Bioreduction of Tempone and spin-labeled gentamicin by gram-negative bacteria: kinetics and effect of ultrasound

The primary objective of this study is the investigation of bioreduction kinetics of hydrophilic spin probes, 2,2,6,6, -tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone), and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tensions, with emphasis on the effect of probe penetration rate. This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into Pseudomonas aeruginosa and Escherichia coli cells. Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR. In cell suspensions, both Tempone and spin-labeled gentamicin were localized predominantly in the aqueous phase surrounding the cells. However, a gradual reduction of the probes in contact with the cells indicated that the probes penetrated through the outer membrane and periplasmic space into the cytoplasmic membrane, where the electron transport chains and other metabolic activities of gram-negative bacteria are localized. The kinetics of probe reduction depended on oxygen tension and presence of electron transport chain blockers. It was found that probe penetration rate through the outer cell membrane affected the rate of probe reduction; damaging the permeability barrier by cell incubation with EDTA or by powerful insonation above the cavitation threshold increased the rate of probe reduction. In contrast, insonation below the cavitation threshold did not affect the rate of probe reduction. These findings imply that the recently observed synergistic effect between hydrophilic antibiotics and low frequency ultrasound in killing gram-negative bacteria did not result from the enhanced antibiotic penetration through bacterial cell walls.