TDK conference 2019

Subclinical mastitis can cause serious financial losses and food hygiene issues in dairy herds worldwide. S. aureus, an important pathogen of subclinical mastitis, often forms biofilm in the udder, a phenomenon that makes eradication of the agent very difficult. Antibiofilm agents, that enhance the efficacy of antibiotics, are essential for successful treatment. The aim of our research was to find active substances that possess antimicrobial and antibiofilm activity, and can help in the management of subclinical mastitis caused by S. aureus. The substances tested in our study were N-acetylcysteine and three substituted cysteine derivates sythesized by our research team, along with three plant-derived essential oils.

In vitro efficacy of the substituted cysteine derivates was determined by crystal violet staining, which describes biomass density. The amount of viable cells embedded in the biofilm was determined by MTS-formazan (methyltetrazolium salt) assay. In the case of essential oils, antibiofilm effect was investigated by MTS-formazan assay, and MIC (minimum inhibitory concentration) determination was used to detect their presumed antimicrobial activity. Our study started with the accurate adaptation of the staining methods. To investigate the effects of the substances against biofilms, bacterial strains were incubated in 96-well microtiter plates for 9 hours at 37ºC to initiate biofilm formation. After the incubation period, investigated substances were added into the wells, and antibiofilm activity was determined after an additional 18 hours of incubation. For testing the antimicrobial effect of essential oils, bacterial strains and oils were put on the plates simultaneously, and after 24 hours of incubation, inhibitory concentrations were determined by the unaided eye.

All the tested essential oils and one of the cysteine derivates possessed pronounced antibiofilm effect against S. aureus biofilms of bovine origin. Furthermore, antimicrobial activity of essential oils might be useful to kill planktonic, free-floating bacterial cells. Usage of these agents may represent a significant improvement in the treatment of S. aureus induced subclinical mastitis.