Słupskie Prace Biologiczne

Nr 15 ss. 59-78 2018

ISSN 1734-0926 Przyjęto: 16.10.2018 © Instytut Biologii i Ochrony Środowiska Akademii Pomorskiej w Słupsku Zaakceptowano: 10.02.2018

THE ANTIBACTERIAL ACTIVITIES OF ETHANOLIC EXTRACTS OBTAINED FROM LEAVES OF SOME THYMUS (LAMIACEAE) REPRESENTATIVES AGAINST β-LACTAMASE PRODUCING PSEUDOMONAS AERUGINOSA STRAIN

Vitaliy Honcharenko 1 Halyna Tkachenko 2 Zbigniew Osadowski 2 Viktor Nachychko 1 Andriy Prokopiv 1 1Ivan Franko National University of Lviv Hrushevsky Str. 4, 79-005 Lviv, Ukraine e-mail: [email protected] 2Department of Zoology and Animal Physiology Institute of Biology and Environmental Protection Pomeranian University in Słupsk Arciszewskiego Str. 22B, 76-200 Słupsk, Poland e-mail: [email protected], [email protected]

ABSTRACT

The Thymus is one of the most widely used genera in folk medicine, where it is popular for its stimulatory action on all organism functions. Many species of this genus are used in the traditional medicine as tonics, carminatives, antitussives, aromatic, expectorant, stomachic, antispasmodic, bronchospasmolytic, diuretic, sedative, diaphoretic, and antiseptics, as well as anti-inflammatory, antioxidant, anthelmintic, hepatoprotective and antitumor agents. Moreover, some of the plants of the genus Thymus were previously reported for their antimicrobial activities. Therefore, the aim of this study was to evaluate the antimicrobial effects of five ethanolic extracts obtained from leaves of some Thymus representatives ( Thymus serpyllum L. emend. Mill., Th. pannonicus All., Th. × porcii Borbás, Th. pulegioides L., Th. alpestris Tausch ex A. Kern.) against β-lactamase producing Pseudomonas aeruginosa strain. Freshly leaves were washed, weighted, crushed, and homogenized in 96% ethanol (in proportion 1:19) at room temperature. The extracts were then filtered and investigated for their antimicrobial activity. Antimicrobial activity was determined using the agar disk dif-

59 fusion assay. The ethano-lic extracts obtained from leaves of Thymus plants showed different antibacterial activities against β-lactamase producing P. aeruginosa strain. The effects varied significantly according to the Thymus taxa. It should be noted that the most antimicrobial effective plant against β-lactamase producing P. aeruginosa was Th. alpestris , being highly active with the ethanolic extract (mean diameter of inhibition zone was 12.8±0.8 mm). The antibacterial activity of extracts was greatest for Th. alpestris followed by Th. pannonicus followed by Th. serpyllum and then by Th. pulegioides . The antimicrobial activity of the crude ethanolic extracts obtained from leaves of Thymus plants may be attributed to a specific compound or a combination of compounds. The knowledge about the chemical profile of the extract helps in explaining the observed activity and designing experiments for activity fractionation for isolation of the active principle. The identification of precise molecular mechanism addressing how these extracts inhibit bacterial growth needs to be explored. The present study lays the basis for future research, to validate the possible use of Thymus species as a candidate in the treatment of infections caused by P. aeruginosa .

Key words: Thymus , β-lactamase producing Pseudomonas aeruginosa strain, leaves, ethanolic extract, antimicrobial activity, agar disk diffusion technique

INTRODUCTION

Pseudomonas aeruginosa is an important bacterial pathogen, particularly as a cause of infections in hospitalized patients, in patients with burn trauma, diffused pan-bronchitis, chronic obstructive pulmonary disease, cystic fibrosis and with im- mune defects (Livermore 2002). P. aeruginosa also remains one of the major causes of nosocomial infections (Rossi Gonçalves et al. 2017). The multivariable analysis presented in a study by Rossi Gonçalves and co-workers (2017) showed that mechanical ventilation, enteral/nasogastric tubes, primary bacteremia with unknown focus, and in- appropriate therapy were independent risk factors associated with bacteremia. These bacteria possess a diversity of resistance mechanisms that may lead to multidrug or even pan-drug resistance (Potron et al. 2015). During the past few decades, multidrug-resistant and ex- tensively drug-resistant lineages of P. aeruginosa have emerged in hospital settings with increasing numbers, including carbapenem resistance and multidrug resistance (Kaiser et al. 2017). Extended-spectrum β-lactamases conferring resistance to broad- spectrum cephalosporins, carbapenemases conferring resistance to carbapenems, and 16S rRNA methylases conferring resistance to all clinically relevant aminoglycosides are the most important causes of concern. Concomitant resistance to fluoroquinolones, polymyxins (colistin) and tigecycline may lead to pan-drug resistance (Potron et al. 2015). The pathogenesis of P. aeruginosa is associated closely with the production of a myriad of extracellular virulence factors and the formation of biofilm (Davies et al. 1998). Several mechanisms are involved in P. aeruginosa resistance to antimicrobial agents, such as chromosomal expression of resistance encoding genes, β-lactamase

60 production, efflux pumps and a decrease in membrane permeability (Doosti et al. 2013). One of the mechanisms of resistance to carbapenem antibiotics in P. aeruginosa is Metallo-β-lactamases (MBL) production that hydrolyzes all carbapenems. The preva-lence of carbapenem resistance mediated by acquired MBL including imipenem (IPM) and Verona integron-encoded Metallo-β-lactamase (VIM), are increasing from different parts of the world (Lepsanovic et al. 2008; Doosti et al. 2013). Medicinal plants are an important resource of bioactive substances, and in the last decade a huge number of works have been dedicated all over the world to the assessment of the antimicrobial properties of plants, providing the possibility of obtaining molecules that could be used as a natural antiseptics and antimicrobial agents in medicine (Hemaiswarya et al. 2008). The emergence of multiresistant strains of microorganisms reinforces the need to search for new compounds able to overcome resistant b a c t e r i a . Antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world. In an effort to expand the spectrum of antibacterial agents from natural resources, genus Thymus L. belonging to Lamiaceae Martinov family has been selected, because, among plant-based antimicrobials, the antimicrobial activity of Thymus species has been well studied. The genus Thymus consists approximately 215 species currently recognized (Morales 2002). These herbaceous perennials and sub-shrubs distributed in Europe, Northwest Africa, Ethiopia, Asia and Greenland (Morales 2002; Bartolucci et al. 2013). It is one of the most widely used genera in folk medicine, where it is popular for its stimulatory action on all organism functions (Viuda-Martos et al. 2011). Many species of this genus are used in traditional medicine as tonics, carminatives, antitussives, aromatic, expectorant, stomachic, antispasmodic, bronchospasmolytic, diuretic, sedative, diaphoretic, and antiseptics, as well as anti-inflammatory, antioxidant, anthelmintic, hepatoprotective and antitumor agents (Khan and Abourashed 2010; Na- bavi et al. 2015). Internally, thyme is used for treatment of acute bronchitis, laryngitis, whooping cough, chronic gastritis, diarrhea, and lack of appetite, while externally in baths to treat rheumatic and skin problems (bruises, sprains, fungal infections) as well as for minor arthritis, gum disease, tonsillitis, etc. (Khan and Abourashed 2010). Moreover, Thymus species have wide application nowadays in food as a culinary herb and food flavoring, as well as in cosmetics and pharmaceutics in toothpaste, soaps, detergents, creams, lotions, and perfumes. Thymus i.e. leaves of Th. vulgaris L. and Th. zygis L. is used as a spice in several foods. Furthermore, Thymus species also have antimicrobial activities against a wide range of Gram-positive and Gram- negative bacteria, yeast, and fungi (Marino et al. 1999; Karaman et al. 2001; Rota et al. 2008; Xu et al. 2008; Palaniappan and Holley 2010; Mathela et al. 2010; Rivas et al. 2010; Pemmaraju et al. 2013; Kavoosi et al. 2013; de Morais et al. 2014; Marchese et al. 2016). These versatile pharmacological effects can be attributed to the secondary plant metabolites, especially to essential oil and polyphenols. Plants from the genus Thymus are rich in different active substances such as thymol, carvacrol, p-cymene, and terpinene (Nabavi et al. 2015). Thymol, and its main natural source, thyme ( Th. vulgaris ), are employed for their positive antioxidant, anti-inflammatory, local anesthetic, antinociceptive, cicatrizing, antiseptic, antibacterial, and antifungal properties as well as for their beneficial ef-

61 fects on the cardiovascular system (Marchese et al. 2016). Xu and co-workers (2008) have revealed that thymol (200 mg/ml) inhibited the growth of Escherichia coli by inducing the permeabilization and depolarization of the cytoplasmic membrane. A report from Palaniappan and Holley (2010) revealed that thymol at 2.5 mM inhibits the growth of Staphylococcus aureus , E. coli and Salmonella typhimurium . Thymol and carvacrol were found to be highly effective in reducing the resistance of S. typhimurium SGI 1 (tet A) to ampicillin, tetracycline, penicillin, bacitracin, erythromycin and novobiocin and resistance of Streptococcus pyogenes ermB to erythromycin. With E. coli N00 666, thymol and cinnamaldehyde were found to have a similar effect in reducing the minimum inhibition concentrations (MIC’s) of ampicillin, tetracycline, penicillin, erythromycin, and novobiocin. Carvacrol, thymol, and cinnamaldehyde were effective against S. aureus blaZ and in reducing the MIC’s of ampicillin, penicillin, and bacitracin (Palaniappan and Holley 2010). The antibacterial activity of fourteen esters of thymol and carvacrol and their esters against four Gram-positive ( Streptococ- cus mutans MTCC 890, S. aureus MTCC 96, Bacillus subtilis MTCC 121, Staphylo- coccus epidermidis MTCC 435) and one Gram-negative ( E. coli MTCC 723) bacteria were screened by Mathela and co-workers (2010). These authors demonstrated that thymol ester derivatives were found to be more effective against Strepto- coccus species. Thymol was found to possess antibacterial activity against selected verocytotoxigenic E. coli strains and other bacterial species and spoilage bacteria (Rivas et al. 2010). The study of Pemmaraju and co-workers (2013) demonstrated the synergistic effect of terpenes (eugenol, menthol, and thymol) with fluconazole on Candida albicans biofilm, which could be future medications for biofilm infections. Thymol (0.12%) possess antifungal activity against C. albicans MTCC 227 biofilm inhibition (Pemmaraju et al. 2013). Gelatin films containing different thymol concentrations (1-8%) produced inhibitory zones ranging from 30 to 46 mm against several bacteria and could be used as a safe and effective source of natural antioxidant and antimicrobial agents with their potential use as modern nano-wound dressing against wounds burn patho- gens. Thymol was more effective against Gram-positive strains. The antibacterial activity of the films containing thymol was greatest against S. aureus followed by B. subtilis followed by E. coli and then by P. aeruginosa (Kavoosi et al. 2013). Thymol derivative named benzoyl-thymol was the best inhibitor against Leishmania infantum chagasi both in vitro and in vivo study, and acetyl-thymol was more active than thymol and the positive control drug amphotericin B (de Morais et al. 2014). A convincing number of studies that reveal that thymol alone or thymol in plants along with other metabolites possess potent antimicrobial, antifungal, antibacterial, and antiparasitic properties prompted us to verify antibacterial effects of four species and one interspecific hybrid of Thymus genus sampled in the western part of Ukraine against β-lactamase producing P. aeruginosa locally isolated. Considering the points highlighted above and based on previous results obtained in our laboratory, in the present study, we propose to evaluate the antimicrobial effects of five ethanolic extracts obtained from leaves of Thymus representatives against β- lactamase producing P. aeruginosa strain.

MATERIALS AND METHODS

Collection of Plant Materials. Samples were harvested in June-August, 2016. Leaves of Thymus serpyllum L. emend. Mill. were collected among grass on sandy soil in the edge of a pine forest (Baymaky village, Bilohirya district, Khmelnytsky region, Ukraine; N 50°03'58,9'', E 26°13'37,5'', 257 m a.s.l.). Leaves of Th. pannonicus All. were harvested among grass in the roadside between the two cultivated fields (Syvky village, Bilohirya district, Khmelnytsky region, Ukraine; N 50°02'09,6'', E 26°13'19,2'', 283 m a.s.l.). Leaves of Th. pulegioides L. were collected among grass nearby land parcels (Syvky village, Bilohirya district, Khmelnytsky region, Ukraine; N 50°02'02,8'', E 26°14'13,9'', 306 m a.s.l.). Leaves of Th.× porcii Borbás (a hybrid between Th. pannonicus and Th. pulegioides ) were sampled in the grass stand, on the side of the footpath of the race track (Medovoi Pechery Str., Lviv, Ukraine; N 49°49'15.1", E 24°05'12.5", 348 m a.s.l.). Leaves of Th. alpestris Tausch ex A. Kern. were harvested on the side of the road below the stream, in mountain valley Shumneska (Kvasy village, Rakhiv district, Zakarpattia region, Ukraine; N 48°09'32.3", E 24°21'26.4", 1259 m a.s.l.). Identification of these five taxa was made according to Nachychko (2014, 2015) and Nachychko and Honcharenko (2016). The voucher herbarium specimens of plants used in this study were deposited at the Herbarium of M.G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine (KW). Plant samples were thoroughly washed to remove all attached material and used to prepare ethanolic extracts. Preparation of Plant Extracts . Freshly leaves were washed, weighted, crushed, and homogenized in 96% ethanol (in proportion 1:19) at room temperature. The extracts were then filtered and investigated for their antimicrobial activity. Bacterial isolates. Clinical specimens submitted for routine culture and antibiotic susceptibility testing of hospitalized patients during the period of September to Octo- ber 2016, at the microbiology laboratory of the Koszalin Hospital were processed. P. aeru-ginosa was isolated and identified based on the conventional biochemical test according to Forbes and co-workers (2002). Confirmation of MBL production and antimicrobial susceptibility testing. All the isolates were tested for MBL production by the imipenem-EDTA disk diffusion test (Yong et al. 2002). Susceptibility testing of the isolates was performed by disk diffusion according to the Guidelines of Clinical and Laboratory Standard Institute (M100- S24 2014). The following antimicrobial agents were used: ceftazidime (30 µg), imipenem (10 µg), meropenem (10 µg), ciprofloxacin (5 µg), aztreonam (30 µg), amikacin (30 µg), gentamicin (10 µg), piperacillin (100 µg), piperacillin-tazobactam (100/10 µg) and cefepime (30 µg). MIC of imipenem, meropenem, ceftazidime, and colistin was determined by E-test strips (according to manufacturer’s instruction) and agar dilution method on all MBLs-producing isolates (the Guidelines of Clinical and Laboratory Standard Institute). P. aeruginosa ATCC 27853 was used as a quality control in the susceptibility testing.

63 Plant Extracts by the Disk Diffusion Method. Strain tested was plated on TSA medium (Tryptone Soya Agar) and incubated for 24 hr at 25°C. Then the suspension of microorganisms was suspended in sterile PBS and the turbidity adjusted equivalent to that of a 0.5 McFarland standard. Antimicrobial activity of extracts was evaluated by using agar well diffusion method (Bauer et al. 1966). Muller-Hinton agar plates were inoculated with 200 µl of standardized inoculum (10 8 CFU/mL) of the bacterium and spread with sterile swabs. Sterile filter paper discs impregnated by extract were applied over each of the culture plates, 15 min after bacteria suspension was placed. The antimicrobial susceptibility testing was done on Muller-Hinton agar by disc diffusion method (Kirby-Bauer disk diffusion susceptibility test protocol). A negative control disc impregnated by sterile ethanol was used in each experiment. After culturing bacteria on Mueller-Hinton agar, the disks were placed on the same plates and incubated for 24 hr at 37˚C. The assessment of antimicrobial activity was based on the measurement of the diameter of the inhibition zone formed around the disks. Statistical analysis. The diameters of the inhibition zones were measured in millimeters and compared with those of the control and standard susceptibility disks. Activity was evidenced by the presence of a zone of inhibition surrounding the well. Each test was repeated six times. All statistical calculation was performed on separate data from each species with STATISTICA 8.0 (StatSoft, Poland) (Zar 1999). The following zone diameter criteria were used to assign susceptibility or resistance of bacteria to the phytochemicals tested: Susceptible (S) ≥ 15 mm, Intermediate (I) = 11- 14 mm, and Resistant (R) ≤ 10 mm (Okoth et al. 2013).

RESULTS

Antimicrobial activity of various ethanolic extracts obtained from leaves of Thy- mus representatives against β-lactamase producing P. aeruginosa measured as inhibition zone diameter is shown in Figs 1 and 2. The present study has shown that ethanolic extracts obtained from leaves of Thymus plants inhibited intermediate activity against β-lactamase producing P. aeruginosa . The mean diameter of inhibition zone for Th. serpyllum was (11.3±0.3) mm, for Th. pannonicus – (11.5±0.5) mm, for Th. ×porcii – (9.8±0.5) mm, for Th. pulegioides – (11.2±0.7) mm, and for Th. alpestris – (12.8±0.8) mm (Figs 1 and 2).

Fig. 1. The mean of diameters of inhibition zone of various ethanolic extracts obtained from leaves of Thymus plants against β-lactamase producing Pseudomonas aeruginosa (n=6) Source: own research

Detailed data regarding the zones of inhibition by the various plant extracts were recorded and presented in Fig. 2.

A B

C D

E Fig. 2. Antimicrobial activity of various ethanolic extracts obtained from leaves of Th. serpyllum (A), Th. pannonicus (B), Th. × porcii (C), Th. pulegioides (D), Th. alpestris (E) against β-lactamase producing Pseudomonas aeruginosa measured as inhibition zone diameter Source: own research

DISCUSSION

The main aim of our study was to evaluate the antimicrobial effects of five ethanolic extracts obtained from leaves of Thymus representatives against β-lactamase producing P. aeruginosa strain. Ethanolic extracts of the plant materials decrease the growth of the strain studied. The effects varied significantly according to the Thymus taxa. It was observed in the present study that ethanolic extracts inhibited the growth of bacteria tested moderately. The highest antimicrobial effect was recorded for Th. alpestris (Figs 1 and 2E). It should be noted that the most antimicrobial effective plant against β-lactamase producing P. aeruginosa was Th. alpestris , being highly active with the ethanolic extract (mean diameter of inhibition zone was 12.8±0.8 mm). The antibacterial activity of extracts was greatest for Th. alpestris followed by Th. pannonicus (11.5±0.5) followed by Th. serpyllum (11.3±0.3 mm) and then by Th. pulegioides (11.2±0.7 mm) (Fig. 1). The antimicrobial activity of Thymus species has been well studied. Many in vitro studies have shown that thymol possesses antibacterial and antifungal properties. A study conducted by Marino and co-workers (1999) have evaluated the antibacterial activity of essential oils obtained from Th. vulgaris against both Gram-negative bacteria ( E. coli , E. coli O157:H7, Proteus mirabilis , P. vulgaris , S. typhimurium , Serratia marcescens , Yersinia enterocolitica , P. fluorescens , P. putida ) and Gram-positive ( Mi- crococcus spp., Sarcina flava , S. aureus , Bacillus licheniformis , B. thuringiensis , Lis- teria innocua ) using a bioimpedance method. The results showed that all the thyme essential oils had bacteriostatic activities against the microorganisms. The antibacterial and antifungal perspectives of the essential oil from aerial parts of Th. revolutus Čelak. were assessed by Karaman and co-workers (2001). The microbial strains ( Bacillus megaterium DSM 32, B. subtilis IMG 22, Bacillus cereus EÜ, E. coli DM, P. aeruginosa DSM 50071, S. aureus Cowan 1, Listeria monocytogenes A, Micrococcus luteus LA 2971, K. pneumoniae FMC 5, Mycobacterium

66 smegmatis RUT, Proteus vulgaris FMC 1 bacteria, and Torulopsis holmii FUC 42, Saccharomyces cerevisiae UGA 102, Candida tropicalis FUC 34, C. albicans CCM 314) were used in their study. Although, at low quantities (0.2-0.4l), the essential oil of Th. revo-lutus showed inhibition zones against many, but not all, bacteria and fungi, a strong antimicrobial and antifungal activity were observed at 1.6 l for the bacteria and fungi tested (Karaman et al. 2001). Antibacterial properties of essential oils of Th. pubescens Boiss. & Kotschy ex Če- lak. and Th. serpyllum collected at pre and flowering stages against E. coli , S. aureus , B. subtilis , K. pneumonia , P. aeroginosa were demonstrated in a study of Rasooli and Mirmostafa (2002). The oils were strongly bactericidal even at higher dilutions with the exception of P. aeruginosa , which showed a static reaction to the oils from Th. pubescens and resistance to the oils from Th. serpyllum . On the basis of the diameter of microbial inhibition zones, oils from Th. pubescens and Th. serpyllum at the MBC dilutions exhibited a 26-41-mm and 15-40-mm range with different bacteria, respectively. The kinetics of bactericidal activity indicated that more than 50% of any bacterial population, with the exception of the resistant genus P. aeruginosa , were ren- dered non-viable in 15 min. One hundred percent lethal effects were observed within 30 min of exposure to the oils. The high carvacrol content of Th. pubescens oil ac- counts for its strong antimicrobial activity. The carvacrol content decreases and p- cymene increases significantly at the flowering stage of Th. pubescens . This change is followed by the increased antibacterial property of Th. pubescens indicating a bactericidal property of p-cymene as well. Aromatic compounds such as carvacrol, thymol (phenols) and p-cymene contributed to more than 75% of the chemical composition of thyme oils with strong antibacterial properties (Rasooli and Mirmostafa 2002). Rota and co-workers (2008) have described the volatile profile and antimicrobial activity of Th. vulgaris (thymol chemotype), Th. zygis subsp. gracilis (Boiss.) R. Morales (thymol and two linalool chemotypes) and Th. hyemalis Lange (thymol, thymol/linalool, and carvacrol chemotypes) essential oils extracted from seven plants cultivated in Murcia (Spain). Antimicrobial activities of the oils were evaluated using the filter paper disc agar diffusion technique for control of growth and survival of 10 pathogenic microorganisms – seven Gram-negative strains ( Salmo- nella enteritidis CECT 4155, S. typhimurium CECT 443, E. coli serovar O157: H7 CECT 4267, E. coli CECT 516, Y. enterocolitica serotype O:8; biotype 1 CECT 4315, Shigella flexneri serovar 2a CECT 585, and Shigella sonnei CECT 457) and three Gram-positive strains ( Listeria monocytogenes serovar 4b CECT 935, L. monocytogenes serovar 1/2c CECT 911, and S. aureus CECT 239). The essential oils showed strong activity (inhibition zone 20 mm), moderate activity (inhibition zone

<20-12 mm) and no inhibition (zone <12 mm). The major effectiveness was achieved by the essential oils from Th. hyemalis (thymol chemotype) followed by Th. zygis (thymol chemotype) and Th. vulgaris . Related to the inhibition of growth, no differ-

67 ences were detected among these essential oils, since all of them showed a strong activity for nine of the ten strains assayed. The minimum inhibitory concentration (MIC; µl oil/mL medium) and minimum bactericidal concentration (MBC; µl oil/mL medium) (against 10 microorganisms) of seven essential oils obtained from 3 Thymus species were also assessed. The essential oils with the most bactericidal and bacteriostatic properties were: Th. hyemalis (thymol and carvacrol chemotypes), Th. zygis

(thymol ch.) and Th. vulgaris (thymol ch.) with MIC 0.2 µl/mL and MBC 0.2

µl/mL, respectively (Rota et al. 2008). S. typhimurium , Y. enterocolitica and S. flexneri were the most susceptible organisms; their growth presented strong inhibition by all essential oils tested with MIC and MBC <0.2 µl/mL, except Th. zygis (82% linalool) and Th. hyemalis (thymol/linalool ch.) with MIC and MBC <0.7 µl/mL for Y. enterocolitica and S. flexneri (Rota et al. 2008). Nejad Ebrahimi and co-workers (2008) have studied the essential oil composition of Th. carmanicus Jalas, an endemic species grown in Iran, at different developmen- tal stages and its coherence with antibacterial activity. Three Gram-negative and four Gram-positive bacteria were used as follows: B. subtilis ATCC 9372, Enterococcus faecalis ATCC 15753, S. aureus ATCC 25923, S. epidermidis ATCC 12228, E. coli ATCC 25922, P. aeruginosa ATCC 27852, K. pneumoniae ATCC 3583. The inhibition zones and MIC values for bacterial strains, which were sensitive to the essential oil of Th. carmanicus , were in the range of 15-36 mm and 0.5-15.0 mg/ml, respectively. The oils of various phenological stages showed high activity against all tested bacteria, of which B. subtilis and P. aeruginosa were the most sensitive and resistant strains, respectively. The ranges of major constituents were as follow: carvacrol (58.9-68.9%), p-cymene (3.0-8.9%), γ-terpinene (4.3–8.0%), thymol (2.4–6.0%) and borneol (2.3-4.0%) (Nejad Ebrahimi et al. 2008). Maksimovi ć and co-workers (2008) have assessed the antimicrobial activity of Th. pannonicus essential oil against Gram-positive bacteria S. aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212), Gram-negative bacteria E. coli (ATCC 25922), P. aeruginosa (ATCC 27853) and two strains of K. pneumoniae (ATCC 29665 and NCIMB 9111), as well as two strains of yeast C. albicans (ATCC 10259 and ATCC 24433). Th. pannonicus essential oil had noteworthy antimicrobial potential against bacteria and yeasts, but no clear correlation between observed activity and the dose applied was observed. The oil exhibited clear bacteriostatic effect against E. coli , S. aureus , and P. aeruginosa . The observed activity cannot be associated with the presence of phenolic constituents, as only low concentrations of thymol, its isomers and/or derivatives were detected (Maksimovi ć et al. 2008). The associations between Moroccan endemic thyme essential oils and classical antibiotics have synergistic interactions against multidrug-resistant bacteria. The use of these combinations is likely to reduce the minimum effective dose of the drugs, thus minimizing their possible toxic side effects and the treatment cost. Fadli and co- workers (2012) have evaluated the antibacterial effect of the association between con-

68 ventional antibiotics (ciprofloxacin, gentamicin, pristinamycin, and cefixime) and essential oils of endemic Moroccan thyme species, Th. maroccanus Ball and Th. broussonetii Boiss., on antibiotic-resistant bacteria involved in nosocomial infections. The results of Fadli and co-workers (2012) indicated that the oils had a high inhibitory activity against tested bacteria, except for P. aeruginosa . Out of 80 combinations tested between essential oils and antibiotics, 71% showed total synergism, 20% had partial synergistic interaction and 9% showed no effect. The combination with carvacrol, the major constituent of Th. maroccanus and Th. broussonetii , showed also an in- teresting synergistic effect in combination with ciprofloxacin. The effect on Gram- positive bacteria was more important than on Gram-negative bacteria (Fadli et al. 2012). The essential oils from Th. moroderi Pau ex Martínez and Th. piperella L. is a source of important bioactive compounds with antibacterial properties. Ruiz-Navajas and co-workers (2012) have determined the chemical composition and antibacterial properties of the essential oils of two aromatic endemic herbs, Th. moroderi and Th. piperella , both endemic to the southeast of Spain, against Listeria innocua , Serratia marcenscens , Pseudomonas fragi , P. fluorescens , Aeromonas hydrophila , Shewanella putrefaciens, Achromobacter denitrificans , Enterobacter amnigenus , Enterobacter gergoviae , Alcali-genes faecalis , and Leuconostoc carnosum . Th. piperella essential oil had an inhibitory effect on 5 of the 11 bacteria assayed, with inhibition halos that ranged from 16.00 mm for A. denitrificans to 45.00 mm for A. hydrophila , and no antibacterial activity against P. fragi , P. fluorescens , S. putrefaciens , E. gergoviae , En- terobacter anmigenus , and L. carnosum . The Th. moroderi essential oil had an inhibitory effect on 4 of the bacteria, with inhibition halos from 15 mm ( L. innocua ) to 24.00 mm ( A. faecalis ). This essential oil showed no activity against P. fragi , P. fluorescens , S. putrefaciens , E. gergoviae , E. anmigenus , A. denitrificans , and L. carnosum (Ruiz- Navajas et al. 2012). The essential oils of Th. zygis , Th. mastichina (L.) L., Th. capitatus (L.) Hoff- manns. & Link (currently treated as a synonym of Thymbra capitata (L.) Cav.) and Th. vulgaris obtained from organic growth, were chemically analyzed by the agar disk diffusion and microdilution assays to determine their antibacterial activity against several bacteria: Listeria innocua CECT 910, Serratia marcescens CECT 854, Pseudomonas fragi CECT 446, P. fluorescens CECT 844, A. hydrophila CECT 5734, Shewanella putrefaciens CECT 5346, Achromobacter denitrificans CECT 449, Enterobacter amnigenus CECT 4078, Enterobacter gergoviae CECT 587, and Alcali-genes faecalis CECT 145. The agar disk diffusion assay, Th. zygis and Th. capitatus essential oils showed inhibitory effects against the ten tested bacteria, while Th. matichina and Th. vulgaris had inhibitory effects against eight and seven tested bacteria, respectively. Th. zygis and Th. capitatus essential oils had higher inhibition halos against all tested bacteria, at higher concentration (40 µL) than Th. matichina and Th. vulgaris essential oils. In Th. mastichina essential oil the major compound was 1,8-cineole (51.94%) whilst in Th. zygis essential oil the main constituent was thymol (48.59%). Th. capitatus essential oil was characterized by a high content in carvacrol (69.83%), while Th. vulgaris essential oils had a high content of linaool (44.00%) (Ballester-Costa et al. 2013).

69 The essential oil extracted in Th. numidicus Poir. showed high antioxidant and antibacterial activities. Adrar and co-workers (2016) have evaluated, by the microdilution method, the antibacterial effects of different combinations of two oils from Th. numidi-cus and Salvia officinalis L. with their major components or antibiotics (cephalosporines) against S. aureus subsp. aureus methicillin resistant or MRSA, ATCC 25923, K. pneumoniae ATCC 1603, E. coli ATCC 25922, and other clinical strains ( Serratia marcescens , P. aeruginosa ) and the antioxidant effect of the same essential oils com- bined with thymol or DL-α-tocopherol against DPPH free radical. Essential oils of Th. numidicus and S. officinalis have a bactericidal effect on all strains. Th. numidicus essential oil exhibited higher activity than S. officinalis essential oil, against all bacterial strains. According to other studies, there is a relationship between the antibacterial activity of essential oils and their chemical composition. Essential oils in combination with other substances exhibit different effects (indifferent, additive, antagonistic and synergistic) against bacteria and DPPH free radical. This is probably due to different mechanisms of action involved in this case. Essential oil of Th. numidicus showed a high phenolic content. Its major component is thymol (57.20-66.31%), followed by linalool (8.62-9.26%), gamma terpinene (6.12-9.19%) and p-cymene (6.20-7.55%). The presence of these components could explain the high activities found against all bacterial strains and the high antioxidant activity of Th. numidicus essential oil (Adrar et al. 2016). Moghimi and co-workers (2016) have provided important new information about the mode of action of antimicrobial nanoemulsions based on essential oils on an important bacterial species. Nanoemulsions may be particularly effective delivery sys- tems for essential oils due to their ability to facilitate antimicrobial application and increase antimicrobial efficacy. In their work, an essential oil ( Th. daenensis Čelak.) was formulated as a water-dispersible nanoemulsion (diameter 143 nm) using high- intensity ultrasound. The antibacterial activity of the essential oil in both pure and nanoemulsion forms was measured against an important food-borne pathogen bacterium, E. coli . Antibacterial activity was determined by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The mechanism of antibacterial activity was investigated by measuring potassium, pro- tein, and nucleic acid leakage from the cells, and electron microscopy. The conver- sion of an essential oil ( Th. daenensis ) into a nanoemulsion greatly enhanced its antibacterial activity against E. coli . The mechanism of action for the antimicrobial nanoemulsion against E. coli involved bringing the essential oil into close proximity with the cell membrane. This enabled the hydrophobic molecules within the essential oil to disrupt the cell membrane, possibly by altering the phospholipid bilayer integ- rity or by interfering with active transport proteins embedded in the phospholipid bilayer. The change in permeability of the disrupted cell membranes led to leakage of nucleic acids, pro-teins, and potassium from the cell interior, which led to cell death within 5 min. A comparison of the antibacterial activity of pure and emulsified essential oil showed that the nanoemulsion was much more effective. Presumably, this is because the small lipid particles within the nanoemulsion were able to bring the essen-

70 tial oil to the cell membrane surface, whereas the pure oil (which has a low water solubility) could not easily interact with the cell membranes (Moghimi et al. 2016). Petrovi ć and co-workers (2016) have investigated antimicrobial and another potential of supercritical extracts of Th. praecox Opiz subsp . polytrichus (A.Kern. ex Borbás) Jalas (synonym Th. balcanus Borbás). For the bioassays eight bacterial strains were used: Gram-positive Bacillus cereus (human isolate), Micrococcus flavus (ATCC 10240), S. aureus (ATCC 6538) and Listeria monocytogenes (NCTC 7973), and Gram-negative E. coli (ATCC 35210), P. aeruginosa (ATCC 27853), Enterobacter cloacae (human isolate), and S. typhimurium (ATCC 13311). In antifungal assay eight fungi were used: Aspergillus fumigatus (ATCC 9197), Aspergillus versicolor (ATCC 11730), Aspergillus ochraceus (ATCC 12066), Aspergillus niger (ATCC 6275), Trichoderma viride (IAM 5061), Penicillium funiculosum (ATCC 10509), Penicillium ochrochloron (ATCC 9112) and Penicillium verrucosum var. cyclopium (food isolate). The results of Petrovi ć and co-workers (2016) showed the significant potential of Th . praecox to be commercially cultivated as Th. vulgaris due to the high content of thymol and strong antimicrobial and antioxidant activity. All investigated extracts showed remarkable antimicrobial activity. Thymol was the major component identified in supercritical extra-cts which showed the strongest antimicrobial potential. In the case of supercritical extracts, the fungi appear to be more sensitive compared to the bacteria. Contrary in case of hexane/ethanol extract, the bacteria appear to be more sensitive compared to the fungi (Petrovi ć et al. 2016). Fadli and co-workers (2016) have defined the antibacterial effect of Th. riatarum Humbert & Maire essential oil on antibiotic-resistant bacteria, with a special focus on its permeabilizing effect on the bacterial membrane and its capability to restore chloramphenicol efficacy by blocking the efflux pump expressed in some isolates. Six Gram-negative bacteria ( E. coli , Salmonella spp., Enterobacter cloacae , Entero- bacter aerogenes , Klebsiella pneumoniae , P. aeruginosa ) and four Gram-positive bacteria ( Bacillus subtilis , Bacillus cereus , Micrococcus luteus , S. aureus ) were used in agar dilution method with Mueller-Hinton agar. The chemical composition of Th. riatarum essential oil identified 20 compounds, representing 78.93% of the essential oil. It was mainly composed of borneol, terpinene-4-on, and trans-caryophyllene representing, respectively, 41.67%, 8.65%, and 7.59%. Results of the disk diffusion experiment revealed that the tested essential oil had a wide antibacterial spectrum. It was active on tested strains by producing inhibition zone diameters varying from 8 to 22 mm. These diameters were lower than those obtained with usual antibiotics. Gram-positive bacteria ( B. cereus , B. subtilis , and M. luteus ) were generally found to be more sensitive than the Gram-negative ones ( E. coli , E. cloacae , K. pneumoniae ), P. aeruginosa being the most resistant. The MIC and the MBC values of Th. riatarum essential oil against the tested strains showed that this essential oil inhibited Gram-negative bacteria (E. coli , E. cloacae , K. pneumoniae ) at a concentration of 7.5 mL/L. In contrast, Gram- positive bacteria ( B. cereus , B. subtilis and M. luteus ) were inhibited with a MIC of 3.75 mL/L. This oil showed low activity on the growth of P. aeruginosa which was only sus-ceptible to high concentration (>7.5 mL/L). It is also important to mention

71 that the MIC was often equivalent to the MBC supporting a bactericidal action of Th. riatarum essential oil (Fadli et al. 2016). Schött and co-workers (2017) have examined the essential oil as well as the en- tire spectrum of the polyphenols in four different pharmacological Thymus species (Th. vulgaris , Th. zygis , Th. serpyllum , and Th. pulegioides ) cultivated under comparable conditions, and the volatile compounds, as well as the polyphenols, were characterized. In addition, the in vitro antibacterial activity against Streptococcus mutans , one of the primary cariogenic bacterial species, as well as of the essential oil and of the polyphenols were investigated. Furthermore, the bacterial viability and its effect on the initial bacterial adhesion under oral conditions were evaluated in situ for the essential oil and the polyphenols. The essential oils of the four investigated Thymus species exhibited an antibacterial activity against S. mutans in vitro , in contrast to the polyphenols of Th. vulgaris . Rinsing with polyphenol-rich infusions re- duced the initial bacterial colonization while the essential oil inhibited the bacterial growth on dental enamel in situ (Schött et al. 2017). The biological activities observed in a study of Vitali and co-workers (2017) may support possible applications of Th. alternans Klokov as a food and cosmetic pre- servative agent and/or as an active ingredient in topical preparations with antimicrobial properties. The essential oil from Th. alternans was tested against S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, C. albicans ATCC 24433. P. aeruginosa was the bacterial species not affected by the Th. alternans essential oil. Conversely, the growth of the other species representatives, namely S. aureus , E. faecalis , and E. coli , was inhibited. The diameters of the inhibition zone were almost comparable, ranging from 9 to 11 mm, and indicated a measurable antimicrobial effect. The antifungal activity was even more remarkable: 10 mg of essential oil produced a 20 mm inhibition zone diameter around the disc, which was higher than the diameter produced by nystatin used as the reference antimicotic compound. Results showed that Th. alternans belongs to the nerolidol chemotype, being rich of this sesquiterpene alcohol (15.8%) which might contribute to the antimicrobial (particularly effective on C. albicans growth) and antioxidant activities observed in a study of Vitali and co-workers (2017). In the organic food industry, no chemical additives can be used to prevent microbial spoilage. As a consequence, the essential oils obtained from aromatic herbs and spices are gaining interest in their potential as preservatives (Ballester-Costa et al. 2013). Semeniuc and co-workers (2017) have compared the antibacterial effects of several essential oils alone and in combination against different Gram-positive and Gram-negative bacteria associated with food products. Parsley, lovage, basil, and thyme essential oils, as well as their mixtures (1:1, v/v), were tested against Bacillus cereus , S. aureus , P. aeruginosa , E. coli , and S. typhimurium . P. aeruginosa is the most susceptible to all essential oils and their combinations. Three essential oils combinations show antagonistic effects against P. aeruginosa (parsley/thyme, lovage/thyme, and basil/thyme essential oils), and the other three combinations indifferent effects (parsley/lovage, parsley/basil, and lovage/basil essential oils). Parsley/lovage, parsley/basil, and parsley/ thyme essential oil mixtures display significantly higher antibacterial activities than the parsley essential oil. Basil essential oil does not significantly affect the antibacte-

72 rial activity of lovage/basil essential oil mixture. Thyme essential oil significantly contributes to the antibacterial activity of parsley/thyme and lovage/thyme essential oil mixtures but does not significantly influence the antibacterial activity of basil/thyme essential oil mixture (Semeniuc et al. 2017). Variation in the chemical profile of extracts could influence their biological activities. Therefore, it was important to know the chemical composition of extracts to correlate with their antimicrobial activities. Most of the antimicrobial activity in essential oils from Thymus genus appears to be associated with high amounts of monoterpenoid phenols (thymol and carvacrol) or monoterpenic alcohols (geraniol and linalool) (Petrovi ć et al. 2016). The thymol is responsible for antimicrobial activity (Marchese et al. 2016). Results of this study are in agreement with other research showing that thyme essential oil (especially thymol chemotype) possesses high activity against both Gram-positive and Gram-negative bacteria (Karaman et al. 2001; Rasooli and Mirmostafa 2002; Rota et al. 2008; Maksimovi ć et al. 2008; Ne- jad Ebrahimi et al. 2008; Ruiz-Navajas et al. 2012; Ballester-Costa et al. 2013; Adrar et al. 2016; Moghimi et al. 2016; Fadli et al. 2012, 2016; Schött et al. 2017; Semeniuc et al. 2017; Vitali et al. 2017). However, it should be noted that ethanolic extracts have a complex composition and their antimicrobial activities were due to a synergist effect between a large number of components present in small amounts in the extracts. The two main bioactive compounds in thyme essential oil are thymol and carvacrol responsible for most therapeutic aspects of the thyme extracts, i.e. antibacterial, antifungal, anti-inflammatory, and antioxidant activities (Petrovi ć et al. 2016). In a study of Rota and co-workers (2008), most of the antimicrobial activity in essential oils from Thymus genus appears to be associated with phenolic compounds (thymol and carvacrol). In Th. hyemalis oil (thymol, thymol/linalool, and carvacrol chemotypes) 49, 51 and 51 components were identified representing about 97%, 98.4% and 86.7% of the total detected constituents. Major components quantified for the thymol chemotype were: thymol (43%) followed by p-cymene (16.0%) and γ- terpinene (8.4%); for Th. hyemalis thymol/linalool chemotype: linalool (16.6%), thymol (16.0%), γ-terpinene (9.8%), 1-8-cineole (5.4%), borneol (4.7%), verbenone (4.8%); and for Th. hyemalis carvacrol chemotype were: carvacrol (40.1%), p-cymene (19.8%), borneol (5.0%) and thymol (2.9%). Attending to the volatile profile of the essential oils, a richer relative concentration of terpenic hydrocarbons ( γ-terpinene), alcohols (linalool, (Z)-verbenol, terpinen-4-ol, α(alpha)-terpineol, geraniol, spathulenol) ketones (camphor, verbenone) and thymol oxygenated derivates (thymol methyl ether) were quantified in Th. hyemalis (thymol ch.) when compared to Th. zygis (thymol ch.) and T. vulgaris (thymol ch.). Results of these authors suggest that it could be a synergistic action among phenolic components and these compounds. Carvacrol is another phenolic component that described the chemotype of Th. hyemalis essential oil. The assays using this essential oil (40% carvacrol) showed bactericidal and bacteriostatic activities similar to Th. hyemalis (43% thymol) since concentrations under 0.2 µl/mL were enough to achieve the MIC and MBC for 9 of the 10 microorganisms assayed in the study of Rota and co-workers (2008). The bacteriostatic properties of this oil are suspected to be associated with the carvacrol content (Rota et al. 2008).

73 Schött and co-workers (2017) have identified 69 volatile compounds in the ethyl acetate extracts, and 49 polyphenols in the aqueous infusion. The comprehensive ex- aminations of the essential oil and the polyphenols in four pharmacologically active Thymus species ( Th. vulgaris , Th. zygis , Th. serpyllum , and Th. pulegioides ) revealed that the main components in both, volatile compounds and polyphenols, were equal. All investigated Thymus species contained thymol for the volatile compounds and rosmarinic acid or luteolin-glucuronide for the polyphenols as major constituents. Further characteristic volatile compounds were p-cymene, γ-terpinene, β- linalool, carvacrol, and β-caryophyllene (Schött et al. 2017). Th. moroderi essential oil, 51 compounds were identified as representing 92.1% of the oil. The main components were camphor (26.74%), 1.8-cineol (24.99%), myr- cene (5.63%) and α-pinene (4.35%). Forty-eight compounds were identified for Th. piperella , representing 90.5% of the essential oil. The major component was carvacrol (31.92%) followed by para-cymene (16.18%), γ-terpinene (10.11%) and, to a lesser extent, 4-terpineol (7.29%) (Ruiz-Navajas et al. 2012). Dall’Acqua and co-workers (2017) have pointed out the great chemical diversity of Th. alternans phytocomplex. The significant cytotoxic activity of some of the isolated triterpenes underlines the possible role of the plant as a potential source of bioactive compounds for therapeutical applications. Phytochemical investigations on Th. alternans allowed identification of the main phytoconstituents of the plant’s aerial parts, showing the presence of glycosylated flavonoids and rosmarinic acid, pentacyclic triterpenes, and essential oil. Luteolin-4'-O-β-d-glucopyranoside (P1), chrysoeriol-7-O-β-d-glucopyranoside (P2), chrysoeriol-5-O-β-d-glucopyranoside (P3), apigenin-7-O-β-d-glucopyranoside (P4), rosmarinic acid (P5), rosmarinic acid-3'-O-β- d-glucopyranoside (P6), caffeic acid-3-O-β-d-glucopyranoside (P7), 3 α-hydroxy-urs- 12,15-diene (T1), α-amyrin (T2), β-amyrin (T3), isoursenol (T4), epitaraxerol (T5), and oleanolic acid (T6) were identified. Triterpenes and essential oil exhibited significant antiproliferative activity against a panel of tumour cell lines. In particular, the pentacyclic triterpenes showed improved activity compared to the reference compounds cisplatin and betulinic acid. (E)-nerolidol, the main volatile component of Th. alternans appeared to afford the major contribution to the cytotoxic activity of the essential oil (Dall’Acqua et al. 2017). It is well documented that the presence of these chemicals is responsible for various medicinal properties of plants. There are many re- ports available to support the role of phytochemicals and their activity against specific diseases.

CONCLUSIONS

The ethanolic extracts obtained from leaves of studied Thymus representatives showed different antibacterial activities against β-lactamase producing Pseudomonas aeruginosa strain. The effects varied significantly according to the Thymus taxa. It should be noted that the most antimicrobial effective plant against β-lactamase producing P. aeruginosa was Th. alpestris , being highly active with the ethanolic extract (mean diameter of inhibition zone was 12.8±0.8 mm). The antibacterial activity of ex-

74 tracts was greatest for Th. alpestris followed by Th. pannonicus followed by Th. serpyllum and then by Th. pulegioides . The antimicrobial activity of the crude ethanolic extracts obtained from leaves of Thymus plants may be attributed to a specific compound or a combination of compounds. The knowledge about the chemical profile of the extract helps in explaining the observed activity and designing experiments for activity fractionation for isolation of the active principle. The identification of precise molecular mechanism addressing how these extracts inhibit bacterial growth needs to be explored. The present study lays the basis for future research, to validate the possible use of Thymus species as a candidate in the treatment of infections caused by P. aeruginosa .

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SUMMARY

Antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world. In an effort to expand the spectrum of antibacterial agents from natural resources, genus Thymus belonging to Lamiaceae family has been selected, because, among plant-based antimicrobials, the antimicrobial activity of Thymus species has been well studied. Thymus species also have antimicrobial activities against a wide range of Gram-positive and Gram-negative bacteria, yeast, and fungi. These versatile pharmacological effects can be attributed to the secondary plant metabolites, especially to essential oil and polyphenols. Plants from the genus Thymus are rich in different active substances such as thymol, carvacrol, p-cymene, and terpinene. A convincing number of studies that reveal that thymol alone or thymol in plants along with other metabolites possess potent antimicrobial, antifungal, antibacterial, and antiparasitic properties prompted us to verify antibacterial effects of four species and one interspecific hybrid of Thymus genus sampled in the western part of Ukraine against β-lactamase producing Pseudomonas aeruginosa locally isolated. Samples were harvested in June-August, 2016. Leaves of Th. serpyllum L. emend. Mill. were collected among grass on sandy soil in the edge of a pine forest (Baymaky village, Bilohirya district, Khmelnytsky region, Ukraine; N 50°03'58,9'', E 26°13'37,5'', 257 m a.s.l.). Leaves of Th. pannonicus All. were harvested among grass in the roadside between the two cultivated fields (Syvky village, Bilohirya district, Khmelnytsky region, Ukraine; N 50°02'09,6'', E 26°13'19,2'', 283 m a.s.l.). Leaves of Th. pulegioides L. were collected among grass nearby land parcels (Syvky village, Bilohirya district, Khmelnytsky region, Ukraine; N 50°02'02,8'', E 26°14'13,9'', 306 m a.s.l.). Leaves of Th. ×porcii Borbás (a hybrid between Th. pannonicus and Th. pulegioides ) were sampled in the grass stand, on the side of the footpath of the race track (Medovoi Pechery Str., Lviv, Ukraine; N 49°49'15.1", E 24°05'12.5", 348 m a.s.l.). Leaves of Th. alpestris Tausch ex A. Kern. were harvested on the side of the road below the stream, in mountain valley Shumneska (Kvasy village, Rakhiv district, Zakarpattia region, Ukraine; N 48°09'32.3", 24°21'26.4", 1259 m a.s.l.). Freshly leaves were washed, weighted, crushed, and homogenized in 96% ethanol (in proportion 1:19) at room temperature. The extracts were then filtered and investigated for their antimicrobial activity. Antimicrobial activity was determined using the agar disk diffusion assay. The present study has shown that ethanolic extracts obtained from leaves of Thymus representatives inhibited intermediate activity against β-lactamase producing P. aeruginosa . The mean diameter of inhibition zone for Th. serpyllum was (11.3±0.3) mm, for Th. pannonicus – (11.5±0.5) mm, for Th. × porcii – (9.8±0.5) mm, for Th. pulegioides – (11.2±0.7) mm, and for Th. alpestris – (12.8±0.8) mm. The effects varied significantly according to the Thymus taxa. It should be noted that the most antimicrobial effective plant against β-lactamase producing P. aeruginosa was Th. alpestris , being highly active with the ethanolic extract (mean diameter of inhibition zone was 12.8±0.8 mm). The antibacterial activity of extracts was greatest for Th. alpestris followed by Th. pannonicus followed by Th. serpyllum and then by Th. pulegioides . The antimicrobial activity of the crude ethanolic extracts obtained from leaves of Thymus plants may be attributed to a specific compound or a combination of compounds.

78 The knowledge about the chemical profile of the extract helps in explaining the observed activity and designing experiments for activity fractionation for isolation of the active principle. The identification of precise molecular mechanism addressing how these extracts inhibit bacterial growth needs to be explored. The present study lays the basis for future research, to validate the possible use of Thymus species as a candidate in the treatment of infections caused by P. aeruginosa .