2.1. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Myrtenol against S. aureus
To assess the antibacterial activity of myrtenol on S. aureus
, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The values are shown in Table 1
. The MIC of myrtenol was 128 µg/mL for all S. aureus
strains used in this study and the MBC was also 128 µg/mL for all strains tested (Table 1
The MIC of myrtenol against S. aureus
indicate the antibacterial activity of myrtenol, which proved to be more potent than the reports found in the literature using the isolated compound against other strains. Al-Mariri et al. [15
] investigated the antibacterial activity of myrtenol isolated from the Myrtus communis
L. essential oil and observed antibacterial activity against different Gram-negative species, with MIC ranging from 25 to 50 µL/mL. İşcan [16
] evaluated the antimicrobial activity of constituents commonly found in essential oils, including myrtenol, and identified antibacterial activity ranging from 1 to 4 mg/mL for Gram-positive and negative bacteria. The MIC determined against S. aureus
(ATCC 43300) was 2 mg/mL. Selvaraj et al. [14
] determined the effect of myrtenol isolated against Methicillin-resistant Staphylococcus aureus
(MRSA) ATCC-33591 strain and clinical isolates, identifying MIC of 600 µg/mL.
The MIC/MBC ratio was 1:1. A drug is considered to exhibit bactericidal activity against a particular isolate when the MBC/MIC ratio is ≤4 [17
]. In this way, myrtenol exhibits antibacterial activity against S. aureus
, acting in a bactericidal way from the MIC concentration. A substance is determined to be bactericidal when it has the ability to kill the bacterial cell, whereas bacteriostatic molecules only inhibit cell growth. Such data is relevant to provide information about the potency of action of the molecule. However, it is a valid concept under the pre-established and controlled conditions of experimentation, as it can vary according to the type of bacteria, amount of inoculum, drug concentration and duration of the test. These changes are therefore seen in clinical practice, where the conditions described are as variable as possible [18
]. For this reason, it is necessary to combine this information with pharmacokinetic and pharmacodynamic data, to provide a more significant prediction of in vivo efficacy.
2.3. Molecular Docking Analysis
Molecular docking was performed in order to identify the possible target of myrtenol in the bacterial cell. The interaction of the substance with penicillin-binding protein 2 (PBP2) is observed, which is a transpeptidase that acts in the synthesis of the cell wall, being the target of some antimicrobial agents. For the validation of protocols performed in molecular docking, redocking was performed and the value of RMSD (root mean standard deviation) was used to analyze the accuracy of molecular docking. To be considered a valid docking, the RMSD values must remain in the range of 0–2 Å [19
]. The RMSD value for the tested enzyme remained in the acceptable values (0.37 Å), Moldock Score −125.2 kcal/mol. Myrtenol showed binding energy −52.3 kcal/mol with PBP2 and, analyzing the interactions with the active site of the enzyme, the hydroxyl of myrtenol performed hydrogen bonding interactions with the residues of Ser403 and Thr600 and Van der Walls hydrophobic interactions with Lys406 (Figure 2
). These interactions are necessary for effective anchoring at the active site of PBP2 and are carried out by β-lactam drugs [20
The antibacterial mechanism of the action of myrtenol is not yet completely elucidated. In this work, the results indicate that PBP2 is a possible target for myrtenol to act against S. aureus
. Thus, the substance would act by interfering in the synthesis of the bacterial cell wall, leading to cell death [19
]. It is important to note, however, that these are preliminary results that help guide future in vitro and in vivo studies in order to clarify the exact mechanisms of action of myrtenol.
2.4. Association Test of Myrtenol with Antibacterial Drugs against S. aureus
Antibacterials are often used in combination, so it is relevant to understand the possible interactions between myrtenol and some commonly used drugs. Synergistic effects were found in the combination of myrtenol with gentamicin and additives in the association with ciprofloxacin, for all strains tested. The association of myrtenol with oxacillin, on the other hand, resulted in an indifferent effect. No antagonistic effects were observed with any drug tested (Table 2
The combination of natural products and antimicrobials has been shown to be quite effective and promising for use in clinical practice. The use of associated substances makes it possible to reduce the required administration dose of each drug, while also reducing dose-dependent toxic effects. In addition, the association of natural products with antimicrobial agents can be an effective strategy to combat resistant strains. These phytoconstituents can work by several strategies, such as inhibition of target modifying and drug degrading enzymes or as efflux pumps inhibitors. Thus, they can act as bacterial resistance modifying agents, restoring the effectiveness of commercial antimicrobials or even have greater potency of action by acting in different mechanisms, achieving effectiveness against resistant strains [21
The results indicate that myrtenol, despite having an indifferent effect when combined with oxacillin, in combination with gentamicin and ciprofloxacin increased the antimicrobial effect in a synergistic or additive way, which suggests the possibility of reducing the viability of the strains using a smaller concentration of these substances, and it is possible, consequently, also to reduce the side effects resulting from the administration of these drugs. It is interesting that association and modulation studies are carried out using strains resistant to multiple drugs, to verify whether the combination of myrtenol with other drugs is able to alter the resistance of these bacteria.
2.5. Antibiofilm Effect of Myrtenol against S. aureus
The antibiotic activity of myrtenol was evaluated based on the ability to inhibit the formation of S. aureus
biofilms in vitro. Myrtenol showed a strong ability to inhibit the formation of the biofilm formed by S. aureus
, in all tested concentrations. Significant differences were observed between the treated groups and the control group without treatment (Figure 3
Myrtenol was able to strongly inhibit biofilm formation by all S. aureus
strains used in this study from MIC (Figure 3
A). In subinhibitory concentrations, myrtenol did not have a stimulating effect on the production of biofilm, being able to reduce the formation of biofilm, even in concentrations below the MIC (Figure 3
After 1/4 MIC, there was more than a 50% reduction in biofilm formation by S. aureus
C,D). From the MIC, up to the maximum concentration used in this study (8× MIC), it is possible to observe that myrtenol promoted an inhibition greater than 90% of biofilm formation by S. aureus
. Kwasny and Opperman [23
] classify as good antibiofilm activity when a substance is capable of inhibiting ≥80% of biofilm growth and inhibit ≥40% of planktonic growth compared to untreated controls. Thus, it is observed that myrtenol has good antibiofilm activity from MIC onwards, in suprainhibitory concentrations, where there is ≥80% inhibition of biofilm growth and 100% inhibition of planktonic growth.
Similar results of myrtenol antibiofilm activity were found by Selvaraj et al. [14
], whose data show that myrtenol attenuates MRSA biofilm considerably, in addition to inhibiting the production of major virulence factors of MRSA, such as lipase, hemolysin and staphyloxanthin. In addition, it affected the slime synthesis, autoaggregation, autolysis, and eDNA production in MRSA.
It has been observed that several classes of antibiotics, including β-lactams, at suboptimal concentrations, increase the potential for biofilm formation by different mechanisms. The induction of biofilm formation in subinhibitory concentrations is a clinically relevant process, because during the treatment of the infection, a part of the microorganism population is exposed to suboptimal concentrations, even when the recommended conditions of use of the drug are followed. Thus, low doses of these drugs can interfere with the course of the infection, complicating the treatment of these diseases [6
]. However, myrtenol was able to reduce biofilm formation, even at concentrations below MIC, and potentially promising antibiofilm of the substance is evidenced, which should be further investigated and evaluated also against other microorganisms, in order to obtain a new clinical agent to combat this relevant virulence factor.
The ability of some microorganisms, such as S. aureus
, to form biofilms, contributes to antibacterial resistance and therapeutic failures [25
], so the development of effective tools to remove biofilms not only improves the treatment of biofilm-related infections, but can also potentially offer benefits to slow the spread of antibiotic resistance [26
]. Since these results show that myrtenol is a potential antibiofilm agent, being able to act even in suboptimal concentrations, it is relevant that studies be further developed, evaluating its activity against biofilms formed by other species.
Since a substance can act by the most diverse mechanisms, such as interfering in key enzymes for biofilm formation, matrix-targeting enzymes, adhesion factors to the substrate, adhesion proteins involved in cell-cell aggregation or the biosynthesis of proteins important for biofilms formation and maturation [27
], it is of great importance that the mechanism of action of myrtenol against biofilms is also elucidated, investigating its performance on possible molecular targets and using this information for future clinical applications.
2.6. In Silico Studies of Myrtenol Lipinski’s Parameters
The theoretical potential of myrtenol as a candidate for a new drug was assessed by the in silico parameters. According to the values expressed in Table 3
, calculated by the online program SwissADME, it is possible to predict whether the myrtenol molecule may be a candidate for a drug, based on the rules of Lipinski [28
], Ghose [29
], Veber [30
], Egan [31
According to Table 3
, the molar mass of myrtenol has a value of less than 500 g/mol, which meets the Lipinski criterion [28
], where there should be no problem regarding the distribution aspect, as it is more easily transported than larger molecules. However, for Ghose parameters [29
], the molecular mass must be between a range of 160–480 g/mol, so the molar mass of myrtenol, which is 152.23 g/mol, violates this rule. Log P is the partition coefficient of a molecule in n-octanol and water. To be considered an important parameter in the preparation of a candidate compound for a drug, Log P is related to the hydrophobicity of the molecule in the drug due to the ability to cross plasma membranes. However, molecules that are too hydrophobic tend to be more toxic, due to their ability to stay longer in the body. According to Table 1
, the value of Log P was 2.40 and met the standards of the Lipinski rules (Log Po/w
≤ 5) [28
], Ghose (Log Po/w
≤ 5.6) [27
] and Egan (Log Po/w
≤ 5.8) [31
The hydrogen acceptor and donor values shown in Table 3
met the parameters of the rule of 5, where the number of hydrogen acceptors must be ≤10 and hydrogen donors must be ≤5. According to all the parameters presented by the Lipinski rule, myrtenol presents an excellent theoretical oral bioavailability. However, according to Veber, molecules that have TPSA values ≤140 Å and the number of rotatable connections ≤10 have a high probability of oral availability. In this way, myrtenol can display a high prospect of being employed orally. Solubility is an important feature for the absorption and distribution of the molecule in the body. Having a soluble compound favors medication planning, especially in formulation and manipulation. The Log S (coefficient of solubility determined by the Ali method [32
]) of myrtenol presented a value of −3.32, indicating that the compound is soluble according to the class shown in Table 1
Based on the above, in silico results show a favorable pharmacokinetic profile for this substance. Myrtenol has characteristics that suggest a good drug candidate and can display a high prospect of being used orally, with excellent theoretical oral bioavailability and good solubility, which can guarantee adequate absorption and distribution in vivo.