Periodontitis is a microorganism-triggered, inflammation-driven disease that primarily affects the tooth-supporting tissues. Impacting over 10% of the global population, periodontitis is one of the leading causes of tooth loss in adults [1,2]. The disease's pathogenesis is intimately linked with alterations in the microbial composition and load of the subgingival and supragingival microbiota [[3], [4], [5]]. In a healthy state, the microbial community within dental plaque is balanced, maintaining periodontal pathogens at minimal levels [[6], [7], [8]]. However, inadequate oral hygiene can lead to plaque accumulation, resulting in gingival inflammation and the initial formation of periodontal pockets [5,9]. These changes in the microenvironment, such as hypoxia and increased crevicular fluid, create an optimal niche for the proliferation of periodontal pathogens, especially the “red complex” bacteria (P. gingivalis, T. forsythia, T. denticola) [8,10]. An increase in the relative abundance and quantity of these pathogenic bacteria intensifies periodontal inflammation, establishing a self-sustaining cycle of microbial imbalance and disease progression [[10], [11], [12]]. Given the critical role of microbial composition and load shifts in the onset and progression of periodontitis, investigating these changes is essential for understanding pathogenic mechanisms and discovering new therapeutic targets [13].

Investigating these shifts requires simultaneous evaluation of both microbial load and community composition. However, the predominant method, relative microbiome profiling (RMP), has inherent limitations due to its normalization process, which confines analysis to shifts in bacterial proportions and neglects changes in microbial load [14,15]. This limitation poses significant risks of misinterpretation when examining microbial dynamics during disease progression [15,16]. For example, if disease activity modifies the total microbial load, a microbial population with a stable quantity might appear to passively increase or decrease in relative abundance due to proportional recalculation. Such artifacts could erroneously associate bacteria with health or disease, potentially obscuring genuine associations with disease status [14,15,17]. The emergence of quantitative microbiome profiling (QMP) addresses these limitations. Unlike qPCR, which quantifies only a limited subset of bacterial species, QMP enables the simultaneous measurement of the absolute quantity changes of all detectable microorganisms within a microbial community [15,18]. By eliminating reliance on proportional data, QMP avoids the false associations inherent in RMP analysis [14,15]. Crucially, the combination of QMP and RMP allows for simultaneous monitoring of both biomass fluctuations and community structure dynamics, which might provide a comprehensive view of microbial behavior in periodontitis and reveal previously inaccessible insights into microbial drivers of disease progression.

Dental plaque, particularly subgingival plaque, is widely acknowledged as the primary initiator of periodontal disease [1,10]. Although its microbial composition and microbial load are directly linked to disease pathogenesis, challenges in sample collection and difficulties in accurately quantifying the mass and volume of samples hinder the application of absolute quantitative sequencing techniques. Conversely, saliva offers practical advantages: it can be non-invasively collected using standardized protocols, allowing precise measurement of volume and mass [19,20]. Furthermore, the salivary bacteriome has demonstrated strong correlations with both subgingival and supragingival plaque bacteriomes [21,22], as well as with periodontal conditions [20,23]. These attributes position saliva as a preferable sample for assessing microbial load and composition shifts in the oral bacteriome during periodontitis development.

Therefore, this study aims to combine QMP and RMP to characterize the salivary microbiota of subjects diagnosed with periodontal health, stage I/II periodontitis, and stage III/IV periodontitis. The null hypothesis is that the salivary microbiota as explored using QMP and RMP do not differ significantly between subjects in periodontal health and in different stages of periodontitis.