The microbiota (née flora) of the healthy oral cavity is highly diverse and both site- and subject-specific (Aas et al., 2005). Microbial communities form multispecies biofilms which generally exist in harmony with the host. Biofilm-host interaction gives important benefits which contribute to overall health and well-being. Within these biofilms, microorganisms live in close proximity with each another, which results in a wide range of either synergistic or antagonistic interactions. Both composition and function of the microbiome are highly influenced by the oral environment. Changes in local conditions can (in part) determine whether the relationship between the biofilm and the host is symbiotic or potentially damaging (dysbiotic). (Marsh 2003, Roberts and Darveau 2015)
In general, superficial keratinized and non-keratinized mucosa (buccal mucosa, keratinized gingiva and hard palate) consist mainly of firmicutes, followed in decreasing order of relative abundance by proteobacteria, bacteroidetes and either actinobacteria or fusobacteria, whereas in deeper sites, as in gingival pockets, the abundance of firmicutes decreases dramatically and that of actinobacteria increases (Segata et al., 2012).
When this study was planned, only limited number of studies regarding oral bacterial presence in atherosclerotic plaques or vascular samples was available.
Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans had been isolated from the vascular samples (Han et al., 2004;
Saito et al., 2008; Kozarov et al., 2005) Since streptococcae are predominant in the supragingival area (Kolenbrander and London, 1993) and Prevotella intermedia is known to enhance the progression of periodontitis (Maeda et al., 1998) bacteria chosen to deal in this review are Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans.
2.1.1 Porphyromonas gingivalis
Porphyromonas gingivalis is a non-motile, asaccharolytic, obligately anaerobic Gram-negative rod. It forms black-pigmented colonies on blood agar plates and has an absolute requirement for iron and amino acids for growth and energy production (How, Song and Chan, 2016). It produces several virulence factors, the expressions of which are regulated by the external environment of the pathogen. These virulence factors include enzymes (hyaluronidase, chondroitin sulfatase), capsule, fimbriae, lipopolysaccharide, exopolysaccharide, outer membrane proteins, gelatinase, collagenase, trypsin-like protease and aminopeptidase (How et al., 2016). Two widely studied peptidases, gingipains K and R, are responsible for 85% of the proteolytic
dental infectious burden, as well as the bacterial content of the gingival pockets of the patients with intracranial aneurysms, were assessed using clinical examination and molecular analyses methods: qPCR and 16S rRNA gene sequencing.
2 REVIEW OF THE LITERATURE
2.1 PERIODONTAL MICROBIOLOGY
The microbiota (née flora) of the healthy oral cavity is highly diverse and both site- and subject-specific (Aas et al., 2005). Microbial communities form multispecies biofilms which generally exist in harmony with the host. Biofilm-host interaction gives important benefits which contribute to overall health and well-being. Within these biofilms, microorganisms live in close proximity with each another, which results in a wide range of either synergistic or antagonistic interactions. Both composition and function of the microbiome are highly influenced by the oral environment. Changes in local conditions can (in part) determine whether the relationship between the biofilm and the host is symbiotic or potentially damaging (dysbiotic). (Marsh 2003, Roberts and Darveau 2015)
In general, superficial keratinized and non-keratinized mucosa (buccal mucosa, keratinized gingiva and hard palate) consist mainly of firmicutes, followed in decreasing order of relative abundance by proteobacteria, bacteroidetes and either actinobacteria or fusobacteria, whereas in deeper sites, as in gingival pockets, the abundance of firmicutes decreases dramatically and that of actinobacteria increases (Segata et al., 2012).
When this study was planned, only limited number of studies regarding oral bacterial presence in atherosclerotic plaques or vascular samples was available.
Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans had been isolated from the vascular samples (Han et al., 2004;
Saito et al., 2008; Kozarov et al., 2005) Since streptococcae are predominant in the supragingival area (Kolenbrander and London, 1993) and Prevotella intermedia is known to enhance the progression of periodontitis (Maeda et al., 1998) bacteria chosen to deal in this review are Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans.
2.1.1 Porphyromonas gingivalis
Porphyromonas gingivalis is a non-motile, asaccharolytic, obligately anaerobic Gram-negative rod. It forms black-pigmented colonies on blood agar plates and has an absolute requirement for iron and amino acids for growth and energy production (How, Song and Chan, 2016). It produces several virulence factors, the expressions of which are regulated by the external environment of the pathogen. These virulence factors include enzymes (hyaluronidase, chondroitin sulfatase), capsule, fimbriae, lipopolysaccharide, exopolysaccharide, outer membrane proteins, gelatinase, collagenase, trypsin-like protease and aminopeptidase (How et al., 2016). Two widely studied peptidases, gingipains K and R, are responsible for 85% of the proteolytic
activity of P. gingivalis at the site of infection: Gingipain R cleaves bonds after arginines and Gingipain K cleaves after lysines (de Diego et al., 2014). In a susceptible host, these virulence factors cause bone resorption, destruction of periodontal tissues, inhibition of host protective mechanisms and induce cytokine production (How et al. 2016).
P. gingivalis is referred to as a “keystone” pathogen: its presence shifts the microbiota in the periodontal pocket to a dysbiotic one (Hajishengallis, Darveau and Curtis, 2012). The count of P. gingivalis correlates with pocket depth and bleeding on probing (Oliveira et al., 2016).
2.1.2 Fusobacterium nucleatum
Fusobacterium nucleatum is a Gram-negative, anaerobic rod of the mammalian mouth and digestive tract (Strauss et al., 2008). It is a crucial microorganism in dental biofilms because of its coordinator role. It coaggregates with both late and early dental biofilm colonizers, working like a bridge between different bacteria.
(Kolenbrander et al., 2002) Invasion and adherence are the most important mechanisms for F.nucleatum to colonize, disseminate, escape from host defense, and to induce host responses (Han 2015). Adhesin (and invasin) FadA has been identified to bind and to invade host cells being the main virulence factor identified from F.
nucleatum (Xu et al., 2007; Han 2015). F.nucleatum creates favourable conditions for other periodontal bacteria (together with Prevotella intermedia). The mechanisms behind this include promoting neutral pH and a capnophilic environment, which induces increased gingival crevicular fluid (GCF) formation and colonization of another more proteolytic but acid-intolerant bacterium, P. gingivalis. (Takahashi, 2005; Huang, Li and Gregory, 2011) The dependence of P.gingivalis on F.nucleatum has been shown in one cell culture study, in which The human gingival epithelial cell line (Ca9-22) and Human Aorta Endothelial Cell (HAEC) were infected by F. nucleatum and P. gingivalis: Coincubation of P. gingivalis ATCC 33277 with F. nucleatum significantly increased P. gingivalis invasion to host cells, resulting in a 2–20-fold increase in invasion efficiencies (Saito et al., 2008). In another in vitro biofilm study, the presence of Actinomyces naeslundii was shown to be important for the growth of F. nucleatum (Periasamy et al., 2009). Intestinal F. nucleatum is associated with colorectal cancer, and reducing it in patients with colorectal cancer may improve their response to chemotherapy and decrease cancer recurrence (Yu et al., 2017).
2.1.3 The Streptococcus mitis group
Normally, streptococci (together with actinomyces) are predominant in the supragingival area in healthy periodontium as a part of the natural bacterial flora.
They can adhere to the saliva-coated tooth surface by attachment between adhesins and receptors (Kolenbrander and London, 1993). The genus Streptococcus is the most abundant in the salivary microbiome (Nasidze et al., 2009). The Streptococcus mitis group includes 13 species of bacteria. They are Gram-positive microbes sharing
common virulence traits and similar strategies with other bacteria from the family Streptococcaceae to escape from the oral niche and to establish a distant infection in other parts of the host, typically infective endocarditis, meningitis, bacteremia/sepsis or toxic shock-like syndrome (Sitkiewicz, 2018). The virulence factors of S. mitis include capsule, pili, lipoteichoic acid, fibronectin-, laminin-, fibrinogen-, and collagen-binding proteins (Nasidze et al., 2009; Sitkiewicz, 2018).
2.1.4 Prevotella intermedia
Prevotella intermedia, a periodontitis-associated member of the orange complex (F.
nucleatum, P. intermedia, Prevotella nigrescens, Parviomonas micra, Eubacterium nodatum and various Camplylobacter species), is among the most frequently encountered species in subgingival plaque (Kamma et al., 2004). It is an obligately anaerobic, Gram negative, moderately saccaharolytic short rod (0.5 by 2 micrometers) (Chen et al., 2010). The main virulence factors are proteases. P.intermedia encodes beta-lactamases and multidrug/efflux transporters providing resistance to antibiotics. Adhesion, competing with surrounding microbes and horizontal gene transfer are the main drive of the evolution of pathogen. (Ruan et.al., 2015) It has been suggested that P.
intermedia may increase the activity of degradative enzymes (such as alkaline phosphatase, esterase, esterase-lipase and chymotrypsin) under certain conditions and enhance the progression of periodontitis (Maeda et al., 1998). Since P. intermedia has a highly dynamic genome and it can take up various exogenous factors through horizontal gene transfer, it has been suggested that P. intermedia and P. nigrescens may serve as “crucial substances” in subgingival plaque, and that P. intermedia along with other members of the “orange complex” could reflect changes in microbial and environmental dynamics in the subgingival microbiome (Zhang et al., 2017).
2.1.5 Aggregatibacter actinomycetemcomitans
Aggregatibacter actinomycetemcomitans is a Gram-negative, facultative anaerobic bacillus that causes periodontal diseases such as localized aggressive periodontitis (née juvenile periodontitis) (Gholizadeh et al., 2017). Its presence is linked to aggressive periodontitis in adolescents (Fine et al., 2007). Virulence factors for A.
actinomycetemcomitans are endotoxin (as in other Gram-negative bacteria; causes a general pro-inflammatory host response), cytolethal distending toxin (Cdt) and a leukotoxin (LtxA) (Åberg et al., 2014). In the local environment, its presence is linked to periodontal bone loss (Fine et al., 2007). Systemically, the lipopolysaccharide of A.
actinomycetemcomitans is responsible for inducing low-grade systemic inflammation via pro-inflammatory mediators such as interleukins (IL) -1β, −6, −8, and tumour necrosis factor (TNF) -α (Bodet, Chandad and Grenier, 2006). As a result of leukotoxin A production, A.actinomycetemcomitans has substantial pro-inflammatory effects on human brain endothelial cells (Dietmann et al., 2013), and it helps A.
actinomycetemcomitans to evade the host immune system by killing neutrophils, lymphocytes, and monocytes (Sampathkumar et al., 2017).
activity of P. gingivalis at the site of infection: Gingipain R cleaves bonds after arginines and Gingipain K cleaves after lysines (de Diego et al., 2014). In a susceptible host, these virulence factors cause bone resorption, destruction of periodontal tissues, inhibition of host protective mechanisms and induce cytokine production (How et al. 2016).
P. gingivalis is referred to as a “keystone” pathogen: its presence shifts the microbiota in the periodontal pocket to a dysbiotic one (Hajishengallis, Darveau and Curtis, 2012). The count of P. gingivalis correlates with pocket depth and bleeding on probing (Oliveira et al., 2016).
2.1.2 Fusobacterium nucleatum
Fusobacterium nucleatum is a Gram-negative, anaerobic rod of the mammalian mouth and digestive tract (Strauss et al., 2008). It is a crucial microorganism in dental biofilms because of its coordinator role. It coaggregates with both late and early dental biofilm colonizers, working like a bridge between different bacteria.
(Kolenbrander et al., 2002) Invasion and adherence are the most important mechanisms for F.nucleatum to colonize, disseminate, escape from host defense, and to induce host responses (Han 2015). Adhesin (and invasin) FadA has been identified to bind and to invade host cells being the main virulence factor identified from F.
nucleatum (Xu et al., 2007; Han 2015). F.nucleatum creates favourable conditions for other periodontal bacteria (together with Prevotella intermedia). The mechanisms behind this include promoting neutral pH and a capnophilic environment, which induces increased gingival crevicular fluid (GCF) formation and colonization of another more proteolytic but acid-intolerant bacterium, P. gingivalis. (Takahashi, 2005; Huang, Li and Gregory, 2011) The dependence of P.gingivalis on F.nucleatum has been shown in one cell culture study, in which The human gingival epithelial cell line (Ca9-22) and Human Aorta Endothelial Cell (HAEC) were infected by F. nucleatum and P. gingivalis: Coincubation of P. gingivalis ATCC 33277 with F. nucleatum significantly increased P. gingivalis invasion to host cells, resulting in a 2–20-fold increase in invasion efficiencies (Saito et al., 2008). In another in vitro biofilm study, the presence of Actinomyces naeslundii was shown to be important for the growth of F. nucleatum (Periasamy et al., 2009). Intestinal F. nucleatum is associated with colorectal cancer, and reducing it in patients with colorectal cancer may improve their response to chemotherapy and decrease cancer recurrence (Yu et al., 2017).
2.1.3 The Streptococcus mitis group
Normally, streptococci (together with actinomyces) are predominant in the supragingival area in healthy periodontium as a part of the natural bacterial flora.
They can adhere to the saliva-coated tooth surface by attachment between adhesins and receptors (Kolenbrander and London, 1993). The genus Streptococcus is the most abundant in the salivary microbiome (Nasidze et al., 2009). The Streptococcus mitis group includes 13 species of bacteria. They are Gram-positive microbes sharing
common virulence traits and similar strategies with other bacteria from the family Streptococcaceae to escape from the oral niche and to establish a distant infection in other parts of the host, typically infective endocarditis, meningitis, bacteremia/sepsis or toxic shock-like syndrome (Sitkiewicz, 2018). The virulence factors of S. mitis include capsule, pili, lipoteichoic acid, fibronectin-, laminin-, fibrinogen-, and collagen-binding proteins (Nasidze et al., 2009; Sitkiewicz, 2018).
2.1.4 Prevotella intermedia
Prevotella intermedia, a periodontitis-associated member of the orange complex (F.
nucleatum, P. intermedia, Prevotella nigrescens, Parviomonas micra, Eubacterium nodatum and various Camplylobacter species), is among the most frequently encountered species in subgingival plaque (Kamma et al., 2004). It is an obligately anaerobic, Gram negative, moderately saccaharolytic short rod (0.5 by 2 micrometers) (Chen et al., 2010). The main virulence factors are proteases. P.intermedia encodes beta-lactamases and multidrug/efflux transporters providing resistance to antibiotics. Adhesion, competing with surrounding microbes and horizontal gene transfer are the main drive of the evolution of pathogen. (Ruan et.al., 2015) It has been suggested that P.
intermedia may increase the activity of degradative enzymes (such as alkaline phosphatase, esterase, esterase-lipase and chymotrypsin) under certain conditions and enhance the progression of periodontitis (Maeda et al., 1998). Since P. intermedia has a highly dynamic genome and it can take up various exogenous factors through horizontal gene transfer, it has been suggested that P. intermedia and P. nigrescens may serve as “crucial substances” in subgingival plaque, and that P. intermedia along with other members of the “orange complex” could reflect changes in microbial and environmental dynamics in the subgingival microbiome (Zhang et al., 2017).
2.1.5 Aggregatibacter actinomycetemcomitans
Aggregatibacter actinomycetemcomitans is a Gram-negative, facultative anaerobic bacillus that causes periodontal diseases such as localized aggressive periodontitis (née juvenile periodontitis) (Gholizadeh et al., 2017). Its presence is linked to aggressive periodontitis in adolescents (Fine et al., 2007). Virulence factors for A.
actinomycetemcomitans are endotoxin (as in other Gram-negative bacteria; causes a general pro-inflammatory host response), cytolethal distending toxin (Cdt) and a leukotoxin (LtxA) (Åberg et al., 2014). In the local environment, its presence is linked to periodontal bone loss (Fine et al., 2007). Systemically, the lipopolysaccharide of A.
actinomycetemcomitans is responsible for inducing low-grade systemic inflammation via pro-inflammatory mediators such as interleukins (IL) -1β, −6, −8, and tumour necrosis factor (TNF) -α (Bodet, Chandad and Grenier, 2006). As a result of leukotoxin A production, A.actinomycetemcomitans has substantial pro-inflammatory effects on human brain endothelial cells (Dietmann et al., 2013), and it helps A.
actinomycetemcomitans to evade the host immune system by killing neutrophils, lymphocytes, and monocytes (Sampathkumar et al., 2017).
2.1.6 Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Prevotella intermedia and Aggregatibacter
actinomycetemcomitans in cardiovascular diseases
Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels and they include: coronary heart disease, peripheral arterial disease, cerebrovascular disease, rheumatic heart disease, congenital heart disease, pulmonary embolism and deep vein thrombosis. CVDs are the number one cause of death globally: more people die annually from CVDs than from any other cause.
There is an association between chronic periodontitis and CVDs (Li et al., 2017).
Abilities of bacteria to invade vascular tissue and intravascular plaque
The ability of F. nucleatum to invade blood vessels has been demonstrated in cell culture studies and animal models (Han et al., 2004; Saito et al., 2008; Velsko et al., 2015; Chukkapalli et al., 2017). In humans, the presence of P. gingivalis, A.
actinomycetemcomitans and F. nucleatum was assessed in atheromatous plaque samples obtained from endarterectomies (n=42) using nested PCR: 78.6% of the samples were positive for P. gingivalis DNA, 66.7% for A. actinomycetemcomitans and 50.0% for F. nucleatum DNA. The simultaneous presence of various bacterial species within the same specimen was a common observation. (Figuero et al., 2011) The presence of A. actinomycetemcomitans (together with P. gingivalis) has been confirmed within the coronary artery plaque by cell culture invasion assays and immunofluorescence microscopy together with positive PCR findings (Kozarov et al., 2005). The bacterial DNA of S. mitis has been found in thrombus aspirates (Pessi et al., 2013) and atherosclerotic plaque (Eberhard et al., 2017). Cocci and rods were seen in scanning electron microscopy or in transmission electron microscopy together with positive next-generation sequencing (ngs) results (Pessi et al., 2013; Armingohar et al., 2014). It has been suggested that high blood pressure at the aortic valve may prevent the adhesion and proliferation of bacterial colonies (Raffaelli et al., 2010).
PCR was used in order to detect periodontal pathogens (Tannerella forshytia, P.
gingivalis, A. actinomycetemcomitans, P. intermedia, F. nucleatum, Campylobacter rectus, Eikenella corrodens and Treponema denticola), but neither 19 aortic valve specimens nor the blood samples were positive for the genoma of these pathogens (Raffaelli et al., 2010).
Abilities of bacteria to invade myocardium
The presence of A. actinomycetemcomitans, P. gingivalis, Tannerella forsythia, T.
denticola, P. intermedia, Parvimonas micra, F. nucleatum, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens and Capnocytophaga species in adult human myocardium has been confirmed, with an immunohistochemically detected impact on atrial and myocardial tissue bacterial recognition molecules, such as CD14, CD68
and lipopolysaccharide binding protein (LBP) (Ziebolz et al., 2018). In children with congenital heart disease, the presence of A. actinomycetemcomitans in myocardial tissue was negative, although 8% of saliva samples were positive (Bozdogan et al., 2016).
Porphyromonas gingivalis in cardiovascular diseases
The presence of P. gingivalis in the gingival pockets has been associated with uncontrolled diabetes and cardiovascular diseases (Norio Aoyama, Suzuki, Kobayashi, et al., 2018). The presence of P. gingivalis has been detected at the sites of atherosclerotic disease in humans by cell culture invasion assays and immunofluorescence microscopy (Kozarov et al. 2005) and in several PCR or 16S rRNA gene-based studies (Mahendra et al., 2015; Szulc et al., 2015; Mahalakshmi, Krishnan and Arumugam, 2017; Mougeot et al., 2017; Atarbashi-Moghadam et al., 2018). It has been shown that P. gingivalis has the ability to invade host cells, providing an escape from the action of the immune system or therapeutic agents.
This characteristic is thought to be a key virulence factor, along with the presence of a bacterial capsule, lipopolysaccharide, and secretion of proteolytic enzymes (gingipains) (Bostanci and Belibasakis, 2012). Gingipains have been shown to be able to cause neurodegeneration and amyloid accumulation, linked to Alzheimer´s disease, in mice (Ilievski et al., 2018) and in humans (Dominy et al., 2019). In a cell culture model, the modulative action of P. gingivalis on the cytokine response in the cells was demonstrated: It disrupts the adhesion activity and the viability through the action of Arg-gingipain and Lys-gingipain and thereby may contribute to the pathogenesis of cardiovascular diseases (Baba et al., 2002). In addition, it has been shown that P. gingivalis can induce high-density lipoprotein (HDL) oxidation, weakening the atheroprotective function of HDL and even making it proatherogenic (H.-J. Kim et al., 2018). The ability of P. gingivalis and F. nucleatum (together with T.
denticola and Tannerella forsythia) to directly and actively invade the aortic adventitial layer of integrin beta 6 -/- mice has been shown by fluorescence in an in situ hybridization study. Along with invasion, alterations in Toll-like receptor (TLR) and nucleotide-binding domain and leucine-rich-repeat-containing receptor (NLR) gene expressions were observed. (Velsko et al., 2015) The invasive properties of P.
gingivalis are partly dependent on other bacteria, as was shown in a cell culture study by Saito and colleagues (Saito et al., 2008). Animal studies have shown the impact of P. gingivalis on cardiac hypertrophy via oxidative stress (Sato et al., 2016) and cardiac rupture after myocardial infarct, as well as its ability to invade the ischemic myocardium and promote cardiomyocyte apoptosis (Shiheido et al., 2016).
In conclusion, there is strong evidence that all these 5 bacteria (F. nucleatum, P.
gingivalis, A. actinomycetemcomitans, S. mitis and P. intermedia) are cabable of invading the human vascular wall.
2.1.6 Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Prevotella intermedia and Aggregatibacter
actinomycetemcomitans in cardiovascular diseases
Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels and they include: coronary heart disease, peripheral arterial disease, cerebrovascular disease, rheumatic heart disease, congenital heart disease, pulmonary embolism and deep vein thrombosis. CVDs are the number one cause of death globally: more people die annually from CVDs than from any other cause.
There is an association between chronic periodontitis and CVDs (Li et al., 2017).
Abilities of bacteria to invade vascular tissue and intravascular plaque
The ability of F. nucleatum to invade blood vessels has been demonstrated in cell culture studies and animal models (Han et al., 2004; Saito et al., 2008; Velsko et al., 2015; Chukkapalli et al., 2017). In humans, the presence of P. gingivalis, A.
actinomycetemcomitans and F. nucleatum was assessed in atheromatous plaque samples obtained from endarterectomies (n=42) using nested PCR: 78.6% of the samples were positive for P. gingivalis DNA, 66.7% for A. actinomycetemcomitans and 50.0% for F. nucleatum DNA. The simultaneous presence of various bacterial species within the same specimen was a common observation. (Figuero et al., 2011) The
actinomycetemcomitans and F. nucleatum was assessed in atheromatous plaque samples obtained from endarterectomies (n=42) using nested PCR: 78.6% of the samples were positive for P. gingivalis DNA, 66.7% for A. actinomycetemcomitans and 50.0% for F. nucleatum DNA. The simultaneous presence of various bacterial species within the same specimen was a common observation. (Figuero et al., 2011) The