# Unlocking the Gates: A Novel Diagnostic Molecule for Quantifying Efflux Levels in Gram‐Positive Bacteria

## TL;DR

This paper introduces a new diagnostic tool using a phenazinium compound to quickly measure efflux levels in Gram-positive bacteria, improving antibiotic treatment decisions.

## Contribution

A novel phenazinium-based molecule and protocol for rapid, real-time efflux quantification in Gram-positive bacteria.

## Key findings

- The selected phenazinium compound shows differential MIC in Staphylococcus efflux mutants.
- The diagnostic protocol successfully categorizes efflux levels as no, weak, or strong.
- In-silico docking and flow cytometry confirm the compound's interaction with the NorA efflux pump.

## Abstract

Efflux‐mediated antibiotic resistance poses a significant global threat, affecting diverse bacterial species. Clinicians recognize the danger of efflux mechanisms during antibiotic treatment, yet precise diagnostic tools remain unavailable. The antibiogram currently infers abnormal efflux pump activity in clinical isolates, which is subsequently confirmed through transcriptomic or genomic analysis. This study harnesses the colorimetric, fluorescence, and solubility properties of phenazinium derivatives to develop a rapid protocol for detecting bacterial efflux. Among several synthesized phenazinium compounds, the compound demonstrating differential MIC in Staphylococcus efflux mutants and exhibiting appropriate physicochemical properties is selected. A diagnostic protocol is developed using the selected compound to assess efflux levels, categorized as no, weak, or strong, through colorimetry and spectroscopy techniques. Testing on Gram‐positive efflux mutants and clinical Staphylococcus isolates further validates the approach. In‐silico docking analysis confirms the interaction between the chosen compound and the NorA efflux pump in S. aureus. Flow cytometry is employed to re‐evaluate the detection assays. The developed molecule and protocol represent the first known method to evaluate efflux levels in any Gram‐positive species through a streamlined and user‐friendly process. This pioneering test significantly advances the epidemiological analysis of efflux mechanisms and enables more informed therapeutic decision‐making, leading to more effective treatment.

Efflux‐mediated resistance compromises antibiotic efficacy, yet rapid detection remains elusive. This study presents a novel phenazinium‐based diagnostic molecule for quantifying bacterial efflux levels in Gram‐positive pathogens. The optimized compound enables precise, real‐time efflux assessment using fluorescence and colorimetric techniques, facilitating clinical decision‐making and antimicrobial stewardship. Molecular docking and flow cytometry further validate its diagnostic potential.