Ispitivanje antibakterijskog dejstva karvakrola, eugenola, cinamaldehida i timola prema sojevima Staphylococcus aureus izolovanih u slučajevima mastitisa krava

Abstract

taphylococcus aureus predstavlja jednog od najznaĉajnijih uzroĉnika mastitisa krava, koji dovodi do velikih zdravstvenih problema i ekonomskih gubitaka u proizvodnji mleka. Imajući u vidu sve prisutniju rezistenciju prema antibioticima sojeva S. aureus izolovanih kod mastitisa, odnosno zabranjenu ili ograniĉenu upotrebu antibiotika u organskoj proizvodnji, etarska ulja imaju sve veći znaĉaj u terapiji obolelih grla. Cilj ovog istraţivanja je bio da se ispita: antibakterijsko delovanje odabranih komponenti etarskih ulja: karvakrola, eugenola, cinamaldehida i timola na sojeve S. aureus izolovanih u sluĉajevima supkliniĉkih mastitisa krava, njihovo sinergistiĉko delovanje u teĉnoj kulturi, kao i delovanje na biofilm S. aureus. Za ispitivanja na kliniĉkim izolatima, korišćeni su uzorci prikupljeni u toku redovne kontrole sa podruĉija koje obuhvata 8500 mleĉnih grla, holštajn-frizijske rase. Osim kliniĉkih izolata u radu su korišćeni i referentni sojevi S. aureus ATCC (American Type Culture Collection) 25923 i meticilin rezistentni S. aureus ATCC 43300. Kao kontrolni soj za produkciju biofilma korišćen je izolat S. aureus sa Instituta za mikrobiologiju, Medicinskog fakulteta, Univerziteta u Beogradu. Da bi se postigli postavljeni ciljevi korišćene su i klasiĉne mikrobiološke metode izolacije identifikacije S. aureus, kao i PCR metoda molekularne detekcije: nuc gena za proizvodnju termostabilne nukleaze koji se pojavljuje u razliĉitim alelnim formama kod S. aureus, S. intermedius i S. hyicus, na ĉemu se zasniva identifikacija vrste, zatim coa gena za produkciju koagulaze i ica A i D alelne forme gena koji kodira produkciju biofilma. Disk difuzionom metodom ispitivana je osetljivost izolata na antimikrobne supstance, zastupljene u terapiji mastitisa u Srbiji. Metodom odreĊivanja minimalnih inhibitornih i baktericidnih koncentracija antimikrobnih supstanci odreĊema je osetljivost izlata S. aureus prema aktivnim komponentama etarskih ulja karvakrola, eugenola, cinamaldehida i timola u koncentracijama od 2560μg/ml do 1,25μg/ml i kontrolnim antibioticima linkomicinu, neomicinu i kloksacilinu. OdreĊivanje meĊusobnog odnosa na antimikrobno dejstvo aktivnih komponenti u vršeno je modifikovanom metodom šahovske table, dok je odreĊivanje duţine antibakterijskog delovanja izvršeno time – kill metodom u mleku u trajanju od 14 dana uz tri dodatne inokulacije bakterija 2., 4. i 6. dana ispitivanja...

Staphylococcus aureus is one of the most important causes of cow mastitis that leads to major health problems and economic losses in milk production. Essential oils and their active components are gaining importance in the treatment of mastitis patients due to growing antimicrobial resistance of S. aureus strains, as well as due to prohibited or limited use of antibiotics in organic production. The aim of this study was to examine: antibacterial activity of selected essential oil components: carvacrol, eugenol, thymol and cinnamaldehyde on S. aureus isolated in cases of subclinical mastitis in cows, their synergistic effects, and the effect on the S. aureus biofilm. To achieve these goals, clinical isolates were used, collected during regular checkups in the areas covered by 8500 dairy cattle of the Holstein-Friesian breed. Beside clinical isolates reference strains of S. aureus ATCC (American Type Culture Collection) 25923 and meticillin-resistant S. aureus ATCC 43300 have been used. Control used for the production of S. aureus biofilm was the strain from the Institute of Microbiology, Faculty of Medicine, University of Belgrade. In order to reach the set objectives, classical microbiological methods of isolation and identification of S. aureus were used as well as the PCR methods of molecular genetic detection: nuc gene for the production of thermostable nuclease that appears in different allelic forms in S. aureus, S. intermedius, and S. hyicus, which is used for identification species, then coa genes for the production of coagulase, and ica A and D allelic forms of the gene coding the production of a biofilm. Disk diffusion method was used in testing of the isolates’ sensitivity to antimicrobial substances, present in the treatment of mastitis in Serbia. Determining the minimum inhibitory and bactericidal concentrations of antimicrobial substances was used to describe S. aureus isolates to the active components of essential oils: carvacrol, eugenol, cinnamaldehyde and thymol in a concentration of 2560μg / ml to 1,25μg / ml and control antibiotics as well lincomycin, neomycin and cloxacillin. Determination of influence between the antimicrobial activities of active components was performed by chessboard modified method, while the length of antibacterial activity was tested by time - kill method in milk for 14 days with three additional reinoculation of bacteria on the 2nd, 4th and 6th days of testing...