Evaluating models and assessment techniques for understanding oral biofilm complexity

Abstract

Oral biofilms are three-dimensional (3D) complex entities initiating dental diseases and have been evaluated extensively in the scientific literature using several biofilm models and assessment techniques. The list of biofilm models and assessment techniques may overwhelm a novice biofilm researcher. This narrative review aims to summarize the existing literature on biofilm models and assessment techniques, providing additional information on selecting an appropriate model and corresponding assessment techniques, which may be useful as a guide to the beginner biofilm investigator and as a refresher to experienced researchers. The review addresses previously established 2D models, outlining their advantages and limitations based on the growth environment, availability of nutrients, and the number of bacterial species, while also exploring novel 3D biofilm models. The growth of biofilms on clinically relevant 3D models, particularly melt electrowritten fibrous scaffolds, is discussed with a specific focus that has not been previously reported. Relevant studies on validated oral microcosm models that have recently gaining prominence are summarized. The review analyses the advantages and limitations of biofilm assessment methods, including colony forming unit culture, crystal violet, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt assays, confocal microscopy, fluorescence in situ hybridization, scanning electron microscopy, quantitative polymerase chain reaction, and next-generation sequencing. The use of more complex models with advanced assessment methodologies, subject to the availability of equipment/facilities, may help in developing clinically relevant biofilms and answering appropriate research questions.

Keywords: assessment; biofilm; dynamic; polymicrobial; three-dimensional model.

Figure 1

Schematic of a typical biofilm in different stages: Biofilm formation occurs in several stages. Within the oral cavity, in the initial stage, salivary glycoproteins adsorb onto the tooth surface, resulting in the formation of an acquired pellicle. Subsequently, aerobic bacteria including cocci and bacilli adhere to the tooth surface and the attachment is reversible. Later facultative anaerobic bacteria attach to each other and the surface of the existing biofilm matrix. Finally, a mature polymicrobial biofilm forms typical mushroom‐shaped towers with pores and water channels.

Figure 2

Biofilm models can be classified as “in vitro” and “in vivo” models (based on the environment of biofilm growth), static and dynamic models (based on the flow of nutrients and waste products), monospecies biofilms, multispecies biofilms, and microcosm models (based on the microbiota in the biofilms) and into biofilms in two‐dimensional and three‐dimensional models (based on the type of substrates).

Figure 3

(a) Confocal image of a 3D melt electrowritten scaffold stained with SYTO 9 (calibration x: 2.47 µm, y: 2.47 µm, z: 15.00 mm). Biofilms were imaged using a confocal laser microscope (Nikon Eclipse Ti confocal Microscope) at 488 and 561 nm. These scaffolds can provide niches for bacteria to grow both in vertical and horizontal dimensions, additionally creating a 3D microenvironment. (b) Scanning electron microscope image of salivary biofilms grown in 3D melt electrowritten scaffolds. The bacteria are seen crisscrossing the fibers of the scaffolds. From data collected during research work towards the degree of Doctor of Philosophy, The School of Dentistry, The University of Queensland. Herston, Australia. https://doi.org/10.14264/b9fe031.

Figure 4

CDC biofilm reactor has eight polypropylene coupon holders (a), with each rod having three slots for placement of coupons (b). Biofilms grow on coupons that fit into slots within these rods (c). CDC, Centre for Disease Control.

Figure 5

Image of a setup for a CDC biofilm reactor infection model. Nutrient media is pumped through the inlet pump into the CDC biofilm reactor and an outlet pump removes waste media into the waste jars. CDC, Centre for Disease Control.

Figure 6

Various stains can be used to visualize biofilms using confocal microscopy and specific dyes/molecular probes. (a) A confocal microscopy image of a 5‐day‐old subgingival plaque microcosm treated with amoxicillin stained with SYTO 9 and propidium iodide (LIVE/DEAD^TM BacLight^TM Bacterial Viability Kit). Green fluorescence indicates live bacteria, whereas red fluorescence indicates dead bacteria. (b) A confocal image of a 10‐day‐old salivary microcosm biofilm grown on a hydroxyapatite disc in an anaerobic box at 37°C stained with SYTO 9 (bacteria stained green) and calcofluor‐white (extracellular matrix of the biofilms stained blue) (calibration x: 2.47 µm, y: 2.47 µm). Biofilms were imaged using a confocal laser microscope (Nikon Eclipse Ti confocal Microscope) at 488 and 561 nm. From data collected during research work towards the degree of Doctor of Philosophy, The School of Dentistry, The University of Queensland, Herston, Australia. https://doi.org/10.14264/b9fe031.

Figure 7

Scanning electron microscopic image of a 2‐day salivary microcosm biofilm on a two‐dimensional medical grade polycaprolactone film grown in a microtitre plate in an anaerobic box at 37°C. The inoculum used consisted of 85% brain heart infusion, 5% defibrinated sheep's blood, and 10% pooled saliva from six periodontally healthy volunteers. Cocci and rod‐shaped bacilli are present. From data collected during research work towards the degree of Doctor of Philosophy, The School of Dentistry, The University of Queensland, Herston, Australia. https://doi.org/10.14264/b9fe031.

Figure 8

A schematic image depicting the hierarchy and the most used levels in bacterial taxonomy. Following next‐generation sequencing, bacteria are presented as kingdom, phylum, class, order, family, genus, and species.

Figure 9

A representative Krona plot depicting the salivary microbiome following 16S rRNA gene sequencing. Unstimulated saliva was collected from six periodontally healthy individuals, and genomic DNA was isolated and subsequently 16S rRNA gene sequencing was carried out. Next‐generation sequencing is a culture‐independent method to qualitatively assess the microbiome. Sequencing provides information on cultivable and noncultivable bacterial species in a biofilm. From data collected during research work towards the degree of Doctor of Philosophy, The School of Dentistry, The University of Queensland, Herston, Australia. https://doi.org/10.14264/b9fe031.