CHAPTER 1.................................................................................................................................. 1
1.4 Quorum sensing inhibition and Pseudomonas aeruginosa
1.4.3 Pseudomonas aeruginosa biofilm production
Biofilms were proposed to be ubiquitous by Costerton and colleagues in 1978, and have since been shown to be ubiquitous in the environment, industrial, engineering, and medical fields (Lee and Yoon, 2017). Many chronic medical conditions can also be attributed to biofilm formation, and biofilm formation on medical devices are untreatable and necessitate the removal of the device (Kim and Lee, 2016).
Biofilms are classified as a sessile mode of growth, until the cells actively detach and once again become planktonic and disperse to infect new environments (Kim and Lee, 2016).
A biofilm can be defined as a complex community of microorganisms attached to a surface and embedded in a self-produced extracellular matrix (ECM) comprised of extracellular polymeric substances (EPS) in the form of extracellular DNA, proteins, dead cells, RNA, lipids, polysaccharides (Kim and Lee, 2016; Zhang and Li, 2016; Lee and Yoon, 2017) and various other biomolecules which give the biofilm a strain-specific phenotype (Rasamiravaka et al., 2015). Biofilms, with their diffusion-limiting ECM have been linked to 65-80% of all microbial infections (Rasamiravaka et al., 2015). The EPS is interspersed with interstitial voids and water channels, allowing for the transportation of water and the diffusion of oxygen into the biofilm and the creation of an oxygen and water gradients (Kim and Lee, 2016; Lee and Yoon, 2017).
The ECM, therefore, keeps the cells attached to the surface (Kim and Lee, 2016) and affords the microorganisms protection from harsh environmental factors, including antimicrobial or
28
disinfection agents, resulting in biofilm-encased cells having a 1000-fold higher resistance to these substances than their planktonic counterparts (Lee and Yoon, 2017).
Biofilm resistance is due to several biofilm-specific bacterial adaptations not available to planktonic cells. These include the ECM which retards the entrance of antimicrobial compounds into the biofilm, thereby acting as a physical barrier of protection, especially to those cells lower down in the matrix (Kim and Lee, 2016; Lee and Yoon, 2017). Within the biofilm also lies a subpopulation of cells termed persister cells which reproduce at a highly reduced rate, or in some cases altogether (Lee and Yoon, 2017). This reduction in respiration affords the cells protection against many classes of antibiotics (Lee and Yoon, 2017). Biofilm- embedded cells have additionally been shown to have increased efflux pump production, further adding to the resistance of these cells (Lee and Yoon, 2017).
Being in a biofilm and therefore attached to a surface also ensures that external forces do not away sweep the encased cells such as those present in a stream or drain (Kim and Lee, 2016). The development of biofilms can be broken down into several stages the process of which are elucidated in Fig. 1.5. These are initial attachment, irreversible attachment, maturation, and detachment (Kim and Lee, 2016; Zhang and Li, 2016). During initial attachment, planktonic cells adhere to a surface (Kim and Lee, 2016). Initial attachment involves many different components, such as hydrophobic interactions and Van der Waals forces, lipopolysaccharides, type IV pili and adhesins (Castillo-Juárez et al., 2017; Lee and Yoon, 2017). During irreversible attachment, microcolonies are formed through subsequent cell division (Kim and Lee, 2016). These microcolonies form a single layer on the surface, using both flagella and pili for attachment (Kim and Lee, 2016; Castillo-Juárez et al., 2017). The attachment become irreversible once microcolonies have been formed and established, maturing through the production of EPS, and developing a highly ordered structure (Kim and Lee, 2016). The release of extracellular DNA in Pseudomonas biofilm formation is mediated by PQS and, in combination with type IV pili and flagella, facilitates the initial and irreversible attachment of the cells to a surface, as well as serving as a cell-to-cell interconnecting compound and nutrient source (Rasamiravaka et al., 2015). During this stage, the cells also undergo complex physiological changes, becoming increasingly resistant to antimicrobials and other stressors (Lee and Yoon, 2017).
29
Figure 1.5: Biofilm formation by Pseudomonas aeruginosa. In Stage I, cells reversibly attach to a surface. This attachment becomes irreversible in Stage II, shortly followed by the production of an extracellular matrix as well as microcolonies in Stage III. The biofilm can be seen to be maturing in Stage IV and lastly planktonic cells will disperse from the biofilm in Stage V to colonize new surfaces (Rasamiravaka et al., 2015).
During the maturation stages, the self-produced EPS begins to form a mushroom shaped, highly porous structure. This structure is supplied with nutrients and oxygen through channels within the ECM, allowing for the growth of the cells within it (Kim and Lee, 2016).
The fully developed biofilms can then be maintained if the environment remains suitable, after which cells will detach to become planktonic and colonize new niches (Kim and Lee, 2016). Detachment can either occur through passive, shear related processed (termed sloughing for losses of large biofilm mass or erosion for small losses) or through an active process termed seeding (Kim and Lee, 2016; (Lee and Yoon, 2017). During seeding, the biofilm releases single planktonic cells or small microcolonies from the centre of the biofilm where the motile cells are located, thereby leaving cavities behind surrounding by the external, non- motile cells (Lee and Yoon, 2017).
Pseudomonas aeruginosa has been described as a model organism for biofilm study, as the protein expression of this organism differed as much as 35% when in a biofilm as compared to planktonic growing cells (Kim and Lee, 2016). Cells of P. aeruginosa within a biofilm undergo spatial phenotypic differentiation, where the cells in the centre of the biofilm remain motile and those on the outskirts do not. The non-motile cells, therefore, form the
30
stalks of the biofilms during maturation, whilst the motile cells climb the stalks using type IV pili-mediated twitching motility, assisted by rhamnolipid production via the rhl QS system (Lee and Yoon, 2017), and aggregate at the top as the mushroom caps (Kim and Lee, 2016).
These motile cells then swim away during active seeding dispersal if the surrounding environment is no longer suitable (Kim and Lee, 2016).
Cells within the P. aeruginosa biofilm are afforded antimicrobial resistance as oxygen only penetrates 50–90 μm from the air interface, thereby reducing the metabolic activity of the cells (Kim and Lee, 2016). For the aerobic cells located in the upper layer of the biofilm, over 50% of the required oxygen is supplied through interstitial voids within the matrix (Kim and Lee, 2016). Because these microorganisms exhibit social or group behaviour (Kim and Lee, 2016), biofilm formation can, therefore, be associated AHL-mediated QS in many Gram- negative organisms, including P. aeruginosa (Banerjee and Ray, 2016; Rekha et al., 2016; Lee and Yoon, 2017). A variety of autoinducers, including AHLs with carbon chains of between four and 14 carbons in length and the AI-2 autoinducer, are present in sites contaminated by biofilms, further cementing the link between QS and biofilm formation (Zhang and Li, 2016).
The las QS system regulates biofilm formation in P. aeruginosa through control of the pel operon, responsible for producing the glucose-rich ECM associated with these biofilms. This thick, protective EPS layer allows P. aeruginosa to outcompete other colonies by securing a nutrient supply (Zhang and Li, 2016).
There are three major components of the EPS which have been identified in P.
aeruginosa biofilms. These are Psl (polysaccharide synthase locus), Pel and alginate (Lee and Yoon, 2017). The Psl component initiates and maintains the biofilm by providing intercellular reactions and surface attachment, as well as interacting with eDNA to provide structural support (Lee and Yoon, 2017). Equally important is Pel, responsible for providing protection against aminoglycoside antibiotics and forming pellicles at the air liquid interface (Lee and Yoon, 2017). Alginate, however, is the most studied EPS component; retaining water and nutrients, providing immune evasion and antibiotic resistance, and differentiating non- mucoid from the alginate rich mucoid biofilm phenotypes in the lungs of CF patients (Lee and Yoon, 2017). The release of eDNA is also mediated through QS-controlled quinolone production (Castillo-Juárez et al., 2017). The PQS system of P. aeruginosa mediates the release of quinolone 2-N-heptyl-4-hydroxyquinoline-N-oxide (HQNO), which then promoted the production of reactive oxygen species, damaging cell membranes and causing cell lysis,
31
allowing for the release of eDNA into the biofilm matrix (Castillo-Juárez et al., 2017).
Pseudomonas aeruginosa is especially virulent when found in biofilms (Singh and Reddy, 2014) and treatment of infections by this organism has been shown to be most effective if achieved prior to biofilm formation (Gellatly and Robert, 2013).