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Rapid antimicrobial susceptibility testing by sensitive detection of precursor rRNA using a novel electrochemical biosensing platform.

Antimicrobial agents and chemotherapy (2012-12-12)
Colin Halford, Rodrigo Gonzalez, Susana Campuzano, Bo Hu, Jane T Babbitt, Jun Liu, Joseph Wang, Bernard M Churchill, David A Haake
ABSTRACT

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 ~1,200 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Chloramphenicol, ≥98% (HPLC)
Sigma-Aldrich
Chloramphenicol, BioReagent, suitable for plant cell culture
Supelco
Chloramphenicol, VETRANAL®, analytical standard
Sigma-Aldrich
Chloramphenicol, meets USP testing specifications
Sigma-Aldrich
Chloramphenicol, γ-irradiated
Chloramphenicol, European Pharmacopoeia (EP) Reference Standard