Methotrexate suppresses growth of Enterococcus faecalis in vitro and shows localized synergistic effects in...

To evaluate the in vitro antimicrobial activity of methotrexate against Enterococcus faecalis and assess its interaction with gentamicin across planktonic growth, biofilm-related endpoints and confocal viability analysis. Methotrexate was tested against E. faecalis using concentration-dependent growth inhibition assays. Methotrexate-gentamicin interactions were assessed by checkerboard assays. Growth kinetics were monitored over 9 h. Biofilm biomass formation and effects on preformed biofilms were evaluated after exposure to methotrexate, gentamicin and selected combinations. Confocal microscopy-based analysis was used to assess treatment-associated biofilm death. Methotrexate showed concentration-dependent inhibitory activity against planktonic E. faecalis, with an MIC of 70 mg/mL and an MBC of 75 mg/mL. Growth kinetics demonstrated reduced bacterial growth by methotrexate alone and selected methotrexate-gentamicin combinations. Interaction mapping revealed an overall Loewe score consistent with an additive to mildly positive interaction. Methotrexate moderately reduced biofilm biomass, while its effects on established biofilm eradication remained limited. Confocal analysis indicated increased biofilm death in treated groups relative to the untreated control. Methotrexate exhibited in vitro anti-E. faecalis activity across multiple experimental endpoints. While broad synergy with gentamicin was not observed, the overall interaction was additive to mildly positive, supporting further investigation of methotrexate as a potential non-antibiotic adjunct candidate within defined concentration ranges. Further mechanistic and strain-level validation is warranted.