Characterization of Mixed-Species Biofilms Formed by Four Gut Microbiota

In natural settings, approximately 40–80% of bacteria exist as biofilms, most of which are mixed-species biofilms. Previous studies have typically focused on single- or dual-species biofilms. To expand the field of study on gut biofilms, we found a group of gut microbiota that can form biofilms well in vitro: Bifidobacterium longum subsp. infantis, Enterococcus faecalis, Bacteroides ovatus, and Lactobacillus gasseri. The increase in biomass and bio-volume of the mixed-species biofilm was confirmed via crystal violet staining, field emission scanning electron microscopy, and confocal laser scanning microscopy, revealing a strong synergistic relationship in these communities, with B. longum being the key biofilm-contributing species. This interaction may be related to changes in the cell number, biofilm-related genes, and metabolic activities. After quantifying the cell number using quantitative polymerase chain reaction, B. longum and L. gasseri were found to be the dominant flora in the mixed-species biofilm. In addition, this study analyzed biological properties of mixed-species biofilms, such as antibiotic resistance, cell metabolic activity, and concentration of water-insoluble polysaccharides. Compared with single-species biofilms, mixed-species biofilms had higher metabolic activity, more extracellular matrix, and greater antibiotic resistance. From these results, we can see that the formation of biofilms is a self-protection mechanism of gut microbiota, and the formation of mixed-species biofilms can greatly improve the survival rate of different strains. Finally, this study is a preliminary exploration of the biological characteristics of gut biofilms, and the molecular mechanisms underlying the formation of biofilms warrant further research.