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Biofilm models for the testing of antimicrobial-releasing materials. Dr Jonathan Pratten Department of Microbial Diseases UCL Eastman Dental Institute A model is a pattern, plan, representation (especially in miniature), or description designed to show the main object or workings of an object, system, or concept. i.e. a simplified system that illustrates or exhibits the same behavior as the more complex, general system Types of models • In vivo • In vitro • In silico (Mathematical) We can control it! Biofilm model Examples of organisms tested Culture Substratum Advantages Annular Reactor Sulfate-reducing bacteria, Pseudomonas aeruginosa Continuous culture Various Versatile surface shear, ASTM Standard Method CDC Biofilm Reactor Gram-negative bacteria Continuous culture Plastic connectors High shear, removable coupons, ASTM Standard Method Constant Depth Film Fermentor (CDFF) Listeria spp., Pseudomonas spp., Staphylococcus spp., P. aeruginosa, multi-species oral biofilms Continuous culture Various Longitudinal studies, intermittent pulsing of the antimicrobial or antibiotic Flow-cell (Flowchamber) streptococci, Pseudomonas alcaligenes Continuous or batch culture Various Direct visualisation MBEC Assay™ P. aeruginosa, Staphylococcus aureus, E. coli, enterococci Batch culture Plastic pegs High throughput and simultaneous MIC Membrane filters Enterococcus faecalis, P. aeruginosa, Escherichia coli Solid medium Membrane filter on agar Simple to grow, high biofilm mass Microtiter plate assay S. aureus, Burkholderia cepacia Batch culture Plastic High throughput, use in standard spectrophotometers Modified Robbins device Porphyromonas gingivalis, P. aeruginosa Continuous or batch culture Various Flow device with removable coupons for antimicrobial testing Sorbarod filter S. aureus, P. aeruginosa, multi-species oral biofilms Continuous culture Filter plug Large biofilm mass, a number of replicates Chemostats • Bradshaw and Marsh 1996 – 2002 • hydroxyapatite disks are immersed in the chemostat for known time periods • oral biofilms of 10 species • advantage of being able to investigate planktonic and biofilm modes of growth within the same system •the large fluid phase is not consistent with the in vivo situation where shear forces and a thin film of fluid are present on the oral surfaces The CDC Biofilm Reactor was developed to provide consistent biofilm samples and growth conditions for evaluation of antimicrobial agents, surface treatments and materials. As described in ASTM 2562-07: “Standard Test Method for Quantification of Pseudomonas aeruginosa Grown with High Shear and Continuous Flow using a CDC Biofilm Reactor”. Annular reactor (formerly the Rototorque) • produces biofilms under shear conditions in a steadystate system Li, 2000 & 2001 The Flow Cell • direct visualization of attachment Stoodley 1997 onwards Flow cell – direct visualization of attachment • • • • • good for direct visualisation limited by the requirement to use a transparent surface and also limited by the thickness of biofilm or time or…use fluorescent viability stains or…remove biofilms for visualisation Modified Robbins Device Larsen & Fiehn Constant Depth Film Fermentor • The advantage of generating a constant depth biofilm is that it is possible to enter a long-term quasisteady-state where some properties of the film do not change significantly with time. •The Constant Depth Film Fermentor was first described by Coombe et al. (1982) to investigate the growth of dental plaque organisms and further developed by Peters and Wimpenny (1988). • Once a steady state has been achieved, perturbing the system becomes easy and unequivocal results can be obtained sampling port air inlet medium inlet area of biofilm growth scraper blade PTFE pan disk of material PTFE plug turntable QVF glass housing drive shaft effluent CDFF peristaltic pump effluent air outlet inoculum vessel • single or mixed species 37°C grow back trap peristaltic pump CDFF 37°C air outlet waste medium container air outlet medium reservoir PTFE angled scraper blade position of main medium inlet position of silicone rubber ring PTFE pan PTFE pan plug stainless steel disc QVF glass cylinder direction of disc revolution • As well as being used for the study of bacterial perturbation (Pratten et al., 1998; Valappil et al., 2008) the CDFF has been used in studies evaluating: – Characterise ecological shifts associated with dental health and disease (Dalwai et al., 2006) – endodontic microleakage (Matharu et al., 2001) – oral malodour generation (Pratten et al., 2003) – the corrosion potential of dental plaque (Wilson et al., 1995) – gene transfer in oral biofilms (Roberts et al., 2001) Endodontics / microleakage x10 x1000 thanks to…. • Everyone at UCL Eastman Dental Institute, especially – Prof Mike Wilson – Dr David Spratt – Dr Derren Ready – Dr Sean Nair – Dr Anne Young • Chemistry Department, UCL • Funding bodies:
