Antibiotic-Resistant Acinetobacter baumannii Is Susceptible to the Novel Iron-Sequestering Anti-infective DIBI In Vitro and in Experimental Pneumonia in Mice

Abstract

Acinetobacter baumannii is a major cause of nosocomial infections especially hospital-acquired pneumonia. This bacterium readily acquires antibiotic resistance traits and therefore, new treatment alternatives are urgently needed. The virulence of A. baumannii linked to iron acquisition suggests a potential for new anti-infectives that target its iron acquisition. DIBI, a 3-hydroxypyridin-4-one chelator, is a purpose-designed, iron-sequestering antimicrobial that has shown promise for treating microbial infection. DIBI was investigated for its in vitro and in vivo activities against clinical A. baumannii isolates. DIBI was inhibitory for all isolates tested with very low MICs (2 μg/ml, equivalent to 0.2 μM), i.e., at or below the typical antibiotic MICs reported for antibiotic-sensitive strains. DIBI inhibition is Fe specific, and it caused an iron-restricted bacterial physiology that led to enhanced antibiotic killing by several discrete antibiotics. DIBI also strongly suppressed recovery growth of the surviving population following antibiotic exposure. A low intranasal dose (11 μmol/kg) of DIBI after intranasal challenge with hypervirulent ciprofloxacin (CIP)-resistant A. baumannii LAC-4 significantly reduced bacterial burdens in mice, and DIBI also suppressed the spread of the infection to the spleen. Treatment of infected mice with CIP alone (20 mg/kg, equivalent to 60 μmol/kg) was ineffective given LAC-4's CIP resistance, but if combined with DIBI, the treatment efficacy improved significantly. Our evidence suggests that DIBI restricts host iron availability to A. baumannii growing in the respiratory tract, bolstering the host innate iron restriction mechanisms. DIBI has potential as a sole anti-infective or in combination with conventional antibiotics for the treatment of A. baumannii pneumonia.

Keywords: anti-infective; antibiotic resistance; iron acquisition; iron sequestration.

FIG 1

A. baumannii strains LAC-4 (A), ATCC 17978 (B), and ATCC 19606 (C) were cultured in deferrated RPMI (open circles), deferrated RPMI + 1 μM iron citrate (solid triangles), deferrated RPMI + 1 μM iron citrate + 0.1μM DIBI (inverted solid triangles), and deferrated RPMI + 1 μM iron citrate + 1 μM DIBI (solid diamonds). Cultures were shaken continuously at 35°C, and growth was measured periodically by measuring the OD₆₀₀. Data points are reported as means ± SEM from at least two independent experiments. *, P < 0.05 for deferrated RPMI + 1 μM iron citrate + 1 μM DIBI versus deferrated RPMI + 1 μM iron citrate at 12, 24, and 36 hpi.

FIG 2

Influence of DIBI on GEN and CIP killing and recovery growth for A. baumannii LAC-4 and ATCC 17978. Cultures of LAC-4 (A and C) or ATCC 17978 (B and D) were inoculated at a final concentration of approximately 5 × 10⁶ CFU/ml in RPMI or RPMI containing GEN (A and B) or CIP (C and D) alone or with DIBI either alone or combined with the antibiotics. Cultures were grown at 35°C, with bacterial counts (CFU/ml) being determined at intervals over 24 h. Symbols: control (open circles), DIBI added (open triangles) at 20 μg/ml, antibiotic added (solid circles, GEN at 32 μg/ml [A] or 1 μg/ml [B] or CIP at 32 μg/ml [C] or 0.5 μg/ml [D]), or DIBI + antibiotic (solid triangles). Data points are reported as means ± SEM. *, P < 0.05 for DIBI plus antibiotic combinations versus DIBI and antibiotic treatments alone.

FIG 3

Influence of DIBI on CST and TGC killing and recovery growth for A. baumannii LAC-4 and ATCC 17978. Cultures of LAC-4 (A and C) or ATCC 17978 (B and D) were inoculated at a final concentration of approximately 5 × 10⁶ CFU/ml in RPMI or in RPMI containing CST (A and B) or TGC (C and D) alone or with DIBI either alone or combined with the antibiotics. Cultures were grown at 35°C with bacterial counts (CFU/ml) being determined at intervals over 24 h. Symbols: control (open circles), DIBI added (open triangles) at 20 μg/ml, antibiotic added (solid circles, CST at 0.5 μg/ml [A] or 0.25 μg/ml [B] or TGC at 0.5 μg/ml [C] or 0.5 μg/ml [D]), or DIBI + antibiotic (solid triangles). Data points are reported as means ± SEM. *, P < 0.05 for DIBI plus antibiotic combinations versus DIBI and antibiotic treatments alone.

FIG 4

Bacterial burdens in the lungs and spleen of DIBI or vehicle-treated BALB/c mice after i.n. challenge with either A. baumannii ATCC 17978 or LAC-4. DIBI (closed bars)- and diluent vehicle (open bars)-treated mice were infected i.n. with approximately 3.6 × 10⁷ CFU of A. baumannii ATCC 17978 or 3.0 × 10⁶ CFU of LAC-4 at 0 h. The mice were treated 3 and 10 h postchallenge i.n. with DIBI (one half at each time of a total treatment of 100 mg/kg = 11 μmol/kg). Bacterial burdens in the lungs and spleen were determined by quantitative bacteriology 24 h after challenge. The data are presented as means ± SEM (n = 5 to 10) and represent one of at least two independent experiments with similar results. The detection limit (dotted lines) for bacterial burdens was 1.3 log₁₀ CFU/organ. *, P < 0.05 versus vehicle-treated mice.

FIG 5

Effect of DIBI and DIBI/CIP treatment on bacterial burdens in the lungs, spleens, and blood of BALB/c mice after i.n. challenge with A. baumannii LAC-4. Groups of five mice were infected i.n. with 3.0 × 10⁶ or 1.7 × 10⁷ CFU of A. baumannii LAC-4 at 0 h. Treatments were administered to mice i.n. 3 and 10 h later, with 100 mg/kg (11 μmol/kg) DIBI, 100 mg/kg DIBI + 20 mg/kg (60 μmol/kg) CIP, 20 mg/kg CIP, or diluent. Bacterial burdens in the lungs, spleens, and blood were determined by quantitative bacteriology 24 h after inoculation. The data are presented as means ± SEM (n = 5 to 10) and represent one of at least two independent experiments with similar results. The detection limits for the tissue and blood are indicated by dotted lines. *, P < 0.05 versus diluent vehicle-treated mice.

FIG 6

Effect of DIBI and DIBI/CIP treatment on the cytokine and chemokine levels in the sera (A) and lungs (B) of BALB/c mice after i.n. challenge with A. baumannii. Groups of BALB/c mice were i.n. inoculated with 1.7 × 10⁷ CFU of A. baumannii LAC-4. Serum and lung homogenate supernatant samples were collected and processed at 24 h, and cytokine and chemokine levels were determined using the mouse panel of 12-plex multiplex kits (Millipore) on a Luminex Magpix instrument. The data are expressed as means ± SEM of five mice. The detection limits of the assays were 2.5 to 15 pg/ml. *, P < 0.05 versus diluent vehicle-treated mice.