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Essential oils, N-acetylcysteine and tris-EDTA against biofilms of Pseudomonas aeruginosa isolated from bovine subclinical mastitis
Börcsök Márk - year 6
University of Veterinary Medicine Budapest, Department of Pharmacology and Toxicology
Supervisors: Dr. Ákos Jerzsele, Dr. Orsolya Juhász


Subclinical mastitis is one of the most significant dairy cattle farm disease worldwide, which causes remarkable economic losses and food hygiene problems. Pseudomonas aeruginosa is a clinically significant pathogen due to its biofilm forming ability, which makes it much more difficult or often impossible to treat the animals. The key to successful treatment is to facilitate the penetration of antibiotics into the deeper layers of biofilms, thus the antibiotics can kill the bacterial cells there. The aim of our study was to look for active agents, which have significant antimicrobial and anti-biofilm properties used alone or in combination with antibiotics. We investigated the antimicrobial and antibiofilm effects of three plant-derived essential oils: geraniol, cinnamaldehyde and thymol, as well as tris-EDTA, N-acetylcysteine (NAC) and a fluoroquinolone antibiotic.

In our study we examined P. aeruginosa strains isolated from subclinical mastitis. Antibiofilm properties of the active substances were investigated by MTS-formazan (methyl-tetrazolium salt) assay, for which the bacterial strains were incubated in 96-well microtiter plates at 37°C for 24 hours to produce biofilms. The mature biofilms were treated with the two-fold dilution series of the active agents and incubated at 37 ° C for 24 hours. After MTS staining microtiter plates were examined spectrophotometrically at 492 nm wavelength and the reduction of the produced biofilms was determined by comparing each well to the positive control. The presumed antimicrobial activity of the active substances was investigated by determining their MIC- (minimum inhibitory concentration) values.

In our in vitro experiments the three plant-derived essential oils, a fluoroquinolone-derivative and tris-EDTA were able to efficiently eradicate the biofilms produced by P. aeruginosa strains of bovine mastitis origin. We expressed the rate of biofilm rupture as a percentage and the EC50 value was determined where the reduction rate reached 50%. The mean EC50 was 1,13mg/ml for geraniol, 0,62mg/ml for cinnamaldehyde, 2,63mg/ml for thymol, 1,00mg/ml for fluoroquinolone antibiotic and 1,38µg/ml for tris-EDTA. MIC values ranged between 1-2µg/ml for fluoroquinolone antibiotic, 0,5-4 mg/ml for essential oils and 1-2 mg/ml for Tris-EDTA. Based on our results, geraniol, thymol, cinnamaldehyde, and tris-EDTA might be effective in the treatment of subclinical mastitis caused by P. aeruginosa strains. The investigated substances in our study are not dangerous for consumers, and they might increase the efficacy of antibiotic treatments, thus reducing the level of drug residues in the food chain.

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