Animal research has provided important information about many aspects of the pathophysiology of h... more Animal research has provided important information about many aspects of the pathophysiology of human disease. Well-performed animal studies can determine the potential benefit of many proposed therapeutic interventions, and experimental results from animal studies have served as the basis for many landmark clinical trials. Many animal research models are described in the research literature, and choosing the appropriate model to answer a research question can be a daunting task. Even more challenging is developing a new model when none of the existing systems are relevant to the proposed question. This article was prepared by members of the SAEM Research Committee to provide an overview of animal modeling. Important considerations in choosing, applying, and developing animal research models are outlined. Practical discussions of potential problems with animal models are also provided.
ASAIO journal (American Society for Artificial Internal Organs : 1992), Jan 23, 2015
Central line-associated bloodstream infections (CLABSIs) are not easily treated and many catheter... more Central line-associated bloodstream infections (CLABSIs) are not easily treated and many catheters (e.g., hemodialysis catheters) are not easily replaced. Biofilms (the source of infection) on catheter surfaces are notoriously difficult to eradicate. We have recently demonstrated that modest elevations of temperature lead to increased staphylococcal susceptibility to vancomycin and significantly soften the biofilm matrix. In this study, using a combination of microbiological, computational, and experimental studies, we demonstrate the efficacy, feasibility, and safety of using heat as an adjuvant treatment for infected hemodialysis catheters. Specifically, we show that treating with heat in the presence of antibiotics led to additive killing of Staphylococcus epidermidis with similar trends seen for Staphylococcus aureus and Klebsiella pneumoniae. The magnitude of temperature elevation required is relatively modest (45-50°C) and similar to that used as an adjuvant to traditional can...
The overall physiologic effect of partial liquid ventilation (PLV) in the setting of unilateral l... more The overall physiologic effect of partial liquid ventilation (PLV) in the setting of unilateral lung injury remains unclear. Therefore, we evaluated the effect of PLV on gas exchange in unilateral lung injury. Left unilateral lung injury was induced in 14 adult dogs by oleic acid instillation into a left pulmonary artery. The animals were divided into two groups: gas ventilation (GV) and PLV. During both GV and PLV, systemic blood gas levels were analyzed. Oxygen consumption (O(2)), carbon dioxide production (CO(2)) and pulmonary blood flow (Q) of both the right lung (uninjured lung) and left lung (injured lung) were measured. During PLV, O(2) of the injured left lung (o(2)-injured), CO(2) of the injured left lung (CO(2)-injured), and Q of the injured left lung (Q-injured) were greater than those in GV (O(2)-injured, 41.6 mL/min vs 23.4 mL/min, p = 0.006; CO(2)-injured, 34.4 mL/min vs 25.5 mL/min, p = 0.026; and Q-injured, 0.47 L/min vs 0.22 L/min, p = 0.002, respectively). However, overall PaO(2) during PLV was less than that during GV, likely due to either a redistribution of Q toward the injured lung (PLV Q-injured, 0.47 L/min vs GV Q-injured, 0.22 L/min; p = 0.002) or reduced gas exchange efficiency in the healthy lung. We conclude that in our model, PLV increases O(2) and VCO(2) in the injured lung. However, over all gas exchange efficiency is reduced.
We evaluate the application of cavitation rheology to characterize the elasticity of biological s... more We evaluate the application of cavitation rheology to characterize the elasticity of biological soft matter that may be confined to volumes as small as 1 µL. Cavitation rheology, a technique developed by Zimberlin et. al (Soft Matter, 3(6), 765-767, 2007), is a simple, rapid method to characterize the mechanical properties of materials that can be approximated as linearly elastic by creating a bubble, or cavity, in a relatively small volume of the material. This technique relates the critical pressure necessary to form the cavity to the material elasticity in a limit where the bubble size is small relative to size of the specimen. For characterization of small volumes of biological soft matter, such as surface adherent bacterial biofilms, this method must be extended to accommodate situations in which the cavitation volume is finite relative to the specimen volume. We therefore investigated the underlying principles of cavitation rheology. From basic elasticity theory, we mathematic...
Biofilm-like bacterial constructs consisting of sub-micron bacterial cells and chitosan are tuned... more Biofilm-like bacterial constructs consisting of sub-micron bacterial cells and chitosan are tuned to create materials with distinct microstructural and mechanical features. While it is known that biofilms are viscoelastic in nature and the colloidal microstructure of biofilms can vary depending on their growth environment, little is known about the contributions of individual biofilm components to biofilm mechanics. Bacterial biofilms can be thought of as composite materials that contain bacterial cells surrounded by matrix materials, such as polysaccharides, proteins and DNA. Here, we use bacteria cells and chitosan—a polysaccharide with physical chemistry similar to a naturally occurring polysaccharide—as building blocks to create biofilm-like composites and probe the contributions of individual components to biofilm structure and mechanics. Using confocal laser scanning microscopy coupled with bacteria identification and dynamic bacteria tracking tools, we characterize the micros...
Microscale cellular clustering and intercellular separation of surface adherent biofilms of the o... more Microscale cellular clustering and intercellular separation of surface adherent biofilms of the opportunistic pathogen Staphylococcus epidermidis are found to be well-described by colloidal measures such as the cluster distribution and the radial distribution function. These measures are found to depend significantly on the environmental stress or antibiotic stress present as the biofilm grows under conditions of steady flow. Bacterial biofilms are bacterial cells encapsulated in an extracellular polymeric substance consisting of polysaccharides, DNA, and proteins. Since bacterial size is on the order of 1 μm, we view bacterial biofilms as a colloidal composite material with the bacterial cells as the particles and the extracellular matrix as a viscoelastic hydrogel. Here we probe the cellular microstructure of S. epidermidis biofilms at varying environmental stress conditions by combining high-resolution confocal microscopy with colloidal image analysis techniques that identify ind...
Biofilms are aggregates of bacteria encapsulated with a protective layer of extracellular polysac... more Biofilms are aggregates of bacteria encapsulated with a protective layer of extracellular polysaccharide (EPS). Staphylococcus epidermidis biofilms are frequent in hospital-related infections associated with contaminated medical devices. Studies have shown that NaCl can contribute to the overexpression of the ica operon, which plays a role in the production of EPS (Conlon, K.M., J. Bacteriology, 2002). This study determined the effect of osmotic stress imparted by high concentrations of NaCl on material and mechanical properties of S. epidermidis RP62A bacterial biofilms. We probed the intercellular microstructure by analysis of 3D image volumes acquired by confocal laser scanning microscopy. We observed that increasing NaCl concentration significantly decreased the cellular density of biofilms grown in a 0.011 Pa shear environment. Parallel plate rheology was used to determine the mechanical properties of S. epidermidis biofilms under conditions of both linear and non-linear deform...
We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be cr... more We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be created through colloidal self-assembly of cells and polymers, and thereby link the complex material properties of biofilms to well understood colloidal and polymeric behaviors. This finding is applied to soften and disassemble staphylococcal biofilms through pH changes. Bacterial biofilms are viscoelastic, structured communities of cells encapsulated in an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, and DNA. Although the identity and abundance of EPS macromolecules are known, how these matrix materials interact with themselves and bacterial cells to generate biofilm morphology and mechanics is not understood. Here, we find that the colloidal self-assembly of Staphylococcus epidermidis RP62A cells and polysaccharides into viscoelastic biofilms is driven by thermodynamic phase instability of EPS. pH conditions that induce phase instability of chitosan produce artificial S. epidermidis biofilms whose mechanics match natural S. epidermidis biofilms. Furthermore, pH-induced solubilization of the matrix triggers disassembly in both artificial and natural S. epidermidis biofilms. This pH-induced disassembly occurs in biofilms formed by five additional staphylococcal strains, including three clinical isolates. Our findings suggest that colloidal self-assembly of cells and matrix polymers produces biofilm viscoelasticity and that biofilm control strategies can exploit this mechanism.
Journal of applied physiology (Bethesda, Md. : 1985), 2001
Systemic complement activation has been noted in a variety of shock states, and there is growing ... more Systemic complement activation has been noted in a variety of shock states, and there is growing evidence that, in addition to being proinflammatory effectors, products of complement activation contribute directly to generalized manifestations of shock, such as hypotension and acidosis. To study the effects of complement activation, we examined responses in rats to systemic activation of complement with cobra venom factor (CVF), including blood pressure, metabolic acidosis, changes in vascular permeability, and lung function. High doses of CVF produced circulatory collapse (mean arterial pressure = 110 +/- 16 and 35 +/- 9 mmHg in control and with CVF, respectively, P < 0.05), metabolic acidosis (HCO concentration = 27.8 +/- 1.7 and 9.6 +/- 3.4 meq/l in control and with CVF, respectively, P < 0.05), extravasation of albumin into the lung and gut, and modest arterial hypoxemia (PO2 = 486 +/- 51 and 201 +/- 36 Torr in control and during 100% O2 breathing, respectively, P < 0.0...
Given the increasing evidence of safe application of elevated temperature in other clinical conte... more Given the increasing evidence of safe application of elevated temperature in other clinical contexts, we consider the potential for supplemental hyperthermia to augment the effects of vancomycin against staphylococci, a major source of post-operative and post-traumatic sepsis. Laboratory reference strains and libraries of clinical blood isolates of S. epidermidis and methicillin resistant S. aureus, both as planktonic cells and as established biofilms, were assessed for thermosensitivity and increased susceptibility to vancomycin in the setting of thermal treatment. In addition to viability measures, patterns of stress gene expression were assessed with qPCR and structural changes were measured using quantitative transmission electron microscopy. Laboratory strains of both species had reduced growth and biofilm viability at 45°C, a temperature commonly employed in other domains such as adjuvant treatments of malignancy. Blood isolates of S. epidermidis were consistent in this regard as well, but significant between-isolate variability in thermosensitivity was seen in blood isolates of S. aureus. Expression profiling and ultrastructural measurements confirmed that elevated temperature was a substantial stressor with or without vancomycin treatment. Our findings suggest that temperature elevations shown to be tolerated in humans in other settings hold the potential to be used as an adjuvant to antibiotic therapy against staphylococcal biofilms.
We present a multi-scale model to study the attachment of spherical particles with a rigid core, ... more We present a multi-scale model to study the attachment of spherical particles with a rigid core, coated with binding ligands and in equilibrium with the surrounding, quiescent fluid medium. This class of fluid-immersed adhesion is widespread in many natural and engineering settings. Our theory highlights how the micro-scale binding kinetics of these ligands, as well as the attractive / repulsive surface potential in an ionic medium effects the eventual macro-scale size distribution of the particle aggregates (flocs). The results suggest that the presence of elastic ligands on the particle surface allow large floc aggregates by inducing efficient inter-floc collisions (i.e., a large, non-zero collision factor). Strong electrolytic composition of the surrounding fluid favors large floc formation as well.
In this work we consider how surface-adherent bacterial biofilm communities respond in flowing sy... more In this work we consider how surface-adherent bacterial biofilm communities respond in flowing systems. We simulate the fluid-structure interaction and separation process using the immersed boundary method. In these simulations we model and simulate different density and viscosity values of the biofilm than that of the surrounding fluid. The simulation also includes breakable springs connecting the bacteria in the biofilm. This allows the inclusion of erosion and detachment into the simulation. We use the incompressible Navier-Stokes (N-S) equations to describe the motion of the flowing fluid. We discretize the fluid equations using finite differences and use a geometric multigrid method to solve the resulting equations at each time step. The use of multigrid is necessary because of the dramatically different densities and viscosities between the biofilm and the surrounding fluid. We investigate and simulate the model in both two and three dimensions. Our method differs from previou...
Measurement of the elastic modulus of soft, viscoelastic liquids with cavitation rheometry is dem... more Measurement of the elastic modulus of soft, viscoelastic liquids with cavitation rheometry is demonstrated for specimens as small as 1 μl by application of elasticity theory and experiments on semi-dilute polymer solutions. Cavitation rheometry is the extraction of the elastic modulus of a material, E, by measuring the pressure necessary to create a cavity within it [J. A. Zimberlin, N. Sanabria-DeLong, G. N. Tew, and A. J. Crosby, Soft Matter 3, 763-767 (2007)]. This paper extends cavitation rheometry in three ways. First, we show that viscoelastic samples can be approximated with the neo-Hookean model provided that the time scale of the cavity formation is measured. Second, we extend the cavitation rheometry method to accommodate cases in which the sample size is no longer large relative to the cavity dimension. Finally, we implement cavitation rheometry to show that the theory accurately measures the elastic modulus of viscoelastic samples with volumes ranging from 4 ml to as low...
Langmuir : the ACS journal of surfaces and colloids, Jan 17, 2015
Changes in temperature were found to affect the morphology, cell viability, and mechanical proper... more Changes in temperature were found to affect the morphology, cell viability, and mechanical properties of Staphylococcus epidermidis bacterial biofilms. S. epidermidis biofilms are commonly associated with hospital-acquired medical device infections. We observed the effect of heat treatment on three physical properties of the biofilms: the bacterial cell morphology and viability, the polymeric properties of the extracellular polymeric substance (EPS), and the rheological properties of the bulk biofilm. After application of a 1 h heat treatment at 45 °C, cell reproduction had ceased, and at 60 °C, cell viability was significantly reduced. Size exclusion chromatography was used to fractionate the extracellular polymeric substance (EPS) based on size. Chemical analysis of each fraction showed that the relative concentrations of the polysaccharide, protein, and DNA components of the EPS were unchanged by the heat treatment at 45 and 60 °C. The results suggest that the EPS molecular const...
The goal of this work is to develop a numerical simulation that accurately captures the biomechan... more The goal of this work is to develop a numerical simulation that accurately captures the biomechanical response of bacterial biofilms and their associated extracellular matrix (ECM). In this, the second of a two-part effort, the primary focus is on formally presenting the heterogeneous rheology Immersed Boundary Method (hrIBM) and validating our model against experimental results. With this extension of the Immersed Bounadry Method (IBM), we use the techniques originally developed in Part I, (Hammond et al. [15]) to treat the biofilm as a viscoelastic fluid possessing variable rheological properties anchored to a set of moving locations (i.e., the bacteria locations). We validate our modeling approach from Part I by comparing dynamic moduli and compliance moduli computed from our model to data from mechanical characterization experiments on Staphylococcus epidermidis biofilms. The experimental setup is described in Pavlovsky et al. (2013) [22] in which biofilms are grown and tested in a parallel plate rheometer. Matlab code used to produce results in this paper will be available at https://github.com/MathBioCU/BiofilmSim.
Real-time assessment of operator performance during procedural simulation is a common practice th... more Real-time assessment of operator performance during procedural simulation is a common practice that requires undivided attention by 1 or more reviewers, potentially over many repetitions of the same case. To determine whether reviewers display better interrater agreement of procedural competency when observing recorded, rather than live, performance; and to develop an assessment tool for pediatric rapid sequence intubation (pRSI). A framework of a previously established Objective Structured Assessment of Technical Skills (OSATS) tool was modified for pRSI. Emergency medicine residents (postgraduate year 1-4) were prospectively enrolled in a pRSI simulation scenario and evaluated by 2 live raters using the modified tool. Sessions were videotaped and reviewed by the same raters at least 4 months later. Raters were blinded to their initial rating. Interrater agreement was determined by using the Krippendorff generalized concordance method. Overall interrater agreement for live review was 0.75 (95% confidence interval [CI], 0.72-0.78) and for video was 0.79 (95% CI, 0.73-0.82). Live review was significantly superior to video review in only 1 of the OSATS domains (Preparation) and was equivalent in the other domains. Intrarater agreement between the live and video evaluation was very good, greater than 0.75 for all raters, with a mean of 0.81 (95% CI, 0.76-0.85). The modified OSATS assessment tool demonstrated some evidence of validity in discriminating among levels of resident experience and high interreviewer reliability. With this tool, intrareviewer reliability was high between live and 4-months&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; delayed video review of the simulated procedure, which supports feasibility of delayed video review in resident assessment.
American journal of physiology. Lung cellular and molecular physiology, 2003
Our objective was to test the effect of inhibition of thromboxane synthase versus inhibition of c... more Our objective was to test the effect of inhibition of thromboxane synthase versus inhibition of cyclooxygenase (COX)-1/2 on pulmonary gas exchange and heart function during simulated pulmonary embolism (PE) in the rat. PE was induced in rats via intrajugular injection of polystyrene microspheres (25 micro m). Rats were randomized to one of three posttreatments: 1) placebo (saline), 2) thromboxane synthase inhibition (furegrelate sodium), or 3) COX-1/2 inhibition (ketorolac tromethamine). Control rats received no PE. Compared with controls, placebo rats had increased thromboxane B(2) (TxB(2)) in bronchoalveolar lavage fluid and increased urinary dinor TxB(2). Furegrelate and ketorolac treatments reduced TxB(2) and dinor TxB(2) to control levels or lower. Both treatments significantly decreased the alveolar dead space fraction, but neither treatment altered arterial oxygenation compared with placebo. Ketorolac increased in vivo mean arterial pressure and ex vivo left ventricular press...
Interdisciplinary Perspectives on Infectious Diseases, 2014
Staphylococcus epidermidis is an important cause of nosocomial infection and bacteremia. It is al... more Staphylococcus epidermidis is an important cause of nosocomial infection and bacteremia. It is also a common contaminant of blood cultures and, as a result, there is frequently uncertainty as to its diagnostic significance when recovered in the clinical laboratory. One molecular strategy that might be of value in clarifying the interpretation of S. epidermidis identified in blood culture is multilocus sequence typing. Here, we examined 100 isolates of this species (50 blood isolates representing true bacteremia, 25 likely contaminant isolates, and 25 skin isolates) and the ability of sequence typing to differentiate them. Three machine learning algorithms (classification regression tree, support vector machine, and nearest neighbor) were employed. Genetic variability was substantial between isolates, with 44 sequence types found in 100 isolates. Sequence types 2 and 5 were most commonly identified. However, among the classification algorithms we employed, none were effective, with CART and SVM both yielding only 73% diagnostic accuracy and nearest neighbor analysis yielding only 53% accuracy. Our data mirror previous studies examining the presence or absence of pathogenic genes in that the overlap between truly significant organisms and contaminants appears to prevent the use of MLST in the clarification of blood cultures recovering S. epidermidis.
Animal research has provided important information about many aspects of the pathophysiology of h... more Animal research has provided important information about many aspects of the pathophysiology of human disease. Well-performed animal studies can determine the potential benefit of many proposed therapeutic interventions, and experimental results from animal studies have served as the basis for many landmark clinical trials. Many animal research models are described in the research literature, and choosing the appropriate model to answer a research question can be a daunting task. Even more challenging is developing a new model when none of the existing systems are relevant to the proposed question. This article was prepared by members of the SAEM Research Committee to provide an overview of animal modeling. Important considerations in choosing, applying, and developing animal research models are outlined. Practical discussions of potential problems with animal models are also provided.
ASAIO journal (American Society for Artificial Internal Organs : 1992), Jan 23, 2015
Central line-associated bloodstream infections (CLABSIs) are not easily treated and many catheter... more Central line-associated bloodstream infections (CLABSIs) are not easily treated and many catheters (e.g., hemodialysis catheters) are not easily replaced. Biofilms (the source of infection) on catheter surfaces are notoriously difficult to eradicate. We have recently demonstrated that modest elevations of temperature lead to increased staphylococcal susceptibility to vancomycin and significantly soften the biofilm matrix. In this study, using a combination of microbiological, computational, and experimental studies, we demonstrate the efficacy, feasibility, and safety of using heat as an adjuvant treatment for infected hemodialysis catheters. Specifically, we show that treating with heat in the presence of antibiotics led to additive killing of Staphylococcus epidermidis with similar trends seen for Staphylococcus aureus and Klebsiella pneumoniae. The magnitude of temperature elevation required is relatively modest (45-50°C) and similar to that used as an adjuvant to traditional can...
The overall physiologic effect of partial liquid ventilation (PLV) in the setting of unilateral l... more The overall physiologic effect of partial liquid ventilation (PLV) in the setting of unilateral lung injury remains unclear. Therefore, we evaluated the effect of PLV on gas exchange in unilateral lung injury. Left unilateral lung injury was induced in 14 adult dogs by oleic acid instillation into a left pulmonary artery. The animals were divided into two groups: gas ventilation (GV) and PLV. During both GV and PLV, systemic blood gas levels were analyzed. Oxygen consumption (O(2)), carbon dioxide production (CO(2)) and pulmonary blood flow (Q) of both the right lung (uninjured lung) and left lung (injured lung) were measured. During PLV, O(2) of the injured left lung (o(2)-injured), CO(2) of the injured left lung (CO(2)-injured), and Q of the injured left lung (Q-injured) were greater than those in GV (O(2)-injured, 41.6 mL/min vs 23.4 mL/min, p = 0.006; CO(2)-injured, 34.4 mL/min vs 25.5 mL/min, p = 0.026; and Q-injured, 0.47 L/min vs 0.22 L/min, p = 0.002, respectively). However, overall PaO(2) during PLV was less than that during GV, likely due to either a redistribution of Q toward the injured lung (PLV Q-injured, 0.47 L/min vs GV Q-injured, 0.22 L/min; p = 0.002) or reduced gas exchange efficiency in the healthy lung. We conclude that in our model, PLV increases O(2) and VCO(2) in the injured lung. However, over all gas exchange efficiency is reduced.
We evaluate the application of cavitation rheology to characterize the elasticity of biological s... more We evaluate the application of cavitation rheology to characterize the elasticity of biological soft matter that may be confined to volumes as small as 1 µL. Cavitation rheology, a technique developed by Zimberlin et. al (Soft Matter, 3(6), 765-767, 2007), is a simple, rapid method to characterize the mechanical properties of materials that can be approximated as linearly elastic by creating a bubble, or cavity, in a relatively small volume of the material. This technique relates the critical pressure necessary to form the cavity to the material elasticity in a limit where the bubble size is small relative to size of the specimen. For characterization of small volumes of biological soft matter, such as surface adherent bacterial biofilms, this method must be extended to accommodate situations in which the cavitation volume is finite relative to the specimen volume. We therefore investigated the underlying principles of cavitation rheology. From basic elasticity theory, we mathematic...
Biofilm-like bacterial constructs consisting of sub-micron bacterial cells and chitosan are tuned... more Biofilm-like bacterial constructs consisting of sub-micron bacterial cells and chitosan are tuned to create materials with distinct microstructural and mechanical features. While it is known that biofilms are viscoelastic in nature and the colloidal microstructure of biofilms can vary depending on their growth environment, little is known about the contributions of individual biofilm components to biofilm mechanics. Bacterial biofilms can be thought of as composite materials that contain bacterial cells surrounded by matrix materials, such as polysaccharides, proteins and DNA. Here, we use bacteria cells and chitosan—a polysaccharide with physical chemistry similar to a naturally occurring polysaccharide—as building blocks to create biofilm-like composites and probe the contributions of individual components to biofilm structure and mechanics. Using confocal laser scanning microscopy coupled with bacteria identification and dynamic bacteria tracking tools, we characterize the micros...
Microscale cellular clustering and intercellular separation of surface adherent biofilms of the o... more Microscale cellular clustering and intercellular separation of surface adherent biofilms of the opportunistic pathogen Staphylococcus epidermidis are found to be well-described by colloidal measures such as the cluster distribution and the radial distribution function. These measures are found to depend significantly on the environmental stress or antibiotic stress present as the biofilm grows under conditions of steady flow. Bacterial biofilms are bacterial cells encapsulated in an extracellular polymeric substance consisting of polysaccharides, DNA, and proteins. Since bacterial size is on the order of 1 μm, we view bacterial biofilms as a colloidal composite material with the bacterial cells as the particles and the extracellular matrix as a viscoelastic hydrogel. Here we probe the cellular microstructure of S. epidermidis biofilms at varying environmental stress conditions by combining high-resolution confocal microscopy with colloidal image analysis techniques that identify ind...
Biofilms are aggregates of bacteria encapsulated with a protective layer of extracellular polysac... more Biofilms are aggregates of bacteria encapsulated with a protective layer of extracellular polysaccharide (EPS). Staphylococcus epidermidis biofilms are frequent in hospital-related infections associated with contaminated medical devices. Studies have shown that NaCl can contribute to the overexpression of the ica operon, which plays a role in the production of EPS (Conlon, K.M., J. Bacteriology, 2002). This study determined the effect of osmotic stress imparted by high concentrations of NaCl on material and mechanical properties of S. epidermidis RP62A bacterial biofilms. We probed the intercellular microstructure by analysis of 3D image volumes acquired by confocal laser scanning microscopy. We observed that increasing NaCl concentration significantly decreased the cellular density of biofilms grown in a 0.011 Pa shear environment. Parallel plate rheology was used to determine the mechanical properties of S. epidermidis biofilms under conditions of both linear and non-linear deform...
We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be cr... more We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be created through colloidal self-assembly of cells and polymers, and thereby link the complex material properties of biofilms to well understood colloidal and polymeric behaviors. This finding is applied to soften and disassemble staphylococcal biofilms through pH changes. Bacterial biofilms are viscoelastic, structured communities of cells encapsulated in an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, and DNA. Although the identity and abundance of EPS macromolecules are known, how these matrix materials interact with themselves and bacterial cells to generate biofilm morphology and mechanics is not understood. Here, we find that the colloidal self-assembly of Staphylococcus epidermidis RP62A cells and polysaccharides into viscoelastic biofilms is driven by thermodynamic phase instability of EPS. pH conditions that induce phase instability of chitosan produce artificial S. epidermidis biofilms whose mechanics match natural S. epidermidis biofilms. Furthermore, pH-induced solubilization of the matrix triggers disassembly in both artificial and natural S. epidermidis biofilms. This pH-induced disassembly occurs in biofilms formed by five additional staphylococcal strains, including three clinical isolates. Our findings suggest that colloidal self-assembly of cells and matrix polymers produces biofilm viscoelasticity and that biofilm control strategies can exploit this mechanism.
Journal of applied physiology (Bethesda, Md. : 1985), 2001
Systemic complement activation has been noted in a variety of shock states, and there is growing ... more Systemic complement activation has been noted in a variety of shock states, and there is growing evidence that, in addition to being proinflammatory effectors, products of complement activation contribute directly to generalized manifestations of shock, such as hypotension and acidosis. To study the effects of complement activation, we examined responses in rats to systemic activation of complement with cobra venom factor (CVF), including blood pressure, metabolic acidosis, changes in vascular permeability, and lung function. High doses of CVF produced circulatory collapse (mean arterial pressure = 110 +/- 16 and 35 +/- 9 mmHg in control and with CVF, respectively, P < 0.05), metabolic acidosis (HCO concentration = 27.8 +/- 1.7 and 9.6 +/- 3.4 meq/l in control and with CVF, respectively, P < 0.05), extravasation of albumin into the lung and gut, and modest arterial hypoxemia (PO2 = 486 +/- 51 and 201 +/- 36 Torr in control and during 100% O2 breathing, respectively, P < 0.0...
Given the increasing evidence of safe application of elevated temperature in other clinical conte... more Given the increasing evidence of safe application of elevated temperature in other clinical contexts, we consider the potential for supplemental hyperthermia to augment the effects of vancomycin against staphylococci, a major source of post-operative and post-traumatic sepsis. Laboratory reference strains and libraries of clinical blood isolates of S. epidermidis and methicillin resistant S. aureus, both as planktonic cells and as established biofilms, were assessed for thermosensitivity and increased susceptibility to vancomycin in the setting of thermal treatment. In addition to viability measures, patterns of stress gene expression were assessed with qPCR and structural changes were measured using quantitative transmission electron microscopy. Laboratory strains of both species had reduced growth and biofilm viability at 45°C, a temperature commonly employed in other domains such as adjuvant treatments of malignancy. Blood isolates of S. epidermidis were consistent in this regard as well, but significant between-isolate variability in thermosensitivity was seen in blood isolates of S. aureus. Expression profiling and ultrastructural measurements confirmed that elevated temperature was a substantial stressor with or without vancomycin treatment. Our findings suggest that temperature elevations shown to be tolerated in humans in other settings hold the potential to be used as an adjuvant to antibiotic therapy against staphylococcal biofilms.
We present a multi-scale model to study the attachment of spherical particles with a rigid core, ... more We present a multi-scale model to study the attachment of spherical particles with a rigid core, coated with binding ligands and in equilibrium with the surrounding, quiescent fluid medium. This class of fluid-immersed adhesion is widespread in many natural and engineering settings. Our theory highlights how the micro-scale binding kinetics of these ligands, as well as the attractive / repulsive surface potential in an ionic medium effects the eventual macro-scale size distribution of the particle aggregates (flocs). The results suggest that the presence of elastic ligands on the particle surface allow large floc aggregates by inducing efficient inter-floc collisions (i.e., a large, non-zero collision factor). Strong electrolytic composition of the surrounding fluid favors large floc formation as well.
In this work we consider how surface-adherent bacterial biofilm communities respond in flowing sy... more In this work we consider how surface-adherent bacterial biofilm communities respond in flowing systems. We simulate the fluid-structure interaction and separation process using the immersed boundary method. In these simulations we model and simulate different density and viscosity values of the biofilm than that of the surrounding fluid. The simulation also includes breakable springs connecting the bacteria in the biofilm. This allows the inclusion of erosion and detachment into the simulation. We use the incompressible Navier-Stokes (N-S) equations to describe the motion of the flowing fluid. We discretize the fluid equations using finite differences and use a geometric multigrid method to solve the resulting equations at each time step. The use of multigrid is necessary because of the dramatically different densities and viscosities between the biofilm and the surrounding fluid. We investigate and simulate the model in both two and three dimensions. Our method differs from previou...
Measurement of the elastic modulus of soft, viscoelastic liquids with cavitation rheometry is dem... more Measurement of the elastic modulus of soft, viscoelastic liquids with cavitation rheometry is demonstrated for specimens as small as 1 μl by application of elasticity theory and experiments on semi-dilute polymer solutions. Cavitation rheometry is the extraction of the elastic modulus of a material, E, by measuring the pressure necessary to create a cavity within it [J. A. Zimberlin, N. Sanabria-DeLong, G. N. Tew, and A. J. Crosby, Soft Matter 3, 763-767 (2007)]. This paper extends cavitation rheometry in three ways. First, we show that viscoelastic samples can be approximated with the neo-Hookean model provided that the time scale of the cavity formation is measured. Second, we extend the cavitation rheometry method to accommodate cases in which the sample size is no longer large relative to the cavity dimension. Finally, we implement cavitation rheometry to show that the theory accurately measures the elastic modulus of viscoelastic samples with volumes ranging from 4 ml to as low...
Langmuir : the ACS journal of surfaces and colloids, Jan 17, 2015
Changes in temperature were found to affect the morphology, cell viability, and mechanical proper... more Changes in temperature were found to affect the morphology, cell viability, and mechanical properties of Staphylococcus epidermidis bacterial biofilms. S. epidermidis biofilms are commonly associated with hospital-acquired medical device infections. We observed the effect of heat treatment on three physical properties of the biofilms: the bacterial cell morphology and viability, the polymeric properties of the extracellular polymeric substance (EPS), and the rheological properties of the bulk biofilm. After application of a 1 h heat treatment at 45 °C, cell reproduction had ceased, and at 60 °C, cell viability was significantly reduced. Size exclusion chromatography was used to fractionate the extracellular polymeric substance (EPS) based on size. Chemical analysis of each fraction showed that the relative concentrations of the polysaccharide, protein, and DNA components of the EPS were unchanged by the heat treatment at 45 and 60 °C. The results suggest that the EPS molecular const...
The goal of this work is to develop a numerical simulation that accurately captures the biomechan... more The goal of this work is to develop a numerical simulation that accurately captures the biomechanical response of bacterial biofilms and their associated extracellular matrix (ECM). In this, the second of a two-part effort, the primary focus is on formally presenting the heterogeneous rheology Immersed Boundary Method (hrIBM) and validating our model against experimental results. With this extension of the Immersed Bounadry Method (IBM), we use the techniques originally developed in Part I, (Hammond et al. [15]) to treat the biofilm as a viscoelastic fluid possessing variable rheological properties anchored to a set of moving locations (i.e., the bacteria locations). We validate our modeling approach from Part I by comparing dynamic moduli and compliance moduli computed from our model to data from mechanical characterization experiments on Staphylococcus epidermidis biofilms. The experimental setup is described in Pavlovsky et al. (2013) [22] in which biofilms are grown and tested in a parallel plate rheometer. Matlab code used to produce results in this paper will be available at https://github.com/MathBioCU/BiofilmSim.
Real-time assessment of operator performance during procedural simulation is a common practice th... more Real-time assessment of operator performance during procedural simulation is a common practice that requires undivided attention by 1 or more reviewers, potentially over many repetitions of the same case. To determine whether reviewers display better interrater agreement of procedural competency when observing recorded, rather than live, performance; and to develop an assessment tool for pediatric rapid sequence intubation (pRSI). A framework of a previously established Objective Structured Assessment of Technical Skills (OSATS) tool was modified for pRSI. Emergency medicine residents (postgraduate year 1-4) were prospectively enrolled in a pRSI simulation scenario and evaluated by 2 live raters using the modified tool. Sessions were videotaped and reviewed by the same raters at least 4 months later. Raters were blinded to their initial rating. Interrater agreement was determined by using the Krippendorff generalized concordance method. Overall interrater agreement for live review was 0.75 (95% confidence interval [CI], 0.72-0.78) and for video was 0.79 (95% CI, 0.73-0.82). Live review was significantly superior to video review in only 1 of the OSATS domains (Preparation) and was equivalent in the other domains. Intrarater agreement between the live and video evaluation was very good, greater than 0.75 for all raters, with a mean of 0.81 (95% CI, 0.76-0.85). The modified OSATS assessment tool demonstrated some evidence of validity in discriminating among levels of resident experience and high interreviewer reliability. With this tool, intrareviewer reliability was high between live and 4-months&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; delayed video review of the simulated procedure, which supports feasibility of delayed video review in resident assessment.
American journal of physiology. Lung cellular and molecular physiology, 2003
Our objective was to test the effect of inhibition of thromboxane synthase versus inhibition of c... more Our objective was to test the effect of inhibition of thromboxane synthase versus inhibition of cyclooxygenase (COX)-1/2 on pulmonary gas exchange and heart function during simulated pulmonary embolism (PE) in the rat. PE was induced in rats via intrajugular injection of polystyrene microspheres (25 micro m). Rats were randomized to one of three posttreatments: 1) placebo (saline), 2) thromboxane synthase inhibition (furegrelate sodium), or 3) COX-1/2 inhibition (ketorolac tromethamine). Control rats received no PE. Compared with controls, placebo rats had increased thromboxane B(2) (TxB(2)) in bronchoalveolar lavage fluid and increased urinary dinor TxB(2). Furegrelate and ketorolac treatments reduced TxB(2) and dinor TxB(2) to control levels or lower. Both treatments significantly decreased the alveolar dead space fraction, but neither treatment altered arterial oxygenation compared with placebo. Ketorolac increased in vivo mean arterial pressure and ex vivo left ventricular press...
Interdisciplinary Perspectives on Infectious Diseases, 2014
Staphylococcus epidermidis is an important cause of nosocomial infection and bacteremia. It is al... more Staphylococcus epidermidis is an important cause of nosocomial infection and bacteremia. It is also a common contaminant of blood cultures and, as a result, there is frequently uncertainty as to its diagnostic significance when recovered in the clinical laboratory. One molecular strategy that might be of value in clarifying the interpretation of S. epidermidis identified in blood culture is multilocus sequence typing. Here, we examined 100 isolates of this species (50 blood isolates representing true bacteremia, 25 likely contaminant isolates, and 25 skin isolates) and the ability of sequence typing to differentiate them. Three machine learning algorithms (classification regression tree, support vector machine, and nearest neighbor) were employed. Genetic variability was substantial between isolates, with 44 sequence types found in 100 isolates. Sequence types 2 and 5 were most commonly identified. However, among the classification algorithms we employed, none were effective, with CART and SVM both yielding only 73% diagnostic accuracy and nearest neighbor analysis yielding only 53% accuracy. Our data mirror previous studies examining the presence or absence of pathogenic genes in that the overlap between truly significant organisms and contaminants appears to prevent the use of MLST in the clarification of blood cultures recovering S. epidermidis.
Uploads
Papers by John Younger