title: Rapid antimicrobial susceptibility testing by sensitive detection of precursor rRNA using a novel electrochemical biosensing platform.
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.
authors.affiliation: Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, USA.
authors.forename: Colin
authors.initials: C
authors.lastname: Halford
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Gonzalez
authors.forename: Susana
authors.initials: S
authors.lastname: Campuzano
authors.forename: Bo
authors.initials: B
authors.lastname: Hu
authors.forename: Jane T
authors.initials: JT
authors.lastname: Babbitt
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Joseph
authors.initials: J
authors.lastname: Wang
authors.forename: Bernard M
authors.initials: BM
authors.lastname: Churchill
authors.forename: David A
authors.initials: DA
authors.lastname: Haake
mesh_terms: D000890:Anti-Infective Agents / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D002701:Chloramphenicol / Q000378:metabolism; D002939:Ciprofloxacin / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D055664:Electrochemical Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D012322:RNA Precursors / Q000032:analysis* / Q000096:biosynthesis / Q000378:metabolism; D012335:RNA, Ribosomal / Q000032:analysis* / Q000096:biosynthesis; D012293:Rifampin / Q000378:metabolism; D059005:Topoisomerase II Inhibitors
chemical_list: D000890:Anti-Infective Agents; D012322:RNA Precursors; D012335:RNA, Ribosomal; D059005:Topoisomerase II Inhibitors; D002939:Ciprofloxacin; D002701:Chloramphenicol; D012293:Rifampin