Unveiling the Antibacterial Potential of Melaleuca cajuputi Essential Oils Against MRSA: Integrating In Vitro Efficacy and In Silico Mechanistic Insights

Abstract

The increasing prevalence of antimicrobial resistance, especially in methicillin-resistant Staphylococcus aureus (MRSA), underscores the need for alternative therapies from natural sources. This study investigated the chemical composition, antibacterial activity, and gene expression modulation of Melaleuca cajuputi essential oils. Gas chromatography–mass spectrometry (GC-MS) identified 91 compounds, with naphthalene (23.90%), guaiol (12.92%), caryophyllene oxide (9.69%), D-limonene 98% (8.59%), and gamma terpinene (7.54%) among the most abundant. In Silico molecular docking against MRSA virulence proteins revealed that alloaromadendrene had the strongest binding to toxic shock syndrome toxin-1 (TSST-1) (−7.948 kcal/mol), suggesting high inhibitory potential, while cyclohexane showed weak binding with staphylococcal enterotoxin A (SEA) (−3.532 kcal/mol). Antibacterial assays demonstrated concentration-dependent inhibition, with the zones ranging from 6.33 ± 0.33 mm to 16.67 ± 0.88 mm. MIC and MBC values ranged from 1.56 to 12.5% and 3.13 to 25%, respectively, with most isolates showing bactericidal effects (MBC/MIC ≤ 2). Gene expression analysis of MRSA isolate 4 indicated that sea was moderately upregulated (FC = 1.44), while sec remained unchanged (FC = 1.02). In contrast, fnbA (FC = 0.72), seb (FC = 0.33), and mecA (FC = 0.23) genes were downregulated, and the tsst-1 gene (FC = 0.05) was nearly silent. These findings highlight M. cajuputi essential oils as a promising candidate with both antibacterial efficacy and regulatory effects on MRSA virulence genes.

1. Introduction

Antibiotics are potent agents used against microbial infections, but their inappropriate or excessive use can cause adverse effects and accelerate the emergence of resistant strains. Antibiotic resistance is now a major global health threat, contributing to treatment failures, prolonged illnesses, increased healthcare costs, and higher mortality rates [1]. Resistance arises when bacteria develop strategies to withstand antibiotic action, including structural modification of target sites, enzymatic degradation or inactivation of drugs, active efflux pumps, and alterations of outer membrane components or receptors that reduce antibiotic uptake [2]. Furthermore, resistance genes can spread rapidly within microbial communities through horizontal gene transfer, amplifying the challenge of effective treatment. These limitations underscore the urgent need to explore alternative therapeutic strategies, including plant-derived natural products, bioactive phytochemicals, and microbial enzymes such as tannase, which have shown promising antimicrobial and synergistic properties against resistant pathogens [3,4].

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most clinically important antibiotic-resistant pathogens and is responsible for a wide range of infections, from skin and soft tissue infections to life-threatening conditions such as pneumonia, sepsis, and endocarditis. It is a major cause of hospital-acquired infections worldwide, posing a significant challenge to public health because of limited treatment options, high morbidity, and increased mortality rates [5]. The resistance mechanism in S. aureus is primarily attributed to the acquisition of the mecA gene located on the staphylococcal cassette chromosome mec (SCCmec). This gene encodes penicillin-binding protein 2a (PBP2a), an altered enzyme with a markedly reduced affinity for β-lactam antibiotics, thereby rendering methicillin, oxacillin, and related antibiotics ineffective. In addition to chromosomal acquisition, resistance traits can spread between S. aureus strains through horizontal gene transfer mediated by mobile genetic elements such as bacteriophages and plasmids, which facilitates the rapid dissemination of resistance [6].

Clinically, MRSA infections are classified into two categories. Hospital-associated MRSA (HA-MRSA) typically occurs in immunocompromised or hospitalized patients, especially those exposed to invasive procedures, medical devices, or prolonged antibiotic therapy, and these strains are often resistant to multiple classes of antibiotics [7]. In contrast, community-associated MRSA (CA-MRSA) arises in healthy individuals without recent healthcare exposure, most frequently presenting as skin and soft tissue infections, although it can also lead to severe invasive diseases. CA-MRSA strains are commonly associated with virulence factors such as Panton–Valentine leukocidin (PVL), which enhances their pathogenicity, and they generally remain susceptible to a broader range of non-β-lactam antibiotics compared to HA-MRSA [8]. The distinct molecular characteristics, virulence factors, and antibiotic susceptibility patterns of HA-MRSA and CA-MRSA strongly influence clinical outcomes and treatment strategies. Effective management of MRSA therefore requires continuous epidemiological surveillance, strict infection control measures, and prudent antibiotic stewardship to minimize resistance development and improve patient outcomes [9].

For centuries, essential oils have been used in different cultures around the world, such as in Chinese, Egyptians and Greek cultures. However, in the modern world, essential oils are still used as a different approach or alternative in the community and present in cosmetics, aromatherapy, perfumes and complementary medicines. Essential oils have a wide range of medicinal applications, including antimicrobial, anti-inflammatory, antioxidant, anticancer and many others [10,11,12,13,14]. Essential oils contain natural compounds that have the ability to inhibit the growth of pathogens. Some of the major compounds are alcohols, terpene (monoterpenes and sesquiterpenes), esters, acids, aromatic hydrocarbons, terpenoids, aldehydes and ketones, and may act together to contribute to their antibacterial activity [15,16,17].

Melaleuca cajuputi (family Myrtaceae) is distributed across Borneo, Cambodia, Java, Peninsular Malaysia, Sumatra, Thailand, and Vietnam. According to the Malaysia Biodiversity Information System (MyBIS), its common names include English (cajeput, paper bark tree, swamp tea tree) and Malay (gelam, kayu putih). This species typically forms colonies and is predominantly found in lowland ecosystems, shallow peat forests, and swampy wetlands [18,19]. Members of the genus Melaleuca exhibit notable ecophysiological adaptability, enabling them to tolerate acidic freshwater conditions, nutrient-poor soils, seasonal flooding, and climate variability [20]. Beyond their ecological role in stabilizing soils, regulating hydrological balance, and supporting wetland biodiversity, M. cajuputi is also valued for its bioactive secondary metabolites, particularly essential oils rich in terpenoids, which are traditionally used in folk medicine, aromatherapy, and as antimicrobial, anti-inflammatory, and antioxidant agents [21,22].

The biological activity of M. cajuputi has been investigated by many researchers, including its antimicrobial and antifilarial effects, among others [23,24,25,26]. Although other subspecies of M. cajuputi have been investigated, no dedicated study has yet explored the antimicrobial potential of M. cajuputi EO against MRSA. The extensive and inappropriate use of antibiotics, which has led to the emergence of antimicrobial resistance, has prompted researchers to search for new bioactive compounds from plants. Medicinal plants contain abundant bioactive compounds that can be used alone or in combination with antibiotics to enhance their antimicrobial efficacy in antimicrobial activity [27]. MRSA remains a significant concern in the region, with persistently high levels in several countries. MRSA is also listed in the high-priority category in the Bacterial Priority Pathogens Lists (BPPL) [28]. According to the National Surveillance of Antimicrobial Resistance (NSAR) report 2023, in Malaysia, from the total S. aureus (34,677) isolated from all clinical samples in 2023, 1893 (5.5%) isolates were confirmed to be MRSA [29]. MRSA poses a significant challenge as it continues to develop resistance to vancomycin, which is considered the last-resort antibiotic. Although cases of vancomycin-resistant MRSA have not yet been documented in Malaysia, a case of vancomycin-resistant S. aureus has been reported in Indonesia [30].

Developing an ideal antibacterial agent for MRSA poses a significant challenge due to limitations of current treatments, including resistance, adverse side effects, suboptimal efficacy against biofilms, and poor tissue penetration. Combination therapies also face multiple challenges, even though they are promising for severe infections. Some of the challenges are resistance, drug interactions, and limited clinical evidence to support the effectiveness of combination therapies [31]. HA-MRSA and CA-MRSA exhibit different antibiotic susceptibility patterns, along with distinct clinical characteristics and molecular profiles, which influence treatment strategies. The majority of MRSA bacteraemia cases are acquired in hospitals (52.7%) or other healthcare facilities (35.6%), a trend that has also been observed across other Southeast Asian countries [9].

Therefore, this study will be conducted to determine the antibacterial effect of M. cajuputi essential oils against MRSA. The objectives are to characterize the phytochemical composition of the essential oil, evaluate its antibacterial activity against MRSA using In Vitro assays, assess its effect on the expression of selected MRSA virulence and resistance genes, and explore its potential mechanism of action through molecular docking with MRSA target proteins. The overall aim of this study is to provide scientific evidence supporting the antibacterial potential of M. cajuputi essential oils as a natural therapeutic alternative or complementary approach for the management of MRSA infections.

2. Materials and Methods

2.1. Plant Collection

The leaves of M. cajuputi were collected from Gong Badak, Kuala Nerus, Terengganu, Malaysia, and subsequently deposited at the Herbarium, Faculty of Bioresources and Food Industry (FBIM), Universiti Sultan Zainal Abidin (UniSZA) for species identification and confirmation (Voucher No. UniSZA/A/000000045).

2.2. Essential Oils Extraction

Briefly, fresh leaves of M. cajuputi were subjected to steam distillation for 6 h per cycle. In this process, water vapor at atmospheric pressure and 100 °C was used to release the oil from the leaves, thereby isolating the essential oil from the crude plant material. The resulting oil–hydrosol emulsion was left to stand for 12 h to allow complete separation of the oil and hydrosol layers. The essential oil was then carefully collected and stored in dark glass vials at room temperature until further use [25]. Subsequently, the oil samples were analyzed by GC–MS.

2.3. GC-MS

Phytochemical characterization of M. cajuputi essential oils was carried out using an Agilent 7890A gas chromatograph (GC) (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 5975C inert mass spectrometer (MS) (Agilent Technologies) equipped with a triple-axis detector and fitted with a DB-5MS UI capillary column (Agilent Technologies) (30 m × 0.25 mm i.d., 0.25 µm film thickness, 5% phenyl methylpolysiloxane stationary phase). The oven temperature was initially set at 50 °C and increased at a rate of 6 °C/min to 280 °C, where it was held for 10 min. The injector temperature was maintained at 250 °C, with helium as the carrier gas at a constant flow rate of 1 mL/min. Sample injection was performed in splitless mode. The MS was operated with an electron multiplier voltage of 1094 eV, while the ion source and interface temperatures were set at 250 °C and 200 °C, respectively. Mass spectra were acquired over the m/z range of 45–600, with a solvent delay of 1.372 min, starting from 6 min to 50.333 min. Compound identification was conducted using MSD ChemStation software (Agilent Technologies, Santa Clara) and peaks were matched against the NIST/EPA/NIH mass spectral library (version 2.0). Identified constituents were subsequently compiled into a peak table [32,33].

2.4. In Silico Molecular Docking Analysis

Molecular docking was carried out to evaluate the interactions of M. cajuputi essential oils phytochemicals with MRSA virulence and resistance proteins, including staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC), toxic shock syndrome toxin-1 (TSST-1), fibronectin-binding protein A (FnBPA), and penicillin-binding protein 2a (PBP2a). Twenty major phytoconstituents identified through GC-MS profiling were selected as ligands. Their 3D structures were retrieved from PubChem, energy-minimized, and converted into PDBQT format using AutoDock MGLTools (The Scripps Research Institute, La Jolla, CA, USA). Protein crystal structures were obtained from the RCSB Protein Data Bank. Receptor preparation involved removing water molecules and heteroatoms, repairing incomplete residues, and adding polar hydrogens and Kollman charges. Docking simulations were performed using AutoDock 4.2.6 under blind docking conditions with the Lamarckian Genetic Algorithm (100 runs, population size 150, 2.5 × 10⁶ energy evaluations, 27,000 generations, mutation rate 0.02, crossover rate 0.8). Binding poses were clustered at 2.0 Å RMSD, and the best conformations were selected based on the lowest binding energies and largest clusters. Ligand–protein interactions were further analyzed in both 2D and 3D representations using Discovery Studio Visualizer (BIOVIA, Vélizy-Villacoublay, France) [14,34].

2.5. Tested Bacteria

Eight clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) obtained from Premier Integrated Labs Sdn Bhd were used as test organisms. Additionally, S. aureus ATCC 25923, used as a reference strain, was sourced from the Microbiology Laboratory culture stock at the Faculty of Medicine, Universiti Sultan Zainal Abidin.

2.6. Antibacterial Assay

2.6.1. Disc Diffusion Method

Antimicrobial activity of M. cajuputi essential oils was determined using the disc diffusion method. Bacterial inocula were prepared in Mueller–Hinton broth (MHB) and standardized to 0.5 McFarland turbidity. The suspensions were streaked uniformly onto Mueller–Hinton agar (MHA) plates using sterile cotton swabs. Sterile 6 mm discs were impregnated with 10 µL of essential oil at concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.13%, and positioned on the agar surface. Two controls were used. Vancomycin (30 µg/mL) was used as the positive control, while 10% DMSO in MHB was used as the negative control. Plates were incubated aerobically at 37 °C for 24 h. All assays were carried out in triplicate, and inhibition zone diameters were measured and recorded [35].

2.6.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The MIC of the M. cajuputi essential oils was determined using a 96-well microdilution assay. Two-fold serial dilutions of the preparation were prepared to achieve final concentrations ranging from 100% to 3.13%. An equal volume of bacterial suspension was added to each well, and the plate was incubated aerobically at 37 °C for 24 h. Following incubation, 50 µL of MTT solution was added to each well, and the plate was further incubated for 2 h. Experiments were performed in triplicate. A yellow coloration indicated the absence of bacterial growth, while a purple coloration reflected active growth of the test organism. To determine the minimum bactericidal concentration (MBC), suspensions from MIC wells showing no visible growth were sub-cultured. Briefly, 50 µL of suspension from each selected well was mixed with 150 µL of fresh broth and incubated at 37 °C for 48 h. Subsequently, 100 µL aliquots were spread onto nutrient agar (NA) plates and incubated at 37 °C for 24 h. The absence of bacterial colonies on NA plates was interpreted as the MBC endpoint [23].

2.7. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)

qRT-PCR was performed to assess the transcriptional response of MRSA virulence and antibiotic resistance genes to M. cajuputi essential oils. MRSA 4 was sub-cultured on nutrient agar and incubated at 37 °C for 24 h. Bacterial suspensions were prepared in Mueller–Hinton broth and adjusted to a 0.5 McFarland standard. Two experimental groups were established: (i) treated (T) with essential oils at a sub-minimum inhibitory concentration (sub-MIC) and (ii) untreated control (C). For the treated group, 100 µL of bacterial suspension was incubated overnight at 37 °C, followed by the addition of 100 µL EO (3.13% v/v) and further incubation under the same conditions. For the control group, 120 µL of bacterial suspension was incubated without essential oils. After overnight incubation, both cultures were diluted 1:50 in MHB and grown for an additional 4 h. Cultures were then centrifuged at 16,000× g for 3 min, and the supernatant was discarded [36]. Bacterial RNA was extracted using an RNA extraction kit (1st BASE, Singapore) and subjected to qRT-PCR using the Toyobo kit (Toyobo Co., Ltd., Osaka, Japan). The specific primers for qRT-PCR are listed in

Table 1. Primers for MRSA and housekeeping genes.

Target Gene	Primer Sequence	Function
sea	F 5′-AGC TTG TAT GTA TGG TGG TGT-3′	Staphylococcal Enterotoxins
	R 5′-ACG TCT TGC TTG AAG ATC CA-3′
seb	F 5′-AGG ACA CTA AGT TAG GGA AT-3′	Staphylococcal Enterotoxins
	R 5′-CTC AGT TAC ACC ACC ATA CA-3′
sec	F 5′-TGT AGG TAA AGT TAC AGG TGG T-3′	Staphylococcal Enterotoxins
	R 5′-TGT CTA GTT CTT GAG CTG TTA C-3′
tsst-1	F 5′-ATG GCA GCA TCA GCT TGA TA-3′	Toxic Shock Syndrome-Toxin-1
	R 5′- TTT CCA ATA ACC ACC CGT TT-3′
fnbA	F 5′-CAC AAC CAG CAA ATA TAG-3′	Fibronectin Binding Protein A
	R 5′-CTG TGT GGT AAT CAA TGT-3′
mecA	F 5′-AAA ATC GAT GGT AAA GGT TGG C-3′	Methicillin Resistance Gene
	R 5′-AGT TCT GCA GTA CCG GAT TTG C-3′
nuc	F 5′-GCG ATT GAT GGT GAT ACG GTI-3′	Housekeeping Gene
	R 5′-AGC CAA GCC TTG ACG AAC TAA AGC-3′

. Quantitative PCR (qPCR) was performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The reaction began with a reverse transcription step at 61 °C for 20 min to synthesize complementary DNA (cDNA) from the RNA template, followed by polymerase enzyme activation at 95 °C for 10 min. The cycling stage consisted of 45 cycles of denaturation at 95 °C for 15 s, annealing for 15 s at temperatures optimized according to the melting temperature (Tm) of each primer, and extension/data collection at 74 °C for 45 s, during which fluorescence was monitored. The final melt-curve analysis included denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, and a gradual increase to 95 °C in 0.3 °C increments to determine the melting temperatures. Normalized relative expression levels were calculated using the 2−ΔΔCT method [37].

2.8. Statistical Analysis

Experiments were conducted in triplicate. Data were analyzed using Microsoft Excel 2019 and expressed as mean ± standard deviation (SD). Statistical significance was determined by one-way ANOVA, followed by Tukey’s post hoc test for multiple comparisons. Differences were considered significant at p < 0.05.

3. Results

3.1. Essential Oils

The dried leaves of M. cajuputi used for essential oil extraction weighed 2060.49 g. The extraction yielded 10 mL of essential oil, corresponding to an oil yield of 0.48%.

3.2. GC-MS Analysis

The GC–MS analysis of M. cajuputi essential oils demonstrated a complex chemical composition dominated by sesquiterpene hydrocarbons and their oxygenated derivatives (

Table 2. Chemical composition of M. cajuputi essential oils analyzed by GC–MS.

Peak No.	RT (min)	Identified Compound (Best Hit)	CAS No.	Area %	Match Quality
1	7.027	Bicyclo[3.1.0]hexane, 4-methylene…	003387-41-5	0.03	91
2	7.166	Bicyclo[3.1.1]heptane, 6,6-dimethyl…	018172-67-3	1.56	97
3	7.443	β-pinene	000127-91-3	0.45	92
4	7.891	α-phellandrene	000099-83-2	1.09	91
5	8.162	1,3-cyclohexadiene, 1-methyl-4-(…)	000099-86-5	1.29	97
6	8.585	D-limonene	005989-27-5	8.59	98
7	8.926	1,3,7-octatriene, 3,7-dimethyl-	000502-99-8	0.06	87
8	9.348	γ-terpinene	000099-85-4	6.98	97
9	10.074	(+)-4-carene	029050-33-7	8.87	98
10	10.124	Benzene, 1-methyl-4-(1-methylethyl-)	001195-32-0	0.23	97
11	10.370	Bicyclo[4.3.0]nonane, 2-methylen…	040954-37-8	0.02	70
12	10.572	3-carene	013466-78-9	0.16	97
13	10.654	3-carene	013466-78-9	0.45	94
14	10.938	3-carene	013466-78-9	0.04	97
15	10.982	3-carene	013466-78-9	0.06	95
16	11.058	3-carene	013466-78-9	0.05	95
17	11.184	1,3,6-octatriene, 3,7-dimethyl-…	003338-55-4	0.14	89
18	11.342	3-carene	013466-78-9	0.03	93
19	11.399	3-carene	013466-78-9	0.16	92
20	12.572	Mandelic acid	90-64-2	0.01	70
21	12.951	γ-terpinene	99-85-4	0.05	93
22	13.026	γ-terpinene	99-85-4	0.08	96
23	13.152	γ-terpinene	99-85-4	0.09	96
24	13.260	γ-terpinene	99-85-4	0.01	96
25	13.279	γ-terpinene	99-85-4	0.05	96
26	13.430	γ-terpinene	99-85-4	0.02	96
27	13.455	γ-terpinene	99-85-4	0.07	96
28	13.821	2,6-dimethyl-1,3,5,7-octatetraene	460-01-5	0.01	83
29	13.872	Benzene, tert-butyl-	98-06-6	0.01	53
30	13.954	2,6-dimethyl-1,3,5,7-octatetraene	460-01-5	0.01	89
31	14.357	3-carene	13466-78-9	0.06	78
32	14.402	3-carene	13466-78-9	0.02	70
33	14.452	(+)-4-carene	29050-33-7	0.03	64
34	14.534	3-carene	13466-78-9	0.01	78
35	14.559	3-carene	13466-78-9	0.01	83
36	14.641	3-carene	13466-78-9	0.04	50
37	14.736	(1R)-2,6,6-trimethylbicyclo[3.1…	7785-70-8	0.09	87
38	15.234	Cyclohexene, 5-methyl-3-(1-methyl…	56816-08-1	0.00	52
39	15.442	6,7-dimethyl-1,2,3,5,8,8a-hexahy…	107914-92-1	0.01	38
40	15.518	1,5,5-trimethyl-6-methylene-cycl…	514-95-4	0.01	90
41	15.764	1,3,6-heptatriene, 2,5,5-trimethyl-	29548-02-5	0.12	81
42	15.840	Cyclohexene, 4-ethenyl-4-methyl-	20307-84-0	0.26	99
43	16.099	α-cubebene	17699-14-8	0.01	97
44	16.263	3,7-dimethyloct-6-enyl ethyl car…	1000373-78-1	0.01	74
45	16.288	Tricyclo [4.4.0.0(2,8)]dec-3-en-5-ol	1000193-38-7	0.01	50
46	16.464	Cyclohexene, 1-methyl-4-(1-methyl…	586-62-9	0.01	64
47	16.616	α-ylangene	1000374-19-0	0.02	99
48	16.767	Copaene	3856-25-5	0.14	99
49	16.912	Cyclohexane, 1-ethenyl-1-methyl-…	110823-68-2	0.19	94
50	17.171	Cyclohexane, 1-ethenyl-1-methyl-…	515-13-9	3.65	90
51	17.449	Naphthalene, 1,2,3,4,4a,5,6,8a-o…	473-13-2	0.04	64
52	17.537	Methyleugenol	93-15-2	0.06	95
53	17.922	Caryophyllene	87-44-5	7.65	99
54	17.960	γ-elemene	29873-99-2	0.33	95
55	18.067	α-guaiene	3691-12-1	0.08	99
56	18.168	Aromandendrene	489-39-4	0.07	99
57	18.319	Naphthalene, decahydro-4a-methyl-…	17066-67-0	0.04	70
58	18.452	(E)-β-farnesene	18794-84-8	0.10	96
59	18.647	Humulene	6753-98-6	4.33	97
60	18.887	2-isopropenyl-4a,8-dimethyl-1,2,…	1000192-43-5	0.34	94
61	19.032	α-muurolene	31983-22-9	0.35	98
62	19.089	1,6-cyclodecadiene, 1-methyl-5-m…	23986-74-5	0.34	98
63	19.291	Naphthalene, 1,2,3,5,6,7,8,8a-o…	10219-75-7	1.12	99
64	19.436	Naphthalene, 1,2,3,4,4a,5,6,8a-o…	473-13-2	1.15	91
65	19.505	Naphthalene, 1,2,3,5,6,8a-hexahy…	483-76-1	0.31	99
66	19.625	Naphthalene, 1,2,3,5,6,7,8,8a-oc…	10219-75-7	0.18	78
67	19.720	γ-muurolene	30021-74-0	0.07	98
68	19.808	Naphthalene, 1,2,3,5,6,8a-hexahy…	483-76-1	0.29	98
69	19.909	Epizonarene	41702-63-0	0.10	89
70	20.092	Neoisolongifolene	1000156-12-4	0.03	83
71	20.199	Epizonarene	41702-63-0	0.03	90
72	20.319	α-calacorene	21391-99-1	0.06	95
73	20.382	Cadala-1(10),3,8-triene	1000140-05-6	0.02	91
74	20.508	cis-Z-α-bisabolene epoxide	1000131-71-2	0.08	78
75	20.647	Naphthalene, 1,2,3,4,4a,5,6,8a-o…	473-13-2	0.16	93
76	20.836	Cyclohexane, 1-ethenyl-1-methyl-…	515-13-9	1.54	95
77	21.007	α-guaiene	3691-12-1	0.14	91
78	21.227	Caryophyllene oxide	1139-30-6	1.96	92
79	21.398	Neoisolongifolene, 8,9-dehydro-	67517-14-0	1.78	70
80	21.492	Alloaromadendrene	25246-27-9	0.83	52
81	21.921	Guaiol	489-86-1	12.92	70
82	21.978	Benzoic acid, 4-[(trimethylsilyl)…	27739-17-9	1.66	38
83	22.035	1H-indene, 1-ethylideneoctahydro…	56362-87-9	0.49	51
84	22.142	1H-benzocycloheptene, 2,4a,5,6,7	3853-83-6	0.93	49
85	22.306	Cyclohexene, 6-ethenyl-6-methyl-…	5951-67-7	1.30	96
86	22.470	2-naphthalenemethanol, 1,2,3,4,4…	1209-71-8	3.20	93
90	23.007	β-panasinsene	1000159-39-0	6.89	94
91	23.051	Naphthalene, decahydro-4a-methyl-	000515-17-3	3.67	90
92	23.757	2-acetyl-5-chloro-3-methylbenzo…	51527-18-5	5.35	72
93	24.136	Trans-farnesol	106-28-5	0.35	64
94	24.357	Naphthalene, 1,2,3,4,4a,5,6,8a-o…	473-13-2	0.11	89
95	24.603	Naphthalene, decahydro-4a-methyl…	017066-67-0	0.05	97
96	24.678	Naphthalene, decahydro-4a-methyl…	017066-67-0	0.02	95
97	24.893	Carbazole, 1,4-dimethyl-	18028-55-2	0.09	55
98	24.912	Carbazole, 1,4-dimethyl-	18028-55-2	0.17	55
99	25.517	2′,3′,4′ trimethoxyacetophenone	13909-73-4	0.02	42
100	25.555	3,5-pyridine-diamidoxime	1000212-04-1	0.03	38
101	25.707	cis-β-farnesene	28973-97-9	0.07	70
102	26.237	(Z,Z)-α-Farnesene	1000293-03-1	0.01	38
103	26.767	Ethylene, 1-nitro-2-[3-hydroxy-4…	1000127-35-7	0.01	46
104	27.101	Hexadecanoic acid, methyl ester (Methyl palmitate)	112-39-0	0.09	98
105	27.246	Pentadecanoic acid, 14-methyl-, methyl ester	5129-60-2	0.01	97
106	27.871	Benzenemethanol, 4-(1,1-dimethylethyl)-	877-65-6	0.00	49
107	28.167	n-hexadecanoic acid (palmitic acid)	57-10-3	0.49	99
108	28.533	n-hexadecanoic acid	57-10-3	0.02	98
109	28.596	n-hexadecanoic acid	57-10-3	0.01	98
110	28.646	n-hexadecanoic acid	57-10-3	0.02	99
111	28.760	n-hexadecanoic acid	57-10-3	0.00	93
112	28.804	n-hexadecanoic acid	57-10-3	0.01	94
113	29.239	n-hexadecanoic acid	57-10-3	0.01	95
114	29.561	Retene	483-65-8	0.11	64
115	29.612	Retene	483-65-8	0.07	68
116	29.694	Retene	483-65-8	0.01	68
117	29.713	Retene	483-65-8	0.02	68
118	29.795	Ethanone, 1-(4,6-dihydroxy-2,3,5-trihydroxyphenyl)-	21987-07-5	0.07	64
119	29.883	Ethanone, 1-(4,6-dihydroxy-2,3,5-trihydroxyphenyl)-	21987-07-5	0.03	52
120	29.984	1H-3a,7-methanoazulene, octahydro-	25491-20-7	0.12	93
121	30.135	Cyclopentaneacetaldehyde, 2-formyl-	5951-57-5	0.08	51
122	30.495	3,6-di(N-pyrrolidinyl)-1,2,4,5-tetrazine	117040-53-6	0.04	38
123	30.571	1H-3a,7-methanoazulene, octahydro-	25491-20-7	0.02	86
124	30.596	1H-3a,7-methanoazulene, octahydro-	25491-20-7	0.02	92
125	30.646	1H-3a,7-methanoazulene, octahydro-	25491-20-7	0.01	94
126	30.672	6-(1-hydroxymethylvinyl)-4,8a-dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene	1000190-51-4	0.02	42
127	30.741	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.02	94
128	30.791	2H-2,4a-ethanonaphthalen-8(5H)-one	032391-46-1	0.01	50
129	31.138	Bicyclo[6.1.0]nonane, 9-(1-methylethenyl)-	056666-90-1	0.04	70
130	31.189	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.03	95
131	31.365	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.01	95
132	31.448	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.02	94
133	31.466	Naphthalene, 1,2,4a,5,6,8a-hexahydro-	483-75-0	0.02	66
134	32.337	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.01	87
135	32.476	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.01	95
136	32.652	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.01	86
137	32.684	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.01	95
138	32.766	1H-cycloprop[e]azulene, 1a,2,3,5,6,7,7a,7b-octahydro-	021747-46-6	0.01	91
139	32.785	6-isopropenyl-4,8a-dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene	1000189-10-2	0.01	93
140	32.867	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.01	89
141	32.924	Bicyclo[7.2.0]undec-4-ene, 4,11-dimethyl-	013877-93-5	0.01	91
142	32.943	Isocaryophillene	1000140-07-2	0.01	93
143	34.072	6-Isopropenyl-4,8a-dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene	1000189-10-2	0.00	90
144	34.129	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.00	94
145	34.211	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.00	89
146	34.255	1H-3a,7-methanoazulene, octahydro-	025491-20-7	0.00	94
147	34.305	Isocaryophillene	1000140-07-2	0.00	66
148	40.027	Zinc, dicyclopentyl-	020525-74-0	0.00	38
149	40.273	Anthracene, 9,10-dihydro-9,9,10-trimethyl-	014923-29-6	0.00	55
150	40.437	9,10-methanoanthracen-11-ol, 9,10-dihydro-	126615-74-5	0.00	62
151	47.087	Methyl abietate	000127-25-3	0.00	11
152	47.118	Di-n-decylsulfone	111530-37-1	0.01	12
153	49.383	Acetic acid, [4-(1,1-dimethylethyl)phenyl] ester	088530-52-3	0.01	38
154	49.497	2-ethylacridine	055751-83-2	0.00	42

; see Supplementary Information for the GC–MS chromatogram of M. cajuputi essential oils).

GC–MS analysis of the sample revealed a total of 154 peaks, corresponding to 96 unique compounds. The chemical profile was dominated by monoterpenes, including β-Pinene, α-phellandrene, D-limonene, γ-terpinene, and 3-carene, which eluted between 7 and 15 min. Sesquiterpenes such as caryophyllene, isocaryophillene, alloaromadendrene, and α-guaiene were detected in the mid-range of 17–34 min. Oxygenated terpenes, including caryophyllene oxide and cis-Z-α-bisabolene epoxide, were present in lower amounts. Aromatic and polycyclic hydrocarbons, including naphthalene, anthracene, and azulene derivatives, were detected in trace quantities. Fatty acids and their esters, such as n-hexadecanoic acid (palmitic acid), hexadecanoic acid methyl ester, and methyl abietate, eluted later between 27 and 49 min. The most abundant compounds were guaiol (12.92%), (+)-4-carene (8.87%), D-limonene (8.59%), caryophyllene (7.65%), and γ-terpinene (6.98%), indicating that monoterpenes and sesquiterpenes are the major constituents. Match quality for most compounds ranged from 70 to 99%, while some minor components exhibited lower values. Overall, the GC–MS profile indicates that the sample is rich in monoterpenes and sesquiterpenes, with minor contributions from fatty acids, esters, and aromatic hydrocarbons, consistent with typical plant-derived essential oils.

The GC–MS analysis of the sample revealed a diverse range of compounds with potential antimicrobial activity. Among the major components, D-limonene was identified and is known to possess strong antibacterial activity, including inhibition of MRSA [38]. Limonene also exhibits synergistic effects when combined with antibiotics such as gentamicin and demonstrates antifungal activity against Candida albicans and Candida tropicalis, including the ability to disrupt biofilms [39].

β-pinene was also detected in the sample and is recognized for its antibacterial properties, particularly against S. aureus. It can act synergistically with other compounds in essential oils to enhance antimicrobial efficacy [40]. The analysis showed the presence of γ-terpinene, which has been reported to exert antibacterial effects against S. aureus and is commonly found in essential oils used for treating infections [41]. Caryophyllene and its oxygenated derivative, caryophyllene oxide, were identified as well; both compounds are known for their antibacterial and antibiofilm activity, including inhibitory effects against MRSA and E. coli [42].

γ-muurolene, a sesquiterpene detected in the sample, has been reported in essential oils with antimicrobial activity against both Gram-positive and Gram-negative bacteria. Additionally, 1H-3a,7-methanoazulene (octahydro-) was present, although there is limited direct evidence of its antimicrobial activity in the literature. Its role in the essential oil may be more modulatory, potentially contributing to synergistic effects alongside other bioactive compounds. The combination of monoterpenes, such as D-limonene, and sesquiterpenes, such as caryophyllene and γ-muurolene, in the sample suggests a synergistic effect that enhances antimicrobial potency [43].

Overall, the chemical composition identified in the GC–MS analysis supports the potential of the sample to exhibit antimicrobial activity, particularly against MRSA and fungal pathogens. The presence of both major and minor bioactive compounds contributes to a broad-spectrum antimicrobial potential, with monoterpenes primarily responsible for antibacterial and antifungal effects and sesquiterpenes enhancing synergistic activity. The results indicate that M. cajuputi essential oils are chemically characterized by a high proportion of sesquiterpene hydrocarbons and oxygenated sesquiterpenes, along with trace amounts of phenolic and aromatic compounds, highlighting its potential as a natural source of bioactive molecules with pharmacological and therapeutic applications.

3.3. In Silico Molecular Docking

Table 3. Docking results of the 20 strongest compounds of M. cajuputi essential oils with the lowest estimated binding energies (kcal/mol) against MRSA SEA, SEB, SEC, TSST-1, FnBPA and PBP2a proteins.

Peak	Ligand	Docking Score/Binding Interaction
		Staphylococcal Enterotoxin A (SEA)	Staphylococcal Enterotoxin B (SEB)	Staphylococcal Enterotoxin C (SEC)	Toxic Shock Syndrome Toxin-1 (TSST-1)	Fibronectin-Binding Protein A (FnBPA)	Penicillin-Binding Protein 2a (PBP2a)
1.	Alpha-guaiene	−5.942 kcal/mol Pi-Alkyl bond = A: PHE58, A: PHE57	−6.759 kcal/mol Alkyl bond = B: LEU11. B: PRO6	−6.022 kcal/mol Alkyl and Pi-Alkyl bond = A: LYS212, A: ALA203, A: TYR213, A: PHE208	−7.578 kcal/mol Alkyl bond = A: LYS110	−7.395 kcal/mol Alkyl bond = B: VAL256, B: PRO219, B: PRO309	−6.827 kcal/mol Alkyl bond = A: LEU224, A: VAL217
2.	Alpha-muurolene	−5.716 kcal/mol Alkyl bond = A: LEU23	−6.425 kcal/mol Pi-Sigma bond = A: PHE47 Pi-Alkyl bond = A: TYR46, B: TYR233	−6.339 kcal/mol Alkyl and Pi-Alkyl bond = A: MET216, A: LYS212, A: TYR213, A: PHE208, A: ALA203	−7.819 kcal/mol Alkyl bond = A: LYS111, A: LYS110	−6.967 kcal/mol Alkyl and Pi-Alkyl bond = B: PHE306, B: PRO219, B: PRO309, B: VAL256	−6.934 kcal/mol Alkyl and Pi-Alkyl bond = A: LYS604, A: TRP616, A: LEU603, A: ILE164, A: ALA601
3.	Alpha-ylangene	−5.827 kcal/mol Alkyl bond = A: LEU23	−6.309 kcal/mol Alkyl and Pi-Alkyl bond = B: ALA203, B: TYR213, B: LYS212	−6.169 kcal/mol Alkyl bond = A: LYS162, A: VAL147	−7.766 kcal/mol Alkyl bond = A: LYS111	−7.763 kcal/mol Alkyl and Pi-Alkyl bond = B: PHE306, B: VAL256	−6.57 kcal/mol Pi-Sigma bond = A: TYR272 Alkyl bond = A: LYS289
4.	Alloaromandendrene	−5.974 kcal/mol Alkyl and Pi-Alkyl bond = A: PRO139, A: ARG161, A: TYR162	−6.207 kcal/mol Pi-Alkyl bond = A: PHE47	−6.177 kcal/mol Alkyl and Pi-Alkyl bond = A: LEU58, A: HIS31	−7.948 kcal/mol Alkyl bond = A: LYS111	−7.249 kcal/mol Alkyl bond = B: VAL256	−6.828 kcal/mol Pi-Alkyl bond = A: TYR272
5.	Caryophyllene	−6.02 kcal/mol Pi-Alkyl bond = A: TYR162	No interaction.	−5.897 kcal/mol Pi-Sigma bond = A: PHE196 Alkyl bond = A: LYS221	No interaction.	−7.006 kcal/mol Alkyl bond = B: VAL256	−7.001 kcal/mol Pi-Alkyl bond = A: TYR272
6.	Copaene	−5.685 kcal/mol Alkyl bond = A: ALA22, A: LEU23, A: LEU18	−5.876 kcal/mol Pi-Alkyl bond = A: PHE47, A: TYR46, B: TYR233	−6.312 kcal/mol Alkyl and Pi-Alkyl bond = A: MET216, A: TYR213, A: HIS12, A: LYS212	−7.314 kcal/mol Pi-Alkyl bond = A: TRP52	−7.915 kcal/mol Alkyl and Pi-Alkyl bond = B: PHE306, B: VAL256	−6.303 kcal/mol Alkyl and Pi-Alkyl bond = A: LEU224, A: VAL217, A: PHE227, A: TYR223
7.	Cyclohexane	−3.532 kcal/mol Alkyl bond = A: LEU131, A: LYS178	−3.582 kcal/mol Alkyl and Pi-Alkyl bond = A: TYR213, A: LYS212, A: ALA203	−3.759 kcal/mol Alkyl and Pi-Alkyl bond = A: TYR213, A: LYS212, A: ALA203	−4.74 kcal/mol Alkyl and Pi-Alkyl bond = A: ILE166, A: PHE212, A: LYS218	−3.729 kcal/mol Alkyl and Pi-Alkyl bond = B: PHE367, B: LYS369, B: ILE409	−4.063 kcal/mol Alkyl bond = A: LEU570, A: LEU455, A: LYS456
8.	D-limonene	−4.939 kcal/mol Alkyl and Pi-Alkyl bond = A: LEU23, A: LYS27, A: PHE175	−4.999 kcal/mol Alkyl and Pi-Alkyl bond = A: ALA203, A: LYS212, A: LYS207, A: PHE208	−5.097 kcal/mol Alkyl and Pi-Alkyl bond = A: ALA203, A: LYS212, A: TYR213, A; PHE208	−6.163 kcal/mol Alkyl bond = A: LYS110, A: LYS111	−6.004 kcal/mol Alkyl and Pi-Alkyl bond = A: VAL256, A: PRO309, A: PHE306, A: PRO219, C: ALA14	−5.66 kcal/mol Alkyl and Pi-Alkyl bond = A: ALA276, A: TYR272, A: LYS273
9.	Napthalene	−5.187 kcal/mol Pi-Pi T-shaped and Amide-Pi Stacked bond = A: TYR162 Pi-Alkyl bond = A: PRO139, A: ARG161	−5.195 kcal/mol Pi-Sigma bond = B: LEU11 Pi-Alkyl bond = B: PRO6	−5.469 kcal/mol Pi-Donor Hydrogen bond = A: ASP209 Pi-Alkyl bond = A: LYS212 Pi-Sigma bond = A: ALA203	−6.829 kcal/mol Pi-Cation bond = A: LYS211 Pi-Alkyl bond = A: LYS211 Pi-Pi Stacked bond = A: TRP52	−6.375 kcal/mol Pi Alkyl bond = A: VAL256, A: PRO309, C: ALA14	−5.655 kcal/mol Pi-Donor Hydrogen bond = TYR344, A: THR399 Pi-Alkyl bond = A: LYS634, A: LYS394, A: ILE614 Pi-Pi T-shaped bond = A: TYR344
10.	1, 6 Cyclodecadiene	−4.723 kcal/mol Pi-Alkyl bond = A: PHE58	−4.82 kcal/mol Alkyl bond = B: ALA203, B: LYS212	−5.097 kcal/mol Alkyl bond = A: ALA203, A: LYS212	−6.428 kcal/mol Alkyl bond = A: ARG108	−5.336 kcal/mol Alkyl bond = A: ILE409	−5.352 kcal/mol Alkyl bond = A: LEU570, A: LYS456
11.	Beta-pinene	−4.795 kcal/mol Alkyl bond = A: LEU23, A:LYS27	−4.796 kcal/mol Alkyl and Pi-Alkyl bond = A: ALA203, A: LYS212, A: TYR213, A: PHE208	−5.344 kcal/mol Alkyl and Pi-Alkyl bond = A:HIS31, A:LYS57, A:LEU58	−6.201 kcal/mol Alkyl and Pi-Alkyl bond = A: ILE166, A: PHE212, A: LYS218	−5.704 kcal/mol Alkyl bond = B:VAL256	−5.265 kcal/mol Alkyl bond = A:VAL217, A:PRO370
12.	Alpha-phellandrene	−4.73 kcal/mol Alkyl & Pi-Alkyl bond = A: LEU23, A:LYS27, A:PHE175 Pi-Donor Hydrogen Bond = A:SER172	−5.287 kcal/mol Pi-Alkyl bond = B: ALA203, B:HIS12, B:TYR213 Pi-Pi T-shaped & Amide-Pi Stacked = B:LYS212, B:TYR213	−5.724 kcal/mol Pi-Alkyl bond = A:ALA203, A:HIS12, A:TYR213, A:LYS212 Pi-Pi T-shaped = A:TYR213	−6.424 kcal/mol Alkyl and Pi-Alkyl bond = A:LEU49, A:TRP52, A:LYS211 Pi-Pi Stacked = A:TRP52 Pi-Cation = A:LYS211	−5.98 kcal/mol Alkyl & Pi-Alkyl bond = B:LYS411 Pi-Sigma bond = B:ILE409	−5.415 kcal/mol Pi-alkyl bond = A:ALA642 Pi-Pi Stacked = A:TYR446
13.	Gamma-terpinene	−4.706 kcal/mol Alkyl & Pi-Alkyl bond = A: TYR162, A:PRO139	−5.068 kcal/mol Alkyl and Pi-Alkyl bond = B:PRO6, B:TYR186, B:LEU11	−5.233 kcal/mol Alkyl and Pi-Alkyl bond = A: HIS12, A:MET216, A:TYR213	−6.299 kcal/mol Alkyl and Pi-Alkyl bond = A:LEU49, A:TRP52, A:LYS211	−5.817 kcal/mol Alkyl & Pi-Alkyl bond = B:LYS369, B:PHE367, B:ILE409	−5.726 kcal/mol Alkyl & Pi-Alkyl bond = A:TYR272, A:ALA276, A:LYS273
14.	Methyleugenol	−4.726 kcal/mol Carbon hydrogen bond = A:GLU165	−5.093 kcal/mol Conventional hydrogen bond = A:TYR46, B:TYR186	−4.991 kcal/mol Conventional hydrogen bond = A:ASP209 Carbon hydrogen bond = A:ALA203, A:LYS207	−5.692 kcal/mol Conventional hydrogen bond = A:LYS111	−5.269 kcal/mol Conventional hydrogen bond = B:SER351 Carbon hydrogen bond = B:His220	−4.797 kcal/mol Conventional Hydrogen bond = A:HIS293
15.	Gamma-elemene	−4.431 kcal/mol Alkyl bond = A:ALA201, A:ILE212	−5.867 kcal/mol Pi-Alkyl bond = A:TYR46	−5.558 kcal/mol Alkyl and Pi-Alkyl bond = A:PHE208, A:LYS212, A:TYR213	−6.768 kcal/mol Alkyl bond = A:LYS110	−6.516 kcal/mol Alkyl bond = B:LYS411, B:ILE409	−5.961 kcal/mol Alkyl bond = A:VAL217
16.	Caryophyllene oxide	−5.813 kcal/mol Conventional hydrogen bond = A:THR59, A:ASP60	−5.919 kcal/mol Carbon hydrogen bond = B:PRO8	−5.909 kcal/mol Conventional hydrogen bond = A:ASN88 Pi-Alkyl bond = A:HIS31	−6.933 kcal/mol Alkyl bond = A:LYS218 Conventional hydrogen & Carbon hydrogen bond = A:THR216	−7.396 kcal/mol Alkyl bond = B:VAL256	−6.577 kcal/mol Conventional Hydrogen bond = A:LYS218 Alkyl Bond = A:VAL217, A:PRO370
17.	α-calacorene	−5.501 kcal/mol Alkyl & Pi-Alkyl bond = A: TYR162, A:ALA210, A:ILE212 Pi-Sigma bond = A:ILE212	−6.157 kcal/mol Pi-Alkyl bond = A:PHE47	−6.214 kcal/mol Alkyl and Pi-Alkyl bond = A: ALA203, A:PHE208, A:LYS212, A:TYR213	−7.893 kcal/mol Alkyl and Pi-Alkyl bond = A:ILE125, A:TRP52, A:LYS211 Pi-Cation bond = A:LYS211 Pi-Pi Stacked = A:TRP52	−7.038 kcal/mol Alkyl & Pi-alkyl bond = A:PRO309, A:VAL256, A:TYR 414, A:VAL365	−7.126 kcal/mol Alkyl & Pi-Alkyl bond = A:TYR272, A:ALA276, A:LYS273, A:LYS289 Pi-Pi T-shaped = A:TYR272
18.	Gamma-muurolene	−5.422 kcal/mol Alkyl bond = A:ARG161	−6.363 kcal/mol Pi-Alkyl bond = A:PHE47, A:TYR46, B:TYR233	−6.17 kcal/mol Alkyl bond = A:LYS162, A:VAL147	−7.417 kcal/mol Alkyl bond = A:LYS111	−7.539 kcal/mol Alkyl & Pi-alkyl bond = B:PRO309, B:VAL256, B:LEU498, B:PRO219, B:PHE306	−6.494 kcal/mol Alkyl & Pi-Alkyl bond = A:TYR272, A:ALA276, A:LYS273, A:LYS289 Pi-sigma = A:TYR272
19.	Guaiol	8.944 kcal/mol Alkyl & Pi-Alkyl bond = A: TYR30, A:LEU26, A:ALA201, A:ILE212, A:LEU209 Unfavorable Bump = A:ILE212, A:ASP197, A:ARG160	−6.481 kcal/mol Alkyl and Pi-Alkyl bond = A:PRO6, A:TYR186, A:LEU11, A:TYR233	−4.821 kcal/mol Alkyl bond = A:MET215	−7.179 kcal/mol Alkyl and Pi-Alkyl bond = A:TRP52, A:LYS111	−7.274 kcal/mol Alkyl bond = B:PRO309, B:PRO219, B:VAL256	−6.922 kcal/mol Alkyl & Pi-Alkyl bond = A:PHE227, A:VAL217, A:LEU190, A:ILE171, A:LEU224
20.	Humulene	−4.573 kcal/mol Alkyl bond = A:ILE212	−5.846 kcal/mol Alkyl bond = B:ALA203, B:LYS212	−5.601 kcal/mol Pi-Alkyl bond = A:PHE196	−6.674 kcal/mol Alkyl bond = A:LYS111	−6.149 kcal/mol Alkyl bond = B:LYS411	−7.009 kcal/mol Pi-alkyl bond = A:TYR272

presents the docking results, showing the 20 compounds from M. cajuputi EO with the strongest predicted binding affinities (lowest binding energies, kcal/mol) against MRSA target proteins, including SEA, SEB, SEC, TSST-1, FnBPA, and PBP2a.

The docking study revealed that hydrophobic interactions dominated the binding of most plant-derived compounds with their respective protein targets, with docking scores ranging from approximately −7.0 to −8.0 kcal/mol. Such scores are generally considered indicative of favorable binding affinities in structure-based drug design studies [44]. Among the tested ligands, alloaromadendrene (−7.948 kcal/mol) and α-calacorene (−7.893 kcal/mol) showed the strongest binding to toxic shock syndrome toxin-1 (TSST-1) through multiple alkyl and π-alkyl interactions, particularly with residues such as LYS111 and TRP52. These residues have previously been implicated in the structural stability and functional activity of TSST-1 [45], suggesting that hydrophobic blockade at these positions could interfere with toxin-receptor recognition and subsequent pathogenic effects.

Similarly, alpha-murolene (−7.819 kcal/mol) exhibited the strongest binding to staphylococcal enterotoxin C (SEC), forming numerous hydrophobic contacts with key residues including MET216, LYS212, and TYR213. Hydrophobic interactions are critical in stabilizing staphylococcal enterotoxins, which are known for their robust tertiary structures [46]. The capacity of terpenoids like alpha-murolene to establish such interactions aligns with prior reports on the antimicrobial efficacy of hydrophobic phytochemicals, which often disrupt protein stability or membrane integrity [47].

Interestingly, caryophyllene oxide (−7.396 kcal/mol) stood out in the case of fibronectin-binding protein A (FnBPA). Unlike most ligands that relied primarily on hydrophobic interactions, caryophyllene oxide also established conventional hydrogen bonds with THR59 and ASP60. Hydrogen bonding is a pivotal determinant of ligand specificity and stability in protein–ligand complexes [48]. The formation of hydrogen bonds by caryophyllene oxide suggests a more directed and specific interaction with FnBPA, which may enhance its inhibitory potential. Previous studies have also highlighted the antimicrobial properties of caryophyllene oxide, including its ability to compromise biofilm formation and bacterial adhesion [49].

For penicillin-binding protein 2a (PBP2a), α-calacorene (−7.126 kcal/mol) and Humulene (−7.009 kcal/mol) emerged as the most promising ligands, displaying favorable hydrophobic interactions with residues such as TYR272, ALA276, and LYS273. PBP2a is a well-characterized mediator of MRSA due to its reduced affinity for β-lactam antibiotics [6]. The ability of terpenoids to interact with their allosteric or active site residues provides a rational basis for further exploration, particularly in the context of overcoming antimicrobial resistance. Natural terpenoids have previously been reported to enhance the susceptibility of MRSA to β-lactams by disrupting cell wall synthesis or interfering with PBP activity [50].

Taken together, the docking results suggest that α-calacorene, alloaromadendrene, α-murolene, caryophyllene oxide, and humulene are the most promising candidates across the four staphylococcal protein targets. Although hydrophobic interactions were the predominant binding mechanism, the presence of hydrogen bonding in some cases, particularly with caryophyllene oxide, indicates stronger and more specific binding [51,52]. These findings underscore the therapeutic potential of plant-derived terpenoids as antibacterial agents, particularly against virulence factors and resistance-associated proteins in S. aureus.

Nevertheless, it is important to note that molecular docking represents a predictive approach and may not fully capture the dynamic nature of protein–ligand interactions. Further validation through rescoring methods, molecular dynamics simulations, and binding free energy calculations is recommended to assess the stability of these complexes under physiologically relevant conditions [53]. Ultimately, experimental assays such as enzyme inhibition, bacterial growth kinetics, and cytotoxicity profiling will be critical to confirm the biological relevance of these interactions.

3.4. Antibacterial Assay

The M. cajuputi essential oils show notable antibacterial activity against MRSA as well as S. aureus. The antibacterial strength can be categorized into four groups, which are weak (<5 mm), moderate (5–10 mm), strong (10–20 mm), and very strong (20–30 mm), based on the zone of inhibition (ZOI) [54]. From the findings, all the MRSA clinical isolates, as well as S. aureus, displayed moderate to strong sensitivity at concentrations ranging from 25% to 100%, with ZOI values ranging from 6.33 ± 0.33 mm to 16.67 ± 0.88 mm. However, certain MRSA clinical isolates and S. aureus show sensitivity at the 12.5% concentration, with ZOI values ranging from 6.00 ± 0.00 mm to 7.00 ± 0.00 mm. At concentrations of 6.25% and 3.13%, all bacteria show no ZOI, which is 6.00 ± 0.00 mm. For the negative control, which is 10% DMSO, all bacteria show no inhibition as measured by the ZOI. In contrast, positive control, which is vancomycin, exhibited higher ZOI across all bacteria, ranging from 15.33 ± 0.33 mm to 18.00 ± 0.58 mm. These findings indicate that the ZOI of the bacteria is concentration-dependent and demonstrates moderate antibacterial activity (

Table 4. Zone of inhibition of disc diffusion method for M. cajuputi essential oils against MRSA clinical isolates.

Tested Bacteria	Zone of Inhibition (mm)
	100%	50%	25%	12.50%	6.25%	3.13%	10% DMSO (Negative Control)	Vancomycin 30 mcg (Positive Control)
S. aureus ATCC25923	12.67 ± 0.67	9.67 ± 0.33	7.67 ± 0.33	7.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	17.67 ± 0.33
MRSA 1	9.67 ± 0.33	7.67 ± 0.33	7.00 ± 0.00	6.67 ± 0.33	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	16.33 ± 0.33
MRSA 2	10.00 ± 0.00	7.33 ± 0.33	6.33 ± 0.33	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	6.60 ± 0.00	18.00 ± 0.58
MRSA 3	12.00 ± 0.00	8.67 ± 0.33	7.33 ± 0.33	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	18.00 ± 0.58
MRSA 4	16.00 ± 0.58	7.67 ± 0.33	7.67 ± 0.33	6.67 ± 0.33	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	17.33 ± 0.33
MRSA 5	16.67 ± 0.88	8.67 ± 0.33	7.67 ± 0.33	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	16.00 ± 0.58
MRSA 6	10.67 ± 0.33	8.33 ± 0.33	7.00 ± 0.00	6.33 ± 0.33	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	16.00 ± 0.00
MRSA 7	9.67 ± 0.33	8.0 ± 0.00	7.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	18.00 ± 0.58
MRSA 8	14.67 ± 0.67	9.67 ± 0.33	8.33 ± 0.33	6.33 ± 0.33	6.00 ± 0.00	6.00 ± 0.00	6.00 ± 0.00	15.33 ± 0.33

Data are means of three replicates (n = 3) ± standard error.

).

The concentration-dependent activity observed in this study is consistent with previous reports on essential oils, in which higher concentrations generally enhance bacterial membrane disruption and increase the availability of bioactive compounds [55]. The strong activity at higher concentrations may be attributed to the high proportion of oxygenated monoterpenes, such as 1,8-cineole, terpineol, and linalool, which are major constituents of M. cajuputi essential oils and have been reported to exert broad-spectrum antibacterial properties. These compounds are known to integrate into lipid bilayers, increasing membrane fluidity and permeability, which ultimately leads to the leakage of vital intracellular components and cell death [56]. In addition to their membrane-disruptive effects, oxygenated monoterpenes can interfere with bacterial enzymatic systems and metabolic pathways, including the inhibition of ATP synthesis and the disruption of quorum-sensing mechanisms [57]. The synergistic interactions among multiple terpenoids and other minor constituents present in M. cajuputi essential oils may further potentiate their antibacterial efficacy, reflecting the complexity of essential oil bioactivity compared to individual isolated compounds. Collectively, these mechanisms highlight the potential of M. cajuputi essential oils as a natural antibacterial agent, particularly in combating multidrug-resistant pathogens.

The MIC values (3.13–12.5%) and MBC values (3.13–25%) obtained in this study further confirm the antibacterial potency of the essential oils (

Table 5. Minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of M. cajuputi essential oils against MRSA clinical isolates.

Tested Bacteria	MIC (%)	MBC (%)	MBC/MIC
S. aureus ATCC25923	3.13	3.13	1
MRSA 1	6.25	12.5	2
MRSA 2	12.5	25	2
MRSA 3	6.25	12.5	2
MRSA 4	6.25	12.5	2
MRSA 5	3.13	3.13	1
MRSA 6	6.25	12.5	2
MRSA 7	3.13	6.25	2
MRSA 8	3.13	3.13	1

Test is performed in triplicate. MBC/MIC = Bactericidal effect was defined as the MBC/MIC ratio less than 4, whereas bacteriostatic effect was defined as the ratio being greater than 4.

). Natural extracts with MIC values below 8 mg/mL, or equivalent low concentrations, are considered promising antibacterial agents [58]. Although the MIC values for M. cajuputi essential oils are higher than those of conventional antibiotics, they are comparable to or better than those of many other essential oils studied against MRSA and S. aureus. For example, Melaleuca alternifolia essential oils exhibited MICs ranging from 0.5% to 2% against MRSA isolates [59], while Cymbopogon citratus essential oils showed MICs of 2–4% [60]. The somewhat higher MIC observed in the present study could be attributed to differences in chemical composition, bacterial strain variability, or the inherent resistance mechanisms of MRSA.

Importantly, the ability of M. cajuputi essential oils to exhibit both inhibitory and bactericidal effects suggest that their bioactive compounds may target multiple bacterial pathways. Essential oils are known to act primarily through the disruption of bacterial cell membranes, leakage of cellular contents, inhibition of enzyme activity, and interference with quorum sensing [61]. The broad range of MBC values (3.13–25%) indicates variability among the bacterial isolates, highlighting potential strain-specific susceptibility.

From a therapeutic perspective, these findings support the potential application of M. cajuputi essential oils as a natural antibacterial agent, particularly in addressing infections caused by antibiotic-resistant pathogens such as MRSA. With the growing global challenge of antimicrobial resistance, the exploration of natural products such as essential oils offers promising alternatives or complementary approaches to conventional antibiotics [62]. However, further investigations are warranted, including in vivo studies, cytotoxicity assessments, and formulation development, to evaluate the safety, efficacy, and potential synergism with existing antibiotics.

M. cajuputi essential oils demonstrate moderate to strong antibacterial activity against MRSA and S. aureus in a concentration-dependent manner, with promising MIC and MBC values. These results underline their potential as a natural antibacterial agent, although optimization and clinical validation are necessary before practical applications can be realized.

3.5. Gene Level Expression

RT-qPCR was performed using OneStepPlus System by Applied Biosystem. In this study, relative quantification (RQ) change was used to assess the gene expression level of several virulence and toxin genes from MRSA, and the results are presented as fold changes. As presented in Figure 1, the sea gene exhibited the highest expression, with a fold change of 1.44, indicating moderate upregulation relative to the control. The sec gene showed a fold change of 1.02, suggesting expression comparable to baseline. The fnbA gene was modestly upregulated, with a fold change of 0.72. In contrast, seb and mecA showed reduced expression, with fold changes of 0.33 and 0.23, respectively. Notably, tsst-1 demonstrated minimal transcriptional activity, with a fold change of 0.05, indicating near absence of expression in this isolate. These results suggest that sea may play a dominant role in MRSA 4’s virulence, whereas tsst-1 appears transcriptionally silent under the tested conditions. Molecular analysis indicated that all treated groups with M. cajuputi essential oils showed a significant decrease in the expression of sea, seb, sec, tsst-1, fnbA, and mecA genes compared to the respective control groups (p < 0.05).

The RT-qPCR analysis provided insights into the transcriptional profile of several virulence- and toxin-associated genes in MRSA. Among the tested genes, sea exhibited the highest expression with a fold change of 1.44, suggesting moderate upregulation relative to the control. Enterotoxin A (sea) is one of the most prevalent staphylococcal enterotoxins and has been strongly linked to foodborne illness and immune modulation in invasive infections [63]. Its upregulation in the present study indicates that sea may play a dominant role in maintaining the virulence potential of this MRSA isolate.

In contrast, sec demonstrated expression close to baseline (1.02-fold change), while seb showed clear downregulation (0.33-fold change). Enterotoxin B (seb) is commonly associated with severe systemic manifestations, including toxic shock-like syndromes [64]. Its reduced expression under the present experimental conditions may reflect strain-specific regulation of superantigenic toxins or possible suppression by environmental or stress-related factors. The fnbA gene, encoding fibronectin-binding protein A, showed a modest upregulation (0.72-fold change). Although the increase was not pronounced, even low-level upregulation of adhesin genes such as fnbA can facilitate bacterial attachment to host extracellular matrix proteins and promote biofilm initiation [65]. Adhesion and biofilm formation are critical for MRSA persistence, immune evasion, and antibiotic tolerance, underscoring the pathogenic relevance of fnbA expression in this isolate.

Interestingly, mecA, the genetic determinant of methicillin resistance, was markedly downregulated (0.23-fold change). While mecA expression is essential for the synthesis of penicillin-binding protein 2a (PBP2a) and resistance to β-lactam antibiotics, its expression can be influenced by environmental cues, regulatory systems, and potential stressors [66]. The observed reduction may indicate conditional modulation of resistance mechanisms, which could have important implications for therapeutic strategies aimed at attenuating resistance phenotypes. The transcriptional silence of tsst-1 (0.05-fold change) is particularly notable. tsst-1 encodes toxic shock syndrome toxin-1, a potent superantigen responsible for toxic shock syndrome. Its minimal expression in this study suggests that this isolate does not rely on tsst-1 for pathogenicity under the tested conditions.

Overall, these findings suggest a differential transcriptional program in MRSA 4, in which sea appears to be the dominant virulence determinant, while seb, mecA, and particularly tsst-1 are suppressed. Such gene expression patterns highlight the complexity of MRSA pathogenicity, where virulence factor expression is often coordinated and influenced by environmental stressors, including exposure to antimicrobial agents and host immune defenses [67]. These results have important implications for understanding the pathogenic potential of MRSA isolates. The upregulation of sea suggests a prominent role for enterotoxin-mediated virulence in this isolate, whereas the downregulation of mecA and seb may indicate potential vulnerabilities that could be exploited therapeutically.

4. Conclusions

This study demonstrates that Melaleuca cajuputi essential oils exhibit significant antibacterial activity against MRSA, as evidenced by concentration-dependent growth inhibition, favorable MIC and MBC values, and pronounced bactericidal effects. GC–MS analysis identified alloaromadendrene and other major compounds as key bioactive constituents, with molecular docking revealing strong interactions between selected terpenoids, particularly alloaromadendrene and MRSA virulence proteins. The M. cajuputi essential oils also modulated key virulence-associated genes, significantly suppressing mecA, seb, fnbA, and tsst-1, suggesting their potential to attenuate pathogenicity through interference with gene expression. Overall, these findings support the therapeutic potential of M. cajuputi essential oils as a natural anti-MRSA agent and highlight the need for in vivo validation, safety assessment, and formulation development to advance their application against antimicrobial-resistant infections.