Potential Anti-Infectious Activity of Essential Oil Chemotypes of Lippia origanoides Kunth on Antibiotic-Resistant Staphylococcus aureus Strains

Staphylococcus aureus infections are prevalent in healthcare and community environments. Methicillin-resistant S. aureus is catalogued as a superbug of high priority among the pathogens. This Gram-positive coccus can form biofilms and produce toxins, leading to persistent infection and antibiotic resistance. Limited effective antibiotics have encouraged the development of innovative strategies, with a particular emphasis on resistance mechanisms and/or virulence factors. Medicinal aromatic plants have emerged as promising alternative sources. This study investigated the antimicrobial, antibiofilm, and antihemolysis properties of three different chemotypes of Lippia origanoides essential oil (EO) against susceptible and drug-resistant S. aureus strains. The chemical composition of the EO was analyzed using GC-MS, revealing high monoterpene concentrations, with carvacrol and thymol as the major components in two of the chemotypes. The third chemotype consisted mainly of the sesquiterpene β-caryophyllene. The MIC values for the two monoterpene chemotypes ranged from 62.5 to 500 µg/mL for all strains, whereas the sesquiterpene chemotype showed activity against seven strains at concentrations of 125–500 µg/mL, which is the first report of its anti-S. aureus activity. The phenolic chemotypes inhibited biofilm formation in seven S. aureus strains, whereas the sesquiterpene chemotype only inhibited biofilm formation in four strains. In addition, phenolic chemotypes displayed antihemolysis activity, with IC50 values ranging from 58.9 ± 3.8 to 128.3 ± 9.2 µg/mL. Our study highlights the importance of L. origanoides EO from the Yucatan Peninsula, which has the potential for the development of anti-S. aureus agents.


Introduction
The discovery of antibiotics is one of the most significant advances in medicine [1].However, the overuse and misuse of antibiotics have become prevalent among outpatients, resulting in drug resistance and ending the gold era of antibiotics [2].Antimicrobial resistance (AMR) is one of the principal public health problems of the 21 century and threatens the effective prevention and treatment of diverse infections caused by parasites, fungi, bacteria, and viruses that are not susceptible to the common drugs used to treat them [3].AMR in bacteria is particularly urgent because, in recent decades, bacterial resistance to new antibiotics has developed in both community and healthcare-associated infections [4].In 2017, the World Health Organization published a list of global priority microbes, including 12 species of bacteria with critical, high, and medium antibiotic resistance, and called upon academic and pharmaceutical scientific communities to conduct investigations to fight AMR [5].One of these species is methicillin-resistant Staphylococcus aureus (MRSA), a Grampositive coccus that spreads in the healthcare environment and community and causes a variety of infections such as suppurative or abscessed lesions, surgical wound infections, bacteremia, pneumonia, osteomyelitis, arthritis, and intravascular catheter-associated infection or urinary catheterization.The main serious complications of staphylococcal bacteremia are septic shock, endocarditis, myocarditis, meningitis, and pneumonia [6].
Numerous in vitro biological studies have been performed using commercially available reference strains, and only a few have included clinical isolates.It is important to perform a bioactivity assay using priority drug-resistant clinical isolates that involve mechanisms such as biofilm-formation and toxin production, which are associated with recalcitrant infections, to test a possible new antibiotic against wild/circulating bacteria [7,8].Currently, the search for novel therapies for treating S. aureus infections is a prevalent area of research, with a particular emphasis on resistance mechanisms and/or virulence factors.Essential oils (EOs) obtained from aromatic plant species have emerged as a promising alternative [9][10][11][12].
Oregano is a spice commonly used in various culinary traditions and has been found to have medicinal properties.There are approximately 60 plant species in the oregano ethnobotanical complex, primarily from the genera Origanum and Lippia, which are characterized by the presence of carvacrol and thymol.Lippia origanoides (botanical synonyms L. graveolens and L. sidoides) is considered an American oregano species [13,14].In traditional medicine, dried or fresh L. origanoides leaves have been used to treat respiratory and gastrointestinal diseases, such as colds, bronchitis, and diarrhea.It is also used to treat uterine inflammation, menstrual cramps, postpartum cramps, vaginal infections, headaches, wounds, mycoses, and pain [15][16][17][18].Recently, EOs from L. origanoides have shown antimicrobial activity, including a reduction in biofilm-formation and antiquorum sensing (QS) activity [12,19,20].
EOs are obtained from aromatic plants via steam distillation and consist of lipophilic, low-molecular-weight volatile compounds, including monoterpenes, sesquiterpenes, phenylpropanes, and oxygenated derivatives [21].The effectiveness of these compounds against pathogens is influenced by their EO composition and is primarily attributed to the most abundant metabolites present at high concentrations, which can exhibit simple, additive, or synergistic effects.The chemical complexity of EOs, which target multiple bacterial cell types simultaneously, reduces the likelihood of resistance in microorganisms [22][23][24][25][26].
The chemical composition of EOs depends on various factors, including the internal and external plants factors.Chemotypes, which are individuals from a plant species with an identical morphology that present EOs with different chemical compositions, are often found in aromatic species [27].The Lippia origanoides EO has significant variability in its chemical composition, with two main phenolic chemotypes characterized by the oxygenated monoterpenes, carvacrol and thymol, and a non-phenolic chemotype with a higher diversity of metabolites [28][29][30].
To gain a more comprehensive understanding of the antimicrobial activity of the native L. origanoides Kunth EO from the Yucatan Peninsula, which is traditionally used to treat fever, asthma, cough, diarrhea, stomach cramps, and pain [31], and explore its potential against resistant strains of S. aureus, we evaluated three EO chemotypes against reference strains and clinical isolates of S. aureus including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA).In this study, we analyzed the effects of these chemotypes on bacterial growth, drug resistance mechanisms, and virulence factors.

Chemical Composition of the Three Chemotypes of the L. origanoides EO
The chemical composition of the L. origanoides EO varied considerably among the three chemotypes.The two phenolic chemotypes were dominated by monoterpenes and oxygenated monoterpenes (<80% chromatogram area), with carvacrol and thymol, respec-tively, as the major components, representing >55% of the total chromatogram area.In contrast, for the sesquiterpene chemotype, in which sesquiterpenes represented 80% of the total chromatogram area, β-caryophyllene, α-humulene, and α-selinene were the most abundant sesquiterpenes (Table 1).

Activity of the L. origanoides EO on Staphylococcal Growth
The antibacterial activity of the L. origanoides EO depends on the interaction between the bacterial strain and the chemotype.The antibacterial activities of the three chemotypes are summarized in Table 2. Clinical isolates (except SAU-UIMY-44) were found to be more susceptible to the three chemotype EOs than all the ATCC reference strains.

Activity of the L. origanoides EO on the Formation of the Biofilm of S. aureus
The effect of L. origanoides EO chemotypes on biofilm formation inhibition coincided with the results of the antibacterial assays.This response depends on the interaction between the S. aureus bacterial strain and EO chemotype.A two-way ANOVA showed a statistically significant interaction between the bacterial strain and EO chemotype (F(16,54) = 20.73,p = 0.001) on the inhibitory effect on biofilm formation.Phenolic chemotypes inhibited biofilm formation with IC50 values of less than 250 µg/mL in all S. aureus strains (except for SAU-UIMY-26 and SAU-UIMY-44).In contrast, the sesquiterpene chemotype presented an IC50 biofilm-inhibition value lower than 250 µg/mL for the strains SAU-UIMY-1, SAU-UIMY-31, SAU-UIMY-39, and S. aureus MRSA ATCC 4330.In these four

Activity of the L. origanoides EO on the Formation of the Biofilm of S. aureus
The effect of L. origanoides EO chemotypes on biofilm formation inhibition coincided with the results of the antibacterial assays.This response depends on the interaction between the S. aureus bacterial strain and EO chemotype.A two-way ANOVA showed a statistically significant interaction between the bacterial strain and EO chemotype (F (16,54) = 20.73,p = 0.001) on the inhibitory effect on biofilm formation.Phenolic chemotypes inhibited biofilm formation with IC 50 values of less than 250 µg/mL in all S. aureus strains (except for SAU-UIMY-26 and SAU-UIMY-44).In contrast, the sesquiterpene chemotype presented an IC 50 biofilm-inhibition value lower than 250 µg/mL for the strains SAU-UIMY-1, SAU-UIMY-31, SAU-UIMY-39, and S. aureus MRSA ATCC 4330.In these four strains, the effect of the sesquiterpene chemotype EO was statistically equal to that of phenolic chemotypes (Table 3).For the EO of the three chemotypes, a negative correlation was observed between the IC 50 values of the EO on biofilm formation and the biofilm biomass (OD values) of the various S. aureus strains.The Spearman rank correlation coefficient (R) values were −0.73 p = 0.025; −0.85 p = 0.004; and −0.93 p = 0.0003 for the carvacrol, thymol, and sesquiterpene chemotypes, respectively.The clinical isolate SAU-UIMY-1 presented the highest biofilm biomass and the lowest IC 50 values of the sesquiterpene (IC 50 = 30.8± 1.5), thymol (IC 50 = 60.6 ± 1.7), and carvacrol (IC 50 = 77.3± 1.7) EOs.A similar trend was observed for the SAU-UIMY-31 and MRSA ATCC 43300 strains, with superior biofilm biomass values and generally lower IC 50 values (Table 3).strains, the effect of the sesquiterpene chemotype EO was statistically equal to that of phenolic chemotypes (Table 3).For the EO of the three chemotypes, a negative correlation was observed between the IC50 values of the EO on biofilm formation and the biofilm biomass (OD values) of the various S. aureus strains.The Spearman rank correlation coefficient (R) values were −0.73 p = 0.025; −0.85 p = 0.004; and −0.93 p = 0.0003 for the carvacrol, thymol, and sesquiterpene chemotypes, respectively.The clinical isolate SAU-UIMY-1 presented the highest biofilm biomass and the lowest IC50 values of the sesquiterpene (IC50 = 30.8± 1.5), thymol (IC50 = 60.6 ± 1.7), and carvacrol (IC50 = 77.3± 1.7) EOs.A similar trend was observed for the SAU-UIMY-31 and MRSA ATCC 43300 strains, with superior biofilm biomass values and generally lower IC50 values (Table 3).

Correlation of the Chemical Composition of the L. origanoides EO and Its Antibiofilm Activity
The nine S. aureus biofilm producing strains presented a significant association between the IC50 values for biofilm inhibition and the concentration (% peak area in chromatogram) of the major chemical components in the EO of L. origanoides chemotypes, as shown by the values of the Spearman rank correlation coefficient (Table 4).
The inhibition of biofilm formation varied according to the S. aureus strain.In two clinical isolates, SAU-UIMY-1 and SAU-UIMY-31, the concentration of the sesquiterpenes β-caryophyllene, α-humulene, α and β selinene, and γ and δ cadinene were significantly correlated with the IC50 values.Higher concentrations of these metabolites in the EO resulted in lower IC50 values.In the remaining seven clinical isolates, the percentages of the monoterpenes, p-cymene, carvacrol, and thymol showed a significant association with biofilm inhibition (Table 4).

Correlation of the Chemical Composition of the L. origanoides EO and Its Antibiofilm Activity
The nine S. aureus biofilm producing strains presented a significant association between the IC 50 values for biofilm inhibition and the concentration (% peak area in chromatogram) of the major chemical components in the EO of L. origanoides chemotypes, as shown by the values of the Spearman rank correlation coefficient (Table 4).The inhibition of biofilm formation varied according to the S. aureus strain.In two clinical isolates, SAU-UIMY-1 and SAU-UIMY-31, the concentration of the sesquiterpenes β-caryophyllene, α-humulene, α and β selinene, and γ and δ cadinene were significantly correlated with the IC 50 values.Higher concentrations of these metabolites in the EO resulted in lower IC 50 values.In the remaining seven clinical isolates, the percentages of the monoterpenes, p-cymene, carvacrol, and thymol showed a significant association with biofilm inhibition (Table 4).

Antihemolysis Activity of the L. origanoides EO on S. aureus
In our study, the carvacrol and thymol chemotypes of the L. origanoides EO exhibited antihemolysis activity against S. aureus ATCC BAA-977 and SAU-UIMY-31.A two-way ANOVA showed a statistically significant interaction between the bacterial strain and the EO chemotype (F [1,8] = 339.9,p = 0.0001) on the IC 50 average value.Among them, the thymol chemotype in strain SAU-UIMY-31 displayed the best activity (IC 50 = 58.9± 3.8 µg/mL), while in the carvacrol chemotype, the lowest IC 50 value was observed in the reference strain ATCC BAA-977 (IC 50 = 78.4± 6.3 µg/mL; Figure 1).On the other hand, the sesquiterpene EO chemotype did not exhibit 50% hemolysis inhibition at its highest concentration (250 µg/mL).9).Highlighted in green are significant negative correlations, indicating that a lower IC50 is associated with a higher concentration of the component percentage in the EO.

Chemical Composition of the Three Chemotypes of the L. origanoides EO
The three chemotypes identified in this study for the L. origanoides EO have been previously reported for this species [20,28,29,32].These chemotypes were also observed in O. vulgare cultivars [33].

Chemical Composition of the Three Chemotypes of the L. origanoides EO
The three chemotypes identified in this study for the L. origanoides EO have been previously reported for this species [20,28,29,32].These chemotypes were also observed in O. vulgare cultivars [33].
Our research found that the majority of L. origanoides EOs were bactericidal agents.This information provides valuable insights into the mechanisms of action of the antibacterial agents.However, no extrapolations have been made to clinical therapeutics.Previous studies have shown that carvacrol and thymol, as phytoconstituents of EO, as well as pure compounds, exhibit bactericidal activity against S. aureus [20,[33][34][35].To the best of our Plants 2024, 13, 1172 7 of 15 knowledge, this is the first report of the bactericidal activity of the sesquiterpene chemotype of L. origanoides.

Activity of the L. origanoides EO on the Formation of the Biofilm of S. aureus
Gómez-Sequeda et al. (2020) reported that the carvacrol and thymol chemotypes of L. origanoides exhibited values of IC 50 = 70 µg/mL and 1200 µg/mL, respectively, against S. aureus ATCC MRSA biofilms, whereas the non-phenolic chemotype showed an IC 50 > 3000 µg/mL against the same strain [20].Martinez et al. 2023 reported that the L. origanoides EO (thymol 32.7% and carvacrol 18.8%) exhibited a biofilm inhibition of 72% at 400 µg/mL against S. aureus ATCC 29213 (MSSA).In addition, these authors reported that EO alters the expression of genes related to biofilm formation and virulence factors associated with the QS system [35].Our study found similar results for the carvacrol and thymol chemotypes.It is important to highlight that the non-phenolic chemotype, with a predominance of sesquiterpenes (β-caryophyllene, α-humulene, and α-selinene), also showed significant biofilm inhibition activity, like that of the phenolic chemotype, particularly against the S. aureus strains SAU-UIMY-1, SAU-UIMY-31, SAU-UIMY-39, and MRSA ATCC 4330.
Our study demonstrated that higher biofilm biomass producers were inhibited by lower concentrations of EO, but the antibiofilm activity of EO against S. aureus strains may not be directly related to the type of biofilm biomass producers.Rather, it may depend on the unique genetic characteristics of each strain [36].The accessory gene regulator (agr) QS system in S. aureus has been found to correlate with biofilm biomass.Staphylococcus aureus strains can be grouped into four categories based on their agr system (agrI-IV).These groups often exhibit different phenotypes, such as varying biofilm formation capacities [37,38].Certain chemotypes of L. origanoides with thymol (32.7%) and carvacrol (18.8%) have been found to reduce the expression of agr in S. aureus [35], suggesting that differences in the type of agr among the strains could affect the biofilm biomass production and IC 50 antibiofilm activity of EO.
The Lippia origanoides EO suppresses the ica operon (ADBC), which is a key regulator of polysaccharide intercellular adhesin (PIA) synthesis in S. aureus biofilms [39]; icaA and icaD are responsible for PIA synthesis [40].Previous research has demonstrated that the coexpression of icaA and icaD in S. aureus is required for strong biofilm production; however, the absence of these genes does not completely inhibit biofilm formation, implying the existence of ica-independent mechanisms [39].These differences may also contribute to discrepancies in antibiofilm activity among EO chemotypes.

Correlation of the Chemical Composition of the L. origanoides EO and Its Antibiofilm Activity
Our results found a relationship between the presence of β-caryophyllene, α-humulene, α and β selinene, γ and δ cadinene p-cymene and carvacrol and the prevention of biofilm formation.Some EO components have been reported to exhibit antibiofilm properties.Studies have frequently reported the antibiofilm effects of the monoterpenes carvacrol and thymol, particularly against S. aureus [9,41].Reichling (2020) discovered that carvacrol and thymol significantly reduced biofilm mass and inhibited the movement of bacteria [9].Similarly, Kim et al. (2022) found that applying carvacrol and thymol at sublethal concentrations can effectively combat S. aureus by impacting its ability to form biofilm [41].Our findings align with those of previous studies, as the L. origanoides EO from the carvacrol and thymol chemotypes showed a significant inhibitory effect on biofilm formation.Carvacrol and thymol have been shown to impede biofilm development by impacting membrane lipids, preventing protein accumulation, and halting the microcolony stage [42][43][44].It also affects cell viability and interacts with transcriptional regulators of QS communication, biofilm formation, and virulence genes [35].The activity of EO components can be influenced by their minor components in complex interactions, which could explain the variations in the anti-S.aureus activity of the L. origanoides EO depending on the bacterial strain and EO composition.The monoterpene p-cymene may enhance the activity of other antimicrobial compounds such as carvacrol and/or thymol, which cause membrane instability [34,45,46] The sesquiterpene β-caryophyllene exhibited anti-S.aureus activity on fusidic acid resistant to the strain [47], which induces apoptosis and the disruption of mitochondrial membrane potential [48] and, using molecular docking analyses, interacts with bacterial DNA gyrase B, suggesting that it acts as an inhibitor [49].The biological activity of βselinene against S. aureus ATCC 25923 has also been reported [50].It has been shown that two or three major components, constituting up to 85% of the chromatogram area, drive biological activity [25,26]; however, often, the minor components also play a role [22,24].Because of the diverse chemical nature of EOs, their components could exert additive, antagonistic, or synergistic effects on each other; therefore, the specific modes of action could be a result of these interactions.Detailed studies with specific experimental designs are needed to deepen our understanding of the interactions between the different metabolites present in the EO of L origanoides and their anti-S aureus effect.Our results provide a basis for designing and conducting such experiments in future research.

Materials and Methods
This project was approved by the Scientific and Ethics Committees National of the Instituto Mexicano del Seguro Social (IMSS), with approval number R-785-2022-009.Figure 2 shows the flowchart of the methodology.

EO Extraction and Chemical Characterization
Lippia origanoides leaves from adult plants were collected from wild populations located in the Yucatan Peninsula [51].The chemotype carvacrol was collected in Chicxulub Puerto Yucatán (21.253 • N 89.573 • W); the thymol chemotype was collected in San Felipe Yucatán (21.561 • N, 88.189 • W); and the sesquiterpene chemotype was collected in Sotuta Yucatán (20.514 • N 88.946 • W).The leaves were dried at 35 • C for 36 h in an air-flux drying oven (NOVATECH HS60, Tlaquepaque, Mexico) and stored at 4 • C until distillation, following the method described in [51].Herbaria voucher specimens were deposited at the Centro de Investigación Científica de Yucatán, Mérida, Mexico (LM Calvo 240, 248, and 258); species identification was performed by the herbaria staff.EO was extracted from 250 g of dried leaves using hydrodistillation in a Clevenger-type apparatus for 1.5 h, with 25:1 mL/g water: plant material and 44 drops per minute as the average distillation rate, with hexane as the collector solvent.The oil-hexane mixture was dried with sodium sulfate, and the solvent was evaporated under a flow of nitrogen.The samples were stored in sealed amber vials at 4 • C until biological assays and chromatographic analysis.
To analyze the chemical composition of the EO in the three L. origanoides chemotypes, GC-MS analyses were conducted using an Agilent 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA), which was connected to an Agilent 5975 mass-selective detector and equipped with G1701DA GC-MSD ChemStation software v.D.03.00.The arithmetic linear retention index (AI) of each peak was determined [52], relative to that of a homologous series of n-alkanes (C8-C40), which was injected in the CG-MS under the same conditions previously described.For a metabolite to be considered tentatively identified, its mass spectrum (MS) would have to match the corresponding mass spectra contained in the library ADAMS 2007 [52] and in the library of the National Institute of Standards and Technology (NIST 11).In addition, the corresponding AI value must coincide with the AI values reported in the literature [52][53][54].When available, the definitive identification of individual metabolites was carried out by comparing their MS with those of commercial standards such as carvacrol, thymol, β-caryophyllene, and eucalyptol (98-99% purity, Sigma-Aldrich, St. Louis, MO, USA) or authentic samples previously purified and identified in our laboratories.The quantification of metabolites was performed using a Varian 430 GC (Varian BV, Santa Clara, CA, USA) equipped with an FID detector and N 2 as the carrier gas, using the same column, temperature program, and conditions described previously.

Anti-Infectious Assays 4.2.1. Staphylococcus aureus Strains
Three reference strains and nine clinical isolates of susceptible and drug-resistant S. aureus were used in the bioassays (Table 5).All strains were maintained at −80 • C in tryptic soy broth (TSB; Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with glycerol (J.T. Baker, Inc., Phillipsburg, NJ, USA).

Activity of EO on Staphylococcal Growth
The Minimal Inhibitory Concentration (MIC) of EO against the S. aureus strains was determined using the resazurin microtiter assay (REMA) broth dilution method.Briefly, bacterial cultures were cultured on Mueller-Hinton agar (MHA; Becton Dickinson Co., Ltd., Franklin Lakes, NJ, USA).Then, two to three bacterial colonies were suspended in 3 mL of Muller-Hinton broth (MHB; Becton Dickinson Co., Ltd., Franklin Lakes, NJ, USA) and incubated at 37 • C for 2-4 h until reaching growth comparable to the turbidity of the 0.5 McFarland standard (DEN-1; Biosan, Riga, Latvia).This suspension was further diluted at 1: 50 to obtain the working inoculum, and 100 µL of this suspension was incubated and cultured with 100 µL of MHB containing the EO at serial dilutions ranging from 500 to 15.62 µg/mL.All assays included a positive control (cultures with antibiotic specific) and negative controls (culture-free wells of the EO or antibiotic (Sigma-Aldrich, St. Louis, MO, USA) and a sterility control (culture broth alone).After incubation at 37 • C for 16 h, 30 µL of resazurin (Sigma-Aldrich, St. Louis, MO, USA) was added, and the microplates were incubated again at 37 • C for 2 h.A well-defined pink color indicated positive bacterial growth, whereas a blue color indicated the absence of growth.The results were expressed as the MIC, which corresponded to the greatest dilution of the EO in which a color shift from blue to pink was not observed.Each assay was performed three times independently in duplicate [55].
The minimal bactericidal concentration (MBC) of the EO was determined by reseeding the bacterial culture according to the method described by de Jesús Dzul-Beh et al. 2023 [56].Succinctly, 5 µL of the EO-treated bacterial suspensions corresponding to MIC, 2× and ½ MIC in MHB in 96-well plates, were transferred to a new microplate containing 195 µL of fresh culture medium per well.In addition, antibiotic-treated bacterial suspensions (positive control), untreated bacterial suspensions (negative control), and culture medium alone (sterility control) were transferred to fresh broth [57].After incubation at 37 • C for 16 h, resazurin was added to the wells, as previously described.MBC corresponded to the minimal EO concentration that did not cause a color shift in cultures re-incubated in the fresh medium.Each assay was performed three times in duplicate, and the MBC/MIC index for the EO was calculated.

Activity of the EO on the Formation of Biofilm
The inhibition of biofilm formation in all strains of S. aureus was evaluated using the crystal violet (CV) staining method in flat-bottom 96-well microplates, as previously described [55].Briefly, the strains were activated and cultured on MHA, and two bacterial colonies were cultured in 3 mL of TSB (Beckton Dickinson Co., Ltd., Franklin Lakes, NJ, USA) at 37 • C for 24 h.Then, an aliquot was transferred into TBS supplemented with glucose 1% (w/v; TSB + G) to match the turbidity of 0.5 McFarland.This suspension was further diluted at 1:50, and 100 µL of this was incubated with 100 µL of TBS + G containing serial dilutions of 500 to 15.6 µg/mL concentrations of the EO.An ethylenediaminetetraacetic acid (Sigma-Aldrich, St. Louis, MO, USA) solution was used as the positive control, whereas the negative control was EO-free wells.The microplate was incubated for 24 h at 37 • C.After incubation, the culture broth was gently aspirated, and each well was washed thrice with sterile distilled water to remove non-adherent cells and dried at 60 • C for 45 min.The biofilm was stained by incubating for 30 min with 200 µL of 0.1% CV (Sigma-Aldrich, St. Louis, MO, USA) solution.Any excess CV was removed with sterile distilled water before adding 200 µL of 40% acetic acid (Fermont, Monterrey, Mexico), and the absorbance was measured at 490 nm using a microplate reader (IMark; Bio-Rad, Hercules, CA, USA), which was related to the amount of biofilm produced.Each assay was performed in triplicate, and the concentration of EO that inhibited 50% (IC 50 ) biofilm formation was calculated using GraphPad Prism ver. 5 software (GraphPad Software Inc., La Jolla, CA, USA).

Antihemolytic Activity of EO
The antihemolytic activity of the EO was assessed using S. aureus ATCC BAA-977 and the clinical isolate SAU-UIMY-31, according to Loges et al., 2020, with minor modifications [58].Briefly, an aliquot on an overnight culture of S. aureus was transferred into fresh TSB to match the turbidity of 0.5 McFarland.A total of 30 µL of the suspension was incubated with 2970 µL of TBS containing serial dilutions of 200 to 25 µg/mL EO and incubated with shaking at 200 rpm and 37 • C for 24 h.After incubation, the bacterial culture supernatant was collected via centrifugation.In total, 100 µL of the bacterial supernatant was incubated with 300 µL of diluted human red blood cells in PBS buffer (Sigma-Aldrich; 330 µL red blood cells/10 mL PBS buffer), with shaking at 200 rpm and 37 • C for 1 h.The mixture was centrifuged at 4000 rpm for 4 min to remove intact red blood cells, and the absorbance of the supernatant was measured at 430 nm using a microplate reader (IMark, Bio-Rad, Hercules, CA, USA).The percentage of hemolysis inhibition for each concentration of EO was calculated.Each assay was performed in triplicate, and the IC 50 hemolysis was calculated using GraphPad Prism ver. 5 software (GraphPad Software, Inc., La Jolla, CA, USA).

Statistical Analysis
The Spearman rank correlation between the IC 50 for biofilm formation inhibition and the biofilm producer index was estimated for the nine biofilm producer strains.Similarly, for each bacterial strain, a Spearman rank correlation was estimated between the IC 50 for biofilm formation inhibition and the concentration of the major components (% area in the chromatogram) in the three chemotypes of the L. origanoides EO (N = 9).Two-way ANOVA was used to analyze the interaction of the bacterial strain and EO chemotype composition on the IC 50 values for biofilm formation inhibition and antihemolytic activity.In all statistical analyses, results with p-values > 0.05 were considered non-significant.

Conclusions
In the present study, the chemical composition of the three chemotypes of the L. origanoides EO varied considerably.The two phenolic chemotypes mainly contained carvacrol and thymol, whereas the third chemotype contained sesquiterpenes, with β-caryophyllene as the major component.All chemotypes exhibited anti-staphylococcal and antibiofilm activities against the reference and clinical isolates of S. aureus strains.The activity in both assays varied depending on the interaction between the bacterial strain and the EO chemotype.However, the carvacrol and thymol chemotypes also showed antihemolytic activity against reference and clinical isolates of S. aureus strains.Our study highlights the potential of chemotypes of the L. origanoides EO from the Yucatan Peninsula as candidates for future research in developing anti-S.aureus agents, and they are even active against the drug-resistant strains MRSA and VRSA.

15 Figure 2 .
Figure 2. Flowchart of the methodology.4.1.EO Extraction and Chemical Characterization Lippia origanoides leaves from adult plants were collected from wild populations located in the Yucatan Peninsula [51].The chemotype carvacrol was collected in Chicxulub Puerto Yucatán (21.253°N 89.573°W); the thymol chemotype was collected in San Felipe
552.The non-polar DB5 5% phenyl-metilpolisiloxane column (60 m, 0.25 mm i.d., film thickness 0.25 µm) was used, with a temperature program of 45 • C for 5 min, 45 to 150 • C at 4 • C/min, 150 • C for 2 min, 150 to 250 • C at 5 • C/min, 250 • C for 5 min, 250 to 275 • C at 10 • C/min, and 275 • C for 15 min.The injector temperature was 280 • C and the detector temperature was 290 • C. The carrier gas was He (1.5 mL/min); the injection volume was 1 µL, and the split ratio was 1:40.Mass spectra (MS) were obtained by electron impact at an energy of 70 eV.The temperatures of the ionization chamber and the transfer line were maintained at 230 • C and 285 • C, respectively.MS, total ionic currents, and extracted ions were obtained with a quadrupole analyzer using automatic radiofrequency scanning (full scan) in the mass range of m/z 35-300.

Table 1 .
Chemical composition of the EO chemotypes of L. origanoides.
Only compounds with a >1% chromatogram area are shown.Car: carvacrol; Thy: thymol; Ses: sesquiterpene; M: monoterpenes; OM: oxygenated monoterpenes; S: sesquiterpenes; OS: oxygenated sesquiterpenes.a AI: Linear retention indices determined experimentally on the DB5 column relative to a series of n-alkanes (C8-C40).b Bibliographic linear retention indices.c Relative contents are given as GC-FID peak areas; for the three most abundant compounds of each chemotype, the contents (% area of chromatogram) are given in bold type; n.d.= not detected.d Identification method: RI = tentative identification based on AI;

Table 2 .
Anti-S.aureus activity of the three chemotypes of the L. origanoides EO.

Table 2 .
Anti-S.aureus activity of the three chemotypes of the L. origanoides EO.

Table 3 .
Activity of the three chemotypes of the L. origanoides EO on S. aureus biofilm formation.

Table 3 .
Activity of the three chemotypes of the L. origanoides EO on S. aureus biofilm formation.

Table 4 .
Spearman rank correlation coefficient values between the IC 50 of antibiofilm activity and the concentration of the major components in the EO of the three chemotypes of L. origanoides.-significant correlation values > 0.05 are shown in italics for each of the nine analyzed S. aureus strains (N = 9).Highlighted in green are significant negative correlations, indicating that a lower IC 50 is associated with a higher concentration of the component percentage in the EO. Non