Essential oils of Eugenia spp. (myrtaceae) show in vitro antibacterial activity against Staphylococcus aureus isolates from bovine mastitis

Highlights

• • The essential oils of the genus Eugenia show significant antibacterial activity.

• • Essential oils combat the formation of Staphylococcus aureus biofilm responsible for bovine mastitis.

• • The essential oils of the genus Eugenia act synergistically with oxacillin.

• • The essential oil of Eugenia stictopetala was analyzed for the first time.

• • The major components interact with penicillin-binding proteins, indicating a possible antibacterial mechanism.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-024-01489-6.

Abstract

Bovine mastitis, an inflammation of the mammary glands, is mainly caused by bacteria such as Staphylococcus aureus. While antibiotics are the primary treatment for this disease, their effectiveness is often diminished due to resistant strains and biofilm formation, creating the need for safer and more efficient therapies. Plant-based oil therapies, particularly those derived from the genus Eugenia, are gaining popularity due to their pharmacological potential and historical use. In this study, we evaluated the antibacterial, antibiofilm, and synergistic potential of essential oils (EOs) from four species of the genus Eugenia (E. brejoensis, E. gracillima, E. pohliana, and E. stictopetala) against S. aureus isolates from bovine mastitis. The EO of E. stictopetala was obtained by hydrodistillation, and its composition was analyzed using gas chromatography coupled with mass spectrometry. The experiment employed seven clinical isolates from mastitis and two control strains: ATCC 33591 (methicillin-resistant S. aureus - MRSA) and ATCC 25923 (methicillin-susceptible and biofilm producer). A broth microdilution assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the EOs and oxacillin. The EO of E. stictopetala contained (E)-caryophyllene (18.01%), β-pinene (8.84%), (E)-nerolidol (8.24%), and α-humulene (6.14%) as major compounds. In the MIC assay, all essential oils showed bactericidal and bacteriostatic effects, especially the species E. brejoensis and E. pohliana, which had MICs ranging from 64 to 256 µg/mL. Regarding the antibiofilm effect, all essential oils were capable of interfering with biofilm formation at subinhibitory concentrations of ½ and ¼ of the MIC. However, they did not significantly affect pre-established biofilms. Additionally, a synergistic interaction was detected between the EOs and oxacillin, with a reduction of 75–93.75% in the antimicrobial MIC. Molecular docking studies indicated that the phytochemicals β-(E)-caryophyllene, (E)-nerolidol, Δ-elemene, and α-cadinol present in the EOs formed more stable complexes with penicillin-binding proteins, indicating a possible mechanism of antibacterial action. Therefore, these results show that the essential oils of Eugenia spp. are promising sources for the development of new therapeutic methods, opening new perspectives for a more effective treatment of bovine mastitis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-024-01489-6.

Introduction

Bovine mastitis is one of the main problems affecting dairy herds worldwide. This disease consists of an inflammation of the mammary gland, commonly caused by microorganisms, particularly bacteria from the genus Staphylococcus. This inflammation results in physical and chemical changes in the milk, as well as physiological alterations in the affected animals [1].

The main etiological agent of bovine mastitis is Staphylococcus aureus, a pathogen of significant concern for both human and animal health, responsible for many nosocomial diseases and infections [2]. The World Health Organization (WHO) has classified this species as a high-priority pathogen for antimicrobial development due to its multidrug resistance [3]. The threat of this pathogen lies in its ability to cause infections even in hosts with a healthy and active immune system, utilizing sophisticated mechanisms to evade and invade host cells [4]. Key virulence factors include the production of toxins (such as hemolysins, exfoliative toxins, and enterotoxins) and biofilm formation [5].

Biofilms are groups of microorganisms surrounded by a self-produced exopolymeric matrix composed of proteins, carbohydrates, genetic material, ions, and water. This structure is primarily produced on surfaces and encompasses the microbial community, enabling microorganisms to survive in hostile and extreme environments while inhibiting the action of drugs [6].

Antibiotic therapy has been the primary strategy to treat mastitis [7]. However, this approach has shown limited efficacy due to increased antimicrobial resistance [8]. In this context, the use of natural products, especially those derived from plants, has emerged as a promising alternative due to their effective pharmacological action and low toxicity [9].

The genus Eugenia L. (Myrtaceae) holds significant economic and pharmacological potential, as highlighted by the diverse applications of its species, especially in the extraction of essential oils [10]. Essential oils are aromatic and volatile liquids containing various chemical compounds produced by the secondary metabolism of several plant organs [11]. Due to the vast variety of compounds present in essential oils, many studies have emphasized their broad-spectrum antibacterial potential, making them into promising candidates for developing new, efficient, and safer therapeutic methods to control bovine mastitis [12].

From this perspective, the objective of this study was to evaluate the in vitro antibacterial and antibiofilm activities, as well as the synergistic potential with oxacillin, of essential oils derived from plants of the genus Eugenia against S. aureus, the causative agent of bovine mastitis. Additionally, an in silico analysis was conducted via molecular docking with the major components of these essential oils to explore potential mechanisms of action on penicillin-binding proteins.

Materials and methods

Plant material and essential oil extraction

The essential oils extracted from the leaves of Eugenia brejoensis (EOEb), Eugenia pohliana (EOEp), Eugenia gracillima (EOEg), and Eugenia stictopetala (EOEs) were evaluated. The entire process of collection, identification, extraction, and chemical composition analysis of essential oils for E. brejoensis, E. pohliana, and E. gracillima was previously described by Mendes et al. [13], Costa et al. [14], and Guedes et al. [15], respectively. The plant material of E. stictopetala was collected in Serra da Madeira in the Municipality of Santa Cruz da Baixa Verde, Pernambuco, Brazil. Plant identification was conducted by a specialist at the Herbarium of the Brazilian Semiarid (HESBRA), where the specimen was deposited under voucher no. HESBRA 3240.

The hydrodistillation method was employed to extract the essential oils (EOEs). Approximately 250 g of crushed leaves were combined with 2.5 L of distilled water in a volumetric flask. This flask was then placed on a heating mantle (160–170 °C) and connected to a modified Clevenger apparatus. The hydrodistillation process was carried out for 3 h, during which the essential oil was collected and treated with anhydrous sodium sulfate P.A. to remove any remaining water. Finally, the essential oil was stored in amber vials at -40 °C until the assays were performed.

Characterization of E. stictopetala essential oil

The qualitative analysis of the essential oil constituents was carried out using gas chromatography coupled with mass spectrometry (GC-MS). This was performed using an Agilent 5975 C GC/MS quadrupole instrument (Agilent Technologies, Palo Alto, CA, USA) equipped with an uncoated fused silica capillary column DB-5 (Agilent J & W, 30 m × 0.25 mm) with a film thickness of 0.25 μm (Agilent Technologies, Palo Alto, CA, USA). A 1 µL injection in split mode (50:1) was made with the injector temperature set to 250 °C for each analysis. The GC oven temperature was initially set at 40 °C for 2 min, then increased to 230 °C at a rate of 4 °C/min, and held for 5 min. Helium gas was used as the carrier at a flow rate of 1 mL/min and a constant pressure of 7.0 psi. The MS source and quadrupole temperatures were set at 230 °C and 150 °C, respectively. Mass spectra were recorded at 70 eV (EI mode) with a scan speed of 1.0 scans per m/z 35–350 range. Identification of individual components was performed by comparing retention index values obtained through co-injection of esterified oil samples and a set of fatty acids, calculated according to the Van den Dool and Kratz Eq. (1963), with previously reported values. The data from 82 MS runs for each component were combined and compared with available data in the mass spectral library of the GC-MS system (MassFinder 4, Scientific Consulting by Dr. Hochmuth, Hamburg, Germany); NIST08 Mass Spectral Library (ChemSW Inc., Fairfield, CA, USA); Wiley Registry™ 9th Edition Mass Spectral Database (Wiley, Hoboken, NJ, USA); and other published mass spectral data [16]. A Thermo Trace GC Ultra gas chromatograph (Thermo Scientific, Milan, Italy) with a flame ionization detector (FID) (Thermo Scientific, Milan, Italy) was used for quantitative analysis of the components present in the essential oil. A ValcoBond fused silica capillary column VB-5 (Valco Instruments Company Inc., 30 m × 0.25 mm ID, film thickness: 0.25 mm) was also employed. Nitrogen was used as the carrier gas at a flow rate of 1 L/min and an inlet pressure of 30 psi. The oven temperature gradient started at 40 °C for the first 2 min, then increased steadily at a rate of 4 °C/min until it reached 230 °C, where it was held for 5 min. The injector temperature was set at 250 °C, and the detector temperature was set at 280 °C. A 1 µL sample of each oil, at a concentration of 2 mg/mL in n-hexane, was injected without separation. The relative amounts of each component were inferred from peak areas and expressed as the percentage of the total chromatogram area. The analyses were performed in triplicate.

Solubilization of essential oils

The dilution protocol for the essential oils was based on Limaverde [17]. Accordingly, the substances were dissolved in 15% dimethyl sulfoxide (DMSO) and sterile distilled water, reaching a final concentration of 2,048 µg/mL. The material was then stored at 8 ºC and protected from light until testing. Oxacillin was solubilized only in sterile distilled water, with a final concentration of 1,024 µg/mL.

S. aureus isolates

Seven clinical isolates of S. aureus belonging to the collection of the Laboratory of Microbiology and Animal Immunology of Federal University of Vale do São Francisco (UNIVASF) (SisGen A12F383) were used. They were previously obtained and characterized by Da Costa Krewer [18]. For the control, two standard S. aureus strains from the American Type Culture Collection (ATCC) were selected: ATCC 33591 (methicillin-resistant) and ATCC 25923 (methicillin-sensitive).

Standard growth of the isolates

The relationship and equivalence between the Optical Density (OD) and the Colony Forming Units (CFU) of the nine isolates, as well as their growth profiles, were determined by standardization according to Baron [19]. For this purpose, the inocula were prepared by adding 5 µL of the stock solution at -20 ºC into Falcon tubes containing 5 mL of Mueller Hinton broth (MH). These tubes were then incubated at 37 ºC for 22 h while being agitated at 180 rpm.

After the incubation period, the OD was measured using a spectrophotometer at an absorbance of 600 nm. Next, 100 µL of each inoculum was added to microtubes containing 900 µL of a saline solution (0.85%). After, dilutions ranging from 10^− 1 to 10^− 10 were obtained. Then, 100 µL of each dilution was spread onto MH agar using glass beads, followed by incubation at 37 ºC for 24 h and the number of CFU was determined. Biological triplicates were performed on different days.

The bacterial inoculums recommended for each test was prepared by inoculating 5 µL of the stock solution into 5 mL of MH broth, followed by incubation at 37 ºC for 24 h. Next, the OD was measured at 600 nm, and adjustments were made according to the standard growth. Based on the growth pattern of each isolate, biological tests were conducted by consistently adjusting the recommended bacterial concentration.

Antibacterial activity assay

The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined using the broth microdilution test as outlined in the Clinical and Laboratory Standards Institute document M100-CLSI [20]. The test consisted of distributing 100 µL of Mueller Hinton broth (MH) in 96-well microplates. Next, 100 µL of the stock solution of the solubilized essential oils were added to the first well. After homogenization, the mixture was transferred to the second well and so forth, thus obtaining concentrations ranging from 1,024 µg/mL to 8 µg/mL. The same test was performed for oxacillin, obtaining concentrations ranging from 1,024 µg/mL to 8 µg/mL for strain ATCC 33591 and from 10.24 µg/mL to 0.08 µg/mL for the other isolates. The bacterial inoculum was adjusted to a concentration of 1.5 × 10⁶ CFU/mL. From this suspension, 10 µL aliquots were inoculated into the microplate wells. Finally, the plates were incubated at 37 °C for 24 h under aerobiosis.

Subsequently, 10 µL aliquots from each well were spread on the surface of the MH agar. After 24 h of incubation at 37 °C, the MBC was defined as the lowest concentration capable of causing bacterial death. Next, 30 µL of 1% 2,3,5-triphenyl-tetrazolium chloride (TTC) was added to each well, and the plate was incubated at 37 °C for one hour. The color change in the wells to pink/red indicated the metabolic viability of the microorganisms. Thus, the MIC was determined by identifying the lowest concentration capable of inhibiting bacterial growth. All assays were performed in technical and biological triplicate. Sterility controls of the medium and essential oils were included, as well as controls of bacterial viability and the antibacterial potential of the diluent.

Biofilm quantification assay

Biofilm quantification of the S. aureus isolates was performed by the microplate adhesion assay, with some modifications [21, 22]. The bacterial inoculum was adjusted to 6 × 10⁶ CFU/mL in Trypticase Soy Broth (TSB) supplemented with 0.25% glucose (TSBg). Then, 195 µL of TSBg was added to the sterile 96-well microplates along with 5 µL of the bacterial suspension. The microplates were then incubated at 37 °C for 24 h. After incubation, the microplates were washed three times with 200 µL of sterile distilled water and dried at room temperature for 5 min. Subsequently, biofilm fixation was performed by adding 150 µL of methanol P.A. to each well for 20 min at room temperature. The contents of the microplates were then discarded, and the plates were dried overnight. Next, the wells were stained with 100 µL of 0.25% gentian violet for 5 min. After staining, all wells were rewashed three times with 200 µL of distilled water. Finally, 200 µL of acetone-ethanol (20:80) was added to each well, and absorbance was measured at 620 nm using an EXPERT PLUS-UV microplate reader. TSBg medium was used as a sterility and negative control. All assays were performed in technical and biological triplicates. Based on the OD values obtained, the isolates were classified following the criteria established by Stepanović et al. [23] as follows: non-biofilm producer (OD_S< OD_NC), weak producer (OD_NC< OD_S<2xOD_NC), moderate producer (2xOD_NC < OD_S<4xOD_NC), or strong biofilm producer (OD_S>4xOD_NC), where OD_S represents the optical density of the sample and OD_NC represents the optical density of the negative control.

Antibiofilm activity assay

The interference protocol on biofilm formation and mature biofilm was based on the methodologies of Merino et al. [21] and Nostro et al. [24], with modifications. The bacterial inoculum in TSBg (0.25%) was adjusted to 3 × 10⁵ CFU/mL. Subsequently, 100 µL of the bacterial suspension was added to a 96-well microplate along with 100 µL of the essential oil solution to obtain the final concentrations of ½ and ¼ MIC. After 24 h of incubation at 37 °C, the microplates were subjected to the same washing, fixation, staining, resuspension, and reading processes as performed in the biofilm quantification assay.

The effect of the essential oils on mature biofilm was determined by preparing a bacterial suspension with 6 × 10⁶ CFU/mL in TSBg. Next, 195 µL of TSBg was added to each well of the microplate along with 5 µL of the bacterial inoculum, followed by incubation for 24 h at 37 °C. After incubation, the microplates were washed three times with 200 µL of sterile distilled water. To achieve final concentrations of 2xMIC, 1xMIC, and ½MIC of essential oils, 200 µL of the appropriate solution was added to the wells. An initial OD reading at 620 nm (OD0h) was taken at 0 h, and a second reading (OD24h) was performed after 24 h of incubation at 37 °C. Interference was determined using the following calculation: (mean OD24h/ mean OD0h) x 100, where results < 100% were classified as ‘with interference’ and results ≥ 100% were considered as ‘without interference’ on the biofilm. The TBSg medium was used as the negative and sterility control. For each tested concentration, a control without the addition of bacterial suspension was conducted to monitor any changes in the OD of this solution after 24 h of incubation. The assays were performed in both technical and biological triplicates.

Checkerboard synergy assay

The synergistic potential of the essential oils with oxacillin was evaluated by performing the checkerboard test according to the methodology of Lee, Jang & Cha [25]. The bacterial suspension was adjusted to 1.5 × 10⁶ CFU/mL in MH broth. Next, 100 µL of MH broth was added to all wells of a 96-well microplate. In column no. 1, 100 µL of oxacillin, diluted to a concentration of 4x the MIC, was added to initiate the serial dilution, transferring 100 µL horizontally until column no. 6. Row “A” received 100 µL of the essential oil solution at a concentration of 2x the MIC and was serially diluted vertically down to row “F”. The final concentrations achieved were 1x, 1/2x, 1/4x, 1/8x, 1/16x, and 1/32x of the MIC value of both substances. Each well of the microtiter plate received 10 µL of the bacterial suspension, and bacterial viability and sterility controls were included. The microplates were incubated for 24 h at 37 °C. After incubation, TTC was added, and the plates were incubated for approximately 1 h before reading the results. The interpretation of the synergistic effect of each antimicrobial and their combinations was determined by the inhibitory fraction index (IFI), using the following equation:

The sum of the IFIs was used to classify the association effects of the substances according to Lee, Jang & Cha [25]: synergistic action (IFI ≤ 0.5), additive action (0.5 < IFI < 1), indifferent action (1 < IFI < 2), and antagonistic action (IFI ≥ 2).

Molecular docking

Based on the chemical composition of the previously mentioned essential oils [13–15], the primary components of EOEb are β-(E)-caryophyllene (31%), δ-cadinene (20%), and bicyclogermacrene (12%). EOEg contains Germacrene D (16.10%), γ-Muurolene-g (15.60%), bicyclogermacrene (8.53%), Germacrene B (7.43%), Δ-cadiene (6.23%), and Δ-elemene (6.06%). Finally, the key components of EOEp are β-caryophyllene (12.56%), δ-cadinene (11.24%), α-cadinol (10.89%), and biciclogermacrene (8.13%). The major constituents of EOEs were selected for analysis in this study. The chemical structures of all these components were obtained in “sdf” format from the PubChem chemical database (https://pubchem.ncbi.nlm.nih.gov/) of the National Institutes of Health. Additionally, a comparative analysis was performed with penicillin G, and the chemical structure was obtained using the same method.

The molecular targets used to evaluate the antibacterial potential were two penicillin-binding proteins, PBP-1 and PBP-2a (Fig. 1). Their crystallographic structures were obtained from RCSB-PBD (www.rcsb.org) under codes PDB ID: 5TRO (unpublished data) and PDB ID: 5M19 [26]. UniProt (https://www.uniprot.org/) was used to confirm the information about these proteins.

Fig. 1.

Secondary structure of Staphylococcus aureus proteins used as molecular targets. Penicillin-binding proteins. (a) PBP-1 and (b) PBP-2a. Graphic representation obtained with UCSF Chimera (2022)

Molecular docking was conducted using Molegro Virtual Docker, v. 6.0.1 (MVD). All water molecules were excluded from the protein and ligand structures, and the software package was run with default parameters: MolDock score as the scoring function, and ligand evaluation included internal ES, internal HBond, and Sp2-Sp2 torsions. All of these were verified. The docking procedure included 10 runs using the MolDock SE search algorithm with a maximum of 1500 interactions, a population size of 50, and 300 steps. The neighbor distance factor was set to 1.00, and the maximum number of poses returned was set to 5. The docking procedure was performed using a GRID radius of 10Å and a resolution of 0.20 to cover the ligand binding site of the structure in question.

Statistical analysis

The results referring to biofilm quantification and the interference of essential oils on biofilm formation underwent statistical analysis using parametric tests with multiple comparisons, two-way ANOVA, and Dunnett’s and Sidak’s post hoc tests. The graphs were constructed using the software GraphPad Prism 7^®, and the results were plotted as the mean ± standard deviation. A significance level of p < 0.05 was adopted for all tests.

Results

Chemical characterization of EOEs

The phytochemical composition of the essential oil of E. stictopetala is described in Table 1. Fifty-nine volatile compounds were identified, accounting for 99.41% of the compounds detected in the GC/MS analysis. The major components are (E)-caryophyllene (18.01%), β-pinene (8.84%), (E)-nerolidol (8.24%), α-humulene (6.14%), bicyclogermacrene (4.99%), and Germacrene B (4.99%).

Table 1.

Chemical compostition of the essential oil obtained from the leaves of E. Stictopetala (EOEs)

Compound	RIa	RIb	%
α-thujene	924	924	0.07
α-pinene	932	931	2.48
camphene	946	944	0.02
β-pinene	974	976	8.84
myrcene	988	990	0.21
α-terpinene	1014	1014	0.06
ο-cymene	1022	1022	0.04
sylvestrene	1025	1026	0.83
(E)-β-ocimene	1044	1047	0.03
γ-terpinene	1054	1057	0.2
terpinolene	1086	1086	0.11
linalool	1095	1098	0.07
α-fenchol	1114	1111	0.03
|cis|-p-menth-2-ene-1-ol	1118	1119	0.03
trans-pinocarveol	1135	1136	0.16
pinocarvone	1160	1160	0.05
borneol	1165	1163	0.07
terpinen-4-ol	1174	1176	0.58
α-terpineol	1186	1189	0.43
myrtenol	1194	1195	0.23
verbenone	1204	1207	0.01
δ-elemene	1335	1337	1.9
α-cubebene	1348	1350	1.09
cyclosativene	1369	1367	0.12
α-ylangene	1373	1371	1.09
α-copaene	1374	1378	2.94
β-bourbonene	1387	1378	0.57
β-elemene	1389	1393	0.72
α-gurjunene	1409	1410	0.27
(E)-caryophyllene	1417	1427	18.01
β-copaene	1430	1431	0.86
γ-elemene	1436	1436	1.64
aromadendrene	1439	1441	0.67
6,9-guaiadiene	1442	1445	0.01
α- humulene	1452	1458	6.14
allo-aromadendrene	1458	1463	1.22
γ-muurolene	1478	1478	2.5
germacrene D	1480	1483	1.68
β-selinene	1489	1490	2.5
trans-muurola-4(14),5-diene	1493	1494	0.63
bicyclogermacrene	1500	1501	4.99
δ-amorphene	1511	1509	0.53
γ-cadinene	1513	1516	0.67
δ-cadinene	1522	1528	4.99
trans-cadina-1,4-diene	1533	1535	0.53
selina-3,7(11)-diene	1545	1544	0.44
elemol	1548	1553	0.6
germacrene B	1559	1562	4.99
(E)-nerolidol	1561	1571	8.24
spathulenol	1577	1584	2.03
guaiol	1600	1604	4.9
ledol	1602	1607	0.48
humulene epoxide II	1608	1613	0.44
1-epi-cubenol	1627	1632	1.64
γ-eudesmol	1630	1636	0.72
β-eudesmol	1649	1654	1.2
bulnesol	1670	1672	2.69
cadalene	1675	1677	0.13
eudesm-7(11)-en-4-ol	1700	1698	0.15
Total			99.41

Antibacterial activity

Regarding the antibacterial effect of the essential oils of the genus Eugenia, the broth microdilution assay was performed to determine the MIC and MBC on the S. aureus isolates. All essential oils tested showed antibacterial activity, displaying both bacteriostatic (MIC) and bactericidal effects (MBC). The MIC ranged from 64 to 256 µg/mL for EOEb and from 64 to 1,024 µg/mL for EOEg, whereas the MBC ranged from 64 to 512 µg/mL for EOEb, and from 256 to 1,024 µg/mL for EOEg. The EOEs showed MIC values ranging from 64 to 512 µg/mL and MBC values ranging from 256 to 1,024 µg/mL. Finally, the EOEp showed MIC and MBC values ranging from 64 to 256 µg/mL. For comparative purposes, the mean MIC and MBC values were plotted in Fig. 2. Thus, the EOEb and EOEp exhibited the best antimicrobial activity, with mean MIC values of 106.7 µg/mL and mean MBC values of 156.4 µg/mL and 128 µg/mL, respectively.

Fig. 2.

Mean values of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the essential oils of Eugenia spp. on Staphylococcus aureus isolates from bovine mastitis

Additionally, the MIC and MBC of oxacillin on S. aureus were determined to assess the resistance profile of the isolates to this antibiotic (Table 2). All clinical isolates from bovine mastitis and ATCC 25923 were sensitive to oxacillin, with MIC values ranging from 0.08 to 0.32 µg/mL, below the cutoff of ≥ 4 µg/mL established by CLSI 2020 [20]. As expected, only the ATCC 33591 strain showed resistance to oxacillin, with MIC values at 64 µg/mL, surpassing the cutoff by 16 times.

Table 2.

Minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC) of oxacillin on Staphylococcus aureus isolates from bovine mastitis and ATCC’s

ID of the isolates	OXACILLIN
	MIC (µg/mL)	MBC (µg/mL)
WE07	0.08	0.08
WE08	0.16	0.16
WE09	0.16	0.32
WE10	0.16	0.16
AL01	0.16	0.16
AL02	0.16	0.16
78	0.16	0.16
ATCC 33591	64	64
ATCC 23923	0.32	0.32

Antibiofilm activity

The study on biofilm production profiles of S. aureus isolates indicated that all strains, except for isolate WE07 (a weak producer), were capable of forming biofilms and were categorized as moderate producers (Supplementary Material: Table S1).

When evaluating the impact of essential oils on biofilm formation by S. aureus isolates, it was observed that all essential oils tested at subinhibitory concentrations (½ and ¼ MIC) partially reduced biofilm formation in most isolates (Fig. 3). Intriguingly, isolate AL02 showed increased biofilm production with all essential oils (Fig. 3B). Similarly, for isolate WE09, the ½ MIC concentration of EOEg promoted greater biofilm formation (Fig. 3B). No statistically significant difference was found in the results for isolates WE07 and AL01 (Fig. 3A).

Fig. 3.

Interference of the essential oils of Eugenia spp. on biofilm formation by Staphylococcus aureus isolates from bovine mastitis. Interference on isolates AL01 and WE07 (A), AL02 and WE09 (B), WE08 and WE10 (C) and 78, ATCC33591, and ATCC25923 (D). OEEg: essential oil of Eugenia gracillima; OEEs: essential oil of Eugenia stictopetala; OEEp: essential oil of Eugenia pohliana. (*): p < 0.05; (**): p < 0.01; (***): p < 0.001; (****): p < 0.00001

The assessment of the essential oils of Eugenia spp. on pre-formed biofilm was satisfactory, but the oils did not achieve the desired performance, as there was no disruption of the biofilms previously formed by the isolates (Supplementary Material: Tables S2, S3, S4, and S5).

Synergistic activity

Table 3 shows the results of the synergy test (checkerboard) between the essential oils and oxacillin against the methicillin-resistant strain ATCC 33591 of S. aureus. This strain was chosen for the test as it was the only oxacillin-resistant isolate. Considering the IFIs of the essential oils of Eugenia spp. in association with oxacillin, only the EOEs and EOEp showed synergistic effects (IFI ≤ 0.5), while EOEg showed an additive effect (0.5 < IFI ≤ 1).

Table 3.

Interaction of the essential oils of Eugenia spp. with oxacillin against Staphylococcus aureus (ATCC 33591)

ID of the isolate	Association	Individual MIC (µg/mL)	MIC of the association (µg/mL)	Individual IFI	∑IFI	Effect	MIC reduction %
ATCC 33591 (MRSA)	OXA +  EOEg	64 / 256	32 / 64	0.50 / 0.25	0.75	Additive	50 / 75
	OXA +  EOEs	64 / 512	16 / 128	0.25 / 0.25	0.5	Synergistic	75 / 75
	OXA +  EOEs	64 / 512	8 / 128	0.125 / 0.25	0.375	Synergistic	87.50 / 75
	OXA +  EOEs	64 / 512	4 / 128	0.0625 / 0.25	0.3125	Synergistic	93.75 / 75
	OXA +  EOEp	64 / 128	8 / 32	0.125 / 0.25	0.5	Synergistic	87.50 / 75
	OXA +  EOEp	64 / 128	4 / 32	0.0625 / 0.25	0.375	Synergistic	93.75 / 75

IFI: inhibitory fraction index; OXA: oxacillin; EOEg: essential oil of Eugenia gracillima; EOEs: essential oil of Eugenia stictopetala; EOEp: essential oil of Eugenia pohliana; % MIC reduction: (individual MIC– combined MIC) x100/ individual MIC. ΣIFI ≤ 0.5: synergism; ΣIFI > 0.5 and ≤ 1: additive; ΣIFI > 1 and ≤ 2: indifferent; ΣIFI > 2: antagonistic

EOEs demonstrated three synergistic concentrations, with a 4-fold reduction in the MIC (from 512 µg/mL to 128 µg/mL) and substantial reductions in the MIC of oxacillin by 4-fold (from 64 µg/mL to 16 µg/mL), 8-fold (from 64 µg/mL to 8 µg/mL), and 16-fold (from 64 µg/mL to 4 µg/mL), representing MIC reductions of 75%, 87.50%, and 93.75%, respectively. EOEp showed two synergistic concentrations, decreasing the MIC by 4-fold (128 µg/mL to 32 µg/mL) and the MIC of oxacillin by 8-fold (64 µg/mL to 8 µg/mL) and 16-fold (64 µg/mL to 4 µg/mL), resulting in MIC reductions of 87.50% and 93.75%, respectively. EOEg showed an additive effect, with a 4-fold reduction in the MIC of the essential oil (from 256 µg/mL to 64 µg/mL) and 2-fold for oxacillin (from 64 µg/mL to 32 µg/mL).

Molecular interaction between the major compounds of the essential oils and penicillin-binding proteins

The molecular docking results showed that the major components of the essential oils from the genus Eugenia interact favorably with the binding site of PBP-1 and PBP-2a, suggesting a possible antibacterial mechanism pathway.

Table 4 presents the best MolDock scores for the interaction of each major component with the target proteins. The components β-(E)-caryophyllene (major in EOEb and EOEp), (E)-nerolidol (major in EOEs), Δ-Elemene (major in EOEg), and α-cadinol (major in EOEp) showed a lower binding energy and consequently a higher affinity to the active protein site compared to penicillin G. All components, except for Δ-cadiene, demonstrated good scores relative to the reference antibiotic. Considering only the best results, the components of the essential oils interact through electrostatic interactions (red lines) with amino acid residues from PBP-1 (Fig. 4) and PBP-2a (Fig. 5). The interactions between β-(E)-Caryophyllene and the target proteins occur between the lysine (194) and glycine (195) residues in chain B and the alanine (196) in chain A of PBP1 (Fig. 4a), and between the tyrosine (165), serine (240), and arginine (151) residues in chain B of PBP2a (Fig. 5a). The (E)-nerolidol interacts with alanine (196) and tryptophan (301) in chain A and the glutamine (185) in chain B of PBP1 (Fig. 4b), and with lysine (322) and glutamine (325) in chain A and asparagine (159) in chain B of PBP2a (Fig. 5b). Δ-Elemene interacts with alanine (196), tryptophan (301), and leucine (197) in chain A and with glutamine (185) in chain B of PBP (Fig. 4c), and with aspartic acid (323), glutamine (325), and leucine (155) in chain A and asparagine (159) in chain B of PBP2a (Fig. 5c). Finally, the interaction between α-cadinol occurs between the leucine (197), tryptophan (301), and alanine (196) residues in chain A and glutamine (185) and alanine (196) in chain B of PBP1 (Fig. 4d), and with the histidine (293), serine (149) and threonine (165) residues in chain B of PBP2a (Fig. 5d). The graphic representations of the formation of the ligand-protein complexes are available in the supplementary material (Fig. S1, S2, S3 and S4).

Table 4.

Score ranking (MolDock) of the best poses obtained from the major components of the essential oils of Eugenia spp.

Compound	PBP-1 (PDB ID 5TRO)	PBP-2a (PDB ID 5M19)
Penicillin G	-73.0329	-72.7931
β-(E)-caryophyllene	-99.6275	-90.6253
(E)-nerolidol	-87.5093	-84.464
Δ-Elemene	-87.3151	-83.9622
α-cadinol	-84.4317	-89.0516
β-pinene	-82.6891	-90.11
δ-cadinene	-81.6872	-83.957
Bicyclogermacrene	-81.634	-83.5364
(E)-caryophyllene	-81.4834	-77.5995
Germacreme B	-81.0782	-80.3505
Germacrene D	-79.3756	-85.2105
γMuurolene-g	-78.6581	-80.6755
αhumulene	-75.5296	-80.6615
Δ-cadiene	-55.9941	-53.8766

PBP (Penicillin Binding Protein)

Fig. 4.

Intermolecular attractions by electrostatic interactions of the major compounds of the essential oils of Eugenia spp. in the crystalline structure of the penicillin-binding protein 1 (5TRO) of Staphylococcus aureus. Components β-(E)-Caryophyllene (a), (E)-nerolidol (b), Δ-Elemene (c), and α-cadinol (d)

Fig. 5.

Intermolecular attractions by electrostatic interactions of the major compounds of the essential oils of Eugenia spp. in the crystalline structure of the penicillin-binding protein 2a (5M19) of Staphylococcus aureus. Components β-(E)-Caryophyllene (a), (E)-nerolidol (b), Δ-Elemene (c), and α-cadinol (d)

Discussion

Previous research on the composition of Eugenia species, including E. biflora, E. egensis, E. flavenscens, E. patrisii, E. polystachya, E. protenta, E. punicifolia, E. brejoensis, and E. klotzschiana, has reported compounds such as caryophyllene, bicyclogermacrene, and germacrene B as primary constituents [27–31]. These sesquiterpenes appear to be inherent to the genus, given their frequent presence in various Eugenia species, even if they are not always dominant compounds.

On a different note, the compound β-pinene was exclusively observed in the composition of E. umbeliflora among the major components, albeit at a higher proportion (13.2%) [32]. It is important to highlight that the composition of E. umbeliflora also includes caryophyllene, α-humulene, bicyclogermacrene, and Germacrene B. However, (E)-nerolidol was found in trace amounts solely in the composition of E. patrisii (0.2%) [30]. Given the limited or negligible presence of β-pinene in the majority of essential oils from Eugenia species described in the literature, it is conceivable that these compounds could be specific to the species, particularly when they are found among the predominant components. However, since this is the first study reporting on the essential oil composition of E. stictopetala leaves, it is important to assess whether the composition is heavily influenced by biotic and abiotic factors.

Studies evaluating the virulence factors and antimicrobial resistance markers in S. aureus isolates from bovine mastitis have demonstrated that the majority of isolates exhibit traits such as biofilm formation, hemolysis, and genotypic and phenotypic resistance to almost all antimicrobial agents (e.g., β-lactams, macrolides, chloramphenicols, tetracyclines, aminoglycosides, fluoroquinolones, and lincosamides), demonstrating an imminent risk posed by infections of animal origin [33]. Furthermore, this scenario causes concerns due to the decline in the discovery of new antimicrobial agents that can reverse and prevent the spread of bacterial resistance [34].

From this perspective, there is a trend towards the development of alternative therapies to mitigate the effects of antimicrobial resistance in the treatment of bovine mastitis [7]. These treatments, based on phytotherapeutic agents such as essential oils, show promising results in controlling mastitis-causing pathogens [35]. In this study, the essential oils extracted from E. brejoensis, E. gracillima, E. stictopetala, and E. pohliana showed in vitro antimicrobial activity against S. aureus isolates from bovine mastitis, particularly E. brejoensis and E. pohliana, which inhibited bacterial growth at concentrations ranging from 64 to 256 µg/mL. Low concentrations such as these are promising as they prevent potential cytotoxic effects and enable the formulation of drugs with low contents of essential oils.

The efficacy of essential oils as antibacterial agents was demonstrated in other studies using the essential oil of Eugenia chlorophylla, which showed antimicrobial activity against 20 bacterial isolates and yeasts, with MIC values ranging from 50 to 1,000 µg/mL [36]. Another result was found when evaluating the antibacterial activity of the essential oils of the leaves of Eugenia uniflora against S. aureus and Listeria monocytogenes, resulting in MIC values of 800 µg/mL and 1,040 µg/mL, respectively [37]. Magina et al. [32] evaluated the antibacterial activity of the essential oils of Eugenia brasiliensis, Eugenia beaurepaireana, and Eugenia astringens and observed effective bactericidal action against S. aureus (ATCC 25923), with MIC values of 156, 119, and 1,110 µg/mL, respectively.

This antibacterial effect of the essential oils of the genus Eugenia is due to a variety of bioactive compounds present in their composition, e.g., terpenoids (monoterpenes and sesquiterpenes), aliphatic compounds, flavonoids, tannins, and cyanidins [38]. Moreover, it has been proposed that the main mechanism of action of these molecules causes irreversible damage to the cell wall and membrane, resulting in the leakage of cell content and cell death [39]. In addition, the lipophilic nature of essential oils also favors their penetration into the phospholipid bilayer, resulting in increased permeability and loss of cell integrity [11]. However, it is also important to consider the impact of these compounds on host cells. Recent studies have shown that these effects can vary depending on the plant species, phytochemical content, and proposed dosage, potentially leading to cytotoxic properties in various organisms [40–43].

Based on our molecular docking results, we can suggest that the components present in the essential oils analyzed herein favorably interact with the target proteins (PBP1 and PBP2a). These proteins have the transglycosylase and transpeptidase functions of the peptidoglycan chains in cell wall synthesis, vital for the survival of S. aureus, and constitute outstanding targets in the development of new antibiotics [44]. Furthermore, a variant of these proteins, PBP-2 A, is commonly found in MRSA and is capable of resuming cell wall synthesis even in the presence of β-lactam antibiotics [45]. Our molecular docking results are corroborated by our in vitro antibacterial activity assay, which revealed that the essential oils of Eugenia spp. show good antibacterial activity in both MRSA (ATCC 33591) and MSSA (ATCC 25923), highlighting their effect regardless of the type of PBP present. However, more studies should be conducted to evaluate other possible molecular targets that can also result in bacterial death.

This study also analyzed the biofilm production profile of the S. aureus isolates from bovine mastitis from the states of Bahia and Pernambuco and determined the antibiofilm activity of the essential oils. All clinical isolates tested showed biofilm-forming ability, with one isolate being classified as a weak producer and the others classified as moderate producers. These results are similar to the findings of Felipe et al. [46], who evaluated biofilm production in 127 S. aureus isolates from bovine mastitis, finding that 45% of the isolates were moderate producers, followed by 35% and 20% categorized as strong and weak producers, respectively.

Regarding the antibiofilm effect of essential oils, it is noteworthy that the essential oils from Eugenia species exerted a significant influence on biofilm formation in most of the isolates tested in this study. A notable exception was observed in one isolate, in which the essential oils had a positive impact on the production of the biofilm structure, a phenomenon deserving further investigation in subsequent research. Although the essential oils tested in this study did not show a significant interference with pre-established biofilms, it is necessary to conduct a more in-depth study regarding their use in combination with compounds capable of disrupting the biofilm [47].

Several studies have also demonstrated that essential oils can interfere with the formation of bacterial biofilms [48]. Biofilms are groups of bacteria generally fixed and surrounded by a self-produced polysaccharide matrix, playing a crucial role in the defense against the host’s immune system [49]. This structure also acts as a physical barrier against the action of antimicrobial agents [50] and facilitates the exchange of resistance genes between the microorganisms contained in the matrix [51]. Infections with the presence of biofilm often become persistent, particularly in the case of mastitis, with biofilm leading to a higher survival rate of the microorganisms contained in the matrix even after using different classes of antimicrobials [52]. Therefore, phytochemical agents capable of preventing the formation or destabilize the structure of biofilms are crucial for ensuring effective treatment and averting possible chronicity and reinfection [53].

In addition to possessing antibacterial potential, essential oils and their phytochemical components have been the subject of recent studies investigating the comprehensive effects of these molecules. Specifically, these research endeavors have focused on the interaction of substances with the ability to inhibit the bacterial communication system known as ‘quorum sensing’ (QS) [54–56]. QS is a genetic regulatory mechanism that depends on cell density and plays a pivotal role in governing various bacterial behaviors, e.g., virulence, pathogenicity, and resistance. This encompasses processes such as biofilm formation, toxin production, spore development, horizontal gene transfer, among others [57, 58]. Inhibiting the ability of bacteria to communicate and trigger mechanisms that lead to biofilm production is critical for research aimed at reducing bacterial virulence and resistance to control infections [59]. This makes bacteria more susceptible to the immune system of the host and can enhance the effectiveness of traditional antibiotics without subjecting them to selective pressure [60, 61]. For instance, Sharifi et al. [62] assessed the anti-QS potential of the essential oil of Satureja hortensis against S. aureus, highlighting that the subinhibitory concentration (0.0312 µl/mL) was capable of inducing negative regulation of up to 5.88 times in the expression of the hld gene compared to untreated biofilms. However, there remains a significant gap in research, particularly concerning the essential oils of Eugenia spp. and their isolated components with regard to QS inhibition. This is a promising area to be explored in future studies.

Considering the combination between essential oils and antibiotics, the essential oils of E. stictopetala and E. pohliana showed synergistic effects with oxacillin, with MIC reductions ranging from 75 to 93.75% (decreasing from 64 to 4 µg/mL) against the MRSA strain ATCC 33,591. We stress that these results are extremely promising since they can reverse the resistance status of an MRSA, eventually decreasing their concentration below the cutoff (≥ 4 µg/mL) proposed by CLSI [20]. Furthermore, we suggest that the chemical components present in the essential oils of Eugenia spp. can bind to an allosteric site of PBP-2a, which modulates the active site, enabling β-lactam antibiotics (such as oxacillin) to regain their bactericidal effect and reversing the susceptibility of MRSA strains [53]. Thus, our study enables the development of therapeutic strategies aiming at the combined use of essential oils and traditional antimicrobials in order to obtain better treatment effectiveness and help solve the bacterial resistance problem [63]. Similar results were found by Aelenei et al. [48], in whose study the essential oil of coriander (Coriandrum sativum L., Apiaceae) showed a synergistic effect with oxacillin against the same strain tested in the present study, reducing the MIC of the antibiotic from 64 to 8 µg/mL (reduction of 87.5%). Therefore, essential oils are, in fact, outstanding candidates for synergistic use with regular antimicrobials, potentiating their bactericidal activity and reducing the concentrations required to achieve the desired effect [9]. Therefore, these products also reduce the selection pressure of resistant pathogens, which is crucial to mitigate the long-term impacts of resistance on animal, human, and environmental health [64].

Given the impact of mastitis on dairy herds, there is an immediate need for alternative therapies due to the high antimicrobial resistance rates, especially by S. aureus [65]. Furthermore, bovine milk is an important food source with high nutrient value. However, consumption without pasteurization can result in serious food infection problems and poses significant risks to public health due to the transmission of resistant pathogens in food products [66]. The essential oils presented herein are promising sources for developing new drugs and antiseptic products, offering new perspectives for effective mastitis treatment [67]. Furthermore, we suggest evaluating the antimicrobial effects of the main components of essential oils in isolation, as these can serve for the chemical synthesis of new components. Finally, studies on the cytotoxic effects and in vivo antimicrobial activities also need to be evaluated.

Conclusions

The chemical composition of the essential oil of E. stictopetala was reported for the first time, revealing that the main chemical constituents are (E)-caryophyllene, β-pinene, (E)-nerolidol, α-humulene, bicyclogermacrene, and Germacrene B. This study also demonstrated that the essential oils of E. brejoensis, E. gracillima, E. stictopetala, and E. pohliana have good in vitro and in silico antibacterial activity against S. aureus from bovine mastitis. The species E. brejoensis and E. pohliana stood out by showing antibacterial activity at concentrations lower than 0.2 mg/mL. Regarding antibiofilm activity, essential oils decreased biofilm formation in 55.5% of the isolates, led to an increase in 11.1%, and had no significant effect in 22.2% of cases. However, it is important to emphasize that no interference against pre-established biofilms was observed in any of the isolates. Finally, the essential oils of E. stictopetala and E. pohliana showed synergistic effects with oxacillin, demonstrating their potential as adjuvants in combined therapy. Our promising results indicate the importance of this segment in research to determine the safety of such products in in vivo models. Given the high resistance of antimicrobials in human and veterinary therapies, natural alternatives with essential oils have significant potential for sustainable animal production.

Electronic supplementary material

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Author contributions

Alisson Teixeira da Silva: Methodology, Validation, Investigation, Data Curation, Writing– Original Draft and Project administration. Danillo Sales Rosa: Formal analysis and Writing– Review & Editing. Marcio Rennan Santos Tavares: Software and Formal analysis. Renata de Faria Silva Souza: Conceptualization, Methodology, Writing– Review & Editing and Supervision. Daniela Maria do Amaral Ferraz Navarro: Methodology and Resources. Júlio César Ribeiro de Oliveira Farias de Aguiar: Methodology and Resources. Márcia Vanusa da Silva: Conceptualization and Resources. Mateus Matiuzzi da Costa: Conceptualization, Resources, Writing– Review & Editing, Supervision, Project administration and Funding acquisition.

Funding

This study was financed in part by the Coordination for the Improvement of Higher Education Personnel– Brazil (CAPES)– Finance Code 001 and by the Science and Technology Support Foundation of Pernambuco (FACEPE) (grant number APQ-0474-5.05/19).

Data availability

Not applicable

Code availability

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Declarations

Consent for publication

All authors agree to publish this work.

Ethical approval

This article did not contain any studies with animals performed by any of the authors.

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Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.