PMC

Accumulation of 20 μg/ml [³H]gentamicin by B. vietnamiensis and 5 μg/ml [³H]gentamicin by P. aeruginosa ATCC 27853. Baseline accumulation was set as 0. MICs of gentamicin are shown in parentheses. Data points represent the averages for at least three biological replicates ± standard errors. *, P < 0.05 compared with B. vietnamiensis D0774 (unpaired Student t test). Bv, B. vietnamiensis; Pa, P. aeruginosa; GEN, gentamicin.

B. vietnamiensis lipid A structural analysis. C8395 (A), C8952 (B), and D0774 (C) are sequential isolates from patient Bv1. D0099 (D) and D2075 (E) are sequential isolates from patient Bv2. (F) D1389 is an isolate from patient Bv3. MICs of gentamicin are shown in parentheses. Purified lipid A was analyzed by MALDI-TOF mass spectroscopy. Tetra- and penta-acylated molecules are identified by m/z 1,444, 1,469, and 1,495 and m/z 1,670 and 1,696, respectively. Lipid A moieties containing Ara4N are identified by m/z 1,575, 1,601, 1,802, and 1,827. Arrows point to changes in acylation between sequential isolates. Bv, B. vietnamiensis; m/z, mass-to-charge ratio.

Permeabilizing effects of gentamicin on B. vietnamiensis and P. aeruginosa ATCC 27853. NPN was added to cells 30 s after initiation of fluorescence readings; antibiotic was added 30 to 90 s later. Final values were taken as averages of those recorded from 200 to 500 s, when a plateau in fluorescence was observed. Fluorescence was measured at least every 10 s. Baseline NPN fluorescence was set to 1. MICs of gentamicin are shown in parentheses. Data points represent the averages for at least three biological replicates plus standard errors. Bv, B. vietnamiensis; GEN, gentamicin.

B. vietnamiensis acquisition of aminoglycoside resistance in vivo and in vitro. Chronic B. vietnamiensis infections in CF patients Bv1 (A), Bv2 (B), and Bv3 (C) were evaluated based on clinical chart data. Patient FEV₁ data are shown as open circles. MICs for the infecting B. vietnamiensis strains are shown as bar graphs and represent triplicate experiments. In cases where multiple isolates from the same day were tested, only the highest MIC is shown (where there was a difference, it was 2-fold). Filled arrows indicate start dates of intravenous tobramycin (TOB) treatment. Open arrows indicate start dates of inhaled tobramycin treatment. A cross refers to the time of patient death. (D) Selection of aminoglycoside resistance in early isolates from patients Bv1 and Bv3 was done under tobramycin pressure in vitro. Isolates were serially passaged every 24 h in medium containing the antibiotic, to concentrations that represent half the MICs of late isolates (16 μg/ml for D0072 and 64 μg/ml for C8395). Susceptibility data for isolates grown at half the MICs of late isolates are not shown due to their lack of viability during analysis.