Uncultured Members of the Oral Microbiome.

Around one-third of oral bacteria cannot be cultured using conventional methods. Some bacteria have specific requirements for nutrients while others may be inhibited by substances in the culture media or produced by other bacteria. Oral bacteria have evolved as part of multispecies biofilms, and many thus require interaction with other bacterial species to grow. In vitro models have been developed that mimic these interactions and have been used to grow previously uncultivated organisms

Cultivation strategies for growth of uncultivated bacteria.

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.This work was supported by The National Institute of Dental and Craniofacial Research of the National Institutes of Health under award R37DE01693

Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and pyrosequencing for the characterization of the dentine caries-associated microbiome

Culture-independent analyses have greatly expanded knowledge regarding the composition of complex bacterial communities including those associated with oral diseases. A consistent finding from such studies, however, has been the under-reporting of members of the phylum Actinobacteria. In this study, five pairs of broad range primers targeting 16S rRNA genes were used in clonal analysis of 6 samples collected from tooth lesions involving dentine in subjects with active caries. Samples were also subjected to cultural analysis and pyrosequencing by means of the 454 platform. A diverse bacterial community of 229 species-level taxa was revealed by culture and clonal analysis, dominated by representatives of the genera Prevotella, Lactobacillus, Selenomonas, and Streptococcus. The five most abundant species were: Lactobacillus gasseri, Prevotella denticola, Alloprevotella tannerae, S. mutans and Streptococcus sp. HOT 070, which together made up 31.6 % of the sequences. Two samples were dominated by lactobacilli, while the remaining samples had low numbers of lactobacilli but significantly higher numbers of Prevotella species. The different primer pairs produced broadly similar data but proportions of the phylum Bacteroidetes were significantly higher when primer 1387R was used. All of the primer sets underestimated the proportion of Actinobacteria compared to culture. Pyrosequencing analysis of the samples was performed to a depth of sequencing of 4293 sequences per sample which were identified to 264 species-level taxa, and resulted in significantly higher coverage estimates than the clonal analysis. Pyrosequencing, however, also underestimated the relative abundance of Actinobacteria compared to culture

Uncultured members of the oral microbiome

Around one-third of oral bacteria cannot be cultured using conventional methods. Some bacteria have specific requirements for nutrients while others may be inhibited by substances in the culture media or produced by other bacteria. Oral bacteria have evolved as part of multispecies biofilms, and many thus require interaction with other bacterial species to grow. In vitro models have been developed that mimic these interactions and have been used to grow previously uncultivated organisms

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

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

Draft whole genome sequences of the periodontal pathobionts Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia contain phase variable restriction modification systems

The periodontal strains used were collected during previous studies performed at the University of Cardiff (strains WW414, WW855 and WW2096), University of Bristol (WW2834, WW2842, WW2866, WW2881, WW28585, WW2903, WW2931, WW2952, WW3039, WW3040, and WW3102), King’s College London (WW5019, WW5127, and WW10960) and Queen Mary University of London (WW11663). Illumina sequencing was performed by the NUCLEUS Genomics Core Facility and data analysis used the Spectre2 and Alice2 High Performance Computing Facility at the University of Leicester. This work was in part funded by a grant from the BBSRC (BB/N002903/1) to MRO.Peer reviewedPublisher PD

Cultivation of a Synergistetes strain representing a previously uncultivated lineage

Subgingival plaque samples obtained from human subjects with periodontitis, shown to include previously uncultivable members of the phylum Synergistetes, were used to inoculate Cooked Meat Medium (CMM). The presence of Cluster A (uncultivable) Synergistetes was monitored by fluorescent in situ hybridization (FISH) and quantitative PCR (Q-PCR). Cluster A Synergistetes were found to grow in CMM in co-culture with other plaque bacteria and growth was stimulated by the addition of mucin and serum. Plaque samples were also used to inoculate Blood Agar (BA) plates and growth of Cluster A Synergistetes was revealed after anaerobic incubation, by colony hybridization with specific probes. Surface growth on the plates in regions identified by colony hybridization was harvested and used to inoculate fresh plates, thus enriching for Cluster A Synergistetes. Cross-streaks of other plaque bacteria were also used to stimulate Synergistetes growth. In the early passages, no discrete Synergistetes colonies were seen, but after eight passages and the use of cross-streaks of other bacteria present in the enriched community, colonies arose, which consisted solely of Cluster A Synergistetes cells, as determined by 16S rRNA gene PCR and cloning. This is the first report of the successful culture of a member of the uncultivable branch of this phylum

As-yet-uncultivated oral bacteria: breadth and association with oral and extra-oral diseases

It has been shown that 40–60% of the bacteria found in different healthy and diseased oral sites still remain to be grown in vitro, phenotypically characterized, and formally named as species. The possibility exists that these as-yet-uncultivated bacteria play important ecological roles in oral bacterial communities and may participate in the pathogenesis of several oral infectious diseases. There is also a potential for these as-yet-uncultivated oral bacteria to take part in extra-oral infections. For a comprehensive characterization of physiological and pathogenic properties as well as antimicrobial susceptibility of individual bacterial species, strains need to be grown in pure culture. Advances in culturing techniques have allowed the cultivation of several oral bacterial taxa only previously known by a 16S rRNA gene sequence signature, and novel species have been proposed. There is a growing need for developing improved methods to cultivate and characterize the as-yet-uncultivated portion of the oral microbiome so as to unravel its role in health and disease

Draft Whole-Genome Sequences of Periodontal Pathobionts Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia Contain Phase-Variable Restriction-Modification Systems.

Periodontal disease comprises mild to severe inflammatory host responses to oral bacteria that can cause destruction of the tooth-supporting tissue. We report genome sequences for 18 clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia, Gram-negative obligate anaerobes that play a role in the periodontal disease process.This work was in part funded by a grant from the BBSRC (BB/N002903/1) to M.R.O

Dental biofilm: ecological interactions in health and disease.

BACKGROUND: The oral microbiome is diverse and exists as multispecies microbial communities on oral surfaces in structurally and functionally organized biofilms. AIM: To describe the network of microbial interactions (both synergistic and antagonistic) occurring within these biofilms and assess their role in oral health and dental disease. METHODS: PubMed database was searched for studies on microbial ecological interactions in dental biofilms. The search results did not lend themselves to systematic review and have been summarized in a narrative review instead. RESULTS: Five hundred and forty-seven original research articles and 212 reviews were identified. The majority (86%) of research articles addressed bacterial-bacterial interactions, while inter-kingdom microbial interactions were the least studied. The interactions included physical and nutritional synergistic associations, antagonism, cell-to-cell communication and gene transfer. CONCLUSIONS: Oral microbial communities display emergent properties that cannot be inferred from studies of single species. Individual organisms grow in environments they would not tolerate in pure culture. The networks of multiple synergistic and antagonistic interactions generate microbial inter-dependencies and give biofilms a resilience to minor environmental perturbations, and this contributes to oral health. If key environmental pressures exceed thresholds associated with health, then the competitiveness among oral microorganisms is altered and dysbiosis can occur, increasing the risk of dental disease