Whether Borrelia burgdorferi, the causative agent of Lyme disease, can persist for long periods in the human body has been a controversial question. The objective of this study was to see if we could find B. burgdorferi in a Lyme disease patient after a long clinical course and after long-term antibiotic treatment. Therefore, we investigated the potential presence of B. burgdorferi antigens and DNA in human autopsy tissues from a well-documented serum-, PCR-, and culture-positive Lyme disease patient, a 53-year-old female from northern Westchester County in the lower Hudson Valley Region of New York State, who had received extensive antibiotic treatments during extensive antibiotic treatments over the course of her 16-year-long illness. We also asked what form the organism might take, with special interest in the recently found antibiotic-resistant aggregate form, biofilm. We also examined the host tissues for the presence of inflammatory markers such as CD3+ T lymphocytes. Autopsy tissue sections of the brain, heart, kidney, and liver were analyzed by histological and immunohistochemical methods (IHC), confocal microscopy, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), and whole-genome sequencing (WGS)/metagenomics. We found significant pathological changes, including borrelial spirochetal clusters, in all of the organs using IHC combined with confocal microscopy. The aggregates contained a well-established biofilm marker, alginate, on their surfaces, suggesting they are true biofilm. We found B. burgdorferi DNA by FISH, polymerase chain reaction (PCR), and an independent verification by WGS/metagenomics, which resulted in the detection of B. burgdorferi sensu stricto specific DNA sequences. IHC analyses showed significant numbers of infiltrating CD3+ T lymphocytes present next to B. burgdorferi biofilms. In summary, we provide several lines of evidence that suggest that B. burgdorferi can persist in the human body, not only in the spirochetal but also in the antibiotic-resistant biofilm form, even after long-term antibiotic treatment. The presence of infiltrating lymphocytes in the vicinity of B. burgdorferi biofilms suggests that the organism in biofilm form might trigger chronic inflammation.

Lyme disease is a tick-borne, multi-systemic disease, caused by the bacterium Borrelia burgdorferi. Though antibiotics are used as a primary treatment, relapse often occurs after the discontinuation of antimicrobial agents. The reason for relapse remains unknown, however previous studies suggest the possible presence of antibiotic resistant Borrelia round bodies, persisters and attached biofilm forms. Thus, there is an urgent need to find antimicrobial agents suitable to eliminate all known forms of B. burgdorferi. In this study, natural antimicrobial agents such as Apis mellifera venom and a known component, melittin, were tested using SYBR Green I/PI, direct cell counting, biofilm assays combined with LIVE/DEAD and atomic force microscopy methods. The obtained results were compared to standalone and combinations of antibiotics such as Doxycycline, Cefoperazone, Daptomycin, which were recently found to be effective against Borrelia persisters. Our findings showed that both bee venom and melittin had significant effects on all the tested forms of B. burgdorferi. In contrast, the control antibiotics when used individually or even in combinations had limited effects on the attached biofilm form. These findings strongly suggest that whole bee venom or melittin could be effective antimicrobial agents for B. burgdorferi; however, further research is necessary to evaluate their effectiveness in vivo, as well as their safe and effective delivery method for their therapeutic use.

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 Borrelia-Chlamydia 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.

Borrelia burgdorferi, the causative agent of Lyme disease, has been recently shown to form biofilm structures in vitro and in vivo. Biofilms are tightly clustered microbes characterized as resistant aggregations that allow bacteria to withstand harsh environmental conditions, including the administration of antibiotics. Novel antibiotic combinations have recently been identified for B. burgdorferi in vitro, however, due to prohibiting costs, those agents have not been tested in an environment that can mimic the host tissue. Therefore, researchers cannot evaluate their true effectiveness against B. burgdorferi, especially its biofilm form. A skin ex vivo model system could be ideal for these types of experiments due to its cost effectiveness, reproducibility, and ability to investigate host–microbial interactions. Therefore, the main goal of this study was the establishment of a novel ex vivo murine skin biopsy model for B. burgdorferi biofilm research. Murine skin biopsies were inoculated with B. burgdorferi at various concentrations and cultured in different culture media. Two weeks post-infection, murine skin biopsies were analyzed utilizing immunohistochemical (IHC), reverse transcription PCR (RT-PCR), and various microscopy methods to determine B. burgdorferi presence and forms adopted as well as whether it remained live in the skin tissue explants. Our results showed that murine skin biopsies inoculated with 1 × 107 cells of B. burgdorferi and cultured in BSK-H + 6% rabbit serum media for two weeks yielded not just significant amounts of live B. burgdorferi spirochetes but biofilm forms as well. IHC combined with confocal and atomic force microscopy techniques identified specific biofilm markers and spatial distribution of B. burgdorferi aggregates in the infected skin tissues, confirming that they are indeed biofilms. In the future, this ex vivo skin model can be used to study development and antibiotic susceptibility of B. burgdorferi biofilms in efforts to treat Lyme disease effectively.

The spirochaete bacterium Borrelia burgdorferisensu lato is the aetiologic agent of Lyme disease. Borrelia is transmitted to mammals through tick bite and is adapted to survive at tick and mammalian physiological temperatures. We have previously shown that B. burgdorferi can exist in different morphological forms, including the antibiotic-resistant biofilm form, in vitro and in vivo. B. burgdorferi forms aggregates in ticks as well as in humans, indicating potential of biofilm formation at both 23 and 37 °C. However, the role of various environmental factors that influence Borrelia biofilm formation remains unknown. In this study, we investigated the effect of tick (23 °C), mammalian physiological (37 °C) and standard in vitro culture (33 °C) temperatures with the objective of elucidating the effect of temperature on Borrelia biofilm phenotypes invitro using two B. burgdorferisensu stricto strains (B31 and 297). Our findings show increased biofilm quantity, biofilm size, exopolysaccharide content and enhanced adherence as well as reduced free spirochaetes at 37 °C for both strains, when compared to growth at 23 and 33 °C. There were no significant variations in the biofilm nano-topography and the type of extracellular polymeric substance in Borrelia biofilms formed at all three temperatures. Significant variations in extracellular DNA content were observed in the biofilms of both strains cultured at the three temperatures. Our results indicate that temperature is an important regulator of Borrelia biofilm development, and that the mammalian physiological temperature favours increased biofilm formation in vitro compared to tick physiological temperature and in vitro culture temperature.