Evaluation of the antimicrobial effect of the Origanum vulgare L essential oil on strains of Klebsiella pneumoniae

Abstract Bacteria may be the initial cause of certain pathologies as well as a secondary agent responsible for the development of complications such as pressure ulcer infections. Pressure ulcers are a persistent health problem, especially in immunocompromised patients, and associated with infection by opportunistic microorganisms with antimicrobial resistance, such as Klebsiella pneumoniae, highlight the need for the development of new antimicrobial approaches. Thus, the aim of this study was to evaluate the antibacterial and anti-adherent activity of Origanum vulgare L. (oregano) essential oil against Klebsiella pneumoniae strains, as well as the effect of its association with synthetic antimicrobials. To this end, the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) analyses were performed on microdilution plates. The assay of the Minimum Inhibitory Adherence Concentration (MIAC), with test tubes. As well as, the association study through the infusion disc method containing ampicillin (AMP), gentamicin (GEN), ciprofloxacin (CIP) and ceftriaxone (CEF). Therefore, it was possible to obtain that the essential oil of oregano presents antimicrobial and bactericidal activity, with MIC ranging between 128μg/mL and 256 μg/mL and MBC between 256 μg/mL and 512 μg/mL, on the tested K. pneumoniae strains. When used in association with ampicillin and gentamicin, oregano essential oil showed synergistic effect for some strains. Therefore, it is observed that the tested essential oil can act as a promising antibacterial in the treatment of diseases caused by K. pneumoniae.

thymol from thyme and others in the treatment of PUs (Aburayan et al., 2020).
Studies show that terpenes are an important category of plant-derived compounds and can interact with each other and with bacterial cells to increase or decrease each other's antibacterial effects.And they observed that eugenol, terpineol, carveol, citronellol, geraniol showed antibacterial activity and linked this effect to the presence of hydroxyl groups (Zengin and Baysal, 2014;Mughal et al., 2024).
Not only, but more specifically, when analyzing the essential oil of oregano, it was possible to observe that oregano is rich in carvacrol and thymol, has as its Origanum vulgare L. essential oil (OEO) and presents activity on digestive, respiratory or dermatological disorders, with antibacterial, antifungal, antiparasitic, antioxidant, antiinflammatory and antitumor effects (Fournomiti et al., 2015;Lombrea et al., 2020).
In this context, this work sought to evaluate the antibacterial and anti-adherent activity and the association study with synthetic antimicrobials and oregano oil against strains of Klebsiella pneumoniae, as a promising strategy for management and infection control of pressure sores.

Test substance
The oregano essential oil (Origanum vulgare) was purchased from Industria Quinari (Ponta Grossa-PR).For the pharmacological tests, the substance was solubilized in DMSO and diluted in distilled water.The concentration of DMSO (dimethyl sulfoxide) used was less than 0.1% v/v.

Analysis of the essential oil by Gas Chromatography Coupled with Mass Spectrometry (GC-MS)
The oregano essential oil was analyzed for its chemical composition using a gas chromatograph coupled to a quadrupole mass spectrometer (GC-MS), Shimadzu model GC-QP2010 (Shimadzu, Kyoto, Japan), using a DB-5 fused silica capillary column (30m x 0.25mm i.d.x 0.25μm).The chromatographic conditions used were: injector at 220 °C and split ratio 1:10 (3.0 min), He carrier gas at 0.6 mL/ min, interface temperature at 250 °C and electron ionization source (35-350 m/z), oven temperature ramp: 40 °C (2 min), with increment of 3 °C/min up to 240 °C for 5 min and injected volume of 1 L (1% solution in dichloromethane).
The identification of the compounds was performed by comparing their fragmentation patterns with the mass spectra from the NIST 14 database (NIST/EPA/NIH Mass Spectral Library, 2014) present in the GC-MS; also, by comparing these with literature data (Adams, 2007), and through their linear retention indices (LRIs), calculated in relation to the retention times of a homologous series of hydrocarbons (C8 to C26) injected under the same conditions as the sample.The linear retention index was based on Van den Dool and Kratz (1963) and Adams (2007).For identification by comparison with spectra available in these databases only matches above 90% were considered.

Introduction
Bacteria are causative of quite simple and even more complex diseases.They present association and are responsible for several disease states, such as urinary tract infections, gastrointestinal diseases, infections: respiratory tract, central nervous system, bones and joints; skin conditions, cursing with acute and chronic conditions as, for example, the chronicity of pressure injury healing, resulting from bacterial invasion and biofilm formation (Baron et al., 2013;Freire et al., 2013;Hannigan et al., 2018;Paula et al., 2020).
In this sense, pressure ulcers (PUs) characterized by discontinuity and interruption of the normal skin structure, by pressure or shear force, present themselves as an open lesion, without protective barrier predisposed to immediate contamination, and can be colonized by the host microflora or by microbes of the environment, exposing an ideal environment for proliferation of pathogenic microorganisms, which after their entry develop and originate infectious processes, such as Klebsiella pneumoniae in ICU environment (Martins, 2010;Coates et al., 2014;Cardoso, 2015;Wert et al., 2020).
In turn, Klebsiella pneumoniae (Kp) is a Gram-negative bacillus, from the Enterobacteriaceae family, facultative aerobic, but with better growth in aerobic conditions.Its colonization in humans probably occurs through contact with various environmental sources and can be found colonizing the oropharynx and feces of healthy people, or as an opportunist in the body of immunocompromised people or with open wounds and scars, leading to infection (Santos, 2007;Almeida, 2013;Seibert et al., 2014).
Moreover, it stands out for being a producer of an enzyme called carbapenemase, and therefore also receives the acronym KPC -Klebsiella pneumoniae carbapenemase.Therefore, it is resistant to antibiotics of the carbapenem class: Meropenem, Ertapenem, and Imipenem, besides being able to inactivate β-lactam agents, such as cephalosporins, penicillins, and monobactams.In addition, it is known that KPC presents a high potential for dissemination, being Klebsiella pneumoniae a bacterium with a high capacity to transfer its genetic material, and consequently, its resistance genes, which is associated with the difficulty of infection control involving this bacterium (Cunha, 2014;Nirwati et al., 2019).
Therefore, the main complication of PUs is infection, which associated with risk factors such as increased age, unconsciousness, urinary and/or intestinal incontinence, poor nutritional status, diabetes mellitus, paralysis and cardiovascular diseases determine the morbidity and mortality of the pathology (Luz et al., 2010;Wert et al., 2020).
Soon PUs are one of the current concerns in hospitals and patient care services, because antimicrobial resistance hinders therapy and consequently increases the risk of morbidity and mortality (Mariano and Berreta, 2019).
Given this scenario, a need for the development of new pharmacological therapies for antibacterial treatment with low or no resistance is recognized.Thus, there are studies exploring the use of herbal medicinal compounds such as curcumin from saffron, aloesin from aloe vera, Brazilian Journal of Biology, 2023, vol.83, e269317 3/10 Antimicrobial effect of the Origanum vulgare Essencial Oil on strains of Klebssiella pneumoniae.

Microorganisms
Klebsiella pneumoniae strains (ATCC 13883, Kp 101, Kp 103, Kp 104 Kp 105 and Kp 110) were used.All strains were maintained on Muller Hinton Agar (MHAG) at 4 °C.The inoculate were obtained from overnight cultures in MH at 37 °C and diluted in sterile saline to obtain a final concentration of approximately 1.5 x 108 colony forming units per mL (CFU/mL), adjusted by turbidity compared to the 0.5 tube of the McFarland scale (Bona et al., 2014).

Culture medium
The culture medium used in the assay to evaluate the antimicrobial activity was Mueller Hinton liquid medium and Muller Hinton agar solid medium, the same purchased from Difco® and prepared according to the manufacturer's instructions.

Determination of the Minimum Inhibitory Concentration (MIC)
The MIC was determined using the microdilution technique in a 96-well plate with a U-shaped bottom.100μL of double concentrated Mueller Hinton broth and 100μL of oregano essential oil at the initial concentration of 2048 μg/ml were added, were dispensed into the wells of the first row of the plate.And by serial dilution in ratio of two, the concentrations of 1024,512,256,128,64,32,16,8 and 4 μg/ml were obtained, so that in the first row of the plate is the highest concentration and in the last, the lowest concentration.
The determination of the MIC was conducted with 10μL of the microorganism in each cavity, approximately 1.5 x 108 CFU/mL A sterility control was also prepared in the penultimate well, with only 200μL of the broth, and in the last well the growth control was performed, containing 100μL of the double concentrated Muller Hinton broth and the microorganism suspension, and the entire assay was performed in duplicate.The plates were incubated at 35±2 ºC for 24 hours, after the incubation time, the first reading of the results was performed.Then, 20μL of sodium resazurin solution (SIGMA) were added, previously solubilized in sterile distilled water at a concentration of 0.01% (w/v), recognized as a colorimetric indicator of oxide-reduction for bacteria, then another incubation was performed at 35±2 ºC.
The reading was done, visually, by the absence or presence of growth of the microorganism due to the formation of a cluster of cells (button) and also by the observation of the change in color of the solution, from blue to pink, indicating its growth.The MIC was determined as the lowest concentration of the product that inhibited the visible growth of the microorganism assayed, verified by a no change in the coloration of the indicator dye (Palomino et al., 2002;Ostrosky et al., 2008;CLSI, 2012;Bona et al., 2014).

Determination of the Minimum Bactericidal Concentration (MBC)
The minimum bactericidal concentration (MBC) of oregano essential oil was also determined for Klebsiella pneumoniae strains.After the MIC reading, inoculations (10μL) of up to three dilutions prior to the MIC value were made into Mueller-Hinton broth medium (100μL/cavity) in sterile microdilution plate, then passed for incubation at 35±2 °C for 24 hours, after which time 20μL of resazurin was added.The assays were incubated at 35±2 ºC for another 24 hours for confirmation of the concentration capable of inhibiting the total growth of the bacterial species, verified by a non-change in the coloration of the indicator dye (Ncube et al., 2008;Guerra et al., 2012).

Determination of the MIAC
The Minimum Inhibitory Adherence Concentration (MIAC) of oregano essential oil was determined in the presence of 5% sucrose, according to Albuquerque et al. (2010) with modifications, using concentrations corresponding to the pure oil up to 1:1024 dilution.From the bacterial growth, the strain of Klebsiella pneumoniae (KP 105) was grown at 35±2 ºC in Mueller Hinton broth (DIFCO, Michigan, United States), subsequently 0.9mL of the subculture was distributed in test tubes and then 0.1mL of the solution corresponding to the compound dilutions was added.Incubation was performed at 35±2 ºC for 24 hours with tubes inclined at 30º.The reading was performed by visual observation of the adherence of the bacteria to the walls of the tube, after shaking it.The assay was performed in duplicate.The same procedure was performed for the positive control, 0.12% chlorhexidine digluconate (Periogard®, Colgate-Palmolive Company, New York, USA).The MIAC was considered the lowest concentration of the agent in contact with sucrose that prevented adherence to the glass tube.

Study of the association of oregano essential oil with synthetic antimicrobial agents
For the association study of oregano essential oil with antimicrobials, it was carried out by means of the disc diffusion technique in solid medium using filter paper discs (Ampicillin-AMP, Gentamicin-GEN, Ciprofloxacin-CIP and Ceftriaxone-CEF).In smooth sterile Petri plates containing Muller Hinton agar medium previously inoculated with the bacteria suspension, discs containing antimicrobials were introduced and 20μL of the MIC of the oregano essential oil were added, then the plates were incubated at 35±2 ºC for 24-48h, followed by their reading.It was considered as synergistic effect, when the microbial growth inhibition halo formed by the association (oil + antimicrobial) had a diameter ≥ than 2mm, when compared to the inhibition halo formed by the action of the isolated antimicrobial, When the formation of the halo of inhibition resulting from the association had a diameter smaller than the one developed by the isolated action of the antimicrobial, it was considered an antagonistic effect, and when the halo of inhibition resulting from the association had a diameter equal to the one resulting from the isolated application of the antimicrobial, it was considered an indifferent effect (Cleeland and Squires, 1991).All tests were performed in duplicate.

Results and Discussion
Infectious diseases are the second leading cause of death in the world, accounting for approximately 15 million deaths each year.This high mortality rate is mainly due to antibiotic resistance.For this reason, natural products have been evaluated as a source of new pharmacological molecules with antimicrobial potential (Aelenei et al., 2016).
In this sense, several essential oils have been evaluated and, in this research, the oregano essential oil was analyzed for its chemical constituents through the retention time presented in each peak of the spectrum, as can be seen in Table 1.
As a result, we identified a total of 9 constituents present in oregano essential oil: alpha-pinene, camphene, myrcene, carene, cymene, gamma-terpinene, linalool, carvacrol and caryophyllene.And from the analysis of the percentages, we verified that carvacrol presents itself as the majority compound, with a high percentage (40.52%).
Corroborating what was described by Penteado et al. (2021), who, in their analyses, found carvacrol also as one of the major components of the oregano essential oil, with a percentage of 69.1%, being this oil also evaluated by gas chromatography coupled to mass spectrometry (GC-MS).Similarly, Gomes et al. (2019), using gas chromatography coupled to mass spectrometry when analyzing the chemical composition of OEO obtained a percentage of 45.74% of carvacrol, and further noted that this is the majority compound of the oil.These variations in percentages are explained from environmental conditions such as altitude and water availability, season, phenological stage of the plant, the type of extraction, among other factors (Lombrea et al., 2020).
The carvacrol as a component of the OEO presents itself as the main compound responsible for the antibacterial activity (Oliveira et al., 2008).Ultee et al. (2002) describes that the carvacrol together with the thymol (present in thyme essential oil), are able to disintegrate the external membrane of gram-negative bacteria.And, more specifically, carvacrol is responsible for depleting the intracellular ATP (adenosine triphosphate) reserve and increasing the permeability of the cytoplasmic membrane for the entry of cations, altering essential cellular processes, resulting in bacterial death (Magi et al., 2015).
Therefore, in the present research, the Minimum Inhibitory Concentration (MIC) of oregano essential oil was evaluated, in liquid medium and at the different concentrations suggested in the methodology.Then, the MIC was determined by the lowest concentration capable of visibly inhibiting bacterial growth.
As shown in Table 2, it was observed that the MIC results for oregano essential oil was 128 μg/mL for three of the six strains of Klebsiella pneumoniae tested.Thus, a MIC50 (lowest concentration capable of inhibiting growth by 50%) of 128μg/mL was considered.
According to Sartoratto et al. (2004), the antimicrobial activity can be classified as strong when it has MIC up to 500μg/mL, moderate for MIC from 600μg/mL to 1500μg/ mL and weak for MIC above 1500μg/mL.Therefore, the present study, has oregano essential oil showing strong antimicrobial activity on Klebsiella pneumoniae strains.
An additional analysis refers to the bactericidal or bacteriostatic characteristic of the tested product.For this, it is necessary to determine the Minimum Bactericidal Concentration (MBC) of oregano EO on Klebsiella pneumoniae strains.Thus, the MBC was determined from the lowest concentration of the oil that resulted in visible inhibition of the growth of the microorganism.According to Table 3, it is observed that the values obtained were the same for some strains and higher for others in relation to the MIC.
Finally, for determination of bacteriostatic or bactericidal activity, the ratio of MBC and MIC is then analyzed.For, according to Hafidh et al. (2011), the compound is considered bactericidal when this ratio is between 1: 1 to 2: 1, and the compound is considered as bacteriostatic when the ratio is greater than 2:1.Therefore, in the present study, oregano EO has bactericidal activity, with MBC ranging between 256 μg/mL and 512 μg/mL and MIC between 128 μg/mL and 256 μg/mL against K. pneumoniae strains.
Corroborating this data, it is possible to observe in the literature, studies such as that of Kozics et al. (2019), in which they obtained that the essential oil of oregano showed an MIC of 0.05% m/v and MBC of 0.05%m/v on K. pneumoniae KMB522 bacterial strains, through the microwell dilution method.Thus, the authors considered oregano essential oil bactericidal, thus effective against K. pneumoniae.Kosakowska et al. (2021), on the other hand, evaluated the essential oil of Oregano vulgare (Greek) on several gram-positive and gram-negative bacterial strains, by the microdilution method.And, they obtained an MIC ranging between 2-8 mg × mL-1 and MBC between 4-16 mg × mL-1 showing the two strains of E. coli ATCC 25922 and micro-well.coli O157:H7ATCC 700728, both gram negative, as the most susceptible strains among the tested strains.That said, they concluded that Oregano vulgare (Greek) showed bacteriostatic and bactericidal activity.
From the point of view of the compounds responsible for the main therapeutic activities identified in the essential oil of oregano, it is observed as more representative the carvacrol and thymol (Bora et al., 2022).Thus, it is verified in the study of Guarda et al. (2011), that the antimicrobial capacity of the microencapsulated carvacrol and thymol was 0.225mg/mL and 0.250 mg/mL respectively, against the microorganisms of Escherichia coli, Staphylococcus aureus, Listeria innocua, Saccharomyces cerevisiae and Aspergillus niger.
In another study, analyzing the activity of oregano essential oil, with carvacrol as the main constituent, on bacteria, fungi and yeast, and by the well microdilution method, it was obtained that bacteria were the most affected, and against these bacterial strains, an MIC between 31.25-128μl/mL was identified (Jan et al., 2020).
And also, in the study by Fratini et al. (2017), when analyzing the minimum inhibitory concentration of oregano essential oil, it was identified that its main constituent was carvacrol (65.93%), and it was obtained from the microdilution assay a MIC ≤ 0.240 mg/mL for eight strains of S. aureus and MIC ≤ 0.480 mg/mL for another five strains also of S. aureus.
Considering the anti-adherent activity, in the present study, it was observed that both oregano essential oil and chlorhexidine digluconate 0.12% failed to inhibit biofilm formation at the tested proportions, as shown in Table 4.
And reinforcing this result, Zapién-Chavarría et al. ( 2019), when analyzing the susceptibility of uropathogenic strains (biofilm-forming and resistant to multiple conventional drugs), to oregano essential oil of Mexican origin, through the glass tube and microplate tests, found bactericidal activity, and absence of bacteriostatic activity.
However, Lee et al. (2017), analyzed the antibiofilm activity of oregano and red thyme essential oil on uropathogenic Escherichia coli strains by confocal laser scanning microscopy.Then, they obtained that the two oils inhibited biofilm formation in more than 60% of the evaluated strains and associated this effect with inhibition of fimbria production and swimming motility.2019), aiming to determine the bioactive compounds of the EOs of Origanum heracleoticum, Origanum vulgare, Thymus vulgaris and Thymus serpyllum, and evaluate their antimicrobial and antibiofilm activity against Salmonella Enteritidis performed the biomass biofilm formation assay and incubated with the EOs and the bioactive compounds of the EOs.To this end, it obtained for Salmonella Enteritidis biofilms, the EO of O. heracleoticum showed the highest effect, followed by O. vulgare, T. vulgaris, carvacrol and thymol, while the EO of T. serpyllum showed the weakest effect regarding prevention of biofilm growth and eradication of pre-formed biofilms.
Moreover, taking into account the bacterial strain, Ramalho et al. (2020), used strains of Klebsiella pneumoniae (kp 102), grown in Mueller Hinton broth in test tubes in order to evaluate the Minimum Inhibitory Concentration of Adherence (MIAC) of the essential oils of Eucalyptus globulus and Eucalyptus citriodora, and as a positive control was employed chlorhexidine digluconate at 0.12%.Thus, when analyzing the results of the anti-adherent activity of the EOs, it was obtained that the EO of Eucalyptus globulus showed good efficiency against biofilm formation by Klebsiella pneumoniae showing results equivalent to the control chlorhexidine digluconate 0.12%, and inhibiting biofilm formation at the same concentration 1:8.However, the analysis of the Eucalyptus citriodora EO showed biofilm formation to the tube walls, so it did not show inhibition against Klebsiella pneumoniae at any concentration.
And Souza et al. (2021), also using strains of Klebsiella pneumoniae (kp 102) grown in Mueller Hinton broth in test tubes, evaluated the anti-adherent activity of the essential oil of Lavandula hybrid Grosso, having as positive control the chlorhexidine digluconate at 0.12%.Thus, it was possible to observe that the EO showed a better property than the 0.12% chlorhexidine digluconate, because it presented a lower concentration capable of inhibiting biofilm adherence to the tube, being 1:32 for Lavandula hybrid Grosso essential oil and 1:8 for 0.12% chlorhexidine digluconate.Then, they concluded that EO can be used as an alternative method for biofilm control in the presence of K. pneumoniae.
Finally, Rajasekharan et al. (2017) investigated the anti-adherent potential of Arctium lappa Linn (burdock root) extract and one of its components, chlorogenic acid, on clinical isolates of Klebsiella pneumoniae.Burdock root extract (BRE) (20-200 μg/ml) or chlorogenic acid (CGA) (0.028-0.28 mmol/l) were added to NB medium supplemented with 0.5% glucose and inoculated with 1% bacterial inoculum, and biofilm growth was quantified by biofilm biomass formation assay (crystal violet).Thus, as a result, they obtained that both the extract and the acid, showed significant anti-adherent activity against K. pneumoniae strains.
Regarding the association of oregano essential oil with the tested antimicrobials, AMP showed synergistic (50%) and indifferent (50%) effect, synergistic (33%) and indifferent (67%) effect with GEN, and found indifferent (67%), synergistic (16.5%) and antagonistic (16.5%) effect in the presence of CEF.With CIP, an indifferent (50%), synergistic (16.7%), and antagonistic (33.3%) effect was observed.This result was achieved from the comparative observation of the halo of inhibition in the presence of antibiotics, and the halo of inhibition in the presence of the association of antimicrobials with oregano essential oil.This result can be seen in Table 5.
In this case, it is verified in literature researches such as Rosato et al. (2020), in which they evaluated the essential oils of Cinnammonum zeylanicum, Mentha piperita, Origanum vulgare, and Thymus vulgaris, regarding their anti-biofilm effect and synergism with common antibiotics such as gentamicin, norfloxacin and oxacillin.Then, the association showed a strong destruction of the bacterial biofilm and synergism of action between the EOs and the evaluated antibiotics.Being the interaction of norfloxacin with EOs the most effective among all tested combinations of EO and antibiotic against the strains of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Staphylococcus epidermidis IG4, and Staphylococcus aureus IG22.Corroborating with the present research, from which we can observe that oregano oil showed synergistic effect with ampicillin and gentamicin.
While, in the association study by Uzair et al. (2017), essential oils with the potential to inhibit the in vitro growth of methicillin-resistant Staphylococcus aureus (MRSA), isolated and in association with amoxicillin, an antibiotic of the penicillin class, were investigated.To carry out the study, the disk diffusion method was applied to evaluate the activity of ten essential oils extracted from traditional plants: Thymus vulgaris L, Mentha pulegium, Ocimum sanctum, Mentha piperita, Cymbopogon citratus, Rosmarinus officinalis L., Cortex cinnamom, Citrus nobilis, Citrus deliciosa, Origanum vulgare and Mentha sp.As results, they observed that the essential oils have both bacteriostatic and bactericidal activity higher than that of synthetic antibiotics when tested in vitro, with higher activity for thyme and cinnamon oil.And oregano and mint (Mentha pulegium) showed strong synergistic activity with amoxicillin.The authors attribute this synergism of oregano due to the high carvacrol content in its composition.And Magi et al. (2015), examined the antibacterial activity of the essential oils of Origanum vulgare, Thymus vulgaris, Lavandula angustifolia, Mentha piperita and Melaleuca alternifolia, and of carvacrol, against 32 erythromycin-resistant and cell-invasive strains of Group A Streptococci (GAS), in isolation, and in association with erythromycin.Overall, they observed that thyme and oregano essential oils demonstrated the greatest antimicrobial activity.And, through quad assays, they obtained a 2 to 2,048-fold reduction in the MIC of erythromycin, with confirmed synergy for carvacrol and erythromycin.Santana et al. (2020), in order to evaluate the antibacterial activity of the essential oil of Lavandula hybrid Grosso, associated with the antimicrobial ampicillin against strains of S. aureus, used the microdilution broth technique to obtain the MIC and then performed the association of essential oil with the antimicrobial ampicillin (10 μg/mL), in disc.As a conclusion, they obtained a MIC of 128 μg/mL of the EO for the strain under analysis and, as for the association test, a synergistic effect of ampicillin with the essential oil of Lavandula hybrid Grosso was observed.
In this scenario, it can be observed that essential oils in association with common antimicrobials, may represent alternative strategies in combating bacteria resistant to common isolated antimicrobials (Magi et al., 2015).

Conclusion
From the results obtained in this study we can conclude that the essential oil of Origanum vulgare L. presents itself as a candidate for the development of new pharmacological therapies for antimicrobial treatment, in order to properly manage pressure lesions infected by Klebsiella pneumoniae.Since, OEO showed bactericidal capacity and synergistic effect when used in association with some conventional antimicrobials on strains of this bacterium.However, further studies are needed to confirm and elucidate the mechanisms and patterns of efficiency and efficacy, as well as to develop a suitable pharmaceutical form for the mentioned application.

Table 1 .
Chemical composition of oregano essential oil.

Table 2 .
Minimum inhibitory concentration (MIC) of oregano essential oil against different strains of Klebsiella pneumoniae.

Table 3 .
Minimum bactericidal concentration (MBC) of oregano essential oil against different strains of Klebsiella pneumoniae.

Table 5 .
Study of the association of oregano essential oil with synthetic antimicrobials.: halo of inhibition in the presence of the antibiotic; OEO: oregano essential oil.Source: research data (2022).