Biofilm models for the testing of antimicrobial-releasing materials. Dr Jonathan Pratten Department of Microbial Diseases UCL Eastman Dental Institute

Types of models In vivo In vitro In silico (Mathematical) 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 We can control it!

Biofilm modelExamples of organisms testedCultureSubstratumAdvantages Annular ReactorSulfate-reducing bacteria, Pseudomonas aeruginosa Continuous culture VariousVersatile surface shear, ASTM Standard Method CDC Biofilm ReactorGram-negative bacteriaContinuous culture Plastic connectorsHigh 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 VariousLongitudinal studies, intermittent pulsing of the antimicrobial or antibiotic Flow-cell (Flow- chamber) streptococci, Pseudomonas alcaligenes Continuous or batch culture VariousDirect visualisation MBEC Assay™P. aeruginosa, Staphylococcus aureus, E. coli, enterococci Batch culturePlastic pegsHigh throughput and simultaneous MIC Membrane filtersEnterococcus faecalis, P. aeruginosa, Escherichia coli Solid mediumMembrane filter on agar Simple to grow, high biofilm mass Microtiter plate assayS. aureus, Burkholderia cepaciaBatch culturePlasticHigh throughput, use in standard spectrophotometers Modified Robbins device Porphyromonas gingivalis, P. aeruginosa Continuous or batch culture VariousFlow device with removable coupons for antimicrobial testing Sorbarod filterS. aureus, P. aeruginosa, multi-species oral biofilms Continuous culture Filter plugLarge 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 : “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 steady- state system Li, 2000 & 2001

direct visualization of attachment Stoodley 1997 onwards The Flow Cell

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 quasi- steady-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 PTFE pan area of biofilm growth turntable scraper blade medium inlet disk of material PTFE plug air inlet drive shaft QVF glass housing effluent

peristaltic pump inoculum vessel single or mixed species CDFF 37°C effluent air outlet

37°C medium reservoir waste medium container air outlet grow back trap peristaltic pump CDFF

position of main medium inlet PTFE pan plug direction of disc revolution QVF glass cylinder PTFE angled scraper blade position of silicone rubber ring stainless steel disc

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: