Biovolume and spatial distribution of foodborne Gram-negative and Gram-positive pathogenic bacteria in mono- and dual-species biofilms

The objective of this study was to characterize the biofilms formed by Salmonella enterica serotype Agona, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) after 12, 48, 72, 120 and 240 h of incubation at 10 °C. Biofilms containing a single species, together with dual-species biofilms in which S. enterica and a Gram-positive bacterium existed in combination, were formed on polystyrene and evaluated by using confocal laser scanning microscopy (CLSM). All strains were able to form biofilm. The greatest biovolume in the observation field of 14,161 μm2 was observed for mono-species biofilms after 72 h, where biovolumes of 94,409.0 μm3 ± 2131.0 μm3 (S. enterica), 58,418.3 μm3 ± 5944.9 μm3 (L. monocytogenes), 68,020.8 μm3 ± 5812.3 μm3 (MRSA) and 59,280.0 μm3 ± 4032.9 μm3 (VRE) were obtained. In comparison with single-species biofilms, the biovolume of S. enterica was higher in the presence of MRSA or VRE after 48, 72 and 120 h. In dual-species biofilms, the bacteria showed a double-layer distribution pattern, with S. enterica in the top layer and Gram-positive bacteria in the bottom layer. This spatial disposition should be taken into account when effective strategies to eliminate biofilms are being developed. •Single- and dual-species biofilms formed by four pathogenic bacteria were studied.•S. enterica had the greatest biovolume in both single-species and mixed biofilms.•The highest biovolume of S. enterica was observed in dual-species biofilms.•S. enterica was located in the upper layer and Gram-positive bacteria in the lower.•The two-layer structure of biofilms should be considered in disinfection treatments.