Variations in biofilms harbouring Listeria monocytogenes in dual and triplex cultures with Pseudomonas fluorescens and Lactobacillus plantarum produced under a model system of simulated meat processing conditions, and their resistance to benzalkonium chloride

Abstract

Contamination of foods with Listeria monocytogenes is a frequent occurrence that results in expensive recalls and on occasion, deadly outbreaks of foodborne listeriosis. Biofilm production is thought to play a key role in the persistence of these bacteria in food processing facilities. To date, research on L. monocytogenes biofilms has predominantly involved pure cultures grown under conditions that do not represent actual food processing environments. In nature, multiple microbial species comprise biofilm communities that develop under constantly changing conditions. This study investigated changes in the characteristics of biofilms harbouring L. monocytogenes (Lm) when in the presence of two different commensal bacteria, Lactobacillus plantarum (Lp) and Pseudomonas fluorescens (Pf), commonly isolated from ready-to-eat (RTE) meat products. Using CDC biofilm reactors, four different strain combinations (Lm monoculture; Lm + Lp; Lm + Pf; and Lm + Lp + Pf) were grown on stainless steel (SS) and high-density-polyethylene (PE-HD) coupons following a dynamic 12-day regimen of simulated meat processing conditions (SMPC) that exposed cells to periods of nutrient cycling and starvation. Viable numbers of L. monocytogenes 568 (Lm568) in pure culture biofilms at the end of the regimen were 5.8 and 6.4 log10 CFU/cm2 on SS and PE-HD, respectively. In dual cultures with P. fluorescens (ATCC 13525), Lm568 biofilm cell numbers rose by 1.0 and 1.8 log on SS and PE-HD, respectively. Enhanced L.monocytogenes numbers were also observed in the triplex biofilms with Lb. plantarum and P. fluorescens (ATCC 1024) (1.4 and 0.5 log for SS and PE-HD, respectively), whereas the same trend was not observed when L. monocytogenes was paired with Lb. plantarum. Imaging biofilms by scanning electron microscopy (SEM) revealed important architectural changes to the biofilms related to the specific strain combinations. Integration of L.monocytogenes cells into P. fluorescens biofilms significantly (P < 0.05) increased the survival of the pathogen to benzalkonium chloride. These results demonstrate the important role other commensal bacteria play in aiding the survival of foodborne pathogens; hence these factors must be considered when evaluating the effectiveness of biofilm mitigation strategies.