Detection of rifampin-resistant bacteria using DNA probes for precursor rRNA.
Ribosomal RNA precursor (pre-rRNA) molecules have terminal domains (tails) which are removed during late steps in rRNA processing, to yield the mature rRNA subunits. Transcriptional inhibitors such as rifampin can deplete pre-rRNA in sensitive cells by inhibiting de novo pre-rRNA synthesis while allowing maturation to proceed. We developed direct DNA probe assays for pre-rRNA tail sequences of Escherichia coli, and evaluated their ability to rapidly distinguish rifampin-resistant from rifampin-sensitive strains in cultures treated with the drug. Pre-rRNA became undetectable in sensitive cells less than a generation time after rifampin exposure, but remained abundant in resistant cells. Resistant cells were detectable by this method against a 100-fold excess of sensitive cells, showing that this method can detect resistant mutants even when present as a small percentage of a pathogen population. Our data indicate that the response of pre-rRNA to antibiotic treatment is sufficient in rate and magnitude to make it a useful metabolic marker for antibiotic sensitivity.
Britschgi
T B
TB
MicroProbe Corporation, Diagnostics Division, Bothell, WA 98021, USA.
Cangelosi
G A
GA
D001483:Base Sequence; D015342:DNA Probes; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D005821:Genetic Techniques; D015151:Immunoblotting / Q000379:methods; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012322:RNA Precursors / Q000032:analysis / Q000096:biosynthesis*; D012329:RNA, Bacterial / Q000096:biosynthesis; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000096:biosynthesis*; D012293:Rifampin / Q000494:pharmacology; D013045:Species Specificity
D015342:DNA Probes; D015345:Oligonucleotide Probes; D012322:RNA Precursors; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012293:Rifampin