Mild magnetic nanoparticle hyperthermia enhances the susceptibility of Staphylococcus aureus biofilm to antibiotics

Abstract

Objective: A critical challenge in the treatment of biofilm infection is the capacity of biofilm-grown bacteria to develop resistance to traditional antimicrobial therapies. The objective of this study was to validate the therapeutic potential of magnetic nanoparticle/alternating magnetic field (MNP/AMF) hyperthermia in combination with conventional antibiotics against biofilm infection.Materials and methods: The impact of MNP/AMF hyperthermia on the viability of S. aureus biofilm in the absence and presence of antibiotics as well as on the bactericidal activity of macrophages were evaluated at varying conditions of MNPs concentration and AMF intensity using in vitro cell culture models.Results: The application of MNP/AMF alone at a CEM43 thermal dose below the threshold for skin tissue exhibited a modest efficacy in the eradication of Staphylococcus aureus (S. aureus) biofilm (<1-log reduction). The treatment of antibiotics (ciprofloxacin, vancomycin) alone at a bactericidal concentration for planktonic S. aureus had no significant effect on the eradication of biofilm phase of S. aureus. However, when the biofilm was pre-exposed to mild MNP/AMF hyperthermia, the treatment of antibiotics could exhibit bactericidal effects against S. aureus biofilm, which was associated with increased uptake of antibiotics to the bacterial cells. Importantly, the application of MNP/AMF could promote the bactericidal activity of macrophages against intracellular bacteria via MNP-dependent generation of reactive oxygen species (ROS).Conclusion: Our results validate that the application of mild MNP/AMF hyperthermia within a safe thermal dose threshold is synergistic with conventional antibiotics as well as aids host innate immune response of macrophages for the clearance of intracellular bacteria.

Keywords: Magnetic nanoparticles; S. aureus biofilm; alternating magnetic field; antibiotics; hyperthermia.

Figure 1.

Effects of MNP/AMF hyperthermia on the CEM₄₃ thermal dose for varying conditions of MNPs concentration and AMF intensity. (A) A schematic diagram on the experimental procedure for the application of MNP/AMF hyperthermia to the biofilm bacteria in vitro. (B) Increase in temperature (ΔT) during the application of MNP/AMF as a function of MNP concentration (1–3 mg/mL) and AMF intensity (18, 24, and 30 kA/m). N = 3 per group. (C) Estimation of CEM₄₃ values in response to MNP/AMF hyperthermia as a function of MNP concentration (1–3 mg/mL) and AMF intensity (18, 24, and 30 kA/m). The dotted red line indicates the reported threshold of the safe thermal dose for the application of hyperthermia to skin tissue [20]. N = 3 per group.

Figure 2.

Efficacy of MNP/AMF hyperthermia alone on the killing of S. aureus biofilm bacteria. (A) The viable CFU numbers of S. aureus in the biofilm following MNP/AMF application as a function of MNPs concentration (1–3 mg/mL) and AMF intensity (18, 22, and 30 kA/m). N = 5–8 per group. N.S: Not Significant (p > .05). #: p < .01. (B) Representative SYTO9/PI fluorescence images following the application of MNP/AMF for varying concentrations of MNPs at 30 kA/m of AMF (green/SYTO9 live cells; red/PI = dead cells). Representative of three independent experiments. (C) Representative flow cytometric plots of forward and side scattering from S. aureus biofilm incubated with varying concentrations of MNPs (0–1 mg/mL) for 2 h. Representative of three independent experiments. (D) The atomic absorption spectroscopy analysis to quantify levels of intracellular iron in the S. aureus bacteria incubated with MNPs (1 mg/mL). N = 3 per group. *p < .05.

Figure 3.

Efficacy of MNP/AMF hyperthermia on the susceptibility of S. aureus biofilm to antibiotics. (A) The susceptibility of planktonic (PLK) and biofilm (BIO) phase of S. aureus to ciprofloxacin (CIP) and vancomycin (VAN). N = 4 per group. (B) A schematic diagram for the experimental protocol to quantify the effect of MNP/AMF on the susceptibility of S. aureus biofilm to antibiotics. (C) The effect of MNP/AMF hyperthermia on the susceptibility of S. aureus biofilm to CIP and VAN for varying conditions of MNP concentration (1, 2, 3 mg/mL) and AMF intensity (18, 24, 30 kA/m). N = 5–9 per group. *p < .05 and #p < .01 vs No antibiotics group.

Figure 4.

Effects of MNP/AMF hyperthermia on the uptake of antibiotics by S. aureus biofilm. (A) The uptake of FL-VAN by planktonic and biofilm phase of S. aureus, quantified by fluorescence emitted from FL-VAN in the bacterial cells. N = 6 per group. #p < .01 vs PLK group. (B) The effect of MNP/AMF hyperthermia on the uptake of FL-VAN by S. aureus biofilm. The fluorescence emitted from FL-VAN in the S. aureus collected from biofilm culture for varying concentrations of MNPs (0–2 mg/mL) in the absence and presence of AMF application (30 kA/m). N = 6 per group. #p < .01 vs No MNP/AMF group. *p < .01. (C) The penetration of CIP (16, 32, 64, and 128 µg/mL) through the S. aureus biofilm in the absence or presence of MNP/AMF application (pre-exposure at 2 mg/mL-MNPs and 30 kA/m-AMF for 6 min) assessed by disk diffusion method. Left: Representative photographic images of TSA plate showing the zone of growth inhibition by CIP. Right: Quantification for the diameter of the zone of growth inhibition by CIP. N = 3 per group. N.S: Not Significant (p > .05).

Figure 5.

Effects of MNP/AMF hyperthermia on the viability, bactericidal activity, and ROS generation of RAW 264.7 macrophages. (A) The viability of RAW 264.7 macrophages for varying concentrations of MNPs (0–3 mg/mL) in the absence or presence of AMF application at the intensity of 30 kA/m, assessed by MTT viability assay. N = 4 per group. (B) The effect of varying concentrations of MNPs (0–2 mg/mL) on the bactericidal activity of RAW 264.7 cells in the absence and presence of AMF application (30 kA/m), assessed by antibiotic protection assay. N = 10–15 per group. (C) The effect of varying concentrations of MNPs (0–2 mg/mL) on the ROS generation in RAW 264.7 cells in the absence and presence of AMF application (30 kA/m). N = 4–6 per group. N.S: Not Significant (p > .05). *p < .05 vs untreated control (without MNP/AMF). #p < .05 vs without AMF group at a given MNP concentration.

Figure 6.

A schematic depicting the therapeutic potential of mild MNP/AMF hyperthermia against biofilm infections. The application of mild MNP/AMF hyperthermia can enhance the susceptibility of S. aureus biofilm to antibiotics by facilitating their uptake to biofilm bacteria. Additionally, when applied to macrophages at lower thermal doses, the treatment of MNP/AMF can promote a macrophage-mediated host response for the clearance of intracellular bacteria via MNP-dependent generation of ROS.