Application of a single‐colony coculture technique to the isolation of hitherto unculturable gut bacteria

Tanaka, Yoshiki; Benno, Yoshimi

doi:10.1111/1348-0421.12220

Cited by 30 publications

(23 citation statements)

References 38 publications

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“…In the present study, we investigated the interactions between probiotic strains and normal intestinal bacteria. For this purpose, we used the in vitro membrane filter method to reveal interbacterial communications between gut bacteria and probiotic strains.…”

Section: Discussionmentioning

confidence: 99%

“…TYG agar (pH 7.0) comprising trypticase (10.0 g), yeast extract (5.0 g), glucose (4.0 g), Tween ® 80 (0.5 g), L‐cysteine·HCl·H 2 O (0.5 g), 1 M MgCl 2 (0.4 mL), 1 M KCl (20.0 mL), 1 M NaCl (30.0 mL), 1 M NH 4 Cl (19.0 mL), 1 M Na 2 SO 4 (0.8 mL), 1 M Na 2 HPO 4 / 1 M NaH 2 PO 4 buffer, pH 7.0 (64.0 mL), agar (15.0 g or 4.0 g), and distilled water (865.8 mL) was used .…”

Section: Methodsmentioning

confidence: 99%

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Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota

Shimizu

Benno

2015

Microbiology and Immunology

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A large number of commensal bacteria inhabit the intestinal tract, and interbacterial communication among gut microbiota is thought to occur. In order to analyze symbiotic relationships between probiotic strains and the gut microbiota, a ring with a membrane filter fitted to the bottom was used for in vitro investigations. Test strains comprising probiotic nitto strains (Lactobacillus acidophilus NT and Bifidobacterium longum NT) and type strains (L. acidophilus JCM1132T and B. longum JCM1217T) were obtained from diluted fecal samples using the membrane filter to simulate interbacterial communication. Bifidobacterium spp., Streptococcus pasteurianus, Collinsella aerofaciens, and Clostridium spp. were the most abundant gut bacteria detected before coculture with the test strains. Results of the coculture experiments indicated that the test strains significantly promote the growth of Ruminococcus gnavus, Ruminococcus torques, and Veillonella spp. and inhibit the growth of Sutterella wadsworthensis. Differences in the relative abundances of gut bacterial strains were furthermore observed after coculture of the fecal samples with each test strain. Bifidobacterium spp., which was detected as the dominant strain in the fecal samples, was found to be unaffected by coculture with the test strains. In the present study, interbacterial communication using bacterial metabolites between the test strains and the gut microbiota was demonstrated by the coculture technique. The detailed mechanisms and effects of the complex interbacterial communications that occur among the gut microbiota are, however, still unclear. Further investigation of these relationships by coculture of several fecal samples with probiotic strains is urgently required.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota

Shimizu

Benno

2015

Microbiology and Immunology

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“…Likewise, Ueda and Beppu [52] co-cultured the syntrophic bacterium Symbiobacterium thermophilum and the Bacillus strain on which it depends for growth, in a two-compartment flask with dialysis membrane separating the two. In another innovative example, included here although it relates to the human gut flora rather than to environmental microorganisms because it uses a similar principle to the above, pairwise symbiotic relationships were detected between co-cultivated bacteria growing in soft agar on either side of a 0.22 μm membrane filter [53]. Morris et al [23, 24] demonstrated that Prochlorococcus taxa are dependent for growth on neighboring hydrogen peroxide-scavenging bacteria for protection from oxidative stress and cellular damage.…”

Section: Cultivation Strategies For Uncultivated Bacteriamentioning

confidence: 99%

Cultivation strategies for growth of uncultivated bacteria

Vartoukian

2016

Journal of Oral Biosciences

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Background The majority of environmental bacteria and around a third of oral bacteria remain uncultivated. Furthermore, several bacterial phyla have no cultivable members and are recognised only by detection of their DNA by molecular methods. Possible explanations for the resistance of certain bacteria to cultivation in purity in vitro include: unmet fastidious growth requirements; inhibition by environmental conditions or chemical factors produced by neighbouring bacteria in mixed cultures; or conversely, dependence on interactions with other bacteria in the natural environment, without which they cannot survive in isolation. Auxotrophic bacteria, with small genomes lacking in the necessary genetic material to encode for essential nutrients, frequently rely on close symbiotic relationships with other bacteria for survival, and may therefore be recalcitrant to cultivation in purity. Highlight Since in-vitro culture is essential for the comprehensive characterisation of bacteria, particularly with regard to virulence and antimicrobial resistance, the cultivation of uncultivated organisms has been a primary focus of several research laboratories. Many targeted and open-ended strategies have been devised and successfully used. Examples include: the targeted detection of specific bacteria in mixed plate cultures using colony hybridisation; growth in simulated natural environments or in co-culture with ‘helper’ strains; and modified media preparation techniques or development of customised media eg. supplementation of media with potential growth-stimulatory factors such as siderophores. Conclusion Despite significant advances in recent years in methodologies for the cultivation of previously uncultivated bacteria, a substantial proportion remain to be cultured and efforts to devise high-throughput strategies should be a high priority.

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“…Bacteria that are metabolically dependent on others may be impossible to grow in pure culture. Conversely, in vitro cultivation of bacteria in consortia can enable the isolation of previously uncultivated bacteria (Vartoukian et al 2010;Tanaka and Benno 2015). In particular, species within biofilm communities, such as dental plaque, may depend on one another for metabolic cooperation and intercellular signals (Vartoukian et al 2010;Stewart 2012;Mihai et al 2015).…”

Section: Introductionmentioning

confidence: 99%

First Cultivation of Health-Associated Tannerella sp. HOT-286 (BU063)

et al. 2016

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Despite significant advances in recent years in culture-independent molecular microbiology methods, the detailed study of individual bacterial species still relies on having pure cultures in the laboratory. Yet, more than a third of the approximately 700 bacterial taxa found in the human oral cavity are as yet uncultivated in vitro. One such taxon, Tannerella sp. , is the focus of much interest since it is associated with periodontal health, while Tannerella forsythia, its closest phylogenetic neighbor, is strongly associated with periodontal disease. HOT-286, however, has remained uncultivated despite the efforts of several research groups, spanning over a decade. The aim of this study was to cultivate Tannerella sp. HOT-286. A heavily diluted sample of subgingival plaque was inoculated onto culture plates supplemented with siderophores (pyoverdines-Fe complex or desferricoprogen) or a neat plaque suspension. After 8 d of anaerobic incubation, microcolonies and colonies showing satellitism were passaged onto fresh culture plates cross-streaked with potential helper strains or onto cellulose-acetate membranes placed over lawn cultures of helper strains. Subcultured colonies were identified by 16S rRNA gene sequencing, and purity was confirmed by sequencing 20 clones per library prepared from a single colony. Three colonies of interest (derived from pyoverdines-and plaque-supplemented plates) were identified as Tannerella sp. HOT-286. The isolates were found to be incapable of independent growth, requiring helpers such as Propionibacterium acnes and Prevotella intermedia for stimulation, with best growth on membranes over "helper" lawns. A representative isolate was subjected to phenotypic characterization and found to produce a range of glycosidic and proteolytic enzymes. Further comparison of this novel "periodontal health-associated" taxon with T. forsythia will be valuable in investigating virulence factors of the latter and possible health benefits of the former.

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Application of a single‐colony coculture technique to the isolation of hitherto unculturable gut bacteria

Cited by 30 publications

References 38 publications

Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota

Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota

Cultivation strategies for growth of uncultivated bacteria

First Cultivation of Health-Associated Tannerella sp. HOT-286 (BU063)

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