Bovine mastitis is one of the most prevalent problem which affects dairy herd production worldwide, and antimicrobial therapy is the primary tool for the treatment. This disease is responsible for a negative effect in the economy of several countries, because of the decrease results in the production level and quality of milk. The most frequent pathogen which caused subclinical mastitis in dairy cows is Staphylococcus aureus (1). This microorganism is characterized by its capacity of select resistance against traditional antimicrobials and some virulence factors as its ability to form biofilms and to invade and survive inside epithelial cells (2).

The emergence of resistance to multiple antibiotics has been favored by abuse in the application of antimicrobials in several areas: medicine, veterinary, agriculture.

At present, the growing number of methicillin-resistant S. aureus (MRSA) strains isolated from both groups, humans, and animals, hazards the efficacy of traditional antimicrobial treatments. The excessive and irrational usage of antimicrobials favored the emergence of resistance to multiple antibiotic families.

It is probably that the introduction of antimicrobials resistant to β-lactamases in the 1960s led to the emergence of MRSA (3). In veterinary medicine literature, the isolation of MRSA in animals has been reported since 1975 (4). This has been generating a dissemination of resistant and multi-resistant strains transmitted between humans and animals (5).

Cloxacillin (CLOX) is a semisynthetic antimicrobial derivate of penicillin that resists being breakdown by the enzyme penicillinase. CLOX is an antibiotic widely used in veterinary medicine against S. aureus (1). It has bactericidal activity against β-lactamase producing S. aureus. However, the increasing occurrence of methicillin-resistance and the consequent failure in therapy induces the search of new therapeutic alternatives. An alternative to face the problem of bacterial resistance is the use of products derived from plant extracts. In the past, natural and phytochemical products were used in the treatment and prevention of infectious diseases. Plants naturally synthesize aromatic chemical compounds and secondary metabolites which serve as a defense mechanism against microbes (6). The biological activity of a medicinal plant is usually based on the presence of one or a set of chemical components located in the tissues of the plant. The compounds with antimicrobial activity are mostly present in essential oils, which can occur in one or more organs depending on the species (7). Essential oils turn out to be the final product of the secondary metabolism of aromatic plants (8).

Essential oils are an interesting kind of herbal extract composed by complex mixtures with high antimicrobial activities. It is useful to take advantage of this property and transfer it to the treatment of bacteria resistant to antibiotics. There are many works that establish the restoration of antimicrobial activity of antibiotics, which have diminished their effectiveness against microorganisms, when they are combined with essential oils (9–12).

The exploitation of essential oils in the prevention of bacterial resistance is promising because those are multi-components with different targets of action, compared to many conventional antimicrobial agents that only have a single target site. Depending on the composition of the essential oils, different mechanisms of action can be attributed, including damage to essential proteins of the pathogen, blocking of membrane enzymes with which the microorganisms can pump out the active principle, changes in the metabolism, permeabilization of their membranes. These oils contain wide ranges of polyphenols and terpenoids, which have strong binding affinity to different molecular structures such as membranes, due to their great lipophilicity, presenting a high potential to penetrate through cell walls and disorganize them, leading to leakage cellular content (13).

Several authors are researching about the application of essential oils as adjuvant to increase the effect of antimicrobials against bacterial species. This is a new concept with high potential. For example Pelargonium graveolens essential oil reduces the minimum effective dose of norfloxacin against Bacillus cereus, Bacillus subtilis, Escherechia coli, and S. aureus (10). Rodrígues et al. (11) reported that the essential oil of leaves of Croton zehntneri is able to enhance the activity of gentamicin by 42.8% against Pseudomonas aeruginosa by gaseous contact, which suggests that the oil has a potential to be used as an adjuvant in antimicrobial therapy (11). Gram positive bacteria as S. aureus are more susceptible to essential oils than Gram negative ones (14), because these last one have an outer layer with lipopolysaccharide that cover the peptidoglycan and limiting the diffusion of hydrophobic compounds through it (15).

The antimicrobial properties of essential oils promote scientific interest in the study of them as a new group of pharmacological compounds. They possess a huge chemical and structural variety, converting them into functionally versatile mixtures (9). Although their activity can be associated with the major components, all of them influence in the pharmacological effect. It is probably that the high concentration of the major compounds masks the effects of the others. However, in terms of antimicrobial activity, every isolated component shows less antibacterial effect than the complete essential oil (16). It has also been shown that the function of the main compounds is regulated by the minority molecules, contributing to the synergic effect (17). Considering the large number of chemical structures that make up the essential oils it is likely that antibacterial properties cannot be attributed to a single chemical compound, and therefore there could be several targets in the microbial cells where these would act resulting in an enhancing influence. That is why it is reasonable to study the essential oils as a whole and take advantage of the synergy of all the components (18).

Melaleuca armillaris (Soland. ex Gaertn.) Sm. is one of the most widely cultivated Melaleuca plants. It is commonly known as Honey Bracelet Myrtle and grows in the form of a large bush or as a small tree. Reports of the literature about M. armillaris essential oil (EO) remain scarce. Investigations by GC-MS of its EO revealed the presence of 1.8-cineole as the major component (19–21). Several activities have been determined for this EO. Rizk et al. (22) obtained positive results in vivo using this EO in the treatment against the parasitoid Schistosoma mansoni, responding to the oxidant activity generated by the pathogen. Inhibitory activity was also found, in vitro, against some bacterial species such as B. subtilis, S. aureus, S. epidermidis, E. coli, and P. aeruginosa (22, 23). The activities observed for the EO of M. armillaris in pure form allow supposing that the good results obtained by themselves can be increased in synergistic systems for the same purpose, such as the improvement of antibiotics to treat infections caused by strains resistant to conventional treatment. No bibliography has been found indicating the use of the essential oil of M. armillaris as an enhancer of antibiotics used in the treatment of microbial infections, particularly against strains of S. aureus. This pathogen is very resistant to antibiotics, and is responsible for high percentage persistent infections. The indiscriminate use of antimicrobials is generating microorganisms resistant to multiple drugs, becoming a serious problem for global health (24).

Our aim was to assess the pharmacodynamic interaction established by combining EO of M. armillaris with CLOX emulating an extracellular and intracellular pH conditions (pH 7.4, 6.5, and pH 5) in search of a synergistic effect that maximizes the activity of the antibiotic against S. aureus.

The whole distillation process lasted 5 h and 550 mL of essential oil were extracted. The analysis of composition revealed the presence of 1.8 cineole (72.3%), limonene (7.8%) and α-pinene (6%) as the most abundant components in the EO (Table 1, Figure 1).

The strains used in this study were identified as members of S. aureus specie after characterizing them biochemically, resulting in Gram positive, coagulase positive, catalase positive, β-hemolytic, glucose fermenting, and growth in saline (7.5%) strains.

The MIC of the CLOX obtained for S. aureus ATCC 29213 at pH 7.4 was 0.125 μg/mL, which is in accordance with the recommendations of the CLSI 2009 (32). The 3 wild strains (named SA13, SA96, and SA139) presented a MIC of 0.5 μg/mL at pH 7.4. In all cases, a decrease in this concentration was observed when acidifying the culture medium (Table 2).

Respect the M. armillaris EO, for S. aureus ATCC 29213 the MIC was 25 μL/mL, while for the 3 wild strains it was 12.5 μL/mL. These values decreased slightly with the acidity of the culture medium (Table 3).

With the evaluation of the antibacterial activity index we could compare the incidence of EO in the CLOX activity, in addition to the effect caused by the pH variation. This is visualized in Figures 2, 3. It is observed how the acidification of the medium enhances the antimicrobial activity of CLOX against S. aureus (for the reference strain and the wild strains). Lower pH values requires a less amount of antibiotic to obtain an E-value lesser than −3 indicating a bactericidal effect. In the graphs it can be seen how the presence of the EO favors the action of the CLOX, since lower E-values are reached with lower concentrations of the antibiotic in comparison with the results obtained for CLOX alone. For the reference strain ATCC 29213, it was achieved a bactericidal effect very close to virtual eradication, since the E-values are close to −4. This was observed from concentrations of 2 the MIC of CLOX in the mixture, at the 3 pHs evaluated (Figure 2). In wild isolates the behavior was similar to that observed for the reference strain (Figure 3).

The chromatographic analysis of our EO revealed the presence of 1.8 cineole as major component (72.3%) and in lesser magnitude limonene (7.8%) and α-pinene (6.0%). Those are commonly present in effective antimicrobial essential oils. The 1.8 cineole is a monocyclic monoterpene with high antimicrobial activity. In several studies (19, 22, 33, 34), the main component reported was also 1.8 cineole, but this result disagree with other authors like Amri et al. (23) because the concentration of this one was markedly low (3.6%). There is evidence that the differences found in the concentration of the components in essential oils, extracted from different plants, would be affected by environmental conditions (35). The main compound of the mixture generally exerts the strongest activity, but can be influenced by the other molecules (36).

In our study, EO alone, without the addition of the antimicrobial, have demonstrated a strong antibacterial activity against S. aureus, this result agree with the research carried out by Amri et al. (23). The MIC against S. aureus ATCC 29213 was 25 μL/mL at pH 7.4 and it decreased 2-fold at pH 5. This result was also observed in wild strains.

A clear synergic effect was observed for the combination EO/CLOX, since FIC values obtained were ≤1 (Table 4). When we faced the EO/CLOX combination against the S. aureus strains, there was a great decreased in the concentration of antibiotic necessary to inhibit the bacterial growth. In EO/CLOX combination the bactericidal effect was maintained even when the pH of the broth was reduced from 7.4 to 5.0, as we have mentioned above when EO was studied without CLOX. And in the same way, when the culture medium was acidified, the concentration of CLOX, in the mixture EO/CLOX, was reduced even up to 10-fold to inhibit the microorganism. As we have mentioned in the introduction, there is still no published works where the pharmacological interaction between the M. armillaris EO and CLOX has been studied. In the bibliography another Melaleuca sp., M. alternifolia (tree of tea), has been widely investigated using the checkerboard technique against S. aureus. However, the results obtained when combining its EO with different antibiotics such as vancomycin (37), tobramycin (38), and ciprofloxacin (39) were indifference or antagonism effect.

The combined activity of EO and CLOX, used in our study, was also evidenced in the bactericidal effect, established by the E index. The decrease in the initial bacterial inoculum -Log₁₀ (CFU/mL)- in a factor of 3 in 24 h marked bactericidal activity observed in both for the CLOX alone and for the mixture. However, the concentration of the antimicrobial in the presence of EO was clearly lower and the E-value obtained was closer to the virtual eradication effect (E = −4) than the concentration needed when applying β-lactam alone. In a study conducted by Nascimento et al. (40) the essential oil of Eremanthus erythropappus was evaluated in combination with ampicillin (β-lactam antibiotic) against S. aureus achieving a synergistic bactericidal effect after 24-h incubation (40).

In conclusion, it was possible to reduce the concentration of the antibiotic needed to inhibit S. aureus by combining CLOX with EO in vitro. Considering that CLOX is an antimicrobial of the group of β-lactams with good activity against S. aureus and with wide use in veterinary medicine, the EO enhance CLOX antibacterial effect even when the intracellular pH were more acid than the extracellular medium. This is important for the treatment of intracellular infections where S. aureus is internalized inside of phagolysosomes because the probability of therapeutic success would be really increased. Our results suggest an increase of the susceptibility to β-lactams due to the acidic pH prevailing in the vacuoles where S. aureus live and prosper, which would be facilitated by the action of M. armillaris EO. The acid pH causes a conformational change of the target protein of action (PBP2a), increasing the affinity of its catalytic center for β-lactam (41). So the synergy we found between CLOX and EO in acid conditions could take place in the inner cell and have an important effect against S. aureus when it is refractory to immunological mechanisms. These findings become into a valuable alternative for the treatment of persistent staphylococcal infections. M. armillaris EO needs to be considered in the design of future formulations to evaluate in vivo effects, in order to maximize the efficacy of current and future antimicrobials.

NM conceived and designed the experiments. DB performed all the experimental assay and statistical analysis. AVB, MM, and FA contributed with experimental assays. AB performed the EO quality assay. All authors contributed to the redaction, revision, and approved the final manuscript.