Characterization of Biofilm Formation by <em>Borrelia burgdorferi In Vitro</em>

<div><p><em>Borrelia burgdorferi</em>, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that <em>Borrelia burgdorferi</em> aggregate formation dramatically changes the <em>in vitro</em> response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied <em>Borrelia burgdorferi</em> for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that <em>Borrelia burgdorferi</em> is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that <em>Borrelia burgdorferi</em> is capable of forming biofilm <em>in vitro.</em> Biofilm formation by <em>Borrelia</em> species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.</p> </div

<i>Borrelia burgdorferi</i> B31 strain large aggregate surrounded by individual spirochetes and several small aggregates stained with the calcium-detecting stain Alizarin.

<p>Red coloration indicates presence of calcium, by differential interference contrast (Panel A) and dark field microscopy (Panel B). White arrows indicate unstained spirochetes and small aggregates. 400× magnification.</p

Three-dimensional AFM images of <i>Borrelia burgdorferi</i> B31 strain early aggregates on agarose substrate.

<p>The preparation of <i>Borrelia burgdorferi</i> cells on mica is described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048277#s4" target="_blank">Materials and Methods</a>. The sample was scanned at 0.3 Hz using contact mode. A: The original AFM image produced with NanoRule© software. B: The AFM dataset was converted to a 3D mesh via a custom meshing utility, cleaned with MeshLab open-source software, then imported to Adobe Photoshop and false-color hand-painted with 3D painting tools. White: spirochete bodies; blue: potential EPS matrix; purple: protrusions; and yellow: small round bodies.</p

Representative images of <i>Borrelia burgdorferi</i> B31 strain aggregates in the early (1^st column, 0 to 2 day), middle (2^nd column, 3 to 6 days) and late (3^rd column, 7 to 21 days) stages of development, observed with dark field (A, B, C – 400× magnification); differential interference contrast (D, E, F - 400× magnification); and FITC-band epifluorescence (G, H, I – 400× magnification).

<p>D, F, and I are extended depth of field composites.</p

Representative image showing Spicer & Meyer aldehyde fuchsine-Alcian blue sequential staining pattern of two <i>Borrelia burgdorferi</i> B31 strain aggregates by differential interference contrast (A and B) and dark field microscopy (C and D).

<p>Fuchsia coloration is indicative of weakly acidic sulfomucin; purple coloration indicates strongly acidic sulfomucins and/or sulfated proteoglycans; blue coloration indicates non-sulfated, carboxylated mucins. 500× magnification.</p

Immunohistochemical staining of a collagen-embedded <i>Borrelia burgdorferi</i> B31 aggregates (Panels A–C and G–I) and individual spirochetal cells (Panels D–F) for alginate (red staining; Panels B andI) and <i>Borrelia</i> antigen (green staining; Panels A,D,G) expression using fluorescent microscopy (see methods for detailed protocol).

<p>Panel H shows the lack of red staining in the absence of the primary antibody for alginate. Panels C, F and I show DAPI - DNA counterstain images. 400× magnification.</p

<i>Borrelia burgdorferi</i> B31 aggregates surrounded by individual spirochetes (marked with white arrows) stained with the DDAO [7-hydroxy-9H-(1, 3-dichloro-9, 9 dimethylacridin-2-one DNA binding fluorescent dye.

<p>A: Dark field image B: Differential interference contrast image, and C: DDAO red stained fluorescent image of the same cellular structures. 400× magnification.</p

Three-dimensional AFM images of a mature aggregate of <i>Borrelia burgdorferi</i> B31 strain after 20 days.

<p>The preparation of <i>Borrelia burgdorferi</i> cells on mica is described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048277#s4" target="_blank">Materials and Methods</a>. The scan was conducted with 0.4 Hz using contact mode. A and B show a pit and a protrusion, respectively, of a large mature aggregate as depicted in C. Images A and C were produced with NanoRule© software; image B was produced with a custom meshing utility and MeshLab open-source software.</p