Evidence of In Vivo Existence of Borrelia Biofilm in Borrelial Lymphocytomas

Abstract

Lyme borreliosis, caused by the spirochete Borrelia burgdorferi sensu lato, has grown into a major public health problem. We recently identified a novel morphological form of B. burgdorferi, called biofilm, a structure that is well known to be highly resistant to antibiotics. However, there is no evidence of the existence of Borrelia biofilm in vivo; therefore, the main goal of this study was to determine the presence of Borrelia biofilm in infected human skin tissues. Archived skin biopsy tissues from borrelial lymphocytomas (BL) were reexamined for the presence of B. burgdorferi sensu lato using Borrelia-specific immunohistochemical staining (IHC), fluorescent in situ hybridization, combined fluorescent in situ hybridization (FISH)-IHC, polymerase chain reaction (PCR), and fluorescent and atomic force microscopy methods. Our morphological and histological analyses showed that significant amounts of Borrelia-positive spirochetes and aggregates exist in the BL tissues. Analyzing structures positive for Borrelia showed that aggregates, but not spirochetes, expressed biofilm markers such as protective layers of different mucopolysaccharides, especially alginate. Atomic force microscopy revealed additional hallmark biofilm features of the Borrelia/alginate-positive aggregates such as inside channels and surface protrusions. In summary, this is the first study that demonstrates the presence of Borrelia biofilm in human infected skin tissues.

Keywords: Lyme disease; alginate; atomic force microscopy; biofilm; mucopolysaccharides.

Fig. 1.

Representative immunohistochemical (IHC) images of borrelial lymphocytoma (BL) tissue sections stained with Borrelia-specific antibody following an IHC protocol as described earlier [41]. Positive Borrelia staining is depicted by red color. 200× magnification, bar: 100 μm

Fig. 2.

Representative images of borrelial lymphocytoma (BL) tissue sections stained with modified Dieterle silver methods and Borrelia-specific IHC methods as described earlier [30]. White arrows in panel A show the silver-stained spirochetes (i and iv) and aggregates (ii and iii), and panel B shows one of the silver-stained aggregates (panel Aiii) stained positive for Borrelia antigen. As a negative control for the immunohistochemical method, panel Bvii shows a no antibody control on consecutive section, and panel Bviii is the DIC image of the panel Bvii section to prove that there is still aggregate on the tissue section. 400× magnification, bar: 200 μm

Fig. 3.

A representative image shows a validation study for our 16S rDNA fluorescent in situ hybridization (FISH) protocol using different bacterial cells. The different bacterial cells placed on microscope slides and fixed and FISH protocols were carried out as described in Materials and methods section. The same FISH protocols were carried out on every experiment for Borrelia afzelii (panels A and B), Borrelia garinii (panels C and D), Treponema denticola (panels E and F), Borrelia burgdorferi (panels G and H), Borrelia hermsii (panels I and J), and Escherichia coli (panels K and L). DAPI nuclear stains depicting the cell morphology (panels B, D, F, H J, and L). 400× magnification, bar: 100 μm

Fig. 4.

Fluorescent in situ hybridization (FISH) of the BL tissues. Borrelia-specific 16S rDNA probe was utilized in these experiments to localize Borrelia DNA (A). As comprehensive negative controls, a competing oligonucleotide (B), DNase-treated samples (C), and a random DNA probe (D) were used on consecutive tissue sections to further show the specificity of the 16S rDNA probe (further details of the experimental conditions are in Materials and methods section). The tissue morphology was demonstrated with DAPI nuclear (E) and differential interphase contrast microscopy (DIC, panel F). 400× magnification, bar: 200 μm

Fig. 5.

Representative agarose gel picture of a Borrelia burgdorferi sensu lato specific 16S rDNA PCR amplification of genomic DNAs obtained from borrelial lymphocytoma skin sections. Lane M: 1 kb DNA Ladder (Life Technologies). Genomic DNA extracted from Borrelia afzelii laboratory strain (positive control, +C) and no template negative control (–C). Genomic DNAs extracted from borrelial lymphocytoma skin sections located in the S1–S6 lanes. The bands seen in lanes of +C and S1–S6 (~450 bp) represent DNA amplified using Borrelia-specific primers which have a target size of 445 bp, indicating the presence of Borrelia burgdorferi sensu lato DNA in the samples. No bands were seen in the negative control (–C)

Fig. 6.

Representative images showing Spicer & Meyer aldehyde fuchsine–alcian blue sequential staining pattern of two aggregates (white arrows) found in the BL tissues via dark field (panels A and C) and by differential interference contrast (panels B and D) microscopy methods. Fuchsia/purple colorations are indicative of sulfomucins and blue coloration indicates non-sulfated, carboxylated mucins. 400× magnification, bar: 200 μm

Fig. 7.

Representative images of IHC analyses of spirochetes and aggregates found in BL tissues costained with Borrelia (green) and alginate (red) antibodies on the same tissue sections (panels A and B as well as D and E) or consecutive tissue section (panels G and H). As negative control, the same IHC experiments were repeated on normal foreskin tissue sections (panels J and K). Differential interference contrast (DIC) images were provided to show the morphology of the tissues (panels C, F, I, and L). 400× magnification, bar: 200 μm

Fig. 8.

Graphical representation of the number of the spirochetes and the number of aggregates observed in 1200 sections of six BL tissues (BL1-BL6; 200 sections/specimen) as well as in commercially purchased normal human foreskin tissue specimens (20 specimens). Y-axes data shows the number of the spirochetes (ranging from 350 to 480 spirochetes/200 sections/specimen) and the number of aggregates (ranging from 4 to 12 aggregates/200 sections/specimen) found in the six BL tissue. There were no detectable spirochetes or aggregates found in any of the control specimens

Fig. 9.

Immunohistochemical staining of BL skin sections showing several different aggregates stained positive with Borrelia (green staining: panels A, D, G, and J) and alginate specific antibodies (red staining: panels B, E, H and K). Differential interference microscopy (DIC) showing the size and tissue morphology of the skin tissues (panels C, F, I, and L). 400× magnification, bar: 200 μm

Fig. 10.

IHC staining of BL skin sections showing two different aggregates stained positive with Borrelia antigen (green staining: panels i and ii) and alginate (red staining: panels iii and iv). Panels v and vi are a merge of the anti-Borrelia and anti-alginate antibody. Panels vii and viii show the bright field image with Sudan Black-B staining. In the merged picture, yellow/light orange indicates the regions where both Borrelia and alginate are present. 1000× magnification, bar: 200 μm

Fig. 11.

Combined FISH and IHC representative image of Borrelia aggregates in BL skin tissues showing that Borrelia-DNA-positive structures identified with FISH experiment using 16S rDNA probe (green staining, panel A) express alginate antibody (blue staining, panel B) as depicted with an independent IHC method. To show that the structure indeed is Borrelia DNA positive, competing oligonucleotide was used as a negative control (panel C). Differential interference microscopy (DIC) showing the size and tissue morphology of the skin tissues (panel D). 400× magnification, bar: 200 μm

Fig. 12.

Three-dimensional atomic force microscopy analyses of Borrelia/alginate-positive aggregates from a BL biopsy tissue section. Panel A shows a representative image from the AFM analyses which was performed using contact mode of the Nanosurf Easyscan 2 AFM with SHOCONG probe (AppNANO™ [30]). Images were processed, and measurements were obtained using Gwyddion software. The individual height and width ranges are indicated on the panels. Panel B shows evidence that the scanned tissue is Borrelia- and alginate-positive by fluorescent IHC analyses (400× magnification). Red arrows represent the same area of the tissue illustrated on panels A and B. Red and green arrowheads indicate potential channels and protrusions in the Borrelia/alginate-positive aggregates, bar: 200 μm