Antibacterial Activity of Positively and Negatively Charged Hematite (α-Fe2O3) Nanoparticles to Escherichia coli, Staphylococcus aureus and Vibrio fischeri

Abstract

In the current study, the antibacterial activity of positively and negatively charged spherical hematite (α-Fe₂O₃) nanoparticles (NPs) with primary size of 45 and 70 nm was evaluated against clinically relevant bacteria Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) as well as against naturally bioluminescent bacteria Vibrio fischeri (an ecotoxicological model organism). α-Fe₂O₃ NPs were synthesized using a simple green hydrothermal method and the surface charge was altered via citrate coating. To minimize the interference of testing environment with NP's physic-chemical properties, E. coli and S. aureus were exposed to NPs in deionized water for 30 min and 24 h, covering concentrations from 1 to 1000 mg/L. The growth inhibition was evaluated following the postexposure colony-forming ability of bacteria on toxicant-free agar plates. The positively charged α-Fe₂O₃ at concentrations from 100 mg/L upwards showed inhibitory activity towards E. coli already after 30 min of contact. Extending the exposure to 24 h caused total inhibition of growth at 100 mg/L. Bactericidal activity of positively charged hematite NPs against S. aureus was not observed up to 1000 mg/L. Differently from positively charged hematite NPs, negatively charged citrate-coated α-Fe₂O₃ NPs did not exhibit any antibacterial activity against E. coli and S. aureus even at 1000 mg/L. Confocal laser scanning microscopy and flow cytometer analysis showed that bacteria were more tightly associated with positively charged α-Fe₂O₃ NPs than with negatively charged citrate-coated α-Fe₂O₃ NPs. Moreover, the observed associations were more evident in the case of E. coli than S. aureus, being coherent with the toxicity results. Vibrio fischeri bioluminescence inhibition assays (exposure medium 2% NaCl) and colony forming ability on agar plates showed no (eco)toxicity of α-Fe₂O₃ (EC₅₀ and MBC > 1000 mg/L).

Keywords: MicrobeJ; antibacterial; confocal; environmental safety; hematite; hydrothermal synthesis; nano-bio interactions; surface charge; α-Fe2O3 nanoparticles.

Figure 1

(a) XRD patterns of α-Fe₂O₃ nanoparticles (NPs): (A) α-Fe₂O₃-p45, (B) α-Fe₂O₃-p70, (C) α-Fe₂O₃-n45, (D) α-Fe₂O₃-n70. The peak positions of the pure rhombohedral phase of α-Fe₂O₃ (ICDD 98-000-0240). (b) Attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectra of α-Fe₂O₃ NPs before and after citrate coating.

Figure 2

SEM observation of α-Fe₂O₃ NPs prepared with different concentrations of Fe (III) nitrate, (A) 0.05 M (α-Fe₂O₃-p45), (B) 0.1 M (α-Fe₂O₃-p70), and after ɑ-Fe₂O₃ NPs coating with the citrate, (C) 0.05 M (α-Fe₂O₃-n45), (D) 0.1 M (α-Fe₂O₃-n70).

Figure 3

TEM images of the α-Fe₂O₃ NPs: (A) uncoated-positively charged (α-Fe₂O₃-p70), (B) citrate coated-negatively charged (α-Fe₂O₃-n70).

Figure 4

Visualization of stability of the α-Fe₂O₃ NPs in DI water and 2% NaCl (see also Table 3).

Figure 5

Viability of Escherichia coli (upper panels) and Staphylococcus aureus (lower panels) after exposure to α-Fe₂O₃ NPs in DI water for 30 min and 24 h at room temperature. Viability was evaluated by the ability of exposed bacteria to yield colonies at the nutrient agar plate as indicated on the panels.

Figure 6

Agar diffusion test using Escherichia coli: (a) α-Fe₂O₃-p45 (b) α-Fe₂O₃-n45. Exposure time 24 h, temperature 30 °C, Mueller–Hinton agar medium (MHA). Agar diffusion test images for Staphylococcus aureus can be found in the Supplementary Material (Figure S2).

Figure 7

Kinetics of the Vibrio fischeri bioluminescence inhibition upon exposure of bacteria to different concentrations of α-Fe₂O₃ NPs. (a) α-Fe₂O₃-p45; (b) α-Fe₂O₃-p70; (c) α-Fe₂O₃-n45; (d) α-Fe₂O₃-n70

Figure 8

Viability of Vibrio fischeri after exposure to α-Fe₂O₃ NPs in 2% NaCl for 30 min and 24 h at room temperature. Viability was evaluated by the ability of exposed bacteria to yield colonies at the Beneckea Harvey (BH) agar plate as indicated on the panels. Blue-green spots are bioluminescent bacterial colonies photographed in the dark; 3,5-dichlorophenol (3,5 DCP) was used as a positive control.

Figure 9

Generation of reactive oxygen species (ROS) measured with fluorescent dye DCF-DA in abiotic conditions (i.e., without of the cells) after 30 min (a) and 24 h (b) exposure to hematite NPs (1–100 mg/L), and Mn₃O₄ NPs—a positive control (0.1–100 mg/L)—in DI water. The values are presented as fold-increase in fluorescence in the presence of the NPs compared to the control (DI water); the mean of 4 experiments is given ± SD. All exposure concentrations are nominal. The dashed horizontal line indicates the ROS level in the DI water.

Figure 10

Generation of ROS measured with fluorescent dye DCF-DA in the biotic conditions after 30 min (a–c) and 24 h (b–d) exposure to hematite NPs (1–100 mg/L), and H₂O₂ as a positive control (1–100 mg/L for Escherichia coli; 1–1000 mg/L for Staphylococcus aureus) in DI water. The values are presented as fold-increase in fluorescence of exposed cells compared to unexposed cells; the mean of 3 experiments is given ± SD. All exposure concentrations are nominal. The dashed horizontal line highlights the ROS level in the control bacteria in DI water. Graphs of ROS generation in biotic conditions after 24 h of NP incubation with bacteria and 30 min after dye adding points can be found in the Supplementary Material (Figure S3).

Figure 11

Representative maximal projections of confocal laser scanning microscopy (CLSM) images (pseudocolored: cellular Syto9 signal—cyan, NPs signal from reflection mode—red) of Escherichia coli (A–F) and Staphylococcus aureus (G–L) exposed to 100 mg/L α-Fe₂O₃-p (left column), α-Fe₂O₃-n (middle column) or incubated without NPs (right column) for 30 min. Merged multichannel images (A–C,G–I), as well as reflection channel signals, are presented (D–F,J–L). Scale bars represent 10 µm. Single-channel and multichannel projection images for 30 min and 24h time points can be found in the Supplementary Material (Figures S4 and S5). A respective quantitative analysis is presented in Figure 12.

Figure 12

Results for quantitative cellular fluorescent and reflective NP signal analysis from maximal projections of CLSM images of bacteria exposed to 100 mg/L NPs or unexposed controls. Significantly more (p < 0.0001; ****) α-Fe₂O₃-p NPs signals were associated with both Escherichia coli and Staphylococcus aureus cellular signals than α-Fe₂O₃-n NPs signals, showing that more positively charged NPs were associated with generally negatively charged bacterial cells than negatively charged NPs and that this effect was larger for of E. coli, which is also in concordance with α-Fe₂O₃-p NPs being more toxic to E. coli than S. aureus. The 24 h time point was excluded from analysis for E. coli due to lethal exposure and thus poor cellular signals. No reflective NPs signals were detected in nonexposed controls.

Figure 13

Quantification of hematite NPs binding to the bacteria Escherichia coli (a) and Staphylococcus aureus (b) by flow cytometry to determine the side-scattered (SSC) signal shift of Syto9-stained cells. Control cells-gated cell population is marked by the dashed red line. The spot test (c) shows that α-Fe₂O₃-p45 NPs at 50 mg/L were toxic to E. coli but not to S. aureus, and α-Fe₂O₃-n45 NPs were not toxic to both bacteria. After exposure to 70% ethanol for 60 min, the colony-forming ability of E. coli and S. aureus was not observed.