In vitro and in vivo antimicrobial activities of gallium nitrate against multidrug-resistant Acinetobacter baumannii

Abstract

Multidrug-resistant Acinetobacter baumannii poses a tremendous challenge to traditional antibiotic therapy. Due to the crucial role of iron in bacterial physiology and pathogenicity, we investigated iron metabolism as a possible target for anti-A. baumannii chemotherapy using gallium as an iron mimetic. Due to chemical similarity, gallium competes with iron for binding to several redox enzymes, thereby interfering with a number of essential biological reactions. We found that Ga(NO(3))(3), the active component of an FDA-approved drug (Ganite), inhibits the growth of a collection of 58 A. baumannii strains in both chemically defined medium and human serum, at concentrations ranging from 2 to 80 μM and from 4 to 64 μM, respectively. Ga(NO(3))(3) delayed the entry of A. baumannii into the exponential phase and drastically reduced bacterial growth rates. Ga(NO(3))(3) activity was strongly dependent on iron availability in the culture medium, though the mechanism of growth inhibition was independent of dysregulation of gene expression controlled by the ferric uptake regulator Fur. Ga(NO(3))(3) also protected Galleria mellonella larvae from lethal A. baumannii infection, with survival rates of ≥75%. At therapeutic concentrations for humans (28 μM plasma levels), Ga(NO(3))(3) inhibited the growth in human serum of 76% of the multidrug-resistant A. baumannii isolates tested by ≥90%, raising expectations on the therapeutic potential of gallium for the treatment of A. baumannii bloodstream infections. Ga(NO(3))(3) also showed strong synergism with colistin, suggesting that a colistin-gallium combination holds promise as a last-resort therapy for infections caused by pan-resistant A. baumannii.

Fig 1

A. baumannii ATCC 17978 growth (A), siderophore production (B), and activity of the basA::lacZ iron-regulated fusion (C). Bacteria were grown in different media for 24 or 48 h, in the absence (white bars) or presence (gray bars) of 100 μM FeCl₃. Data are the means (± standard deviations [SD]) of triplicate experiments.

Fig 2

Growth (A) and siderophore production (B) in a collection of 58 A. baumannii strains grown for 24 and 48 h in M9-DIP. Boxes represent medians and second and third interquartiles; whiskers represent range of 58 strains. White boxes, M9-DIP without added iron; gray boxes, M9-DIP supplemented with 100 μM FeCl₃. For each strain, the mean value of two independent microtiter plate assays was considered.

Fig 3

Inhibitory activity of Ga(NO₃)₃ on a collection of 58 A. baumannii strains grown in chemically defined medium. Plots show the Ga(NO₃)₃ concentrations (μM) required to inhibit the growth of each isolate by 50% (IC₅₀) and 90% (IC₉₀) at 24 and 48 h in M9-DIP. Boxes represent medians and second and third interquartiles; whiskers represent range of 58 strains. For each strain, the mean value of two independent microtiter plate assays was considered.

Fig 4

Effect of iron on the inhibitory activity of Ga(NO₃)₃. A. baumannii strains ACICU (A), A376 (B), A399 (C), and A451 (D) were grown for 48 h in M9-DIP supplemented with increasing FeCl₃ (0 to 50 μM) and Ga(NO₃)₃ (0 to 128 μM) concentrations. Growth is expressed as percentage relative to growth (OD₆₀₀) of control cultures in M9-DIP.

Fig 5

Effect of Ga(NO₃)₃ on the activity of the Fur-Fe(II)-regulated basA::lacZ transcriptional fusion. A. baumannii ATCC 17978 carrying plasmid pMP220::PbasA was grown in M9-DIP supplemented or not supplemented with increasing FeSO₄ (1.25, 2.5, 5, or 10 μM) and/or Ga(NO₃)₃ (6.25 or 12.5 μM) concentrations. LacZ activity was measured at 48 h and expressed in Miller units (37). Data are the means (±SD) of triplicate experiments.

Fig 6

Inhibitory activity of Ga(NO₃)₃ on a collection of 57 A. baumannii strains grown in human serum. Strains were grown for 48 h in serum supplemented with increasing Ga(NO₃)₃ concentrations (0 to 128 μM). Plots show the Ga(NO₃)₃ concentrations (μM) required to inhibit the growth of each isolate by 50% (IC₅₀) and 90% (IC₉₀) at 48 h. Boxes represent medians and second and third interquartiles; whiskers represent range of 57 strains. For each strain, the mean value of two independent microtiter plate assays was considered.

Fig 7

Effect of Ga(NO₃)₃ on the viability of G. mellonella caterpillars infected with a lethal dose of the following A. baumannii strains: ATCC 17978 (∼ 5 × 10⁶ CFU/larva) (A), RUH 5875 (∼ 1 × 10⁷ CFU/larva) (B), AYE (∼ 5 × 10⁶ CFU/larva) (C), ACICU (∼ 7 × 10⁶ CFU/larva) (D), 50C (∼ 1 × 10⁶ CFU/larva) (E), and A371 (∼ 6 × 10⁵ CFU/larva) (F). Solid lines, Ga(III)-untreated larvae; dotted lines, Ga(III)-treated larvae.