Standard operating procedure (SOP) for disk diffusion-based quorum sensing inhibition assays

Abstract

Introduction: The emergence of multidrug-resistant bacterial strains is a severe global health issue, which is worsened by the inability of new antibiotics. Virulence inhibition is one of the novel strategies that have been proposed to combat bacterial pathogens more effectively, without the risk of exerting selection pressure on these microorganisms. Inhibition of bacterial cell-cell communication (quorum sensing; QS) is a promising approach however, rapid and cost-effective screening for compounds with QS-inhibitory activity is not yet well-established. Aims: The aim of the present study is to determine the ideal experimental conditions for the disk-diffusion based QS-inhibitory assay with the most frequently used QS-signal molecule-producing and reporter strains. Methods: In our study, the effects of growth characteristics, incubation time, temperature and the used culture media were studied on the used bacterial strains and results of the disk-diffusion based QS-inhibitory assay. Results: Based on our results, the ideal experimental setting includes a modified Luria-Bertani medium (LB*; complemented with nutrients and microelements), incubation at room temperature (25 °C) for 48 hours before the reading of results, where the density of the starting inocula has less influence of the results of the assay. Conclusion: Establishing standard operating procedures (SOPs) is a way to help carry out various operations, aiming to increase precision and efficiency. Adherence to the experimental settings defined based on our results may aid in improving the reproducibility, comparability and reliability of results obtained by this method.