Antibacterial activity of Thymus vulgaris (thyme) essential oil against strains of Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus saprophyticus isolated from meat product

Abstract Meat products represent an important component of the human diet and are a good source of nutrients. Food-borne microorganisms are the main pathogens that cause human diseases as a result of food consumption, especially products of animal origin. The objective of the present research was to verify the antibacterial activity of the essential oil of Thymus vulgaris against strains of Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus saprophyticus isolated from meat products. For this, the analyses of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were performed in microdilution plates. The association of the product with antimicrobials was also studied using disk diffusion. And the anti-adherent activity, which was determined in the presence of sucrose, in glass tubes. Thyme oil showed a strong inhibitory activity against K. pneumoniae, P. aeruginosa and S. saprophyticus, with the MIC values ranging from 64 to 512 μg/mL, and bactericidal effect for most strains, with MBC values ranging from 256 to 1,024 μg/mL. T. vulgaris oil exhibited varied interactions in association with the antimicrobials, with synergistic (41.67%), indifferent (50%) and antagonistic (8.33%) effects. Regarding the anti-adherent activity, the test product was effective in inhibiting the adherence of all bacterial strains under study. Therefore, thyme oil presents itself as an antibacterial and anti-adherent agent against K. pneumoniae, P. aeruginosa and S. saprophyticus, being a natural product that can represent an interesting alternative in the efforts to combat foodborne diseases.

pharmaceutical drugs as well as herbal remedies derived from medicinal plants (Li et al., 2020).
Essential oils are composed 'of a mixture of terpenes, terpenoids, phenylpropanoids, and various low molecular weight compounds (Wińska et al., 2019).They are isolated from leaves, bark, flowers, shoots, seeds, roots, stems, and fruits of different aromatic plants (Maurya et al., 2021), having their bioactive components of volatile nature, which provide a well-built odor and have the ability to release aroma or flavor (Smith et al., 2005).They are isolated from different plants, including those in the families Asteraceae, Lamiaceae, Cyperaceae, Zingerberaceae, Piperaceae, Apiaceae, Myrtaceae, Solanaceae, Apocynaceae, and Lauraceae (Nuzhat and Vidyasagar, 2013).
Thymus vulgaris is one of the most important species of the Lamiaceae family, native to the Mediterranean region and used worldwide as a seasoning in cooking and in liquors, in addition to therapeutic use (Morales, 2002).The main active agent of thyme herb is the volatile oil, which has several constituents (such as thymol, γ-terpinene, p-cymene, carvacrol and linalool) (György et al., 2020).
Thus, due to the antibacterial activity verified in thyme spice against some species (Millezi et al., 2012;Fournomiti et al., 2015;Miladi et al., 2016;Ozogul et al., 2020), in addition to the increasing consumer demand for effective, safe and natural products (Hammer et al., 1999), quantitative data about the activity of the essential oil of this vegetable becomes urgent.Based on this, the present study tested the antibacterial and anti-adherent potential of T. vulgaris essential oil against Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus saprophyticus strains.

Obtaining the essential oil
The Thymus vulgaris (thyme) essential oil used was from the brand QUINARÍ® (Ponta Grossa -Paraná).It was solubilized in the presence of Tween 80 and DMSO, and diluted in distilled water (Allegrini et al., 1973).

Microorganisms
We used strains of Klebsiella pneumoniae (Kp 42 and Kp 44), Pseudomonas aeruginosa (Pa 43 and Pa 44) and Staphylococcus saprophyticus (Sa 41 and Sa 45), isolated from meat products from the Microbiology Laboratory of the Central Laboratory of the UACB -UFCG.
These strains were preserved in Muller-Hinton Agar (MHAG) and glycerin at 4ºC.
The inocula were obtained from these overnight cultures in AMH at 37 ºC and diluted in sterile 0.9% saline solution to obtain a final concentration of approximately 1.5 x 108 colony forming units per mL (CFU/mL), adjusted by turbidity compared to a suspension of barium sulfate and sulfuric acid in tube # 0.5 of the McFarland scale (Bauer et al., 1966;Cleeland and Squires, 1991).
The laboratory tests were performed in the Microbiology Laboratory of the Central Laboratory of the Academic Unit

Introduction
Meat products represent an important component of the human diet, and their consumption has registered a global increase in recent years (Ursachi et al., 2020).These foods are a good source of essential nutrients, high quality protein, fat and minerals (Aminzare et al., 2019).Safety and quality assessment of animal source foods are necessary in order to reduce losses, mitigate health risks, and ensure consumer safety (Ahmed et al., 2018).
Foodborne microorganisms are major pathogens that affect food safety and cause human illness worldwide as a result of consumption of food, mainly, animal products, contaminated with vegetative pathogens or their toxins (Abebe et al., 2020).
Waterborne and foodborne diseases impede socioeconomic development, burdening health systems and damaging national economies, tourism, and trade (WHO, 2015).An estimated 600 million -nearly 1 in 10 people worldwide -fall ill after eating contaminated food and 420,000 die each year (WHO, 2015).
Of the biological hazards, bacterial pathogens are the most serious concern regarding meat safety issues for consumers (Zelalem et al., 2019).Among these pathogens, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus saprophyticus are species that have been found in meat products (Calbo et al., 2011;Charmpi et al., 2020;Silva et al., 2022).
The Klebsiella pneumoniae species is Gram-negative bacillary, belonging to the Enterobacteriaceae family (Li et al. 2023), and is an opportunistic pathogen capable of causing a variety of infections, such as pneumonia, urinary tract infections, and bacteremia (Choby et al., 2020).Pseudomonas aeruginosa is a Gram-negative bacillary species belonging to the family Pseudomonadaceae and is an important human pathogen that is often associated with hospital infections, affecting mainly immunosuppressed patients (Urganci et al., 2022).And, Staphylococcus saprophyticus is a coccoid-shaped, Gram-positive bacterium, belonging to the Staphylococcaceae family, found in the human microbiome, which causes hospital infections, and is especially important in young women and those of reproductive age (Oliveira et al., 2023).
Synthetic antimicrobials consist of one of the main drugs used to treat bacterial infections, however the overuse of antibiotics in animals and humans, antibiotics sold without prescription, increased international travel, lack of sanitation/hygiene, and release of unmetabolized antibiotics or their residues into the environment through manure/feces, has caused the emergence of multidrugresistant bacteria (Aslam et al., 2018).Currently, the introduction of a new antibiotic into the market is almost immediately followed by the emergence of resistant bacterial strains, which are responsible for infections that are more difficult to treat, requiring the use of more toxic and more expensive drugs (Serwecińska, 2020), making alternative routes in the treatment of bacterial diseases necessary, entering in this context the medicinal plants.
Plants are living chemical factories for the biosynthesis of a huge array of secondary metabolites and, in fact, it is these metabolites that form the basis of many commercial Brazilian Journal of Biology, 2023, vol.83, e275306 3/9 Antibacterial activity of thyme essential oil against strains bacterials isolated from meat product of Biological Sciences (UACB) of the Federal University of Campina Grande (UFCG) / Patos -PB.

Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration was determined using the microdilution technique in a 96-well plate with a U-shaped bottom.In a 96-well plate, 100 μl Mueller Hinton broth, doubly concentrated, and 100 μl of thyme essential oil were added at concentrations of 1024 to 16 μg/ ml.The determination of the MIC was conducted with 10 μl of the microorganism in each well, approximately 1.5 x 108 CFU/ml.The penultimate well containing 200 μl of the broth was inoculated with the microorganism suspension, being the positive growth control, and the last well received only 200 μl of the broth, being the negative control.The assay was performed in duplicate.The plates were incubated at 35 °C for 24 hours.After the proper incubation time of the assays with the bacteria, the first reading of the results was performed.Then, 20 μl of sodium resazurin solution (SIGMA) in sterile distilled water at a concentration of 0.01% (w/v), recognized as a colorimetric oxide-reduction indicator for bacteria, was added.The reading was done, visually, by the absence or presence of growth of the microorganism by the formation of a cluster of cells (button).And also by observing the change in color of the solution, from blue to pink, indicating growth.A new incubation was performed at 37 ºC.The MIC was determined as the lowest concentration of the essential oil that inhibited the visible growth of the microorganism and also by observing the change in the coloration of the solution, from blue to pink, indicating growth of the microorganism (Palomino et al., 2002;Ostrosky et al., 2008;CLSI, 2012;Bona et al., 2014).

Determination of the Minimum Bactericidal Concentration (MBC)
After reading the results, inoculations (10 μL) of three dilutions from the MIC were made into Mueller-Hinton broth medium (100 μL/cavity) in sterile microdilution plate for the determination of the MBC.After incubation at 37 °C for 24 hours, 20 μL of resazurin was added.The assays were incubated at 37 ºC for another 24 hours for confirmation of the concentration capable of inhibiting the total growth of the bacterial species, verified by a nonchange in the coloration of the indicator dye (Ncube et al., 2008;Guerra et al., 2012).

Association study of the product with antimicrobials
To study the association of the product with antimicrobials, the solid medium disk diffusion technique using filter paper disks was used (Bauer et al., 1966;Oliveira et al., 2006).An aliquot of 20 μL of the MIC of the test product was transferred to the discs containing the antimicrobials in their respective concentrations, and then placed in smooth sterile Petri dishes (140 x 15) containing the AMH medium, which were previously inoculated with sterile swabs, an approximate volume of 1 mL of the bacterial suspensions.Subsequently, the plates were incubated at 37 °C for 24-48 h, followed by reading (Oliveira et al., 2006;Koneman et al., 2008;Ostrosky et al., 2008).The interfering effect of the combination of the product plus antimicrobials, was evaluated according to the methodology described by Cleeland and Squires (1991).It was considered as synergistic effect, when the occurrence of halo of inhibition of microbial growth formed by the combined application of the essential oil (EO) plus the antimicrobial (AB) with diameter ≥ than 2mm, when compared with the halo of inhibition formed by the action of the AB alone.When the formation of a halo of inhibition resulting from the combined action of AB and OE had a smaller diameter than the one developed by the action of AB alone, it was considered an antagonistic effect.It was considered an indifferent effect when a halo of inhibition was observed as a consequence of the combined application of AB and OE with a diameter equal to that of the application of AB alone.

Determination of the Minimum Inhibitory Adherence Concentration (MIAC)
The Minimum Inhibitory Adherence Concentration (MIAC) of the compound was determined in the presence of 5% sucrose, according to Albuquerque et al. (2010), using concentrations corresponding to the compound up to 1:1024 dilution.From the bacterial growth, the bacterial strain was grown at 37 ºC in Mueller Hinton broth, then 0.9 mL of the subculture was dispensed into test tubes and then 0.1 mL of the solution corresponding to the compound dilutions was added.Incubation was done at 37 ºC for 24 hours with tubes tilted at 30º.The reading was done 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.

Results
The results of antibacterial activity of T. vulgaris essential oil with MIC and MBC values are shown in Table 1 and the activity was measured in terms of the presence of microorganism growth.
By analyzing Table 1, it is observed that thyme essential oil shows MIC values between 64 μg/mL and 512 μg/mL against the growth of different strains of Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus saprophyticus.Thus, the oil shows strong inhibitory activity.It also shows bactericidal activity for most of the strains, where the MBC ranges between 256 μg/mL and 1,024 μg/mL.
Table 2 shows the interference of thyme essential oil on the antibacterial action of clinical use antimicrobials.Considering the comparison of the diameters of the halos of inhibition of bacterial growth in the assays with the antimicrobials alone and in association with the essential oils, it can be observed, in some interactions, the occurrence of interference of the essential oils on the antibacterial power of the antimicrobials.This interference occurs with modulations for synergism, indifference and antagonism, being respectively 41.67%, 50% and 8.33%.
Gentamicin and ceftadizime were the antimicrobials that had the greatest interference of the essential oils, being observed synergism in the three bacterial species under study, in at least one type of strain in each species.Ampicillin and ciprofloxacin showed indifferent effect in most associations.
Regarding the results of the minimum adherence inhibitory concentration, the essential oil of T. vulgaris was effective in inhibiting the adherence of the bacterial strains of K. pneumoniae, P. aeruginosa, and S. saprophyticus in the presence of sucrose.The minimum adherence inhibitory concentrations of this herbal agent are shown in Table 3.The antibacterial agent chlorhexidine digluconate 0.12%, also shows a significant inhibition in the adhesion effect against S. saprophyticus, while for K. pneumoniae and P. aeruginosa there is no inhibition.Antibacterial activity of thyme essential oil against strains bacterials isolated from meat product

Discussion
The results of the MIC indicate strong inhibitory activity of thyme essential oil, since, when the MIC is from 50 to 500 μg/mL it is considered that the test substance presents a strong activity; from 600 to 1,500 μg/mL a moderate activity, and, above 1,500 μg/mL a weak activity (Sartoratto et al., 2004).
According to Hafidh et al. (2011), a compound can be considered bactericidal or bacteriostatic and, for that, the ratio of MBC and MIC is analyzed, when this ratio is between 1:1 to 2:1, the compound is considered bactericidal and, to be considered as bacteriostatic the ratio should be higher than 2:1.In this case, the oil shows bactericidal activity for most strains and bacteriostatic for Sa 45.
These results of strong inhibitory activity and bactericidal/bacteriostatic effect of thyme essential oil against strains of K. pneumoniae, P. aeruginosa and S. saprophyticus may be related to the high content of the compound p-cymene, which alone has inhibitory effects on microorganisms (Van Zyl et al., 2006), promoting a decrease in the viability of biofilms (Silva et al., 2012), besides causing damage to cell membranes, suggesting that it is the main mechanism of action of this compound (Andrews et al., 1980).Phenolic compounds, such as thymol, present in this oil, confer strong antibacterial action against many pathogenic bacteria (Sienkiewicz et al., 2012), presenting a mode of action that involves interference with cytoplasmic membrane functions, including proton motive forces and active transport (Eklund, 1985).
Most studies on the action of essential oils against food spoilage organisms and foodborne pathogens have observed that essential oils are relatively more active against Grampositive than Gram-negative bacteria (Lambert et al., 2001).In the present research, it was possible to verify this through the results of the S. saprophyticus Sa 45 strain, which has the minimum inhibitory concentration of 64 μg/mL and minimum bactericidal concentration of 256 μg/mL.
Research has revealed that thyme oil against Klebsiella pneumoniae also showed antibacterial activity.Using the disk diffusion technique, using standard bacteria against thyme essential oil, Hammer et al. (1999) observed a MIC of 0.25% (v/v) for K. pneumoniae.Ozogul and collaborators (2020), using standard strain (ATCC) of K. pneumoniae, through the agar disc diffusion method, obtained an average of the inhibition halos of 23.4 mm, and, through the minimum inhibitory concentration and the minimum bactericidal concentration, had a value of 3.13 mg/mL for both.
Through the well diffusion assay technique, Al-Dosary (2018), observed a zone of inhibition of 7 mm for standard K. pneumoniae (ATCC) and resistance for five strains of K. pneumoniae isolated from patients in a hospital in Saudi Arabia.Whereas, using the minimum inhibitory concentration method, a MIC of 0.75 μg/mL was observed for the standard strain (ATCC) and, again, resistance for all five strains of K. pneumoniae (Al-Dosary, 2018).Fournomiti et al. (2015), in a research conducted with strains isolated from patients in a hospital in Greece and with standard strain (NCTC) of K. pneumoniae, using the broth microdilution method, observed the mean MIC value of 9.5 μg/mL for thyme oil.Using microwell dilution method, Al-Bayati (2008) observed that thyme essential oil proved active against K. pneumoniae obtained from Department of Biology, College of Science (University of Mosul, Iraq), with MIC value of 500 μg/mL.Damjanović-Vratnica et al. (2015), using K. pneumoniae strains isolated from clinically treated or hospitalized patients in a Medical Health Center (Podgorica), using the agar disc diffusion method, recorded a 38 mm diameter of the halo of inhibition at the lowest concentration of the oil (4.5 μg).
Other studies performed with thyme oil against Pseudomonas aeruginosa also showed effective antibacterial activity.Hammer and collaborators (1999), conducted a research using standard bacteria against thyme essential oil through the disk diffusion technique, and had as a result an MIC of > 2.0% (v/v).In another study, also with standard strains (ATCC), the minimum inhibitory concentration of the essential oil of T. vulgaris against P. aeruginosa was 5%, and at this concentration the formation of a halo of inhibition averaged 6.5 mm, and at the highest concentration (50%), the average halo was 8.83 mm (Millezi et al., 2012).Galovičová et al. (2021), using standard strain of P. aeruginosa, through the disk diffusion method, obtained moderate antibacterial activity (mean zone of inhibition of 10.67 mm), while for MIC and MBC, the values were 103.28 μL/mL and 169.19 μL/mL, respectively.
Only, Al-Bayati (2008), using microwell dilution method, observed that thyme essential oil was not active against P. aeruginosa obtained from Department of Biology, College of Science (University of Mosul, Iraq).Damjanović-Vratnica et al. (2015), using P. aeruginosa strains of reference (ATCC) and isolated from clinically treated or hospitalized patients in a Medical Health Center (Podgorica), using the disc diffusion method on agar, recorded a diameter of 18 mm and 14 mm of the halo of inhibition at the oil concentration of 9 μg, respectively, while at the lowest concentration (4.5 μg) of the oil, there was resistance of both strains.
The essential oil of T. vulgaris was tested against two standard Staphylococcus aureus strains in combination with the antimicrobial clindamycin and showed a potent inhibitory activity against both strains with MIC ranges between 125-500 μg ml -1.The synergistic activity was confirmed, suggesting that the use of an antimicrobialplant combination may be a successful technique to reduce antimicrobial consumption, which would overcome antimicrobial resistance or delay its onset (Mayyas et al., 2021), corroborating the data of the present research regarding gentamicin and ceftadizime, which showed synergistic effect when associated with thyme oil.
Regarding the data of the anti-adherence activity of the oil in the present research, it is observed that the thyme essential oil was effective in inhibiting the adherence of all strains, presenting a better result for P. aeruginosa.Al-Shuneigat et al. (2014), also obtained good results regarding this species using the essential oil of T. vulgaris, which was able to inhibit P. aeruginosa strains adhering to polystyrene surface at a subinhibitory level, where a very small amount was sufficient to eliminate biofilms and planktonic cells.
Previous research has suggested that Gram-positive bacteria are more susceptible to essential oils than Gramnegative bacteria (Snoussi et al., 2018;Bordoni et al., 2019).However, there were no differences between Gram-positive and Gram-negative bacteria with respect to the anti-adherent effect.
Other studies have also observed the anti-stick effect of thyme oil against various bacteria.Jafri et al. ( 2014), observed that the essential oil of T. vulgaris acts effectively inhibiting biofilm formation in Staphylococcus aureus strains.Miladi and collaborators (2016), reported that thyme essential oil acts against Salmonella typhimurium, through the XTT [2, 3-bis (2-methyloxy 4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide] reduction assay method, in which they visualized inhibitory effect on biofilm formation of reference and food isolated strains.Kryvtsova et al. (2019) found that T. vulgaris essential oil exhibits high antibiofilm activity against clinical strains of S. aureus.Alibi et al. (2020) demonstrated that thyme essential oil reduced biofilm formation on biomaterial surfaces against clinical strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Citrobacter freundii, Klebsiella oxytoca, Salmonella enteridis, Salmonella typhimurium, Salmonella zanzibar, Salmonella livingstone, Salmonella derby, Salmonella heidelberg, Corynebacterium striatum and Staphylococcus aureus.Oliveira et al. (2021) also detected antibiofilm effect of T. vulgaris against the standard Streptococcus mutans strain in the initial biofilm formation of the salivary microcosm.
Bacteria in the biofilm are known to be much more resistant to antimicrobial agents than free-living cells (Lebert et al., 2007) and are medically important due to their implication in the pathogenesis of numerous bacterial infections that are difficult to eradicate with antimicrobials (Snoussi et al., 2018).In this regard, phytochemicals with antibiofilm properties may increase the efficacy of antimicrobials, allowing to reduce their use (Langeveld et al., 2014).

Conclusion
Thyme oil showed antibacterial potential, with a strong inhibitory activity against Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus saprophyticus, and bactericidal for most strains tested.It proved to be an alternative route in the search for compounds that potentiate the action of antimicrobial drugs, because it showed synergistic effect when combined with ampicillin, gentamicin, ceftadizime, and ciprofloxacin.In addition to presenting an anti-adherent effect against the tested strains.Therefore, the T. vulgaris oil presents itself as a natural product that can represent an interesting alternative in the efforts to combat foodborne diseases.

Table 3 .
Minimum adhesion inhibitory concentration of Thymus vulgaris and chlorhexidine against K. pneumoniae, P. aeruginosa and S. saprophyticus bacteria.

Table 1 .
Antibacterial activity of Thymus vulgaris essential oil against K. pneumoniae, P. aeruginosa and S. saprophyticus isolated from meat products.

Table 2 .
InterferenceLegend: HI: diameter of the growth inhibition zone determined by the antimicrobial alone.HITHY: diameter of the growth inhibition zone determined by the association of the antimicrobial and Thymus vulgaris oil.↑ = Synergistic effect.↓ = Antagonistic effect.* = Indifferent effect.
of Thymus vulgaris essential oil in association with clinical antimicrobials against strains of K. pneumoniae, P. aeruginosa and S. saprophyticus.Average of duplicates.Brazilian Journal of Biology, 2023, vol.83, e275306 5/9