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.

Objective Lyme disease is a tick-borne, multisystemic disease caused by Borrelia burgdorferi. Standard treatments for early Lyme disease include short courses of oral antibiotics but relapses often occur after discontinuation of treatment. Several studies have suggested that ongoing symptoms may be due to a highly antibiotic resistant form of B. burgdorferi called biofilms. Our recent clinical study reported the successful use of an intracellular mycobacterium persister drug used in treating leprosy, diaminodiphenyl sulfone (dapsone), in combination therapy for the treatment of Lyme disease. In this in vitro study, we evaluated the effectiveness of dapsone individually and in combination with cefuroxime and/or other antibiotics with intracellular activity including doxycycline, rifampin, and azithromycin against Borrelia biofilm forms utilizing crystal violet biofilm mass, and dimethyl methylene blue glycosaminoglycan assays combined with Live/Dead fluorescent microscopy analyses. Results Dapsone, alone or in various combinations with doxycycline, rifampin and azithromycin produced a significant reduction in the mass and protective glycosaminoglycan layer and overall viability of B. burgdorferi biofilm forms. This in vitro study strongly suggests that dapsone combination therapy could represent a novel and effective treatment option against the biofilm form of B. burgdorferi.

Outer membrane vesicles (OMVs) are spherical bodies containing proteins and nucleic acids that are released by Gram-negative bacteria, including Borrelia burgdorferi, the causative agent of Lyme disease. The functional relationship between B. burgdorferi OMVs and host neuron homeostasis is not well understood. The objective of this study was to examine how B. burgdorferi OMVs impact the host cell environment. First, an in vitro model was established by co-culturing human BE2C neuroblastoma cells with B. burgdorferi B31. B. burgdorferi was able to invade BE2C cells within 24 h. Despite internalization, BE2C cell viability and levels of apoptosis remained unchanged, but resulted in dramatically increased production of MCP-1 and MCP-2 cytokines. Elevated secretion of MCP-1 has previously been associated with changes in oxidative stress. BE2C cell mitochondrial superoxides were reduced as early as 30 min after exposure to B. burgdorferi and OMVs. To rule out whether BE2C cell antioxidant response is the cause of decline in superoxides, superoxide dismutase 2 (SOD2) gene expression was assessed. SOD2 expression was reduced upon exposure to B. burgdorferi, suggesting that B. burgdorferi might be responsible for superoxide reduction. These results suggest that B. burgdorferi modulates cell antioxidant defense and immune system reaction in response to the bacterial infection. In summary, these results show that B. burgdorferi OMVs serve to directly counter superoxide production in BE2C neurons, thereby ‘priming’ the host environment to support B. burgdorferi colonization.

Background: Infections by bacterial or viral agents have been hypothesized to influence the etiology of neurodegenerative diseases. Objective: This study examined the potential presence of Borrelia burgdorferi spirochete, the causative agent of Lyme disease, in brain autopsy tissue of patients diagnosed with either Alzheimer’s (AD) or Parkinson’s diseases. Methods: Brain tissue sections from patients with age-matched controls were evaluated for antigen and DNA presence of B. burgdorferi using various methods. Positive Borrelia structures were evaluated for co-localization with biofilm and AD markers such as amyloid and phospho-tau (p-Tau) using immunohistochemical methods. Results: The results showed the presence of B. burgdorferi antigen and DNA in patients with AD pathology and among those, one of them was previously diagnosed with Lyme disease. Interestingly, a significant number of Borrelia-positive aggregates with a known biofilm marker, alginate, were found along with the spirochetal structures. Our immunohistochemical data also showed that Borrelia-positive aggregates co-localized with amyloid and anti-phospho-tau markers. To further prove the potential relationship of B. burgdorferi and amyloids, we infected two mammalian cell lines with B. burgdorferi which resulted in a significant increase in the expression of amyloid-β and p-Tau proteins in both cells lines post-infection. Conclusion: These results indicate that B. burgdorferi can be found in AD brain tissues, not just in spirochete but a known antibiotics resistant biofilm form, and its co-localized amyloid markers. In summary, this study provides evidence for a likely association between B. burgdorferi infections and biofilm formation, AD pathology, and chronic neurodegenerative diseases.

Borrelia burgdorferi, the causative agent of Lyme Disease, is known to be able to disseminate and colonize various organs and tissues of its hosts, which is very crucial for its pathogenicity and survival. Recent studies have shown the presence of B. burgdorferi DNA in various breast cancer tissues, in some with poor prognosis, which raises the question about whether B. burgdorferi can interact with mammary epithelial cells and could have any effect on their physiology, including tumorigenic processes. As the model in this study, we have used MCF 10A normal and MDA-MB-231 tumorigenic mammary epithelial cells and infected both cell lines with B. burgdorferi. Our immunofluorescence and confocal microscopy results showed that B. burgdorferi is capable of invading normal epithelial and breast carcinoma cell lines within 24 h; however, the infection rate for the breast carcinoma cell lines was significantly higher. While the infection of epithelial cells with B. burgdorferi did not cause any changes in cell proliferation rates, it showed a significant effect on the invasion and migratory capacity of the breast cancer cells, but not on the normal epithelial cells, as determined by Matrigel invasion and wound healing assays. We have also found that the levels of expression of several epithelial–mesenchymal transition (EMT) markers (fibronectin, vimentin, and Twist1/2) changed, with a significant increase in tissue remodeling marker (MMP-9) in MDA-MB-231 cells demonstrated by quantitative Western blot analyses. This observation further confirmed that B. burgdorferi infection can affect the in vitro migratory and invasive properties of MDA-MB-231 tumorigenic mammary epithelial cells. In summary, our results suggest that B. burgdorferi can invade breast cancer tumor cells and it can increase their tumorigenic phenotype, which urges the need for further studies on whether B. burgdorferi could have any role in breast cancer development.