Inertial-based Fluidic Platform for Rapid Isolation of Blood-borne Pathogens.

INTRODUCTION: Bacterial sepsis is a life-threatening disease and a significant clinical problem caused by host responses to a microbial infection. Sepsis is a leading cause of death worldwide and, importantly, a significant cause of morbidity and mortality in combat settings, placing a considerable burden on military personnel and military health budgets. The current method of treating sepsis is restricted to pathogen identification, which can be prolonged, and antibiotic administration, which is, initially, often suboptimal. The clinical trials that have been performed to evaluate bacterial separation as a sepsis therapy have been unsuccessful, and new approaches are needed to address this unmet clinical need. MATERIALS AND METHODS: An inertial-based, scalable spiral microfluidic device has been created to overcome these previous deficiencies through successful separation of infection-causing pathogens from the bloodstream, serving as a proof of principle for future adaptations. Fluorescent imaging of fluorescent microspheres mimicking the sizes of bacteria cells and blood cells as well as fluorescently stained Acinetobacter baumannii were used to visualize flow within the spiral. The particles were imaged when flowing at a constant volumetric rate of 0.2 mL min-1 through the device. The same device was functionalized with colistin and exposed to flowing A. baumannii at 0.2 mL h-1. RESULTS: Fluorescent imaging within the channel under a constant volumetric flow rate demonstrated that smaller, bacteria-sized microspheres accumulated along the inner wall of the channel, whereas larger blood cell-sized microspheres accumulated within the center of the channel. Additionally, fluorescently stained A. baumannii displayed accumulation along the channel walls in agreement with calculated performance. Nearly 106 colony-forming units of A. baumannii were extracted with 100% capture efficiency from flowing phosphate-buffered saline at 0.2 mL h-1 in this device; this is at least one order of magnitude more bacteria than present in the blood of a human at the onset of sepsis. CONCLUSIONS: This type of bacterial separation device potentially provides an ideal approach for treating soldiers in combat settings. It eliminates the need for immediate pathogen identification and determination of antimicrobial susceptibility, making it suitable for rapid use within low-resource environments. The overall simplicity and durability of this design also supports its broad translational potential to improve military mortality rates and overall patient outcomes.

Magnetic Extraction of Acinetobacter baumannii Using Colistin-Functionalized γ-Fe2O3/Au Core/Shell Composite Nanoclusters.

Acinetobacter baumannii is a Gram-negative bacterium of increasing concern due to its virulence and persistence in combat and healthcare environments. The incidence of both community-acquired and nosocomial A. baumannii infections is on the rise in foreign and domestic healthcare facilities. Treatment options are limited due to the acquisition of multidrug resistance to the few effective antibiotics. Currently, the most effective pharmaceutically based treatment for multidrug-resistant A. baumannii infections is the antibiotic colistin (polymyxin E). To minimize side effects associated with administration of colistin or other toxic antimicrobial agents, we propose the development of a nanotechnology-mediated treatment strategy. In this design-based effort, colistin-functionalized multilayered, inorganic, magnetoplasmonic nanoconstructs were fabricated to bind to the surface of A. baumannii. This result, for the first time, demonstrates a robust, pharmaceutical-based motif for high affinity, composite nanoparticulates targeting the A. baumannii surface. The antibiotic-activated nanomaterials demonstrated cytocompatibility with human cells and no acute bacterial toxicity at nanoparticle to bacterial concentrations <10 000:1. The magnetomotive characteristics of the nanomaterial enabled magnetic extraction of the bacteria. In a macroscale environment, maximal separation efficiencies exceeding 38% were achieved. This result demonstrates the potential for implementation of this technology into micro- or mesofluidic-based separation environments to enhance extraction efficiencies. The future development of such a mesofluidic-based, nanotechnology-mediated platform is potentially suitable for adjuvant therapies to assist in the treatment of sepsis.

Dynamic Computational Model of Symptomatic Bacteremia to Inform Bacterial Separation Treatment Requirements.

The rise of multi-drug resistance has decreased the effectiveness of antibiotics, which has led to increased mortality rates associated with symptomatic bacteremia, or bacterial sepsis. To combat decreasing antibiotic effectiveness, extracorporeal bacterial separation approaches have been proposed to capture and separate bacteria from blood. However, bacteremia is dynamic and involves host-pathogen interactions across various anatomical sites. We developed a mathematical model that quantitatively describes the kinetics of pathogenesis and progression of symptomatic bacteremia under various conditions, including bacterial separation therapy, to better understand disease mechanisms and quantitatively assess the biological impact of bacterial separation therapy. Model validity was tested against experimental data from published studies. This is the first multi-compartment model of symptomatic bacteremia in mammals that includes extracorporeal bacterial separation and antibiotic treatment, separately and in combination. The addition of an extracorporeal bacterial separation circuit reduced the predicted time of total bacteria clearance from the blood of an immunocompromised rodent by 49%, compared to antibiotic treatment alone. Implementation of bacterial separation therapy resulted in predicted multi-drug resistant bacterial clearance from the blood of a human in 97% less time than antibiotic treatment alone. The model also proposes a quantitative correlation between time-dependent bacterial load among tissues and bacteremia severity, analogous to the well-known 'area under the curve' for characterization of drug efficacy. The engineering-based mathematical model developed may be useful for informing the design of extracorporeal bacterial separation devices. This work enables the quantitative identification of the characteristics required of an extracorporeal bacteria separation device to provide biological benefit. These devices will potentially decrease the bacterial load in blood. Additionally, the devices may achieve bacterial separation rates that allow consequent acceleration of bacterial clearance in other tissues, inhibiting the progression of symptomatic bacteremia, including multi-drug resistant variations.

Colistin-Functionalized Nanoparticles for the Rapid Capture of Acinetobacter baumannii.

Gold nanoparticles (AuNPs) were functionalized for rapid binding of Acinetobacter baumannii (A. baumannii), a Gram-negative bacterium. AuNPs were functionalized with colistin (Col), a polycationic antibiotic, using a two-step self-assembly process, in which heterobifunctional polyethylene glycol (PEG) was used as a linker. Colistin was successfully conjugated to the AuNPs (Col-PEG-AuNP), as validated by dynamic light scattering (DLS) and proton nuclear magnetic resonance (H1 NMR). High angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images, acquired simultaneously with X-ray energy dispersive spectroscopy (EDS) data, confirmed binding of Col-PEG-AuNPs to the cell envelope of A. baumannii. Results generated from a binding assay indicated that Col-PEG-AuNP complexation with A. baumannii occurred rapidly and reached half-maximum saturation in approximately 7 minutes, on average, for all A. baumannii strains evaluated. Quantitative measurement of the kinetics of Col-PEG-AuNP binding to A. baumannii is essential to inform the design of colistin-functionalized magnetic nanoparticles for magnetic separation of nanoparticle-bound A. baumannii.