Gram-negative rough mutants used as test bacteria can increase sensitivity of direct bioautography

Abstract

Currently, the antimicrobial activity of essential oils (EOs) is an outstanding research field due to antibiotic resistance of microorganisms. Thin-layer chromatography‒direct bioautography (TLC‒DB) is an effective, fast method to find components with antimicrobial activity in a mixture of plant compounds, e.g., in EOs. The volatility and hydrophobic characters of EOs require special experimental conditions, and disc diffusion assay is not appropriate to explore the antimicrobial activity of them. The aim of this study was to use “R” mutants, which are more sensitive to synthetic antimicrobial drugs, in DB to increase the sensitivity of this method. Our hypothesis was that these mutants show sensitivity to some EOs (thyme, clove, and peppermint) as well. The chemical composition of our tested EOs was measured with gas chromatography‒mass spectrometry (GC‒MS). The main compounds (39.8% thymol, 78.8% eugenol, and 50.4% menthol) of EOs showed notable antibacterial activity in TLC‒DB. Based on our results, we suggest to use Salmonella minnesota Re595 rough strain as test bacterium in bioautography, because it showed the highest sensitivity to the tested antibiotics (gentamicin and cephalexin) and EOs. Furthermore, this rough mutant could make TLC‒DB more faster, because only 4 h incubation time was enough to detect the inhibition zones of the active compounds used in this study.