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Analytical Procedures

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

Colin Halford, Rodrigo Gonzalez, Susana Campuzano, Bo Hu, Jane T. Babbitt, Jun Liu, Joseph Wang, Bernard M. Churchill, David A. Haake
Colin Halford
aVeterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, USA
bDepartments of Urology, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, California, USA
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Rodrigo Gonzalez
aVeterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, USA
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Susana Campuzano
eDepartment of Nanoengineering, University of California, San Diego, La Jolla, California, USA
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Bo Hu
fDepartment of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, Texas, USA
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Jane T. Babbitt
aVeterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, USA
cMedicine, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, California, USA
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Jun Liu
fDepartment of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, Texas, USA
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Joseph Wang
eDepartment of Nanoengineering, University of California, San Diego, La Jolla, California, USA
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Bernard M. Churchill
bDepartments of Urology, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, California, USA
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David A. Haake
aVeterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, USA
bDepartments of Urology, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, California, USA
cMedicine, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, California, USA
dMicrobiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, California, USA
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DOI: 10.1128/AAC.00615-12
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    Fig 1

    Regions targeted by pre-rRNA probe pairs. The structures of the 16S and 23S pre-rRNA molecules are shown, including locations of mature rRNA termini (arrows) and regions targeted by electrochemical sensor probe pairs for the 16S 5′ tail (blue letters) and 16S and 23S splice sites (red letters).

  • Fig 2
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    Fig 2

    Comparison of pre-rRNA probe pairs. Probe pairs were tested for detection of E. coli in the log and stationary phases of growth. Log- and stationary-phase cells are expected to have high and low levels of pre-rRNA, respectively, yielding a high signal ratio for log-phase versus stationary-phase cells. Probe pairs for 16S pre-rRNA had good sensitivity for log-phase cells, but the signal ratio for log-phase versus stationary-phase cells was low. Some probe pairs targeting the splice sites at the termini of mature 16S and 23S rRNA had higher signal ratios for log-phase versus stationary-phase cells. The probe pair targeting the splice site at the 3′ terminus of 23S rRNA had the best combination of sensitivity and high signal ratio of log-phase versus stationary-phase cells.

  • Fig 3
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    Fig 3

    Variations in pre-rRNA and rRNA levels during E. coli growth. (A) Changes in mature rRNA, pre-rRNA, and their ratio were measured in overnight cultures that were subsequently inoculated into fresh MH growth medium and incubated for 7 h at 37°C. (B) Comparison of the mature/pre-RNA ratio and growth rate during different phases of growth. The growth rate curve is a weighted average determined from the change in CFU during each 30-min time period. Error bars estimated the standard deviations.

  • Fig 4
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    Fig 4

    E. coli cell volume versus rRNA copy number during different growth phases. (A) Correlation between the cell volume and rRNA copy number per cell at densities below an OD600 of ≤1.0. (B) Electron micrographs demonstrating progressively smaller cells over incubation time from log phase (2.5 h) to stationary phase (7 h). Error bars estimated the standard deviations.

  • Fig 5
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    Fig 5

    (A to D) Response of mature rRNA and pre-rRNA to antibiotics. Antibiotics have differential effects on rRNA and pre-rRNA. Rifampin and ciprofloxacin selectively inhibited transcription of new pre-rRNA, while the addition of chloramphenicol and gentamicin resulted in a selective decrease in mature rRNA. Error bars estimated the standard deviations.

  • Fig 6
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    Fig 6

    Comparison of ciprofloxacin-susceptible and -resistant E. coli strains. rRNA and pre-rRNA were measured in cultures of an E. coli clinical isolate susceptible to ciprofloxacin (EC103) and three ciprofloxacin-resistant isolates (EC135, EC139, and EC197). The amount of pre-rRNA in strain EC103 was significantly lower than that of the ciprofloxacin-resistant isolates within 15 min after addition of the antibiotic. Error bars estimated the standard deviations.

Tables

  • Figures
  • Table 1

    DNA oligonucleotides used in this study

    Probe nameaSequenceb
    Pre16S 15 m 48D5′-TTTTTCGTCTTGCGA-F
    Pre16S 15 m 63C5′-B-GAGACTTGGTATTCA
    Pre16S 15 m R63D5′-F-GAGACTTGGTATTCA
    Pre16S 15 m R48C5′-TTTTTCGTCTTGCGA-B
    Pre16S 17 m R63D5′-F-TTGAGACTTGGTATTCA
    Pre16S 17 m R46C5′-TTTTTCGTCTTGCGACG-B
    Pre16S 19 m R63D5′-F-TCTTGAGACTTGGTATTCA
    Pre16S 19 m R44C5′-TTTTTCGTCTTGCGACGTT-B
    Pre16S 21 m R63D5′-F-ACTCTTGAGACTTGGTATTCA
    Pre16S 21 m R42C5′-TTTTTCGTCTTGCGACGTTAA-B
    Pre16S 17 m R60D5′-F-AGACTTGGTATTCATTT
    Pre16S 17 m R43C5′-TTCGTCTTGCGACGTTA-B
    Pre16S 19 m R60D5′-F-TGAGACTTGGTATTCATTT
    Pre16S 19 m R41C5′-TTCGTCTTGCGACGTTAAG-B
    Pre16S 21 m R60D5′-F-CTTGAGACTTGGTATTCATTT
    Pre16S 21 m R39C5′-TTCGTCTTGCGACGTTAAGAA-B
    Pre16S 17 m R66D5′-F-CTCTTGAGACTTGGTAT
    Pre16S 17 m R49C5′-TCATTTTTCGTCTTGCG-B
    Pre16S 19 m R66D5′-F-CACTCTTGAGACTTGGTAT
    Pre16S 19 m R47C5′-TCATTTTTCGTCTTGCGAC-B
    Pre16S 21 m R66D5′-F-TTCACTCTTGAGACTTGGTAT
    Pre16S 21 m R45C5′-TCATTTTTCGTCTTGCGACGT-B
    Pre16S 19 m 5′JxnD5′-TTTGATGCTCAAAGAATTA-F
    Pre16S 21 m 5′JxnC5-S-TCAAACTCTTCAATTTAAAAG
    Pre16S 21 m R5′JxnD5-F-TCAAACTCTTCAATTTAAAAG
    Pre16S 19 m R5′JxnC5′-TTTGATGCTCAAAGAATTA-S
    Pre16S 17 m 3′JxnD5′-GAGGTGATCCAACCGCA-F
    Pre16S 20 m 3′JxnC5-S-GAACGCTTCTTTAAGGTAAG
    Pre16S 20 m R3′JxnD5-F-GAACGCTTCTTTAAGGTAAG
    Pre16S 17 m R3′JxnC5′-GAGGTGATCCAACCGCA-S
    Pre23S 17 m 3′JxnDc5′-AAGCCTCACGGTTCATT-F
    Pre23S 14 m 3′JxnCc5′-S-GGCGTTGTAAGGTT
    Pre23S 14 m R3′JxnD5′-F-GGCGTTGTAAGGTT
    Pre23S 17 m R3′JxnC5′-AAGCCTCACGGTTCATT-S
    Mature rRNA 18 m 1484D5′-GTTACGACTTCACCCCAG-F
    Mature rRNA 19 m 1502C5′-S-GTTCCCCTACGGTTACCTT
    Synthetic target oligonucleotides
    Pre-rRNA 31 m5′-AATGAACCGTGAGGCTTAACCTTACAACGCC
    Mature rRNA 37 m5′-CTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAAC
    • ↵a m, number of nucleotides; C, capture probe; D, detector probe; Jxn, splice site; R, reverse orientation.

    • ↵b F, FITC; B, biotin; S, thiol.

    • ↵c Capture and detector probe pair selected based on its high signal-to-noise ratio.

  • Table 2

    Pre-rRNA and mature rRNA quantitation during E. coli growth phases

    Time point (min)OD600CFU/mlRateaDoublingbGeneration time (min)rRNAcPre-rRNAcRatioGrowth phase
    Overnight culture2.3076.67E + 096,00922,720Stationary phase
    005.90E + 055,94211054Lag phase
    3005.70E + 0528,42745762
    600.0038.63E + 050.181.2050.0855,27869679Log phase
    900.0082.30E + 060.432.8321.1978,74197081
    1200.0246.20E + 060.432.8621.0091,049120076
    1500.0681.91E + 070.493.2418.5198,782523189
    1800.1756.53E + 070.533.5516.8861,571235262
    2100.4092.30E + 080.553.6416.5038,033208183
    2400.7075.27E + 080.362.3925.1429,801114260Early stationary phase
    2701.0951.17E + 090.352.3026.1419,61042466
    3001.4662.33E + 090.302.0030.0013,16225536
    3301.6863.23E + 090.140.9463.747,60811665Stationary phase
    3601.8344.43E + 090.140.9165.875,7348723
    3901.9575.03E + 090.060.37163.814,8997706
    4202.0515.73E + 090.060.38159.686,23061,049
    • ↵a Growth rate in log units per 30 min.

    • ↵b Doublings per hour.

    • ↵c Copies per cell.

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Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor rRNA Using a Novel Electrochemical Biosensing Platform
Colin Halford, Rodrigo Gonzalez, Susana Campuzano, Bo Hu, Jane T. Babbitt, Jun Liu, Joseph Wang, Bernard M. Churchill, David A. Haake
Antimicrobial Agents and Chemotherapy Jan 2013, 57 (2) 936-943; DOI: 10.1128/AAC.00615-12

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Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor rRNA Using a Novel Electrochemical Biosensing Platform
Colin Halford, Rodrigo Gonzalez, Susana Campuzano, Bo Hu, Jane T. Babbitt, Jun Liu, Joseph Wang, Bernard M. Churchill, David A. Haake
Antimicrobial Agents and Chemotherapy Jan 2013, 57 (2) 936-943; DOI: 10.1128/AAC.00615-12
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