Methicillin resistant Staphylococcus aureus adhesion to human umbilical vein endothelial cells demonstrates wall shear stress dependent behaviour
© Viegas et al; licensee BioMed Central Ltd. 2011
Received: 25 August 2010
Accepted: 22 March 2011
Published: 22 March 2011
Methicillin-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.
Human 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).
Under 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.
This 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.
Infections of the cardiovascular system, including those involving prostheses and devices, are a globally recurring problem. Vascular infections are often life-threatening, spread easily, and costly to treat. Furthermore, infection is a common problem affecting the success of biomedical implants, such as vascular stents . Bacteria can be introduced through surgical interventions, travel through the bloodstream and infect the endothelial cells lining the blood vessels. Cardiovascular disease has also been linked to microbial infection [2, 3], with attachment of bacterial pathogens to endothelium or extracellular matrix being an initial step in the process .
Staphylococcus aureus is a highly adaptable microbial pathogen that is considered to be a leading cause of various community- and hospital-acquired infections . The frequency of S. aureus infection has risen dramatically over the last few decades in accordance with the number of patients receiving vascular implants, such that S. aureus accounts for the majority of device-related infections . In addition, S. aureus is the primary cause of endovascular and endocardial infections that are extremely difficult to treat, with mortality rates between 40 and 50% at one year . Of particular concern is the ability of S. aureus to become tolerant to antibiotic therapies, and to generate antibiotic resistant strains.
One such strain is Methicillin resistant Staphylococcus aureus (MRSA). In addition to its intrinsic resistance to methicillin, MRSA is also insensitive to other common antibiotics such as oxacillin, penicillin and amoxicillin . MRSA was first identified in 1961 and is now the most common antibiotic-resistant bacteria worldwide . MRSA infection rates have continued to grow in recent decades: in 1974, 2% of S. aureus infections in US intensive-care units were caused by MRSA, in 1995 this number was 22%, and in 2003, 64% . Approximately 94,000 Americans suffer each year from invasive MRSA infections, and an estimated 19,000 of these incidences (20%) result in death . Globally, MRSA is known to be associated with longer hospital stays, increased mortality, morbidity and much higher costs of treatment.
The location, strength, and magnitude of vascular bacterial adhesion, along with subsequent cellular interactions involved in disease progression, are likely to be highly dependent on the hydrodynamic environments experienced within affected vessels. Once in the bloodstream, bacteria contact the vascular endothelium. Endothelial cells mediate many vascular functions including inflammatory responses and transendothelial migration of nutrients, biological molecules, and leukocytes into the surrounding tissue . These functions occur under the dynamic conditions of blood flow, and are strongly affected by hemodynamic forces. Shear stress levels ≥1 Pa, which generally occur through straight portions of the arteries, typically correspond to healthy, disease resistant regions of the vasculature, while low shear stress (≤ 0.4 Pa), which is found at arterial branch points and areas of curvature, often corresponds to areas susceptible to both atherosclerosis and microbial adhesion [12–14]. Receptor-ligand adhesive bonds are strongly affected by flow conditions. While it would seem from intuition and theory that receptor-ligand bonds should form less frequently and break more quickly under flow, experimental evidence has shown that in some cases the opposite occurs. We previously showed monocyte adhesion to HUVEC to be shear stress dependent, with stronger adhesion occurring with increased shear stress , possibly due to increased cell or microvilli deformation . However, the bonds themselves may respond differently depending on the level of force (from shear stress, atomic force microscopy, or other methods). Adhesive bonds are characterized as 'slip' bonds if bond lifetimes decrease under increased force and catch bonds if the opposite occurs. While many studies have focused on P- and L-selectins involved in leukocyte adhesion [17, 18], a catch bond has been identified in Escherichia coli [19, 20]. Therefore it is essential to observe adhesive interactions under physiologically relevant conditions. This may enable establishment of more accurate models of vascular infection in the human body and evaluation of specific adhesion mechanisms. A few studies have investigated S. aureus adhesion to endothelium under flow [21–23], however none of these involve MRSA.
In vitro systems have been developed that allow cultured endothelial cells to be exposed to well-defined flow conditions, and include orbital shakers , cone and plate viscometers [25, 26], and parallel-plate flow chambers (PPFC) [15, 16, 27–31]. PPFC enable establishment of fully developed laminar flow within the channel of the chamber and theoretically should allow exposure of cells to uniform flow fields. However, many PPFCs have been found to not provide uniform shear stresses across the surface of the chamber [32–34]. Therefore, cell responses may not be consistent over the chamber area .
This study investigated the hypothesis that shear stress inhibits MRSA adhesion and subsequent infection of vascular endothelium. The objectives of this study were to first evaluate the performance of a newly designed PPFC for its ability to provide defined flow and uniform shear stress to cultured cells, and second, to use this PPFC to analyze the influence of shear stress on MRSA infection of endothelium. Human umbilical vein endothelial cells (HUVEC), were selected since they represent a good endothelial model and are widely used in the field [15, 16, 31]. Further, since many S. aureus experiments have been performed with HUVEC or the HUVEC cell line EA hy926, this enables closer comparisons with previous data [22, 23, 35–37].
The bacterial strain used for all experiments, MRSA UC18, a hospital-acquired isolate, was kindly provided by Dr. Howard Ceri (University of Calgary). MRSA was routinely cultured in Tryptic Soy Broth (TSB) at 37°C and 100 rpm. Bacterial growth was quantified using optical density (OD) at a spectrophotometer light wavelength of 600 nm and viable plate counts. Cells passaged from actively growing cultures were maintained at 37°C under constant rotation, and harvested at an OD of 0.50, after ~3 h of incubation. As determined by growth curves, this point corresponded to mid-exponential phase. All bacterial ODs were correlated to colony counts (measured in colony forming units; CFU). Prior to adhesion studies, bacteria were labelled for 30 minutes at 37°C with a 1:1000 dilution of SYTO 9 (Invitrogen, Grand Island, NY, USA) in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2% Fetal Bovine Serum (FBS). After labelling, cells were washed twice with TBS and diluted in DMEM to a concentration of 107 CFU/mL.
Endothelial cell culture
Pooled Human Umbilical Vein Endothelial Cells (HUVEC; Lonza, Walkersville, MD, USA) were cultured from supplied stock and expanded up to a maximum of passage 6. Tissue culture flasks and glass slides were coated with 0.1% gelatin (Difco, Becton, Dickinson, Sparks, MD, USA) in M199 (Sigma-Aldrich) prior to cell seeding. Slides were usually confluent and ready for use after 2 days.
In vitro flow model
According to the PIV analysis (see below), the height of the HUVEC and extracellular matrix layer was 20.1 ± 3.9 μm . This height was subtracted from the channel height (254 μm ) to determine the height of the flow path, h. The density and viscosity of the culture media were 0.9852 g·cm-3 and 0.831 cP (determined using a capillary viscometer (Cannon-Fenske; Cannon Instrument Company State College, PA, USA), respectively. These values yield shear stresses of 0.24 Pa and 1.2 Pa (at shear rates of 293s-1 and 1460s-1 and Reynolds numbers of 6.57 and 32.9, respectively) for the conditions used in the infection study. The Reynolds numbers less than 1200 indicate that flow is in the laminar regime. Physiological values of Reynolds numbers (200-6000) could not be reproduced due to the high flow rates needed .
where ψ is the steady state concentration near the surface, C is the concentration of bacteria in the bulk fluid (1 × 107 cells ml-1), x is the distance from the inlet (2.3 cm), u is the velocity and h' is the height of fluid (taken to be one cell diameter, 1 μm [41, 43]). The velocity, u, was determined using the method of Goldman et al.  to take into account the lower velocity of a neutrally buoyant sphere near the wall. The dimensionless concentration was normalized against the value determined for the static condition then multiplied by the experimentally determined values as in previous work .
where r is the cell diameter (1 μm) and t is the shear stress. F x * is equal to 1.7005 for the case where the bacterium is touching the surface .
An Olympus Fluoview FV1000 Confocal Scanning Laser Microscope (CSLM) was used to take fluorescent image stacks using a water-immersed, coverslip-corrected 60× objective and an Argon laser. For each condition, a minimum of three replicates were analyzed. For each experiment, between 4 and 12 confocal stacks (thickness of slice = 0.5 μm) were taken randomly along the flow channel. This generated data encompassing adhesion to a minimum of 100 HUVEC per experiment. Within each image stack taken, all fully visible HUVEC were analyzed, with any cell only partially contained in the field of view excluded. All HUVEC with one or more adherent MRSA were considered infected. The number of HUVEC associated MRSA were counted on a per-cell basis. The area analyzed in each picture was 0.001 cm-2.
The velocity gradient was estimated using a three-point fit. The overall measurement uncertainty is estimated to be ± 2% and ± 6% for the local velocity and wall shear stress, respectively, based on pixel resolution and repeatability.
where n is the refractive index of the fluid between the micro-fluidic device and the objective lens; λ0 is the wavelength of light, in vacuum, imaged by the optical system; NA is the numerical aperture of the objective lens; θ is the light collection angle and d p is the particle diameter. The resulting measurement depth uncertainty is about 20 μm.
For the PPFC without cells, a uniform velocity profile was used at the inlet and a no slip boundary condition was applied at the walls. Outflow boundary condition was used at the outlet: the flow variable gradients normal to the boundary are set to zero. For the PPFC with endothelial cells, the inlet velocity profile measured experimentally was used at the beginning of the solution domain. No slip boundary conditions were applied at the lateral walls and endothelial cell surfaces. Additional details of the CFD analysis are provided in Dol et al. .
Data is reported as the mean +/- the standard error of the mean (SEM). Systat (v. 13, Systat Software Inc, Chicago, IL) was used to perform analysis of variance (ANOVA). A Tukey post-test analysis was performed on data pairs. P-values < 0.05 (corresponding to a 95% confidence interval) were considered statistically significant.
MRSA adhered to HUVEC and was internalized
Flow chamber provided uniform flow field
Endothelial cells increased shear variations
Effect of shear stress on MRSA infection of HUVEC
MRSA infection under flow varied with time
In order to correct for differences in bacterial concentration near the endothelial surface with increasing shear stress, the dimensionless cell concentration was determined and used to normalize the data. The number of bacteria per area was determined for each condition. This data had similar trends to the number of MRSA per HUVEC data already presented. When the experimentally determined bacteria per area was corrected for the dimensionless cell concentration near the surface, there was an increase in MRSA per area from static conditions to a shear rate of 293s-1 (0.24 Pa) and a decrease in MRSA per area at 1460s-1 (1.2 Pa) at the time point of 30 minutes (Figure 10C).
This study demonstrated that MRSA adheres to human endothelial cells in a manner dependent on fluid flow. The highest adhesion was found at the low shear stress condition (0.24Pa), which is a level found in arterial bifurcations. As fluid forces increased to levels seen in straight portions of arterial vessels, normalized MRSA adhesion decreased. Results also showed the capabilities of micro-PIV and CFD in determining the flow field over endothelial cells and the wall shear stress distribution over irregular cell surfaces.
Through analysis of image stacks obtained from confocal microscopy, MRSA infection of HUVEC was found to be heterogeneous (with some HUVEC experiencing a high degree of infection compared to others) and localized. Internalized bacteria were found as early as 15 minutes. For all conditions, most of the adherent bacteria were found around the periphery of the HUVEC. Becker et al.  made the same observation and hypothesized that this could be attributed to the fact that these pericellular regions are rich in fibronectin. Based upon our numerical simulations, PIV results and adhesion assays, we predict that there are also micro-scale shear stress effects at play during adhesion under fluid flow. The shear stress over the surface of each endothelial cell reaches a minimum in the junctional areas and a maximum above the cell nucleus. This leads to fluid velocity variations near the monolayer surface that impact cell trajectories. These factors may explain why we observed an increase in MRSA adherence in the area between adjacent cells.
Before normalization, our results showed that the number of MRSA per HUVEC decreased after 30 minutes of steady flow at 293 s-1 (0.24 Pa) as compared to behaviour under static conditions, and decreased further with increasing shear rate and stress (1.2 Pa; 1460s-1). These results are similar to those of Shenkman et al.  who found a 2.5 fold decrease in S. aureus 8325-4 adhesion to a HUVEC cell line after 20 minutes at a shear rate of 200s-1 compared to static conditions. The larger fold decrease found by Shenkman et al. may be due to strain-specific differences in adhesion. The same group found no change in S. aureus RN6390 adhesion to endothelium after 20 minutes at a shear rate of 200s-1 compared to static ; indicating a dependence on strain of S. aureus. Further, Reddy and Ross found no adhesion for S. aureus 8325-4 to Bovine Aortic Endothelial Cells for any shear rate (1s-1- 200s-1) ; indicating a possible dependence on type and species of endothelial cell. However, there may be other factors contributing to the different results seen by Shenkman et al. and Reddy and Ross [23, 21]. The growth phase for maximal adherence is strain specific . Reddy and Ross used bacteria in the early exponential phase while Shenkman et al. used bacteria in the stationary phase. At later growth phases, S. aureus may increase levels of an adhesin important for adhesion under flow. Further, Reddy and Ross counted surface bound bacteria, whereas Shenkman et al. measured radioactivity and thus intracellular as well as attached bacteria. In our work, we analyzed stacks obtained from confocal microscopy to quantify intracellular and extracellular bacteria, which may explain why our results agree more closely with Shenkman et al. .
When our results are corrected for differences in cell flux to the endothelial surface under flow, bacteria per area was shown to increase with low levels of shear (293 s-1; 0.24 Pa), then decrease at the higher shear condition (1460 s-1; 1.2 Pa). Lower levels of adhesion may be observed at high shear rates because adhesin-receptor bonds are less likely to form at the shortened contact times . A similar trend was seen by Boks et al (2008) for Staphylococcus epidermidis on glass . An analysis of the drag forces on the attached bacteria shows values of 1.9 pN to 9.6 pN for the conditions of this study. This force was calculated for a bacterium attached to the highest point on the endothelial cell and therefore represents a maximum. Lower forces would be present in the valleys between the cells as discussed previously. The force required to detach S. aureus from a collagen coated surface was found by others to be much greater than 3.9 pN and on a fibronectin coated surface to be between 15-26 pN [51, 52]. Therefore significant cell detachment may not be occurring under the conditions of this study. Catch bonds, which increase in strength with increasing shear stress, could be forming more frequently at the lower shear stress condition (0.24 Pa) leading to increased cell adhesion under these conditions [53, 54].
MRSA exposure time affected the extent of infection under both static and flow conditions. For static experiments, endothelial binding sites may have been saturated, as there was no increase in the number of MRSA per HUVEC between 30 minutes and 60 minutes, and no significant change in the percent HUVEC infected was observed over the time course. This agrees with findings of Tompkins et al.  who saw saturation within 60 minutes for static adhesion using similar bacterial inoculum sizes. Under flow at 293s-1, a very different result was found. There was no change in the number of MRSA per HUVEC between 15 and 30 minutes, but there was a 260% increase between 30 and 60 minutes. The presence of flow appears to be exacerbating the endothelial response to infection at the one hour time point, perhaps leading to modulation of surface properties that increase bacterial uptake. Further studies are needed to investigate the mechanisms involved.
This study demonstrated that MRSA adheres to endothelium in a shear dependent manner which may be affected by the non-uniform shear stress distribution over an undulating endothelial monolayer. MRSA adhered to and invaded HUVEC, with a large degree of heterogeneity. The time course of adhesion under flow conditions varied greatly as compared to static. Therefore, the mechanisms of adhesion under flow may differ from those seen under static conditions and thus warrant further investigation. During establishment of blood borne infections, bacteria contact endothelial cells throughout the vasculature. Our results suggest that at increasing time points, MRSA will adhere in increasing numbers under low flow, an effect not seen in static culture. Further, adhesion appears to be dependent on shear stress magnitude. The number of MRSA increased between the static and low flow conditions. A further increase in shear stress led to a decrease in MRSA numbers. Therefore, MRSA appear to adhere and invade endothelial cells preferentially in regions of low shear stress, such as those found in vascular branches and areas of curvature. Taken together, these findings suggest that shear stress and time each influence MRSA adhesion to, and internalization by, endothelial cells. While steady flow was assessed in this work, further studies investigating the role of flow pulsatility and recirculation should provide further insights into the role of fluid dynamics on bacterial adhesion in the vasculature.
The authors would like to thank Dr. Howard Ceri (University of Calgary) for the culture of MRSA UC18, Dr. Elena Di Martino (University of Calgary) for assisting with the statistical analysis, and Carol Chan for measurement of fluid properties. K. Viegas and M. Salek would like to acknowledge the National Science and Engineering Research Council of Canada (NSERC) and Alberta Ingenuity Fund (now part of Alberta Innovates Technology Futures), respectively, for postgraduate scholarships. K. Rinker and R. Martinuzzi would like to acknowledge support through their NSERC Discovery grants.
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