Antibiofilm Activity of Essential Oils Against Escherichia coli on Lettuce Leaf Surfaces

Fresh leafy vegetables are widely consumed because of their high nutritional value but are also recognized as important vehicles for foodborne pathogens. Escherichia coli readily attaches to lettuce surfaces and forms biofilms, enhancing bacterial persistence and reducing the effectiveness of conventional washing procedures. This study evaluated the antibiofilm activity of dill, basil, winter savory, and peppermint essential oils (EOs) against E. coli during initial bacterial attachment and against preformed biofilms on polystyrene surface and lettuce leaves.

Biofilm formation was assessed using Congo Red agar, the crystal violet microtiter plate assay, and a bacterial attachment assay on lettuce leaves, while biofilm architecture and the effects of EO treatment were examined by scanning electron microscopy (SEM). Antibacterial activity was determined by the broth microdilution method, and antibiofilm activity on initial bacterial attachment and preformed biofilms was evaluated at 0.5 × MIC, MIC, and 2 × MIC using the crystal violet assay and bacterial attachment assay on lettuce leaves, respectively.

The examined E. coli isolates predominantly exhibited the rdar morphotype and produced stronger biofilms at 25 °C than at 37 °C. All tested EOs significantly reduced initial bacterial attachment and disrupted preformed biofilms in a concentration-dependent manner. Dill EO showed the strongest antibacterial and antibiofilm effects, followed by basil, winter savory, and peppermint EOs. At 2 × MIC, the tested EOs reduced initial bacterial attachment by 74.17-84.63%, while dill EO reduced preformed biofilm on polystyrene by 59.90% after 60 min. SEM analysis confirmed extensive disruption of biofilm architecture and pronounced morphological damage to bacterial cells following EO treatment.

These findings demonstrate that essential oils, particularly dill EO, effectively inhibit both the establishment and persistence of E. coli biofilms on polystyrene and lettuce leaves, highlighting their potential as natural antibiofilm agents for improving the microbiological safety of fresh produce.