Antibacterial efficacy of cinnamon bark essential oil against multi-resistant <i>Enterococci</i> using LEGO® MINDSTORMS®-based automated sterilization equipment

The LEGO® coding platform, widely used in education for its modularity and accessibility, has had limited application in laboratory automation. To address limitations of conventional disk diffusion methods, this study developed an automated flame sterilization and inoculation system using LEGO® MINDSTORMS® Education EV3. The system matched the performance of manual methods while improving consistency and cost-effectiveness. Validation through repeated tests confirmed its reliability and reproducibility. Fifteen presumptive multidrug-resistant Enterococci (MRE) isolates were obtained from sewage samples in Korea. PCR analysis targeting 13 resistance genes revealed frequent detection of ermB, poxtA, tetM, and tetL. Species identification via 16S rRNA sequencing and MALDI-TOF MS confirmed all isolates belonged to the genus Enterococcus, grouped into four species. The antibacterial activity of cinnamon bark essential oil (CBEO) was tested using both methods. Inhibition zones ranged from 26.0 to 43.3 mm (mean 31.7 mm), with only a 0.1 mm difference between methods, indicating high reproducibility. Minimum bactericidal concentrations ranged from 1.6% to 12.5%, confirming bactericidal potential. In some cases, CBEO showed stronger inhibition than standard antibiotics such as ampicillin and kanamycin. These findings suggest CBEO's potential as a natural antimicrobial agent against MRE. Further studies are needed to evaluate its efficacy against broader isolate collections and in various applications, including healthcare and environmental settings. Additionally, this study presents the potential of low-cost, LEGO-based automated laboratory equipment for scalable experimental use.