Biofilms and microbiology
Phenotypic resistance and biofilms
It has long been recognised that the surface characteristics and physiology of bacterial cells are greatly influenced by the cellular growth rate, imposition of specific nutrient deprivations and adherence to surfaces. Over the years, the group has concentrated its attentions on phenotypic changes brought about through the growth conditions by developing the use of continuous culture techniques to model the in-vivo environment. Studies have concentrated on the relationships between cell-envelope permeability, envelope structure/immunogenicity and antibiotic/biocide resistance.
The group has developed techniques that allow the control of growth rate and nutrient availability within bacterial biofilms. These methods enable the biofilms to be perfused with antibiotics in a manner equivalent to that experienced by the cells during antibiotic therapy. The developed techniques offer the potential to evaluate and develop novel delivery systems for antibiotics, and of therapeutic regimes (pharmacokinetic profiling), associated with chronic infections of membrane surfaces and implanted devices. Such techniques enable the effects of antibiotics to be evaluated upon biofilm formation, stability and elimination. These approaches are currently being deployed to investigate not only current therapeutic practice for both single and mixed species infections, but also to optimise biocide use in industrial applications such as food and paper manufacturing.
Multiple Antibiotic Resistance operon
The Multiple Antibiotic Resistance operon (Mar) is chromosomal, and encodes for permease proteins (AcrB), which actively export a wide range of xenobiotics from bacterial cells. Mar is widely distributed. Recent reports show that Mar can be regulated not only by exposure to sub-MIC levels of antibiotic, but also through slow growth rate, the stringent response and a number of other unrelated stimuli. It is not regulated through homoserine lactone but does appear to be part of a global regulatory system that also controls exopolymer biosynthesis. The group is particularly interested in this operon since it is likely that this would be switched on in biofilms and might be a major factor in the high-level antibiotic resistance observed in biofilms.
Bacterial persisters
Bacterial persisters can be considered as small, randomly located pockets or sub-sets of a biofilm population, characterised by their resistance to a wide range of antimicrobial agents. Although the genomic expression of this sub-set is currently subject to much speculation, it is highly probably that persisters are quiescent cells that form in response to an environmental stress induced by conditions of extreme starvation. This general stress response (GSR) process can occur in biofilms that have an abundant supply of nutrients, since regions exist where nutrients are scarce or even absent. Under such conditions a small proportion of the cells present within a mature biofilm will be expressing the GSR regulator and will be relatively recalcitrant to inimical agents.
Over the past 50 years the biocide literature has been punctuated with reports of low-level persistent survival of antimicrobial treatments (tailing) where the agent has not been quenched and where survivors do not demonstrate resistance when re-cultured or cloned. Recent evidence suggests that such cells, rather than being resistant to the agent, are defective in mechanisms of programmed (apoptotic) cell death. Following removal of an inimical stress, these damaged persisters would grow rapidly in the presence of nutrients released from their lysed community partners and the community would become restored. The group is interested in gene expression in the persister population, drawing comparisons to the bulk biofilm population and to planktonically grown cells as a means of understanding biofilm recalcitrance.
The biofilm matrix
The extracellular matrix is a complex and extremely important component of all biofilms, providing architectural structure and mechanical stability to the attached population. It is composed of cells, water and secreted/released extracellular macromolecules. In addition, a range of enzymic and regulatory activities can be found within the matrix.
The different components and activities found within the matrix are likely to interact, thereby creating a series of local environments that can co-exist as a functional consortium. In addition, the matrix is also subject to a number of extrinsic, environmental factors. Together, these intrinsic and extrinsic factors combine to produce a dynamic, heterogeneous environment for the attached and enveloped cells.
As such, in many instances the persistant and problematic nature of biofilms is attributed to the surrounding matrix. It is somewhat surprising that for such a ubiquitous and important occurrence, relatively little is known about the structure, formation and properties of the biofilm matrix. Some of the unanswered questions of interest to Dr David Allison are "What are the 'key' polymers involved in maintaining matrix structure? Which cells produce the polysaccharide skeleton? For how long do such cells secrete matrix materials? Are homoserine lactones involved in matrix production? Which cells are active in changing matrix composition and structure?
Antimicrobial agents
Mechanisms: Study of the action and resistance mechanisms for non-antibiotic antibacterial agents has concentrated upon agents employed in medicine as antiseptics and in the general environment as industrial biocides. The agents studied have had actions either at the cell envelope or with essential thiols associated with the cell envelope and cytosol. Such study has elucidated molecular bases for the actions at the cell envelope of bisbiguanides (alexidine, chlorhexidine, polyhexamethylene biguanides), monophenylethers (phenoxyethanol) and quaternary ammonium compounds (cetrimide, benzalkoniums) and have demonstrated biochemical bases for the activities and associated mammalian cell toxicities of thiol interactive agents (bronopol, isothiazolones). The group's work has enabled the design and synthesis of novel antimicrobial substances that potentially circumvent normal resistance mechanisms and minimise toxicity.
Tolerance development
Work actively under study involves evaluation and design of antimicrobial systems for combating biofilms associated with not only infected medical implants but also industrial plant and water supplies. Such research is evaluating patterns of tolerance development in the biofilm mode of growth, and in response to chronic exposure to sub-effective levels of such agents (i.e. triclosan, quats, hypochlorite). The results of such research are likely to influence use-patterns for such biocides and might have implications in the trend of increased antibiotic resistance.