Correlation of tobramycin-induced inhibition of protein synthesis with postantibiotic effect in Escherichia coli

Abstract

Scant data exist on intracellular events during aminoglycoside-induced postantibiotic effect (PAE). We examined DNA, RNA, and protein syntheses after tobramycin exposure using [3H]thymidine, [14C]uracil, and [14C]alanine incorporation in a clinical Escherichia coli strain. Late-log-phase bacteria in oxygenated minimal salts medium at 37 degrees C were exposed to tobramycin (7.5 micrograms/ml) (twice the MIC) for 30 min. Tobramycin caused a kill of 2 log10 CFU/ml prior to drug removal by filtration and a 5-h PAE, measured by viable counts. Excess amounts of labelled precursors were added to tobramycin-exposed organisms during, immediately after, and at various intervals following exposure. In the presence of tobramycin, DNA, RNA, and protein syntheses were sequentially inhibited within 1 generation time. Following drug removal, both DNA and RNA syntheses promptly resumed, suggesting readily dissociable nonspecific binding to DNA and RNA. However, total protein synthesis did not resume until 4 h later. beta-Galactosidase activity, a measure of functional enzymatic protein synthesis, was also inhibited for 4 h after drug removal. Bacterium length, measured by confocal microscopy, increased during PAE. Two distinct populations eventually emerged: one that returned to control dimensions and one that remained excessively elongated by the end of PAE (2.5 microns versus 4.0 microns; P < 0.05). We hypothesize that only viable cells return to the control morphology. Flow cytometry showed enhanced DNA complexity during PAE, consistent with either impaired cellular protein synthesis in viable cells or perturbations in dying cells. In summary, duration of PAE correlated with inhibition of total and functional protein synthesis but not DNA or RNA synthesis.