Antimicrobial Activity of Gallium Protoporphyrin IX against Acinetobacter baumannii Strains Displaying Different Antibiotic Resistance Phenotypes

Abstract

A paucity of effective, currently available antibiotics and a lull in antibiotic development pose significant challenges for treatment of patients with multidrug-resistant (MDR) Acinetobacter baumannii infections. Thus, novel therapeutic strategies must be evaluated to meet the demands of treatment of these often life-threatening infections. Accordingly, we examined the antibiotic activity of gallium protoporphyrin IX (Ga-PPIX) against a collection of A. baumannii strains, including nonmilitary and military strains and strains representing different clonal lineages and isolates classified as susceptible or MDR. Susceptibility testing demonstrated that Ga-PPIX inhibits the growth of all tested strains when cultured in cation-adjusted Mueller-Hinton broth, with a MIC of 20 μg/ml. This concentration significantly reduced bacterial viability, while 40 μg/ml killed all cells of the A. baumannii ATCC 19606(T) and ACICU MDR isolate after 24-h incubation. Recovery of ATCC 19606(T) and ACICU strains from infected A549 human alveolar epithelial monolayers was also decreased when the medium was supplemented with Ga-PPIX, particularly at a 40-μg/ml concentration. Similarly, the coinjection of bacteria with Ga-PPIX increased the survival of Galleria mellonella larvae infected with ATCC 19606(T) or ACICU. Ga-PPIX was cytotoxic only when monolayers or larvae were exposed to concentrations 16-fold and 1,250-fold higher than those showing antibacterial activity, respectively. These results indicate that Ga-PPIX could be a viable therapeutic option for treatment of recalcitrant A. baumannii infections regardless of the resistance phenotype, clone lineage, time and site of isolation of strains causing these infections and their iron uptake phenotypes or the iron content of the media.

FIG 1

Molecular structure of gallium protoporphyrin IX (Ga-PPIX).

FIG 2

Ga-PPIX disk diffusion assays. A. baumannii bacteria were first seeded onto CAMH agar and then disks impregnated with 50 μg (A) or 100 μg (B) of Ga-PPIX were deposited on the surfaces of the plates. Growth inhibition halos were measured after incubation for 24 h at 37°C. Data are expressed as means ± standard errors of the means (SEM) (error bars) from experiments done twice in triplicate. The insets show the growth inhibition halos observed when the CAMH agar plates were seeded with A. baumannii ATCC 19606^T and incubated for 24 h at 37°C.

FIG 3

Ga-PPIX time-kill assays. Ga-PPIX time-kill kinetics for A. baumannii ATCC 19606^T (A) and ACICU (B) was determined after 0, 2, 4, 6, and 24 h of incubation by CFU count after exposure to 0 μg/ml (0× MIC), 10 μg/ml (0.5× MIC), 20 μg/ml (1× MIC), or 40 μg/ml (2× MIC) Ga-PPIX.

FIG 4

Ga-PPIX toxicity toward A549 human cells and G. mellonella larvae. (A) Submerged A549 cell monolayers containing 4 × 10⁵ cells were incubated with increasing concentrations of Ga-PPIX dissolved in DMSO and added to DMEM containing 10% heat-inactivated fetal bovine serum without antibiotics. Cell viability was determined after 24 h of incubation at 37°C in the presence of 5% CO₂. Responses to the presence of Ga-PPIX were compared to those detected with cells cultured in the absence of this metalloporphyrin derivative. Values that are significantly different from the value for the control (0 μg/ml Ga-PPIX) are indicated by asterisks as follows: ***, P = 0.0004; ****, P < 0.0001. (B) Mass-matched G. mellonella larvae were injected with 5 μl of twofold dilutions ranging from 0.2 mM to 25 mM Ga-PPIX solubilized in 0.5 N NaOH and 1.5% DMSO. Larvae injected with 0.5 N NaOH, 1.5% DMSO, or PBS or not injected (No Inj) served as controls. The inset shows the mean masses of all animal groups with error bars showing standard errors.

FIG 5

Antibacterial activity of Ga-PPIX against experimental infections. Monolayers of A549 human alveolar epithelial cells (A and B) and G. mellonella larvae (C and D) were infected with A. baumannii ATCC 19606^T strain (A and C) or ACICU strain (B and D) in the absence or the presence of either 20 μg/ml or 40 μg/ml of Ga-PPIX. Data shown in panels A and B represent the means (± SEM) of experiments performed twice in octuplet (n = 16) using fresh biological samples each time. Responses to the presence of Ga-PPIX were compared to those detected with bacteria cultured in the absence of this metalloporphyrin derivative. Values that are significantly different from the value for the control (0 μg/ml Ga-PPIX) are indicated by asterisks as follows: *, P = 0.037; **, P = 0.014; ***, P = 0.0005.