	2			2
BRM 050764	ESEO	1,000	15.62	1.02	Additive	64
	CLOX	4	4			1
BRM 064788	ESEO	1,000	15.62	1.02	Additive	64
	CLOX	1	1			1

Although only additive effects were observed with cloxacillin in this study, similar resistance‐modulating mechanisms may still be involved. The FICi was 0.56–1.13 for OVEO and 0.63–1.03 for ESEO. These interactions may involve major constituents, such as 1,8‐cineole and α‐pinene, that are known to increase membrane permeability and interfere with efflux pump activity [41, 42].

These findings also have practical implications for animal production. The reductions in the MIC of cloxacillin observed (up to 16‐fold) indicate that effective antimicrobial activity can be achieved with lower doses when this antibiotic is combined with essential oils. In the context of small ruminant husbandry, this could translate to reduced treatment costs, lower antimicrobial use, and decreased selective pressure for the development of resistance [1]. Furthermore, improved drug performance at lower doses is advantageous for diseases such as CL, where lesion structure limits antimicrobial diffusion.

These results support the potential of these combinations to optimize the achievement of PK/PD targets and promote the rational use of antimicrobials in veterinary medicine. However, some limitations must be considered. The antibacterial activity observed in vitro may not directly translate to therapeutic efficacy in vivo since essential oils are susceptible to degradation, volatilization, and variations in bioavailability. Additionally, encapsulated abscesses characteristic of CL represent a significant barrier to antimicrobial agent penetration, which could limit the efficacy of essential oil constituents at the site of infection. Additionally, the pharmacokinetics, safety, and optimal administration of these compounds in small ruminants are poorly understood. Therefore, additional in vivo studies and formulation strategies aimed at improving stability and tissue penetration are necessary before these combinations can be used in practice.

3. Conclusions

In conclusion, the EOs exhibited antioxidant and antibacterial effects against C. pseudotuberculosis, with OVEO showing greater potency. When combined with cloxacillin, predominantly additive interactions were observed, as reflected by reduced MIC values. These results suggest that EOs are sustainable natural mixtures of agroecological interest. However, further studies, including in vivo models, are required for better assessing their applicability for CL control.

4. Experimental Section

4.1. Chemical Characterization of Essential Oils

The essential oils were commercially acquired from SAMIA (São Paulo, Brazil) and Ferquima (São Paulo, Brazil), respectively. The major components were purchased from Sigma‐Aldrich, São Paulo, Brazil. For chemical characterization, each EO was first diluted in chloroform for injection into gas chromatography mass spectrometer (Shimadzu QP‐2010) under the following conditions: Rtx‐5MS capillary column (Crossbond 5% diphenyl/95% dimethylpolysiloxane), dimensions 30 m × 0.25 mm × 0.25 µm) fused silica coating; helium as the carrier gas (24.2 mL/min), at a constant linear velocity; injector temperature at 250°C (split mode 1:100); detector temperature at 250°C. The temperature program started at 35°C, rising at 4°C/min up to 180°C, followed by 17°C/min up to 250°C, where it was held for 10 min. This generated a chromatogram relating the retention time to the sample peaks. Mass spectra were obtained by electron impact ionization at 70 eV. The equipment suggested possible compounds based on comparison with a mass spectra library. For definitive identification of the oil components, the obtained mass spectra and retention indices (RI) were compared with those reported in the literature, according to the NIST and Adams [43]. Experimental RI values were calculated by linear regression, interpolating the retention times and known RI values of the standard compounds observed in the chromatogram.

4.2. Antioxidant Activity

The antioxidant activity was determined using the free radical 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) (Sigma‐Aldrich, P.A.—ACS, 100% purity), as described by Torres‐Martínez et al. [44] with modifications. Each well of 96‐well microplates received 100 µL of a methanolic DPPH solution (0.1 µmol/mL; 0.004 g/100 mL) and 100 µL of EO diluted in methanol (Dynamics, P.A., ACS grade, 99.8% purity) ranging from 31.25 to 4000 µg/mL. The Ascorbic acid, at an initial concentration of 16 mg/mL, served as the positive control. The DPPH stock solution (0.06 µmol/mL) was used as the negative control, while methanol alone was used as the blank.

The microplates were kept at 20°C in the dark for 1 h, and the absorbance was measured at 517 nm using a microplate reader (Molecular Devices, Model SpectraMax Paradigm). The percentage of free radical scavenging was calculated using the following equation:

$$\mathrm{DPPH}\mathrm{scavenging}\left(\%\right)=[({\mathrm{A}}_{\mathrm{DPPH}}-{\mathrm{A}}_{\mathrm{sample}})/{\mathrm{A}}_{\mathrm{DPPH}}]\times 100$$

where A_DPPH is the concentration of DPPH solution and A_sample is the OE absorbance at different concentrations mixed with DPPH solution. The IC₅₀ (concentration required to inhibit 50% of the DPPH radicals) was calculated using the linear regression equation obtained from the calibration curve (EO concentrations versus corresponding DPPH scavenging %). For comparison, natural antioxidants (quercetin and gallic acid) and synthetic antioxidants (BHT—butylated hydroxytoluene and trolox) were also tested.

4.3. Strains and Growth Conditions

The strains, which were previously isolated from goat and sheep abscesses in various geographic regions of northeastern Brazil, were used in this study. They are part of the Collection of Pathogenic Microorganisms for Goats and Sheep (CMPCO) at Embrapa Goats and Sheep in Sobral, Brazil. The strains were preserved at ‐80°C in Brain Heart Infusion (HIMEDIA, São Paulo, Brazil) broth containing 20% glycerol. Due to the slow‐growing nature of the bacterium, a 10 µL aliquot of the stock culture was cultivated on blood agar plates for 72 h at 37°C. Then, the isolated colonies were transferred to BHI and incubated at 37°C for 24 h. Prior to the biological assays, the turbidity of the bacterial suspension was adjusted to the 0.5 McFarland standard (∼10⁸ CFU/mL), and the suspension was diluted in fresh TSB to achieve an appropriate cell concentration for each experimental method.

4.4. Disk Diffusion Test

The disk diffusion method was used as a screening tool to evaluate the antimicrobial activity. A bacterial suspension of ∼10⁸ CFU/mL was spread on BHI agar plates using a sterile swab. Paper disks (≈6 mm in diameter) were impregnated with 10 µl of pure EO, placed on the plates, and incubated at 37°C for 24 h. The diameters of the inhibition zones were measured in millimeters. The antibacterial activity of the essential oils was classified as follows: inhibition zones <10 mm were considered very low, 20–40 mm moderate, 40–60 mm high, and >60 mm very high [21].

4.5. Manipulations of Antimicrobial Agents

ESEO and OVEO stock solutions were prepared at 32 000 µg/mL in sterile BHI with 0.1% Tween 80 (Sigma‐Aldrich, São Paulo, Brazil). Based on the oil densities (0.916 and 0,940 g/mL for OEES and OEOV, respectively), EOs were first mixed with Tween 80 (15 µL), and volume was brought to 2 mL with fresh BHI. The control group (untreated cells) in the experimental assays was used only with broth media plus Tween 80 in an equivalent amount for each EO concentration. Cloxacillin sodium salt was purchased from Thermo Scientific (cat. No: J64314.03) and prepared in BHI at a concentration of 256 µg/mL. Each solution was previously inoculated onto BHI agar plate to verify any contamination 24 h before tests.

4.6. Microdilution Test

The antibacterial activity of the essential oils was evaluated using the broth microdilution method, as described by Santos et al. [45]. MIC was determined in 96‐well polystyrene microplates (KASVI, Paraná, Brazil). Serial dilutions of EOs were prepared in Mueller‐Hinton (MHB, Kasvi, Paraná, Brazil) medium to obtain 100 µL of EO at concentrations ranging from 31.25 to 4000 µg/mL. Then, 100 µL of bacterial suspension (adjusted to 1 × 10⁶ CFU/mL) was added to each well. The plates were incubated at 37°C for 48 h, and bacterial growth was visually assessed. The MIC was defined as the lowest EO concentration that inhibited visible growth after 24 and 48 h of incubation compared to the negative control (untreated cells). Additionally, to determine the MBC, 10 µL from wells with no visible bacterial growth was inoculated onto MHA plates and incubated at 37°C for 24 h. Following incubation, the MBC was defined as the lowest concentration of the compounds that resulted in no bacterial growth on the agar surface.

4.7. Time‑Kill Curve

The bactericidal effect of essential oils was evaluated by the time‐kill curve assay, following Quirino et al. [46] with modifications. The assay was performed using MBC and 2× MBC concentrations for each C. pseudotuberculosis strain. Three test solutions were prepared: a bacterial suspension at 2 × 10⁶ CFU/mL in BHI broth; OVEO in BHI broth with bacterial suspension; and ESEO in BHI broth with bacterial suspension, both at MBC and 2xMBC concentrations. Each solution added 1:1 (v:v) ratio to microtubes and incubated at 37°C. At 0, 8, 24, 30 and 48 h, 20 µL aliquots were removed and serially diluted in 180 µL of 0.85% saline, plated on BHI agar, and incubated at 37°C for 48 h. Colony counts were then performed. Bactericidal activity was defined as a 99.9% reduction (>3 log₁₀) in CFU/mL compared to the initial inoculum.

4.8. Combinatorial Assay

The synergistic interaction between EOs and Cloxacillin (CLOX) was assessed by the checkerboard assay, as described by Owen et al. [47]. Serial dilutions of each antimicrobial agent were prepared in BHI medium in 96‐well microplates: 50 µL of EO (1:1, v/v) were added across the rows, and CLOX across the columns, starting from their respective MICs. The combinations were mixed, resulting in each well containing a unique EO‐CLOX ratio. Then, 100 µL of bacterial inoculum (1 × 10⁶ CFU/mL) were added to each well and incubated at 37°C for 24 h under aerobic conditions. Results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines as follows: synergism (FICI ≤ 0.5, combined effect greater than individual agents), additive (FICI > 0.5 to ≤ 1, combined effect equals the sum of individual agents); indifference (FICI > 1 to < 2, no enhancement or reduction), antagonism (FICI ≥ 2, combined effect less than individual agents) [48].

4.9. Statistical Analysis

All experiments were performed using independent biological replicates and technical replicates, appropriate for each assay. Antioxidant activity (DPPH assay) was evaluated using five independent replicates (n = 5). MIC and MBC determinations were performed in triplicate for each strain and treatment condition. Time–kill curve assays were conducted in three independent experiments. Data are expressed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 8.4.3 (GraphPad Software, Inc., San Diego, CA, USA), applying one‐way ANOVA followed by Tukey's post hoc test for time–kill assays and Bonferroni's test for antioxidant activity. Values of p < 0.01 were considered statistically significant.

Author Contributions

Wanderléia de Aguiar Policarpo: investigation, methodology, resources. Data curation, Writing – original draft. Felipe José Negreiros de Carvalho: methodology. Benise Ferreira da Silva: methodology. Wildson Max Barbosa da Silva: methodology. Victor Alves Carneiro: methodology, writing – original draft, funding acquisition, project administration, writing – review & editing. Renata Albuquerque Costa: conceptualization, data curation, formal analysis, project administration, writing – review & editing. Patrícia Yoshida Faccioli‐Martins: conceptualization, data curation, formal analysis, funding acquisition, project administration, writing – review & editing. Selene Maia de Morais: conceptualization, data curation, formal analysis, funding acquisition, project administration, writing – review & editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: cbdv71523‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.