Genomic characterization and antibiotic susceptibility of biofilm-forming Borrelia afzelii and Borrelia garinii from patients with erythema migrans
Background: Borrelia afzelii and B. garinii are the most common cause of Lyme borreliosis (LB) in Europe. Persistent forms of LB can be associated with biofilm-like aggregates.  Methods: Whole genome sequencing (WGS) analysis was conducted on 7 B. afzelii and 5 B. garinii isolates from skin biopsy specimens of patients with erythema migrans. Biofilm was characterized by the quantitative assessment of biomass, amyloid fiber production, and extracellular DNA (eDNA). A phenol red metabolic assay was performed to evaluate the minimum inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) of amoxicillin, azithromycin, ceftriaxone, and doxycycline.  Results: Core genome analysis revealed significant portions of core genes in B. afzelii (44.5%), B. garinii (43.4%), and B. burgdorferi (52.5%). The distribution of Cluster of Orthologous Groups (COG) between B. afzelii and B. garinii suggests species-specific metabolic adaptations. All isolates possessed a comparable number of genes implicated in adhesion and biofilm. Phenotypically, both B. afzelii and B. garinii displayed similar levels of biofilm formation, significantly correlated with eDNA production. No antimicrobial resistance genes (ARGs) were detected in B. afzelii or B. garinii isolates. Minimum inhibitory concentration (MIC) values for amoxicillin, azithromycin, ceftriaxone, and doxycycline were 0.25 µg/mL, 0.125 µg/mL, 0.25 µg/mL, and 0.5 µg/mL, respectively, with no significant differences between B. afzelii and B. garinii isolates. However, minimal biofilm inhibitory concentration (MBIC) values were significantly higher. For amoxicillin, the MBIC was 2 µg/mL (8-fold increase), for azithromycin 2 µg/mL (16-fold increase), for ceftriaxone 16 µg/mL (64-fold increase), and for doxycycline >16 µg/mL (>32-fold increase).  Conclusions: B. afzelii and B. garinii demonstrate the ability to form biofilm-like structures, significantly reducing the activity of all antibiotics tested, particularly ceftriaxone and doxycycline. This underscores the urgent need for developing new and more effective therapeutic strategies to combat biofilm-related infections in LB.