Diversity, quantification and polymicrobial culture of oral TM7 bacteria

The oral cavity contains a diverse range of bacteria with many, such as those classed as TM7 bacteria, shown by molecular studies to be present but not yet cultured within the laboratory. Objectives: This study aimed to quantitatively and qualitatively evaluate, and attempt in vitro culture, of TM7 bacteria from dental plaque of patients undertaking orthodontic treatment. Methods: Dental plaque samples were taken from 20 consenting patients aged 11-14 before, during and after orthodontic treatment in the Bristol Dental Hospital. Molecular methods targeting the 16S rRNA gene were used for TM7 quantification, evaluation of diversity by Denaturing Gradient Gel Electrophoresis (DGGE), cloning and sequencing, and Fluorescence In Situ Hybridization (FISH). Plaque samples known to contain TM7 bacteria from one patient were cultured in Fastidious Anaerobe Broth (FAB) medium under anaerobic conditions. Results: Real-time PCR analyses of plaque samples indicated an increase in proportion of TM7 bacteria in gingival margin plaque samples adjacent to bonded tubes but not molar bands during treatment, with reduction to pre-treatment levels three months post-treatment. DGGE, and cloning, sequencing and subsequent phylogenetic analyses, indicate a single individual carries up to seven TM7 phylotypes orally, with shifts in relative prevalence during treatment. Successive sub-culture of plaque bacteria over many months in FAB in anaerobic conditions allowed growth of some, but not all, TM7 phylotypes in mixed culture with strains of Actinomyces viscosus, Fusobacterium nucleatum, Prevotella denticola, Shuttleworthia satelles, Streptococcus gordonii and Veillonella parvula. FISH and live/dead experiments confirmed the presence of very large, viable TM7 cells in mixed culture, and suggested intimate interactions between TM7 and other strains. Conclusions: TM7 bacteria increase in proportion and diversity in plaque during orthodontic treatment, although returning to pre-treatment levels after removal of appliances. Some TM7 phylotypes grow anaerobically in vitro with a defined consortium of other oral bacterial species.