Ex Vivo Murine Skin Model for B. burgdorferi Biofilm

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.

Development of zebrafish (Danio rerio) as an in vivo model for Borrelia burgdorferi infection

“…Finally, tissues were embedded horizontally in molten paraffin. Embedded zebrafish blocks (both infected and uninfected) were sectioned at 4 μm, deparaffinized, then stained with a Borrelia polyclonal antibody diluted to 1:50 in 1x PBS (ThermoFisher, Waltham, MA, USA; FITC-PA1-73005) according to a previously established protocol [31]. Negative control slides omitted the primary Borrelia antibody.…”

Speciality Grand Challenge for “Biofilms”

“…While there is some utility to pursue the development of non-mammalian models of biofilm-infection such as Drosophila melanogaster , Galleria mellonella or Danio rerio (Zebrafish) ( Lebeaux et al., 2013 ), identification of ex vivo models that recreate the infectious environment should be encouraged. Among recently developed ex vivo models are, for example, an ex vivo pig lung biofilm model used for understanding antibiotic tolerance of P. aeruginosa biofilms ( Hassan et al., 2020 ) or an ex vivo murine skin biopsy model to characterize B. burgdoferi biofilms, the etiological agent of Lyme disease ( Torres et al., 2020 ). Last but not least with the development of reconstituted organs such as organoids or organ-on-a-chip, one could anticipate that adaptations of these models to the understanding of the physiopathology of biofilm infections, interactions of biofilms with the immune response or biofilm behaviors in the presence of antimicrobials would provide important information relevant to biofilms in infectious contexts ( Jimi et al., 2017 ; Choi et al., 2020 ; Yuan et al., 2020 ).…”

Pathogenicity and virulence of Borrelia burgdorferi

“…These results suggest that round bodies might play a certain role in LD pathogenesis [ 268 ]. Using an ex-vivo murine skin model, it was observed that Borrelia can also form biofilm-like colonies made by spiral-shaped bacteria [ 274 ]. Biofilm represents an alternative lifestyle in which the microbes grow as structrured aggregates and adopt a multicellular behaviour that facilitates their survival under unfavourable conditions.…”