Mature Listeria monocytogenes Biofilms Exhibit Reduced Susceptibility to Sanitizers – Relevance to the (Leafy Green) Fresh Food Supply Chain

Salads and herbs are important for a healthy diet but during their processing and packaging, they may be exposed to environmental contamination from foodborne pathogens. Of particular concern is Listeria monocytogenes, a Gram-positive, facultative anaerobe, found ubiquitously in the environment. L. monocytogenes can survive and grow under harsh conditions such as refrigeration temperatures, low oxygen or nutrient concentrations, which is why it is a problem in the fresh food supply chain. Infection with L. monocytogenes can result in listeriosis, potentially fatal in immunocompromised patients, pregnant women, newborn babies, and the elderly. This study aims to evaluate the efficacy of common sanitizing methods used in the fresh food supply chain, where biofilm formation has raised concerns, using appropriately developed laboratory models of Listeria biofilms. L. monocytogenes Scott A, L. monocytogenes CECT 936, and L. innocua NCTC 12210 biofilms were grown at 20 °C or 4 °C, on stainless steel coupons for 7 days, and treated with high concentrations of chlorine (up to 300 ppm) or peracetic acid (up to 500 ppm) on days 1, 3, 5, and 7. Coupons were then processed for culturable cell counts on treatment days, and imaged using episcopic differential interference microscopy, coupled with epi-fluorescence microscopy (EDIC/EF) on day 7 of growth. This determined that temperature affects biofilm growth on stainless steel, as biofilm growth reached ∼8 log₁₀ CFU/cm² at 20 °C, but was significantly lower at 4 °C (∼4 log₁₀ CFU/cm²) - highlighting the importance of maintaining a cold chain. Chlorine and peracetic acid were shown to be effective at treating Listeria in the planktonic form but were not effective at treating aged biofilms at both temperatures and the high concentrations of sanitizers used. This work provides important information on sanitizing efforts in the fresh food supply chain, concerning factory temperature, processing surfaces, and the age of biofilm.