Evaluation of single and multispecies biofilm formed in the static and continuous systems

Biofilms consisting of multiple species of bacteria compared to biofilms of single species are common in natural environments including food contact surfaces. The objective of this study was to understand the biofilm formation and the efficiency of sodium hypochlorite (50 ppm/5 mins) on the single and multiple species biofilm of Pseudomonas fluorescens, Staphylococcus aureus, and Listeria monocytogenes formed on stainless steel surfaces in static and continuous systems. The cell concentration of Listeria in the dual and triple species biofilm in the continuous system (7.3-8.4 log CFU/cm²) was higher compared to the static system (4.7-4.9 log CFU/cm²) while the concentration remained consistent in the single species biofilm (6.4-6.7 log CFU/cm²) for both systems. Biofilm formed in the static system was significantly (p < 0.001) more susceptible to sodium hypochlorite than biofilm formed in the continuous system. This observation agrees with the exopolysaccharide concentration which was found to be higher in the continuous system (8.0-15.6 μg/cm²) than in the static system (3.2-6.3 μg/cm²) indicating a positive correlation between EPS production and sanitizer resistance. Epifluorescence microscopy images showed the formation of interstitial voids within the three-species biofilm and filaments in the single and dual species Listeria biofilms in the continuous system which were absent in the static system. Overall, results showed that the biofilm formation and sanitizer resistance vary due to multispecies interaction and the presence of flow and should be considered an important variable in multispecies sanitizer resistance studies.