Potencial antimicrobiano e antibiofilme da combinação dos óleos essenciais de Origanum vulgare e Thymus vulgaris para o tratamento da mastite bovina

Abstract

Bovine mastitis is the most frequent and most expensive disease that affects dairy herds. It is the inflammation of the bovine mammary gland, commonly caused by a bacterial infection. Staphylococcus aureus is one of the main causative agents of bovine mastitis, a microorganism capable of forming biofilms and often resistant to several antimicrobial classes. Thus, the search for new products with antimicrobial activity against resistant microorganisms is necessary for the control of mastitis. This study evaluated the antimicrobial and antibiofilm effects of the combination of essential oils (EOs) from Origanum vulgare (oregano) and Thymus vulgaris L. (thyme) against important bovine mastitis pathogens, aiming at the incorporation of these OEs in a semi-solid pharmaceutical formulation for the treatment of bovine mastitis. To this end, chemical characterization of essential oils was carried out using the gas chromatography method coupled to the mass spectrometer. The compounds carvacrol and thymol were identified as major components of the OEs of O. vulgare and T. vulgaris, respectively. The antimicrobial activity of the EOs and the major components against the control strains Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853), was demonstrated by the disk diffusion technique. The broth microdilution technique was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the EOs. For O. vulgare EO, the values of MIC and CBM against the four control strains ranged between 0.195 - 3.125 µL and 0.390 - 3.125 µL, respectively. Against thirty S. aureus strains isolated from bovine mastitis, the MIC ranged from 0.097 - 0.781 µL and the MBC from 0.781 - 1.562 µL. For Thymus vulgaris EO, MIC and CBM values ranged from 0.195 - 12.500 µL against the four selected control strains, while for clinical isolates the MIC ranged from 0.390 - 1.562 µL and MBC from 0.781 - 1.562 µL. From the CBM values, the fractional inhibitory concentration (FIC) of the EOs against the control lines was determined, using the checkerboard method. An additive interaction between the EOs was observed against S. aureus, for the other control strains the interaction was synergistic. The FIC value established for the S. aureus strain (ATCC 25923) was used to evaluate the effect of the combined EOs on the disarticulation of the biofilm formed by the clinical isolates. All clinical isolates formed biofilm, being classified as low adherent (70.00%) or moderate adherence (30.00%), and the average absorbance of the formed biofilm was 0.192. The concentration of 1X FIC significantly reduced the biofilm formed by 31.61% (0.132) and the concentration of 2X FIC reduced the biofilm by 43.09% (0.110). From the FIC value for the S. aureus strain (ATCC 25923) a semi-solid pharmaceutical formulation was designed containing the combination of EOs. Three active formulations were prepared, with EO concentrations equivalent to 2.5% w/w, 3.5% w/w and 5.0% w/w, named as FA1, FA2 and FA3, respectively. The concentrations of the OEs were added in the same proportion of the FIC of the OEs against the control strain S. aureus. The formulations were evaluated for physical-chemical and microbiological stability and were found to be adequate in all criteria. The antimicrobial activity of the formulations was evaluated against the control strain S. aureus, and the three formulations showed efficacy against this strain. Therefore, FA1 was chosen for further tests on S. aureus strains isolated from mastitis, considering that this formulation presented the best cost-benefit, given the lower amount of EOs used. The efficacy of FA1 was evaluated against S. aureus strains isolated from bovine mastitis, and it was effective against 76.66% of clinical isolates. FA2 was evaluated against the strains for which FA1 was not effective, and in this new scenario, FA2 was effective against 100% of the strains. From the results it can be inferred that the FA2 formulation, prepared with the combination of the additive effect of the EOs of O. vulgare and T. vulgaris, can be effective for the intended purpose, treatment of bovine mastitis caused by S. aureus.