Bactericidal effects of Cinnamon cassia oil against bovine mastitis bacterial pathogens

• Cinnamon cassia oil has bactericidal effects against bovine mastitis pathogenic isolates.
• C. cassia oil treatment impairs membrane integrity of bovine mastitis pathogens.
• C. cassia oil suppresses universal quorum sensing signal, autoinducer-2 production.
• C. cassia oil is an alternative organic antimicrobial to ensure milk safety.

Organic food production is expanding rapidly. However, this industry is hampered by the lack of effective antimicrobial agents which can be used in organic food production. This study examined the antimicrobial activity of Cinnamon cassia oil against major pathogens causing bacterial bovine mastitis, its miscibility in milk and possible antimicrobial mechanisms. C. cassia oil had inhibitory activity against all tested pathogen isolates from bovine mastitis. We conducted disk diffusion assay and found that discs with 20 μL of 2% (v/v) C. cassia oil solution resulted in inhibition zones of 29.6, 19.1, 27.0, 33.3 and 30.7 mm for Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus xylosus and Escherichia coli 29, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of C. cassia oil was 0.00625% and 0.025% (v/v) for S. hyicus, 0.025% and 0.10% (v/v) for E. coli 29, and 0.0125% and 0.05% for S. aureus, S. epidermidis and S. xylosus, respectively. We selected two common mastitis pathogens, a representative S. aureus isolate and E. coli 29 for further analyses. Based on time-kill assay in LB broth with 0.15% agar, 2MBC of C. cassia oil generated bactericidal effects on S. aureus and E. coli 29 within 30 min, and 4MBC caused 6 log reduction of S. aureus and E. coli 29 within 30 min. In milk, C. cassia oil at 4MBC reduced ∼6.0 Log10 CFU/ml of S. aureus and E. coli 29 to undetectable level within 8 h. Using propidium iodide staining, we observed membrane damage on both S. aureus and E. coli 29 cells during incubation with C. cassia oil. In addition, C. cassia oil treatment at MIC impaired membrane integrity of E. coli and S. aureus, which was followed by a decrease in ATP synthesis. Bacterial extracellular signaling quorum sensing orchestrates important events related to bacterial pathogeneses through excreting autoinducer (AI). Sub-inhibitory concentration of C. cassia oil repressed AI-2, a universal signal molecule mediating quorum sensing, production in S. aureus and E. coli 29 isolates. Collectively, our data show that C. cassia oil provides an exciting potential to be used as an alternative antimicrobial for bovine mastitis in organic dairy farms.