Abstract
BackgroundSeveral in vitro oral biofilm growth systems can reliably construct oral microbiome communities in culture, yet their stability and reproducibility through time has not been well characterized. Long-term in vitro growth of natural biofilms would enable use of these biofilms in both in vitro and in vivo studies that require complex microbial communities with minimal variation over a period of time. Understanding biofilm community dynamics in continuous culture, and whether they maintain distinct signatures of health and disease, is necessary to determine the reliability and applicability of such models to broader studies. To this end, we performed next-generation sequencing on biofilms grown from healthy and disease-site subgingival plaque for 80 days to assess stability and reliability of continuous oral biofilm growth.ResultsBiofilms were grown from subgingival plaque collected from periodontitis-affected sites and healthy individuals for ten eight-day long generations, using hydroxyapatite disks. The bacterial community in each generation was determined using Human Oral Microbe Identification by Next-Generation Sequencing (HOMINGS) technology, and analyzed in QIIME. Profiles were steady through the ten generations, as determined by species abundance and prevalence, Spearman’s correlation coefficient, and Faith’s phylogenetic distance, with slight variation predominantly in low abundance species. Community profiles were distinct between healthy and disease site-derived biofilms as demonstrated by weighted UniFrac distance throughout the ten generations. Differentially abundant species between healthy and disease site-derived biofilms were consistent throughout the generations.ConclusionsHealthy and disease site-derived biofilms can reliably maintain consistent communities through ten generations of in vitro growth. These communities maintain signatures of health and disease and of individual donors despite culture in identical environments. This subgingival oral biofilm growth and perpetuation model may prove useful to studies involving oral infection or cell stimulation, or those measuring microbial interactions, which require the same biofilms over a period of time.
Highlights
Several in vitro oral biofilm growth systems can reliably construct oral microbiome communities in culture, yet their stability and reproducibility through time has not been well characterized
Murine oral co-infection with Tannerella forsythia and Fusobacterium nucleatum [9] or with Porphyromonas gingivalis and Treponema denticola [10] induces greater alveolar bone resorption than either species alone, while a polybacterial inoculum of P. gingivalis, T. denticola, T. forsythia, and F. nucleatum elicits a unique immune/inflammatory response and physiologic changes compared to individual oral infection with each of the species [11,12,13,14,15,16]
Peripheral blood mononuclear cells (PBMCs) from localized aggressive periodontitis (LAP) patients and healthy patients respond uniquely to stimulation with bacterial surface components, intact complex oral biofilms, and dispersed complex oral biofilms [17] indicating that the choice of stimulant in immunological assays can skew the conclusions drawn about cellular responses in infectious diseases
Summary
Several in vitro oral biofilm growth systems can reliably construct oral microbiome communities in culture, yet their stability and reproducibility through time has not been well characterized. Understanding biofilm community dynamics in continuous culture, and whether they maintain distinct signatures of health and disease, is necessary to determine the reliability and applicability of such models to broader studies. To this end, we performed nextgeneration sequencing on biofilms grown from healthy and disease-site subgingival plaque for 80 days to assess stability and reliability of continuous oral biofilm growth. Differences in immunological and physiological responses to stimulation with bacterial structures such as LPS, single bacterial species, and multiple bacterial species in vitro and in vivo have demonstrated that it is necessary to study polymicrobial-induced diseases with complex communities to better understand disease etiology. Unique subgingival plaque community profiles correlate with distinct gingival crevicular fluid cytokine profiles in generalized aggressive periodontitis patients and healthy patients [18], chronic periodontitis patients, and clinically healthy sites in chronic periodontitis patients [19]
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