Early Events of Lethal Action by Tobramycin in Pseudomonas aeruginosa

The immediate activities of the aminoglycoside antibiotic, tobramycin, were investigated in Pseudomonas aeruginosa PAO1. The influence of carbon growth substrate and the antibiotic exposure environment in the magnitude of activity were examined. Lethality by 8 ug/ml tobramycin occurred rapidly (1 to 3 minutes). The release of specific cellular component into the supernatant was associated with lethality. This material was initially detected as an increase in UV-absorbance. Magnesium in the reaction mixture provided protection against lethality and leakage, but did not reverse lethal damage after a 3 minute tobramycin treatment. Also, uptake of ³H-tobramycin was reduced in the presence of magnesium. Cells grown with glucose as a carbon source were more susceptible than organic acid grown cells as was the rapidity and amount of cell damage. Analyses of the leakage material revealed a 2-fold increase of protein in the supernatant after a 1-3 minute treatment which paralleled lethality. A prominent 29kDa protein was observed by SDS-PAGE in the released material, which has been identified as the periplasmic enzyme, β-lactamase. Tobramycin also elicited an increase of certain amino acids in the supernatant, particularly, basic amino acids. The immediate activities of tobramycin did not involve i) release of overall cell protein, ii) massive loss of total pool amino acids, iii) cell lysis, iv) inhibition of proline uptake, v) release of lipopolysaccharide, or vi) leakage of ATP. Electron microscopy showed no apparent damage after a 3 minute exposure. Forty percent inhibition of protein synthesis had occurred by 3 minutes of exposure, while release of UV-absorbing material and lethality were detectable after only 1 minute. These data suggest that leakage occurs at least simultaneously, if not prior, to ribosomal interference. Resistant cystic fibrosis isolates of P. aeruginosa did not leak under the same experimental conditions, but one of two susceptible strains examined did show increased UV-absorbance following treatment.