Examining the viability of five Salmonella enterica subsp. enterica in thymol at 4°C and 25°C using flow cytometry

Salmonella spp., a major cause of foodborne illness, requires effective control strategies to improve food safety. Thymol, an antibacterial agent derived from natural essential oils, has been assessed for use as an antimicrobial agent and preservative in the food industry, due to its safety and low cost. This study used flow cytometry and Tryptic Soy Agar (TSA) plate counts, to assess the viability of five serotypes of Salmonella enterica subsp. enterica over 56 days in thymol at 4°C and 25°C, during long-term storage in distilled water. The minimum inhibitory concentration (MIC) of thymol against Salmonella serotypes was found to be 256 µg/mL at both 25°C and 4°C for all serotypes at the initial time point (day 0) and after 154 days of incubation in water. Flow cytometry successfully counted viable cells in the control group, which contained 2% ethanol and 128 µg/mL thymol. However, plate count numbers completely declined after day 7 at both 25°C and 4°C for all thymol concentrations. After exposure to sub-MIC levels and subsequent spiking with 256 µg/mL at 25°C and 4°C, neither flow cytometry nor plate counts detected viable cells. These findings emphasize the importance of advanced techniques such as flow cytometry for the detection of microorganisms and demonstrate thymol's potential as an environmentally friendly solution in food safety strategies to reduce Salmonella contamination in water sources over extended periods.