We developed a method to grow Staphylococcus epidermidis bacterial biofilms and characterize their rheological properties in situ in a continuously fed bioreactor incorporated into a parallel plate rheometer. The temperature and shear rates of growth modeled bloodstream conditions, a common site of S. epidermidis infection. We measured the linear elastic (G′) and viscous moduli (G″) of the material using small-amplitude oscillatory rheology and the yield stress using non-linear creep rheology. We found that the elastic and viscous moduli of the S. epidermidis biofilm were 11 ± 3 Pa and 1.9 ± 0.5 Pa at a frequency of 1 Hz (6.283 rad per s) and that the yield stress was approximately 20 Pa. We modeled the linear creep response of the biofilm using a Jeffreys model and found that S. epidermidis has a characteristic relaxation time of approximately 750 seconds and a linear creep viscosity of 3000 Pa s. The effects on the linear viscoelastic moduli of environmental stressors, such as NaCl concentration and extremes of temperature, were also studied. We found a non-monotonic relationship between moduli and NaCl concentrations, with the stiffest material properties found at human physiological concentrations (135 mM). Temperature dependent rheology showed hysteresis in the moduli when heated and cooled between 5 °C and 60 °C. Through these experiments, we demonstrated that biofilms are rheologically complex materials that can be characterized by a combination of low modulus (~10 Pa), long relaxation time (~103 seconds), and a finite yield stress (20 Pa). This suggests that biofilms should be viewed as soft viscoelastic solids whose properties are determined in part by local environmental conditions. The in situ growth method introduced here can be adapted to a wide range of biofilm systems and applied over a broad spectrum of rheological and environmental conditions because the technique minimizes the risk of irreversible, non-linear deformation of the microbial specimen before analysis.

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 constituents are not significantly degraded by the temperature treatment. However, some aggregation on the scale of 100 nm was found by dynamic light scattering at 60 °C. Finally, relative to control biofilms maintained at 37 °C, we observed an order of magnitude reduction in the biofilm yield stress after 60 °C temperature treatment. No such difference was found for treatment at 45 °C. From these results, we conclude that the yield stress of bacterial biofilms is temperature-sensitive and that this sensitivity is correlated with cell viability. The observed significant decrease in yield stress with temperature suggests a means to weaken the mechanical integrity of S. epidermidis biofilms with applications in areas such as the treatment of biofilm-infected medical devices.

Background: Aerosolized delivery of antibiotics is hindered by poor penetration within distal and plugged airways. Antibacterial perfluorocarbon ventilation (APV) is a proposed solution in which the lungs are partially or totally filled with perfluorocarbon (PFC) containing emulsified antibiotics. The purpose of this study was to evaluate emulsion stability and rheological, antibacterial, and pharmacokinetic characteristics. Methods: This study examined emulsion aqueous droplet diameter and number density over 24 hr and emulsion and neat PFC viscosity and surface tension. Additionally, Pseudomonas aeruginosa biofilm growth was measured after 2-hr exposure to emulsion with variable aqueous volume percentages (0.25, 1, and 2.5%) and aqueous tobramycin concentrations (C a ¼0.4, 4, and 40 mg/mL). Lastly, the time course of serum and pulmonary tobramycin concentrations was evaluated following APV and conventional aerosolized delivery of tobramycin in rats. Results: The initial aqueous droplet diameter averaged 1.9 -0.2 lm with little change over time. Initial aqueous droplet number density averaged 3.5 -1.7 · 10 9 droplets/mL with a significant ( p < 0.01) decrease over time. Emulsion and PFC viscosity were not significantly different, averaging 1.22 -0.03 · 10 -3 Pa$sec. The surface tensions of PFC and emulsion were 15.0 -0.1 · 10 -3 and 14.6 -0.6 · 10 -3 N/m, respectively, and the aqueous interfacial tensions were 46.7 -0.3 · 10 -3 and 26.9 -11.0 · 10 -3 N/m ( p < 0.01), respectively. Biofilm growth decreased markedly with increasing C a and, to a lesser extent, aqueous volume percentage. Tobramycin delivered via APV yielded 2.5 and 10 times larger pulmonary concentrations at 1 and 4 hr post delivery, respectively, and significantly ( p < 0.05) lower serum concentrations compared with aerosolized delivery. Conclusions: The emulsion is bactericidal, retains the rheology necessary for pulmonary delivery, is sufficiently stable for this application, and results in increased pulmonary retention of the antibiotic.

Abstract. 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.Key words. Navier-Stokes equation, biofilm, immersed boundary method, computational fluid dynamics, viscoelastic fluid 1. Introduction. The goal of this work is to develop a numerical simulation method that accurately captures the biomechanical response of bacterial biofilms and their associated extracellular matrix (ECM). In this second paper, we show that the model and simulation method developed in part I [15], can be used to predict material properties of a biofilm and that the simulated results mimic experimentally measured results. The underlying mathematical technique is an adaptation of the Immersed Boundary Method (IBM) that takes into account the finite volume of bacteria, and variable material parameters found in biofilms whose variation is anchored to the positions of bacteria in a biofilm. We call this method the heterogeneous rheology Immersed Boundary Method (hrIBM). A key feature of our results is that the simulations are initialized with experimentally measured position data providing the locations of bacteria in live S. epidermidis biofilms. This removes ambiguity about how to represent the biofilm computationally. When using this data, the bulk physical properties estimated through simulation match experimental results. We also verify that when using different position data sets that possess similar spatial statistics, the physical properties of the biofilm do not change significantly. We also provide quantitative results on the periodic rotation of suspended aggregates of bacteria in shear flow.In recent years, much work has been done to develop detailed mathematical models that capture the biomechanical response of bacterial biofilms to physical changes [1,2,9,15,16]. In general, the physical properties governing the growth, attachment, and detachment of a biofilm are dependent on the ECM, a viscous mixture of polysaccharides and other biological products excreted b...

Measurement of the elastic modulus of soft, viscoelastic liquids with cavitation rheometry is demonstrated for specimens as small as 1 ll 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 as 1 ll. V C 2014 AIP Publishing LLC.[http://dx.doi.org/10.1063/1.4896108]The linear elastic modulus of a soft material is a mechanical property measurable by techniques such as mechanical rheometry, microrheology, and atomic force microscopy (AFM). 1,2 Needs for both in vivo characterization of linear elasticity (such as in tissue viability) as well as rapid measurement (such as in quality control applications) have driven recent methods development. [3][4][5] Mechanical rheometry typically requires approximately milliliter sample volumes and, if sample loading and testing durations are considered, requires as much as 5 min to test one specimen at one deformation frequency. Passive microrheology is a widely used technique to study the mechanical properties of small volumes (between $3 and 50 ll) of soft matter. One method of microrheology-which uses the multiple scattering technique of diffusing wave spectroscopy-requires as much as an hour of measurement time. This method can probe elastic moduli up to $2000 Pa. 1,6,7 Microrheology measurements can also be impacted by the stability of the dispersed probes and the heterogeneity of the material studied. 8,9 AFM can also be used to characterize the elastic modulus of very small volumes (<1 ll) of material; however, this technique requires long durations for measurements and sample preparation time. 3,10 The duration of these techniques makes them challenging for high throughput applications, while their lack of portability complicates their use as in vivo diagnostics.The cavitation rheometry technique of Zimberlin et al. characterizes the linear elastic modulus of soft matter with Young's modulus in the range of 0.12 kPa < E < 40 kPa. 11,12 It is an inexpensive, fast, and portable method that estimates the elastic modulus by measurement of the critical pressure (P c ) required for internal cavitation. Cavitation is induced by air pumped through a needle inserted into the sample. The critical pressure predicts the elastic modulus, E, through the theory of cavitation in an incompressib...