Diversity assessment of Listeria monocytogenes biofilm formation: Impact of growth condition, serotype and strain origin

• Temperature was the most important factor influencing biofilm production.
• Nutrient-poor rather than nutrient-rich medium enhanced biofilm production.
• Effect of serotype on biofilm production was significant and influenced by medium.
• Biofilm production was not significantly affected by the origin of isolation.
• Poor correlation of CV staining and viable biofilm cells due to non-viable cells and eDNA

The foodborne pathogen Listeria monocytogenes has the ability to produce biofilms in food-processing environments and then contaminate food products, which is a major concern for food safety. The biofilm forming behavior of 143 L. monocytogenes strains was determined in four different media that were rich, moderate or poor in nutrients at 12 °C, 20 °C, 30 °C and 37 °C. The biofilm formation was mostly influenced by temperature, resulting in decreased biofilm formation with decreasing temperature. Biofilm formation was enhanced in nutrient-poor medium rather than in nutrient-rich medium, and especially in nutrient-poor medium significantly enhanced biofilm production was observed early in biofilm maturation underlining the effect of medium on biofilm formation rate. Also serotype had a significant effect on biofilm formation and was influenced by medium used because strains from both serotype 1/2b and 1/2a formed more biofilm than serotype 4b strains in nutrient-rich medium at 20 °C, 30 °C and 37 °C, whereas in nutrient-poor medium the biofilm production levels of serotype 1/2a and 4b strains were rather similar and lower than serotype 1/2b strains. The strains used originated from various origins, including dairy, meat, industrial environment, human and animal, and the level of biofilm formation was not significantly affected by the origin of isolation, irrespective of medium used and temperature tested. A linear model was used to correlate crystal violet staining of biofilm production to the number of viable cells within the biofilm. This showed that crystal violet staining was poorly correlated to the number of viable cells in nutrient-poor medium, and LIVE/DEAD staining and DNase I treatment revealed that this could be attributed to the presence of non-viable cells and extracellular DNA in the biofilm matrix. The significant impact of intrinsic and extrinsic factors on biofilm production of L. monocytogenes underlined that niche-specific features determine the levels of biofilm produced, and insights in biofilm formation characteristics will allow us to further optimize strategies to control the biofilm formation of L. monocytogenes.