Multispecies Biofilm Development of Marine Bacteria Implies Complex Relationships Through Competition and Synergy and Modification of Matrix Components

Abstract

Microbial communities composition is largely shaped by interspecies competition or cooperation in most environments. Ecosystems are made of various dynamic microhabitats where microbial communities interact with each other establishing metabolically interdependent relationships. Very limited information is available on multispecies biofilms and their microhabitats related to natural environments. The objective of this study is to understand how marine bacteria isolated from biofilms in the Mediterranean Sea interact and compete with each other when cultivated in multispecies biofilms. Four strains (Persicivirga mediterranea TC4, Polaribacter sp. TC5, Shewanella sp. TC10 and TC11) with different phenotypical traits and abilities to form a biofilm have been selected from a previous study. Here, the results show that these strains displayed a different capacity to form a biofilm in static versus dynamic conditions where one strain, TC11, was highly susceptible to the flux. These bacteria appeared to be specialized in the secretion of one or two exopolymers. Only TC5 seemed to secrete inhibitory molecule(s) in its supernatant, with a significant effect on TC10. Most of the strains negatively impacted each other, except TC4 and TC10, which presented a synergetic effect in the two and three species biofilms. Interestingly, these two strains produced a newly secreted compound when grown in dual-species versus mono-species biofilms. TC5, which induced a strong inhibition on two of its partners in dual-species biofilms, outfitted the other bacteria in a four-species biofilm. Therefore, understanding how bacteria respond to interspecific interactions should help comprehending the dynamics of bacterial populations in their ecological niches.

Keywords: competition; marine bacteria; matrix components; multispecies biofilm; synergy.

FIGURE 1

Surface coverage of the four marine bacteria in static conditions. P. mediterranea TC4, Polaribacter sp. TC5, Shewanella sp. TC10, and Shewanella sp. TC11 were inoculated in ASW at an OD_{600 nm} of 0.1 (A) and 0.3 (B) on glass coverslips placed in 24-well plates. After 3, 24, and 48 h of incubation, the bacteria were stained with DAPI, and the epifluorescence pictures were analyzed by an algorithmic method to define the percentage of coverage. Statistical significance was accepted from p < 0.05; ^∗p < 0.05; ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 2

Biofilm formation of the four marine bacterial strains in dynamic conditions. The formation of biofilm was monitored in ASW on flow chamber glass surfaces using a flux of 3 mL/h with P. mediterranea TC4, Polaribacter sp. TC5, Shewanella sp. TC10, and Shewanella sp. TC11. After 3, 24, and 48 h of incubation, the bacteria were stained with Syto 61 or directly imaged for TC10 pX5-GFP. (A) The percentages of coverage of the most occupied slice were defined over time by the COMSTAT algorithm. (B) The biovolumes were calculated at the same time points by the COMSTAT algorithm. (C) Representative pictures of the biofilm formed after 3, 24, and 48 h of incubation are shown. Values are the mean of three replicates, errors bars represent standard deviation and significant differences between incubation times (p-value <0.05) are indicated by different letters (A,B) in a same alphabet (A≠a≠α).

FIGURE 3

Matrix exopolymers secreted by the marine bacterial strains in static and single species conditions. Exopolymeric substances secreted in the biofilm for each of the four strains were assessed in ASW after 48 h. Different fluorescent markers were used: Concanavalin A for exopolysaccharides of glucose and/or mannose, WGA lectins for exopolysaccharides of sialic acids, PNA lectins for exopolysaccharides containing glycoproteins, TOTO3 for eDNA and Sypro Ruby for proteins. (A) The biovolumes of each matrix compound were evaluated by the COMSTAT algorithmic method. Representative pictures of the observed secretion of matrix compounds are presented for P. mediterranea TC4 (B), Polaribacter sp. TC5 (C), Shewanella sp. TC10 (D), and Shewanella sp. TC11 (E). A closer view of the TC4 biofilm labeled with TOTO3 (F), the TC5 biofilm labeled with ConA (G), the TC10 biofilms labeled with WGA (H), and Sypro Ruby (I) are presented. The white arrows indicate the localization of the matrix compounds. Values are the mean of three replicates, errors bars represent standard deviation.

FIGURE 4

Antibiofilm activity of the TC5 supernatant on the marine bacteria biofilms. The biofilm formation of P. mediterranea TC4, Polaribacter sp. TC5, Shewanella sp. TC10, and Shewanella sp. TC11 was evaluated by the Crystal Violet staining approach in VNSS with or without (VNSS Reference) the addition of the TC5 supernatant harvested in stationary growth phase after 48 h in static conditions. Values are the mean of three replicates, error bars represent standard deviations and asterisks indicate significant difference to the VNSS reference (^∗p < 0.05 and ^∗∗P < 0.01).

FIGURE 5

Biovolumes and spatial patternings of bacteria in dual-species biofilms. The two species biofilm formations were analyzed through each combination of the four bacterial strains in ASW after 24 or 48 h of incubation. (IA–D) Images of the single-species biofilms with P. mediterranea TC4, Polaribacter sp. TC5, Shewanella sp. TC10, and Shewanella sp. TC11 in ASW. (IIA–F) Spatial patterning and biovolumes of each strain in dual-species biofilms compared to when cultivated alone. Values are the mean of three replicates, error bars represent standard deviations and asterisks indicate significant difference to the strain incubated alone (^∗p < 0.05, ^∗∗ P < 0.01, and ^∗∗∗P < 0.001).

FIGURE 6

Matrix Exopolymers secreted in the dual-species biofilm of TC4 and TC10. The exopolymeric substances secretion in the TC4 and TC10 biofilm was assessed in ASW after 48 h of incubation. The exopolymeric components were tagged using the following fluorochromes: exopolysaccharides of sialic acids with WGA lectins (I), proteins with Sypro Ruby (II), eDNA with TOTO3 (III), exopolysaccharides containing glycoproteins using PNA lectins (IV), and exopolysaccharides of glucose and/or mannose using Concanavalin A (V).

FIGURE 7

Biovolumes and spatial patternings of bacteria in three-species biofilms. The three-species biofilm formation selected including P. mediterranea TC4, Shewanella sp. TC10, and Shewanella sp. TC11 was analyzed in ASW after 24 or 48 h of incubation. Representative images of the single-species biofilms with P. mediterranea TC4 (A), Shewanella sp. TC10 (B), and Shewanella sp. TC11 (C). (D) Biovolumes of each strain in three species biofilms compared to when cultivated alone. (E) Representative spatial patterning images after 24 or 48 h of bacteria in three species biofilms. Values are the mean of three replicates, error bars represent standard deviations and asterisks indicate significant difference to the strain incubated alone (^∗∗p < 0.01 and ^∗∗∗ P < 0.001).

FIGURE 8

Biovolumes and spatial patternings of bacteria in four-species biofilms. The four species biofilm formation including P. mediterranea TC4, Shewanella sp. TC10, Shewanella sp. TC11, and Polaribacter sp. TC5 was analyzed in ASW after 24 or 48 h of incubation. Representative mages of the single-species biofilms with P. mediterranea TC4 (A), Polaribacter sp. TC5 (B), Shewanella sp. TC10 (C), and Shewanella sp. TC11 (D). (E) Representative spatial patternings after 24 or 48 h of development in four-species biofilms. (F) Biovolumes of each strain in four species biofilms compared to when incubated alone. Values are the mean of three replicates, error bars represent standard deviations and asterisks indicate significant difference to the strain incubated alone (^∗∗p < 0.01 and ^∗∗∗p < 0.001).