Dynamic nature of multispecies biofilm under stress : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology at Massey University, Manawatu Campus, New Zealand

Abstract

Multispecies biofilms are an arrangement of sessile communities of multiple species of bacteria, embedded within the 3D structure of the extracellular matrix produced by multiple microorganisms in the community. These biofilms can be found in many natural environments, such as food, clinical, and natural surfaces. When multiple species of bacteria come together, the interactions between these bacteria define the evolution of the biofilm, including pathogenicity and response to cleaning and sanitizing agents. The existing research focus has been extensively on single-species biofilm, as these are simpler to study. However, in a natural environment, multi-species biofilm is the most common. The variables present in the environment can impact the complex interactions in multispecies biofilms. This study hypothesized that multispecies biofilms have different biofilm properties compared to their single-species counterparts, and common variables encountered in the food industry, such as nutrients and flow, significantly impact both single and multispecies biofilm formation. For the first research objective, single, dual, and triple species biofilm from a combination of spoilage and pathogenic bacteria relevant to food processing, P. fluorescens, S. aureus, and L. monocytogenes, were studied for changes in the interactions under different nutrient environments on polystyrene surfaces. The crystal violet values (O.D₅₉₀ nm) and interaction equations showed that the interactions between the bacteria can develop into competition under nutrient limitation (10% TSB), in contrast to synergy under nutrient abundance (full-strength TSB). These observations were supported by higher biofilm formation based on total biomass and higher exopolysaccharide under nutrient-abundant conditions. Since the cell concentrations between the bacteria formed under both nutrient conditions were comparable, it may be possible that under low nutrient conditions, the bacteria are focused on survival rather than on matrix overproduction as observed in nutrient-abundant conditions (Chapter 3). All three bacteria involved showed increased tolerance against sodium hypochlorite sanitizer in the multispecies arrangement compared with their single-species counterparts, more so prominent in L. monocytogenes with 1.1 log₁₀ reduction in multispecies compared to its single species tolerance (4.3 log₁₀ reduction), indicating added protection in multispecies biofilm. The next objective examined biofilm formation on an industrially relevant surface, stainless steel, under the influence of turbulent flow (Reynold number > 4000). The impact of flow in single and multispecies biofilm formation was analysed using the Centre for Disease Control and Prevention (CDC) bioreactor. Both single and multispecies biofilm formation were significantly impacted by the turbulent flow, with higher exopolysaccharide (EPS) and increased tolerance to sodium hypochlorite sanitizer in biofilm formed under flow compared to the static system. Epifluorescence microscopy revealed structural variation, with interstitial voids present in the three-species biofilm, which was not observed in the static system (Chapter 4). The impact of turbulence was more pronounced for single and dual species combinations of S. aureus and L. monocytogenes, with observations such as macroscopic threads and microscopic filaments and low cell concentrations in both single and dual species biofilm. In the presence of P. fluorescens, both these structures were absent, and the cell concentrations increased significantly (p<0.05) in the multispecies biofilm. The filamentous cell formation in single species and its absence, followed by a significant increase in L. monocytogenes cells in the dual species biofilm with P. fluorescens under turbulent flow, were further analysed. Preliminary analysis found that the cell concentration of L. monocytogenes in a single species was limited to 5.1 log₁₀ CFU/cm² after 48 h, where filamentous cells (27.7 µm in length) were observed. In dual species with P. fluorescens, the cell concentration reached 8.7 log₁₀ CFU/cm², and no filaments were observed. Gene expression analysis showed significant (p<0.001) downregulation of motB (motility), sigB (stress), and cell division (ftsX and ftsW), and upregulation of mpl (adhesion) and rodA (rod shape), indicating L. monocytogenes adaptation to shear stress (Chapter 5). While the gene expression and motility studies provide insight into stress adaptation and filament formation, further insights into the significantly higher cell concentration of L. monocytogenes in dual species biofilm with P. fluorescens can be understood through sequential biofilm formation. For the final chapter, the sequence of attachment and biofilm formation in a dual species biofilm of P. fluorescens and L. monocytogenes under both static and turbulent flow conditions was investigated to further understand why L. monocytogenes cells show a spike in cell concentration under turbulent flow in dual-species biofilms. Results showed that this significant spike can be observed within 48 h in 2 conditions: when both bacteria are coinoculated, and secondly, when L. monocytogenes was added into the 48 h preformed P. fluorescens biofilm. The preformed P. fluorescens biofilm also correlated with higher attachment (30 min) of L. monocytogenes cells (Chapter 6). Interestingly, the conditioning of the stainless-steel surfaces by freeze-dried and rehydrated exopolysaccharides (0-27.5 µg/mL) extracted from P. fluorescens biofilm did not affect the attachment of L. monocytogenes, indicating other factors, such as biofilm structure, other biofilm components, or the presence of P. fluorescens cells, could be at play. While the initial attachment of P. fluorescens was negatively affected by the Listeria preformed biofilm formed under flow, the overall biofilm (cell concentration) was not affected by either the flow or the colonisation order. This chapter provides insight into the significantly higher growth of L. monocytogenes in multispecies biofilm under turbulent flow, also observed in the previous chapter. Overall, the presence of multiple bacteria in the three species biofilm selected for this study altered the cell composition of the biofilm, the exopolysaccharide concentration, stress adaptation, and the tolerance to sanitizers, indicating the importance of studying multispecies biofilm as the natural state of biofilm in the environment. This is the first reported study on biofilms in these three species to examine the nutrient and flow variables and to provide insights into how the interaction between bacteria varies in response to these external variables.