Antimicrobial Activity of Origanum vulgare L. And Salvia rosmarinus Spenn (syn Rosmarinus officinalis L.) Essential Oil Combinations Against Escherichia coli and Salmonella typhimurium Isolated from Poultry

Abstract

The ban on antibiotic growth promoters (AGPs) in poultry farming has prompted the search for effective, natural alternatives. Essential oils (EOs), such as those from oregano (Origanum vulgare: OVEO) and rosemary (Salvia rosmarinus: ROEO), possess antimicrobial and antioxidant properties that may contribute to intestinal health and pathogen control. This study evaluated the antibacterial activity of OVEO and ROEO, individually and combined, against six Escherichia coli and six Salmonella typhimurium strains isolated from healthy poultry via cloacal swabs, as well as E. coli ATCC 25922 and S. typhimurium ATCC 14028 strains. The minimum inhibitory concentrations (MIC) were determined at five pH levels (7.4–5) simulating avian gastrointestinal tract conditions. EO composition was determined by GC-FID-MS. Checkerboard assays revealed partial or full synergistic effects at most pH, especially under acidic environments (pH ≤ 5.5), where the fractional inhibitory concentration (ΣFIC) values often indicated synergy. No antagonistic interactions were observed. These results suggest that OVEO and ROEO combinations are promising candidates to replace AGPs in poultry, particularly because of their enhanced efficacy under gastrointestinal pH. The strategic use of EO blends may reduce pathogen load, support performance, and limit antimicrobial resistance development, suggesting their potential as natural alternatives to AGPs under One Health principles.

1. Introduction

The excessive and inappropriate use of antimicrobials in both human and veterinary medicine has significantly contributed to the global rise of antimicrobial resistance (AMR) [1]. In animal production, antimicrobials have traditionally been employed not only for therapeutic and prophylactic purposes but as antimicrobial growth promoters (AGPs) administered at subtherapeutic levels to improve feed conversion and weight gain. However, the emergence of multiresistant and panresistant bacterial strains has led to the implementation of stricter regulations, including the ban of AGPs in the European Union since 2006 [2,3] and growing restrictions in other countries, such as Argentina.

The reduction in the use of AGP poses a significant challenge for poultry production, which must maintain high efficiency and economic viability to meet growing global food demand [4]. Consequently, alternative strategies to promote growth and health in poultry flocks are urgently needed. Among these alternatives, plant-derived bioactive compounds, particularly essential oils (EOs), have gained attention due to their antimicrobial, antioxidant, and growth-promoting properties [5,6,7,8].

The gastrointestinal tract (GIT) of broiler chickens is a complex ecosystem. A significant portion of digestion and nutrient absorption takes place in the duodenum and jejunum. The integrity of the intestinal epithelium and a balanced microbiota are essential for efficient absorption and immune function. Beneficial bacteria contribute to digestion and pathogen resistant control, but stress, malnutrition, and poor management in intensive systems may promote the proliferation of pathogenic microorganisms. This can lead to inflammation of the intestinal epithelium, thickening of the lamina propria, impaired absorptive capacity, and ultimately impairing nutrient absorption and productive performance [9].

Multiple, often unpredictable factors influence the equilibrium between commensal and pathogenic microorganisms. Intestinal commensals play an important role not only in nutrient absorption and immune modulation but in preventing colonization by resistant strains, especially when antimicrobial pressure is high [10]. One widely adopted strategy to maintain this balance has been the use of in-feed antimicrobials [11], which reduce pathogen levels and support beneficial flora, thereby improving feed conversion, daily weight gain, and reducing disease-related mortality [12]. Microbial distribution along the digestive tract is closely related to regional pH, with differences of up to 1.8 units between the crop and the rectum [13]. Colonization resistance by the commensal microbiota is a key protective factor in chicks and adult birds, involving both competitive exclusion and immune modulation [14]. The development of the intestinal microbiome starts during embryogenesis, and its composition at hatch can be influenced by conditions in the incubator and egg hygiene [15]. However, certain commercial practices compromise this microbial development, increasing vulnerability to infections by Salmonella gallinarum, Escherichia coli, and Clostridium perfringens. The latter causes necrotic enteritis by displacing beneficial Lactobacillus aviarius [16]. Salmonella spp. can colonize the intestinal mucosa from contaminated feed, while E. coli overgrowth or emergence of pathogenic strains is often linked to hygiene failures or hatchery contamination [17].

Consequently, controlling these pathogens is critical for animal health, public safety, and production efficiency. In this context, international agencies such as World Alliance Against Antibiotic Resistance (WAAAR), World Health Organization (WHO), and Food and Agriculture Organization of the United Nations (FAO) advocate for the restriction of AGPs and the implementation of alternative strategies [18]. Among such strategies, EOs have shown great promise due to their multiple bioactive properties and reduced risk of antimicrobial resistance development [5,6,8].

Essential oils derived from Origanum vulgare L. (oregano) and Salvia rosmarinus Spenn., syn. Rosmarinus officinalis L. (rosemary) are rich in bioactive compounds such as carvacrol, thymol, and 1,8-cineole, which are well-documented for their ability to disrupt microbial membranes and interfere with cellular metabolism [19,20]. In poultry, these oils have been associated not only with antimicrobial effects but with improved gut morphology, enzyme activity, and immune responses. For example, oregano oil combined with organic acids has been reported to increase villus height, enhance trypsin and chymotrypsin activity, and elevate IgA levels in the ileal mucosa [21,22]. Additionally, it delays lipid oxidation in meat products, improving shelf life and quality [23].

Previous studies have highlighted the potential of essential oil (EO) blends to improve poultry performance. For instance, Koiyama et al. [24] and Mathlouthi et al. [19] reported that combinations including cinnamon, thyme, clove, ginger, copaiba resin, rosemary, and oregano improved weight gain and feed conversion in broilers, achieving effects comparable to those of antibiotic growth promoters such as virginiamycin.

Since EOs act at multiple cellular targets and pathways, they may reduce the risk of resistance development [5]. Thus, selecting appropriate EO types and concentrations could offer a viable alternative to AGP, sustaining performance indicators and animal health.

Interestingly, EO efficacy is pH-dependent, with enhanced activity often observed under acidic conditions [13,25]. Considering the pH gradient along the avian digestive tract, in vitro studies simulating these environments are essential for predicting EO behavior in vivo.

The present study aimed to evaluate the antibacterial activity, both individual and combined, of oregano and rosemary EOs against E. coli and S. typhimurium strains isolated from poultry. The investigation was conducted across a range of pH values representative of different GIT compartments, assessing potential synergistic effects via the checkerboard method.

2. Materials and Methods

2.1. Essential Oil Extraction and Characterization

Essential oils (EOs) of oregano (OVEO) and rosemary (ROEO) were obtained by steam distillation from fresh leaves and herbaceous branches collected in San Javier, Córdoba, Argentina (latitude −31.949838 and longitude −65.066850). Voucher specimens were deposited in the LPAG herbarium at the Faculty of Agrarian and Forestry Sciences, UNLP [26]. The EOs were extracted at three time points: 5 November 2021 (spring), 15 November 2022 (spring), and 8 May 2023 (autumn). Meteorological conditions during these harvests were 12.5–30.7 °C, 12.4–34.5 °C, and 6.6–24.5 °C, respectively, under clear skies. Plants harvested in late spring (2021 and 2022) were in a postflowering stage with residual floral structures, whereas those collected in autumn were senescent and flowerless.

After extraction, the oils were dried over anhydrous sodium sulfate, filtered through cotton funnels, and stored in amber glass bottles at –20 °C until use. To minimize variability, fractions from each collection were pooled into a single batch per species, ensuring batch-to-batch consistency.

The chemical composition was analyzed by gas chromatography coupled with flame ionization detection and mass spectrometry (GC-FID-MS) using a PerkinElmer Clarus 500 system (PerkinElmer, Inc., Waltham, MA, USA). This instrument was equipped with a single split/splitless injector (split ratio 1:100). Through a flow divider, the system was connected to two fused silica capillary columns: (a) a polyethylene glycol column with an approximate molecular weight of 20.000 (DB-Wax, J&W Scientific, Folsom, CA, United States) and (b) a column consisting of 5% phenyl and 95% dimethylpolysiloxane (DB-5, J&W Scientific). Both columns were 60 m in length, with an internal diameter of 0.25 mm and a stationary phase thickness of 0.25 μm. The polar column was coupled to a flame ionization detector (FID), whereas the nonpolar column was connected to both a second FID and a quadrupole mass detector (operated at 70 eV) via an MSVent™ system. Helium served as the carrier gas at a constant flow of 1.87 mL/min. The oven temperature program was set from 90 °C to 225 °C at 3 °C/min, followed by an isothermal step of 15 min. The injector and FIDs were maintained at 255 °C and 275 °C, respectively. A 0.2 µL sample of pure oil was injected. For the MS analysis, the transfer line and ion source were kept at 180 °C and 150 °C, respectively, and a scanning mass range of 40–300 Da was used (10 scan/s). Compound identification was based on comparison of linear retention indices (calculated relative to a homologous C8–C20 alkane series) obtained from both columns with those of authentic references. Additionally, the acquired mass spectra were compared with commercial electronic libraries [27,28] as well as a reference collection of spectra compiled in the laboratory from authenticated standards and previously characterized oils. The relative composition of the samples was calculated using the peak area normalization method, without correction for response factors or use of internal standards. For each compound, the lowest response obtained from both columns was considered.

2.2. Microorganism Isolation from Poultry and Identification

Cloacal swabs were collected from broiler chickens (36 days old) from three commercial farms in Buenos Aires province (two in La Plata city and one in Lujan). Twenty-five birds per farm were sampled. The sampling procedure involved catching the animal by gently holding it from the back with both hands. While gently restraining the bird, a sterile swab was introduced through the cloaca until reaching the coprodeum. The protocol followed the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Federation of Animal Science Societies—FASS) and was approved by the Institutional Committee (CICUAL) of the Faculty of Veterinary Sciences, National University of La Plata (89-4-18T, 6 December 2018).

For the isolation of E. coli, the swabs were transported at 4 °C in soft nutrient agar and then plated on EMB agar (which contains eosin and methylene blue). Iridescent green colonies were selected as presumptive E. coli colonies, which were confirmed by IMViC tests (indole, methyl red, Voges–Proskauer, and citrate) and Gram staining.

To isolate S. typhimurium, the swabs were transported at 4 °C in soft brain–heart infusion agar (Biokar Diagnostics, France) and plated on Salmonella–Shigella agar. Characteristic black colonies were confirmed by TSI (triple sugar iron agar), Simmons citrate, and SIM (sulfide, indole, and mobility) tests, along with Gram staining.

After biochemical typing, twenty-six (n = 26) E. coli isolates and twenty-three (n = 23) S. typhimurium isolates were obtained. All isolates were preserved at −80 °C in cryovials with trypticase soy broth (TSB) (Biokar Diagnostics, France) enriched with 20% glycerol (Sintorgan, Argentina).

All culture media were supplied by Britania (Argentina), unless otherwise indicated.

2.3. Minimum Inhibitory and Bactericidal Concentrations

Six isolates per species were selected. The minimal inhibitory concentrations (MICs) of ROEO and OVEO were determined by broth microdilution using 96-well polystyrene microplates with Mueller–Hinton broth (MHB) (Biokar Diagnostics, France) supplemented with 0.5% Tween 80 (Biopack, Argentina). The pH of the broth was adjusted to 5, 5.5, 6, 6.5, and 7.4 by adding hydrochloric acid (Anedra, Argentina). The EO concentrations tested ranged from 100 to 0.18 μL/mL. For each EO, each well was inoculated with a final bacterial concentration of 5 × 10⁵ CFU/mL. The microplates were incubated at 37 °C for 18–24 h. The MIC was established as the lowest concentration that inhibited bacterial growth. Each determination was performed in triplicate. For quality control, E. coli ATCC 25922 and S. typhimurium ATCC 14028 were used [29]. Positive and negative controls containing MHB with Tween 80 (0.5%) were included in the test. The concentration of Tween 80 used in the culture medium did not affect bacterial growth [25].

The minimum bactericidal concentration (MBC) was determined by inoculating 10 μL of each well that did not show evident bacterial growth (after establishing the MIC) onto nutrient agar plates for colony counting after incubation at 37 °C for 18–24 h. The MBC was established as the first antimicrobial concentration that produced a 99.9% drop with respect to the initial inoculum.

MIC and MBC are reported as µL/mL to preserve continuity with our previous work; densities are provided to allow conversion to mass units (ROEO density = 0.8612 g/mL; OVEO density = 0.8915 g/mL). Conversions to mg/mL may be performed by multiplying µL/mL by the corresponding density (g/mL) and converting g to mg.

2.4. EO Interaction Assay

The interactions of the EOs at all the pH values mentioned above were established using the checkerboard technique [30] against the same six isolates per species. Synergistic interactions were assessed at pH 5, 5.5, 6, 6.5, and 7.4.

The microplate design consisted of a row with a double serial dilution of ROEO and a column with a double serial dilution of OVEO (MIC control of the EOs). The intermediate wells contained combinations of ROEO/OVEO in different proportions. The bacterial inocula of E. coli and S. typhimurium were 5 × 10⁵ CFU/mL per well and the incubation was carried out at 37 °C for 18–24 h. The MIC was established as the combination that inhibits bacterial growth.

The interpretation of the results was similar for the MIC of the individual EOs but considering them as a mixture. The fractional inhibitory concentration (ΣFIC) index was determined using the following equation:

ΣFIC = (A/MIC_a) + (B/MIC_b)

where A and B are the concentrations of each EO in the mixture. Interaction was interpreted as synergism (S) if ΣFIC ≤ 0.5, partial synergism (PS) if 0.5 < ΣFIC < 1, indifference or addition (I) if 1 ≤ ΣFIC < 2, and antagonism (A) if ΣFIC ≥ 2 [30].

2.5. Statistical Analysis

MIC values were analyzed as a function of pH using the Friedman test for repeated measures (Statgraphics Centurion XVII, Statgraphics Technologies, Inc., VA, USA). For ΣFIC data, the frequency of synergism (ΣFIC ≤ 0.5), partial synergism (0.5 < ΣFIC ≤ 1), and indifference (1 < ΣFIC ≤ 2) was summarized by pH. Proportions were calculated and 95% confidence intervals (CI) were estimated using the Wilson method.

3. Results

3.1. Essential Oil Extraction and Characterization

The essential oils from oregano (OVEO) and rosemary (ROEO) were successfully extracted by steam distillation and characterized using gas chromatography with flame ionization detection and mass spectrometry (GC-FID-MS). The extraction yield was 0.3% v/w for the three OVEO samples and 0.9% v/w for all ROEO distilled fractions. All the fractions of each EO were combined in a single flask for further processing. The total identified components accounted for 93.5% of OVEO and 94.5% of ROEO, indicating a well-defined and rich chemical composition.

As shown in

Table 1. Chemical composition of OVEO determined by GC-FID-MS. Data are expressed as a relative percentage of detected compounds.

Tr	LRIN	COMPOUND	%	Tr	LRIN	COMPOUND	%
8.6	933	α-Thujene	1.1	13.6	1099	trans-Sabinene hydrate	4.0
8.9	945	α-Pinene	0.9	14.3	1105	Linalool	0.4
9.3	961	Camphene	0.1	14.9	1110	3-Octen-1-yl acetate	0.3
9.7	971	3-Octen-1-ol	1.0	16.4	1197	Terpinen-4-ol	10.6
9.8	985	Sabinene	5.4	17.0	1211	α-Terpineol	1.0
9.9	992	β-Myrcene	2.0	18.3	1243	Carvacrol methyl ether	5.9
10.0	994	ß-Pinene	0.4	20.7	1295	Thymol	2.0
10.6	1017	α-Phellandrene	0.4	21.1	1298	Carvacrol	4.5
10.9	1025	α-Terpinene	7.5	25.7	1437	ß-Caryophyllene	5.0
11.1	1034	p-Cymene	7.5	27.8	1458	allo-Aromadendrene	0.2
11.2	1038	Limonene	0.9	28.0	1496	Germacrene D	1.0
11.3	1043	ß-Phellandrene	1.2	28.6	1508	Bicyclogermacrene	1.2
11.4	1048	trans-ß-Ocimene	0.3	28.7	1510	ß-Bisabolene	0.8
12.1	1060	γ -Terpinene	24.0	32.1	1596	Spathulenol	0.1
12.5	1067	cis-Sabinene hydrate	0.9	32.2	1601	Caryophyllene oxide	0.1
12.9	1095	Terpinolene	2.8
		TOTAL					93.5

Note: Order of elution in the nonpolar column. LRIN: Experimental linear retention indices in the nonpolar column. Tr: retention time in nonpolar column.

, γ-terpinene (24.0%), terpinen-4-ol (10.6%), carvacrol methyl ether (5.9%), carvacrol (4.5%), and thymol (2.0%) were identified as the major constituents of OVEO. These molecules are known for their broad-spectrum antimicrobial activity, particularly carvacrol and thymol, which disrupt bacterial membranes and impair essential cellular functions. In contrast, ROEO (

Table 2. Chemical composition of rosemary essential oil (ROEO) determined by GC-FID-MS. Data are expressed as a relative percentage of detected compounds.

Tr	LRIN	COMPOUND	%	Tr	LRIN	COMPOUND	%
8.6	933	α-Thujene	3.3	12.0	1060	γ-Terpinene	3.3
8.7	937	Tricyclene	0.1	12.9	1095	Terpinolene	0.8
8.9	945	α-Pinene	13.3	13.2	1105	Linalool	0.7
9.4	961	Camphene	5.4	15.4	1141	Camphor	7.6
9.8	985	Sabinene	0.3	16.2	1163	Borneol	1.0
9.9	992	β-Myrcene	29.8	16.4	1197	Terpinen-4-ol	0.9
10.0	994	ß-Pinene	4.1	17.5	1204	Verbenone	0.7
10.6	1017	α-Phellandrene	0.4	16.9	1211	α-Terpineol	0.8
10.9	1025	α-Terpinene	1.3	20.1	1285	Bornyl acetate	0.3
11.1	1034	p-Cymene	0.5	25.7	1437	ß-Caryophyllene	1.0
11.2	1038	Limonene	2.7	27.0	1455	α-Humulene	0.9
11.4	1040	Eucalyptol (1,8-Cineole)	15.2	32.2	1601	Caryophyllene oxide	0.1
		TOTAL					94.5

Note: Order of elution of the nonpolar column. LRIN: Experimental linear retention indices in the nonpolar column. Tr: retention time in nonpolar column.

) was characterized by high levels of myrcene (29.8%), eucalyptol (15.2%), and α-pinene (13.3%), which are typically associated with moderate antimicrobial efficacy and potent antioxidant effects. The presence of eucalyptol, in particular, has been associated with the inhibition of microbial enzymes and modulation of efflux pumps [31].

The chromatographic profiles of both EOs (Figure 1 and Figure 2) confirmed a high content of monoterpenes and phenolic derivatives. The distinct chemical profiles of OVEO and ROEO suggest their potential as complementary agents when used in combination, offering diverse antimicrobial mechanisms and improved functional properties.

3.2. Minimum Inhibitory and Bactericidal Concentrations

The MICs of both EOs were determined against six E. coli and six S. typhimurium isolates, as well as ATCC control strains for both bacterial species, at five different pH values (7.4, 6.5, 6.0, 5.5, and 5.0). The results showed that OVEO exhibited greater antimicrobial activity than ROEO across all pH conditions and for both bacterial species (

Table 3. Minimum inhibitory and bactericidal concentration (MIC and MBC) values (µL/mL) and MBC/MIC ratio (R) of OVEO and ROEO, against E. coli strains at different pH conditions; minimum inhibitory concentration values (µL/mL) of OVEO and ROEO mixed; and ΣFIC index per the checkerboard method.

		* OVEO E. coli			* ROEO E. coli			MIC ROEO/OVEO (µL/mL/µL/mL)	ΣFIC	Interaction
Strain	pH	MIC (µL/mL)	MBC (µL/mL)	R MBC/MIC	MIC (µL/mL)	MBC (µL/mL)	R MBC/MIC
3HE1	7.4	6	6	1	50	100	2	6/3	0.62	PS
	6.5	3	3	1	12.5	50	4	1.5/1.5	0.62	PS
	6.0	3	3	1	6	25	4	3/0.7	0.73	PS
	5.5	3	3	1	6	12.5	2	3/0.7	0.73	PS
	5.0	6	12.5	2	12.5	50	4	1.5/1.5	0.37	S
5HE1	7.4	6	6	1	25	100	4	1.5/3	0.56	PS
	6.5	6	6	1	25	100	4	1.5/3	0.56	PS
	6.0	3	3	1	12.5	50	4	3/1.5	0.74	PS
	5.5	6	6	1	12.5	50	4	1.5/1.5	0.37	S
	5.0	3	3	1	12.5	12.5	1	1.5/1.5	0.50	S
7HE1	7.4	6	6	1	25	100	4	1.5/3	0.56	PS
	6.5	3	3	1	25	100	4	12.5/1.25	0.92	PS
	6.0	6	6	1	12.5	50	4	3/1.5	0.49	S
	5.5	3	3	1	3	25	8	1.5/1.5	1.00	I
	5.0	6	6	1	12.5	50	4	3/1.5	0.49	S
13HE1	7.4	3	3	1	3	100	8	1.5/3	0.74	PS
	6.5	3	3	1	12.5	100	8	0.15/0.3	0.07	S
	6.0	3	3	1	6	25	4	1.5/1.5	0.75	PS
	5.5	3	3	1	6	12.5	2	1.5/1.5	0.75	PS
	5.0	6	6	1	3	6	2	1.5/1.5	0.08	S
16HE1	7.4	6	6	1	6	50	8	1.5/3	0.75	PS
	6.5	12.5	12.5	1	12.5	100	8	1.5/12.5	1.12	I
	6.0	3	6	2	6	25	4	3/1.5	1.00	I
	5.5	6	6	1	12.5	25	2	1.5/1.5	0.37	S
	5.0	6	6	1	12.5	12.5	1	1.5/1.5	0.37	S
17HE1	7.4	6	6	1	6	25	4	1.5/3	0.75	PS
	6.5	25	25	1	12.5	25	2	1.5/6	1.25	I
	6.0	12.5	12.5	1	12.5	25	2	3/6	0.48	S
	5.5	25	25	1	12.5	50	4	1.5/6	0.36	S
	5.0	6	6	1	12.5	12.5	1	1.5/1.5	0.37	S
ATCC 25922	7.4	3	6	2	6	25	4	1.5/1.5	0.50	S
	6.5	3	3	1	6	100	8	1.5/3	0.75	PS
	6.0	3	3	1	12.5	100	8	1.5/3	1.12	I
	5.5	6	6	1	12.5	12.5	1	1.5/1.5	0.37	S
	5.0	3	3	1	12.5	12.5	1	1.5/1.5	0.72	PS

FIC interpretation: S (synergism) ≤ 0.5; PS (partial synergism) = 0.5–1; I (indifference) = 1–2; * ROEO density = 0.8612 g/mL and OVEO density = 0.8915 g/mL.

and

Table 4. Minimum inhibitory and bactericidal concentration (MIC and MBC) values (µL/mL) and MBC/MIC ratio (R) of OVEO and ROEO, against S. typhimurium strains at different pH conditions; minimum inhibitory concentration values (µL/mL) of OVEO and ROEO mixed; and ΣFIC index per the checkerboard method.

		* OVEO S. typhimurium			* ROEO S. typhimurium			MIC ROEO/OVEO (µL/mL/µL/mL)	ΣFIC	Interaction
Strain	pH	MIC (µL/mL)	MBC (µL/mL)	R MBC/MIC	MIC (µL/mL)	MBC (µL/mL)	R MBC/MIC
1HS1	7.4	12.5	12.5	1	50	>100	>2	1.5/6	0.51	PS
	6.5	12.5	12.5	1	50	>100	>2	1.5/6	0.51	PS
	6.0	12.5	12.5	1	100	>100	>1	1.5/6	0.49	S
	5.5	12.5	12.5	1	100	>100	>1	1.5/12.5	1.02	I
	5.0	6	6	1	100	>100	>1	1.5/6	1.02	I
5HS1	7.4	12.5	12.5	1	50	100	2	1.5/6	0.51	PS
	6.5	12.5	12.5	1	25	100	4	0.7/6	0.50	PS
	6.0	12.5	12.5	1	100	>100	>1	0.3/0.6	0.05	S
	5.5	6	6	1	50	100	2	3/6	0.56	PS
	5.0	12.5	12.5	1	50	50	1	1.5/6	0.51	PS
6HS1	7.4	12.5	12.5	1	50	100	2	1.5/6	0.51	PS
	6.5	12.5	12.5	1	50	100	2	0.7/1.5	0.13	S
	6.0	12.5	12.5	1	100	>100	>1	1.5/6	0.50	S
	5.5	6	6	1	50	100	2	3/6	1.06	I
	5.0	12.5	12.5	1	50	100	2	1.5/6	0.51	PS
13HS1	7.4	125	12.5	1	100	>100	>1	1.5/6	0.50	S
	6.5	6	12.5	2	50	100	2	0.7/6	1.01	I
	6.0	12.5	12.5	1	100	>100	>1	1.5/6	0.50	S
	5.5	6	6	1	50	100	2	1.5/6	0.54	PS
	5.0	125	12.5	1	100	100	1	3/6	0.51	PS
15HS1	7.4	12.5	12.5	1	100	100	1	1.5/6	0.50	PS
	6.5	6	12.5	1	50	100	2	0.7/6	1.01	I
	6.0	12.5	12.5	1	100	>100	>1	1.5/6	0.50	S
	5.5	6	6	1	50	100	2	1.5/6	1.03	I
	5.0	12.5	12,5	1	100	100	1	1.5/6	0.49	S
24HS1	7.4	12.5	12.5	1	100	>100	>1	1.5/6	0.50	S
	6.5	6	12.5	2	50	50	1	1.5/6	1.03	I
	6.0	12.5	12.5	1	100	>100	>1	3/6	0.51	PS
	5.5	6	12.5	2	100	>100	>1	3/6	1.03	I
	5.0	12.5	12.5	1	100	>100	>1	1.5/6	0.49	S
ATCC 14028	7.4	6	12.5	2	25	50	2	12.5/3	1.00	PS
	6.5	6	6	1	25	25	2	0.7/6	1.01	I
	6.0	12.5	12.5	1	50	>100	>2	1.5/6	0.51	PS
	5.5	6	6	1	12.5	12.5	1	1.5/3	0.62	PS
	5.0	6	6	1	25	50	2	1.5/6	1.06	I

FIC interpretation: S (synergism) ≤ 0.5; PS (partial synergism) = 0.5–1; I (indifference) = 1–2; * ROEO density = 0.8612 g/mL and OVEO density = 0.8915 g/mL.

).

For E. coli, MIC values for OVEO ranged from 3 to 12.5 µL/mL, while ROEO MICs varied from 3 to 50 µL/mL. Against S. typhimurium, OVEO MICs ranged from 6 to 12.5 µL/mL, whereas ROEO values extended from 25 to 200 µL/mL. Lower MICs were frequently observed under acidic conditions (pH 5.0–5.5), suggesting enhanced activity in environments resembling the distal gastrointestinal tract. Indeed, both EOs showed improved activity at acidic pH, but statistical comparisons revealed no significant differences for OVEO or ROEO against E. coli (p = 0.725 and p = 0.556, respectively), nor for OVEO against S. typhimurium (p = 0.010). Only ROEO displayed pH-dependent variation against S. typhimurium (p = 0.003), but none of these effects remained significant when applying the stringent threshold (p < 0.001, 99.9% CI).

MBC values followed a similar trend, with bactericidal concentrations typically one to two dilution steps higher than the MICs for OVEO against both E. coli and S. typhimurium (Table 3 and Table 4), indicating that this EO exhibited both inhibitory and bactericidal effects. However, using the ROEO, the MBC/MIC ratios ranged 1 to 8 for E. coli and from 1 to >16 for S. typhimurium (Table 3 and Table 4). Thus, the antimicrobial effect varied between bactericidal and bacteriostatic depending on the strain evaluated.

3.3. Antimicrobial Interaction Between OVEO and ROEO

The antimicrobial activity of OVEO and ROEO combinations was also evaluated against E. coli (n = 6) and S. typhimurium (n = 6) isolates and both ATCC control strains across five different pH levels (7.4, 6.5, 6.0, 5.5, and 5.0), mimicking the conditions of various sections of the poultry gastrointestinal tract. Table 3 and Table 4 summarize the MICs and fractional inhibitory concentration (ΣFIC) indices obtained using the checkerboard method.

OVEO exhibited stronger intrinsic antimicrobial activity (MICs 3–12.5 µL/mL) than ROEO (MICs 3–50 µL/mL) against E. coli isolates, with lower MICs observed at acidic pH. Synergistic interactions (ΣFIC ≤ 0.5) were most frequently detected at pH 5.0 and 5.5, particularly in isolates 5HE1, 16HE1, 17HE1, and the ATCC reference strain. Partial synergism (ΣFIC between 0.5 and 1) was predominant at neutral pH, whereas indifference was rarely observed and antagonistic (ΣFIC ≥ 2) was never detected (Figure 3A). Quantitatively, synergism in E. coli increased from 14% (95% CI: 3–51%) at pH 7.4 to 86% (95% CI: 49–97%) at pH 5.0, while partial synergism predominated at neutral pH but declined under acidic conditions (Figure 4).

Similarly, for S. typhimurium, OVEO exhibited better antimicrobial efficacy than ROEO. Synergistic effects were most frequent at pH 6.0 and 5.0, with ΣFIC values as low as 0.05 (isolate 5HS1 at pH 6.0) and 0.13 (isolates 6HS1 at pH 6.5). Partial synergism was common across all pH values, while indifference appeared sporadically. Isolates 5HS1, 6HS1, 13HS1, and 24HS1 displayed synergism or partial synergism in most conditions. The ATCC strain exhibited partial synergism at acidic pH but indifference under neutral or slightly acid conditions. Overall, the OVEO/ROEO combination produced either synergistic or additive effects under most conditions (Figure 3B) with synergism favoured at acidic pH levels. For S. typhimurium, synergy peaked at pH 6.0 (71%, 95% CI: 36–92%) and at pH 5.0 (29%, 95% CI: 8–64%), while indifference was more frequent at pH 5.5 and 5.0 (57% and 29%, respectively). Importantly, antagonism was never observed (Figure 4).

4. Discussion

4.1. Essential Oil Composition

The chemical composition of the essential oils used in this study, determined by GC-FID-MS, revealed that OVEO was rich in γ-terpinene (24.0%), terpinen-4-ol (10.6%), methyl carvacryl ether (5.9%), carvacrol (4.5%), and thymol (2.0%). Carvacrol and thymol, in particular, are well known for their strong antimicrobial activity by disrupting bacterial membranes and causing leakage of cellular contents [20,32]. In contrast, ROEO was dominated by monoterpenes such as myrcene (29.8%), eucalyptol (15.2%), and α-pinene (13.3%), which have moderate antimicrobial activity but contribute antioxidant effects [19,23]. This ROEO corresponds to a myrcene-rich chemotype previously reported in South American populations, including Argentina, Uruguay, and Brazil, where the predominance of myrcene aligns with its strong radical scavenging capacity [33]. Additionally, the eucalyptol content in our sample, consistent with values reported for Argentinean rosemary oils, further supports the observed antimicrobial activity and highlights how EO composition can vary with genotype and environmental factors. Overall, the total identified components accounted for over 93% of each oil’s composition, in line with previous reports for these species [34,35].

4.2. Antimicrobial Activity and pH-Dependency

In agreement with earlier studies, OVEO demonstrated greater antimicrobial activity than ROEO when tested individually against E. coli and S. typhimurium [19,20,34]. This enhanced activity was likely attributable to the higher proportion of oxygenated phenolic compounds in OVEO, especially carvacrol and thymol. Both carvacrol and thymol are phenolic isomers. Their hydroxyl group facilitates membrane partitioning and disrupts membrane integrity, which helps explain their higher activity relative to monoterpene hydrocarbons (such as those present in ROEO) [36]. The hydroxyl group component has been proven to play an important role to depolarize membrane potential [37]. For instance, Hernández-Hernández et al. [38] reported MIC values of 2–4 µL/mL for OVEO against E. coli, in agreement with our results under acidic conditions. Conversely, the lower activity of ROEO is consistent with its reduced content of potent antimicrobial monoterpenes [35]. These differences underline the importance of chemical composition in determining EO effectiveness.

The pH-dependent behavior of EO activity observed in this study aligns with other reports indicating that acidic environments enhance the antimicrobial properties of phenolic compounds [25,32]. Acidic conditions appear to enhance EO solubility and diffusion across bacterial membranes, leading to more efficient penetration and activity in the acidic regions of the avian digestive tract [39]. Morales [13] also emphasized that the avian gastrointestinal pH gradient plays a critical role in shaping microbial colonization, which may explain why EOs showed higher efficacy at pH values simulating crop and proventriculus environments. This observation supports the relevance of simulating in vivo conditions when evaluating EO efficacy for poultry applications.

4.3. Antimicrobial Interaction Between OVEO and ROEO

The checkerboard assays revealed that the combination of OVEO and ROEO resulted in additive or synergistic interactions under most of the conditions tested. Full synergy (ΣFIC ≤ 0.5) was predominantly observed at acidic pH levels (5.0 and 5.5), particularly in E. coli isolates, where the frequency of synergism increased markedly from 14% at pH 7.4 to 86% at pH 5.0. This aligns with the hypothesis that combining EOs with complementary bioactive profiles may enhance antimicrobial activity via distinct but cooperative mechanisms of action, with phenolic compounds disrupting membranes while monoterpenes increase permeability or targeting intracellular processes [40].

For S. typhimurium, partial synergism was more common, although isolates such as 6HS1 and 24HS1 displayed strong synergy under acidic conditions, with ΣFIC values as low as 0.05–0.13. The peak frequency of synergism was detected at pH 6.0 (71%), followed by pH 5.0 (29%), while indifference was observed sporadically, particularly at pH 5.5 and 5.0. These findings suggest a species-specific response, likely linked to structural differences in the outer membrane or efflux activity. Importantly, no antagonistic effects (ΣFIC ≥ 2) were detected under any condition, demonstrating the compatibility of OVEO and ROEO and the absence of inhibitory interference between their bioactive compounds.

This supports the potential use of both EOs together in formulations without risk of reducing their efficacy.

Overall, the combined results of MIC, MBC, and FIC analyses indicate that OVEO–ROEO mixtures can achieve improved antimicrobial effects in acidic environments resembling the upper avian gastrointestinal tract. The predominance of synergistic and partially synergistic outcomes strengthens the rationale for using these oils together in poultry feed formulations.

4.4. Implications for Poultry Production and AGP Alternatives

Beyond their antimicrobial properties, oregano and rosemary essential oils may offer additional benefits relevant to poultry production. The increased antimicrobial activity of EO combinations under acidic conditions is particularly promising for poultry applications, considering the pH gradient present along the gastrointestinal tract. In the crop and proventriculus (pH 4.5–5.5), where pathogenic bacteria can initiate colonization, these combinations may exert maximal antimicrobial effects, thereby improving gut health and reducing pathogen loads that reach the lower intestine.

These results also align with prior findings that EO blends can improve feed conversion, intestinal mucosal integrity, and digestive enzymatic activity in broilers, sometimes reaching performance levels comparable to those of AGPs [21,24]. These effects are attributed to compounds such as carvacrol, thymol, and 1,8-cineole, which exert both direct antimicrobial activity and indirect modulation of intestinal health. Although further in vivo studies are needed, our results support this potential, particularly in the context of AGP withdrawal, as synergistic and partially synergistic interactions between the oils were predominant under simulated gut conditions. This suggests that EO combinations may provide functional benefits comparable to antibiotic growth promoters while minimizing the risk of resistance development.

While the in vitro findings are promising, further research is needed to evaluate the in vivo effects of these EO combinations on animal performance and microbiota modulation. Encapsulation technologies and targeted delivery systems may also enhance their stability and efficacy throughout the gastrointestinal tract. Importantly, the use of EOs as feed additives must consider organoleptic impacts, cost-effectiveness, and regulatory approvals in different countries. Nevertheless, our results reinforce the feasibility of using plant-based antimicrobials to support poultry health and reduce reliance on conventional antibiotics, contributing to global efforts to combat antimicrobial resistance.

5. Conclusions

This study demonstrated that essential oils from oregano and rosemary, both individually and in combination, possess relevant antibacterial activity against Escherichia coli and Salmonella typhimurium strains isolated from broiler chickens. The chemical composition of the essential oils, particularly the presence of carvacrol, thymol, and eucalyptol, provided a strong chemical foundation for the observed effects.

The results confirmed that oregano oil was more active than rosemary oil when tested alone; however, their combination frequently resulted in synergistic or partially synergistic interactions. Notably, synergy reached up to 86% of combinations in E. coli and 71% in S. typhimurium under acidic conditions (pH 5.0–6.0), which simulated the upper compartments of the avian gastrointestinal tract. Importantly, antagonism was never observed, confirming the compatibility of both oils. These findings highlight the importance of considering physiological parameters, such as gastrointestinal pH, when evaluating the in vitro efficacy of natural antimicrobial agents intended for oral administration.

The enhanced performance of the studied EOs in acidic environments suggest potential application in early gut compartments, where controlling pathogen colonization is critical for poultry health and productivity.

Considering the increasing global restrictions on antibiotic growth promoters (AGPs), this study supports the strategic use of OVEO and ROEO as promising natural alternatives to maintain gut health and reduce the prevalence of enteric pathogens in poultry production. Additional in vivo trials are necessary to assess practical applications, optimize dosage, enhance delivery methods, ensure formulation stability, investigate potential synergistic effects under commercial farming conditions, and evaluate palatability, organoleptic impacts, and cost-effectiveness.