In Vitro Cultivation of 'Unculturable' Oral Bacteria, Facilitated by Community Culture and Media Supplementation with Siderophores

PLoS One. 2016 Jan 14;11(1):e0146926. doi: 10.1371/journal.pone.0146926. eCollection 2016.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacteria / classification
  • Bacteria / drug effects
  • Bacteria / genetics
  • Bacteria / growth & development*
  • Bacteriological Techniques
  • Culture Media / chemistry*
  • Humans
  • Mouth / microbiology*
  • Siderophores / chemistry*
  • Siderophores / pharmacology

Substances

  • Culture Media
  • Siderophores