The ability of Listeria to form biofilms on surfaces like stainless steel makes it a persistent threat in food production environments, and is especially dangerous in ready-to-eat (RTE) foods and fresh produce like leafy greens, for which cooking is not used as a final kill step.

In this context, researchers from the University of Southampton sought to better understand the ability of Listeria monocytogenes in biofilms to withstand commonly used sanitizers in leafy greens food production environments. The study was designed using species, sanitizers, and temperatures that are relevant to the fresh leafy greens supply chain. Worst-case scenarios were modeled by applying sanitizers without prior cleaning, simulating neglected niches within a facility.

Specifically, the researchers evaluated the antimicrobial efficacy of chlorine and peracetic acid (PAA) sanitizers against biofilms formed by “appropriately developed laboratory models of Listeria biofilms” comprising L. monocytogenes Scott A (serotype 4b), L. monocytogenes CECT 936 (serotype 1/2b), and avirulent L. innocua NCTC 12210. Biofilms were grown on stainless steel at 20 °C and 4 °C. A static model was chosen to simulate hard-to-clean areas, or areas that experience minimal flow of fluid, in food production facilities.

Key findings and conclusions from the study include:

• Temperature: Biofilms grew significantly denser at 20 °C, at approximately 8 log₁₀ colony forming unit per square centimeter (CFU/cm²), in comparison to 4 °C, at approximately 4 log₁₀ CFU/cm², underscoring the importance of maintaining cold chain integrity.
• Sanitizers against biofilms: High concentrations of chlorine, up to 300 parts-per-million (ppm), and PAA, up to 500 ppm, were ineffective against mature biofilms. This finding provides evidence of reduced susceptibility of biofilms to chlorine and PAA, when compared to the efficacy of sanitizers against planktonic cells.
• Sanitizers against planktonic cells: Planktonic results showed complete eradication of L. monocytogenes Scott A at 50 ppm chlorine and 25 ppm PAA. L. monocytogenes CECT 936 was eradicated at 50 ppm chlorine, and L. innocua at 25 ppm chlorine and 50 ppm PAA.
• Stainless steel surface: Episcopic differential interference microscopy/epi-fluorescence microscopy (EDIC/EF) imaging showed that L. monocytogenes preferentially adhered to cracks and troughs in stainless steel, which, in the real world, would complicate cleaning efforts and enable long-term persistence.

When seeking to explain the biofilms’ sanitizer resistance, the researchers hypothesized that:

• Extracellular polymeric substances (EPS) in biofilms may hinder sanitizer penetration, contributing to microbial survival. EPS, which encapsulate and provide structure to a biofilm, act as a physical barrier from the environment and have been shown to prevent sanitizer entry and sequester toxins.
• Residual organic matter within a biofilm may quench oxidizing agents, reducing sanitizer effectiveness. In the leafy greens sector, bacteria would be supplied with organic matter from wash water, acting not only as a carbohydrate source, but also reducing the efficacy of chlorine or PAA disinfection.
• The upregulation of stress response genes may occur following sanitizer exposure.

While single-agent sanitization proved ineffective in the present study, prior research has demonstrated that combination treatments (e.g., PAA with saturated steam) may reduce biofilm viability.

Overall, the findings emphasize that mature Listeria biofilms are highly tolerant to standard sanitization protocols. Effective control requires integrated cleaning strategies combining mechanical disruption and chemical treatment, tailored to surface material, temperature conditions, and biofilm maturity.

The study was published in the Journal of Food Protection. The lead and corresponding author is Lucy Sutton, Ph.D.