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.

Xanthones Active against Multidrug Resistance and Virulence Mechanisms of Bacteria

“…For the QS tests, all the bacteria used were Gram-negative. The bacteria used were Chromobacterium violaceum wild type 85 (wt85), characterized by the AHL signal molecule-mediated production of the purple violacein pigment, capable of endogenous QS-signal molecule production (N-hexanoyl- l -HSL), C. violaceum CV026 (CV026), a Tn5 transposase-mutant, AHL-signal molecule indicator strain (produces purple violacein pigment in the presence of AHL), which is incapable of endogenous QS-signal molecule-production, but useful in the detection of external stimuli, Sphingomonas paucimobilis Ezf 10-17 (EZF), AHL-producing-strain (used with C. violaceum CV026), and Serratia marcescens AS-1, characterized by the AHL signal molecule-mediated production of the orange–red pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosin), capable of endogenous QS-signal molecule production (N-hexanoyl- l -HSL), were applied [ 57 ].…”

Metabolites from Marine-Derived Fungi as Potential Antimicrobial Adjuvants

“…For the QS tests, all the bacteria used were Gram-negative. The bacteria used were Chromobacterium violaceum wild type 85 (wt85), characterized by the AHL signal molecule-mediated production of the purple violacein pigment, capable of endogenous QS-signal molecule production ( N -hexanoyl- l -HSL), C. violaceum CV026 (CV026), a Tn5 transposase-mutant, AHL-signal molecule indicator strain (produces purple violacein pigment in the presence of AHL), which is incapable of endogenous QS-signal molecule-production, but useful in the detection of external stimuli, Sphingomonas paucimobilis Ezf 10–17 (EZF), AHL-producing-strain (used with C. violaceum CV026), and Serratia marcescens AS-1, characterized by the AHL signal molecule-mediated production of the orange–red pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosin), capable of endogenous QS-signal molecule production ( N -hexanoyl- l -HSL), were applied [ 49 ].…”

“…The agar plates were incubated at room temperature (20 °C) for 24–48 h. The QS inhibition was accessed visually, through the inhibition of pigment production. The discolored, but intact, bacterial colonies were measured with a ruler [ 45 , 49 , 59 ].…”

<p>Contribution of the AbaI/AbaR Quorum Sensing System to Resistance and Virulence of <em>Acinetobacter baumannii</em> Clinical Strains</p>

“…AHL-producing bacteria were screened by using two AHL-sensing bacterial biosensors, Chromobacterium violaceum CV026 13 and Agrobacterium tumefaciens KYC55, 14 which respond to short-chain and long-chain AHLs, respectively, as described previously. 15 , 16 Briefly, fresh cultures of QS biosensor strains were inoculated on LB agar plates. KYC55 was grown in LB medium supplemented with 100 μg/mL tetracycline and 100 μg/mL spectinomycin at 28 °C.…”