Infectious diseases constitute a significant threat to human health and are responsible for high morbidity and mortality. These diseases are often treated with antibiotics, which have led to the emergence of microbial resistance and the development of adverse side effects (Brower, 2018). The emergence of antibiotic resistance problems with severe clinical and economic consequences for health and government health agencies (Brower, 2018; Aljeldah, 2022). Among the several strains that have emerged and spread in hospitals and communities in different countries, methicillin-resistant Staphylococcus aureus (MRSA), which causes meningitis and pneumonia, and Pseudomonas aeruginosa, which affects individuals with compromised immunity (AIDS, cancer, transplant or cystic fibrosis), have been a worldwide public health problem that needs new treatments (Reddy, 2022). Furthermore, from an epidemiological point of view, MRSA is constantly changing, and circulating clones and their antibiotic resistance profiles vary considerably by region and country (Romero and Cunha, 2021). Considering these aspects, natural products are one of the most promising sources of antibacterial agents that can be associated with antibiotics to enhance the therapeutic potential by inhibiting infectious mechanisms (Qadri et al., 2022; Ramata-Stunda et al., 2022).

Ocimum gratissimum L. belonging to the family Lamiaceae is popularly known as basil and is a shrub found in tropical and subtropical regions of Asia, Africa, and South America (Padalia et al., 2013). It is used as a condiment in cooking and as a sedative and anxiolytic, and in traditional medicines for headache and abdominal pain, cough, cold, and bronchitis (Silva et al., 2015a; Ashokkumar et al., 2020). Its essential oil has been used as an anesthetic (Silva et al., 2012; Silva et al., 2015a), antinociceptive (Rabelo et al., 2003; Sahouo et al., 2003; Paula-Freire et al., 2013), anti-inflammatory (Sahouo et al., 2003), antioxidant (Pereira and Maia, 2007; Vasconcelos et al., 2021), insecticide (Nguemtchouin et al., 2013), and antimicrobial against bacteria and fungi (Vasconcelos et al., 2021; Franco et al., 2007; Matasyoh et al., 2007; Adjou et al., 2013; Dambolena et al., 2010).

The essential oil of O. gratissimum (EOGT) is composed of eugenol as a major constituent (Ashokkumar et al., 2020; Vasconcelos et al., 2021; Matasyoh et al., 2007). Eucalyptol (1.8-cineol), methyl eugenol, germacrene D, β-ocimene, and caryophyllene, among others, have been described in the literature (Ashokkumar et al., 2020; Vasconcelos et al., 2021; Matasyoh et al., 2007). The antibacterial effect of EOGT has been attributed to eugenol, which can inhibit the growth of Gram-negative and Gram-positive bacteria (Catherine et al., 2012; Marchese et al., 2017; Ulanowska and Olas, 2021). However, even in lower concentrations, the other components can contribute to antimicrobial activity (Hendry et al., 2009; Mak et al., 2019).

EOGT has been identified as a promising antibacterial agent, and its association with antibiotics can increase therapeutic efficacy. This study evaluated the chemical composition and efficacy of the antibiotic effect induced by EOGT against S. aureus and P. aeruginosa.

Plant and essential oil extraction

O. gratissimum was cultivated in the medicinal garden of the Faculty  of  Pharmacy  of  the  Federal  University  of  Juiz  de  Fora (UFJF), Minas Gerais state, Brazil (latitude 21° 41' 20” S and longitude 43° 20' 40” W). A voucher (CESJ 48248) was deposited at the Herbarium Leopold Krieger/UFJF. After collecting the fresh leaves (400 g) on 10 February, 2014, the essential oils were extracted by hydro-distillation in a Clevenger apparatus for 2 h.

Gas chromatography-mass spectrometry (GC-MS) analysis

GC-MS analyses of EOGT were conducted on a Perkin Elmer Turbomass Quadrupole mass spectrometer (PerkinElmer, Shelton, USA) fitted with a DB-5 capillary column (30 m × 0.32 mm, 0.25 μm film thickness; Supelco Bellefonte, PA, USA). The oven column temperature was programmed from 60 to 240°C with a heating rate of 3°C/min. Helium was used as carrier gas with a 1.0 mL/min flow. The injection volume was 1 μL (1% EO/dichloromethane v/v) with a split ratio of 1:50. The injector and ion source temperatures were 250°C. The ionization energy was 70 eV (EI), with a mass scan range of 40 to 400 m/z, at a sampling speed of 1.0 scan/s. The compounds were identified by comparing the experimental mass spectra with the Pherobase database and their linear retention index (LRI), which were calculated following Van Den Dool and Kratz (1963) after injecting a homologous series of n-alkanes (C7–C26), under the conditions mentioned earlier and compared with the literature (Adams, 2007).

Gas chromatography with a flame ionization detector (GC-FID) method

EOGT was analyzed using a GC-FID with a capillary column of fused silica and 5% phenyl dimethyl polysiloxane (HP-5MS) with a size of 30 m × 0.25 mm × 0.25 µm. Hydrogen was used as carrier gas with a flow of 1.5 mL/min. The temperature program and injection procedures were the same as the GC-MS analysis (60-240°C at 3°C/min). The percentage composition of the compounds was determined by normalization in triplicate.

Microorganism standards

S. aureus (ATCC 6538) and P. aeruginosa (ATCC 25619) were the bacterial species used in this study. These microorganisms were provided by the Instituto Nacional de Controle de Qualidade em Saúde (INCQS) of the Fundação Oswaldo Cruz.

Determination of minimum inhibitory concentration

The minimal inhibitory concentration (MIC) was determined using the microdilution method described by the Clinical and Laboratory Standards Institute Standard M7-A6 (CLSI, 2006). EOGT (solubilized in 0.2% polysorbate 80 in BHI) was diluted in sterile BHI broth, followed by serial dilutions in a sterile 96-well U microplate (100 µL per well). Then, 100 μL of bacterial inoculum (2 × 10⁶ CFU) in 0.9% NaCl was added to each test well. Two positive controls (BHI and BHI containing 0.2% polysorbate 80) were used with bacteria, while the negative control was prepared without bacteria, and the EOG concentration ranged from 58.6 to 7500 μg/mL. The microplate was incubated for 16 to 20 h in an oven at 35 ± 2°C. After incubation, inhibition of bacterial growth was evidenced by the addition of 20 µL of triphenyltetrazolium chloride (TTC) (5% v/v in distilled water) with a change in color to red, indicative of bacterial growth. The MIC was defined as the lowest concentration of EOG without visible bacterial growth.

Evaluation of effectiveness of antibiotics associated with EOGT

The study of the effectiveness of antibiotics associated  with  EOGT was carried out according to the methodology described by Oliveira et al. (2006) with some modifications. Petri dishes with 150 mm in diameter and 20 mm in height were prepared with two different layers of TSA (lower layer with 36 mL of sterile agar and the upper layer containing 12 mL of agar inoculated with 1% of the inoculum containing 10⁸ CFU/mL). Antibiogram multi discs (Laborclin®, soaked with 10 μL of the MIC obtained from the EOGT) were inserted onto the surface of the inoculated medium. After incubation for 24 h at 35 ± 2°C, bacterial growth inhibition was measured using a caliper.

The effectiveness of the association between antibiotics and EOGT was classified as synergistic effect (when the zone of inhibition of the association has a diameter ≥ 2 mm compared to the antibiotic alone), antagonistic effect (when the zone of inhibition of the association is smaller than that of the isolated antibiotic), and indifferent effect (when the zone of inhibition of the association is the same with the combined or isolated antibiotic) (Oliveira et al., 2006).

Determination of the fractional inhibitory concentration index

The antibiotic effectiveness data selected those with promising results to determine the fractional inhibitory concentration index (FICI). For this, two microplates were used in each bacterial strain. The BHI medium (50 μL per well) was added to the first microplate with successive dilutions of EOGT and the second with successive dilutions of the promising antibiotics. In the first microplate, after serial dilution, 50 μL of the MIC of the antibiotic was added, and the MIC of the EOGT in combination with the antibiotic was verified. In the second microplate, 50 μL of EOGT MIC was added to obtain the MIC result of the antibiotic in combination with EOGT. After plate preparation, 100 μL of the inoculum suspension (2 × 10⁶ CFU) was added, followed by incubation for 24 h. Bacterial growth was evidenced by adding 20 μL of 2% TTC. The effect of the combination was calculated using FICI, which is equal to the sum of the Fractional Inhibitory Concentration (FIC) of EOGT and the FIC of the antibiotic. The FIC is defined by the MIC of the substance in combination divided by the MIC of the isolated substance (antibiotic). The result is considered a synergistic effect when FICI ≤ 0.5; an additive or indifferent effect when FICI is > 0.5 and < 1; and an antagonistic effect when FICI > 1 (Malik et al., 2011).

Identification of compounds

After hydro-distillation, the fresh leaves of O. gratissimum produced 0.5% essential oil. The GC-MS and GC-FID analyses showed that eugenol (74.2%), cis-β-ocimene (17.2%), germacrene D (2.3%), and trans-β-ocimene (1.6%) were the major components of EOGT (Table 1). Our results showed a higher eugenol content (74.2%) in EOGT when compared with the studies published by Ribeiro et al. (2016) and Chimnoi et al. (2018). This compound is essential for EOGT chemotype and is a basis for its antibacterial activity (Silva et al., 2015b; Melo et al., 2019; Rodrigues et al., 2020). Like eugenol, other compounds, such as terpineol-4, γ-muurolene, (Z, E)-α-farnesene, α-trans-bergamotene, 1,8-cineole, β-selinene, γ-terpinene, p-cymene, thymol, and myrcene, have also been identified in the essential oil of O. gratissimum (Matasyoh et al., 2007; Chimnoi et  al.,  2018;  Rodrigues et al., 2020; Yayi et al., 1999).

Effectiveness of antibiotics associated with EOGT against S. aureus

In the microdilution test, EOGT showed a MIC of 468.8 μg/mL against S. aureus and P. aeruginosa, highlighting a promising antibacterial activity against the tested strains, according to the criterion described by Aligiannis et al. (2001). Using the same strain (S. aureus, ATCC 6538), Melo et al. (2019) the MIC was 1000 μg/mL, twice as high as the value obtained in the present study. This difference may be related to the variations in the identified components.

As expected, the tested antibiotics inhibited the growth of S. aureus (Table 2). However, antibiotics associated with EOGT potentiated the antibacterial action of rifampicin, tetracycline, and ciprofloxacin showing a synergistic effect. Furthermore, when associated with EOGT, there was an antagonistic effect of cefepime and penicillin G, while with the other antibiotics, there was an indifferent effect (Table 2). The synergistic effect induced by EOGT may be related to different mechanisms against S. aureus since eugenol is capable of causing membrane rupture by inhibiting ATPase activity, inhibiting efflux pump, and reducing several virulence factors at sub-inhibitory concentrations (Langeveld et al., 2014).

Effectiveness of antibiotics associated with EOGT against P. aeruginosa

Considering the data in Table 3, the antibiotics ampicillin, amoxicillin-clavulanic acid, cephalothin, and cefoxitin showed no growth inhibition zone against P. aeruginosa (ATCC 25619), which demonstrates the presence of resistance mechanisms for these drugs. This resistance phenomenon is common in the genus Pseudomonas since it is a Gram-negative bacteria, and the major mechanisms involved include low permeability of the outer membrane, expression of efflux pumps, and production of antibiotic-inactivating enzymes (Pang et al., 2019).

The antibiotic action of cefepime and ciprofloxacin was evaluated in the presence of EOGT, demonstrating a synergistic effect (Table 3). The activity against the growth of P. aeruginosa has been attributed to eugenol (Lou et al., 2019) that may be associated with the competitive binding of this compound to a QS receptor (LasR), which promotes significant repression of genes associated with QS in addition to VF genes (Rathinam et al., 2017). Furthermore, the synergistic effect is related to the ability of eugenol to increase cell membrane permeability (Hemaiswarya and Doble, 2009).

In addition to the synergistic effect, amikacin, ceftazidime, cefuroxime, gentamicin, meropenem, and sulfazotrim   had  an  indifferent  effect  since  the  growth inhibition zone was the same with the combined or isolated antibiotic (Oliveira et al., 2006). Because they are ineffective in inhibiting growth, ampicillin, amoxicillin-clavulanic acid, cephalothin, and cefoxitin were considered resistant to P. aeruginosa.

Fractional inhibitory concentration and fractional inhibitory concentration index

From the results of Tables 2 and 3, ciprofloxacin and tetracycline were used to evaluate the fractional inhibitory concentration (FIC) and the Fractional Inhibitory Concentration Index (FICI) using the MIC of these antibiotics  and  their  combination  with  EOGT (Table 4). Thus, ciprofloxacin reduced MIC to 0.6 and 0.03 µg/mL for S. aureus and P. aeruginosa, respectively, while the MIC of tetracycline was 0.028 µg/mL (S. aureus) and 4 µg/mL (P. aeruginosa). Using the Checkerboard method to obtain FICI values, the synergistic effect of EOGT was confirmed for ciprofloxacin and tetracycline on the growth of S. aureus and tetracycline against P. aeruginosa (Table 4).

As demonstrated in Table 4, in the presence of EOGT, ciprofloxacin, and tetracycline, the MIC was reduced by 99.9 and 92.8%, respectively, against S. aureus and P. aeruginosa, which confirmed the synergistic effect shown in Tables 2 and 3. In addition, tetracycline decreased MIC by 97% against P. aeruginosa (synergistic effect), but ciprofloxacin    showed    an    antagonistic    effect    after combination with EOGT (Table 4). As a major compound with antibacterial action, eugenol should contribute to the potentiation of antibiotic inhibitory activity (Yadav et al., 2015).

EOGT consists of eugenol as the major component and potentiates the antibiotic efficacy of ciprofloxacin and tetracycline against S. aureus and P. aeruginosa via growth inhibition and reduction of MIC, which may serve as a promising agent for the treatment of infections caused by these pathogens.

This study was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Pró-Reitoria de Pós-Graduação e Pesquisa (PROPP-UFJF).

The authors have not declared any conflict of interests.