The Perils of Excess: High EDTA Inactivates Phages and Hinders Antibiofilm Synergy

Background and Aims

Chronic and biofilm-based infections particularly those caused by multidrug-resistant (MDR) Escherichia coli, are clinically challenging due to the protective extracellular matrix and resistome. Bacteriophages offer targeted lysis, while EDTA (Ethylenediaminetetraacetic acid) chelates metal ions to destabilise biofilms. This study investigated the effects of phage-EDTA synergy against E.coli biofilms, aiming to define the optimal therapeutic window and assess cytocompatibility with human fibroblasts.

Methods

24 and 48-hour MDR E.coli biofilm destrcution was determined using a modified MBEC assay with EDTA (0.15–5 mM) and two phages as a cocktail (vB_EcoM_ZYD PQ110661.1 and vB_EcoM_Maryam-PQ799574.1). Antibiofilm activity was quantified via metabolic activity (AlamarBlue), biomass (crystal violet), and viable counts. Synergy was determined using the Bliss independence model. Fibroblast safety was evaluated by LDH release and metabolic activity.

Results

At 24 and 48 hrs strong synergy emerged between 0.5–1.0 mM EDTA and phage MOI 10-100, with biofilm destruction ranging from 61-63%, rspectiviely. However antagonistic and or false synergy was observed at higher EDTA levels and in combination with high phage concentrations. This was more prominent in 48 hr biofilms. Furthermore, concentrations of EDTA exceeding 1  mM inhibited phage activity. Fibroblast cell assays showed no significant cytotoxicity when EDTA-Phage were used at optimum concentrations

Conclusion

Phage-EDTA synergy is mechanism-dependent on biofilm maturity. However, the therapeutic window is narrow due to a toxicity threshold: high EDTA concentrations are directly detrimental to both human fibroblasts and the phages themselves. The lack of synergy at elevated EDTA levels is driven by chelation of ions during phage adsorption and propagation. Low-dose combinations are essential for safe and effective antibiofilm reduction