Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor rRNA Using a Novel Electrochemical Biosensing Platform

Precursor rRNA (pre-rRNA) is an intermediate stage in the formation of mature rRNA and is a useful marker for cellular metab-olism and growth rate. We developed an electrochemical sensor assay for Escherichia coli pre-rRNA involving hybridization of capture and detector probes with tail sections that are spliced away during rRNAmaturation. A ternary self-assembled mono-layer (SAM) prepared on gold electrode surfaces by coassembly of thiolated capture probes with hexanedithiol and posttreat-ment with 6-mercapto-1-hexanol minimized the background signal andmaximized the signal-to-noise ratio. Inclusion of inter-nal calibration controls allowed accurate estimation of the pre-rRNA copy number per cell. As expected, the ratio of pre-rRNA to mature rRNAwas low during stationary phase and high during log phase. Pre-rRNA levels were highly dynamic, ranging from 2 copies per cell during stationary phase to1,200 copies per cell within 60 min of inoculation into fresh growthmedium. Speci-ficity of the assay for pre-rRNAwas validated using rifampin and chloramphenicol, which are known inhibitors of pre-rRNA synthesis and processing, respectively. The DNA gyrase inhibitor, ciprofloxacin, was found to act similarly to rifampin; a decline in pre-rRNAwas detectable within 15 min in ciprofloxacin-susceptible bacteria. Assays for pre-rRNA provide insight into cellu-lar metabolism and are promising predictors of antibiotic susceptibility. There is an urgent need for the development of rapid and con-venient methods for detection and identification of bacterial pathogens in clinical specimens to guide the diagnosis and treat-ment of infectious diseases. Electrochemical biosensors are mo