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

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

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

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

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

Significance and potential of marine microbial natural bioactive compounds against biofilms/biofouling: necessity for green chemistry

Natural products from the unique environments of sea water and oceans represent a largely unfamiliar source for isolation of new microbes, which are potent producers of secondary bioactive metabolites. These unique life-forms from the marine ecosphere have served as an important source of drugs since ancient times and still offer a valuable resource for novel findings by providing remedial treatments. Therefore, it can be expected that many naturally bioactive marine microbial compounds with novel structures and bioactivities against those from terrestrial environments may be found among marine metabolites. Biofilms in aquatic environment possess serious problems to naval forces and oceanic industries around the globe. Current anti-biofilm or anti-biofouling technology is based on the use of toxic substances that can be harmful to their surrounding natural locales. Comprehensive research has been done to examine the bioactive potential of marine microbes. Results are remarkably varied and dynamic, but there is an urgent need for bioactive compounds with environmentally friendly or “green” chemical activities. Marine microbes have the potential as upcoming and promising source of non-toxic compounds with sustainable anti-biofouling/anti-biofilm properties as they can produce substances that can inhibit not only the chemical components required for biofilm production but also the attachment, microorganism growth, and/or cell–cell communication

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

Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under awards R37DE016937 and R01DE 024468

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 reviewe

The oral microbiome – an update for oral healthcare professionals

For millions of years, our resident microbes have coevolved and coexisted with us in a mostly harmonious symbiotic relationship. We are not distinct entities from our microbiome, but together we form a 'superorganism' or holobiont, with the microbiome playing a significant role in our physiology and health. The mouth houses the second most diverse microbial community in the body, harbouring over 700 species of bacteria that colonise the hard surfaces of teeth and the soft tissues of the oral mucosa. Through recent advances in technology, we have started to unravel the complexities of the oral microbiome and gained new insights into its role during both health and disease. Perturbations of the oral microbiome through modern-day lifestyles can have detrimental consequences for our general and oral health. In dysbiosis, the finely-tuned equilibrium of the oral ecosystem is disrupted, allowing disease-promoting bacteria to manifest and cause conditions such as caries, gingivitis and periodontitis. For practitioners and patients alike, promoting a balanced microbiome is therefore important to effectively maintain or restore oral health. This article aims to give an update on our current knowledge of the oral microbiome in health and disease and to discuss implications for modern-day oral healthcare

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes