Advanced amperometric respiration assay for antimicrobial susceptibility testing

Area of Science:

• Microbiology
• Electrochemistry
• Biotechnology

Background:

• Traditional antimicrobial susceptibility testing (AST) methods can be time-consuming.
• Developing rapid and accurate AST methods is crucial for effective treatment and infection control.
• Electrochemical assays offer potential for rapid biological measurements.

Purpose of the Study:

• To adapt a ferricyanide-based electrochemical cell respiration assay for broad-spectrum antimicrobial susceptibility testing (AST).
• To develop a quicker and more convenient procedure for AST compared to traditional cell culture methods.
• To validate the assay's performance with clinically relevant bacteria, including a highly virulent pathogen.

Main Methods:

• Adapted a ferricyanide-based electrochemical assay to measure total bacterial respiration via ferrocyanide amperometry.
• Modified the assay protocol to sequential drug exposure, cell separation, and resuspension in drug-free buffer to overcome interference.
• Validated the assay using Escherichia coli (E. coli) and Burkholderia pseudomallei with six antimicrobial agents.

Main Results:

• The assay demonstrated linearity with bacterial cell counts for E. coli.
• An initial assay version produced incorrect susceptibility classifications due to drug interference.
• The revised sequential assay procedure yielded results consistent with cell culture-based AST for E. coli.
• The assay showed potential for testing antimicrobial resistance in B. pseudomallei.

Conclusions:

• A modified ferricyanide-based electrochemical assay provides a rapid and accurate method for antimicrobial susceptibility testing.
• The revised protocol effectively mitigates interference from electroactive drugs or respiration-affecting compounds.
• This electrochemical approach holds promise for AST, especially for challenging pathogens like Burkholderia pseudomallei.