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Dispersal of P. aeruginosa biofilm
Mature biofilms of P. aeruginosa exhibit a distinct and particularly dramatic form of seeding dispersal, accounting for about 50 to 80% of the detached biofilm95,102. The process is characterized by evacuation of numerous motile single cells from clusters contained in voids at the interior of microcolonies. The cells at the periphery of microcolonies appear to become motile and highly agitated as a prelude to dispersal, after which, they escape through local ‘breakout’ points at random locations in the outer walls of the colonies94. After seeding, the emptied voids do not replenish themselves with new cells nor do they exhibit further growth in size95. Only a small proportion of detached cell clusters, about 1 to 3%, consist of aggregates with over 10 cells102. Detachment from the substratum is mediated by flagella and pili which interact in a well-coordinated sequence to change the orientation from horizontal to vertical and then detach; vertical orientation appears to facilitate surface detachment55. P. aeruginosa also disperses by migrating over the substratum. Surface translocation of bacilli to new sites, away from their inoculation point, is achieved through twitching, swimming and swarming motility54,55,67.
The innate mechanisms that regulate dispersal in P. aeruginosa biofilm are still unclear and the study of these mechanisms is ongoing. Research has unveiled numerous interrelated factors that are associated with biofilm dispersal which include EPS matrix degrading enzymes, changes in the intracellular levels of cyclic diguanosine monophosphate (c-di-GMP), activation of genes responsible for cell motility, lytic bacteriophages, changes in nutrient levels and changes in microbial growth status7.
In vitro studies suggest that the matrix-degrading enzymes are involved in active biofilm dispersal118. Mucoid strains of P. aeruginosa produce alginate polysaccharide and alginate lyase, an enzyme that degrades alginate64,118. Alginate is important in that it promotes surface attachment, contributes to the structure of the biofilm EPS matrix and is required to efficiently retain the bacteria with the biofilm58. When expressed by the alginate lyase gene (algL), the enzyme causes enzymatic degradation of alginate which disrupts the EPS structure and induces biofilm dispersal through sloughing of cells118.
The intracellular nucleotide, c-di-GMP, is a ubiquitous second messenger found in many bacterial species, where it is known to influence a wide range of physiological processes119,120. Intracellular c-di-GMP levels may be increased or decreased by the antagonistic activities of diguanylyl cyclases and phosphodiesterases, respectively121. It has been shown that these changes in c-di-GMP levels influence bacterial choice of lifestyle in P. aeruginosa; accumulation promotes biofilm formation and sessile mode, while a decrease favours a planktonic existence and biofilm dispersal119,120. Much attention has been drawn to the involvement of secreted quorum-sensing signals and a central biofilm-regulating role has been ascribed to c-di-GMP95,120,122. Elevated levels of c-di-GMP stimulate production of EPS and adhesins, reduce cell motility, up-regulate virulence factors and induce biofilm formation. Low levels of c-di-GMP have the opposite effect and trigger biofilm dispersal (Figure 6)
CHAPTER 1: INTRODUCTION
CHAPTER 2: LITERATURE REVIEW
2.1Early studies on surface-bound (benthic) bacteria
2.2The biofilm
2.3The biofilm theory of disease
2.4Biofilm life cycle and structure
2.5The chemical composition of bacterial exopolymeric substance (EPS)
2.6The functions of the exopolymeric substance
2.7Bacterial resistance to antimicrobial agents
2.8Antimicrobial resistance and tolerance in bacterial biofilms
2.9Biofilm infections in clinical practice
2.10Management of biofilms in clinical practice and clinical research
2.11Anti-biofilm actions of EPS matrix degrading enzymes
2.12Rationale for the study of fungal-derived commercial enzymes
2.13Aim
2.14Objectives
CHAPTER 3: MATERIALS AND METHODS
3.1Research and ethics approval
3.3In vitro cytotoxicity testing of Pectinex on human cell cultures and isolated cells
3.3.1HeLa cell growth and Pectinex microtitre challenge
3.3.2Isolation of lymphocytes and neutrophils and Pectinex microtitre challenge
3.4Examination of human cell cultures by polarization-optical transmitted light differential interference contrast microscopy (PlasDIC) after exposure to Pectinex
3.5Scanning electron microscopy (SEM) of bacterial biofilms after exposure to antibiotics and Pectinex
3.6Statistical analyses and presentation of data
CHAPTER 4: RESULTS
4.1Growth and viability of planktonic bacteria
4.2Effect of Pectinex, amoxicillin-clavulanate and ciprofloxacin on biofilm formation, growth and removal
4.2.1Biofilm biomass and viability after 6 h incubation of bacterial cultures with Pectinex and antibiotics
4.2.2Biofilm biomass and viability after 24 h incubation of bacterial cultures with Pectinex and antibiotics
4.2.3Biofilm biomass and viability after 18 h incubation of 6 h-old bacterial cultures with Pectinex and antibiotics
4.2.4Biofilm biomass and viability after 24 h incubation of 24 h-old bacterial cultures with Pectinex and antibiotics
4.3In vitro cytotoxicity assay of Pectinex on HeLa cells, lymphocytes and neutrophils
4.4PlasDIC microscopy of human cell cultures
4.5SEM of bacterial biofilm
CHAPTER 5: DISCUSSION
CHAPTER 6: CONCLUSION
