Molecular investigation of the postantibiotic effects of clarithromycin and erythromycin on Staphylococcus aureus cells

Abstract

The kinetics of recovery after inhibition of growth by erythromycin and clarithromycin were examined in Staphylococcus aureus cells. After inhibition for one mass doubling by 0.5 microg of the antibiotics/ml, a postantibiotic effect (PAE) of 3 and 4 h duration was observed for the two drugs before growth resumed. Cell viability was reduced by 25% with erythromycin and 45% with clarithromycin compared with control cells. Erythromycin and clarithromycin treatment reduced the number of 50S ribosomal subunits to 24 and 13% of the number found in untreated cells. 30S subunit formation was not affected. Ninety minutes was required for resynthesis to give the control level of 50S particles. Protein synthesis rates were diminished for up to 4 h after the removal of the macrolides. This continuing inhibition of translation was the result of prolonged binding of the antibiotics to the 50S subunit as measured by 14C-erythromycin binding to ribosomes in treated cells. The limiting factors in recovery from macrolide inhibition in these cells, reflected as a PAE, are the time required for the synthesis of new 50S subunits and the slow loss of the antibiotics from ribosomes in inhibited cells.

FIG. 1

Growth rates and viable-cell numbers for control and macrolide-treated cells. The increase in cell density (□) and cell number (●) was measured for control (A) and erythromycin (Ery) (B)- or clarithromycin (Clr) (C)-treated cells before and after antibiotic removal. The duration of antibiotic treatment is indicated by a bracket. The cell viability results are the averages of five experiments, with a standard error of ±20%.

FIG. 2

Sucrose gradient profiles of ribosomal subunits labeled with [³H]uracil before and after antibiotic removal. Profiles of clarithromycin-treated cells before (A) and at 30 (B) and 60 (C) min after washing and resuspension are shown. (D) Profile of untreated cells after washing and resuspension. Sedimentation is from left to right.

FIG. 3

Kinetics of 50S ribosomal subunit re-formation. The percentages of the total gradient cpm in the 30S and 50S subunit regions of the sucrose gradients are shown for untreated cells (■) and for cells recovering from erythromycin (▴) and clarithromycin (●) treatments. 30S subunit levels at 0 and 180 min are also shown (□). The results are the averages of five experiments, with a standard error of ±4%.

FIG. 4

Rates of ³⁵S amino acid incorporation into proteins in control and antibiotic-treated cells before and after antibiotic removal. (A) Protein synthesis rates in control cells at 0 (□), 60 (◊), 90 (○), and 180 (▵) min. (B) Protein synthesis rates in erythromycin-treated cells at 0 (□), 60 (◊), 90 (○), and 180 min (▵). (C) Protein synthesis rates in clarithromycin-treated cells at 0 (□), 60 (◊), 90 (○), and 180 min (▵). (D) Relative rates of protein synthesis (³⁵S cpm/10 min) for untreated cells (□) and for erythromycin (◊)- and clarithromycin (○)-treated cells following antibiotic removal. The results are the averages of four experiments, with a standard error of ±4%.

FIG. 5

Sucrose gradient profiles of cells labeled with ¹⁴C-erythromycin ([¹⁴C]ery) (●) before washing and resuspension and with [³H]uracil (□) afterwards. (A) Cells collected before erythromycin removal. (B) Cells collected 15 min after erythromycin removal. (C) Cells collected after 30 min. (D) Cells collected after 60 min. (E) Cells collected after 120 min. (F) Cells collected after 180 min.

FIG. 6

Kinetics of ¹⁴C-erythromycin ([¹⁴C]ery) retention and loss from cells and ribosomes after washing and resuspension. Relative ¹⁴C-erythromycin cpm in cells (■) and in culture medium (●) and loss of erythromycin (Ery) bound to 50S subunits (▴) are shown.