| Summary: | 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: <i>Bifidobacterium longum</i> subsp. <i>infantis</i>, <i>Enterococcus faecalis</i>, <i>Bacteroides ovatus</i>, and <i>Lactobacillus gasseri</i>. 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 <i>B. longum</i> 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, <i>B. longum</i> and <i>L. gasseri</i> 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.
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