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Słupskie Prace Biologiczne 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 ge- nus are used in the traditional medicine as tonics, carminatives, antitussives, aromatic, expectorant, stomachic, antispasmodic, bronchospasmolytic, diuretic, sedative, dia- phoretic, and antiseptics, as well as anti-inflammatory, antioxidant, anthelmintic, hepa- toprotective 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 in- hibition 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 antim- icrobial 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 knowl- edge about the chemical profile of the extract helps in explaining the observed activity and designing experiments for activity fractionation for isolation of the active princi- ple. The identification of precise molecular mechanism addressing how these extracts inhibit bacterial growth needs to be explored. The present study lays the basis for fu- ture 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 pre- sented 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 re- sistance (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 assess- ment of the antimicrobial properties of plants, providing the possibility of obtaining molecules that could be used as a natural antiseptics and antimicrobial agents in medi- cine (Hemaiswarya et al. 2008). The emergence of multiresistant strains of microor- ganisms 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 antimicro- bial 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, antitus- sives, 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, car- vacrol, 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 mem- brane. 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
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