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of gingival epithelium in response to subgingival biofilms. Mol Oral Microbiol 2015;30:323–335.

40Thurnheer T, Belibasakis G: Incorporation of staphylococci into titanium-grown biofilms: an in vitro “submucosal” biofilm model for peri-implantitis. Clin Oral Implants Res 2016;27:890–895.

      Thomas Thurnheer

      Division of Oral Microbiology and Immunology, Clinic of Conservative and Preventive Dentistry

      Center of Dental Medicine, University of Zurich

      Plattenstrasse 11, CH–8032 Zurich (Switzerland)

      [email protected]

       Biofilm Models

      Published online: December 21, 2020

      Eick S (ed): Oral Biofilms. Monogr Oral Sci. Basel, Karger, 2021, vol 29, pp 38–52 (DOI: 10.1159/000510198)

      ______________________

Biofilm Models for the Evaluation of Dental Treatment

      Sigrun Eick

      Department of Periodontology, Laboratory of Oral Microbiology, School of Dental Medicine, University of Bern, Bern, Switzerland

      ______________________

      Abstract

      When analyzing the activity of antimicrobial agents, it should be considered that microorganisms mainly occur in biofilms. Data obtained for planktonic bacteria cannot be transferred non-critically to biofilms. Biofilm models should consider both the relevant microorganisms and the conditions present in the environment. The selection of the model depends on the question to be answered. In dentistry, single species, multispecies, or microcosms originating from saliva or dental biofilm are used to culture biofilms. Microorganism selection depends on the focus of the study, for example caries biofilms mostly include Streptococcus mutans, an endodontic biofilm consists mostly of Enterococcus faecalis, and defined anaerobes are used in periodontal/peri-implant biofilms. In contrast to single-species biofilm models in medicine, where the lowest concentration of the antimicrobial that kills microorganisms is measured, the common analyzed variables are counts of colony-forming units or the percentage of dead bacteria determined by confocal laser scanning microscopy after applying a differentiating stain. All the models are helpful to evaluate new antimicrobial treatment options. Conclusions regarding the antimicrobial activity tendency of the therapeutics can be drawn. However, there are limitations of the model and ultimately a new therapy has to be proven in randomized controlled clinical trials.

      © 2021 S. Karger AG, Basel

      The activity of antimicrobial agents is still mainly evaluated by agar diffusion tests or by determining minimal inhibitory concentrations against planktonic bacteria in medical microbiology. It is increasingly recognized that in many medical conditions microorganisms occur in biofilms and data obtained for planktonic bacteria cannot be transferred non-critically to biofilms. Thus, there is a need to develop methods to estimate the activity of antimicrobials in biofilms.

Biofilm models should consider both the relevant microorganisms and the conditions present in the environment. Furthermore, the selection of the model depends on the question to be answered. Microtiter plates are often used in testing antimicrobials, where the biofilms are formed on polystyrene surfaces. These are static systems and do not guarantee a continuous supply of new nutrients and metabolites. However, most the nutrient medium is exchanged at defined times. For testing antimicrobials in particular, the so-called “Calgary device” is available [1]. The lid of the well plates contains pegs and biofilms are formed on the surface of the pegs. After formation of the biofilms, those can easily be transferred into other well plates containing antimicrobials in defined concentrations for a set time. The lowest concentration of the antimicrobial killing microorganisms in biofilms is the so-called minimal biofilm eradication concentration.

Continuous flow systems guarantee a permanent supply of nutrients and a removal of metabolic products. Several devices have been developed in laboratories. The “CDC” biofilm reactor allows standardized biofilms to be grown [2] on different surfaces and to apply antimicrobial compounds for testing [3].

Meanwhile, in vivo models are also used. Examples include catheter infections generated in rabbits to test potential antibiotics [4], and testing of the potential of implant material to prevent periprosthetic infections with methicillin-resistant Staphylococcus aureus [5].

According to the clinical relevance, most staphylococci as single species were used in biofilm models focusing on catheter-related or periprosthetic infections [4, 5]. Dual-species biofilms consisting of Alloicoccus otidis and Haemophilus influenzae representing an otitis media model were formed to evaluate their susceptibility to amoxicillin or ciprofloxacin [6].

In dentistry, the first models used either single-species biofilms [7–9] or microcosms [9, 10]. Then multispecies models using defined strains were introduced [11, 12]. “Microcosm” biofilms are formed ex vivo and in vivo. For ex vivo formation, saliva or plaque is sampled and cultured with laboratory conditions [13, 14]. Continuous flow systems such as the constant depth film fermenter allowing a biofilm formation of a defined depth were introduced [15].

Studying the effect of an antimicrobial therapy on the microcosm existing in the oral cavity became a topic of interest. Two different approaches exist, either sampling biofilm and culturing ex vivo [16] or whereby volunteers wear appliances allowing the biofilm formation, before the respective treatment is applied [17]. New developments in biofilm models also include interaction with host cells [18] which might be an interesting approach for evaluating treatments in the near future.

      Meanwhile, plenty of in vitro studies evaluating antibiofilm activity in dentistry have been published. The following summary can only touch on examples. Most identical or very similar models of the same research group are considered only once.

      The complete elimination of an oral biofilm by antimicrobials seems to be impossible. Therefore, the minimal biofilm eradication concentration is not measured. Instead, common methods of analysis are counts of colony-forming units (CFU) or the percentage of dead bacteria determined by confocal laser scanning microscopy after applying the differentiating stain.

      The

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