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Do bacteria need stiff substrates to form biofilms?

Biofilms developing on any submerged surface represent a significant interest in many applications, but the conditions of their formation are still unsufficiently understood. A parameter of bacterial colonization which remained little explored so far is the stiffness of the substrate.

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Biofilms are communities of micro-organisms that adhere together and to a surface. They grow on all surfaces, generally in the presence of water. In marine environments, attachment

of bacteria can lead to colonization of boat hulls, underwater pipes and all submerged surfaces by algae and invertebrates, which represent a hindrance to navigation and many other human activities: it is thus very important to understand the processes involved in their formation.

Adhesion of bacteria is the first step; it results from the interaction of attractive or repulsive forces at various scales and depends on factors related to the bacteria themselves, the environment and the substrate. Among the latter, previous studies dealt with the impact of chemical composition, hydrophobe character, electrical charge and roughness, but that of stiffness (defined as the resistance to a deformation) remains poorly known. This work explores the influence of stiffness on the retention of two bacterial strains involved in marine biofilms, one of Pseudoalteromonas (strain D41) and one of Bacillus (strain 4J6).

This was achieved by putting them in contact with two substrates of different stiffness. The susbtrates were hydrogels whose different concentrations of agarose (0.75 and 3 %) conditions their stiffness. Various tests confirmed that the two gels differed only by stiffness, and not by other factors like agarose molecular weight (which affects their mechanical properties), surface homogeneity at microscopic and macroscopic scales, or thermodynamic parameters. For the adhesion tests, gels are incubated with 20 ml of bacterial suspension for 3 h at 20°C, then rinsed three times to eliminate non-adherent bacteria. This procedure is repeated three times for each substrate with independent gels and bacterial cultures.



Scanning electron microscopy images of the two bacterial strains (left: 4J6, right: D41

After staining of the culture, the distribution of adherent bacteria on the gel surface is observed by a technique that provides a tridimensional representation (confocal laser scanning microscopy). Gels are then crushed in order to separate the bacteria and form a supension, an extract of which is incubated on a culture medium. Each bacteria develops into a colony; counting colonies after 48h makes possible to estimate the number of bacteria within the extract and deduce the number which adhered on the agarose gel.

Finally, a proteomic approach is conducted to characterize the proteins which were synthetized in each biofilm. The proteins extracted from the colonies were separated on gel plates (electrophoresis technique), where their distribution is studied through image analysis. Protein spots are detected and quantified, then the protein corresponding to each spot is identified by analytical chemistry methods (liquid chromatography coupled to mass spectrometry).

Motility tests showed that strain D41 is mobile owing to its flagella, whereas 4J6 is not able to swim in the chosen experimental conditions. The adhesion of bacteria on the gels depends on the strain: D41 adheres more on the gel with 3% agarose (135,000 per cm²) than on the gel with 0.75% (35,000 per cm²); however no difference is observed among gels for strain 4J6 (34,000 per cm²). The image analysis confirms these results but shows that the bacterial film of D41 is homogeneous on both gels whereas that of 4J6 presents cell clusters on the 0.75 % gel (but not on the other one).


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Microscopy images of stained bacterial films of both strains on both substrates

The proteomic comparison among gels was done for strain D41. The two-dimensional electrophoresis shows an average of 120 spots, 22 of which are significantly different among gels. The 21 proteins identified by sequencing are involved in primary cellular metabolic functions. One of them, belonging to the group of porins, is located in the outer cell membrane; because of its exposition at the cell surface, it could be involved in the process of adhesion to a substrate.

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Spots formed by the proteins of strain D41 on the electrophoresis plate.White/grey labels respectively indicate the proteins significantly more/less abundant in the 3 % gel than in the 0.75 % gel

As bacteria are in the same size range as colloidal particles, their adhesion has originally been studied by physicochemical models describing the interaction of a colloid particle with a surface. But this approach cannot provide a complete understanding, particularly because bacteria have appendages (polysaccharide chains, proteinous fibers) which have an important bridging function between cells and the substratum.

This study reached three main conclusions:

- bacteria are able to adhere on agarose hydrogels; their low numbers however confirm the low adhesive properties of these gels

- the stiffness of hydrogels impacts bacterial adhesion, through cell number and film morphology

- adhered bacteria adapt their physiology to the stiffness of the hydrogels. 21 proteins, most of them involved in key metabolic pathways, are differently regulated according to which gel D41 adheres to. One outer membrane protein is strongly involved, but a possible surface-sensing role of this protein is a hypothesis requiring further studies.


The paper

Guégan C., Garderes J., Le Pennec G., Gaillard F., Fay F., Linossier I., Herry J.-M., Bellon Fontaine M.-N., Vallée Réhel K., 2014. Alteration of bacterial adhesion induced by the substrate stiffness. Colloids and Surfaces B: Biointerfaces 114 : 193– 200.

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The authors

This work was done in collaboration between members of the Laboratoire de Biotechnologie et Chimie Marines(Lorient, laboratory associated to IUEM), of the Metabomer Platform (Station biologique de Roscoff) and the laboratory Micalis (Massy).


The journal

ublished by Elsevier since 1993, Colloids and Surfaces B: Biointerfaces is an international journal devoted to fundamental and applied research on colloid and interfacial phenomena in relation to systems of biological origin, with particular relevance to the medical, pharmaceutical, biotechnological, food and cosmetic fields.



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