DEVELOPMENTAL STAGES OF BIOFILM FORMATION

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Typical sites where biofilms can form on the human body include the teeth (where biofilms form dental plaque that leads to tooth decay), contact lenses where bacteria produce eye irritations and inflammation that may lead to corneal infections, and cystic fibrosis patients harbouring numerous numbers of P. aeruginosa that produce large amounts of polymers which prevent antibiotic diffusion. Biofilms can also be formed in water-retention devices/systems and in air-cylinders where pathogenic bacteria may hide to evade antimicrobial onslaught. Biofilms also form on prosthetic medical devices such as urine catheters and respirators inserted into the body, and they also form on containers of medical products (Figure 1). Confocal microscopy is the best microscopical technique for studying biofilm formation on surfaces. Biofilms are surfaced attached community of bacterial cells. The formation of biofilm in vivo or in ex-vivo occurs in a number of defined steps or processes including the formation of conditioning layers, bacterial adhesins, bacterial growth and finally bacterial community expansion. These processes of biofilm formation are highlighted in this section.

Figure 1. Possible sites of biofilm formation in the body.Biofilmsarea group of microorganisms in which microbial cells stick to each other over a particular surface including animate and inanimate surfaces. These microbial cells (adherent cells) adhere to these surfaces within a self-produced matrix of extracellular polymeric substance (EPS) – that are secreted by the organisms in the biofilm community.

Biofilms form when bacteria adhere to surfaces (biotic or abiotic) in aqueous environments and begin to excrete a slimy, glue-like substance that can permanently anchor them to all kinds of materials or surfaces. Bacterial biofilms can form in any aqueous environment including on the human teeth, rivers, sinks in the kitchen and hospital environments and even on bathtubs and sinks or washing bowls. As bacteria begin to settle upon a particular surface and on each other, biofilm formation is initiated. The formation of biofilm usually requires the corporation of not just one bacterium but that of many bacteria, and this may involve the direct cooperation of different types of bacteria through QS.

Biofilms usually occur when one bacterial species attaches specifically or non-specifically to a surface, and then secretes carbohydrate slimes (or exopolymers) that imbeds the bacteria and attracts other microbes to the biofilm for better protection and nutritional advantages. Some of the indigenous bacteria are able to construct biofilms on a tissue surface, or they are able to colonize a biofilm built by another bacterial species. Many biofilms are a mixture of microbes, although one member of the bacterial community is responsible for maintaining the biofilm and may predominate over the other members of the community. Biofilm formation is usually completed following few hours of their initial attachment to a suitable surface.

Then later, the biofilms formed undergo additional adaptations to their new surfaces; and these adaptations may include: (1) the mass production of EPS, (2) structural differentiation, (3) increased production of AIs to enhance communication, and finally (4) the development of resistance to the onslaught of antimicrobial agents. Bacterial cells become aware of their number in a given environment prior to the formation of biofilm through the amount of AIs released. The number of signaling molecules received by a bacterium usually depends on how many other bacterial cells are present and ready to form biofilms. These signaling molecules (e.g., AIs) released by bacterial cells helps them to effectively kick-start and co-ordinate the biofilm formation processes. As the signaling molecules increases and reaches a certain threshold (owing to the increased number of bacterial cells present), changes in bacterial gene regulations are triggered up and this culminates to the reorganization of cellular components in the bacterial cells to begin an effective biofilm formation process. Bacterial cells produce large enough EPS and pili for attachment to their surfaces, substrates and to each other.

Twitching motility is a process which allows bacterial cells to adjust their positions in a biofilm community using pili, a bacterial appendage. This adjustment by twitching motility allows bacterial cells in biofilms to come close to each other and move along surfaces effectively. Twitching motility helps bacterial cells to form microcolonies. After the formation of microcolonies, bacterial cells secrete slimy extracellular matrix that comprises nucleic acids, carbohydrates and proteins, and which helps in reinforcing the bacterial cells in a biofilm together. This type of reinforcement provides formidable support and it helps the bacterial cells in a biofilm to be protected against any external aggression such as the onslaught of antimicrobial agents. Finally, an interacting community of bacterial cells in a biofilm (which comprises extracellular polymers, signaling channels and complex bacterial colony) will be formed. Biofilms are formed on biotic and abiotic surfaces in different modes. The different patterns involved in the formation of biofilms by bacteria shall be elaborated in this section (Figure 2). 

Figure 2. Schematic illustration of the different stages of biofilm development. A= Adhesion of planktonic organisms or bacteria to moist surface. This stage is known as the initial attachment phase; B= formation of monolayer and production of slime which makes the surface slimy or oily. Attached cells becomes firm and cannot revert back to their original form at this stage; C= a community of microorganisms known as microcolony is formed, and this is usually accompanied by the development of multilayered cells on the attached surface. The bacterial cells in the community actively divides and, multiplies in size at this stage; D= mature biofilm community is formed. It is at this stage that the biofilm forms; E= the bacterial cells in the microcolony begins to detach from the community to form new biofilms, and this usually completes the life cycle of biofilm development by bacterial cells. This stage is generally known as the dispersion stage, and it is characterized by the flight of the individual organisms that form the biofilm to new habitats.

STAGES OF BIOFILM DEVELOPMENT

Biofilm formation in bacteria usually involves five (5) different stages as follows:

  1. INITIAL ATTACHMENT: This is the first stage of biofilm formation on surfaces (biotic or abiotic environment), and it is usually reversible after initiation. In this stage that usually takes splits of seconds, bacterial cells that are usually free-swimming rests on a specific surface and arrange themselves in clusters on the surfaces. They start producing chemicals and substances that promote their firm attachment to each other. The attachment of bacterial cells to surfaces is very critical to the development or formation of biofilms on either abiotic or biotic environments. Bacterial attachment to surfaces (especially abiotic environments) can be prevented by simply coating them with antimicrobial substances and/or chemical agents that block or disrupt the microbial arrangement or attachment of the cells. Such measures make the environment to be harsh for microbial population to settle and thrive successfully.
  • IRREVERSIBLE ATTACHMENT: Irreversible attachment is the next stage that follows bacterial attachment to surfaces; and this stage is usually characterized by the production of self-synthesized extracellular polymeric substance (EPS) by the bacterial community. It takes the bacterial cells some couple of minutes to establish this stage. EPS grow attached to the surfaces that bacterial cells have initially stuck to. A bridge-like structure or mesh is formed by the EPS, pili, fimbriae, and flagella of the bacterial cells in the community. Compounds that interfere with and disrupt the formation of EPS can be used to coat surfaces to prevent this stage from taking off.
  • MATURATION I: Growth and division of the bacterial population is the next stage that follows an initial and irreversible attachment of bacterial cells to surfaces and it takes hours and days to establish. In order to successfully form a biofilm, bacterial cells in a community must be able to proliferate and expand their population. Bacterial cells in community signal one another through the formation of signaling molecules, and this allows them to multiply and replicate in order to form a micro-colony of cells. This signaling between the bacterial cells is called quorum sensing – a course of action that allows bacterial cells to communicate and exchange vital information amongst each other especially in such a way that is beneficial to them. Maturation I is the foundation for the establishment of biofilm, but it can be disrupted by delivering signal blockers to threatened areas of the bacterial community in order to abort biofilm formation.  
  • MATURATION II: It is at this stage that biofilm actually forms. Maturation II which takes days and even months usually occur after the successful communication amongst bacterial cells to reproduce and form micro-colony of microorganisms. At this stage, the attachment of other microorganisms to the biofilm formed also occurs. This stage of biofilm formation is characterized by the co-existence of diverse species of microbial cells, and peak metabolic activities amongst the organisms. The use of multiple doses of antimicrobial agents is required to undermine the varied survival strategies of the biofilm cells formed in this stage.
  • DISPERSION: Dispersion is often the last stage that characterizes biofilm formation, and it is the phase at which some bacterial cells return to their initial free-living environment and escape in order to form new biofilms in different or newly found habitats. Several factors can spur the dispersion of microbial cells in a biofilm. Some of these factors include oxygen depletion, nutrient depletion and the accumulation of toxic substances in their environment. As the dispersion continues, microbial cells move to new areas where they combine to form biofilms. Biotic and abiotic surfaces where biofilms had initially formed should be properly swabbed and treated with antimicrobial agents to prevent future assemblage of microbial cells on such surfaces.

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

Gilligan P.H, Shapiro D.S and Miller M.B (2014). Cases in Medical Microbiology and Infectious Diseases. Third edition. American Society of Microbiology Press, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

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