title: In Vitro Cultivation of 'Unculturable' Oral Bacteria, Facilitated by Community Culture and Media Supplementation with Siderophores.
abstract: Over a third of oral bacteria are as-yet-uncultivated in-vitro. Siderophores have been previously shown to enable in-vitro growth of previously uncultivated bacteria. The objective of this study was to cultivate novel oral bacteria in siderophore-supplemented culture media. Various compounds with siderophore activity, including pyoverdines-Fe-complex, desferricoprogen and salicylic acid, were found to stimulate the growth of difficult-to-culture strains Prevotella sp. HOT-376 and Fretibacterium fastidiosum. Furthermore, pyrosequencing analysis demonstrated increased proportions of the as-yet-uncultivated phylotypes Dialister sp. HOT-119 and Megasphaera sp. HOT-123 on mixed culture plates supplemented with siderophores. Therefore a culture model was developed, which incorporated 15 μg siderophore (pyoverdines-Fe-complex or desferricoprogen) or 150 μl neat subgingival-plaque suspension into a central well on agar plates that were inoculated with heavily-diluted subgingival-plaque samples from subjects with periodontitis. Colonies showing satellitism were passaged onto fresh plates in co-culture with selected helper strains. Five novel strains, representatives of three previously-uncultivated taxa (Anaerolineae bacterium HOT-439, the first oral taxon from the Chloroflexi phylum to have been cultivated; Bacteroidetes bacterium HOT-365; and Peptostreptococcaceae bacterium HOT-091) were successfully isolated. All novel isolates required helper strains for growth, implying dependence on a biofilm lifestyle. Their characterisation will further our understanding of the human oral microbiome.
authors.affiliation: Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
authors.forename: Sonia R
authors.initials: SR
authors.lastname: Vartoukian
authors.affiliation: Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Adamowska
authors.affiliation: Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
authors.forename: Megan
authors.initials: M
authors.lastname: Lawlor
authors.affiliation: King's College London Dental Institute, London, United Kingdom.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Moazzez
authors.affiliation: The Forsyth Institute, Cambridge, United States of America.|Harvard School of Dental Medicine, Boston, United States of America.
authors.forename: Floyd E
authors.initials: FE
authors.lastname: Dewhirst
authors.affiliation: Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
authors.forename: William G
authors.initials: WG
authors.lastname: Wade
mesh_terms: D001419:Bacteria / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D001431:Bacteriological Techniques; D003470:Culture Media / Q000737:chemistry*; D006801:Humans; D009055:Mouth / Q000382:microbiology*; D017262:Siderophores / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D003470:Culture Media; D017262:Siderophores