M. Mehdi Salek

Staphylococcus aureus biofilm formation and tolerance to antibiotics in response to oscillatory shear stresses of physiological levels

Bacterial infections in the blood system are usually associated with blood flow oscillation generated by some cardiovascular pathologies and insertion of indwelling devices. The influence of hydrodynamically induced shear stress fluctuations on the Staphylococcus aureus biofilm morphology and tolerance to antibiotics was investigated. Fluctuating shear stresses of physiologically relevant levels were generated in wells of a six-well microdish agitated by an orbital shaker. Numerical simulations were performed to determine the spatial distribution and local fluctuation levels of the shear stress field on the well bottom. It is found that the local biofilm deposition and morphology correlate strongly with shear stress fluctuations and maximum magnitude levels. Tolerance to killing by antibiotics correlates with morphotype and is generally higher in high shear regions.

The influence of flow cell geometry related shear stresses on the distribution, structure and susceptibility of Pseudomonas aeruginosa 01 biofilms

The effects of non-uniform hydrodynamic conditions resulting from flow cell geometry (square and rectangular cross-section) on Pseudomonas aeruginosa 01 (PAO1) biofilm formation, location, and structure were investigated for nominally similar flow conditions using a combination of confocal scanning laser microscope (CSLM) and computational fluid dynamics (CFD). The thickness and surface coverage of PAO1 biofilms were observed to vary depending on the location in the flow cell and thus also the local wall shear stress. The biofilm structure in a 5:1 (width to height) aspect ratio rectangular flow cell was observed to consist mainly of a layer of bacterial cells with thicker biofilm formation observed in the flow cell corners. For square cross-section (1:1 aspect ratio) flow cells, generally thicker and more uniform surface coverage biofilms were observed. Mushroom shaped structures with hollow centers and wall breaks, indicative of ‘seeding’ dispersal structures, were found exclusively in the square cross-section tubes. Exposure of PAO1 biofilms grown in the flow cells to gentamicin revealed a difference in susceptibility. Biofilms grown in the rectangular flow cell overall exhibited a greater susceptibility to gentamicin compared to those grown in square flow cells. However, even within a given flow cell, differences in susceptibility were observed depending on location. This study demonstrates that the spanwise shear stress distribution within the flow cells has an important impact on the location of colonization and structure of the resultant biofilm. These differences in biofilm structure have a significant impact on the susceptibility of the biofilms grown within flow channels. The impact of flow modification due to flow cell geometry should be considered when designing flow cells for laboratory investigation of bacterial biofilms.

Methicillin resistant Staphylococcus aureus adhesion to human umbilical vein endothelial cells demonstrates wall shear stress dependent behaviour

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prevalent pathogen capable of causing severe vascular infections. The goal of this work was to investigate the role of shear stress in early adhesion events.MethodsHuman umbilical vein endothelial cells (HUVEC) were exposed to MRSA for 15-60 minutes and shear stresses of 0-1.2 Pa in a parallel plate flow chamber system. Confocal microscopy stacks were captured and analyzed to assess the number of MRSA. Flow chamber parameters were validated using micro-particle image velocimetry (PIV) and computational fluid dynamics modelling (CFD).ResultsUnder static conditions, MRSA adhered to, and were internalized by, more than 80% of HUVEC at 15 minutes, and almost 100% of the cells at 1 hour. At 30 minutes, there was no change in the percent HUVEC infected between static and low flow (0.24 Pa), but a 15% decrease was seen at 1.2 Pa. The average number of MRSA per HUVEC decreased 22% between static and 0.24 Pa, and 37% between 0.24 Pa and 1.2 Pa. However, when corrected for changes in bacterial concentration near the surface due to flow, bacteria per area was shown to increase at 0.24 Pa compared to static, with a subsequent decline at 1.2 Pa.ConclusionsThis study demonstrates that MRSA adhesion to endothelial cells is strongly influenced by flow conditions and time, and that MSRA adhere in greater numbers to regions of low shear stress. These areas are common in arterial bifurcations, locations also susceptible to generation of atherosclerosis.

Methicillin resistant Staphylococcus aureus biofilm susceptibility in response to increased level of shear stresses and flow agitation

The appearance of highly resistance bacterial strains or “super bugs” is a major challenge in both the community and hospital environment. Manipulating laboratory based devices in order to generate the correct environmental conditions to study these biofilms requires a through comprehension of the hydrodynamics surrounding the biofilm. In this study, MBEC™ biomedical devices were used to study biofilm antibiotic response and susceptibility at different rotational speeds of an orbital shaker. The fluid motion was modeled with higher shear stresses corresponding to higher rotational speeds which result in an increased susceptibility of the biofilm to antimicrobial agent.

Numerical Simulation of Fluid Flow and Hydrodynamic Analysis in Commonly Used Biomedical Devices in Biofilm Studies

Biofilms are microbial communities which can form on most biotic or abiotic surfaces including glass, metal, plastic, rocks, and live tissues. These colonies begin with individual planktonic bacterial cells that attach to a surface and then start to generate a sticky Extracellular Polymeric Substance (EPS). This complex polysaccharide matrix contributes to a modification of the phenotypic status of bacteria and protects them against the detrimental changes in the microenvironment surrounding the biofilms. These phenotypic changes typically confer increased resistance to antibiotics or to the host defence system in patients. This enhanced tolerance is associated with significant problems, such as hospital acquired infections, equipment damage, and energy losses (Trachoo, 2003; Percival et al., 2004), making biofilms a major concern in different industries. In health care, biofilms are responsible for 65% of hospital acquired infections, adding more than

Micro-PIV and CFD studies show non-uniform wall shear stress distributions over endothelial cells

1 billion annually for treatment costs in United States (Percival et al., 2004). Hospital acquired infections are the fourth leading cause of death in the U.S. accounting for 2 million death annually (Wenzel, 2007). Almost all types of biomedical devices and tissue engineering constructs are susceptible to biofilm formation (Bryers & Ratner, 2004; Bryers, 2008). Biofilms are particularly associated with a variety of bloodstream infections related to indwelling medical devices (e.g. urinary and cardiovascular catheters, vascular and ocular prostheses, prosthetic heart valves, cardiac pacemakers, cerebrospinal fluid shunts and other types of surgical devices). They are also responsible for chronic infections and recalcitrant diseases such as cystic fibrosis and periodontal diseases (Castelli et al.,2006; MacLeod et al., 2007; Meda et al., 2007; Presterl et al., 2007; Murray et al., 2007; Bryers, 2008; Phillips et al., 2008). In industrial applications, biofilms can clog filters, block pipes and induce corrosion. They are responsible for billions of dollars yearly in equipment damage, energy losses, and water system contamination (Geesey & Bryers 2000). Additional costs associated with biofilm contaminations include disinfection, preventive maintenance, mitigation and replacement of contaminated materials.

Numerical Simulation of Fluid Flow and Oxygen Transport in the Tube Flow Cells Containing Biofilms

Wall shear stress acting on arterial walls is an important hemodynamic force determining vessel health. A parallel-plate flow chamber with a 127 μm-thick flow channel is used as an in vitro system to study the fluid mechanics environment. It is essential to know how well this flow chamber performs in emulating physiologic flow regimes especially when cultured cells are present. Hence, the objectives of this work are to computationally and experimentally study the characteristic of the flow chamber in providing a defined flow regime and shear stress to cultured cells and to map wall shear stress distributions in the presence of an endothelial cell layer. Experiments and modeling were performed for the nominal wall shear stresses of 2 and 10 dyn/cm2 . Without endothelial cells, the flow field is uniform over 95% of the chamber cross-section and the surfaces are exposed to the target stress level. Using PIV velocity data, the endothelial cell surfaces were re-constructed and flow over these surfaces was then simulated via FLUENT. Once endothelial cells are introduced, local shear variations are large and the velocity profiles are no longer uniform. Due to the velocity distribution between peaks and valleys, the local wall shear stresses range between 47–164% of the nominal values. This study demonstrates the non-uniform shear stress distribution over the cells is non-negligible especially in small vessels or where blockage is important.Copyright

Intermittent turbulence in flowing bacterial suspensions.

The hydrodynamics in flow systems is known to induce phenotypic changes associated with bacterial biofilms, including increased tolerance to antimicrobial agents and biocides. Results obtained in flow cells commonly used in biological and medical studies on the influence of flow on biofilm behavior and antimicrobial susceptibility are sometimes contradictory. It is thus hypothesized that discrepancies in the results may be related to the flow cell geometry. In this study, the shear stress distribution and substrate concentration were numerically simulated inside long rectangular and square tubes. The fluid was Newtonian and a uniform distribution of biofilms, which consume the substrate from the medium, was assumed on the walls. The consumption of oxygen by biofilms was assumed to follow the Monod kinetics. The effects of flow velocity, flow cell geometry, and substrate diffusivity on wall shear stress and substrate concentration distributions were investigated. Based on simulation results, differences observed in the morphology and response of biofilms can be directly related to hydrodynamic changes caused by the flow cell configuration.Copyright

Energy Redistribution Between the Mean and Pulsating Flow Field in a Separated Flow Region

Dense suspensions of motile bacteria, possibly including the human gut microbiome, exhibit collective dynamics akin to those observed in classic, high Reynolds number turbulence with important implications for chemical and biological transport, yet this analogy has remained primarily qualitative. Here, we present experiments in which a dense suspension of Bacillus subtilis bacteria was flowed through microchannels and the velocity statistics of the flowing suspension were quantified using a recently developed velocimetry technique coupled with vortex identification methods. Observations revealed a robust intermittency phenomenon, whereby the average velocity profile of the suspension fluctuated between a plug-like flow and a parabolic flow profile. This intermittency is a hallmark of the onset of classic turbulence and Lagrangian tracking revealed that it here originates from the presence of transient vortices in the active, collective motion of the bacteria locally reinforcing the externally imposed flow. These results link together two entirely different manifestations of turbulence and show the potential of the microfluidic approach to mimic the environment characteristic of certain niches of the human microbiome.

What do biofilms sense in agitated well plates? A combined CFD and experimental study on spatial and temporal wall shear stress distribution

One of the main features of the backward-facing step (BFS) low frequency pulsatile flow is the unsteadiness due to the convection of vortical (coherent) structures, which characterize the flow dynamics in the shear layer. The physics of the flow field is analyzed by looking at energy redistribution between the mean and pulsating flow field obtained via a particle image velocimeter (PIV) using the concept of a triple decomposition. The total fluctuating kinetic budget is calculated and discussed for a mean Reynolds number of 100 and for 0.035 ≤ St ≤ 2.19. The effects that these coherent structures have on the fluctuating kinetic energy production, dissipation, and transport mechanism are examined. The results provide insight into the physics of the flow and suggest reasons for vortex growth and decay. Fluctuating kinetic energy is generally produced at the separated shear layers and transported towards the core flow and then to the upper and lower walls where viscosity dissipates the energy. The remaining energy is transported streamwise and decays as it is convected downstream (St = 0.4 and 1 cases). It was also found that the pressure-velocity correlation diffusion plays a significant role in the transport of kinetic energy and Reynolds stresses, especially in the separated shear layer. More energy was dissipated at the walls for the high Strouhal number case St = 2.19 due to the transverse pressure diffusion term being increasingly dominant. This could be the reason why the convected primary vortices were much smaller in size and weaker with no upper wall vortices formed at this pulsation Strouhal number. The shear production for St = 0.035 was very minimal; thus, the vortices died down quickly even before the shedding could happen. Finally, the pressure-strain correlation term was found to be significant in redistributing the kinetic energy from u-component to v-component.