Activity of Gallium Meso- and Protoporphyrin IX against Biofilms of Multidrug-Resistant Acinetobacter baumannii Isolates

Abstract

Acinetobacter baumannii is a challenging pathogen due to antimicrobial resistance and biofilm development. The role of iron in bacterial physiology has prompted the evaluation of iron-modulation as an antimicrobial strategy. The non-reducible iron analog gallium(III) nitrate, Ga(NO₃)₃, has been shown to inhibit A. baumannii planktonic growth; however, utilization of heme-iron by clinical isolates has been associated with development of tolerance. These observations prompted the evaluation of iron-heme sources on planktonic and biofilm growth, as well as antimicrobial activities of gallium meso- and protoporphyrin IX (Ga-MPIX and Ga-PPIX), metal heme derivatives against planktonic and biofilm bacteria of multidrug-resistant (MDR) clinical isolates of A. baumannii in vitro. Ga(NO₃)₃ was moderately effective at reducing planktonic bacteria (64 to 128 µM) with little activity against biofilms (≥512 µM). In contrast, Ga-MPIX and Ga-PPIX were highly active against planktonic bacteria (0.25 to 8 µM). Cytotoxic effects in human fibroblasts were observed following exposure to concentrations exceeding 128 µM of Ga-MPIX and Ga-PPIX. We observed that the gallium metal heme conjugates were more active against planktonic and biofilm bacteria, possibly due to utilization of heme-iron as demonstrated by the enhanced effects on bacterial growth and biofilm formation.

Keywords: Acinetobacter baumannii; biofilm; gallium mesoporphyrin IX; gallium protoporphyrin IX; multidrug-resistant.

Figure A1

Effect of heme iron and non-heme iron sources on biofilm formation in vitro. A. baumannii strain 17978, was grown for up to 24 h in MHB II (0.3 g/L) supplemented with increasing concentrations of (A) human hemoglobin or haptoglobin (0–512 µg/mL) or (B) FeCl₃, or Fe₂(SO₄)₂ (0 to 50 μM). Biofilm biomass, is reported as the measured absorbance of solubilized crystal violet of stained biofilms at 570 nm (CV at OD₅₇₀), Values represent the mean ± SEM of three independent experiments.

Figure A2

Screening of biofilm formation amongst clinical isolates of A. baumannii used in this study. (A) A. baumannii isolates were grown in MHB II broth at 37 °C and then incubated in the MBEC device overnight as previously described in the methods used in 4.5. Results shown are colony counts recovered from individual pegs after sonication of biofilms on MBEC plates and expressed as Log₁₀; (B) screening of biofilm formation amongst the clinical isolates assessed by quantitating biofilm biomass by measuring the absorbance of ethanol solubilized crystal violet at 570 nm (CV at OD₅₇₀) of previously stained biofilms. Bars represent the mean ± SEM of three independent experiments.

Figure 1

Effect of heme iron and non-heme iron sources on bacterial growth and biofilm formation in vitro. A. baumannii strain 17978 was grown for up to 24 h in MHB II (0.3 g/L) supplemented with increasing concentrations of (A) human hemoglobin; (B) haptoglobin (0–512 µg/mL) or (C) FeCl₃, or (D) Fe₂(SO₄)₃ (0 to 50 μM). Growth over time was expressed as the mean log value ± SD. Percentage of biofilm formation of A. baumannii strain 17978, as determined by the crystal violet method, relative to untreated controls, in the presence of heme iron (E) and non-heme iron sources (F) after 24 h of growth in MHB II (0.3 g/L) under static conditions. Values are expressed as the mean ± SD. Experimental assays are representative of at least three independent experiments. * p < 0.001 and ** p < 0.00001 relative to the mean value of biofilm formation for strains grown under non-supplemented conditions.

Figure 2

Differential effects of iron sources on biofilm formation of clinical isolates of A. baumannii. Mean increases of biofilm formation of clinical isolates of A. baumannii (n = 12), as determined by the crystal violet method, relative to untreated controls, in the presence of heme iron (1–512 µg/mL; (A) and (B) and non-heme iron sources (1–512 µM; C and D) after 24 h. Values are representative of the mean ± SD, of relative % increase of biofilm formation of each of the individual strains to untreated control groups. Experimental assays are representative of at least three independent experiments. * p < 0.001 and ** p < 0.00001 relative to the mean value of biofilm formation for strains grown under non-supplemented conditions.

Figure 3

Inhibitory activities of Ga-MPIX and Ga-PPIX on a planktonic MDR A. baumannii isolates. Genotypically distinct isolates of A. baumannii (n = 13) were grown in the iron deplete medias, MHB II (0.3 g/L) (un-shaded boxes) or RPMI1640 (shaded boxes), and exposed to different concentrations (0–512 µM) of (A) Ga(NO₃)₃; (B) Ga-MPIX; or (C) Ga-PPIX for 24 h. Plots show the concentrations (μM) required to reduce viable bacteria of individual isolates by 50% (IC₅₀) and 90% (IC₉₀). Boxes represent medians; whiskers represent range of strains tested.

Figure 4

Activity of Ga-MPIX and Ga-PPIX against biofilms of MDR A. baumannii isolates. Biofilms of isolates of A. baumannii (n = 13) were grown on individual pegs of an MBEC device for 24 h followed by exposure to different concentrations (0–512 µM) of (A) Ga(NO₃)₃; (B) Ga-MPIX; or (C) Ga-PPIX for 24 h in the iron deplete medias, MHB II (0.3 g/L) (un-shaded boxes) or RPMI1640 (shaded boxes). Plots show the concentrations (μM) required to reduce viable bacteria within biofilms of individual isolates by 50% (MBEC₅₀) and 90% (MBEC₉₀) to untreated controls. Boxes represent medians; whiskers represent range of strains tested.

Figure 5

Evaluation of the toxicity of gallium porphyrins on primary human fibroblasts. Human fibroblasts were exposed to different concentrations (0–512 µM) of (A) Ga(NO₃)₃; (B) Ga-MPIX; or (C) Ga-PPIX for 24 h. Cell viability was measured using the cell titer flour assay and viability was expressed as a percentage compared to untreated control groups. Cytotoxicity was evaluated by measuring the amount of lactate dehydrogenase (LDH) in culture supernatants of cells (A_490nm) treated with the gallium compounds. Values are expressed as the mean ± SD of three independent experiments. * p < 0.01 relative to the untreated control group.