# Synergistic Inhibitory Effect and Mechanism of Action of Cinnamaldehyde‐Carvacrol Nanoemulsion on the Staphylococcus aureus and Escherichia coli Mixed Biofilms Formation

## TL;DR

This study shows that a nanoemulsion of cinnamaldehyde and carvacrol can effectively inhibit mixed biofilms of Staphylococcus aureus and Escherichia coli, both in lab and mouse models.

## Contribution

The novel contribution is the development and testing of a CA/CV nanoemulsion with synergistic biofilm inhibition in a mixed bacterial infection model.

## Key findings

- CA/CV nanoemulsion significantly inhibited mixed biofilm formation at 64 μg/mL by reducing adhesion, motility, and PIA synthesis.
- The nanoemulsion inhibited LuxS/AI-2 expression and reduced bacterial burden in a mouse implant infection model.
- The synergistic effect of CA and CV was confirmed with a Q value of 2.28.

## Abstract

The formation of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) mixed biofilms challenges the treatment of bacterial infections. In this study, the synergistic effect of plant essential oils cinnamaldehyde (CA) and carvacrol (CV) on inhibiting S. aureus and E. coli mixed biofilms formation was investigated. The Q value of CA and CV combination on inhibiting mixed biofilms formation was 2.28, suggesting their synergistic effect. Furthermore, CA/CV nanoemulsion was prepared, which further enhanced the inhibitory effect. CA/CV nanoemulsion could significantly inhibit the mixed biofilms formation at 64 μg/mL by reduction of bacterial adhesion and motility, polysaccharide intercellular adhesin (PIA) synthesis, and the inhibition of LuxS/AI‐2 expression. In the S. aureus and E. coli infected implant model of mice, CA/CV NEs demonstrated prominent inhibitory effect on the formation of S. aureus and E. coli biofilms, and simultaneously reduced the bacterial burden.

The study prepared Cinnamaldehyde‐Carvacrol nanoemulsions (CA/CV NEs) through high‐energy emulsification. In a mouse infection model, CA/CV NEs exhibited a synergistic inhibitory effect on the formation of Staphylococcus aureus–Escherichia coli mixed biofilms, reducing PIA, LuxS/AI‐2, adhesion, athletic ability, and extracellular protein.