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Biofilm models for the testing of antimicrobial-releasing materials.
Dr Jonathan PrattenDepartment of Microbial DiseasesUCL Eastman Dental Institute
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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!
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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 (Flow- chamber)
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
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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
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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”.
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Annular reactor (formerly the Rototorque)
• produces biofilms under shear conditions in a steady-state system
Li, 2000 & 2001
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• direct visualization of attachmentStoodley 1997 onwards
The Flow Cell
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Flow cell – direct visualization of attachment
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• 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
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Modified Robbins Device
Larsen & Fiehn
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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
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sampling port
PTFE pan
area of biofilm growth
turntable
scraper blade
medium inlet
disk of material
PTFE plug
air inlet
drive shaftQVF glass housing
effluent
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peristaltic pump
inoculum vessel• single or mixed species
CDFF
37°C
effluent
air outlet
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37°C
medium reservoirwastemediumcontainer
air outlet air outlet
grow back trap
peristaltic pump
CDFF
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position of mainmedium inlet
PTFE pan
PTFE panplug
direction of disc revolution
QVF glass cylinder
PTFE angled scraper blade
position of silicone rubber ring
stainless steel disc
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• 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)
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Endodontics / microleakage
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x10 x1000
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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: