Biofilm formation

Bacteria in aquatic environments are rarely found in the planktonic or free-swimming phase.

Rather, they are found in association with a solid surface in a sessile state. The first observation of surface adherent bacteria was made by Anthony van Leeuwenhoek in 1684, when he observed the plaque of his own teeth and discovered what would later be known as “bacterial biofilm”. The term “biofilm”, however was not used until 1978 and in 1999 Costerton and co-authors defined the biofilm as “a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface” [97-101]. Biofilms are found everywhere from drinking water to medical devices and cause the most problematic bacterial infections such as urinary tract infections, dental plaque and upper respiratory tract infections [102-105].

Biofilms can be either single or multilayered and can contain either homologs or heterologs population of bacteria. In most biofilm formations, unicellular organism come together to form a community. This community become attached to either biotic or abiotic surfaced and embedded in a self-produced matrix. The matrix is generally referred to as extracellular polymeric substance (EPS) which is a mixture of polysaccharides, proteins (composed primarily of D-amino acids), fatty acid and extracellular nucleic acids (eDNA) [99, 106, 107]. Most of the biomasses of a biofilm is composed of more EPS (90%) than microbial cells (10%) [108]. EPS is built of water channels that facilitate exchange of nutrients, waste products and oxygen to all parts of the structure. EPS is also involved in facilitating surface adherence, aggregation and maintaining the three-dimensional architecture of the biofilm. Furthermore, the EPS surrounding the biofilm serves as a barrier protecting the bacterial cells against various stress factors, such as antimicrobial compounds, host immune systems, oxidation and metallic cations, hence enhancing growth and survival by providing nutrients and protecting from predators. Thus, the biofilm is the preferred lifestyle among vibrios and other microbes, providing several advantages such as virulence in V.

cholerae, V. vulnificus and V. parahaemolyticus, and host colonization by A. fischeri [108-112].

Figure 5 demonstrates the stages of biofilm formation, that can be divided into four stages (i) surface attachment; (ii) microcolony formation; (iii) biofilm maturation; and (iv) dispersal and detachment [106].

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Figure 5 The steps of biofilm life cycle. Biofilm formation is a multistage process that involves (i) attachment of cells to a surface, (ii) secretion of adhesins and EPS that result in irreversible attachment of the biofilm and the growth of cells (iii) maturation of the biofilm into mushroom structure (iv) dispersal of single cells that return to a planktonic phase.

Surface attachment

Surface attachment is the turning point from a planktonic lifestyle to the biofilm mode and could be categorized as a two-stage process: initial reversible attachment and irreversible attachment.

When planktonic cells come in contact with a surface, they adhere either by using a physical force of by bacterial appendages such as pili or flagella. This surface attachment is termed “reversible attachment”. The initial attachment to the surface is dynamic and can be reversed due to weak interaction between bacteria and the surface. In this case the bacteria can detach and rejoin the planktonic population if perturbed by repulsive forced or in response to nutrient availability.

There are several interaction forces that help the bacteria to adhere to a surface such as hydrophobic interaction, protein adhesion, electrostatic interactions and Van der Waal force.

When the attractive force is greater that the repulsive force, some of the attached cells become immobilized and attach irreversibly [107, 113-115].

Microcolony formation

Following the irreversible attachment, surface associated bacterial cells come together and start to proliferate and produce biofilm matrix components, forming small aggregates to generate multi-layer microcolonies. At this stage bacterial cells enhance the production of EPS and repress flagellar-mediated swimming motility [114, 116].

14 Biofilm maturation

The multi-layer microcolonies undergo a maturation process involving two stages: stage I involves inter-cell communication and the production of autoinducer signal molecules such as AHLs. In stage II the microcolonies grow through cell proliferation, increase in size and gradually mature forming macrocolonies. At this stage the macrocolonies are encased in a self-produced EPS matrix that stabilizes the biofilm network and is essential to build the three-dimensional

mushroom-like structure [107, 117].

Dispersal and detachment

Inside the mature biofilm, bacteria exchange and share products that play an essential role in maintaining the biofilm structure and providing a suitable and favorable environment for the bacterial colony. As the biofilm matures, resources such as nutrients and oxygen become limited and at the same time toxic products accumulate. In order to survive, expand, get nutrients and eliminate stress, the dispersal become an option and some cells of the biofilm disperse and return to a planktonic lifestyle and may subsequently colonize other surfaces to form new biofilms. For example, Pseudomonas putida biofilms can dissolve rapidly once the medium flow in the chambers stops, suggesting that nutrient limitation leads to biofilm dispersal [118]. In addition to nutrient limitation previous studies show that increase in nutrient availability can lead to dispersal of parts of a biofilm. For example Pseudomonas aeruginosa induces dispersal with increasing nutrient availability in the environment [119]. The dispersal stage is the final stage of biofilm life cycle as well as the start of a new cycle through dispersal. This can occur passively through dynamic forces or actively through the production of matrix-degrading enzymes and induction of flagella motility.

In general, the mature biofilm is built of two distinct layers. The base film layer where the bacterial cells exist and the surface film layer where the bacterial cells get dispersed into their surroundings. Hence the dispersal could occur in the whole biofilm or just in a part of it [114, 117].

Biofilm formation is a highly regulated process, in which bacteria have to synchronize their gene expression to be able to create the overall biofilm structure. To achieve this, bacteria use several regulatory mechanisms such as QS, c-di-GMP signaling, alternative sigma factors, sRNAs and two-component regulators.

QS is associated with almost all stages of biofilm development from attachment to dispersal. For some species bacterial QS systems regulate flagellar activity and adhesion, which in turn influences the attachment of bacteria to surface and microcolony aggregation [120]. For example, in Staphylococcus aureus the agr QS system regulates surface adhesion, which influence the attachment to the host [121]. In P. aeruginosa QS regulates other aspects of biofilm formation,

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including biofilm structure. The lasI mutant, which is defective in the synthesis of N-3-oxo-dodecanoyl-L-homoserine lactone (3OC12-HSL), formed thin (about 20% of the wild type thickness) and densely packed biofilms lacking water channels and mushroom structure [66, 122]. Similarly, in V. cholerae QS tightly regulate the transcription of genes involved in the production of exopolysaccharides which is necessary for biofilm maturation and the formation of the three-dimensional architecture [123]. Furthermore, QS plays a critical role in dispersal of detached bacteria from mature biofilm to trigger a new cycle of biofilm formation [124].

An intracellular second messenger, bis-(3-5)-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a critical role in several stages of the bacterial biofilm formation. At early stages, the high intercellular concentration c-di-GMP is involved in the bacterial decision between remaining as planktonic cells or entering the biofilm lifestyle [125]. In V. cholerae and other bacterial spp.

the increased level of c-di-GMP enhance biofilm formation and at the same time represses motility, while the low level of c-di-GMP inhibit biofilm formation and promote motility [126, 127]. C-di-GMP has been shown to be controlled by QS, where changes in cell density is one of the environmental factors sensed by the second messenger [128].

Alternative sigma factors and their role in QS and biofilm formation will be presented later in this thesis, whereas other regulatory factors are beyond the scope of this work.