Resolving the mechanism of bacterial inhibition by plant secondary metabolites employing a combination of whole-cell biosensors

Abstract

Tightening regulations regarding the use of biocides have stimulated interest in investigating alternatives to current antimicrobial strategies. Plant essential oils and their constituent compounds are promising candidates as novel antimicrobial agents because of their excellent ability in killing microbes while being non-toxic to humans at antimicrobially-active concentrations. Allyl isothiocyanate (AIT), carvacrol, cinnamaldehyde (CNAD), citral, and thymol were investigated for their antibacterial activity against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The five compounds were screened via disc diffusion assay and broth microdilution method, by which inhibition zone diameters, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs) were determined. AIT and CNAD displayed the greatest inhibitory effects against all species tested, with AIT yielding MICs of 156.25mg/L and MBCs of 156.25 to 312.5mg/L, and CNAD yielding MICs of 78.125 to 156.25mg/L and MBCs of 78.125 to 312.5mg/L. Based on these results, AIT and CNAD were selected for closer examination of their toxic effects. Two complementary bioluminescence-based bacterial biosensors, E. coli HB101_pUCD607_lux and Acinetobacter baylyi ADP1_recA_lux, were employed to examine the dose-response relationships and mechanism of action of AIT and CNAD. This is the first reported study to employ a lux-based biosensor assay coupled with parallel plate count experiments to demonstrate that AIT and CNAD not only damaged cell membranes, but also disrupted cellular metabolism and energy production in bacteria. It is also the first to use genotoxicity-sensing whole-cell bioreporters to demonstrate that neither AIT nor CNAD induced expression of the universal DNA repair gene, recA. This suggests that AIT and CNAD were not genotoxic. As an antimicrobial agent, it is advantageous that the compound be genetically non-damaging so that toxicity towards higher multicellular organisms and resistance development can be minimized. Thus, AIT and CNAD may be of high value as novel antimicrobial agents.