Iron and Acinetobacter baumannii Biofilm Formation

Abstract

Acinetobacter baumannii is an emerging nosocomial pathogen, responsible for infection outbreaks worldwide. The pathogenicity of this bacterium is mainly due to its multidrug-resistance and ability to form biofilm on abiotic surfaces, which facilitate long-term persistence in the hospital setting. Given the crucial role of iron in A. baumannii nutrition and pathogenicity, iron metabolism has been considered as a possible target for chelation-based antibacterial chemotherapy. In this study, we investigated the effect of iron restriction on A. baumannii growth and biofilm formation using different iron chelators and culture conditions. We report substantial inter-strain variability and growth medium-dependence for biofilm formation by A. baumannii isolates from veterinary and clinical sources. Neither planktonic nor biofilm growth of A. baumannii was affected by exogenous chelators. Biofilm formation was either stimulated by iron or not responsive to iron in the majority of isolates tested, indicating that iron starvation is not sensed as an overall biofilm-inducing stimulus by A. baumannii. The impressive iron withholding capacity of this bacterium should be taken into account for future development of chelation-based antimicrobial and anti-biofilm therapies.

Figure 1

Effect of different iron chelators on planktonic growth of A. baumannii ATCC 17978. Bacteria were grown for 48 h at 37 °C in 96-wells microtiter plates containing 100 μL M9 supplemented with the indicated iron chelator at different concentrations: 32 μM (light grey bars), 128 μM (dark grey bars) or 128 μM chelator + 100 μM DIP (black bars). Growth was measured as OD₆₀₀ and expressed as percentage relative to the untreated control (i.e., OD₆₀₀ in M9). The average of the OD₆₀₀ in control M9 was 0.318 ± 0.008 and represents 100% of growth (white bar). Relative growth in M9 supplemented with 100 μM FeCl₃ is reported (striped bar). Data represent the average of three independent experiments ± standard deviation. h-TF, tranferrin; CIT, citrate; DFX, deferasirox; DFO, desferrioxamine; DFP, deferiprone.

Figure 2

Growth and biofilm formation by selected A. baumannii strains in different iron-poor media. Bacterial cells were inoculated at OD₆₀₀ of 0.01 in 100 μL of the different growth media, dispensed in a 96-wells microtiter plate, and grown at 37 °C without shaking for 24 and 48 h. Growth (circles) was measured spectrophotometrically (OD₆₀₀) and biofilm formation (bars) was evaluated using the CV staining assay [36]. Dark grey, TSBD; light grey M9; white M9 supplemented with 100 μM DIP. Data represent the average of three independent experiments ± standard deviation.

Figure 3

Regulatory mechanism and activity of the basA::lacZ iron biosensor in the reference A. baumannii strain ATCC 17978. (A) Schematic of the regulatory mechanism the basA::lacZ iron-regulated transcriptional fusion carried by plasmid pMP220::P_basA [26]. Under iron proficient conditions (left), the Fur repressor protein binds the P_basA promoter and inhibits β–galactosidase (LacZ) expression; under iron deficient conditions Fur repression is relieved and the LacZ enzyme is expressed. (B) Activity of the basA::lacZ iron-regulated fusion in A. baumannii ATCC 17978 grown for 24 and 48 h in different media, as indicated, in the absence (white bars) or presence (black bars) of 100 μM FeCl₃. Data are the means (±standard deviations (SD)) of triplicate experiments.

Figure 4

Seven-days biofilm of selected A. baumannii strains grown in TSBD. (A) Confocal microscope images (x-y plane and side view) of A. baumannii biofilms stained with acridine orange, a fluorescent dye which labels double-stranded nucleic acids (prevalently DNA) in green, and single-stranded nucleic acids (prevalently RNA) in red. (B) A. baumannii biofilms stained with the calcofluor white for exopolysaccharide labelling [19,28], and analyzed by fluorescence microscopy. Scale bar: 50 μm.

Figure 5

Growth and biofilm formation in a representative collection of 54 A. baumannii strains from clinical and veterinary sources. (A) Growth of 54 A. baumanni strains for 48 h in 96-wells microtiter plates containing 100 µL TSBD supplemented (black circles) or not (white circles) with 100 μM FeCl₃, as indicated. (B) Absolute values of biofilm formation by the same isolates shown in panel A, evaluated by the CV staining assay (OD₆₀₀). Grey circles (B) represent the values for strains that in either or both conditions yielded negative biofilm values, and were excluded from calculations in panel C. (C) Relative values of biofilm formation (Biofilm formation (OD₆₀₀)/Growth (OD₆₀₀)) for a subset of 50 biofilm-producing isolates. The line bar represents the median value for each group. Values for each strain are the average of three independent experiments.

Figure 6

Effect of DFX on A. baumannii biofilm formation. (A) A. baumannii strains were grown statically for 48 h in microtiter plates containing 100 μL TSBD supplemented with DFX at indicated concentrations, or 128 µM DFX plus 100 µM FeCl₃. Biofilm formation (OD₆₀₀ in the CV staining assay) was normalized by the growth yield (OD₆₀₀ of the culture) and expressed as percentage relative to the DFX-untreated control (TSBD). Boxes represent medians, second and third interquartiles; whiskers represent range of 50 isolates tested. (B) Relative biofilm levels produced by individual isolates in presence of 128 µM DFX, expressed as % of the untreated control in TSBD. With reference to Figure 5C, the bar filling denotes: isolates in which biofilm production was significantly enhanced by iron deficiency (white, 21 isolates), or significantly reduced (black, 12 isolates), or in which iron had no effect on biofilm formation (grey, 17 isolates). In both panels data represent the average of three independent experiments.