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. 2012 Jan;78(1):194-203.
doi: 10.1128/AEM.06813-11. Epub 2011 Nov 4.

New Approaches for Isolation of Previously Uncultivated Oral Bacteria

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New Approaches for Isolation of Previously Uncultivated Oral Bacteria

M V Sizova et al. Appl Environ Microbiol. .
Free PMC article

Abstract

A significant number of microorganisms from the human oral cavity remain uncultivated. This is a major impediment to the study of human health since some of the uncultivated species may be involved in a variety of systemic diseases. We used a range of innovations previously developed to cultivate microorganisms from the human oral cavity, focusing on anaerobic species. These innovations include (i) in vivo cultivation to specifically enrich for species actively growing in the oral cavity (the "minitrap" method), (ii) single-cell long-term cultivation to minimize the effect of fast-growing microorganisms, and (iii) modifications of conventional enrichment techniques, using media that did not contain sugar, including glucose. To enable cultivation of obligate anaerobes, we maintained strict anaerobic conditions in most of our cultivation experiments. We report that, on a per cell basis, the most successful recovery was achieved using minitrap enrichment (11%), followed by single-cell cultivation (3%) and conventional plating (1%). Taxonomically, the richest collection was obtained using the single-cell cultivation method, followed by minitrap and conventional enrichment, comprising representatives of 13, 9, and 4 genera, respectively. Interestingly, no single species was isolated by all three methods, indicating method complementarity. An important result is the isolation and maintenance in pure culture of 10 strains previously only known by their molecular signatures, as well as representatives of what are likely to be three new microbial genera. We conclude that the ensemble of new methods we introduced will likely help close the gap between cultivated and uncultivated species from the human oral cavity.

Figures

Fig 1
Fig 1
Minitrap used for in vivo cultivation of oral microorganisms. (Left) Basic design (explanations in the text). (Right) General view of the subjects' dental appliance, with the minitrap glued to a window cut in the appliance.
Fig 2
Fig 2
HOMIM-enabled microbial composition of two subgingival plaque samples from subject 3. Dark green and light green correspond to the samples from the right and left sides of the mouth, respectively. The sizes of the bars reflect the relative band intensities of hybridization with target bacterial species.
Fig 3
Fig 3
Accumulation of growth events in long-term cultivation experiments presented as a fraction of wells positive (PW) for growth. (A) Experiment 1; (B) experiment 2.
Fig 4
Fig 4
Minimum evolution phylogenetic tree of 16S rRNA gene sequences of anaerobic bacteria isolated via anaerobic enrichment. Shown in boldface are strains sharing less than 99% gene sequence homology with the closest named species.
Fig 5
Fig 5
Overlap between culture collections obtained by three different cultivation approaches for a single subject (subject 3). Values in the center of each circle represent the total number of isolated oral taxa by method; values in the overlapping areas represent the numbers of coisolated oral taxa.

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