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Antimicrob Agents Chemother. 2013 Feb;57(2):936-43. doi: 10.1128/AAC.00615-12. Epub 2012 Dec 10.

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

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1
Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, USA.

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.

PMID:
23229486
PMCID:
PMC3553690
DOI:
10.1128/AAC.00615-12
[Indexed for MEDLINE]
Free PMC Article
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