Leaves of E. brejoensis were collected at Parque Nacional do Catimbau (Pernambuco, Brazil) on dry season (September, 2015). All the plant material was processed following the usual techniques in taxonomy and deposited in the Herbarium of Instituto Agronomico de Pernambuco (voucher access number: IPA 84.033). The EO was obtained from leaves of E. brejoensis (EbEO) by hydrodistillation as previously reported (Da Silva et al., 2015). The oil used in this study was characterized by gas chromatography–mass spectrometry (GC/MS) and the chemical profile was published by Da Silva et al. (2015).

The standard strain S. aureus ATCC 29312 was used in most of the assays. The antimicrobial activity of EbEO was further analyzed against a collection of clinical isolates of S. aureus deposited in the Microbial Collection of Departamento de Antibioticos from Universidade Federal de Pernambuco (UFPEDA). The antibiotic resistance profile of each strain is shown in Table 1. The strains used in the present study are part of Dr. Anders Løbner-Olesen collection at University of Copenhagen. The expression of virulence- and SOS-related genes (hla, spa, or recA) was performed using strains carrying the targeted genes fused with lacZ (which encodes for β-galactosidase) (Nielsen et al., 2010; Gottschalk et al., 2013). The strains in Table 1 were kindly shared by Prof. Hanne Ingmer, Copenhagen University.

The antimicrobial activity of EbEO was determined against the standard strains S. aureus ATCC 29312 and clinical isolates (Table 1). Serial dilutions (1024 to 2 μg/mL) of EbEO were prepared in 96-wells plates containing Luria–Bertani (LB) broth. Each well received 10 μL of a microbial suspension [bacterial load of approximately 1.0 × 10⁷ colony forming units per milliliter (CFU/mL) for each well]. The plates were incubated at 37°C, and after 24 h each well received 30 μL of 0.03% resazurin sodium solution (Sigma–Aldrich^®). Following, the plates were incubated for 40 min and the minimum inhibitory concentration (MIC) was defined as the lowest concentration capable of inhibiting bacterial growth (as evaluated color change).

Overnight cultures of S. aureus ATCC 29312 were diluted 1:100 in LB broth and placed in a shaking water bath at 37°C until an optical density at 600 nm (OD₆₀₀) of 0.1 was reached. This microbial suspension was distributed in fresh LB broth containing EbEO (128 or 1024 μg/mL; corresponding to 2 × MIC or 8 × MIC, higher concentrations was applied to verify action of concentrations greater than MIC50). Bacteria treated with ciprofloxacin (2 μg/mL; 2 × MIC) or without treatment were used as positive and negative controls, respectively. Cell growth was monitored in specific time points (0, 1, 2, 3, 4, and 5 h) by plating 4 μL of 10-fold-diluted suspensions from each tube in quadruplicate. The plates were incubated at 37°C for 24 h. After this period, the colonies were counted for the calculation of CFU/mL.

The interaction between EbEO and some important clinical used drugs (ampicillin—25 μg/mL, ciprofloxacin—0.78 μg/mL, chloramphenicol—12.5 μg/mL, erythromycin—0.39 μg/mL, and kanamycin—6.25 μg/mL) were evaluated using checkerboard assay against S. aureus ATCC 29312. Fractional inhibitory concentration index (FICI) was assessed algebraically using the following equation:

Where “EbEO” is the concentration (μg/mL) of EbEO in a given well, and MIC_EbEO represents the control MIC of EbEO alone. “D” is the concentration of the tested drug in a given well, and MIC_D represents the MIC of the tested drug alone. The interactions were defined as: (i) synergistic if FICI ≤ 0.5; (ii) additive if 0.5 < FICI ≤ 1; (iii) non-interaction if 1 < FICI < 4; and (iv) antagonistic if FICI ≥ 4 (Da Silva et al., 2016).

The effects of EbEO on the expression of genes related to virulence and SOS pathways were performed using S. aureus 8325-4 derivative strains carrying the targeted genes (hla, spa, or recA) fused with lacZ (which encodes for β-galactosidase) (Vestergaard et al., 2015). In all assays, each strain was exponentially grown in LB medium until an OD₆₀₀ between 0.1 and 0.2. Cells were treated with EbEO (64 μg/mL, 0.5 × MIC), and permeabilizated by toluene (1 mL) after 3 h. The β-galactosidase activity was measured using ONPG (ortho-nitrophenyl-β-galactoside; Sigma–Aldrich). In the assays for recA expression, ciprofloxacin (0.5 μg/mL) was used as positive control.

Overnight cultures of S. aureus ATCC 29312 were diluted (1:100) in LB medium and samples (1 mL) of this suspension were incubated with sub-inhibitory concentrations of EbEO (64, 32, and 16 μg/mL, corresponding, respectively, to 0.125 × MIC, 0.25 × MIC, and 0.5 × MIC). After overnight incubation at 37°C, the tubes were centrifuged (10,000 r/min for 10 min), suspended with 1 mL of phosphate-buffered saline (PBS), and re-centrifuged. Bacteria pellets were then photographed. Following the total carotenoid pigments [including staphyloxanthin (STX)] in each pellet were extracted using in methanol (0.2 mL) and incubated for 3 min at 55°C. The methanol phase (supernatant) and cell debris were separated by centrifugation (10,000 r/min for 10 min) and the pellets were submitted to entire pigment extraction procedure three more times. Finally, the absorbance was determined at 465 nm (Silva et al., 2017).

Microbial suspension (standardized at OD₆₀₀ = 1.0; Liu et al., 2005) prepared from overnight cultures of S. aureus ATCC 29312 were incubated with sub-inhibitory concentrations of EbEO (64, 32, and 16 μg/mL, corresponding, respectively, to 0.125 × MIC, 0.25 × MIC, and 0.5 × MIC). Each culture received H₂O₂ to reach a final concentration of 1.5% (v/v) for 60 min and were incubated at 37°C. The percentage of cells surviving the stresses was calculated as CFU/mL remaining after each stress divided by the initial CFU/mL.

Staphylococcus aureus ATCC 29312 overnight cultures were diluted at 1:100 in fresh LB and cultured with or without sub-inhibitory concentrations of EbEO (64 and 32 μg/mL) at 37°C. After 16 h (to guarantee a constant number of bacteria present in experiment), 50 μL of culture supernatant was added to 1 mL of 3% human erythrocytes solution. The mixture was incubated at 37°C for 1 h with 250 r/min shaking. The supernatant was collected by centrifugation at 10,000 r/min for 10 min, and the optical density was measured at 540 nm.

To verify whether EbEO could enhance the antimicrobial action in whole blood, the S. aureus ATCC 29312 (diluted in LB medium; 1:100) was grown in the presence of EbEO (64 and 32 μg/mL) at 37°C with shaking of 250 r/min. After 6 h, the S. aureus suspension (62.5 μL) with EbEO (64 or 32 μg/mL) were mixed with freshly drawn human whole blood (0.5 mL) and re-incubated in the conditions described above for 2 h. The samples were plated in LB agar and the survival was measured by counting viable colonies (expressed as CFU/mL) (Lee et al., 2013).

The infection model was performance using C. elegans AU37, a temperature-sensitive sterile strain [sek-1(km4); glp-4(bn2) I; MAPK kinase deficiency] (Jakobsen et al., 2013). The strain was propagated on nematode growth medium (NGM) containing Escherichia coli OP50 as food source. Prior each assay, the worms were age-synchronized by bleaching with alkaline hypochlorite and sodium hydroxide. The released embryos were placed on NGM plates at 25°C (this temperature does not allow the reproduction) until reached the young adult stage. At this time, 15 larvae were transferred to 24-wells plates containing the M9 liquid medium and overnight culture S. aureus ATCC 29312 (grown in LB medium containing 10 μg/mL cholesterol) in a ratio of 4:1 (v/v) (Kong et al., 2014). EbEO (128, 64, and 32 μg/mL) was added and the worm longevity was assessed every day. OP 50 was used with positive control (worm and E. coli) and S. aureus was used with negative control (worm and ATCC 29312). The animals were classified as dead when they did not present spontaneous movement or response after stimulation with a platinum loop. All experiments were performed according to Wormbook (Stiernagle, 2006).

Galleria mellonella larvae (∼200 mg) were randomly distributed in three experimental groups (n = 10) with or without oil treatment. Two groups were infected by injection of 10 μL of a recent S. aureus ATCC 29312 suspension (1.0 × 10⁵ CFU; different bacterial concentrations was previously optimized to guarantee an inicial sublethal load to this assay), into the last left proleg, followed by incubation at 37°C. After 2 h, one of these groups received 10 μL of 128 μg/mL EbEO (resulting in dose of 6.4 mg/kg). Larvae treated with PBS (vehicle) were used as positive control.

Galleria mellonella larvae were infected with S. aureus ATCC 29312 and treated with EbEO as described above. Each day, a total of five larvae were cut in the cephalocaudal direction with a scalpel blade and squeezed to remove the hemolymph. Each sample was 10-fold-diluted in PBS and 4 μL was plated on LB agar. After 24h-incubation at 37°C, the colonies were enumerated, and the results expressed as CFU/mL.

Melanization test concerns the production of this pigment under stress conditions. G. mellonella (n = 10) were infected with S. aureus ATCC 29312 (10⁶ cells/larva) aimed to evaluate melanization as a response to higher concentrations of bacteria (stress) and to quantify this response as a function of the melanin/time correlation after being treated with 6.4 mg/kg EbEO. After the incubation (1 or 3 h), the hemolymph was collected and diluted (1:10; v/v) in cold PBS. The cells suspensions were centrifuged at 12,000 r/min and the absorbance of each supernatant was determined at 465 nm.

All assays were performed in triplicate in at least two independent experiments. Statistical analyses were performed using the software GraphPad Prism version 7.¹ Data were analyzed by one-way, two-way analysis of variance (ANOVA), and Tukey test. A p-value of < 0.05 was considered as statistically significant. Differences in the survival were determined using the Kaplan–Meier method and log-rank test was used to compare survival curves.

Initially, we performed a microdilution-based assay to evaluate antimicrobial activity of EbEO against all tested S. aureus strains, including those with multidrug resistance profile (MDR strains). This oil inhibited the standard S. aureus ATCC 29312 with an MIC of 128 μg/mL, while the MIC values for the other strains ranged from 8 to 516 μg/mL. The MIC₅₀ (concentration able to inhibit 50% of the tested strains) was 128 μg/mL (Table 1).

Following, a time-kill study was performed using EbEO at 128 μg/mL (2 × MIC) or 1024 μg/mL (8 × MIC). Both oil concentrations were able to inhibit the growth of S. aureus ATCC 29312 without any significant differences among them. After 3 h, the oil induced reductions of 50% in the number of viable colonies when compared to untreated cells. The oil exhibited a profile, similar to the observed from ciprofloxacin (0.5 μg/mL) (Figure 1). Further, the action of EbEO was not associated with an increased expression of recA, the first gene related with the activation of SOS response, a pathway involved in the DNA repair (Vestergaard et al., 2015) (Figure 2).

Next, we determined whether EbEO could improve the action of some antibiotics (from different classes) toward S. aureus. EBEO synergistically increased the action of ampicillin (ΣFIC: 0.45), chloramphenicol (ΣFIC: 0.15), and kanamycin (ΣFIC: 0.075), while it had additive effects with ciprofloxacin (ΣFIC: 0.6) and erythromycin (ΣFIC: 0.6).

Following, we evaluated the effects of EbEO on the expression of two gene of this system (hla and spa that encode alpha-hemolysin and protein A, respectively). It is expected that a quorum sensing inhibitor (QSI) reduces the expression of hla and increases the transcriptional levels of spa (Nielsen et al., 2010). Based on this, the results indicated the EbEO (at 0.5 × MIC) could alter the expression of hla (downregulation) and spa (upregulation), suggesting that this oil is QSI (Figures 3A,B).

We further analyzed whether EbEO could decrease the hemolytic activity of S. aureus and its ability to survive in human blood. The treatment with EbEO (0.5 × MIC or 0.25 × MIC) strongly inhibited the hemolysis-mediated by S. aureus (around 90% of inhibition) (Figure 3C). Similarly, the survival of this pathogen on blood was impaired by subinhibitory concentrations (Sub-MIC) values of EbEO (Figure 3D).

Figure 4A shows that EbEO strongly reduced the levels of staphyloxanthin in a dose-dependent manner (95.63 ± 0.98 and 89.38 ± 1.49% for EbEO at 64 or 32 μg/mL, respectively). In addition, the treatment with EbEO (at 64 or 32 μg/mL; 0.5 × MIC or 0.25 × MIC) potentialized the toxicity of hydrogen peroxide (1.5% v/v) toward S. aureus (Figure 4B). Similar levels of viability reduction were observed for both concentrations (24.12 ± 4.19 and 20.87 ± 0.87% for EbEO at 64 and 32 μg/mL, respectively), when compared to cells treated with hydrogen peroxide alone (p < 0.05). These results suggest that the inhibition of staphyloxanthin production by EbEO impaired the antioxidant system of S. aureus and increased its susceptibility to oxidant attack. Based on these findings, we decided to evaluate the anti-infective efficacy of EbEO using two alternative experimental models: C. elegans and G. mellonella.

The infection of C. elegans by S. aureus resulted in a reduction of worm viability and 8 days post-infection (dpi) the survival ratio of this group was 30%, while the uninfected animals showed 90% survival at this day (Figure 5A). The median survival of S. aureus-infected worms was 7 days. This scenario was radically changed by the treatment with EbEO at 128 or 64 μg/mL. The animals treated with 128 μg/mL EbEO showed survival curve similar to control group (p > 0.05). In the end (8 dpi), the survival of worms treated with EbEO were around 60 and 80%, for concentrations of 64 or 128 μg/mL, respectively (Figure 5A). In addition, the C. elegans larvae treated with 128 μg/mL EbEO (MIC) also exhibited lower bacterial load than those incubated with 64 μg/mL EbEO (Figure 5B). It is important to highlight that the tested EbEO concentrations did not significantly reduce the viability of C. elegans when compared with PBS-treated group (data not shown).

The antibacterial activity of EbEO was further analyzed using G. mellonella larvae. EbEO treatment did not show any toxicity to these larvae, resulting in survival curves similar to those observed to PBS-treated animals (data not shown). The larvae infected with S. aureus showed 100% mortality in the third day and this group exhibited a median survival of 1 day. On the other hand, the treatment with a single dose of EbEO (6.4 mg/kg) protected 70% of the larvae at the end of the experiment (4 dpi) (Figure 6A).

Interestingly, EbEO treatment induced small effects on the bacterial load when compared to initial bacterial inoculum (time 0), suggesting a bacteriostatic action (Figure 6B). In relation to S. aureus-infected larvae, EbEO-treated animals exhibited significantly lower levels of bacteria in hemolymph in all days evaluated (around 3 log CFU/mL reductions for all days; p < 0.05).

Finally, we evaluated whether EbEO could reduce the overproduction of melanin induced by S. aureus infection (Figures 6C,D). We observed that the levels of melanin in the hemolymph of S. aureus-infected larvae were significantly increased when compared to PBS-treated group (approximately eightfolds after 1 and 3 h of infection; Figures 6C,D). The treatment with EbEO also significantly reduced the melanin content in hemolymph of G. mellonella larvae at both tested period (reductions of 42.19 and 55.41% after 1 and 3 h of infection, respectively; Figures 6C,D).