Characterization of Mixed-Species Biofilms Formed by Four Gut Microbiota

Abstract

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.

Keywords: Bacteroides; Bifidobacterium; Enterococcus; Lactobacillus; gut microbiota; mixed-species biofilms; synergistic interaction.

Figure 1

(a) Biofilm formation of the four strains. The numbers marked in the histogram represent different strains: 1—B. longum subsp. Infantis FJND16M4, 2—B. ovatus FTJS5K9, 3—L. gasseri FHNFQ11L7, and 4—E. faecalis E1. Lowercase letters above bars represent significant differences in biofilm mass between combinations (p < 0.05) after one-way ANOVA. (b) The biomass of mixed biofilms composed of B. longum FJND16M4, B. ovatus FTJS5K9, L. gasseri FHNFQ11L7, and E. faecalis E1 at different time points (4 h/8 h/12 h/16 h/20 h/24 h). The asterisks above the bar represent significant difference in biofilm mass between this point and the previous time point (p < 0.05) after one-way ANOVA. Error bars in the figure represent ±SEM of biological replicates.

Figure 2

Strain specificity of the mixed-species biofilm. The uppercase letters A–H represent different strains of these four species: A—B. longum FJND16M4, B—B. longum FBJCP1M11, C—E. faecalis E1, D—E. faecalis JNGMMB7, E—B. ovatus FTJS5K9, F—B. ovatus FNXYCHL6K1, G—L. gasseri FHNFQ11L7, and H—L. gasseri FHNFQ14L5. Different strains of these four species were co-cultured in various combinations to verify the interspecies universality of the mixed-species biofilm formation ability. Error bars in the figure represent ±SEM of biological replicates. Lowercase letters above bars represent significant differences in biofilm mass between combinations (p < 0.05) after one-way ANOVA.

Figure 3

Effect of cell-free supernatant on the formation of mixed-species biofilms. Numbers marked with “sup” represent the cell-free supernatant. The numbers 1–4 represent different species: 1—B. longum FJND16M4, 2—B. ovatus FTJS5K9, 3—L. gasseri FHNFQ11L7, and 4—E. faecalis E1. For each sample, diluted cultures and cell-free supernatants of these strains were inoculated into microplates at an equal ratio (v/v). Error bars in the figure represent ±SEM of biological replicates. Lowercase letters above bars represent significant differences in biofilm mass between combinations (p < 0.05) after one-way ANOVA.

Figure 4

Cell count of the four species in suspension and mixed-species biofilms. (a) B. longum FJND16M4, (b) E. faecalis E1, (c) B. ovatus FTJS5K9, and (d) L. gasseri FHNFQ11L7. The green line represents the cell number of each species in suspension and the blue line represents the number of each species in mixed-species biofilms. The number of cells was measured every four hours and error bars in the line chart represent the mean value of biological replicates ± SD.

Figure 5

Metabolic activity and exopolysaccharide concentration of mono- and mixed-species biofilms. Numbers in these graphs from 1 to 4 represent the following species: 1—B. longum FJND16M4, 2—L. gasseri FHNFQ11L7, 3—E. faecalis E1, and 4—B. ovatus FTJS5K9. Error bars in these graphs represent mean ± SEM of biological replicates. Lowercase letters above the bars represent statistically significant differences between these combinations (p < 0.05) after one-way ANOVA. (a) Metabolic activity of biofilms. (b) The concentration of exopolysaccharide of biofilms.

Figure 6

Formation ability of mono- and mixed-species biofilms when exposed to environmental stress. Numbers in these graphs from 1 to 4 represent the following strains: 1—B. longum FJND16M4, 2—E. faecalis E1, 3—B. ovatus FTJS5K9, and 4—L. gasseri FHNFQ11L7. Error bars in these graphs represent mean ± SEM of biological replicates and asterisks above the bars mean significant statistical difference between treated groups and control groups (p < 0.05) after one-way ANOVA. (a) Formation ability of biofilms in response to acid (pH = 4, 4.5, 5, 5.5, 6, and 6.2). (b) Formation ability of biofilms in response to nutrient concentration (glucose concentration = 1%, 1.5%, 2%, 2.5%, and 3%). (c) Formation ability of biofilms in response to osmotic pressure (sodium chloride concentration = 0 mol/L, 0.015 mol/L, 0.05 mol/L, 0.1 mol/L, 0.15 mol/L, and 0.2 mol/L).

Figure 7

Field emission scanning electron microscopy (FESEM) images of mono- and mixed-species biofilms. All images were obtained at 5000× magnification. (a) B. longum; (b) E. faecalis; (c) B. ovatus; (d) L. gasseri; (e) B. longum + E. faecalis; (f) B. longum + B. ovatus; (g) B. longum + L. gasseri; (h) E. faecalis + B. ovatus; (i) E. faecalis + L. gasseri; (j) B. ovatus + L. gasseri; (k) B. longum + E. faecalis + B. ovatus + L. gasseri.

Figure 8

CLSM images of mono- and mixed-species biofilms. (a) B. longum; (b) E. faecalis; (c) B. ovatus; (d) L. gasseri; (e) B. longum + E. faecalis; (f) B. longum + B. ovatus; (g) B. longum + L. gasseri; (h) E. faecalis + B. ovatus; (i) E. faecalis + L. gasseri; (j) B. ovatus + L. gasseri; (k) B. longum + E. faecalis + B. ovatus + L. gasseri; (l) Bio-volume of mono- and mixed-species biofilms. 1—B. longum, 2—E. faecalis, 3—B. ovatus and 4—L. gasseri. Lowercase letters above the bars represent statistically significant differences between these groups (p < 0.05) after one-way ANOVA.