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

Abstract

f Precursor rRNA (pre-rRNA) is an intermediate stage in the formation of mature rRNA and is a useful marker for cellular metabolism 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 rRNA maturation. A ternary self-assembled monolayer (SAM) prepared on gold electrode surfaces by coassembly of thiolated capture probes with hexanedithiol and posttreatment with 6-mercapto-1-hexanol minimized the background signal and maximized the signal-to-noise ratio. Inclusion of internal calibration controls allowed accurate estimation of the pre-rRNA copy number per cell. As expected, the ratio of pre-rRNA to mature rRNA was low during stationary phase and high during log phase. Pre-rRNA levels were highly dynamic, ranging from 2 copies per cell during stationary phase to ϳ1,200 copies per cell within 60 min of inoculation into fresh growth medium. Specificity of the assay for pre-rRNA was 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-rRNA was detectable within 15 min in ciprofloxacin-susceptible bacteria. Assays for pre-rRNA provide insight into cellular metabolism and are promising predictors of antibiotic susceptibility.

New and developing diagnostic technologies for urinary tract infections

“…In a variation of this biosensor-based AST, the level of precursor rRNAs (pre-rRNAs), an intermediate state in the formation of mature rRNA and a marker for cell growth, is assessed. In this assay, a decrease in pre-rRNAs indicates reduced growth and sensitivity to antibiotics in the culture media 123 .…”

Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

“…Precursor rRNA (pre-rRNA) is an intermediate stage in the formation of mature rRNA and has been used as a marker for bacterial metabolism, viability, and growth rate. 42 Antibiotic resistance genes (e.g., mecA gene for MRSA; vanA/B genes for VRE; TEM, SHV, OXA, and CTX-M gene families for ESBLs; 43,44 and KPC, NDM, OXA-48, VIM, and IMP genes for CREs 45 ), as well as antibiotic-responsive mRNA transcripts, 46 have been widely used as markers for rapid AST. Compared with DNA targets, bacterial RNA markers are more abundant in the cell, and can potentially be indicative of viability to differentiate live and dead bacteria, but are more prone to degradation.…”

Tailoring Sensitivity in Electrochemical Nucleic Acid Hybridization Biosensing: Role of Surface Chemistry and Labeling Strategies

“…The use of ternary mixed monolayers comprising two different thiols as diluents/spacers together with an appropriate SHCP self‐assembled on gold electrodes provides interfaces with extremely attractive characteristics for electrochemical biosensing of target nucleic acids hybridization,,,, and aptasensing …”

“…Wang's research group was pioneer in the development of ternary DNA SAM‐interfaces prepared by adding, instead of a short monothiol, a short dithiol as co‐adsorbent/backfiller to the common binary SHCP+MCH SAM ,,,,. These ternary SAMs were prepared by co‐immobilization of a short cyclic or linear,, dithiol with the SHCP, followed by chemisorption of MCH in a subsequent step, and provided large signal‐to‐blank ratios. This behavior was attributed to an efficient passivation of the free gold surface, probably related to a lying flat positioning of the dithiol covering cavities that are strong unspecific adsorption points, without sacrificing the electron transfer through the sensing layer.…”