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.

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.

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.

Fretibacterium fastidiosum gen. nov., sp. nov., isolated from the human oral cavity.

SGP1(T), a strain belonging to a lineage of the phylum Synergistetes with no previously cultivated representatives was subjected to a comprehensive range of phenotypic and genotypic tests. For good growth the strain was dependent on co-culture with, or extracts from, selected other oral bacteria. Cells of strain SGP1(T) were asaccharolytic and major amounts of acetic acid and moderate amounts of propionic acid were produced as end products of metabolism in peptone-yeast extract-glucose broth supplemented with a filtered cell sonicate of Fusobacterium nucleatum subsp. nucleatum ATCC 25586(T) (25 %, v/v). Hydrogen sulphide was produced and gelatin was weakly hydrolysed. The major cellular fatty acids were C(14 : 0), C(18 : 0) and C(16 : 0). The DNA G+C content of strain SGP1(T) was 63 mol%. Phylogenetic analysis of the full-length 16S rRNA gene showed that strain SGP1(T) represented a novel group within the phylum Synergistetes. A novel species in a new genus, Fretibacterium fastidiosum gen. nov., sp. nov., is proposed. The type strain of Fretibacterium fastidiosum is SGP1(T) ( = DSM 25557(T) = JCM 16858(T)).

Strategies for culture of 'unculturable' bacteria.

Molecular ecology methods are now well established for the culture-independent characterization of complex bacterial communities associated with various environmental and animal habitats and are revealing the extent of their diversity. By comparison, it has become clear that only a small minority of microorganisms are readily cultivated in vitro, with the majority of all bacteria remaining 'unculturable' using standard methods. Yet, it is only through the isolation of bacterial species in pure culture that they may be fully characterized, both for their physiological and pathological properties. Hence, the endeavour to devise novel cultivation methods for microorganisms that appear to be inherently resistant to artificial culture is a most important one. This minireview discusses the possible reasons for 'unculturability' and evaluates advances in the cultivation of previously unculturable bacteria from complex bacterial communities. Methods include the use of dilute nutrient media particularly suited for the growth of bacteria adapted to oligotrophic conditions, and the provision of simulated natural environmental conditions for bacterial culture. This has led to the recovery of 'unculturables' from soil and aquatic environments, likely to be due to the inclusion of essential nutrients and/or signalling molecules from the native environment.

Pyramidobacter piscolens gen. nov., sp. nov., a member of the phylum 'Synergistetes' isolated from the human oral cavity.

Four strains of anaerobic, Gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group distinct from any species with validly published names. 16S rRNA and 23S rRNA gene sequence analyses and DNA-DNA reassociation data revealed that the strains constituted a novel group within the phylum 'Synergistetes' and were most closely related to Jonquetella anthropi. Two libraries of randomly cloned DNA were prepared from strain W5455(T) and were sequenced to provide a genome survey as a resource for metagenomic studies. A new genus and novel species, Pyramidobacter piscolens gen. nov., sp. nov., is proposed to accommodate these strains. The genus Pyramidobacter comprises strains that are anaerobic, non-motile, asaccharolytic bacilli that produce acetic and isovaleric acids and minor to trace amounts of propionic, isobutyric, succinic and phenylacetic acids as end products of metabolism. P. piscolens gen. nov., sp. nov. produced hydrogen sulphide but was otherwise largely biochemically unreactive. Growth was stimulated by the addition of glycine to broth media. The G+C content of the DNA of the type strain was 59 mol%. The type strain of Pyramidobacter piscolens sp. nov. is W5455(T) (=DSM 21147(T)=CCUG 55836(T)).

The division "Synergistes".

The "Synergistes" group of organisms are a phylogenetic cluster of Gram-negative anaerobes related to Synergistes jonesii, sufficiently distinct from all other phyla to be considered a distinct phylum or Division. They are widely distributed in nature although normally only a minor constituent of the bacterial community in each habitat. They have evolved to adapt to each habitat, and therefore exhibit a wide range of physiological and biochemical characteristics, although all cultivable taxa so far studied have the ability to degrade amino acids. They are found in the human mouth where they appear to be more numerous in tooth and gum disease than health. They have also been found in the human gut and soft tissue infections. Their role in human disease has yet to be established but improved knowledge of the characteristics that enable their identification should increase the likelihood of their recognition when present at diseased sites.

Does the referral and selection for NHS-funded dental implant treatment in the UK follow National Guidelines?

INTRODUCTION: The Royal College of Surgeons of England (RCSE) has provided a national guideline document detailing specific selection criteria for National Health Service (NHS) dental implant therapy in the UK. The aim of this study was to investigate whether these guidelines are being observed both in the referral and selection of patients. PATIENTS AND METHODS: Information was collected prospectively from 8 consecutive implant assessment clinics at Guy's and St Thomas' Hospital Trust over a 6-month period, via a questionnaire concerning the reason for referral for implant treatment, dental/smoking/radiotherapy status, and the consultant decision on whether an application would be made for NHS implant funding. RESULTS: Of the 103 referred patients who attended the appointment, 83 (80.6%) fell within the established priority groups for NHS implant therapy: in particular, hypodontia and teeth lost through trauma. However, up to a quarter of patients had untreated caries or periodontitis (n = 23, 22.3% and n = 26, 25.2%, respectively), a contra-indication for NHS implant therapy. The proportion of patients with caries was more than double for general dental practitioner-referred (28.3%) than hospital-referred subjects (12.5%) with the difference approaching statistical significance (P = 0.061). Selection for implant funding application was highly dependent on whether or not a patient belonged to a priority group of the national guidelines (P < 0.001). CONCLUSIONS: Although in most respects there is good adherence to the RCSE national guidelines both in the referral and selection of patients for NHS implant therapy, about a quarter of patients particularly those referred from general dental practitioners rather than hospitals, would be contra-indicated from NHS therapy because of untreated caries or periodontitis.