Impact of Early Colonizers on <i>In Vitro</i> Subgingival Biofilm Formation

<div><p>The aim of this study was to investigate the impact of early colonizing species on the structure and the composition of the bacterial community developing in a subgingival 10-species biofilm model system. The model included <i>Streptococcus oralis</i>, <i>Streptococcus anginosus</i>, <i>Actinomycesoris</i>, <i>Fusobacterium nucleatum</i> subsp. <i>nucleatum</i>, <i>Veillonella dispar</i>, <i>Campylobacter rectus</i>, <i>Prevotella intermedia</i>, <i>Porphyromonas gingivalis</i>, <i>Tannerella forsythia</i>, and <i>Treponema denticola</i>. Based on literature, we considered <i>Streptococcus oralis</i>, <i>Streptococcus anginosus</i>, and <i>Actinomyces oris</i> as early colonizers and examined their role in the biofilms by either a delayed addition to the consortium, or by not inoculating at all the biofilms with these species. We quantitatively evaluated the resulting biofilms by real-time quantitative PCR and further compared the structures using confocal laser scanning microscopy following fluorescence <i>in situ</i> hybridisation. The absence of the early colonizers did not hinder biofilm formation. The biofilms reached the same total counts and developed to normal thickness. However, quantitative shifts in the abundances of individual species were observed. In the absence of streptococci, the overall biofilm structure appeared looser and more dispersed. Moreover, besides a significant increase of <i>P. intermedia</i> and a decrease of <i>P. gingivalis</i> , <i>P. intermedia</i> appeared to form filamented long chains that resembled streptococci. <i>A. oris</i>, although growing to significantly higher abundance in absence of streptococci, did not have a visible impact on the biofilms. Hence, in the absence of the early colonizers, there is a pronounced effect on <i>P. intermedia</i> and <i>P. gingivalis</i> that may cause distinct shifts in the structure of the biofilm. Streptococci possibly facilitate the establishment of <i>P. gingivalis</i> into subgingival biofilms, while in their absence <i>P. intermedia</i> became more dominant and forms elongated chains.</p>