Alternating magnetic fields and antibiotics eradicate biofilm on metal in a synergistic fashion

The study addresses the challenge of infections on metal implants such as prosthetic joints, fixation hardware, and dental implants, where biofilm formation renders pathogens highly resistant to antibiotics and immune clearance. Conventional management of prosthetic joint infections (PJI) often requires multiple invasive surgeries with significant morbidity, substantial failure rates (>10%), and high economic burden. Biofilms, comprising bacteria embedded in extracellular polymeric substances (EPS), can confer up to 1000-fold increased antibiotic tolerance. The authors investigate a noninvasive strategy using intermittent alternating magnetic fields (iAMF) to heat metal implants, hypothesizing that brief, controlled heating combined with antibiotics will synergistically eradicate biofilm while minimizing tissue damage. The purpose is to demonstrate efficacy across pathogens and antibiotics, characterize dosing parameters (temperature, exposure duration, intervals), and explore mechanisms underlying synergy, aiming ultimately for a clinically translatable approach to treat metal implant infections without implant removal.

Prior physical strategies for biofilm eradication include electrical currents, ultrasound, heat, and shock waves, each with limitations for use on metal implants. Alternating magnetic fields (AMF) can heat metal implants via induced surface currents and have previously reduced biofilm within minutes, but required sustained high temperatures (50–80 °C) and often led to regrowth if eradication was incomplete. Earlier in vitro work suggested combining AMF with antibiotics (e.g., ciprofloxacin) yields greater and more durable reductions than either alone. Brief, intermittent AMF exposures can reduce collateral tissue injury compared with prolonged heating, as shown in murine models. These lines of evidence motivate evaluating iAMF as an antibiotic adjuvant against biofilms on metal surfaces and optimizing dose structures for safety and efficacy.

Design and simulation: A custom in vitro iAMF system comprising 32 identical 6-turn copper solenoid coils (~2.5-inch diameter, 1 cm pitch) produced a uniform magnetic field (measured 10.2 ± 0.3 mT; simulated 11.2 ± 0.4 mT) at ~507–522 kHz. Stainless-steel rings were positioned axially within 50 mL conical tubes using 3D-printed holders to ensure consistent alignment. iAMF dosing comprised multiple short AMF exposures (seconds) within a dose block (15–60 min), separated by rest intervals to allow the ring to cool, with dose blocks repeated (e.g., at 0 and 12 h). Thermal simulations (initial 37 °C; properties in Supplementary Table 1; ~186,634 mesh elements) predicted uniform ring surface heating (≤2 °C SD circumferentially) and minimal heating of surrounding media for short exposures. Safety was evaluated using cumulative equivalent minutes at 43 °C (CEM43); predicted values did not exceed 240 min at 2 mm from the ring at Tmax = 80 °C and at 1 mm for 12 exposures at Tmax = 65 °C, suggesting low risk of tissue damage at those distances. Biofilm preparation: Biofilms were grown on stainless-steel rings using Pseudomonas aeruginosa PAO1, multidrug-resistant (MDR) P. aeruginosa MB689, and Staphylococcus aureus strains (including UAMS1 and another laboratory strain). Rings were incubated at 37 °C (typically 48 h; extended to 7 days for aged biofilms) with media replenishment (hourly or every 24 h depending on protocol). For MDR experiments, MB689 biofilms were similarly prepared. Treatment protocols: Rings were incubated in media with antibiotics and subjected to iAMF doses at specified times (commonly 0 and 12 h; MDR experiments included 0, 24, and 48 h assessments). Target peak temperatures (Tmax) included 50, 65, and 80 °C. Dose durations per block were 15, 30, or 60 min, with exposure bursts (Δtexp) of a few seconds repeated at fixed intervals (e.g., every 5 min within a dose). Antibiotics and concentrations: ciprofloxacin 0.5 μg/mL (PAO1); ceftriaxone 2 μg/mL or linezolid 2 μg/mL (S. aureus); meropenem 32 or 64 μg/mL and ciprofloxacin 64 μg/mL for MDR MB689 (MICs 64 μg/mL for both). Controls included antibiotics alone, iAMF alone, and plastic rings exposed to iAMF to verify metal dependence of the effect. Outcome measurements: At defined timepoints (immediately post-dose, 12 h pre/post second dose, 24 h; MDR up to 48 h), rings were rinsed, sonicated, and biofilm CFU/cm² quantified via serial dilution and plating on blood agar. Three biological replicates with 2–3 technical replicates were used. Imaging: Confocal laser scanning microscopy of GFP-expressing PAO1 biofilms with EPS stained by Concanavalin-A Alexa Fluor 647 assessed structural and morphological changes 12 h after treatments (iAMF alone, antibiotic alone, combination). Scanning electron microscopy (SEM) examined MDR MB689 biofilms 12 h after treatments (iAMF alone; ciprofloxacin or meropenem alone; combinations) at ~35,000×. Statistics: Two-way ANOVA with Tukey’s multiple comparisons; p < 0.05 considered significant.

- iAMF alone produced immediate 1–2 log10 CFU/cm² reductions in P. aeruginosa PAO1 biofilms after each dose, but CFU rebounded to baseline between doses. - Ciprofloxacin alone (0.5 μg/mL) reduced PAO1 biofilm by ~3 log10 within 12 h, followed by a plateau. - iAMF plus ciprofloxacin synergistically reduced PAO1 biofilm beyond either monotherapy, achieving >3-log reductions at 24 h (p < 0.0001) and often approaching the limit of detection across varying iAMF parameters (Tmax 50–80 °C; different dose durations and exposure counts). - Dose dependence: With Tmax = 65 °C and 5-min spaced exposures, longer iAMF dose blocks (15, 30, 60 min) yielded greater immediate CFU reductions; combinations achieved >5-log reductions by 24 h, versus ~2.7-log with ciprofloxacin alone (two-way ANOVA p = 0.0318 for 15 min; p < 0.0001 for 30 and 60 min). - S. aureus biofilms exhibited strong responses: an initial iAMF 15-min dose (Tmax = 65 °C) elicited >3-log reduction versus ~0.96 log in PAO1 under the same iAMF, and combination with ceftriaxone (2 μg/mL) or linezolid (2 μg/mL) further decreased CFU compared with monotherapies, approaching detection limits after two doses. - MDR P. aeruginosa (MB689; MIC 64 μg/mL for ciprofloxacin and meropenem) showed mechanism-dependent synergy: iAMF plus meropenem significantly reduced CFU to near LOD at MIC and enhanced killing at sub-MIC meropenem (32 μg/mL; p < 0.001), whereas iAMF plus ciprofloxacin did not outperform monotherapies, consistent with porin (oprD) loss mediating carbapenem resistance and gyrA/parC mutations mediating fluoroquinolone resistance. - Imaging indicated morphological changes consistent with membrane perturbation under combination therapy, supporting a hypothesis of heat-induced membrane disruption enhancing antibiotic uptake. - Thermal modeling and measurements showed uniform ring heating with minimal heating of surrounding media during short exposures; CEM43 estimates suggested low risk of collateral tissue damage beyond 1–2 mm from the metal surface under tested regimens. - iAMF–antibiotic efficacy was observed in younger (2-day) and older (7-day) biofilms, though details varied by organism and antibiotic; overall, combinations substantially outperformed single agents.

The findings demonstrate that brief, intermittent heating of metal surfaces by iAMF synergizes with antibiotics to eradicate bacterial biofilms more effectively than either approach alone. Heat alone transiently reduces biofilm burden but allows rapid regrowth, and antibiotics alone are limited by biofilm-mediated tolerance. In combination, iAMF likely disrupts bacterial membranes and alters biofilm structure, enhancing antibiotic penetration and activity, which explains the deeper and more durable reductions observed across diverse dosing parameters and temperatures. Mechanism-dependent rescue in an MDR P. aeruginosa isolate—synergy with meropenem (porin-mediated resistance) but not with ciprofloxacin (gyrase/topoisomerase mutations)—supports a model where membrane effects of iAMF sensitize bacteria to antibiotics that require outer membrane/porin passage, whereas target-based chromosomal resistance is not overcome. This approach could transform management of metal implant infections by enabling noninvasive, on-implant biofilm eradication while minimizing tissue injury via short, controlled heat exposures. Safety modeling (CEM43) suggests that appropriately dosed iAMF can be delivered without significant damage to adjacent tissues, and the multi-coil system shows the feasibility of controlled, uniform heating in vitro.

This study establishes that intermittent alternating magnetic fields (iAMF) combined with antibiotics synergistically eradicate biofilms on metal substrates, achieving multi-log reductions and frequently approaching detection limits across multiple pathogens (P. aeruginosa, S. aureus), antibiotics (ciprofloxacin, ceftriaxone, linezolid, meropenem), and dosing regimens. Mechanistic data support membrane disruption as a contributor to synergy and indicate potential to rescue activity of antibiotics against MDR organisms depending on resistance mechanisms. These results highlight a noninvasive, metal-targeted adjuvant strategy with clinical potential for treating implant-associated infections. Future work should optimize iAMF dosing (temperature, exposure counts, intervals), validate safety and efficacy in relevant in vivo/large animal models, refine coil design for implant geometries and positioning variability, and further delineate mechanistic pathways (membrane effects, stress responses) to guide antibiotic selection.

- In vitro study: Translation to in vivo settings remains unproven; tissue heterogeneity, blood flow, and implant positioning may alter heating and efficacy. - Synergy quantification: Defining synergy with a thermal dose (iAMF) is complex; complementary water-bath experiments support but do not fully resolve interaction modeling. - Non-thermal effects: Potential contributions from non-heat components of AMF were not excluded. - Implementation challenges: Achieving uniform, reproducible heating on patient-specific implant geometries and positions requires sophisticated, possibly customized coil designs; optimal target temperatures, dose numbers, and intervals need determination for durable responses with safety. - Textual inconsistencies in strain identifiers and parameters in some figure references suggest the need for standardized reporting in future studies.