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 chl

Contamination of foods with Listeria monocytogenes (Lm) is a frequent occurrence that results in expensive recalls and on occasion, deadly outbreaks of foodborne listeriosis. In fact, since 2005, L. monocytogenes has been the number one microbiologic reason for recalls in the USA. A hallmark feature of L. monocytogenes is its ability to attach to surfaces and produce biofilm, a structural community of cells encompassed in a self-produced matrix of extracellular polymeric substances (EPS). Cells residing within biofilms display enhanced protection and resistance to antimicrobial agents and environmental stressors. Therefore, biofilm production plays a key role in the persistence of these bacteria in food processing facilities. To date, most research on L. monocytogenes biofilms has been conducted using 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. Clearly, to develop effective mitigation strategies to combat biofilms in food processing plants, a greater understanding is required regarding the interactions between resident microorganisms relevant to these environments, and thus a polymicrobic approach to this research is required. Previously, we used CDC biofilm reactors to develop a model system to study single, dual, and tri-species biofilm formation by commensal meat spoilage bacteria under such nutrient flux over a 12-day cycle of simulated meat processing conditions (SMPC). Ultimately, we aim to create a standardized baseline system where biofilms can be produced under conditions that mimic those found in a food industry of interest; a system where combinations of relevant microorganisms can be introduced and parameters can be adjusted to meet the specifications of a process. The production of biofilms under standardized conditions for a specific industry would greatly improve confidence in the efficacy of sanitization trials for traditional and novel cleaning procedures. In the present study, we employed the same baseline system to examine the impact two of these commensals, Lactobacillus plantarum (Lb.) and Pseudomonas fluorescens (Pf) have on the integration of L. monocytogenes into dual and triplex biofilms and subsequently the effectiveness of a common sanitizing agent (i.e., benzalkonium chloride) on these cultures. Four different strain combinations (Lm alone; Lm + Lp; Lm + Pf; and Lm + Lp + Pf) were grown on two materials relevant to food processing lines, stainless steel (SS) and high-density-polyethylene (PE-HD), following the 12-day SMPC regimen. Relative to its pure cultures, the number of viable L. monocytogenes in the biofilms increased by 10-fold and 60-fold on SS and PE-HD, respectively, when it was paired with P. fluorescens in dual species cultures. Moreover, in the tri-species biofilms Listeria numbers were 25 times on SS and 3.5 times greater on the plastic surfaces. However, in dual species biofilms with Lb. plantarum the same increases were not observed for the pathogen. Imaging biofilms by scanning electron microscopy (SEM) revealed important architectural changes to the biofilms related to the specific strain combinations. In the presence of P. fluorescens biofilms were substantially thicker with copious amounts of associated EPS produced. This allowed for the integration and subsequent stratification of L. monocytogenes cells to deeper levels into the biofilms. Consequently, these cells were afforded better protection from the sanitizing agent, benzalkonium chloride, resulting in a 10-fold increase in the minimum concentration required to kill the pathogen residing in the biofilm. These results demonstrate the important role other commensal bacteria play in aiding the survival of L. monocytogenes; hence its own biofilm-forming abilities may not be as important as a determinant to its persistence as once thought. These factors must