# Action of Betulinic Acid in the Inhibition of Efflux Pump NorA in Staphylococcus aureus Strains: In Vitro and In Silico Approaches

## TL;DR

This study shows that betulinic acid can inhibit the NorA efflux pump in Staphylococcus aureus, potentially improving antibiotic effectiveness.

## Contribution

The novel finding is that betulinic acid acts as an efflux pump inhibitor in S. aureus, despite lacking direct antibacterial activity.

## Key findings

- Betulinic acid reduced the minimum inhibitory concentration of norfloxacin and ethidium bromide in S. aureus strains.
- The compound increased membrane permeability and fluorescence emission, indicating efflux pump inhibition.
- In silico and in vitro approaches confirmed betulinic acid's potential as an efflux pump inhibitor.

## Abstract

Antibiotic resistance poses a serious challenge to public health, particularly in the case of Staphylococcus aureus, a Gram‐positive bacterium that employs multiple resistance mechanisms, including efflux pumps such as NorA, which extrude antimicrobial compounds from the cell and reduce antibiotic efficacy. Therefore, the search for substances capable of inhibiting these mechanisms represents a promising strategy to combat bacterial resistance. Betulinic acid (BA), a pentacyclic triterpene of the lupane type, commonly found in different parts of plants, has demonstrated various pharmacological activities, including antibacterial effects. This study investigated, through in vitro and in silico analyses, the inhibitory action of BA on the NorA efflux pump in S. aureus strains SA‐1199 and SA‐1199B. The minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. Subsequently, their effects on efflux pump‐mediated antibiotic resistance were evaluated by reducing the MIC of the antibiotic and ethidium bromide (EtBr), while fluorimetry and permeability potential tests were carried out using the SYTOX Green fluorescence method. Although BA did not show intrinsic antibacterial activity, showing MIC ≥ 1024 µg/mL, it was able to decrease the MIC of norfloxacin and EtBr, as well as influence membrane permeability and increase fluorescence emission. The results, therefore, indicate that BA has considerable potential as an efflux pump inhibitor and could help in the treatment of resistant bacterial infections.