The oral microbiota is attracting increased attention because of probable associations with systemic and metabolic disorders [21–23]. The associations between the oral microbiota and systemic/metabolic disorders can be explained in the following two aspects. On the one hand, microbial dysbiosis in the oral cavity is a source of systemic inflammation, which could lead to chronic low-grade inflammation and adversely affects the systemic health of the host [6]. On the other hand, the oral microbiota can influence the composition of the gut microbiome, which plays important roles in systemic health [24–27]. Due to the anatomical position, about 1011 bacteria are swallowed from the oral cavity to the stomach every day [25], cultivation and sequencing techniques have also substantiated the association between the oral and gut microbiomes: Arimatsu et al. reported that oral administration of P. gingivalis significantly altered the Firmicutes/Bacteroidetes ratio, a significant index to evaluate the health status of the gut microbiome [26]; Li et al. found that the oral microbiota could overcome physical barriers and colonize the gut in gnotobiotic mice [27]. These findings acknowledged that the oral microbiota plays an important role in the development of systemic and metabolic diseases via “oral-gut axis”. As for MAFLD, although some oral resident microbes have been associated with the development of it [7, 8], there has been no microbiome-wide association study of the association between the development of MAFLD and oral microbial ecology. Shaped by the health status of the host, supragingival plaque has been related to various metabolic disorders. For example, Hintao et al. reported significant differences in the microbial profiles of supragingival plaque between subjects with and without diabetes [28]; La Monte et al. found that metabolic syndrome was significantly associated with supragingival plaque (odds ratio = 1.74; 95% confidence interval = 1.22–2.50) [29]. Considering supragingival plaque can be obtained with minimal discomfort and risk [9], it was collected in this study to explore the ecological shifts of oral microbiota in MAFLD patients. By screening with strict inclusion/exclusion criteria and matching of confounding factors, the differences among the participants were minimized as much as possible in order to focus on compositional and structural differences of the supragingival microbiota in MAFLD patients.
The diversity of the supragingival microbiota of each group was determined using alpha diversity estimators. It is generally acknowledged that microbial diversity reflects the health status of the host. For example, decreased diversity of gut microbiota indicates functional or metabolic disorders in the host [30], while increased diversity of oral microbiota is reported to imply poor oral [31, 32] and holistic health [21–33] because in a state of poor oral health, gingival bleeding provides a richer nutrient source [25]. In the present study, increased diversity (lower Simpson index and higher Shannon index) of the supragingival microbiota in the MAFLD group was observed, suggesting possible alterations to the nutritional status of supragingival plaque in MAFLD patients.
Consistent with previous studies [9, 34], the core phyla identified in the present study included Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, and Fusobacteria, which accounted for 93.83% and 92.37% of the supragingival microbiomes of the control and MAFLD groups, respectively. Similarly, although the proportions differed, the majority of the observed genera (including Capnocytophaga, Leptotrichia, Corynebacterium, Actinomyces, Streptococcus, Fusobacterium, Prevotella, Veillonella, Neisseria, and Comamonas) existed in both groups, thereby also supporting the core genera of the supragingival microbiota [9]. A lower Firmicutes/Bacteroidetes ratio is considered as a healthy trait in both the oral cavity and gut [22]. In the present study, the Firmicutes/Bacteroidetes ratio was lower in the supragingival plaque of the control group as compared to the MAFLD group (61.41% vs. 72.38%, respectively), indicating dysbiosis of the supragingival microbiota of the MAFLD group.
The PCoA and PLS-DA results demonstrated differences in the community compositions between the two groups (Adonis, P = 0.0120). The discriminatory taxa between two groups were identified using LEfSe. At the genus level, Actinomyces and Prevotella 2 had the highest LDA scores in the MAFLD group. Actinomyces spp. are normal resident bacteria of the oral cavity, which exert important roles in biofilm formation [35]. Actinomyces spp. have been associated with the severity of chronic periodontitis [36]. Prevotella 2 is a genus of Gram-negative, anaerobic bacteria that exist in the gut and are relevant to multiple disease states, including an increased lifetime risk of cardiovascular disease [30], ankylosing spondylitis [37], and increased levels of C-reactive protein [38]. Considering the consistency between the oral and gut microbiotas [25–27], the prevalence of Prevotella 2 in the oral cavity is proposed as a potential marker of systematic diseases including MAFLD. In healthy participants, the genera Neisseria and Bergeyella had the highest LDA scores. Neisseria spp. are among the most abundant taxa in the oral cavity [39]. A predominance of Neisseria spp. in the oral cavity indicates healthy conditions of the oral cavity [40, 41, 42]. Bergeyella spp. are Gram-negative, aerobic bacteria [43]. In the present study, Bergeyella spp. were more prevalent in the control group, suggesting a negative correlation to MAFLD.
Co-occurrence networks were used to predict inter-genera correlations of supragingival plaque between the two groups. As shown in Fig. 3, there were significant differences in the interaction patterns of the two groups. In the MAFLD group, there were stronger and more complex interactions within the main cluster, but weaker and sparser correlations among the genera outside of the main cluster. Reportedly, an increase in interaction strength among taxa not only excludes other taxa, but decreases the stability of the microbial community [44]. Therefore, it could be speculated that the supragingival microbial community of the NAFLD group was more unstable.
Inhibition of hepatic glucose production, increased accumulation of lipids in the liver, and IR are vital to the development of MAFLD [45]. It is currently believed that IR is an independent risk factor for the severity of MAFLD [45]. As first proposed by Matthews et al. in 1985, HOMA-IR is both practical and highly efficient for the evaluation of IR in both clinical and scientific studies [12, 46]. In the present study, HOMA-IR was beyond the normal range (normal range ≤ 1) in the MAFLD group and significantly higher than that in the control group (P = 0.0013) suggesting that IR is prevalent in patients with MAFLD. It was believed that chronic low-grade inflammation resulting from dysbiosis of the oral microbiota can reportedly aggravate IR [47]. In this study, Spearman’s correlation analysis revealed that the presence of Granulicatella, Veillonella, Streptococcus, and Scardovia spp. was positively correlated with HOMA-IR. The congregation of Granulicatella spp. with Aggregatibacter actinomycetemcomitans [48] has been positively correlated to periodontitis [49], as well as serious infections outside of the oral cavity, such as infective endocarditis [50]. Veillonella is a genus of Gram-negative anaerobic bacteria mainly found in the oral and gastrointestinal tracts. The presence of Veillonella spp. in the oral cavity has been correlated to increased production of pro-inflammatory cytokines [47, 51] and periodontal infections [41]. Streptococcus and Scardovia spp. are resident bacteria of the oral cavity that are closely related to caries formation [52]. Although relatively few studies have investigated the relationship between caries-related bacteria and IR, patients with IR tend to have more decayed teeth [53].
In a state of chronic low-grade inflammation [54], obesity is a contributor to various metabolic dysfunctions, such as MAFLD and type 2 diabetes [55]. As compared to BMI, visceral adiposity, as measured by waist circumference, has been closely linked to the severity of lipid deposition in the liver [54], which is consistent with the results of the present study, which found an increase in waist circumference in MAFLD patients (P = 0.0020). In addition, multiple studies have verified the influence of obesity on the microbial profile of the oral cavity [56, 57]. In this study, genera positively correlated with obesity mainly included Streptococcus, Oslenella, Scardovia, and Selenomonas. Streptococcus and Scardovia spp. were also positively correlated to IR, supporting the positive association between obesity and IR [54]. The involvement of Oslenella spp. in endodontic infections [58] and periodontal inflammation [22] have been well documented. In the Veillonellaceae family, Selenomonas is a genus of Gram-negative anaerobic bacteria. Members of Veillonellaceae family are considered to act as pro-inflammatory mediators [59] and putative periodontal pathogens [60]. These results support the presumption that obesity is positively correlated to the abundance of bacteria associated with infectious diseases of the oral cavity [61].
Dyslipidemia is a common clinical manifestation of MAFLD, especially hypertriglyceridemia and low serum HDL-C [14, 15, 62], which were also verified in this study (P = 0.0083 for TG; P = 0.0011 for HDL-C). Reportedly, oral infectious diseases and dyslipidemia could have a two-way relationship without a clear cause-and-effect relationship [63]. Actinomyces spp. have been positively correlated to TG levels as a potential indicator of MAFLD-related metabolic dysfunction. A surprising result was that the presence of Aggregatibacter spp. was negatively correlated with TG levels, but positively correlated with HDL-C levels, which might indicate good health, challenging the mainstream concept that the presence of Aggregatibacter spp. (especially A. actinomycetemcomitans) is related to dyslipidemia and other metabolic diseases [63]. Sampling sites may explain this discrepancy because Aggregatibacter spp. are anaerobic bacteria with growth behaviors that may change in response to aerobic conditions (supragingival habitats). However, the exact reasons for this paradox remain unclear.
Known as indicators of hepatocellular damage, elevated serum levels of transaminases and transpeptidases are also main clinical manifestations of MAFLD [2]. Moreover, a decreased AST/ALT ratio is regarded as biomarker of progressive MAFLD [2]. In this study, a decreased AST/ALT ratio (P = 0.0142) as well as elevated GGT (P = 0.0084) were prevalent in MAFLD patients, suggesting the enrolled MAFLD patients had different degrees of hepatocellular damage. Capnocytophaga is a genus of Gram-negative anaerobic bacteria reportedly associated with periodontitis [36] and hyperglycemia [64]. In this study, an abundance of Capnocytophaga spp. was negatively correlated to the AST/ALT ratio, suggesting it could be a potential biomarker of MAFLD progression.
Metagenomic predictions based on PICRUSt2 revealed that functional changes between the control and MAFLD groups mainly involved metabolism (KEGG pathway level 2). Among the KEGG pathways level 3, metabolism of sugars (mainly free sugars, including starch and sucrose, fructose and mannose, and galactose) was more prevalent in subjects with MAFLD, revealing that supragingival plaque in MAFLD patients can easily obtain nutrients, which could explain the increased microbial diversity observed in the supragingival plaque of the MAFLD group (Table 2). Pathways related to aerobic respiration (including oxidative phosphorylation, pyruvate metabolism, and the citrate cycle) were more abundant in the supragingival plaque of the control group, suggesting that the proportion of aerobic bacteria in the supragingival plaque is higher in healthy people. Produced by oral anaerobic bacteria, volatile sulfur butanoate compounds (VSCs) have been positively correlated with halitosis [65] and as indicators of the severity of oral infectious diseases and other disorders of the digestive system [66]. The results of the present study showed that the metabolic pathways of VSC precursors (cysteine and methionine) were significantly over-represented in healthy individuals, which may alleviate halitosis and maintain good oral health. However, a deficiency of predicting functions based on taxa composition is that bacterial functions can change with the health status of the host [65]. Consequently, metatranscriptomics and metabolomics of the microbiota may provide more realistic functional profiles.
As this was a pilot study with matching of confounding factors, some intriguing findings surfaced, but still need to be verified in future studies with larger sample sizes. In addition, with the increasing attention to the functions of the oral microbial community, it is essential to identify changes to the actual functional profiles of the supragingival microbiota in MAFLD by metatranscriptomics and metabolomics.