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Authors: E. Sapi, K. Gupta, K. Wawrzeniak, G. Gaur, J. Torres, K. Filush, A. Melillo and B. Zelger

Our research group has recently shown that Borrelia burgdorferi, the Lyme disease bacterium, is capable of forming biofilms in Borrelia-infected human skin lesions called Borrelia lymphocytoma (BL). Biofilm structures often contain multiple organisms in a symbiotic relationship, with the goal of providing shelter from environmental stressors such as antimicrobial agents. Because multiple co-infections are common in Lyme disease, the main questions of this study were whether BL tissues contained other pathogenic species and/or whether there is any co-existence with Borrelia biofilms. Recent reports suggested Chlamydia-like organisms in ticks and Borrelia-infected human skin tissues; therefore, Chlamydia-specific polymerase chain reaction (PCR) analyses were performed in Borrelia-positive BL tissues. Analyses of the sequence of the positive PCR bands revealed that Chlamydia spp. DNAs are indeed present in these tissues, and their sequences have the best identity match to Chlamydophila pneumoniae and Chlamydia trachomatis. Fluorescent immunohistochemical and in situ hybridization methods demonstrated the presence of Chlamydia antigen and DNA in 84% of Borrelia biofilms. Confocal microscopy revealed that Chlamydia locates in the center of Borrelia biofilms, and together, they form a well-organized mixed pathogenic structure. In summary, our study is the first to show BorreliaChlamydia mixed biofilms in infected human skin tissues, which raises the questions of whether these human pathogens have developed a symbiotic relationship for their mutual survival.

Open access
Authors: P. A. S. Theophilus, M. J. Victoria, K. M. Socarras, K. R. Filush, K. Gupta, D. F. Luecke and E. Sapi

Lyme disease is a tick-borne multisystemic disease caused by Borrelia burgdorferi. Administering antibiotics is the primary treatment for this disease; however, relapse often occurs when antibiotic treatment is discontinued. The reason for relapse remains unknown, but recent studies suggested the possibilities of the presence of antibiotic resistant Borrelia persister cells and biofilms.

In this study, we evaluated the effectiveness of whole leaf Stevia extract against B. burgdorferi spirochetes, persisters, and biofilm forms in vitro. The susceptibility of the different forms was evaluated by various quantitative techniques in addition to different microscopy methods. The effectiveness of Stevia was compared to doxycycline, cefoperazone, daptomycin, and their combinations. Our results demonstrated that Stevia had significant effect in eliminating B. burgdorferi spirochetes and persisters. Subculture experiments with Stevia and antibiotics treated cells were established for 7 and 14 days yielding, no and 10% viable cells, respectively compared to the above-mentioned antibiotics and antibiotic combination. When Stevia and the three antibiotics were tested against attached biofilms, Stevia significantly reduced B. burgdorferi forms. Results from this study suggest that a natural product such as Stevia leaf extract could be considered as an effective agent against B. burgdorferi.

Open access
Authors: E. Sapi, K. Balasubramanian, A. Poruri, J. S. Maghsoudlou, K. M. Socarras, A. V. Timmaraju, K. R. Filush, K. Gupta, S. Shaikh, P. A. S. Theophilus, D. F. Luecke, A. MacDonald and B. Zelger

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.

Open access