Biofilm models for the testing of antimicrobial-releasing materials.

Dr Jonathan PrattenDepartment of Microbial DiseasesUCL 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 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

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 steady-state system

Li, 2000 & 2001

• direct visualization of attachmentStoodley 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 shaftQVF glass housing

effluent

peristaltic pump

inoculum vessel• single or mixed species

CDFF

37°C

effluent

air outlet

37°C

medium reservoirwastemediumcontainer

air outlet air outlet

grow back trap

peristaltic pump

CDFF

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

• 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: