Development of Listeria monocytogenes biofilms in a CDC biofilm reactor and investigation of effective strategies for biofilm control in food processing environments

Abstract

Listeria monocytogenes is one of the most concerning threat for the food industry. This pathogen has shown niche adaptation and ability to form biofilms. Even if sanitation practices can minimize the risk of pathogen survival, difficult-to-clean sites remain high-risk areas. Chemical sanitizers combined with non-thermal processing technologies might represent an effective way to control L. monocytogenes biofilm formation. The main objectives of this study were to: 1) understand L. monocytogenes biofilm formation ability under different conditions of material, temperature, and nutrients; 2) evaluate the effect of several chemical sanitizers alone or in combination with UV light to control biofilms; and 3) study the transcriptional response of L. monocytogenes biofilm to sublethal conditions of chemical sanitizers. A CDC Biofilm Reactor was used to grow 4-days-old multi-strain L. monocytogenes biofilms on stainless steel and polycarbonate coupons under dynamic conditions using TSBYE (Tryptic Soy Broth + 0.6% Yeast Extract) or BHI (Brain Heart Infusion) as media culture at 30 °C or 37 °C incubation temperature. Biofilms grown at 30 °C in TSBYE on stainless steel reached higher cell counts (8.14 log CFU/cm²). These conditions were selected for subsequent experiments. Biofilm survivability was investigated after 10 min exposure to lactic acid (4%), peracid (200 ppm), quaternary-ammonium (400 ppm) alone or in combination with UV light (254 nm) for 15 or 30 min. Sequential treatments effect was also evaluated. Control biofilms reached 6 log CFU/cm² . Reductions ranging from 2.6 to 3.6 log CFU/cm² were observed with chemical sanitizers, while a maximum of 1.8 log CFU/cm² reduction was recorded after UV-C treatment alone. Combined treatments showed enhanced effect and their application sequence was significant for lactic acid and peroxyacid (P < 0.05). Finally, biofilm RNA was preserved for transcriptomic analysis. The present research represents an initial framework to develop L. monocytogenes biofilms under dynamic condition using the CDC Biofilm Reactor. Also, it offers a preliminary understanding of L. monocytogenes biofilm response to chemical sanitizers and UV light supporting the development of effective intervention strategies to control this pathogen in food processing environments.