Targeting Pathogenic Infection and Virulence Genes of Enterococcus Faecalis Using Lactoferrin Bismuth Nano-form

Enterococcus faecalis (E. faecalis) is an opportunistic pathogen associated with severe infections, particularly in hospital settings. Increasing antibiotic resistance and diverse virulence factors highlights the urgent need for innovative, effective therapeutic approaches. In this study, a lactoferrin-Bismuth Nanoform (LF-Bi-NF) was synthesized and comprehensively characterized using UV-visible spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) mapping, dynamic light scattering (DLS), and Fourier-transform infrared (FTIR) spectroscopy. However, gamma radiation negatively impacted the stability of LF-Bi-NF. The LF-Bi-NF was evaluated against pathogenic E. faecalis isolates through in vitro assays, in addition to in vivo investigations in orally infected mice. The LF-Bi-NF exhibited antibacterial activity, with inhibition zones of 20.0 ± 0.10, 21.0 ± 0.15, 18.0 ± 0.25, and 15.0 ± 0.31 mm against E. faecalis_1, E. faecalis_2, E. faecalis_3, and E. faecalis ATCC 29212, respectively. While both lactoferrin and bismuth salts alone exhibited no detectable activity. The LF-Bi-NF demonstrated minimum inhibitory concentrations (MICs) of 3.125, 1.56, 6.25, and 12.5 µg/mL and minimum bactericidal concentrations (MBCs) of 12.50, 3.125, 25, and 25 µg/mL against the respective isolates, along with inhibition of biofilm formation at sub-MIC levels. Mechanistic investigations indicated disruption of cell wall integrity, increased membrane permeability, and protein leakage, particularly in the strong biofilm-forming isolate E. faecalis_2. Additionally, gene expression analysis showed that the antibiotic resistance associated genes norA, ermC, and EF3314 were downregulated by 64%, 59%, and 87%, respectively. In vivo studies demonstrated that LF-Bi-NF treatment promoted recovery in infected mice, improved immunological biomarkers (IL-12, IL-10, MPO, and IFN-γ), and significantly reduced bacterial burden in the bloodstream to undetectable levels within four days. In conclusion, LF-Bi-NF represents a promising nano-therapeutic candidate for the treatment of E. faecalis infections, exhibiting strong antibacterial, anti-biofilm, anti-virulence, and immunomodulatory properties.