Sub-Inhibitory Hydrogen Peroxide Enhances Nitrofurantoin Activity and Reduces Biofilm-Associated Phenotypes in a Selected Enterococcus faecalis Isolate In Vitro

Infection with antibiotic-resistant Enterococcus faecalis is a major public health concern. Enhancing antibiotic efficacy is a key therapeutic strategy. This study investigates whether sub-inhibitory concentrations (sub-MICs) of hydrogen peroxide (H2O2), an antibiofilm agent, enhance nitrofurantoin (NIT) efficacy while reducing bacterial virulence by decreasing biofilm formation and adherence. Ten E. faecalis isolates were recovered from inpatients with wound infections. The MICs of NIT and H2O2, biofilm formation, bacterial adhesion to human oral mucosal epithelial cells (OMECs), and esp and gelE gene expression were evaluated. The interaction between H2O2 and NIT was evaluated using the fractional inhibitory concentration index (FICI). The isolation frequency of E. faecalis among sampled wound swabs was 8.19%. Biofilm formation positively correlated with the MICs of NIT and H2O2 (Spearman’s rank correlation coefficient: 0.615 and 0.701, respectively; p < 0.05). Sub-MICs of NIT and H2O2 were associated with reduced attached biofilm biomass and decreased bacterial adhesion in a concentration-dependent manner; at 1/2 and 1/4 MIC, growth-normalized analysis indicated that this reduction exceeded what would be expected from slowed bacterial growth alone. Both agents also downregulated esp and gelE expression in a concentration-dependent manner (p < 0.05). H2O2 reduced the MIC of NIT, with synergistic to additive interaction (FICI: 0.458–0.75); the combined exposure created greater inhibition of biofilm production, adhesion, and esp and gelE gene expression than either agent alone. These in vitro findings indicate that sub-MICs H2O2 potentiates NIT efficacy against a highly resistant, strong biofilm-forming isolate of E. faecalis, providing proof-of-concept evidence, rather than a generalizable property of the species that antibiofilm agents can improve antibiotic effectiveness; direct clinical extrapolation of the H2O2-specific findings is not implied given its known cytotoxicity at effective concentrations. This wound-derived isolate collection was used as an in vitro model system to investigate the biological interaction between NIT and H2O2; the findings should not be interpreted as evidence supporting the clinical use of NIT for wound infections. The mechanistic experiments (checkerboard assay, combined biofilm assay, and gene-expression analysis) were performed on a single selected nitrofurantoin-nonsusceptible, strong biofilm-producing isolate; validation in a larger and phenotypically diverse isolate collection is required before these findings can be generalized.