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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.

Evidence of in vivo existence of Borrelia biofilm in borrelial lymphocytomas