Fluid Flow Induces Biofilm Formation in Staphylococcus epidermidis Polysaccharide Intracellular Adhesin-Positive Clinical Isolates
- aDepartment of Biomedical Engineering, University of California, Los Angeles, Los Angeles, California, USA
- bCalifornia NanoSystems Institute, University of California, Los Angeles, Los Angeles, California, USA
- cDepartment of Pediatrics, Division of Neonatology and Developmental Biology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
- dDepartment of Microbiology, Immunology, and Molecular Genetics (MIMG), David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
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Fig 1Schematic and operation of the microfluidic biofilm assay. (A) The microchannel design consists of 4 parallel channels (black) with culture chambers placed halfway between inlet a and outlet c. These are connected by a seeding channel (red and black) from b to d. (B) Prior to shear culture, cells are seeded in the device by creating pinched flow in each chamber. (C) Subsequently, b and d are blocked, and the channels are converted to perfusion mode, in which chamber 1 (Ch1) has the lowest resistance and highest wall shear stress. There is significantly lower flow in the seeding channels per the design of the device. Red streaks in panels B and C are 1-μm fluorescent polystyrene beads in flow. (D) The wall shear stress spans over an order of magnitude in a single experiment, covering ranges seen in capillaries and venules as well as those calculated for catheter lumens. Shear stresses presented are for an inflow of 18 μl/min.
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Fig 2S. epidermidis ATCC strains are distinguishable by biofilm phenotype when grown under flow. (A) Panels showing the fluorescence from WGA staining both before shear (0 h) and after 6 h of shear for 35984 (ica+, biofilmc), and 12228 (lacking ica, biofilm−). (B) Higher-magnification images (×40) illustrate the architectural differences between 35984 biofilms grown under different shear stresses. Higher shear results in streamer formation. (C) CLSM images (magnification, ×63) of 35984 biofilms after 12 h of shear indicate that these differences are enhanced over time (green, PIA). (D) Quantification of the subpanels in panel A, showing measurable differences in biofilm phenotype after 6 h. (E) Quantitative differences continue after 12 and 24 h of culture. 12228 decreases at high shear, indicating a release of cells from the surface, and maintains the initial inoculum levels at lower shears, while 35984 flourishes at all shear stresses.
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Fig 3Fluid flow results in differential biofilm formation among clinical isolates. (A) Strain A-26 forms strong biofilms under all shear stresses, whereas A-10 is unable to form biofilms under any shear after 24 h. (B) Strain A-5, after a lag phase of at least 12 h, forms significant biofilm when exposed to fluid flow and shear, with the most biofilm at 0.26 Pa. (C) Strains W-166 and Z-173 are able to form biofilms under a broader range of flow rates than A-5. (D) WGA staining of the biofilm matrix for A-5 after 12 and 24 h of culture under flow. (E) Live/dead staining of the same time points from panel D, showing live cells present on the channel surface after 12 h. These cell numbers drastically increase after 24 h. One asterisk indicates a P value of <0.01 and two asterisks indicate a P value of <0.005 measured against 1. Green, PIA; blue, DAPI; red, EtHDI.
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Fig 4The formation of biofilms under flow by strain A-5 has a biphasic dependence on shear. (A) Stitched images with a ×20 magnification of WGA-stained A-5 biofilms across a broad range of shear stresses. Images are representative of each shear stress value, using devices with two inlet flow rates of 18 μl/min and 4.5 μl/min. (B) Quantification of the fold increase in biofilm in panel A. (C) Images with ×40 magnification of both the PIA matrix and live and dead cells. The panels for A-10 after 24 h show the presence of live cells on the channel surface, indicating that not all of the cells unable to form biofilm are washed out of the chamber during flow. **, P < 0.05, measured against the value of A-5 at 0.016 Pa. Green, PIA; blue, DAPI; red, EtHDI.
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Fig 5Fluid flow and shear induce the formation of biofilms in S. epidermidis A-5. (A) Images with ×40 magnification showing increased PIA matrix deposition with increasing wall shear in low-flow-rate regimes. Bottom panels show the presence of live cells on the surface, even when no visible matrix is present. (B) Images with ×40 magnification from no-flow controls in PDMS/glass wells, showing increased PIA matrix upon EtOH induction. There are live cells present in both cases. (C) Quantification of the ×40-magnified images from panels A and B, presented as the PIA matrix normalized to the area of the cells present. The ratio of the PIA matrix to cell area increases with increasing wall shear and also with increasing EtOH without fluid shear. (D) Representative images of microtiter plate assay screening clones isolated from the microfluidic biofilm assay. HQ1 and HQ2 are clones collected from independent flow-induced biofilms. (E) Quantification of the absorbance of microtiter plate assays at 570 nm. Black bars are the wild type, and the black/red bars are the averages of six clones collected from two separate flow experiments. One asterisk indicates a P value of <0.05 and two asterisks indicates a P value of <0.01 measured against respective minimum values.
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