Research Article

In Vitro Essential Oils (Thymus vulgaris and Mentha piperita) as an Antibacterial Activity Against Staphylococcus aureus Isolated from Ovine Mastitis

Hammad Mohammad Allawi*, Orooba M.S. Ibrahim

1Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq; 2Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq.

Received | November 30, 2025; Accepted | February 18, 2026; Published | July 07, 2026

*Correspondence | Hammad Mohammad Allawi, Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq; Email: [email protected]

Citation | Allawi HM, Ibrahim OMS (2026). In vitro essential oils (Thymus vulgaris and Mentha piperita) as an antibacterial activity against Staphylococcus aureus isolated from ovine mastitis. J. Anim. Health Prod. 14(3): 1007-1018.

DOI | https://dx.doi.org/10.17582/journal.jahp/2026/14.3.1007.1018

ISSN (Online) | 2308-2801

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Mastitis in sheep is considered a major concern due to its detrimental effects on animal welfare, the substantial economic losses associated with reduced milk yield and quality, and its potential risks to human health. Because sheep milk is predominantly utilized in traditional cheese production, preventing mastitis and ensuring optimal hygienic quality of milk are fundamental goals in dairy-sheep management and in systems aimed at producing both milk and lambs. Antimicrobial agents continue to play a key role in mastitis prevention and control programs (Yass et al., 1992; Saleem et al., 2021).

Antimicrobial resistance poses a serious challenge in the treatment of mastitis, often leading to treatment failure and chronic infections. In many regions, pathogens such as Staphylococcus aureus isolated from mastitic milk show high resistance rates to commonly used antibiotics, including penicillin and ampicillin. As resistance spreads, traditional antibiotic therapy becomes increasingly unreliable, highlighting the urgent need for alternative control strategies and stricter antibiotic stewardship in dairy farms (Sharifi et al., 2023).

Staphylococcus aureus is a common commensal organism in many mammalian species. However, under favorable conditions, it can act as an opportunistic pathogen, causing infections that range from mild to severe, including mastitis. Consequently, S. aureus is among the most prevalent pathogens associated with both clinical and subclinical ovine mastitis worldwide (Safana, 2003; Gharaibeh et al., 2025).

Essential oils (EOs) are composed of a complex mixture of volatile molecules, which are specific to each plant, including their range of bioactivities; these molecules include alkaloids, monoterpenes, carotenoids, flavonoids, isoflavones, phenolic acids, oxygen-containing and non-oxygenated terpene hydrocarbons, and aldehydes (Maleš et al ., 2022). The mechanisms of antimicrobial activity of EOs include the degradation of the cell wall and cytoplasmic membrane, cytoplasm coagulation, the inhibition of toxic bacterial metabolites, and the inhibition of the bacterial efflux system. However, the efficacy of antimicrobial activity can vary depending on the pathogen and the composition of the EO (Zhang et al., 2018; Salman et al., 2024).

This study aims to investigate the in vitro effectiveness of Thymus vulgaris and Mentha piperita essential oils as antibacterial agents against Staphylococcus aureus isolated from mastitic sheep.

Materials and Methods

Bacterial isolation and identification

Milk sampling

Twenty-five lactating Awassi ewes (>2 years) in Latifa, Baghdad were sampled for S. aureus isolation. From each, 10 ml milk was collected based on clinical signs of mastitis characterized by hard udder by palpation, pain during milking in addition restricted movement, congested mucous membranes, and watery milk appearance and the California Mastitis Test was performed using 5 ml milk mixed with 5 ml reagent on a plastic paddle (Al-Rasheed et al., 2022).

Bacteriological examination

Culturing

Milk samples were cultured on nutrient agar, and suspected S. aureus colonies were purified and confirmed by hemolysis on blood agar and growth on mannitol salt agar (Markey et al., 2014).

Microscopic examination

A single blood agar colony was gram-stained, heat-fixed, and examined under a light microscope (Olympus, Japan) (100x) to observe bacterial cells (Markey et al., 2014).

Biochemical tests

All test were done according to (Alshammary and Galfoori, 2013).

Mannitol salt agar

A suspected S. aureus colony from nutrient agar was inoculated onto mannitol salt agar, a selective and biochemical medium.

Coagulase test

For the coagulase test, a colony was mixed with rabbit plasma on a slide; agglutination within 10 seconds indicated a positive result.

Catalase test

A blood agar colony was mixed with 3% H₂O₂ on a slide; bubble formation indicated a positive catalase reaction.

Plant extracts preparation

T. vulgaris and M. piperita plant materials were obtained from a certified herbal supplier in Baghdad, where each batch was accompanied by information on origin, harvest period, and handling conditions. The botanical identity of both plants was confirmed by a specialist in plant taxonomy at the College of Agriculture/University of Baghdad through morphological. Both plants were identified and classified by the Al-Razi Center for Alternative Medicine under reference numbers 230 and 231, respectively, on 9/9/2025.

Essential oil extraction

T. vulgaris and M. piperita oils were extracted from 100 g of dried leaves using hydrodistillation with a Clevenger (Jeulin, France) unit, mixing with 1000 mL water in a 2 L flask and heating at 100 °C (El-Kharraf et al., 2020; Kadhim, 2025).

Analysis of essential oil by gas chromatography–mass spectrometry

GC/MS was used to analyze T. vulgaris and M. piperita essential oils for phytochemicals with therapeutic effects. Samples (1 μL) were injected using a Thermo Trace GC Ultra/TSQ Quantum GC-MS with an Agilent HP-5ms column. The phytochemical analysis was performed using an Agilent HP-5ms Ultra Inert capillary column (30 m × 0.25 μm film thickness). The temperature program consisted of four ramps: ramp 1 at 60 °C with a 3-minute hold; ramp 2 from 60–180 °C with a 7-minute hold; ramp 3 from 180–280 °C with an 8-minute hold; and ramp 4 at 280 °C with a 3-minute hold. Operating conditions included helium (99.99%) as the carrier gas, with both injector and detector temperatures set to 250 °C. The chemical constituents of the thyme and peppermint essential oils were identified by comparing the obtained GC–MS spectra and retention times with those in the NIST database (Yassin et al., 2020; Al-Tememmi et al., 2025).

Determination of the MIC of T. vulgaris and M. piperita oil extract

The micro-dilution assay (Veiga et al., 2019) was performed in 96-well plates with Mueller-Hinton broth. Serial dilutions of T. vulgaris and M. piperita oils (100–0.781 µg/ml) and amoxicillin (amoxicillin salt, USA) (µg/ml) were prepared across wells. Each well received S. aureus inoculum (5×105 cfu/ml), while column 12 served as the negative control with broth only. Plates were incubated at 37 °C for 24 h, then treated with resazurin dye to detect bacterial growth (Najem and Abed, 2017).

Antibacterial activity

Preparation of different concentration of plant extract

Stock solutions were made by dissolving 1 g of oil in few drops of DMSO (0.20 µm filtered), then diluted with phosphate buffer to the concentration 1, 2, 4, 6, and 8 µg/ml. The method of oil homogenisation by vortex to ensure uniform exposure. The final concentration of DMSO in the medium was controlled, and a separate DMSO control was included to rule out any inherent inhibitory or toxic effects.

Sensitivity test

The antibacterial activity of T. vulgaris and M. piperita was tested using a well diffusion assay. S. aureus (5×105 CFU/ml) was mixed into Mueller-Hinton agar, poured into plates, and wells were filled with extract concentrations (1–8 µg/ml). After 2 h diffusion, plates were incubated at 37 °C for 24 h, and inhibition zones were measured (Alsterholm et al., 2010; Zhang et al., 2017).

Time kill curve kinetics

The time-kill and post-antibacterial effects of T. vulgaris, M. piperita oil, and amoxicillin against S. aureus were evaluated following NCCLS guidelines. Bacterial suspensions (10⁶ CFU/ml) were exposed to 0.25–4× MIC concentrations and incubated at 37 °C for 24 h. Colony counts were taken at 0, 2, 4, 8, 12, and 24 h, and the area under the killing curve (AUC) was calculated.

Post antibacterial effect (PAE)

The post-antibacterial effect (PAE) and post-antibacterial sub-MIC effect (PA-SME) of T. vulgaris, M. piperita oil, and amoxicillin were measured by exposing S. aureus to MIC and 0.5× MIC for 90 min, removing the agents, washing, and resuspending in fresh broth. Cultures were incubated at 37 °C, and Spectrophotometer (Scot – Tech, Germany) OD₆₀₀ was monitored for 28 h. PAE/PA-SME was calculated as the delay in reaching 50% of the control OD (Odenholt, 1993; Miles et al., 1938; Chiou, 1978).

Statistical analysis

The Statistical Package for the Social Sciences (SPSS, 2019) was used to analyze the effects of different treatments and time periods on the study parameters. Means were compared using two-way ANOVA followed by the Least Significant Difference (LSD) test to determine significant differences.

Results

Isolation of S. aureus

The results of isolation and culturing of bacteria including biochemical tests showed that a total of 25 milk samples which were collected from ewes were diagnosed with clinical mastitis based on clinical signs in different local breeders in Latifiya district/ Baghdad governorate, 18 specimens, exact number of isolates 3 specimens of each test indicated positive results for the existence of S. aureus with percentage 72% (Table 1).

 

Table 1: Prevalence of S. aureus in collected samples.

Microorganism	No. of sample	Percentage %
Staphylococcus aureus	18	72
Other	7	28
Total	25	100

 

Identification of S. aureus

Culturing milk samples on blood agar revealed β-hemolysis, characterized by clear zones surrounding convex, round, mucoid, greyish-yellow colonies (Figure 1). All Staphylococcus aureus isolates fermented mannitol, producing acidic byproducts that changed the phenol red indicator to yellow (Figure 2). Microscopic examination showed Gram-positive cocci, violet in color, arranged in clusters (Figure 3).

The catalase test confirmed the presence of S. aureus, as air bubbles appeared immediately after adding 3% hydrogen peroxide (H₂O₂) to the colonies. This reaction occurs because Staphylococci produce the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen (Figure 4).

The coagulase test was used to differentiate pathogenic from non-pathogenic staphylococci. Production of coagulase, a hallmark of pathogenic S. aureus, was confirmed by the formation of clumps of agglutinated particles within 90 seconds after mixing the isolates with rabbit plasma (Figure 5).

 

 

Extraction of T. vulgaris and M. piperita oil

Thymus vulgaris and Mentha piperita oils were extracted by steam distillation, and their yields were calculated using the following equation.

Yield (%) = EO (g)/Dry matter (g) × 100

The yield of T. vulgaris essential oil was 1.2% (v/w) from 100 g of dried herb, producing a pale-yellow oil with a strong spicy odor, while M. piperita yielded 0.5% (v/w) of a colorless to light yellow greenish oily with menthol odor (Table 2).

 

 

 

Table 2: Yields of the essential oils extract.

Essential oils	Yield (٪)	Oil specifications
T. vulgaris oil	1.20	Pale yellow color and a strong spicy odor
M. piperita oil	0.5	Light yellow greenish oily with menthol odor

 

Analysis of T. vulgaris and M. piperita oil extract by chromatography-mass spectrometry (GC-MS)

Using GC/MS analysis showed that T. vulgaris oil was mainly composed of thymol (78.49%), carvacrol (6.18%), caryophyllene (3.89%), cymene (3.48%), γ-terpinene (1.76%), and thymoquinone (1.35%) (Table 3, Figure 6). While M. piperita oil contained menthol (31.3%), menthone (23.2%), cyclohexene (14.97%), eucalyptol (10.39%), menthyl acetate (8.19%), caryophyllene (3.95%) and β-pinene (3.70%), (Table 4, Figure 7).

 

Table 3: Analysis of the phytochemicals in T. vulgaris essential oil.

Compounds	Retention time	Percentage of total
Thymol	12.415	78.49
Carvacrol	23.908	6.18
Caryophyllene	14.536	3.86
Cymene	6.711	3.48
Gamma.-Terpinene	7.335	1.76
Thymoquinone	11.342	1.35
Olean-12-ene	28.704	1.30
Terpinen-4-ol	9.862	0.94
Hydrazinecarboxylic acid	8.243	0.78
Diethyl sulfate	7.611	0.72

 

Pharmacodynamic analysis

Sensitivity and antibacterial activity

The results of MIC showed that T. vulgaris oil extract relatively had low MIC (3.125 µg/ml) against S. aureus compare to (6.25 µg/ml) and (25 µg/ml) for M. piperita oil and amoxicillin respectively (Figure 8). The MIC was determined based on the presence or absence of purple color; the lowest concentration that showed no color development was considered the MIC.

Different concentrations of Thymus vulgaris and Mentha piperita oils were tested using the agar well diffusion assay, producing varying degrees of inhibition against Staphylococcus aureus. The size of the inhibition zones increased proportionally with higher concentrations of the oil extracts (Table 5, Figure 9).

 

Table 4: Analysis of the phytochemicals in M. piperita essential oil.

Compounds	Retention time	Percentage of total
Menthol	9.440	31.30
Menthone	11.830	23.20
Cyclohexene	7.603	14.97
Eucalyptol	5.231	10.39
Menthyl acetate	10.018	8.19
Caryophyllene	12.485	3.95
Beta.-Pinene	4.392	3.70
Pulegone	9.100	1.83
Gamma-Terpinene	5.638	0.60
1,6-Octadien-3-ol, 3,7-dimethyl	6.443	0.57
Beta.-Farnesene	12.961	0.53

 

 

 

 

Table 5: Inhibitory zone of T. vulgaris and M. piperita oil antibacterial efficacy at various concentration against S. aureus.

Concentration (µg/ml)/ Groups	Zone of inhibition (mm)
	1.0 µg	2.0 µg	4.0 µg	6.0 µg	8.0 µg
T. vulgaris oil	19 ±1.26Ad	22.2 ±1.56Ac	26 ±2.15Ab	27 ±2.41Ab	29.8 ±2.63Aa
M. piperita oil	18 ±0.76Ac	20.5 ±1.07Ab	22 ±0.94Bab	23 ±1.57Ba	24.5 ±2.19Ba

 

LSD: 2.071; Means having with the different capital letters in same column and small letters in same row differed significantly (P≤0.05). Values are mean ± SD, n = 5.

 

 

 

Post antibiotic effect

As shown in Figures 10–12, the post-antibiotic effect (PAE) of T. vulgaris and M. piperita oils was evaluated based on the time required for treated cultures to reach 50% of the ODₘₐₓ of the control. Exposure of S. aureus to MIC and sub-MIC concentrations of the oils significantly (P < 0.05) increased the regrowth time. The PAE times recorded after removal of T. vulgaris oil and M. piperita oil were 7.1 ± 0.38, 6.0 ± 0.25, 5.8 ± 0.23, and 4.4 ± 0.19 hours, respectively, which were higher than those observed for amoxicillin-treated cultures (3.8 ± 0.17 and 3.1 ± 0.11 hours) (Table 6).

 

 

Table 6: Post Antibacterial duration of T. vulgaris and M. piperita oil and amoxicillin against S. aureus.

Groups	MIC (µg/ml)	Post Antibacterial duration (hours)
		MIC level	Sub - MIC level
T. vulgaris	3.125	7.1 ±0.38Aa	6.0 ±0.25Aa
M. piperita	6. 25	5.8 ±0.23Ba	4.4 ±0.19Bb
Amoxicillin	25.0	3.8 ±0.17Ca	3.1 ±0.11Ca

 

LSD: 1.283; Means having with the different capital letters in same column and small letters in same row differed significantly (P≤0.05). Values are mean ± SD, n = 5

 

Discussion

Culturing S. aureus on blood agar allows observation of hemolysis for identification and provides a rich, non-selective medium for microbial growth (Srisrattakarn et al., 2024). Bacterial colonies on blood agar show α (partial), β (complete), or γ (no) hemolysis. The double β-hemolysis zone around S. aureus colonies results from α- and β-hemolysin activity (Al-Izzi et al.,1989; Turista and Puspitasari, 2019).

Staphylococcus aureus is a Gram-positive bacterium that retains the crystal violet stain, appears purple or blue under a light microscope due to its thick peptidoglycan cell wall (Yu et al., 2023; Latif and Yousif, 2025).

A key virulence factor of S. aureus is coagulase, which converts fibrinogen to fibrin, forming plasma clots that protect the bacteria and may delay phagocytosis in tissues (Javed et al., 2024).

These results align with previous studies: T. vulgaris hydrodistillation yields ranged from 0.62% to 1.40% (Shallal and Ahmed, 2022), while M. piperita yields varied from 0.63–1.36% depending on conditions (Ibrahim et al., 2021). Steam distillation efficiency is influenced by water quality, equipment, temperature, and duration (Shrestha et al., 2025).

Thymol, a phenolic monoterpene, is well documented as the principal antimicrobial and antioxidant constituent of thyme oil. Its high abundance confirms that the oil belongs to the “thymol chemotype,” which is particularly effective against Gram-positive bacteria, including Staphylococcus aureus, a major causative agent of ovine mastitis. The presence of γ-terpinene and p-cymene is also significant, since they act as precursors in thymol biosynthesis and may enhance the antimicrobial efficacy (Rani et al., 2022).

Menthol and menthone, key components of peppermint oil, provide analgesic, cooling, and antibacterial effects. Eucalyptol enhances anti-inflammatory action, while caryophyllene and β-pinene add antimicrobial and antioxidant properties. Variations in composition depend on origin, climate, harvest time, and extraction method (Burt, 2004;Yassin et al., 2020).

Pharmacodynamic studies help optimize antibiotic dosing (Yoo and Lee, 2020). In this study, T. vulgaris oil showed MIC of 3.125 µg/ml against S. aureus, consistent with Sienkiewicz et al. (2012), who reported MICs of 0.25–1.0 µg/ml for resistant strains. Thymus digenesis also showed strong activity against S. aureus (MIC 6.25 µg/ml) due to flavonoids (Rashid et al., 214). M. piperita oil had higher MICs, in agreement with Li et al. (2011), who reported 64–256 µg/ml against clinical S. aureus isolates. Singh et al. (2015) demonstrating MIC values of essential oil of M. piperita indicate that S. aureus is more susceptible which record 5µg/ml.

Based on our results showing a proportional increase in the inhibition zones with higher concentrations of T. vulgaris (Thyme) and M. piperita (Peppermint) oils against S. aureus, it is important to note the technical limitations of the agar well diffusion assay for these specific substances (Donaldson et al., 2005). Inhibition zones size increased proportionally with higher extract concentrations. T. vulgaris oil showed strong activity against S. aureus, mainly due to aromatic monoterpenes such as thymol, carvacrol, and p-cymene (Daouk et al., 1995).

Thyme oil’s effectiveness is due to hydrophobic thymol and carvacrol, which disrupt bacterial membranes, increasing permeability (Chaichi et al., 2021). Peppermint oil showed moderate inhibition against S. aureus, attributed to secondary metabolites like essential oils, glycosides, and flavonoids (Okmen et al., 2017). Its activity aligns with studies showing plant oils rich in phenols, terpenes, and aldehydes are potent antimicrobials (Di Matteo et al., 2024).

Pharmacodynamic parameters like the post-antibiotic effect (PAE) are vital for understanding antimicrobial action and optimizing dosing. Many antibiotics induce PAE and SME in various bacteria (Yoo and Lee, 2020). PAE estimation best reflects in vivo antibiotic behavior. PAE likely results from non-lethal bacterial damage or drug persistence at binding sites (Zhanel et al., 1991). Its duration depends on the drug type, bacterial strain, concentration, and assessment method (Farooq et al., 2025). Thymol showed strong antibacterial activity (Mith et al., 2014). This study confirmed its MIC and half-MIC effects and, Moderate claims and contextualize with previous literature, revealed PAE and PA-SME against S. aureus. Agents lacking PAE require more frequent dosing (Zarrini et al., 2010).

Conclusions

This study demonstrated that Thymus vulgaris and Mentha piperita essential oils exhibit strong in vitro antibacterial activity against Staphylococcus aureus isolated from mastitic Awassi ewe milk. Both oils showed concentration-dependent inhibitory effects, as evidenced by measurable inhibition zones, low MIC values, and significant bacterial reduction in time-kill assays. GC–MS analysis revealed the presence of bioactive constituents that likely contribute to the observed antibacterial activity. Additionally, the post-antibacterial effect (PAE) and post-antibacterial sub-MIC effect (PA-SME) assays confirmed their ability to delay bacterial regrowth under controlled laboratory conditions. However, these findings are limited to in vitro experiments. Further in vivo studies, including safety assessments, pharmacokinetic evaluations, formulation optimization, and controlled clinical trials in ewes with mastitis, are necessary to validate the therapeutic potential of these essential oils.

Acknowledgements

The authors would like to thank members of the Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq, Veterinary medicine, Baghdad university, Iraq for their collaboration.

Novelty Statement

This study provides a comprehensive pharmacodynamic evaluation of Thymus vulgaris and Mentha piperita essential oils against Staphylococcus aureus isolated from ovine mastitis. By combining GC–MS profiling with MIC, time-kill kinetics, and post-antibiotic effect analyses, it demonstrates that T. vulgaris exhibits superior antibacterial activity compared with M. piperita and amoxicillin, supporting its potential as a natural therapeutic alternative

Authors’ Contribution

HMA and OMS designed the study. HMA conducted the experimental work and collected the samples. NAR performed the statistical analysis and interpretation of data. HMA wrote the manuscript. All authors read and approved the final version of the manuscript. The final manuscript draft was reviewed by all authors.

Funding

The authors received no specific funding for this work.

Generative AI and AI assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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