An in vitro biofilm model of subgingival plaque

Over 100 years ago Robert Koch made one of the most important conceptual breakthroughs in microbiology with the discovery of methods for the production of solid nutrient media and the ability to isolate microorganisms in pure culture. Since this development, the training of microbiologists and the study of microbiology have been based, to a significant degree, on the elucidation of the properties of a microorganism cultivated in a pure culture. Although it has long been acknowledged that pure cultures of bacteria are virtually absent in nature, it has been only in the past few years that the biofilm-mode of growth has been recognized as the default state for most bacteria. It has become accepted that biofilm-grown bacteria express different phenotypes and often exhibit totally different characteristics than do the same bacteria grown planktonically. Bacteria that are sessile (attached to a surface) express different genes and, so, behave differently from free-floating or planktonic bacteria. Notable among these differences is the increased resistance to antimicrobial agents that can be 100- to 1000-fold greater for a species in a mature biofilm relative to that same species grown planktonically. Most common infections of the oral cavity, e.g. caries, gingivitis and periodontitis, are the result of the accumulation of biofilms. However, treatment and control of these diseases has been based on the in vitro study of pure cultures and their response to various antimicrobial agents. Thus, to better control such infections, it is imperative to understand oral biofilm formation and maturation. Various supragingival plaque biofilm models have been employed for the study of plaque formation, structure and antimicrobial susceptibility. Guggenheim has described a defined multi-species model designed to mimic the composition of the supragingival plaque and has used this model to study structure and antimicrobial susceptibility (9–11). Several investigators have utilized in-mouth splints in healthy subjects in which supragingival plaque formed over time on the splints (2, 45, 46). Wimpenny (44) has described several different laboratory biofilm models that make use of a constant depth film fermenter using a plaque inoculum. The constant depth film fermenter models have been used to study the structure (27, 29, 45) and spatial distribution (2, 15) of viable and non-viable supragingival plaque bacteria. Attempts to obtain realistic subgingival plaque biofilms have been made by placing various insert materials into the periodontal pockets of periodontitis patients and then analysing the bacterial components that colonized the inserts (37, 42). Most recently, Hope and Wilson have described the development of subgingival plaque on hydroxyapatite disks in a constant depth film fermenter (16). This model used a plaque inoculum and reached a steady state after 4 days. Although this is an excellent model for the study of subgingival plaque structure and viability, the apparatus for maintaining an anaerobic constant depth film fermenter is somewhat complex. Although all of the above models have greatly increased our understanding of plaque formation and development, none have directly addressed the question of how subgingival plaque matures over time and the sequence of events that leads up to a steady-state or climax biofilm. For our studies, we needed a simple, inexpensive model that was reproducible and that mimicked the in vivo composition of the subgingival plaque. One requirement was that a sufficient period needed to exist before the establishment of a climax biofilm so that the sequel of colonization could be reasonably followed. Ideally this period would be somewhat similar to what occurs in vivo. It was also felt that the model should be applicable to studying the development of subgingival plaque associated with both diseased and non-diseased sites and be able to demonstrate differences in the bacterial composition. We describe an in vitro multi-species biofilm model of subgingival plaque that closely mimics the composition and proportions of the cultivable bacterial taxa recovered from the gingival crevice and/or periodontal pocket and that is relatively representative of the bacteria recovered from both healthy and diseased periodontal sites. In addition, the effect that saliva and subgingival plaque from the same subject (homologous) vs. plaque or saliva from different subjects (heterogeneous) had on biofilm formation was determined.