Advancement of the 10-species subgingival Zurich Biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms

Abstract

BackgroundPeriodontitis is caused by a highly complex consortium of bacteria that establishes as biofilms in subgingival pockets. It is a disease that occurs worldwide and its consequences are a major health concern. Investigations in situ are not possible and the bacterial community varies greatly between patients and even within different loci. Due to the high complexity of the consortium and the availability of samples, a clear definition of the pathogenic bacteria and their mechanisms of pathogenicity are still not available. In the current study we addressed the need of a defined model system by advancing our previously described subgingival biofilm model towards a bacterial composition that reflects the one observed in diseased sites of patients and analysed the structure of these biofilms.ResultsWe further developed the growth media by systematic variation of key components resulting in improved stability and the firm establishment of spirochetes in the 10-species subgingival Zurich biofilm model. A high concentration of heat-inactivated human serum allowed the best proliferation of the used species. Therefore we further investigated these biofilms by analysing their structure by confocal laser scanning microscopy following fluorescence in situ hybridisation. The species showed mutual interactions as expected from other studies. The abundances of all organisms present in this model were determined by microscopic counting following species-specific identification by both fluorescence in situ hybridisation and immunofluorescence. The newly integrated treponemes were the most abundant organisms.ConclusionsThe use of 50% of heat-inactivated human serum used in the improved growth medium resulted in significantly thicker and more stable biofilms, and the quantitative representation of the used species represents the in vivo community of periodontitis patients much closer than in biofilms grown in the two media with less or no human serum. The appearance of T. denticola, P. gingivalis, and T. forsythia in the top layer of the biofilms, and the high abundance of T. denticola, reflects well the microbial situation observed at diseased sites. The improved model biofilms will allow further investigations of interactions between individual species and of the effects of atmospheric or nutritional changes, as well as interactions with tissue cells.

A reproducible microcosm biofilm model of subgingival microbial communities

“…The original peptone based contains 10% heat‐inactivated fetal horse serum. On the other hand, the use of 50% of heat‐inactivated human serum improves the stability of the biofilms resulting in significantly thicker biofilms. Because the optimal serum concentration was unclear “a priori,” a pilot experiment was performed to test 10%, 30% and 50% of heat‐inactivated fetal bovine serum.…”

Integration of non-oral bacteria into in vitro oral biofilms

“…37,38 Pre-hybridization (15 min, 46 C) was performed in 500 ml hybridization buffer in the absence of any oligonucleotide probes. Thereafter, 500 ml of hybridization buffer was used for each biofilm, supplemented with probes at a concentration of 10 ng/ml.…”

Phenalen-1-One-Mediated Antimicrobial Photodynamic Therapy and Chlorhexidine Applied to a Novel Caries Biofilm Model

“…Furthermore, S. mutans colonizes in vitro much less efficiently on streptococcal biofilms than on A. naeslundii biofilms [Wang et al, 2011]. As compared to the Zurich biofilm model of supragingival plaque and its various modifications comprising up to 11 bacterial or fungal species [Guggenheim et al, 2001;Shapiro et al, 2002;Ammann et al, 2012;Belibasakis and Thurnheer, 2014], this biofilm model is less complex in terms of species diversity but, on the other hand, allows easy and high-throughput screening while still being robust to antimicrobial challenge. Treatment with CHX 2.0% for 20 min served as a positive control leading to total eradication of biofilm bacteria below detection limit.…”

“…preferentially utilize glucose for their metabolism via the Embden-Meyerhof-Parnas pathway [Takahashi and Yamada, 1999]. Proportions of mFUM, saliva, and FBS in the BCM were adjusted to 50, 40, and 10%, respectively, for increasing biofilm density and sturdiness [Ammann et al, 2012]. In contrast to the Zurich biofilm model, biofilms were cultured in 96-well plates rather than on hydroxyapatite discs placed in 24-well plates for ensuring optimal experimental conditions for antimicrobial evaluation of light-based approaches like aPDT.…”