Mild magnetic hyperthermia is synergistic with an antibiotic treatment against dual species biofilms consisting of S. aureus and P. aeruginosa by enhancing metabolic activity

Figures & data

Figure 1. The susceptibility of dual species biofilm consisting of S. aureus and P. aeruginosa to ciprofloxacin in vitro. (A) Log (CFU/mL) reduction of S. aureus and P. aeruginosa in single species biofilms (Single-S. aureus and Single-P. aeruginosa) and dual species biofilms (Dual-S. aureus and Dual-P. aeruginosa) following the treatment of increasing concentrations of ciprofloxacin. (B) Biomass reduction in single species biofilms (Single-S. aureus and Single-P. aeruginosa) and dual species biofilms (Dual-S. aureus + P. aeruginosa) following the treatment of increasing concentrations of ciprofloxacin. The dual biofilms data is analysis of S. aureus or P. aeruginosa from the dual biofilm. N = 3 per group. *p < 0.05 and #p < 0.01.

Figure 2. Effects of MNP/AMF hyperthermia on the CEM₄₃ thermal dose. (A) An experimental setup for the measurment of temperature in MNP (Ferumoxytol) solution during the application of AMF (B) Increase of temperature (△T) during the application of AMF as a function of MNP concentration (1–3 mg/mL). N = 3 per group. (C) Estimation of MNP/AMF-induced CEM₄₃ (min) as a function of MNP concentration (1–3 mg/mL). the dotted red line indicates the reported threshold of safe thermal dose for the application of hyperthermia to skin tissue²⁴. N = 3 per group.

Figure 3. Effects of MNP/AMF hyperthermia on the killing of S. aureus and P. aeruginosa in dual species biofilm in combination with ciprofloxacin for varying thermal dose. (A) An experimental setup for the application of MNP/AMF hyperthermia to the culture of dual species biofilm consisting of S. aureus and P. aeruginosa. (B) The effect of MNP/AMF hyperthermia alone or in combination with ciprofloxacin (CIP, 50 µg/mL) for varying CEM₄₃ thermal doses (CEM₄₃=1 and 20 min) on the log reduction of S. aureus and P. aeruginosa numbers (CFU/mL) in dual species biofilm. N = 3 per group. *p < 0.01.

Figure 4. The in vivo efficacy of combined treatment of mild MNP/AMF hyperthermia with ciprofloxacin in wounds of db/db mice infected with dual species biofilm. (A) An experimental procedure for the application of mild MNP/AMF and CIP to the wounds of db/db mice infected with dual species biofilm consisting of S. aureus and P. aeruginosa. The SEM image shows the presence of S. aureus (spherical shape, white arrow) and P. aeruginosa (rod shape, yellow arrow) in the biofilm collected from wounds of db/db mice day 2 post-infection. (B) Temperature increases in wound surface and rectum of db/db mice measured during the application of MNP/AMF measured by fiber optic temperature probe. (C) The effect of combined mild MNP/AMF hyperthermia and CIP treatment on the reduction in CFU numbers of S. aureus and P. aeruginosa in wounds of db/db mice at day 3 post-infection. N = 3 per group. *p < 0.05 and **p < 0.01.

Figure 5. The effects of mild MNP/AMF hyperthermia on the metabolism of S. aureus biofilm. (A) Volcano plot showing the gene level analysis to select differentially expressed genes after treatment with mild MNP/AMF (CEM₄₃=6 min). the data for all genes are plotted as fold change versus − log10 of the adjusted p-value. Green, gray and red correspond to genes with < −2-fold, −2 to 2-fold and > 2-fold differential expression, respectively. (B) Changes in the expression of the genes for anaerobic fermentation (ldhA, adhE, pflA and pflB) and genes for aerobic glycolysis/TCA pathway (Pyr, pckA, fda, and odhA) in response to mild MNP/AMF hyperthermia (CEM₄₃=6 min) in S. aureus biofilm. The values listed in the figures are the calculated means of fold change from three parallel microarrays. (C) Changes in metabolic activity in S. aureus biofilm in response to MNP/AMF (CEM₄₃=6 min) quantified by resazurin assay. N = 8 per group. *p < 0.05.

Data availability statement

The data that support the findings of this study are available from the corresponding author (MK) upon reasonable request.