Antibiotic Augmentation of Thermal Eradication of Staphylococcus epidermidis Biofilm Infections.

Staphylococcus epidermidis is a major contributor to bacterial infections on medical implants, currently treated by surgical removal of the device and the surrounding infected tissue at considerable morbidity and expense. In situ hyperthermia is being investigated as a non-invasive means of mitigating these bacterial biofilm infections, but minimizing damage to the surrounding tissue requires augmenting the thermal shock with other approaches such as antibiotics and discerning the minimum shock required to eliminate the biofilm. S. epidermidis biofilms were systematically shocked at a variety of temperatures (50-80 °C) and durations (1-10 min) to characterize their thermal susceptibility and compare it to other common nosocomial pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Biofilms were also exposed to three classes of antibiotics (ciprofloxacin, tobramycin and erythromycin) separately at concentrations ranging from 0 to 128 µg mL-1 to evaluate their impact on the efficacy of thermal shock and the subsequent potential regrowth of the biofilm. S. epidermidis biofilms were shown to be more thermally susceptible to hyperthermia than other common bacterial pathogens. All three antibiotics substantially decreased the duration and/or temperature needed to eliminate the biofilms, though this augmentation did not meet the criteria of synergism immediately following thermal shock. Subsequent reincubation, however, revealed strong synergism on a longer timescale.

Thermal susceptibility and antibiotic synergism of methicillin-resistant Staphylococcus aureus biofilms.

Methicillin-Resistant Staphylococcus aureus (MRSA) biofilms are among the most dangerous infections on medical implants, typically requiring surgical explantation and replacement. This study investigated the thermal susceptibility of MRSA biofilms to thermal shocks from 60 to 80 °C for 1-30 min as well as the effect of various antibiotics (most notably methicillin) on thermal mitigation. Pre- and post-shock exposure to three different classes of antibiotics (ciprofloxacin, tobramycin, and methicillin) at concentrations ranging from 0.25 to 128 µg mL-1 were investigated. MRSA biofilms exhibited thermal susceptibility comparable to other common nosocomial pathogens, such as Pseudomonas aeruginosa, though with greater variability. Exposure to antibiotics of any class significantly decreased the degree of thermal shock required for reliable mitigation, including at subclinical concentration. These combined treatments reduced biofilm population more than the sum of thermal and chemical treatments alone, demonstrating synergism, while also indicating a critical population drop of ∼4.5 log10 beyond which the biofilms typically became non-viable.

Thermal Shock and Ciprofloxacin Act Orthogonally on Pseudomonas aeruginosa Biofilms.

Bacterial biofilm infections are a major liability of medical implants, due to their resistance to both antibiotics and host immune response. Thermal shock can kill established biofilms, and some evidence suggests antibiotics may enhance this efficacy, despite having an insufficient effect themselves. The nature of this interaction is unclear, however, complicating efforts to integrate thermal shock into implant infection treatment. This study aimed to determine whether these treatments were truly synergistic or simply orthogonal (i.e., independent). Pseudomonas aeruginosa biofilms of different architectures and stationary-phase population density were subjected to various thermal shocks, antibiotic exposures, or combinations thereof, and examined either immediately after treatment or after subsequent reincubation. Population decreases from the combination treatment matched the product of the decreases of individual treatments, indicating their orthogonality. However, reincubation showed binary behavior, where biofilms with an immediate population decrease beyond a critical factor (~104) died off completely during reincubation, while biofilms with a smaller immediate decrease regrew. This critical factor was independent of the initial population density and the combination of treatments that achieved the immediate decrease. While antibiotics do not appear to enhance thermal shock directly, their contribution to achieving a critical population decrease for biofilm elimination can make the treatments appear strongly synergistic, strongly decreasing the intensity of thermal shock needed.

Thermal shock susceptibility and regrowth of Pseudomonas aeruginosa biofilms.

Biofilms on implanted medical devices cause thousands of patients each year to undergo multiple surgeries to remove and replace the implant, driving billions of dollars in increased health care costs due to the lack of viable treatment options for in situ biofilm eradication. Remotely activated localised heating is under investigation to mitigate these biofilms; however, little is known about the temperatures required to kill the biofilms. To better understand the required parameters this study investigated the thermal susceptibility of biofilms as a function of their fluidic and chemical environment during growth, as well as their propensity for regrowth following thermal shock. Pseudomonas aeruginosa biofilms were cultured in shaker plate fluidic conditions in four different growth media, then thermally shocked at various temperatures and exposure times. Biofilms were re-incubated to determine their regrowth potential following thermal shocks of various intensities. Results indicate that growth media has little impact on thermal susceptibility, while fluidic conditions strongly influence susceptibility to modest thermal shocks. This effect disappears, however, with increasingly aggressive shocks, reducing biofilm populations by up to 5 orders of magnitude. Regrowth studies indicate a critical post-shock bacterial loading (∼103 CFU/cm2) below which the biofilms were no longer viable, while biofilms above that loading slowly regrew to their previous population density.

BioMEMS for biosensors and closed-loop drug delivery.

The efficacy of pharmaceutical treatments can be greatly enhanced by physiological feedback from the patient using biosensors, though this is often invasive or infeasible. By adapting microelectromechanical systems (MEMS) technology to miniaturize such biosensors, previously inaccessible signals can be obtained, often from inside the patient. This is enabled by the device's extremely small footprint which minimizes both power consumption and implantation trauma, as well as the transport time for chemical analytes, in turn decreasing the sensor's response time. MEMS fabrication also allows mass production which can be easily scaled without sacrificing its high reproducibility and reliability, and allows seamless integration with control circuitry and telemetry which is already produced using the same materials and fabrication steps. By integrating these systems with drug delivery devices, many of which are also MEMS-based, closed loop drug delivery can be achieved. This paper surveys the types of signal transduction devices available for biosensing-primarily electrochemical, optical, and mechanical-looking at their implementation via MEMS technology. The impact of MEMS technology on the challenges of biosensor development, particularly safety, power consumption, degradation, fouling, and foreign body response, are also discussed.

Synergistic effects of heat and antibiotics on Pseudomonas aeruginosa biofilms.

Upon formation of a biofilm, bacteria undergo several changes that prevent eradication with antimicrobials alone. Due to this resistance, the standard of care for infected medical implants is explantation of the infected implant and surrounding tissue, followed by eventual reimplantation of a replacement device. Recent studies have demonstrated the efficacy of heat shock for biofilm eradication. To minimize the heat required for in situ biofilm eradication, this study investigated the hypothesis that antibiotics, while ineffective by themselves, may substantially increase heat shock efficacy. The combined effect of heat and antibiotics on Pseudomonas aeruginosa biofilms was quantified via heat shock in combination with ciprofloxacin, tobramycin, or erythromycin at multiple concentrations. Combined treatments had synergistic effects for all antibiotics for heat shock conditions of 60°C for 5 min to 70°C for 1 min, indicating an alternative to surgical explantation.

Prevascularized silicon membranes for the enhancement of transport to implanted medical devices.

Recent advances in drug delivery and sensing devices for in situ applications are limited by the diffusion-limiting foreign body response of fibrous encapsulation. In this study, we fabricated prevascularized synthetic device ports to help mitigate this limitation. Membranes with rectilinear arrays of square pores with widths ranging from 40 to 200 µm were created using materials (50 µm thick double-sided polished silicon) and processes (photolithography and directed reactive ion etching) common in the manufacturing of microfabricated sensors. Vascular endothelial cells responded to membrane geometry by either forming vascular tubes that extended through the pore or completely filling membrane pores after 4 days in culture. Although tube formation began to predominate overgrowth around 75 µm and continued to increase at even larger pore sizes, tubes formed at these large pore sizes were not completely round and had relatively thin walls. Thus, the optimum range of pore size for prevascularization of these membranes was estimated to be 75-100 µm. This study lays the foundation for creating a prevascularized port that can be used to reduce fibrous encapsulation and thus enhance diffusion to implanted medical devices and sensors. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1602-1609, 2016.

Thermal mitigation of Pseudomonas aeruginosa biofilms.

Bacterial biofilms infect 2-4% of medical devices upon implantation, resulting in multiple surgeries and increased recovery time due to the very great increase in antibiotic resistance in the biofilm phenotype. This work investigates the feasibility of thermal mitigation of biofilms at physiologically accessible temperatures. Pseudomonas aeruginosa biofilms were cultured to high bacterial density (1.7 × 10(9) CFU cm(-2)) and subjected to thermal shocks ranging from 50°C to 80°C for durations of 1-30 min. The decrease in viable bacteria was closely correlated with an Arrhenius temperature dependence and Weibull-style time dependence, demonstrating up to six orders of magnitude reduction in bacterial load. The bacterial load for films with more conventional initial bacterial densities dropped below quantifiable levels, indicating thermal mitigation as a viable approach to biofilm control.

Added release time in diffusion/dissolution coupled release.

While increasingly sophisticated models have been developed to more accurately predict dispersed solute release from complex systems, distillation of their results into quantitative trends has been difficult. Here, the numerically calculated release profiles of coupled diffusion/dissolution systems are quantified by their cumulative release time (CRT) and compared against corresponding diffusion-controlled limits. The increase in CRT due to a finite dissolution rate was found to vary inversely with the second Damköhler number across several orders of magnitude, and also vary linearly with the amount of solid drug loaded in the system. The analytical nature of the relationship provides new physical insights into the system and appears to be indifferent to the form of the secondary rate-limiting step. This work provides a simple analytical expression with which one can not only predict the mean release time for a given set of parameter values, but understand precisely how each parameter value will affect it. The simplicity of the correlation and the lack of apparent limits to its validity also suggest the existence of an analytical pathway for its derivation, which may yield additional insights into the effect of secondary rate processes on controlled release.

Magnetic nanoparticle/polymer composites for medical implant infection control.

New potential medical applications for magnetic nanoparticle/polymer composite coatings, including deactivation of bacterial biofilms, require much higher power densities than can be supplied by previously developed polymer composites. These coatings in turn require much higher nanoparticle concentrations, where particle-particle and particle-polymer interactions play a significant role in the material's performance. This paper investigates the effect of several key design parameters on the resulting specific absorption rate of magnetite nanoparticle composites. Hydrophobic (poly(styrene), (PS)) and hydrophilic (poly(vinyl alcohol), (PVA)) polymer composite coatings were compared in both aqueous and non-aqueous solvents at multiple nanoparticle loadings and film thicknesses. Heating rates up to 717 W g-1 Fe were observed in a typical (2.32 kA m-1, 302 kHz) alternating magnetic field (AMF), achieving heating power densities up to 7.5 W cm-2. To estimate in vivo power requirements, electrical resistance heating beneath a tissue mimic heat sink indicated a peak power requirement of only 4.5 W cm-2 to achieve an 80 °C surface temperature in 15 s, demonstrating that these composites can exceed the power densities needed for applications such as treating bacterial infections on medical implants in situ. Polymer identity, solvent identity, and especially orientation within the magnetic field were shown to strongly affect the power density with effects that are interrelated.

BioMEMS in drug delivery.

The drive to design micro-scale medical devices which can be reliably and uniformly mass produced has prompted many researchers to adapt processing technologies from the semiconductor industry. By operating at a much smaller length scale, the resulting biologically-oriented microelectromechanical systems (BioMEMS) provide many opportunities for improved drug delivery: Low-dose vaccinations and painless transdermal drug delivery are possible through precisely engineered microneedles which pierce the skin's barrier layer without reaching the nerves. Low-power, low-volume BioMEMS pumps and reservoirs can be implanted where conventional pumping systems cannot. Drug formulations with geometrically complex, extremely uniform micro- and nano-particles are formed through micromolding or with microfluidic devices. This review describes these BioMEMS technologies and discusses their current state of implementation. As these technologies continue to develop and capitalize on their simpler integration with other MEMS-based systems such as computer controls and telemetry, BioMEMS' impact on the field of drug delivery will continue to increase.

Hard and soft micro- and nanofabrication: An integrated approach to hydrogel-based biosensing and drug delivery.

We review efforts to produce microfabricated glucose sensors and closed-loop insulin delivery systems. These devices function due to the swelling and shrinking of glucose-sensitive microgels that are incorporated into silicon-based microdevices. The glucose response of the hydrogel is due to incorporated phenylboronic acid (PBA) side chains. It is shown that in the presence of glucose, these polymers alter their swelling properties, either by ionization or by formation of glucose-mediated reversible crosslinks. Swelling pressures impinge on microdevice structures, leading either to a change in resonant frequency of a microcircuit, or valving action. Potential areas for future development and improvement are described. Finally, an asymmetric nano-microporous membrane, which may be integrated with the glucose-sensitive devices, is described. This membrane, formed using photolithography and block polymer assembly techniques, can be functionalized to enhance its biocompatibility and solute size selectivity. The work described here features the interplay of design considerations at the supramolecular, nano, and micro scales.

Top-down and bottom-up fabrication techniques for hydrogel based sensing and hormone delivery microdevices.

We review a set of studies dealing with molecular (glucose) sensing and hormone delivery, in which the swelling and shrinking of a hydrogel as a function of glucose concentration play a central role. Confining hydrogels in microfabricated structures permits transduction of their chemomechanical behaviors. Prototype microdevices for wireless glucose sensing and closed loop insulin delivery control have been designed using hydrogels containing phenylboronic acid sidechains. While these devices exhibit desired responses, improved response time is needed, warranting further miniaturization. In a separate application, geometric confinement of glucose oxidase by a pH-sensitive hydrogel membrane sets up a nonlinear feedback loop which enables rhythmic swell/shrink cycles when the system is exposed to a constant glucose concentration. The latter system may be applied to delivery of gonadotropin release hormone, for which rhythmicity of secretion is essential for therapeutic function.

Hydrogel-based microsensors for wireless chemical monitoring.

We report fabrication and characterization of a new hydrogel-based microsensor for wireless chemical monitoring. The basic device structure is a high-sensitivity capacitive pressure sensor coupled to a stimuli-sensitive hydrogel that is confined between a stiff porous membrane and a thin glass diaphragm. As small molecules pass through the porous membrane, the hydrogel swells and deflects the diaphragm which is also the movable plate of the variable capacitor in an LC resonator. The resulting change in resonant frequency can be remotely detected by the phase-dip technique. Prior to hydrogel loading, the sensitivity of the pressure sensor to applied air pressure was measured to be 222 kHz/kPa over the range of 41.9-51.1 MHz. With a pH-sensitive hydrogel, the sensor displayed a sensitivity of 1.16 MHz/pH for pH 3.0-6.5, and a response time of 45 minutes.

Composite block polymer-microfabricated silicon nanoporous membrane.

Block polymers offer an attractive route to densely packed, monodisperse nanoscale pores. However, their fragility as thin films complicates their use as membranes. By integrating a block polymer film with a thin (100 microm) silicon substrate, we have developed a composite membrane providing both nanoscale size exclusion and fast transport of small molecules. Here we describe the fabrication of this membrane, evaluate its mechanical integrity, and demonstrate its transport properties for model solutes of large and small molecular weight. The ability to block large molecules without hindering smaller ones, coupled with the potential for surface modification of the polymer and the microelectromechanical system style of support, makes this composite membrane an attractive candidate for interfacing implantable sensing and drug-delivery devices with biological hosts.

Integration of hydrogels with hard and soft microstructures.

Hydrogels, i.e., water-swollen polymer networks, have been studied and utilized for decades. These materials can either passively support mass transport, or can actively respond in their swelling properties, enabling modulation of mass and fluid transport, and chemomechanical actuation. Response rates increase with decreasing hydrogel dimension. In this paper, we present three examples where incorporation of hydrogels into solid microstructures permits acceleration of their response, and also provides novel functional capabilities. In the first example, a hydrogel is immobilized inside microfabricated pores within a thin silicon membrane. This hydrogel does not have a swelling response under the conditions investigated, but under proper conditions it can be utilized as a part of an electrolytic diode. In the second example, hydrogels are polymerized under microcantilever beams, and their swelling response to pH or glucose concentration causes variable deflection of the beam, observable under a microscope. In the third example, swelling and shrinking of a hydrogel embedded in a microfabricated valve structure leads to chemical gating of fluid motion through that valve. In all cases, the small size of the system enhances its response rate.

A hydrogel-based implantable micromachined transponder for wireless glucose measurement.

In this paper, we report on the design and characterization of a new hydrogel-based implantable wireless glucose sensor. The basic device structure is a passive [inductor/capacitor (LC)] micromachined resonator coupled to a stimuli-sensitive hydrogel, which is confined between a stiff nanoporous membrane and a thin glass diaphragm. As glucose molecules pass through the nanoporous membrane, the hydrogel swells and deflects the flexible glass diaphragm, which is the movable plate of the variable capacitor in the totally integrated passive LC resonator. The corresponding change in resonant frequency can be remotely detected. A glucose- sensitive phenylboronic acid-based hydrogel was loaded into the microtransponder, and its sensitivity and time response were measured. Prior to hydrogel loading, the sensitivity of the pressure sensor to applied air pressure was measured to be -222 kHz/kPa over the frequency range 51-->42 MHz. The sensor showed a sensitivity of -34.3 kHz/mM over the glucose concentration range 0-20 mM (at pH 7.4), and a response time of 90 min. The dynamic response, although unacceptable at such values, can be easily improved by decreasing the hydrogel thickness and reducing the sensor and porous membrane thicknesses. The transponder's overall dimensions were 5x5x0.8 mm3, small enough for subcutaneous implantation.

A polymer membrane containing Fe(0) as a contaminant barrier.

A poly(vinyl alcohol) (PVA) membrane containing iron (Fe(0)) particles was developed and tested as a model barrier for contaminant containment. Carbon tetrachloride, copper (Cu2+), nitrobenzene, 4-nitroacetophenone, and chromate (Cr04(2-)) were selected as model contaminants. Compared with a pure PVA membrane, the Fe(0)/PVA membrane can increase the breakthrough lag time for Cu2+ and carbon tetrachloride by more than 100-fold. The increase in the lag time was smaller for nitrobenzene and 4-nitroacetophenone, which stoichiometrically require more iron and for which the PVA membrane has a higher permeability. The effect of Fe(0) was even smaller for CrO4(2-) because of its slow reaction. Forty-five percent of the iron, based on the content in the dry membrane prior to hydration, was consumed by reaction with Cu2+ and 15% by reaction with carbon tetrachloride. Similarly, 25%, 17%, and 6% of the iron was consumed by nitrobenzene, 4-nitroacetophenone, and CrO4(2-), respectively. These percentages approximately double when the loss of iron during membrane hydration is considered. The permeability of the Fe(0)/PVA membrane after breakthrough was within a factor of 3 for that of pure PVA, consistent with theory. These results suggest that polymer membranes with embedded Fe(0) have potential as practical contaminant barriers.