Ethical review and approval were waived for this study because no live animals were used or experimentally manipulated. The study was conducted using previously obtained bacterial isolates from clinical mastitis cases provided by a diagnostic laboratory, in accordance with institutional and national guidelines for research ethics.

This study was conducted from February to December 2024 at the Clinical Pathology Laboratory, Universidade Estadual do Centro-Oeste (UNICENTRO), Guarapuava, Paraná, Brazil using bacterial isolates obtained from bovine mastitis cases in dairy herds from Paraná State, Brazil. Laboratory analyses were performed in collaboration with the Animal Products Inspection and Bacteriology Laboratories of the Universidade Estadual de Londrina (Brazil).

This study was designed as an in vitro experimental investigation to evaluate the antibacterial activity of EOs against reference strains and phenotypically characterized clinical isolates of mastitis-associated bacteria. The study included antimicrobial susceptibility testing and determination of MIC and MBC.

Reference strains E. coli American Type Culture Collection (ATCC) 25922, Klebsiella pneumoniae ATCC 13883, S. aureus ATCC 25923, and methicillin-resistant S. aureus ATCC 43300 were used. Clinical isolates of S. agalactiae and S. uberis, obtained from bovine mastitis cases in dairy herds from Paraná State, Brazil, were provided by the Animal Products Inspection and Bacteriology Laboratories of the Universidade Estadual de Londrina (Brazil).

Clinical isolates were previously identified by standardized biochemical profiling (API 20 Strep system) and hemolysis patterns on blood agar (HiMedia Laboratories, Mumbai, India). Although molecular confirmation was not performed in this study, identification followed standardized biochemical procedures routinely employed in a reference veterinary diagnostic laboratory.

All strains were stored at −80°C in tryptic soy broth supplemented with 20% (v/v) glycerol. Before testing, strains were subcultured twice on Mueller–Hinton agar (Oxoid, Basingstoke, UK), for E. coli, K. pneumoniae, and S. aureus or Columbia agar (HiMedia Laboratories) supplemented with 5% defibrinated sheep blood (E&O Laboratories Limited, Bonnybridge, UK) for S. agalactiae and S. uberis and incubated at 35 ± 1°C for 18–24 h.

Antimicrobial susceptibility was determined using the Kirby–Bauer disk diffusion method on Mueller–Hinton agar (Oxoid), following Clinical and Laboratory Standards Institute (CLSI) M100 guidelines [8]. For S. agalactiae and S. uberis, Mueller–Hinton agar supplemented with 5% (v/v) defibrinated sheep blood (E&O Laboratories) was used.

The antimicrobial disks (Oxoid) included ampicillin (10 µg), ceftiofur (30 µg), enrofloxacin (5 µg), gentamicin (10 µg), neomycin (30 µg), penicillin (10 IU), oxacillin (1 µg), sulfamethoxazole/ trimethoprim (25 µg), and tetracycline (30 µg).

Bacterial suspensions were prepared in sterile 0.85% saline and adjusted to 0.5 McFarland standard (approximately 1.5 × 10⁸ colony-forming units (CFU)/mL) using spectrophotometric verification (OD600 = 0.08–0.10). Plates were incubated at 35 ± 1°C for 18–24 h under aerobic conditions. Inhibition zones were measured in millimeters and interpreted according to CLSI breakpoints [8]. E. coli ATCC 25922 and S. aureus ATCC 25923 were used as quality control strains.

Tea tree (M. alternifolia) and copaiba (C. officinalis) EOs were obtained from Via Aroma® (Porto Alegre, Brazil), certified as 100% pure and produced by steam distillation. Chemical composition was based on the manufacturer’s gas chromatography–mass spectrometry report (Table 1).

EOs were stored in amber glass vials at 4°C in the dark until used to minimize oxidation and degradation.

MIC values were determined by broth microdilution in sterile 96-well polystyrene microplates according to CLSI M07-A11 guidelines [9], with methodological adaptations to ensure appropriate dispersion and evaluation of hydrophobic EOs.

Cation-adjusted Mueller–Hinton broth (CAMHB; Oxoid, Basingstoke, UK) was used as the test medium. EOs were emulsified in 1 % (v/v) Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) to ensure stable and homogeneous dispersion in the aqueous medium.

Two-fold serial dilutions of each EO were prepared to obtain final concentrations ranging from 0.49 to 250 mg/mL in a final well volume of 100 µL. Bacterial suspensions were adjusted to 0.5 McFarland standard and subsequently diluted to achieve a final concentration of approximately 5 × 10⁵ CFU/mL in each well.

Each assay included the following controls: growth control (broth + bacterial inoculum without EO), sterility control (broth only), and treatment control (broth + EO without bacterial inoculum) to exclude contamination or intrinsic color interference. All controls were included in each independent experiment.

Plates were incubated at 35 ± 1°C for 18–24 h. After incubation, 20 µL of resazurin solution (0.01%) was added to each well and incubated for an additional 2 h. MIC was defined as the lowest concentration that prevented color change from blue to pink, indicating inhibition of bacterial metabolic activity [10]. No reference antibiotic control was included in the MIC assay because the objective was to evaluate the intrinsic antibacterial activity of the EOs rather than to establish comparative antimicrobial potency under standardized CLSI-adapted conditions.

MBC was determined from wells showing no visible growth in the MIC assay. A 100 µL aliquot from each selected well was plated onto Plate Count Agar (Oxoid, Basingstoke, UK) and incubated at 35 ± 1°C for 24 h under aerobic conditions.

MBC was defined as the lowest EO concentration resulting in a ≥99.9% reduction in CFU compared to the initial inoculum [9]. The highest concentration evaluated in the MIC assay was 250 mg/mL.

MIC and MBC assays were performed in triplicate across three independent experiments. As MIC values are determined using two-fold serial dilutions and represent ordinal rather than continuous data, results are presented as modal values (most frequently observed concentration). Inter-assay variation remained within one two-fold dilution step, indicating high reproducibility of the measurements. When minor variation occurred, the most frequently observed value across replicates was reported. Bactericidal activity was defined as an MBC/MIC ratio ≤ 4.

The antimicrobial susceptibility profiles of the tested bacterial strains are presented in Table 2 and were interpreted according to CLSI M100 guidelines [8].

E. coli ATCC 25922 was susceptible to ampicillin, ceftiofur, and tetracycline, resistant to enrofloxacin, gentamicin, and neomycin, and showed intermediate susceptibility to sulfamethoxazole/trimethoprim. K. pneumoniae ATCC 13883 was resistant to ampicillin and susceptible to all other tested antimicrobials.

S. aureus ATCC 25923 demonstrated resistance to enrofloxacin, oxacillin, and sulfamethoxazole/ trimetho-prim, with intermediate susceptibility to gentamicin, neomycin, and tetracycline. The methicillin-resistant S. aureus ATCC 43300 strain exhibited resistance to ampicillin, ceftiofur, gentamicin, neomycin, penicillin, and oxacillin, confirming an MDR phenotype.

The clinical isolate of S. agalactiae showed resistance to gentamicin, neomycin, oxacillin, and sulfamethoxazole/trimethoprim, with intermediate susceptibility to enrofloxacin, supporting its classification as MDR. In contrast, S. uberis remained susceptible to most tested antimicrobials, with intermediate susceptibility observed only for enrofloxacin.

The MIC and MBC values of M. alternifolia and C. officinalis EOs are presented in Table 3. M. alternifolia inhibited the growth of E. coli ATCC 25922 (MIC = 10 mg/mL), with bactericidal activity observed at the same concentration (MBC = 10 mg/mL, MBC/MIC = 1). Inhibitory activity was also detected against the clinical isolates of S. uberis (MIC = 15 mg/mL) and S. agalactiae (MIC = 31 mg/mL); however, MBC values (125–250 mg/mL) resulted in MBC/MIC ratios > 4, indicating predominantly bacteriostatic effects under the tested conditions. Limited inhibitory activity was observed against S. aureus ATCC 25923 (MIC = 25 mg/mL), without bactericidal effect. No inhibitory activity was detected against K. pneumoniae ATCC 13883 or methicillin-resistant S. aureus ATCC 43300 at the tested concentrations.

C. officinalis exhibited lower antibacterial activity overall. Growth inhibition was observed against S. uberis (MIC = 10 mg/mL) and S. agalactiae (MIC = 7.5 mg/mL). However, MBC values were either substantially higher than MIC values or not achieved at the highest tested concentration (250 mg/mL), resulting in MBC/MIC ratios > 4 and indicating absence of bactericidal activity under the experimental conditions. No inhibitory activity was detected against E. coli, K. pneumoniae, or S. aureus strains within the tested concentration range.

Overall, M. alternifolia demonstrated broader inhibitory activity than C. officinalis, particularly against Gram-positive mastitis-associated clinical isolates.

The emergence of MDR pathogens in bovine mastitis represents a growing therapeutic challenge, reinforcing the need to investigate alternative or adjunct antimicrobial strategies. In the present study, the methicillin-resistant S. aureus ATCC 43300 strain demonstrated resistance to multiple antibiotic classes, confirming its MDR phenotype and supporting concerns regarding limited therapeutic options in dairy systems. Similarly, the clinical isolate of S. agalactiae exhibited multidrug resistance, consistent with reports of resistant streptococcal strains in Brazilian dairy herds [11].

EOs have been proposed as potential alternatives due to their complex chemical composition and multimodal mechanisms of action. Among the tested oils, M. alternifolia demonstrated broader antibacterial activity, particularly against streptococcal isolates. However, bactericidal activity (MBC/MIC ≤ 4) was observed only against E. coli ATCC 25922. For S. uberis and S. agalactiae, MBC/MIC ratios exceeded 4, indicating predominantly bacteriostatic effects under the tested conditions. This distinction is important for interpreting the translational potential, as bacteriostatic activity may still contribute to therapeutic efficacy, particularly in intramammary infections where host immune mechanisms assist bacterial clearance or when combined with conventional antimicrobials. Moreover, growth inhibition observed in vitro does not necessarily imply effective bacterial eradication, highlighting the relevance of pharmacodynamic interpretation.

The MIC values observed for M. alternifolia (modal values ranging from 10–31 mg/mL) were higher than those reported in some previous studies evaluating tea tree oil against mastitis-associated pathogens, which ranged approximately from 0.78 to 25 mg/mL under in vitro conditions [12, 13]. Differences in MIC values may be attributed to several factors, including strain variability, chemotype composition, geographic origin of the EO, and methodological differences such as emulsification procedures and broth composition. The present study evaluated clinical isolates obtained from dairy herds, which may exhibit greater phenotypic resilience compared to laboratory-adapted reference strains. Additionally, variations in terpinen-4-ol concentration and the relative abundance of monoterpenes may influence antibacterial potency.

C. officinalis EO exhibited more limited antibacterial activity. Inhibitory effects were detected against S. uberis and S. agalactiae, but bactericidal activity was not achieved at the tested concentrations. Previous studies have reported variable antimicrobial activity for copaiba oils, often influenced by the predominance of sesquiterpenes such as β-caryophyllene [14, 15]. In contrast, the EO evaluated in this study was characterized by higher proportions of α-pinene, β-thujene, and D-limonene, which may partly explain differences in antibacterial performance compared to other reports.

Among Gram-negative strains, M. alternifolia inhibited E. coli ATCC 25922 but showed no activity against K. pneumoniae ATCC 13883. The intrinsic structural barriers of Gram-negative bacteria, particularly the lipopolysaccharide-rich outer membrane, may reduce EO penetration [16]. Additionally, the presence of a polysaccharide capsule in K. pneumoniae may further limit susceptibility [17]. Such variability underscores the importance of species-specific evaluation when considering plant-derived antimicrobials.

The methodology followed CLSI recommendations for disk diffusion testing, including blood supplemen-tation for streptococcal isolates. For broth microdilution assays, CAMHB without blood supplementation was used, as commonly adopted in antimicrobial screening studies involving natural products. Although blood supplementation may be recommended in certain clinical MIC determinations for streptococci, the use of unsupplemented CAMHB allows standardized comparison across bacterial species and minimizes potential interference with EO dispersion and resazurin-based viability assessment. Therefore, the results should be interpreted within the context of exploratory in vitro screening rather than formal clinical breakpoint determination.

Some limitations should be acknowledged. First, molecular confirmation of the clinical streptococcal isolates was not performed; identification relied on standardized biochemical profiling performed in a reference veterinary diagnostic laboratory with established quality control procedures. Second, the study was limited to in vitro antibacterial assays, and the relatively high concentrations required for inhibition raise considerations regarding formulation feasibility, tissue tolerance, and potential cytotoxicity in intramammary applications. Cytotoxicity and selectivity assays using mammary epithelial cell models are currently being conducted as part of an ongoing research line to further evaluate the translational potential of these EOs. Strategies such as nanoemulsion systems or combination approaches may also enhance antibacterial performance while reducing required concentrations [18, 19].

To our knowledge, few studies have directly compared these two EOs under identical experimental conditions against clinically characterized MDR streptococcal isolates from Brazilian dairy herds. Overall, the findings indicate that M. alternifolia EO exhibits broader antibacterial activity than C. officinalis under the tested conditions, particularly against streptococcal mastitis pathogens. Although bactericidal effects were limited, the observed inhibitory activity supports continued investigation into optimized formulations and combinatory strategies for mastitis control.

Future investigations should also explore potential synergistic interactions between EOs and conventional antimicrobials, as well as antibiofilm activity against mastitis-associated pathogens.