Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

A biofilm can be defined as a community of microorganisms adhering to a surface and surrounded by a complex matrix of extrapolymeric substances. It is now generally accepted that the biofilm growth mode induces microbial resistance to disinfection that can lead to substantial economic and health concerns. Although the precise origin of such resistance remains unclear, different studies have shown that it is a multifactorial process involving the spatial organization of the biofilm. This review will discuss the mechanisms identified as playing a role in biofilm resistance to disinfectants, as well as novel anti-biofilm strategies that have recently been explored.

Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.

Bacillus cereusis a food-borne pathogen and a frequent contaminant of food production plants. The persistence of this pathogen in various environments results from the formation of spores and of biofilms. To investigate the role of theB. cereusflagellar apparatus in biofilm formation, we constructed a non-flagellated mutant and a flagellated but non-motile mutant. Unexpectedly, we found that the presence of flagella decreased the adhesion of the bacterium to glass surfaces. We hypothesize that this decrease is a consequence of the flagella hindering a direct interaction between the bacterial cell wall and the surface. In contrast, in specific conditions, motility promotes biofilm formation. Our results suggest that motility could influence biofilm formation by three mechanisms. Motility is necessary for the bacteria to reach surfaces suitable for biofilm formation. In static conditions, reaching the air–liquid interface, where the biofilm forms, is a strong requirement, whereas in flow cells bacteria can have access to the bottom glass slide by sedimentation. Therefore, motility is important for biofilm formation in glass tubes and in microtitre plates, but not in flow cells. Motility also promotes recruitment of planktonic cells within the biofilm by allowing motile bacteria to invade the whole biofilm. Finally, motility is involved in the spreading of the biofilm on glass surfaces.