The Biology of Biofilm Growth
Fundamentals to Prevention
Background on Biofilms
Biofilms are a successful long-term survival strategy employed by bacteria in the environment in the presence of hostile conditions and antibiotic treatment. More specifically, they can be defined as a layer of cells covered in polysaccharides that grow and spread on the skin, or other surfaces, causing infection. Such layers of extracellular polymer matrices provide various advantages for biofilm growth.
Biofilms are the root cause of many human infections. In fact, 80% of human infections are caused by pathogenic biofilms. Such infections include, but are not limited to, dental plaque, urinary tract infections, cystic fibrosis, otitis media, infective endocarditis, and tonsillitis. These fall under categories that will be discussed further later on. However, biofilm formation not only occurs in humans, but on medical devices as well. The biofilms that form on these implants and devices grow in non-sterile and unsanitary environments where bacteria can be present in the air.
Regarding biofilm formation itself at a cellular level, there are 5 crucial steps:
- Adherence: the bacteria migrate and adhere to a surface; biofilm formation begins with an initial coating of small exopolymeric material
- Early Attachment: the bacteria secrete extracellular polymeric substance and stick to the surface; leads to polymeric matrix production which promotes microbial colonization and cell clustering
- Young Biofilm: biofilms work towards full development through the formation microcolonies, water channel structures, and become heavily layered
- Mature Biofilm: fully mature biofilms start to reach maximum density and begin to function as 3D communities
- Dispersal: mature biofilms send bacterial microcolonies (process called dispersion) to different areas to spread the infection through signal transduction pathways. Antibacterial medication cannot easily penetrate the matrix of these biofilms
There are a number of bacterial factors that assist biofilm formation known as extrapolymeric substances. For instance, the polysaccharides, that every bacterial species is composed of, provide mechanical stability and the ability to spread and attach to surfaces in the early stages of formation. Additionally, the EPS also contains specific cell-surface-associated proteins and extracellular carbohydrate-binding proteins that play significant roles. Also, eDNA provides significant structural integrity, serves as an adhesive (attach to surfaces) molecule, as well as an intercellular (between cells) connector. Adhesins, which are present at the tips of extracellular appendages (pili, flagella, and fimbriae), are secreted by the bacteria and determine if the bacteria can recognize and bind to surfaces and other cells. Essentially, the higher the production of EPS in the bacteria, the more effective its biofilm formation is. The biofilm matrix at the end of formation helps cocoon the bacteria with the help of these exopolysaccharides, proteins and extracellular DNA (eDNA).
As mentioned previously, biofilm infections (involving humans or devices) can be categorized into different types of infections since biofilm growth occurs differently depending on the context.
Regarding burns, gram-positive bacteria (i.e. Staphylococci) colonize the surface of the wound and are resistant to thermal damage. Further contamination within burn wounds could lead to more serious disease states, such as sepsis. Burn wounds are especially difficult to penetrate using antimicrobial drugs.
The oral cavity is the perfect environment for biofilm growth as it provides warm temperatures, high humidity, and rich nutrients. Pathogenic oral biofilms arise because of complications between microorganisms, host, and diet. Oral biofilms form on the surface of tooth, soft tissue, tongue, gingival groove, palates, cheeks, and tonsils. Oral biofilms especially have a unique ability to deal with difficulties that no other microbiome has encountered due to frequent contact with external environment. Oral biofilm formation depends on the host’s diet (if it is sugar-rich) which boosts the aggregation of extracellular polymeric substances (EPS) that leads to highly resistant microbiota.
The next most common surface that supports biofilm formation involves medical devices and implants, as mentioned earlier. In medical settings, there is a much greater chance of biofilm formation on medical devices especially in unsanitized conditions. Such specialized biofilms can form on catheters, heart valves, pacemakers, artificial joints, voice prostheses, and contact lenses. For successful growth, bacteria rely on the type of device, the duration that it is implemented, as well as the level of nutrition the environment provides. For example, biofilms that form on cathers inhabit specific locations on the device depending on the duration of the insertion. If the duration is shorter, they form on the outer surface but form on the inner lumen/surface if the duration is longer.
Relevance to Pseudomonas Aeruginosa
This is an introduction to some of my research since the main focus of my project is on the effectiveness of quorum sensing inhibition on the P. Aeruginosa biofilm formation. Here, I will specifically talk about quorum sensing which is the primary mechanism that P. Aeruginosa biofilms employ. In 2017, P. Aeruginosa was recognized as one of the most life-threatening bacteria according to the World Health Organization.
P. aeruginosa uses multiple interconnected signal transduction pathways known as quorum sensing. This allows bacteria to communicate between the individual cells which is enabled by changes in cell density and environmental cues. More specifically, quorum sensing involves the production, secretion, and accumulation of autoinducers (signalling molecules), virulence factors, and swarming motility.
Firstly, autoinducers are a significant signalling molecules that are vital to the success of quorum sensing. Autoinducers are sensed by transcriptional regulators which lead to gene expression. AHLs (small diffusible signal molecules) that are produced by the bacteria bind to and activite transcriptional regulators. Pseudomonas Aeruginosa has two AHL-based qourum sensing systems: the lasIR and rhlIR systems. The lasIR system contains a lasI gene that encodes the autoinducer, LasI, which synthesizes C12-HSL, a molecule that binds to the LasR reception. The lasIR system essentially activates the expression of genes that produce virulence factors. The rhlIR systsem produces the autoinducer rhlIR that encodes the enzyme RHlI. This produces the signaling molecule C4-HSL that binds to the RhlR receptor, activating certain target genes that produce certain virulence factors.
Furthermore, virulence factors have been consistently coming up in the discussions above. Virulence factors help the bacteria invade the host, cause disease, and evade host defenses. They can be categorized as surface appendages, outer membrane components, secretion system, exopolysaccharides, siderophores, proteases, and toxins. All of these are vital in the formation of a biofilm as they provide specific aspects
Lastly, swarming motility is specific coordinated behavior employed by bacteria within a biofilm involving the movement and collection of bacterial cells across surfaces. Such behavior is facilitated by flagella and other surface-associated appendages. Because swarming motility leads to an increased amount of virulence traits in bacteria, it to further inheritance of more cells that contribute to a stronger and more protected environment, making it harder for antibiotics to penetrate the biofilm.
Pseudomonas Aeruginosa is one of the many bacterial strains that is considerably dangerous to humans. My research specifically focuses on this bacterial strain in order to determine the most effective compound that best prevents biofilm formation. More on this is provided below.
Prevention Techniques
As of now, the discussion has largely consisted of the factors behind biofilm formation. However, it is just as important to discuss the importance of detection and destruction of biofilms.
There are three main types of therapy (destruction methods): mechanical destruction, the use of chemicals, bacteriophage therapy, and antibiotic therapy (drug delivery). Biofilms are significantly resistant to antibiotic therapy because of its complex structure composed of an aggregate of EPS. This resistance to antibiotic therapy makes it just as important to make improvements and develop newer techniques to overcome these difficulties.
Many scientists establish biofilm models for research and experimentation because they allow scientists to form a biofilm and test compounds against them in a lab setting. The model that most scientists use to test this is called the microtitration-based model using 96-well plates. More specifically, scientists use this method to grow a layer of biofilms of cultured bacteria on the bottom and sides of the wells in the 96-well plate (shown below). Scientists then establish another plate with a grown biofilm but with the addition of antimicrobial compounds. Using a device called the microplate reader, scientists can collect data that represents the number of cells that are present in each plate. This allows them to determine the effectiveness of compounds against the biofilm by comparing the number of cells (Optical Density) in the plate with just the cells versus the plate with cells treated with compounds.
Antimicrobial compounds can come in various forms including quorum sensing inhibitors, enzymes, metal chelators, and antimicrobial peptides. Each of these have specific abilities that target and inhibit certain aspects of the biofilm. For example, quorum sensing inhibitors essentially inhibit the quorum sensing mechanism (consisting of communication, spreading, attachment, and growth) employed by biofilms. Delivery of such compounds into biofilms requires precision because the EPS in biofilms act as a physical barrier that generally make it quite difficult for such compounds to cross through.
Lastly, bacteriophage therapy is another quite interesting yet effective method of biofilm destruction. It essentially involves the use of viruses to infect and replicate within the bacterial cell in order to lyse (break apart the host cell). Phages are quite effective because they employ specificity which is the ability to only attach to specific organisms while ignoring any others.
Conclusion
Biofilms require more attention in the scientific community as the consequential infections can be quite deadly to the vulnerable population. This discussion of biofilms not only helps to raise awareness but to promote further research looking into effective techniques that improve current therapies.