Two Mechanisms of Killing of Pseudomonas aeruginosa by Tobramycin Assessed at Multiple Inocula via Mechanism-Based Modeling

Abstract

ABSTRACTBacterial resistance is among the most serious threats to human health globally, and many bacterial isolates have emerged that are resistant to all antibiotics in monotherapy. Aminoglycosides are often used in combination therapies against severe infections by multidrug-resistant bacteria. However, models quantifying different antibacterial effects of aminoglycosides are lacking. While the mode of aminoglycoside action on protein synthesis has often been studied, their disruptive action on the outer membrane of Gram-negative bacteria remains poorly characterized. Here, we developed a novel quantitative model for these two mechanisms of aminoglycoside action, phenotypic tolerance at high bacterial densities, and adaptive bacterial resistance in response to an aminoglycoside (tobramycin) against threePseudomonas aeruginosastrains. At low-intermediate tobramycin concentrations (<4 mg/liter), bacterial killing due to the effect on protein synthesis was most important, whereas disruption of the outer membrane was the predominant killing mechanism at higher tobramycin concentrations (≥8 mg/liter). The extent of killing was comparable across all inocula; however, the rate of bacterial killing and growth was substantially lower at the 108.9CFU/ml inoculum than that at the lower inocula. At 1 to 4 mg/liter tobramycin for strain PAO1-RH, there was a 0.5- to 6-h lag time of killing that was modeled via the time to synthesize hypothetical lethal protein(s). Disruption of the outer bacterial membrane by tobramycin may be critical to enhance the target site penetration of antibiotics used in synergistic combinations with aminoglycosides and thereby combat multidrug-resistant bacteria. The two mechanisms of aminoglycoside action and the new quantitative model hold great promise to rationally design novel, synergistic aminoglycoside combination dosage regimens.