Research ArticleDrug Development

Gallium disrupts bacterial iron metabolism and has therapeutic effects in mice and humans with lung infections

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Science Translational Medicine  26 Sep 2018:
Vol. 10, Issue 460, eaat7520
DOI: 10.1126/scitranslmed.aat7520
  • Fig. 1 Expectorated sputum from CF patients is iron-limited.

    (A) Effect of adding iron trichloride (FeCl3) on the growth rate and cell yield of P. aeruginosa in sputum supernatants. Results are representative of five sputum samples (see figs. S1 and S2). Error bars indicate SEM. *P < 0.01 versus no iron addition, Student’s t test. (B and C) Effect of adding iron trichloride (FeCl3) on the expression of the pyoverdine biosynthetic gene pvdA (B) and on pyoverdine production (C) by P. aeruginosa in CF sputum. Results are mean of two replicates. Error bars indicate SEM. *P < 0.01 versus no iron addition, Student’s t test (also see fig. S1 for experiments with sputum from another subject). OD600, optical density at 600 nm; RFU, relative fluorescence units.

  • Fig. 2 Gallium inhibits P. aeruginosa growth in CF sputum.

    Gallium’s effect on P. aeruginosa growth and cell yield in CF sputum supernatants that were not (A) and were (B) supplemented with iron trichloride (FeCl3). Results are mean of three replicates. *P < 0.01 versus no gallium, Student’s t test; #P < 0.01 versus no added iron, Student’s t test.

  • Fig. 3 Gallium inhibits P. aeruginosa catalase and ribonucleotide reductase, but not SOD or aconitase activity.

    Effect of gallium on the activity of ribonucleotide reductase (A), aconitase (B), catalase (C), and SOD (D). Results shown are representative of 3 to 37 experiments and are mean enzyme activity measurements relative to bacteria not treated with gallium. Error bars indicate SEM. *P < 0.05 versus no gallium, analysis of variance (ANOVA).

  • Fig. 4 Gallium increases P. aeruginosa sensitivity to peroxides.

    Effect of subinhibitory concentrations of gallium on P. aeruginosa’s sensitivity to oxidants generating peroxide, including H2O2 (A) and tert-butyl hydroperoxide (tert-butyl) (B); and superoxide, including PQ (C) and PMS (D). Subinhibitory gallium increased sensitivity to killing by peroxides. Data are mean values of three to four replicate experiments. Error bars indicate SEM. *P < 0.01, Student’s t test. CFU, colony-forming units.

  • Fig. 5 Continuous passaging increases gallium and antibiotic resistance.

    Effect of passaging wild-type (A to C) and ΔhitAB P. aeruginosa (D) in gallium (A and D), aztreonam (B), and tobramycin (C). The mean fold change in highest drug concentration that permitted growth (of 12 replicate cultures) is plotted as a function of the passaging day. Error bars indicate SEM. *P < 0.01 versus the highest drug concentrations that permitted growth before passaging (see Methods), Wilcoxon matched-pairs signed-rank test. #P < 0.01 versus the fold change of wild-type P. aeruginosa after passaging in gallium, Mann-Whitney. §P < 0.01 versus the fold change of ΔhitAB P. aeruginosa after passaging in gallium, Mann-Whitney.

  • Fig. 6 Gallium has synergistic activity with antibiotics.

    Combined effect of gallium with colistin (A, D, and G), piperacillin/tazobactam (B, E, and H), and tobramycin (C, F, and I). Photographs (A to C) show disc diffusion assays. The yellow dashed lines represented the expected activity (in preventing P. aeruginosa growth) of the antibiotic in the absence of gallium. Graphs (D to F) show time-kill assays using subinhibitory concentrations of gallium and inhibitory concentrations of antibiotics. Error bars indicate SEM. *P < 0.05, Student’s t test. Isobolograms (G to I) show results of checkerboard assays presented as the fractional inhibitory concentrations (FICs) of the two factors in combination. Calculations are described in Methods. Experiments were repeated two to four times, each with similar results. Abs, antibiotics.

  • Fig. 7 Parenteral gallium treats murine lung infections.

    (A) Effect of a single intraperitoneal (IP) dose of gallium-free vehicle (red line) or gallium [50 μl of 250 mM Ga(NO3)3] administered 3 hours (blue line) or 12 hours after (green line) intratracheal infection with P. aeruginosa (n = 7 mice for gallium and n = 8 mice for vehicle). *P < 0.001 versus vehicle control, Fisher test. (B) P. aeruginosa counts in bronchoalveolar lavage (BAL) fluid and blood 12 hours after mice were infected by the intratracheal route and treated with vehicle (intraperitoneal) or gallium (intraperitoneal) 3 hours after infection (n = 4 mice for vehicle alone and n = 5 mice for gallium). *P < 0.001 versus vehicle control, Fisher test. (C) Effect of intranasal (IN) iron-free vehicle (blue line) or iron (10 μl of 2 mM FeCl3) (black line) on the antibiotic effect of intraperitoneal gallium. *P < 0.001 versus vehicle control, Fisher test; #P < 0.05 versus vehicle control, Fisher test. The red line shows mouse survival without gallium (vehicle administered intraperitoneally and intranasally) (n = 6 mice in each group).

  • Fig. 8 Intravenous gallium produces sustained blood and sputum concentrations and improves lung function.

    Plasma (A) and sputum (B) gallium concentrations in CF subjects treated with intravenous gallium for 5 days; boxes show 25th to 75th percentiles, hatches shows means, and whiskers show minimum and maximum values. Data shown are for cohorts 1 and 2 (100 mg/m2 per day and 200 mg/m2 per day, respectively) combined (see fig. S8 for data from each cohort separated). Mean change in lung function (as measured by FEV1 in mls) by study day for both cohorts pooled together (C) and cohorts 1 (D) and 2 (E) separately (100 mg/m2 per day and 200 mg/m2 per day, respectively). Bars represent the 95% confidence intervals (CIs). P values are indicated on graphs. Inferential analyses of the change from baseline tested the null hypothesis that the change is equal to zero using paired t tests with normally distributed data with 95% CIs.

  • Table 1 Frequency of spontaneous P. aeruginosa mutants.
    Antibacterial agentMutation frequency
    Gallium (64 μM)2.90 × 10−8 ± 1.35 × 10−8
    Tobramycin (4 μg/ml)3.52 × 10−8 ± 1.80 × 10−8
    Colistin (16 μg/ml)5.80 × 10−8 ± 3.00 × 10−9
    Ciprofloxacin (4 μg/ml)8.48 × 10−8 ± 2.13 × 10−8
  • Table 2 Lung function and microbiology in CF patients at baseline, day 14, and day 28. NA, not applicable; IQR, interquartile range.
    EndpointMean and median
    baseline (day 0)
    Mean and median
    change from
    days 0–14
    PMean and median
    change from
    days 0–28
    P
    All patients (n = 20)
    FEV1 (liter)
    Mean (SD)
    Median (IQR)
    2.24 (0.84)
    2.1 (1.68 to 2.58)
    0.13 (0.17)
    0.075 (0.015 to 0.22)
    0.0041
    0.0064
    0.10 (0.14)
    0.085 (0.00 to 0.18)
    0.0041
    0.0042
    Proportion with 5%
    change in FEV1
    (95% CI)
    NA50%
    (27 to 73%)
    45%
    (23 to 69%)
    FVC (liter)
    Mean (SD)
    Median (IQR)
    3.58 (1.0)
    3.53 (3.14 to 4.04)
    0.13 (0.15)
    0.12 (0.01 to 0.23)
    0.0010
    0.0007
    0.16 (0.15)
    0.15 (0.045 to 0.24)
    0.0001
    0.0001
    Sputum P. aeruginosa
    (million CFU/gm)
    n = 19n = 190.5949n = 170.5171
    Mean (SD)
    Median (IQR)
    117 (200)
    48.4 (5.5 to 137.5)
    −5.51 (218)
    −1.83 (−87 to 74)
    0.9134−29.8 (171)
    −3.9 (−105 to 55)
    0.4825

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/460/eaat7520/DC1

    Methods

    Fig. S1. Expectorated sputum from CF patients is iron limited.

    Fig. S2. Iron increases bacterial growth rate in CF sputum.

    Fig. S3. Gallium inhibits P. aeruginosa growth in CF sputum.

    Fig. S4. Transposon mutations in PA5248 or the pvdA-fpvI intergenic region modestly increases gallium resistance.

    Fig. S5. Gallium’s activity used in combination with antibiotics.

    Fig. S6. Gallium does not attenuate P. aeruginosa killing by human macrophages.

    Fig. S7. Intravenous gallium produces minimal changes in red blood cell parameters.

    Fig. S8. Intravenous gallium produces sustained blood and sputum concentrations.

    Table S1. Results of transposon mutagenesis screening for gallium resistant mutants.

    Table S2. Full inclusion and exclusion criteria for clinical trial.

    Table S3. Enrollment table.

    Table S4. Safety laboratory assessments in CF patients at baseline, day 6, day 14, and day 28.

    Table S5. Pharmacokinetics of gallium.

    Table S6. Lung function and microbiology in CF patients at baseline, day 14, and day 28 by dosing cohort.

    Table S7. Correlation of change in lung function with sputum gallium pharmacokinetics.

    References (4752)

  • This PDF file includes:

    • Methods
    • Fig. S1. Expectorated sputum from CF patients is iron limited.
    • Fig. S2. Iron increases bacterial growth rate in CF sputum.
    • Fig. S3. Gallium inhibits P. aeruginosa growth in CF sputum.
    • Fig. S4. Transposon mutations in PA5248 or the pvdA-fpvI intergenic region modestly increases gallium resistance.
    • Fig. S5. Gallium’s activity used in combination with antibiotics.
    • Fig. S6. Gallium does not attenuate P. aeruginosa killing by human macrophages.
    • Fig. S7. Intravenous gallium produces minimal changes in red blood cell parameters.
    • Fig. S8. Intravenous gallium produces sustained blood and sputum concentrations.
    • Table S1. Results of transposon mutagenesis screening for gallium resistant mutants.
    • Table S2. Full inclusion and exclusion criteria for clinical trial.
    • Table S3. Enrollment table.
    • Table S4. Safety laboratory assessments in CF patients at baseline, day 6, day 14, and day 28.
    • Table S5. Pharmacokinetics of gallium.
    • Table S6. Lung function and microbiology in CF patients at baseline, day 14, and day 28 by dosing cohort.
    • Table S7. Correlation of change in lung function with sputum gallium pharmacokinetics.
    • References (4752)

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