Gary C. Armitage

Bacterial diversity in the oral cavity of 10 healthy individuals.

The composition of the oral microbiota from 10 individuals with healthy oral tissues was determined using culture-independent techniques. From each individual, 26 specimens, each from different oral sites at a single point in time, were collected and pooled. An 11th pool was constructed using portions of the subgingival specimens from all 10 individuals. The 16S ribosomal RNA gene was amplified using broad-range bacterial primers, and clone libraries from the individual and subgingival pools were constructed. From a total of 11 368 high-quality, nonchimeric, near full-length sequences, 247 species-level phylotypes (using a 99% sequence identity threshold) and 9 bacterial phyla were identified. At least 15 bacterial genera were conserved among all 10 individuals, with significant interindividual differences at the species and strain level. Comparisons of these oral bacterial sequences with near full-length sequences found previously in the large intestines and feces of other healthy individuals suggest that the mouth and intestinal tract harbor distinct sets of bacteria. Co-occurrence analysis showed significant segregation of taxa when community membership was examined at the level of genus, but not at the level of species, suggesting that ecologically significant, competitive interactions are more apparent at a broader taxonomic level than species. This study is one of the more comprehensive, high-resolution analyses of bacterial diversity within the healthy human mouth to date, and highlights the value of tools from macroecology for enhancing our understanding of bacterial ecology in human health.

Diagnosis of periodontal diseases.

This position paper on the diagnosis of periodontal diseases was prepared by the Research, Science and Therapy Committee of the American Academy of Periodontology. It is intended for the information of the dental profession and other interested parties. The purpose of the paper is to provide the reader with a general overview of the important issues related to the diagnosis of periodontal diseases. It is not intended as a comprehensive review of the subject. J Periodontol 2003;74:1237-1247.

Evidence of a robust resident bacteriophage population revealed through analysis of the human salivary virome.

Viruses are the most abundant known infectious agents on the planet and are significant drivers of diversity in a variety of ecosystems. Although there have been numerous studies of viral communities, few have focused on viruses within the indigenous human microbiota. We analyzed 2 267 695 virome reads from viral particles and compared them with 263 516 bacterial 16S rRNA gene sequences from the saliva of five healthy human subjects over a 2- to 3-month period, in order to improve our understanding of the role viruses have in the complex oral ecosystem. Our data reveal viral communities in human saliva dominated by bacteriophages whose constituents are temporally distinct. The preponderance of shared homologs between the salivary viral communities in two unrelated subjects in the same household suggests that environmental factors are determinants of community membership. When comparing salivary viromes to those from human stool and the respiratory tract, each group was distinct, further indicating that habitat is of substantial importance in shaping human viromes. Compared with coexisting bacteria, there was concordance among certain predicted host–virus pairings such as Veillonella and Streptococcus, whereas there was discordance among others such as Actinomyces. We identified 122 728 virulence factor homologs, suggesting that salivary viruses may serve as reservoirs for pathogenic gene function in the oral environment. That the vast majority of human oral viruses are bacteriophages whose putative gene function signifies some have a prominent role in lysogeny, suggests these viruses may have an important role in helping shape the microbial diversity in the human oral cavity.

Comparison of the clinical features of chronic and aggressive periodontitis

Overall, while most clinicians would agree that aggressive forms of periodontitis exist as clinical entities, the clinical distinction between chronic and aggressive periodontitis (especially generalized) is not clear cut. This may not be all that significant from a treatment perspective, in so far as individualized anti-infective therapies are effective for both forms of the disease. However, from a research perspective, it is essential that these diseases be clearly distinguished in order to gain a complete understanding of their etiology and pathogenesis. The relative lack of clinical inflammation often associated with the localized molar-and-incisor form of aggressive periodontitis has been commented on for almost 100 years, and it is generally accepted that this form of the disease is associated with a thin biofilm, at least in its early stages. In contrast, the presence of clinical inflammation in generalized aggressive periodontitis appears to be similar to that observed in chronic periodontitis, and in this situation age of onset and family history are important additional criteria for either diagnosis or classification. It is also generally recognized that chronic periodontitis may subsequently be superimposed on both localized and generalized forms of aggressive periodontitis. While this may have little bearing on the treatment of such cases, it could have an enormous impact on both the design and interpretation of research studies, whether basic science or clinical. This highlights the essential difference between a diagnosis and a classification, whereby a diagnosis is the clinicians best guess, leading on to a treatment plan, whereas a classification does not allow such flexibility, requiring non-overlapping case definitions for research purposes if the underlying etiology of these diseases is ever to be fully elucidated.

Prevalence of Bacteria of Division TM7 in Human Subgingival Plaque and Their Association with Disease

ABSTRACT Members of the uncultivated bacterial division TM7 have been detected in the human mouth, but little information is available regarding their prevalence and diversity at this site. Human subgingival plaque samples from healthy sites and sites exhibiting various stages of periodontal disease were analyzed for the presence of TM7 bacteria. TM7 ribosomal DNA (rDNA) was found in 96% of the samples, and it accounted for approximately 0.3%, on average, of all bacterial rDNA in the samples as determined by real-time quantitative PCR. Two new phylotypes of this division were identified, and members of the division were found to exhibit filamentous morphology by fluorescence in situ hybridization. The abundance of TM7 rDNA relative to total bacterial rDNA was higher in sites with mild periodontitis (0.54% ± 0.1%) than in either healthy sites (0.21% ± 0.05%, P < 0.01) or sites with severe periodontitis (0.29% ± 0.06%, P < 0.05). One division subgroup, the I025 phylotype, was detected in 1 of 18 healthy samples and 38 of 58 disease samples. These data suggest that this phylotype, and the TM7 bacterial division in general, may play a role in the multifactorial process leading to periodontitis.

Microbiota-targeted therapies: an ecological perspective.

Insights from ecology will help to drive the development of microbiota-targeted therapeutics. The connection between disease and the disruption of homeostatic interactions between the host and its microbiota is now well established. Drug developers and clinicians are starting to rely more heavily on therapies that directly target the microbiota and on the ecology of the microbiota to understand the outcomes of these treatments. The effects of those microbiota-targeted therapies that alter community composition range in scale from eliminating individual strains of a single species (for example, with antibacterial conjugate vaccines) to replacing the entire community with a new intact microbiota (for example, by fecal transplantation). Secondary infections linked to antibiotic use provide a cautionary tale of the unintended consequences of perturbing a microbial species network and highlight the need for new narrow-spectrum antibiotics with rapid companion diagnostics. Insights into microbial ecology will also benefit the development of probiotics, whose therapeutic prospects will depend on rigorous clinical testing. Future probiotics may take the form of a consortium of long-term community residents: “a fecal transplant in a capsule.” The efficacy of microbiota-targeted therapies will need to be assessed using new diagnostic tools that measure community function rather than composition, including the temporal response of a microbial community to a defined perturbation such as an antibiotic or probiotic.

Comparison of the microbiological features of chronic and aggressive periodontitis

Any discussion of the comparative microbiology of chronic and aggressive forms of periodontitis must begin with the premise that our knowledge of the microbiota associated with these infections is incomplete. This does not mean that the field of oral microbiology has neglected the study of periodontal infections. On the contrary, the microbiota associated with periodontal health and disease has been intensely studied for well over a century by several generations of skilled scientists and clinicians (9, 15, 23, 42, 47, 48, 60, 65, 82, 101, 102, 105, 113, 114, 122, 123, 135, 145, 147, 149, 171, 175, 177, 181, 184, 206). The basic problem is that the oral microbiota is an enormously complex and dynamic entity that is profoundly affected by perpetually changing local environments and host-mediated selective pressures. In addition, the microorganisms live in hard-to-study biofilms comprising organized polymicrobial communities that are elegantly adapted to thriving and surviving in the multiple micro-ecosystems of the oral cavity. Another complication in any discussion of periodontal microbiology is the educational bias regarding the nature of infectious diseases that healthcare professionals develop during their training. Most dentists and physicians were taught the classical features of infectious diseases in their basic courses in medical microbiology. The dominant paradigm taught in these courses is that an exogenous pathogen overcomes the innate and adaptive immune defenses of the host, replicates within the body, and causes disease through a variety of virulence factors. Diagnosis of these classical infections often involves submitting a clinical specimen obtained from the infected patient to a clinical laboratory for isolation and identification of the pathogen by growing it in pure culture on artificial medium. These cultures are then used to determine the sensitivity of the isolated pathogen to a panel of antibiotics. Treatment of the infection involves administration of an appropriate regimen of antibiotics that is intended to suppress or eliminate the pathogen. This model for infectious diseases is certainly not the only one that is taught at most medical or dental schools, as it is widely known that some infections can be caused by multiple bacteria (i.e. mixed infections) and some diseases can be due to commensal opportunistic pathogens (82). Nevertheless, the one pathogen ⁄ one disease model dominates the thinking of most physicians and many dentists. Unfortunately, this dominant paradigm does not apply in cases of periodontal infections. These infections are caused by an extremely diverse consortium of microorganisms that are part of the endogenous microbiota of most people (82, 157). Many individuals who are periodontally healthy carry or harbor some periodontal pathogens as part of their normal supragingival and subgingival microbiota (4, 26, 32, 35, 43, 61, 62, 77, 94, 119, 129, 143, 150, 153, 155, 179, 180, 189, 196, 200, 203, 207). When detectable at healthy sites, the pathogens are usually present in very low numbers and show a limited range of phylotypes. It is quite clear that many individuals can harbor potential pathogens for long periods of time without developing periodontal disease. Carriage of putative periodontal pathogens at low levels as part of the commensal or normal oral microbiota is probably beneficial, since it is likely that their

Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time

Viruses may play an important role in the evolution of human microbial communities. Clustered regularly interspaced short palindromic repeats (CRISPRs) provide bacteria and archaea with adaptive immunity to previously encountered viruses. Little is known about CRISPR composition in members of human microbial communities, the relative rate of CRISPR locus change, or how CRISPR loci differ between the microbiota of different individuals. We collected saliva from four periodontally healthy human subjects over an 11- to 17-mo time period and analyzed CRISPR sequences with corresponding streptococcal repeats in order to improve our understanding of the predominant features of oral streptococcal adaptive immune repertoires. We analyzed a total of 6859 CRISPR bearing reads and 427,917 bacterial 16S rRNA gene sequences. We found a core (ranging from 7% to 22%) of shared CRISPR spacers that remained stable over time within each subject, but nearly a third of CRISPR spacers varied between time points. We document high spacer diversity within each subject, suggesting constant addition of new CRISPR spacers. No greater than 2% of CRISPR spacers were shared between subjects, suggesting that each individual was exposed to different virus populations. We detect changes in CRISPR spacer sequence diversity over time that may be attributable to locus diversification or to changes in streptococcal population structure, yet the composition of the populations within subjects remained relatively stable. The individual-specific and traceable character of CRISPR spacer complements could potentially open the way for expansion of the domain of personalized medicine to the oral microbiome, where lineages may be tracked as a function of health and other factors.

Global oral health inequalities: task group--periodontal disease.

Periodontal diseases constitute one of the major global oral health burdens, and periodontitis remains a major cause of tooth loss in adults worldwide. The World Health Organization recently reported that severe periodontitis exists in 5-20% of adult populations, and most children and adolescents exhibit signs of gingivitis. Likely reasons to account for these prevalent diseases include genetic, epigenetic, and environmental risk factors, as well as individual and socio-economic determinants. Currently, there are fundamental gaps in knowledge of such fundamental issues as the mechanisms of initiation and progression of periodontal diseases, which are undefined; inability to identify high-risk forms of gingivitis that progress to periodontitis; lack of evidence on how to prevent the diseases effectively; inability to detect disease activity and predict treatment efficacy; and limited information on the effects of integration of periodontal health as a part of the health care program designed to promote general health and prevent chronic diseases. In the present report, 12 basic, translational, and applied research areas have been proposed to address the issue of global periodontal health inequality. We believe that the oral health burden caused by periodontal diseases could be relieved significantly in the near future through an effective global collaboration.

Principles in prevention of periodontal diseases Consensus report of group 1 of the 11th European Workshop on Periodontology on effective prevention of periodontal and peri-implant diseases

AIMS In spite of the remarkable success of current preventive efforts, periodontitis remains one of the most prevalent diseases of mankind. The objective of this workshop was to review critical scientific evidence and develop recommendations to improve: (i) plaque control at the individual and population level (oral hygiene), (ii) control of risk factors, and (iii) delivery of preventive professional interventions. METHODS Discussions were informed by four systematic reviews covering aspects of professional mechanical plaque control, behavioural change interventions to improve self-performed oral hygiene and to control risk factors, and assessment of the risk profile of the individual patient. Recommendations were developed and graded using a modification of the GRADE system using evidence from the systematic reviews and expert opinion. RESULTS Key messages included: (i) an appropriate periodontal diagnosis is needed before submission of individuals to professional preventive measures and determines the selection of the type of preventive care; (ii) preventive measures are not sufficient for treatment of periodontitis; (iii) repeated and individualized oral hygiene instruction and professional mechanical plaque (and calculus) removal are important components of preventive programs; (iv) behavioural interventions to improve individual oral hygiene need to set specific Goals, incorporate Planning and Self monitoring (GPS approach); (v) brief interventions for risk factor control are key components of primary and secondary periodontal prevention; (vi) the Ask, Advise, Refer (AAR) approach is the minimum standard to be used in dental settings for all subjects consuming tobacco; (vii) validated periodontal risk assessment tools stratify patients in terms of risk of disease progression and tooth loss. CONCLUSIONS Consensus was reached on specific recommendations for the public, individual dental patients and oral health care professionals with regard to best action to improve efficacy of primary and secondary preventive measures. Some have implications for public health officials, payers and educators.