{"paper_id":"1efa6535-b768-45c6-be3b-55815a4d8f40","body_text":"Staphylococcus aureus  is one of the major pathogens credited as being responsible for nosocomial and community acquired infections in humans [ 1 , 2 ]. Colonizing the normal commensal flora of humans and many animals, this opportunistic bacterium is considered the main cause of hospital-acquired infections, generating a variety of symptoms, from mild localized infection to potentially fatal sepsis [ 3 ].  S. aureus  is characterized by a variety of virulence factors, such as secretion of various enzymes and toxins [ 4 ], allowing it to avoid the host’s immune defense system and participate in tissue invasion and destruction, along with its ability to form biofilms on medical devices and biomaterials [ 5 ].\nS. aureus  is one of the biofilm-producing bacteria with high ability to survive in hostile environments and medical or industrial systems [ 6 ]. Inside this biological matrix, bacteria became resistant to different classes of antibiotics and ether-disinfectants, making biofilm-related infections more prone to relapse [ 7 , 8 ]. Across years, synthetic antibiotics are used to overcome  S. aureus  antimicrobial resistance. However, such agents showed high toxicity as well as several side effects in patients [ 9 ]. This is one of the factors making antibacterial resistance an increasingly serious threat to global public health. In clinical practice, the ineffectiveness of single-component medications in treating microbiological infections caused by resistant bacteria can be avoided using a combination of two or more antibiotics, which increases their potency and aids in treatment [ 10 , 11 ].\nAntibacterial drugs’ principal modes of action include inhibition of cell-wall formation and interference with the ribosome, which cause inhibition of protein synthesis. Antibiotics also alter cell membrane activities, interfere with nucleic acid production (inhibition of DNA gyrase and RNA polymerase), and impede metabolic pathways [ 12 ]. Changes in the bacterial genome are responsible for bacterial antibiotic resistance [ 13 ]: primarily genetic mutations that modify genomic DNA, result in new resistant strains, as well as transfer of genetic material such as plasmids or mobile genetic components containing antibacterial-resistance genes.\nThe search for new anti-infection agents from natural resources, with different modes of action and competitive effects, has become a necessity [ 14 ]. Numerous lines of research are developing new classes of molecules aimed at new “targets” of action in bacteria, in order to circumvent bacterial resistance mechanisms [ 15 ]. For instance, Quorum sensing (QS) has been identified as one of the alternate approaches for combating multidrug-resistant pathogens [ 16 ]. Through the QS process, several pathogenic bacteria monitor their own population density and control their virulence factors, antibiotic production, biofilm formation, mobility, and swarming [ 17 ].\nEssential oils (EOs) from medicinal and aromatic plants are of major interest due to their rich bioactive compounds and potent antimicrobial activity [ 18 ]. For instance,  Rosmarinus officinalis  is well known for is medicinal properties against respiratory diseases, headaches, illnesses, and neuropsychiatric disorders [ 19 ]. It was largely used for culinary purposes [ 20 ] and food preservation [ 21 ]. Moreover,  Myrtus communis  is a wild aromatic plant with a wide range of biological properties such as antioxidant, anti-inflammatory, and antimicrobial [ 22 ].\nGiven the large number of chemical compounds present in EOs, there are several targets in the bacterial cell exposed to these molecules [ 23 , 24 ]. Closely related to their chemical composition, numerous mechanisms of action of EOs were documented, including: alteration of the cell wall, degradation of the cytoplasmic membrane and alteration of membrane proteins, leakage of cell contents, coagulation of the cytoplasm, depletion of the proton motility force, and induction of oxidative stress in the bacterial cell [ 25 , 26 ]. It was postulated that EOs induce oxidative stress in treated cells [ 19 ]. EOs inhibit bacterial growth by enhancing the production of reactive oxygen species (ROS) in the cells, which consequently inhibit certain essential biological processes [ 20 ]. In this context, the present study was undertaken to evaluate the antibacterial properties and mode of action of  R. officinalis  and  M. communis  EOs against the opportunistic pathogen  S. aureus .\n\nThe results of chemical analysis of  R. officinalis  and  M. communis  EOs by GC/MS and GC/FID are presented in  Table 1 .\nA total of 56 compounds are identified, representing 96.39% and 96.57% of tested EOs. The major constituent of  R. officinalis  EO was found to be 1,8-cineole with a prevalence of 37.56%. This molecule belongs to the class of monoterpene oxides. The major compound of  M. communis  EO was the α-Pinene, with a percentage of 45.3%. This compound belongs to the class of monoterpene hydrocarbons.\nThe antibacterial effects of both EOs and their combination are reported as ‘in vitro’ activity as MIC and MBC and summarized in  Table 2 .\nBoth tested agents exerted a bacteriostatic effect against the  S. aureus  strain, with MICs values ranging between 0.7 and 11.25 mg/mL, for  R. officinalis  and  M. communis  respectively ( p  < 0.05). MIC obtained from the combination of EOs was better than that of  R. officinalis  EOs. All tested substances showed bacteriostatic activity against  S. aureus  (MBC/MIC > 4).\nTo evaluate the antibiofilm effect of EOs, an established  S .  aureus  biofilm was treated with various concentrations (MIC, MIC × 2, MIC × 4) of tested agents ( Figure 1 ).\nOur results showed that EO1 and EO2 were more effective against the development of preformed biofilm at low concentrations (MIC and 2 × MIC), with percentage reduction values higher than 50%. Whereas, the combination of EOs was more active against  S .  aureus  biofilm, with percentage reduction value exceeding 74% at high concentration of 4 × MIC ( p  < 0.05). At the same dose (4 × MIC) EO1 and EO2 showed lower antibiofilm activities ( p  < 0.05) that did not exceed 50%.\nThe effects of tested EOs against  S. aureus  hemolysin, DNase, and protease production was evaluated by determining the diameter of inhibition halos, in comparison with the untreated strain ( Table 3 ).\nOur results showed that the most significant anti-hemolytic effect was obtained with a combination of EOs, resulting in an inhibition diameter equal to 9 ± 0.8 mm after treatment with a concentration of MIC × 4 ( p  < 0.05). At the same concentration, both EO1 and EO2 exhibited lower anti-hemolytic activities ( p  > 0.05).\nRegarding the anti-DNase activity, EO of  M. communis  showed more potent effects when compared to other agents without significant difference ( p  > 0.05). Furthermore, tested EOs were found to be effective in the inhibition of  S. aureus  protease activities with a concentration-dependent manner. The most significant anti-protease activity was obtained with the combination of EOs (MIC × 4), with an inhibition diameter reaching 11.6 ± 0.5 mm ( p  < 0.05). At the same concentration, EO1 was found to be less effective against the protease activity of  S. aureus  ( p  < 0.05).\nIn this part of our study, we tested the implication of EOs in  S. aureus  oxidative stress through ROS generation ( Figure 2 ).\nWe have found that tested EOs as well as their association caused an increase in the production of reactive oxygen species compared to the control ( p  < 0.05). It was deduced that equal proportion of  R. officinalis  (50%) and  M. communis  (50%) strongly induced oxidative stress in an  S. aureus  strain treated with a MIC × 4, triggering a high production of ROS in a dose-dependent manner. At the same concentration (MIC × 4), both EO1 and EO2 showed high production of ROS ( p  > 0.05) when compared to their combination (EO1 + EO2).\nAfter treatment of an  S. aureus  strain with different concentrations of EOs, we measured the catalase activity, since this enzyme is involved in the defense of the bacterial cell against oxidative stress. Results of this test are presented in  Figure 3 .\nOur results showed that the highest catalase activity (9750 U/mg protein) was observed in an untreated cell. Treatment with various MIC of EOs and their combination revealed a significant decrease in this anti-oxidant activity, in a dose dependent manner ( p  < 0.05). At high concentration (MIC × 4) of tested agents, EO1 showed the lowest catalase activity ( p  < 0.05).\n\nThe first part of our investigation was conducted to analyze the chemical composition of  R. officinalis  and  M. communis  EOs. Our results showed that the major constituent of  R. officinalis  EO was found to be 1,8-cineole, which is in agreement with the study of Badreddine et al. [ 27 ], having reported a similar value of major compounds of  R. officinalis  EO, which is 1,8-cineole (34.8%). Another recent study conducted by Moumni et al. [ 28 ], showed that Tunisian  R. officinalis  EOs are characterized with a high percentage of 1,8-cineole (37.6% to 47.2). Regarding the  M. communis  EO, it was shown that its major compound was found to be the α-Pinene (35.9%) [ 29 ], while the study of Cherrat et al. [ 30 ] showed the presence of another major compound, Myrtenyl acetate (49.3%). This variability of the chemical composition, even among the same specimens of EOs, is often related to the site of collection as well as geographical provenance. In fact, all these components play a major role in the plant adaptation to the ecology and the environment [ 31 ].\nIn the second part of our investigation, we evaluated the anti-staphylococcal activities of both EOs and their combination. The obtained results showed that all tested substances showed bacteriostatic activity against  S. aureus . This finding was confirmed by previous studies having shown that EO of  R. officinalis , with a high amount of 1,8-cineole, excreted a bacteriostatic effect against  S. aureus  strains [ 32 ]. Additionally, the same effect was previously reported with  M. communis  EO against  Escherichia coli  and  S. aureus  strains [ 33 ]. EOs exert various cytotoxic actions on bacterial strains, acting on several cellular structures. Thus, by crossing the bacterial wall and the plasma membrane, the aromatic compounds of EOs permeabilize the cell membrane and disrupt its function. They can make it permeable to protons and various ions, and inhibit the production of ATP. Eventually, this can lead to lysis of the bacteria. EOs can also reduce membrane fluidity, which impairs the proper functioning of the bacteria [ 34 ]. It has been shown that Gram-positive bacteria are more sensitive to EOs than Gram-negative ones. Indeed, it seems that the outer membrane of Gram-negative bacteria, rich in lipopolysaccharides, is more complex and represents an obstacle for aromatic molecules to reach the cytoplasm of this bacterium. However, these active compounds can more easily integrate into the cytoplasm of Gram-positive bacteria given the absence of the complex extra-membrane system, resulting in higher antibacterial activity [ 35 ].\nSince microbial biofilms increase bacterial resistance to various antimicrobial agents, investigation of antibiofilm effects of bioactive substances from natural resources, such as EOs, remain of interest. Our results revealed that tested EOs and their association displayed high potency against the development of biofilm performed by  S. aureus . Previously, the effectiveness EOs against performed methicilin-resistant  S .  aureus  (MRSA) biofilms was reported [ 36 , 37 ]. Generally, the effect of EOs on bacterial biofilm depends on their compositions and on the bacterial strains tested. For example, a previous study reported that  M. communis  EO exhibited significant anti-biofilm activity against  S. aureus  strains, which could be attributed to their α-Pinene richness [ 38 ]. Another study conducted by Jardak et al. [ 39 ]. showed that Tunisian  R. officinalis  EO exerted a significant  S. epidermidis  biofilm eradication percentage of 67%. Similarly, another study showed that two varieties of Tunisian  Laurus nobilis  EO are capable of inhibiting biofilms of oral  S. aureus  strains, with eradication percentages ranging from 50 to 79% [ 31 ]. EOs affect biofilm formation through damage to the outer envelope of this bacterial structure, resulting in the loss of integrity of this layer. Similarly, EOs can also cause inhibition of biofilm synthesis proteins, preventing the development and maturation phase [ 40 ]. Within a biofilm, cell-cell interactions and communications have been described. These interactions involve chemical signals, such as quorum sensing (QS) self-inducers [ 41 ].\nQS is an intercellular communication system that plays an essential role in biofilm formation and virulence-factor production in several bacterial species [ 17 ]. Thus, this communication mechanism is used by these microbes to express various survival or virulence traits leading to increased resistance of bacteria [ 40 ]. Numerous biosynthetic pathways are regulated by QS, including the production of metabolites [ 42 ], biosurfactants [ 43 ], and antimicrobials [ 44 ]. Interestingly, molecules that interfere with the QS system attenuate bacterial pathogenicity [ 45 ]. The activity of tested EOs against  S. aureus  hemolysin production showed that the most significant anti-hemolytic effect was obtained with combination of EOs. Oher findings reported the same hemolytic activity of two EOs of  Dennettia tripetala  on sheep red blood cells [ 46 ]. In addition, EOs from  Lippia origanoides  and  Thymus vulgaris  has been demonstrated to have significant effects on hemolytic activity of  S. aureus  ATCC 29213 [ 47 ]. Apart their roles of increasing the ability of the infection to establish and remain in humans, various types of  S. arures  hemolysins are produced and associated with a possible activation of QS, prior to biofilm formation [ 48 , 49 ]. Furthermore, our results showed the effectiveness of tested EOs in the inhibition of  S. aureus  DNase and protease activities with a concentration-dependent manner. Therefore, valorization of EOs seems to be an effective strategy to control virulence factors of pathogenic bacteria such as  S. aureus .\nThe Implication of EOs in  S. aureus  oxidative stress through ROS generation was investigated. We have found that tested EOs, as well as their association, caused an increase in the production of reactive oxygen species compared to the control, in a dose-dependent manner. Our results are in agreement with a recent study showing that Chamomile EO generated oxidative stress in  S. aureus  ATCC 29213, which may be the main mode of anti-staphylococcal action of this oil [ 50 ]. Similarly, oxidative stress was also detected with an increased level of ROS in bacterial cells of  Klebsiella pneumoniae  BAA-1705 and  E. coli  ATCC 25922 treated with Lavender EO [ 25 ]. The generation of ROS, including superoxide anions (O 2− ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (OH) that are highly reactive, and can lead to oxidative stress if the cell’s antioxidant mechanisms are overcome by pro-oxidant agents [ 51 ]. These species are highly reactive, cause oxidative damage and alter the structure and function of macromolecules, such as DNA/RNA, lipids and proteins [ 52 , 53 ]. Even for untreated bacterial cells, ROS production is a natural side effect of aerobic respiration [ 54 ], which explains their presence in weak proportions in negative controls.\nTo protect against the damaging effect of ROS, bacteria are able to produce enzymes such as catalase (CAT) and superoxide dismutase (SOD), to detoxify ROS and accelerate the spontaneous dismutation reaction of H 2 O 2 , along with regulatory mechanisms to counteract their damage [ 55 , 56 ]. After treatment of the  S. aureus  strain with different concentrations of EOs, we measured the catalase activity. Results of this test showed that the highest catalase activity was registered in an untreated cell, while treatment with various MIC of EOs revealed significant decrease in this anti-oxidant activity. In line with our findings, other studies showed decrease in catalase activity in  S. aureus  bacterium exposed to various phyto-compounds such as  Leonurus cardiaca  extract [ 57 ], allylpyrocatechol [ 58 ], silibin [ 59 ], and Catechin [ 60 ]. Catalase produced by bacteria facilitates cellular detoxification that allows them to repair or escape oxidative damage from H 2 O 2  [ 61 ]. Reduction of catalase activity caused by biologically active substances might result in increased H 2 O 2  level and lead to oxidative stress-mediated toxicity in bacterial cells [ 60 ]. However, a recent study conducted by Mohammed et al. [ 62 ] showed antioxidant capabilities of  Artemisia judaica  EO increasing the content of CAT and SOD enzymes in treated bacteria. It has been reported that a single chemical compound can function as both an antioxidant and a prooxidant [ 63 ]. Furthermore, it has been shown in different bacterial species that a short exposure to various antimicrobial agents leads to an increase of catalase enzyme activity in response to this external stress. Whereas, with increasing exposure time (12 to 24 h), bacteria lose their ability to detoxify these antibacterial agents and mitigate the induced stress, leading to a decreases of catalase activity [ 64 , 65 ].\n\nRosmarinus officinalis  and  Myrtus communis  essential oils were purchased commercially from a local producer (KG Flower, Diar ben Salem Béni khiar, Tunisia) after hydrodistillation of fresh aerial parts. For each species, 3 samples of the obtained EO were stored at 4 °C until analysis was attempted. The bacterial strain  Staphylococcus aureus  ATCC 25923 was obtained from the American Type Culture Collection (ATCC). To ensure optimal growth, the bacterial strain was sub-cultured twice, in Brain heart infusion (BHI) broth, and incubated at 37 °C for 24 h before each treatment.\nQuantitative and qualitative analyses of all the chemical composition of studied essential oils were determined by Gas chromatography with flame-ionization detector (GC/FID) and Gas chromatography-mass-spectrometry (GC/MS) as previously described [ 28 ].\nThe minimum inhibitory concentration (MIC) of  R. officinalis  (EO1) and  M. communis  (EO2) EOs against the  S. aureus  strain, as well as their combination (50% EO1 + 50% EO2), was determined by the broth dilution method according to standard protocols [ 66 ]. Various concentrations ranging between 0.05 mg/mL and 50 mg/mL of the tested agents were aseptically prepared in 96-well microtiter plates containing Muller Hinton broth (MH) and dimethyl sulfoxide (DMSO). Then inocula (0.5 McFarland) of the tested  S. aureus  strain was added to each well. To determine the minimum bactericidal concentration (MBC) values, MH plates were inoculated with 10 µL from each well medium that had no apparent growth and then incubated for 24 h at 37 °C. MBC was defined as the lowest concentration that killed 99% of the treated bacteria [ 67 ].\nThe antibiofilm activity of EO1 and EO2, as well as their combination (EO1 + EO2), were assessed by crystal violet (CV) staining test as described previously [ 31 ]. An established  S. aureus  biofilm (48 h) on a sterile 96 microtiter plate was treated with various concentrations of tested agents (1 × MIC, 2 × MIC, and 4 × MIC per well), prepared in DMSO and BHI broth. After incubation for 24 h, the plate was stained with CV (1%) and the biofilm’s biomass was quantified at 570 nm using the microplate reader. The percentage of biofilm eradication was determined by the following formula: [(OD growth control − OD sample)/OD growth control] × 100. Where control is untreated biofilm with EOs or their combination.\nThe hemolytic activity of the treated  S. aureus  strain was assessed on bacteriological agar supplemented with 5% sheep’s blood for alpha or beta-hemolysin production, and DNase Test Agar Base (DTAB) for the detection of deoxyribonuclease activity [ 68 ]. The overnight bacterial culture grown in trypticase soy broth (TSB, Bio-Rad) was diluted (1:100) with the new TSB medium. Then 100 µL of these dilutions were introduced into uniform wells of 6 mm diameter, which were aseptically perforated in the blood and DTAB agar. Following 24 h incubation of each plate at 37 °C, the diameters of the clear zones around the wells were determined [ 69 ]. The anti-hemolysin and anti-DNase activities were performed as described below, with the addition of EOs at different concentrations (1 × MIC, 2 × MIC, and 4 × MIC) per well. All the assays were carried out in triplicate.\nTo check the inhibition of the protein-digesting enzyme protease, the bacterial cells were incubated with varying concentrations (MIC/2; MIC; MIC × 2 and MIC × 4) of EOs and their combination. Then, 10 µL from each treated bacterial cultures were spotted on Bacto agar containing casein (5%), and incubated at 37 °C for 24 h [ 70 ]. The cleared zone surrounding the colony was measured and compared to the measured zone obtained from the control (untreated cells).\nThe production of ROS by the  S. aureus  strain exposed to EOs was performed using a peroxynitrite indicator, 20–70-dichlorodihydrofluorescein diacetate (DCFH-DA) (SigmaAldrich, UK), which can detect a broad range of ROS [ 70 ]. The adjusted bacterial culture (0.5 McF) was treated with different concentrations of EOs and their combination (corresponding to MIC/2, MIC, 2 × MIC, and 4 × MIC), in presence of DCFH-DA at a final concentration of 5 mM in 0.85% saline, and incubated at 37 °C aerobically for 24 h. Untreated bacterial culture was served as a negative control. The fluorescence emission of DCFH-DA was measured at 525 nm using a Tecan microtiter plate reader with an excitation wavelength of 485 nm [ 71 ]. Experiment was carried out in triplicate.\nThe catalase (CAT) enzyme activity was determined after treatment of overnight  S. aureus  cultures with different concentrations of EOs and their combination (MIC/2, MIC, 2 × MIC, and 4 × MIC). Following incubation for 24 h at 37 °C, the treated bacterial culture was centrifuged at 3000 rpm for 10 min, and the resultant pellet was washed twice with PBS. For enzyme assay, the bacterial extract was prepared by resuspending the pellet in 500 μL of cell lysate buffer (10 Mm Tris-HCl, 1 mMEDTA, 0.1% Triton-X-100 and 150 mM NaCl). After incubation at 37 °C for 1 h, the contents were then centrifuged (3000 rpm for 10 min) and the supernatant was collected for enzyme assay [ 72 ]. CAT activity in the bacterial extract was determined according to Acuna et al. [ 73 ]. In a quartz cuvette, 780 µL of phosphate buffer (KH 2 PO 2 /K 2 HPO 4 , pH7) were introduced to 200 µL H 2 O 2  (20 mM), to which 20 µL of bacterial cell lysate was added. Then, optical density of the mixture in each bacterial cell was monitored for 60 s (t = 0 s and = 60 s) at a wavelength of 240 nm. One unit (U) of enzyme activity is defined as the amount of enzyme required to convert 1 µmol of H 2 O 2  in one second.\nAll the experiments were carried out in triplicate and the data obtained were presented as means ± standard deviations. Data were further analyzed using the one-way analysis of variance (ANOVA) test to calculate the significance of the results:  p  values less than 0.05 were considered significantly statistically different.\n\nIn the context of fighting against pathogenic bacteria, using biological methods, we investigated through this study the anti-staphylococcal activities of two Eos of  R. officinalis  and  M. communis . Our results displayed the potent efficacy of both Eos and their combination on  S. aureus , as a representative of Gram-positive bacteria. Additionally, we highlight the anti-virulence properties of tested agents, due to their active compounds. Of significance, we showed EOs exerting considerable oxidative stress internally within cells, which is coupled with reduction of catalase activity, contributing to their antagonistic effect against  S. aureus.  To overcome chemical degradation and prevent the volatilization of bioactive compounds of tested substances, their encapsulation in nanometric systems could offer a promising intervention.","source_license":"CC-BY-4.0","license_restricted":false}