Antibiofilm activity of eugenol against methicillin resistant Staphylococcus aureus (MRSA) isolated in retail milk, milk products and hand swabs

A total of 315 samples comprising of raw milk, traditional sweets, frozen milk products and hand swabs were col- lected from retail shops and vendors from the Mathura district of Uttar Pradesh, India. The prevalence of Staphylococcus aureus and methicillin resistant S. aureus (MRSA) was found to be 78.0%, 21.3%, 15.0%, 32.7 % and 24.0%, 6.6%, 5.0% and 16.3% in retail raw milk, traditional sweets, frozen milk products and hand swabs, respectively. The overall prevalences of S. aureus and MRSA were 31.1% and 10.7% from all the studied sample sources. The confirmed MRSA isolates were analysed for biofilm formation capability by three different methods, namely: Tissue culture plate (TCP), Tube method (TM) and Congo red agar (CRA) assay, and strong biofilm forming MRSA was detected by these methods in 61.7%, 38.2% and 20.5% samples, respectively. The tissue culture plate method was found to be the most sensitive assay of the three. The strong biofilm forming MRSA strains (n=21) were analysed to find the minimum inhibitory con- centration (MIC) values against eugenol by the microdilution method. The MIC values of eugenol ranged from 3.125 to 0.190 (v/v) for the MRSA strains. The antibiofilm effect of eugenol on the established biofilms of the MRSA strains was evaluated after challenging with MIC and 2×MIC concentrations of eugenol of 0.19 and 0.39 (v/v). After treatment with eugenol, at the 0.19 (v/v) concentration, the Optical Density (OD) values decreased and ranged from 0.612 to 0.912, while at the 0.39 (v/v) concentration, the OD values decreased and ranged from 0.301 to 0.663. Under Scanning electron microscopy (SEM) analysis, the MRSA biofilms treated with MIC and 2×MIC of eugenol showed loss of cell-to-cell connections, disruption of the organized structures of the biofilms, loss of normal morphology, the surface became rough and the bacterial cells collapsed, possibly due to exudation of the contents. The outcomes of MIC and SEM revealed that eugenol had a remarkable antibiofilm effect on the biofilm of the MRSA strains. This study concluded that eugenol oils exert remarkable antibiofilm effects on MRSA biofilms and can be applied to the surfaces of equipment, food containers, utensils and other materials to inhibit the growth of MRSA biofilms.