Abstract
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.
Highlights
Lyme disease is a vector-borne illness that is caused by B. burgdorferi sensu lato, a bacterial spirochete that is transmitted by Ixodes ticks [1]
Several culture conditions were tested to evaluate their effect on the morphology of B. burgdorferi in the murine skin tissues
Those conditions were selected based on experimental data from previously published ex vivo models for B. burgdorferi [54,55,56]
Summary
Lyme disease is a vector-borne illness that is caused by B. burgdorferi sensu lato, a bacterial spirochete that is transmitted by Ixodes ticks [1]. B. burgdorferi disseminates in the skin, and the most common manifestation of infection is a red rash called erythema migrans [2]. The other well-studied dermatological conditions of Lyme disease are Borrelial lymphocytoma (BL), which appears in the early phase of Borrelia infection, and acrodermatitis chronica atrophicans (ACA), which is the late onset cutaneous manifestation [3,4]. Frontline treatments for early Lyme disease cases involve using antibiotics such as doxycycline, amoxicillin, cefuroxime, and ceftriaxone [7,8,9,10,11]. Many studies have suggested that, in late stages of Lyme diseases, B. burgdorferi can persist in the body following
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