The Unculturables: targeted isolation of bacterial species associated with canine periodontal health or disease from dental plaque.

Ian J Davis,Stephen Harris,Alexander Horsfall,Christopher Bull,Ian Morley

doi:10.1186/1471-2180-14-196

Abstract

BackgroundThe current inability to culture the entirety of observed bacteria is well known and with the advent of ever more powerful molecular tools, that can survey bacterial communities at previously unattainable depth, the gap in our capacity to culture and define all of these species increases exponentially. This gap has essentially become the rate limiting step in determining how the knowledge of which species are present in a sample can be applied to understand the role of these species in an ecosystem or disease process. A case in point is periodontal disease, which is the most widespread oral disease in dogs. If untreated the disease results in significant pain, eventual loss of the dentition and potentially an increased risk of systemic diseases. Previous molecular based studies have identified the bacterial species associated with periodontal disease in dogs; however without cultured strains from many of these species it has not been possible to study whether they play a role in the disease process.ResultsUsing a quantitative polymerase chain reaction (qPCR) directed approach a range of microbiological media were screened and optimized to enrich for previously uncultivated target species. A systematic screening methodology was then employed to isolate the species of interest. In cases where the target species were not cultivable in isolation, helper strains grown underneath a nitrocellulose membrane were used to provide the necessary growth factors. This guided media optimization approach enabled the purification of 14 species, 8 of which we had previously been unable to cultivate in isolation. It is also applicable to the targeted isolation of isolates from species that have previously been cultured (for example to study intra-species variation) as demonstrated by the successful isolation of 6 targeted isolates of already cultured species.ConclusionsTo our knowledge this is the first time this combination of qPCR guided media optimization, strategic screening and helper strain support has been used successfully to isolate previously uncultured bacteria. This approach can be applied to any uncultured bacterial species where knowledge of their nutritional requirements or low relative abundance impedes their isolation.

Highlights

The current inability to culture the entirety of observed bacteria is well known and with the advent of ever more powerful molecular tools, that can survey bacterial communities at previously unattainable depth, the gap in our capacity to culture and define all of these species increases exponentially
With the advent of next-generation molecular tools to identify bacterial species, the number of phyla currently stands in excess of 85, the majority of which are unculturable [2,3]
Examples of quantitative polymerase chain reaction (qPCR) data demonstrating the drop in Ct values as the relative prevalence of three target species (COT-064, COT-107 & COT-227) increased through the screening process is given in the supplementary data (Additional files 1 and 2) along with the 16S rRNA sequence generated to confirm their identity (Additional file 3)

Summary

Introduction

The current inability to culture the entirety of observed bacteria is well known and with the advent of ever more powerful molecular tools, that can survey bacterial communities at previously unattainable depth, the gap in our capacity to culture and define all of these species increases exponentially. This gap has essentially become the rate limiting step in determining how the knowledge of which species are present in a sample can be applied to understand the role of these species in an ecosystem or disease process. Studies have demonstrated that between 44% and 63.6% of dogs are affected by the disease [5,6,7,8].Variation in prevalence estimates are likely to be due to the different age and breed compositions of the study groups and the criteria used to establish diagnosis of periodontal disease

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