In Vitro Efficacy of Clove Oil and Eugenol against Staphylococcus spp and Streptococcus mutans on Hydrophobicity, Hemolysin Production and Biofilms and their Synergy with Antibiotics

Abstract

The present study aimed to evaluate Syzygium aromaticum (clove) plant extract, clove oil and eugenol for their antibacterial activity and their potential to eradicate bacterial biofilms alone and in combination with antibiotics. Anti-bacterial efficacy of S. aromaticum extract, clove oil and eugenol was evaluated as minimum inhibitory concentration (MIC) and subsequently sub-MICs was selected for inhibition of virulence factors against test bacterial strains. Biofilm cultivation and eradication was assayed using XTT reduction in 96-well microtiter plate. Checkerboard method was used to study the interaction between essential oils and antibiotics. Staphylococcus aureus MTCC3160, Staphylococcus epidermidis MTCC435, Staphylococcus sciuri (SC-01), Staphylococcus auricularis (SU-01) and Streptococcus mutans MTCC497 were found strong biofilm former among all the test bacterial strains. The potency of test agents was found in the order of eugenol > clove oil > S. aromaticum methanolic extract. Sub-MIC (0.5 × MIC) of clove oil and eugenol showed a significant reduction in cell surface hydrophobicity (p S. auricularis (SU-01), S. epidermidis MTCC435 and S. mutans MTCC497 compared to planktonic MIC (PMIC). Antibiotics (vancomycin and azithromycin) exhibited upto 1000-folds increased in SMIC compared to PMIC against all the test bacterial strains. Synergy was observed between eugenol and antibiotics (vancomycin/azithromycin) against all the test bacterial strains in both planktonic and sessile mode. Highest synergy was exhibited between eugenol and azithromycin in planktonic mode (FICI value 0.141). Further, microscopy also confirmed the spectacular effect of combination treatment on pre-formed S. aureus MTCC3160 and S. mutans MTCC497 biofilms. These findings highlighted the promising role of clove oil and eugenol alone and in combination on pathogenic bacterial biofilms.