Membrane Depolarization Effects of Phytochemicals in Gram-Positive Bacteria

In recent decades, antimicrobial resistance (AMR) has become a significant threat to human health and the global economy. The rise of AMR in various bacterial species, including Staphylococcus aureus, is mainly caused by the overexpression of efflux pumps (EPs). The NorA efflux pump is one of the most effective resistance mechanisms against S. aureus. Efflux pump inhibitors (EPIs) are essential to prevent resistance and enhance bacterial susceptibility to antibiotics by blocking drug extrusion. Antimicrobial activities of prenylated isoflavonoids and indole alkaloids against S. aureus have been observed. Compared to indole alkaloids, prenylated isoflavonoids exhibit higher antibacterial activity. If an EPI has potent antimicrobial activity, it becomes difficult to determine whether bacterial killing results from efflux pump inhibition or from the direct antibacterial effect of the EPI. Therefore, the ideal EPI should be less active against bacteria. Based on this, further research is needed to assess whether brassinin and camalexin have a greater capacity to function as NorA EPIs compared to the evaluated prenylated isoflavonoids. An important aspect of studying a compound's efflux pump inhibition potential is whether it selectively inhibits efflux pumps. NorA EP utilize proton motive force (PMF), which can be disrupted by membrane depolarization. Measuring PMF disruption is necessary to prevent false-positive results regarding EPI activity. This study tested the membrane depolarization effects of indole alkaloids, camalexin, and brassinin. To assess PMF disruption in S. aureus, a method using the membrane potential-sensitive dye DiSC3(5) was developed and validated with valinomycin as a positive control. Following valinomycin addition, fluorescence increased as expected. At each tested concentration, the fluorescence signals of brassinin and camalexin were compared. Brassinin showed less fluorescence than camalexin at all concentrations. At the lowest two concentrations, both compounds displayed consistently low fluorescence signals. These indole alkaloids were previously identified as AcrB EPIs in E. coli. Further studies are necessary to verify their potency as NorA EPIs. However, since the indole alkaloids showed minimal DiSC3(5) accumulation in the NorA over-expressing strain, it can be concluded that these compounds are less likely to cause membrane depolarization in gram-positive bacteria.