Assessing the Effect of Ultrasonic Application Combined with Antimicrobial Solutions on C. difficile Spores and Microbial Contamination in Spinach

The combined use of chemical and physical methods can increase the rate of microbial inactivation resulting in a low risk of foodborne diseases and good preservation of food quality. The aim of this study is to assess the inhibitory effects of various active-ingredient washing solutions on Enterobacteriaceae, total aerobic mesophilic bacteria (TAMB), and Clostridioides difficile spores both with and without the application of ultrasonic treatment. In this respect, spinach samples were washed with sodium hypochlorite (NaOCl), green tea extract–acetic acid (GTE-AA), and a natural disinfectant solution (NDS) for 3 and 6 minutes, either with or without ultrasonic treatment. Washing spinach with the mentioned antibacterial solutions resulted in reductions of 1.3–2.3, 2.23–4.05, and 2.38–3.52 log CFU/g for C. difficile, TAMB, and Enterobacteriaceae, respectively. NaOCl exhibited the highest antimicrobial effect against all microorganisms tested, while the lowest inhibition was observed in samples washed with GTE-AA. Ultrasound treatment alone did not result in a significant reduction in microbial levels on spinach; instead, high-frequency applications appeared to increase the detectable microbial load, likely due to the disruption of microbial clusters or biofilms that made cells more accessible for enumeration. No synergistic interaction was observed between antimicrobial washing treatments and ultrasonic application in terms of microbial reduction. These findings highlight the need for further studies to refine ultrasound parameters and explore optimized combinations with antimicrobial agents in order to enhance microbial inactivation on fresh produce.