Changes in Periodontal Health Status Are Associated with Bacterial Community Shifts as Assessed by Quantitative 16S Cloning and Sequencing

Abstract

The gingival sulcus contains a complex ecosystem that includes many uncultivated bacteria. Understanding the dynamics of this ecosystem in transitions between health and disease is important in advancing our understanding of the bacterial etiology of periodontitis. The objective of this longitudinal study was to examine the stability of bacterial colonization in the gingival crevice and to explore the relationship between shifts in microbial composition and changes in periodontal health status using a comprehensive, quantitative, cultureindependent approach. Subgingival plaque samples and periodontal data were collected from 24 subjects over 2 years. Baseline and 2-year plaque samples were analyzed using quantitative ribosomal 16S cloning and sequencing. Ten subjects remained periodontally healthy over 2 years, the periodontal health of seven subjects worsened, and seven subjects showed clinical improvement. Bacterial stability was greatest among healthy, clinically stable subjects and lowest for subjects whose periodontal status worsened (P ‫؍‬ 0.01). Higher numbers of species lost or gained were also observed for subjects whose clinical status changed (P ‫؍‬ 0.009). This provides evidence that a change in periodontal status is accompanied by shifts within the bacterial community. Based on these data, measures of microbial stability may be useful in clinical diagnosis and prognosis. Regarding individual species, increases in levels of the uncultivated phylotype Veillonella sp. oral clone X042, a gram-negative bacterium and the most common member of the subgingival bacterial community, were associated with periodontal health (P ‫؍‬ 0.04), suggesting that this is an important beneficial species. Filifactor alocis, a gram-positive anaerobe, was found at higher levels in subjects with disease (P ‫؍‬ 0.01).The gingival sulcus contains a complex ecosystem formed by resident and transient bacteria, and the critical role of bacteria in the etiology of chronic periodontitis is well established. The etiology is widely thought to be polymicrobial (16), but the role of individual species and their complex interactions with the host is not well understood. The earliest studies were conducted using open-ended cultivation methods. These were followed by a generation of studies employing molecular detection methods such as hybridization assays and PCR to target bacteria identified by cultivation. These less cumbersome methods allowed much larger scale studies to be carried out. The most recent advance has been cloning and sequencing of 16S rRNA genes. This approach has once again allowed an open-ended exploration of bacterial populations and has revealed the presence of many uncultivated species (18).

Dysbiosis and Alterations in Predicted Functions of the Subgingival Microbiome in Chronic Periodontitis

“…As in previous studies (30,31,48), a difference in microbial composition was apparent between diseased and control sites at all taxonomic levels from phylum to species. Of 457 bacterial taxa, 50 were identified as being significantly enriched in healthy sites by using the LEfSe approach.…”

Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique

“…The exposure of host cells to P. gingivalis invasion resulted in an apparent cell rounding, probably due to cleavage of cadherins and integrins by gingipains, as reported by Sheets et al (2005). These observations support the notion that P. gingivalis is present in small numbers when compared with the total microbiota of the periodontal pockets (Kumar et al, 2006) and, as it remains in the intracellular environment, can multiply and spread to neighboring cells (Tribble and Lamont, 2010), obtaining not only a privileged position to modulate the immune response, causing a 'dysbiosis' between the host and the subgingival microbiota (Darveu et al, 2012), but also a location that allows P. gingivalis to escape from the host eliminating mechanisms and from mechanical periodontal treatment, perpetuating the chronicity of the periodontitis. Furthermore, the existence of overloaded cells supports the ability of the host cells to withstand an extensive P. gingivalis colonization without losing viability, due to the induction of antiapoptotic mechanisms by P. gingivalis as a survival strategy (Nakhjiri et al, 2001).…”

<b>PCR-dipstick DNA chromatography for profiling of a subgroup of cariesassociated bacterial species in plaque from healthy coronal surfaces and periodontal </b><b>pockets </b>

“…While V. parvula appeared to be ubiquitous in intraoral sites (17,38), this species lost the dominant role in periodontally diseased plaque. This result corresponds well with previous investigations that suggested V. parvula as a periodontal health-associated species (14,15,29). S. mutans, commonly taken as a caries initiator and a typical supragingival plaque resident, was (as expected) detected in the vast majority (93.8%) of the supragingival plaque pools, although this frequency was higher than that detected in previous reports (1,2,10,25).…”