5.4 Study IV
5.4.2 The association of clinical parameters with alpha diversity
The associations between all the recorded clinical parameters (presence and amount of periapical lesions, gingival pockets, deep carious lesions, vertical pockets, furcation lesions, tooth brushing frequency and age) and alpha diversity (chao1 index) were assessed. The p-values were calculated with regression analysis (continuous variables), Mann-Whitney test or t-test (discerete variables). The results are presented in Table 6.
The patients who reported brushing their teeth twice a day had the lowest alpha diversity (chao1 index) in the samples, and the patients who brushed their teeth once a day or less frequently had the highest diversity (Figure 8) (p=0.0002; R2 = 34%;
Regression analysis). The regression analyses were repeated with the covariates age and sex [p(adj)=0.09; R2=11%]. To assess in detail which bacterial taxa differed significantly between the groups, differential abundant analysis (DAA) was performed. The abundance of two fusobacteria (both belonging to Leptotrichia -genera) and one bacteroidetes (genus Prevotella) decreased significantly along with tooth brushing frequency (p=0.0001, p=0.0007, and p=0.004, respectively). The sixteen most significant (p<0.1) taxa are presented in Table 7. A trend towards higher alpha diversity in patients than in controls was observed in every diversity index, but no significant difference was observed, as shown in Figure 9.
Figure 7. The relative abundances of major phyla (class level) detected from the gingival pocket crevicular fluid samples of 23 aneurysm patients using 16S rRNA gene sequencing based taxonomy assessment
5.4.2 The association of clinical parameters with alpha diversity
The associations between all the recorded clinical parameters (presence and amount of periapical lesions, gingival pockets, deep carious lesions, vertical pockets, furcation lesions, tooth brushing frequency and age) and alpha diversity (chao1 index) were assessed. The p-values were calculated with regression analysis (continuous variables), Mann-Whitney test or t-test (discerete variables). The results are presented in Table 6.
The patients who reported brushing their teeth twice a day had the lowest alpha diversity (chao1 index) in the samples, and the patients who brushed their teeth once a day or less frequently had the highest diversity (Figure 8) (p=0.0002; R2 = 34%;
Regression analysis). The regression analyses were repeated with the covariates age and sex [p(adj)=0.09; R2=11%]. To assess in detail which bacterial taxa differed significantly between the groups, differential abundant analysis (DAA) was performed. The abundance of two fusobacteria (both belonging to Leptotrichia -genera) and one bacteroidetes (genus Prevotella) decreased significantly along with tooth brushing frequency (p=0.0001, p=0.0007, and p=0.004, respectively). The sixteen most significant (p<0.1) taxa are presented in Table 7. A trend towards higher alpha diversity in patients than in controls was observed in every diversity index, but no significant difference was observed, as shown in Figure 9.
Table 6. The significances of the associations between all the recorded clinical parameters (presence and amount of periapical lesions, gingival pockets, deep carious lesions, vertical pockets, furcation lesions, tooth brushing frequency and age) and alpha diversity (chao1 index). The p-values were calculated with regression analysis (continuous variables), Mann-Whitney test or t-test (discerete variables).
Variable p-value
Periapical lesions yes/no 0.18 6mm and deeper pockets
yes/no 0.40
Number of 6mm and deeper pockets
(min 0; max 14)
0.42
Number of 4-5mm pockets
(min 0; max 11) 0.65
4-5mm pockets yes/no 0.73 Number of deep caries
lesions (min 0; max 5)
0.04
Number of vertical pockets
(min 0; max 2) 0.91
Number of furcation lesions
(min 0; max 4) 0.67
Number of periapical lesions
(min 0; max 6) 0.05
Tooth brushing frequency (less than every day; once a day; twice a day)
0.0002
Age (min 23 years; max 68 years) 0.82
Figure 8. The effect of tooth brushing frequency on the alpha diversity index of the gingival pocket microbiome of the aneurysm patients. Numbers below the x-axis represent tooth brushing frequency (0=less than every day, 1=once a day, 2=two times a day).
Table 6. The significances of the associations between all the recorded clinical parameters (presence and amount of periapical lesions, gingival pockets, deep carious lesions, vertical pockets, furcation lesions, tooth brushing frequency and age) and alpha diversity (chao1 index). The p-values were calculated with regression analysis (continuous variables), Mann-Whitney test or t-test (discerete variables).
Variable p-value
Periapical lesions yes/no 0.18 6mm and deeper pockets
yes/no 0.40
Number of 6mm and deeper pockets
(min 0; max 14)
0.42
Number of 4-5mm pockets
(min 0; max 11) 0.65
4-5mm pockets yes/no 0.73 Number of deep caries
lesions (min 0; max 5)
0.04
Number of vertical pockets
(min 0; max 2) 0.91
Number of furcation lesions
(min 0; max 4) 0.67
Number of periapical lesions
(min 0; max 6) 0.05
Tooth brushing frequency (less than every day; once a day; twice a day)
0.0002
Age (min 23 years; max 68 years) 0.82
Figure 8. The effect of tooth brushing frequency on the alpha diversity index of the gingival pocket microbiome of the aneurysm patients. Numbers below the x-axis represent tooth brushing frequency (0=less than every day, 1=once a day, 2=two times a day).
Table 7. The sixteen most significantly differing taxa (p<0.1) between the tooth brushing frequency groups (twice a day, once a day, less than everyday)
Phylum Class Genus p-value(adj)
Fusobacteria Fusobacteriia Leptotrichia 0.00010
Fusobacteria Fusobacteriia Leptotrichia 0.00059
Bacteroidetes Bacteroidia Prevotella 0.00378
Fusobacteria Fusobacteriia Leptotrichia 0.05961
Fusobacteria Fusobacteriia Leptotrichia 0.05961
Fusobacteria Fusobacteriia Fusobacterium 0.06110
Bacteroidetes Bacteroidia Prevotella 0.07841
Firmicutes Negativicutes Mitsuokella 0.07841
Bacteroidetes Bacteroidia Prevotella 0.07841
Fusobacteria Fusobacteriia Fusobacterium 0.07841
Bacteroidetes Bacteroidia Prevotella 0.09420
Fusobacteria Fusobacteriia Fusobacterium 0.09420 Firmicutes Negativicutes Veillonellaceae_[G-1] 0.09420
Bacteroidetes Bacteroidia Prevotella 0.09420
Bacteroidetes Bacteroidia Porphyromonas 0.09420
Firmicutes Bacilli Lactobacillus 0.09420
Figure 9. Comparison of the alpha diversity of the gingival pocket microbiome between 23 aneurysm patients and 10 voluntary healthy controls
Table 7. The sixteen most significantly differing taxa (p<0.1) between the tooth brushing frequency groups (twice a day, once a day, less than everyday)
Phylum Class Genus p-value(adj)
Fusobacteria Fusobacteriia Leptotrichia 0.00010
Fusobacteria Fusobacteriia Leptotrichia 0.00059
Bacteroidetes Bacteroidia Prevotella 0.00378
Fusobacteria Fusobacteriia Leptotrichia 0.05961
Fusobacteria Fusobacteriia Leptotrichia 0.05961
Fusobacteria Fusobacteriia Fusobacterium 0.06110
Bacteroidetes Bacteroidia Prevotella 0.07841
Firmicutes Negativicutes Mitsuokella 0.07841
Bacteroidetes Bacteroidia Prevotella 0.07841
Fusobacteria Fusobacteriia Fusobacterium 0.07841
Bacteroidetes Bacteroidia Prevotella 0.09420
Fusobacteria Fusobacteriia Fusobacterium 0.09420 Firmicutes Negativicutes Veillonellaceae_[G-1] 0.09420
Bacteroidetes Bacteroidia Prevotella 0.09420
Bacteroidetes Bacteroidia Porphyromonas 0.09420
Firmicutes Bacilli Lactobacillus 0.09420
Figure 9. Comparison of the alpha diversity of the gingival pocket microbiome between 23 aneurysm patients and 10 voluntary healthy controls
6 DISCUSSION
6.1 GENERAL CONSIDERATIONS
About 50% of SAH patients die during the first year (Korja et al, 2013b). The survivors have an excess mortality compared with general population (Korja et al., 2013b).
Those, who survive commonly have deficits in memory, executive function, and language. These cognitive impairments interact to affect patients’ daily functioning and quality of life. Deficits in cognition and day-to-day functioning are further compounded by depression, anxiety, fatigue, and sleep disturbances (Al-Khindi et al., 2010).
It has been shown in many studies that cerebro-cardiovascular diseases are related to inflammatory states (Frösen et al., 2004; Tulamo et al., 2006; Shimonaga et al., 2018), in which oral bacteria play a significant role (Aoyama et al., 2017; Liljestrand et al., 2018; Bagavad et al., 2019). In addition to the studies showing evidence that (oral) bacteria are an independent risk factor for vascular diseases, opposite results exist as well. There is a rather recent study in which a multinomial regression revealed that circulating antibodies against P. gingivalis and A. actinomycetemcomitans correlated with cardiovascular diseases before adding covariates to the model, but after adjusting with covariates, only increasing body mass index, previous smoking and increasing age remained correlated with cardiovascular disease (Damgaard et al., 2017). There is strong, but not established, evidence that periodontal bacteria are linked to the pathogenesis of cardiovascular diseases. Cerebro-cardiovascular diseases and systemic low-grade inflammation share risk factors such as dietary habits, ageing and smoking. Periodontal pathogens have been shown to be correlated with pre-term birth and low birthweight (Daalderop et al., 2018), cardiovascular diseases (Yang et al., 2018) and periodontitis (Oliveira et al., 2016), which are all medical states that basically are driven by a collagen degradation caused by MMP-activation in different sites of the human body (Yabluchanskiy et al., 2013; Chen and Khalil, 2017; L. Zhang et al., 2018).
In all four studies, F. nucleatum appeared to play a role in the results: This bacterium was the second most abundant species in both ruptured and unruptured aneurysm tissue samples (Studies I, II). Along with total bacterial DNA, the amount of DNA from F. nucleatum differed significantly between the groups (gingival pocket depth less than 6mm or more than 6mm), as demonstrated with qPCR (Study III). In the 16S rRNA gene-based metagenomic study (Study IV), differential analysis of abundance (DAA) revealed that the tooth brushing frequency significantly affected two species of the genus Fusobacterium. F. nucleatum is an early colonizer of the gingival pocket (Mark Welch et al., 2016), and has the ability to invade the vessel wall (Velsko et al., 2015). The other main oral bacterial group, streptococci (early colonizers of the oral cavity), was also detected in high numbers (Studies I, II).
Streptococci, as early colonisers of the oral cavity, provide a matrix for all other
6 DISCUSSION
6.1 GENERAL CONSIDERATIONS
About 50% of SAH patients die during the first year (Korja et al, 2013b). The survivors have an excess mortality compared with general population (Korja et al., 2013b).
Those, who survive commonly have deficits in memory, executive function, and language. These cognitive impairments interact to affect patients’ daily functioning and quality of life. Deficits in cognition and day-to-day functioning are further compounded by depression, anxiety, fatigue, and sleep disturbances (Al-Khindi et al., 2010).
It has been shown in many studies that cerebro-cardiovascular diseases are related to inflammatory states (Frösen et al., 2004; Tulamo et al., 2006; Shimonaga et al., 2018), in which oral bacteria play a significant role (Aoyama et al., 2017; Liljestrand et al., 2018; Bagavad et al., 2019). In addition to the studies showing evidence that (oral) bacteria are an independent risk factor for vascular diseases, opposite results exist as well. There is a rather recent study in which a multinomial regression revealed that circulating antibodies against P. gingivalis and A. actinomycetemcomitans correlated with cardiovascular diseases before adding covariates to the model, but after adjusting with covariates, only increasing body mass index, previous smoking and increasing age remained correlated with cardiovascular disease (Damgaard et al., 2017). There is strong, but not established, evidence that periodontal bacteria are linked to the pathogenesis of cardiovascular diseases. Cerebro-cardiovascular diseases and systemic low-grade inflammation share risk factors such as dietary habits, ageing and smoking. Periodontal pathogens have been shown to be correlated with pre-term birth and low birthweight (Daalderop et al., 2018), cardiovascular diseases (Yang et al., 2018) and periodontitis (Oliveira et al., 2016), which are all medical states that basically are driven by a collagen degradation caused by MMP-activation in different sites of the human body (Yabluchanskiy et al., 2013; Chen and Khalil, 2017; L. Zhang et al., 2018).
In all four studies, F. nucleatum appeared to play a role in the results: This bacterium was the second most abundant species in both ruptured and unruptured aneurysm tissue samples (Studies I, II). Along with total bacterial DNA, the amount of DNA from F. nucleatum differed significantly between the groups (gingival pocket depth less than 6mm or more than 6mm), as demonstrated with qPCR (Study III). In the 16S rRNA gene-based metagenomic study (Study IV), differential analysis of abundance (DAA) revealed that the tooth brushing frequency significantly affected two species of the genus Fusobacterium. F. nucleatum is an early colonizer of the gingival pocket (Mark Welch et al., 2016), and has the ability to invade the vessel wall (Velsko et al., 2015). The other main oral bacterial group, streptococci (early colonizers of the oral cavity), was also detected in high numbers (Studies I, II).
Streptococci, as early colonisers of the oral cavity, provide a matrix for all other
bacteria, promoting overall bacterial growth (Kolenbrander et al., 2002). Since most of the bacterial DNA detected from the aneurysm tissue samples was from early colonizers of oral cavity or gingival pockets, the results from Studies I and II suggest that the role of bacteria in the pathogenesis of intracranial aneurysm disease might not be correlated with the severity of periodontal disease. On the other hand, in study III, it was shown that the patients with intracranial aneurysm had significantly deeper gingival pockets than the normal population. A recent Finnish study confirmed these results (Hallikainen et al. 2019). The results from Study I suggest that the bacterial DNA found from the aneurysm tissue could be from bacterial invasion, because simultaneous expression of the bacterial receptors CD14 and TLR2 was observed. TLRs (together with their co-receptors, e.g. CD14) function as important signal transducers mediating innate immune and inflammatory responses to pathogens (Hajishengallis et al.,2002). The alternative options to bacteria playing an active role in the pathogenesis of the aneurysm rupture (bacteria being only innocent bystanders, or DNA being from bacteria degraded by macrophages) are more unlikely.
The results of Study IV support the hypothesis that increase in tooth brushing frequency has a positive effect on the subgingival pocket microbiome, by decreasing the alpha diversity of the gingival pocket microbiome. The effect of tooth brushing could be considered as a long-term effect. Short-term effects on supragingival plaque can be caused by dietary factors, such as high sugar intake and occasional use of mouthwash. However, if tooth brushing frequency remains high enough during a patient’s life time, the bacterial plaque has limited opportunity to invade the subgingival space. When ”invasion to the subgingival space” has occurred, it is no longer possible to disturb the maturation process of the plaque using a tooth brush.