DETECTION OF RIFAMPIN-RESISTANT BACTERIA USING DNA PROBES FOR PRECURSOR RIBOSOMAL-RNA

Ribosomal RNA precursor (pre-rRNA) molecules have terminal domains (fails) 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.