Enterococcus faecalis thrives in dual-species biofilm models under iron-rich conditions

Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) are pathogenic strains that often coexist in intestinal flora of humans and are prone to cause biofilm-associated infections, such as gastrointestinal tract and urinary tract infections. Earlier studies have demonstrated that E. faecalis biofilm can metabolize ferrous ions in iron-rich environments and promote biofilm growth under in-vivo conditions. However, the influence of iron transporters on dual-species biofilm growth and the nature of molecular-level interactions between iron transporter proteins and Fe²⁺ remains unknown. Therefore, in this work, co-culture studies were performed and the study indicates that Fe²⁺ at concentrations of 50-150 µM promotes the colonization of E. coli, and Fe²⁺ concentrations of 50-200 µM promote the growth of E. faecalis and dual-species colonies. Atomic absorption spectroscopy results reveal that Fe²⁺ ion augmentation in bacterial cells was increased to 4 folds in the single-species model and 11 folds in the dual-species model under iron-supplemented conditions. Furthermore, Fe²⁺ augmentation increased the antibiotic resistance of E. faecalis in both single- and dual-species bacterial cultures. In addition, in-silico docking were performed to determine a three-dimensional (3D) structure of ferrous iron-transporter proteins FeoB of E. faecalis and its affinity to extracellular Fe²⁺. Our model suggests that the FeoB facilitates the Fe²⁺ uptake in E. faecalis cells in the absence of iron chelator, 2,2-bipyridyl.