Mobile Microbiome

Abstract

The link between oral infections and adverse systemic conditions has attracted much attention in the research community. Several mechanisms have been proposed, including spread of the oral infection due to transient bacteremia resulting in bacterial colonization in extra-oral sites, systemic injury by free toxins of oral pathogens, and systemic inflammation caused by soluble antigens of oral pathogens. Mounting evidence supports a major role of the systemic spread of oral commensals and pathogens to distant body sites causing extra-oral infections and inflammation. We review here the most recent findings on systemic infections and inflammation complicated by oral bacteria, including cardiovascular disease, adverse pregnancy outcomes, rheumatoid arthritis, inflammatory bowel disease and colorectal cancer, respiratory tract infections, and organ inflammations and abscesses. The recently identified virulence mechanisms of oral species Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, and Campylobacter rectus are also reviewed. A pattern emerges indicating that only select subtype(s) of a given species, e.g., F. nucleatum subspecies animalis and polymorphum and S. mutans non-c serotypes, are prone to extra-oral translocation. These findings advocate the importance of identification and quantification of potential pathogens at the subtype levels for accurate prediction of disease potential.

Introduction

The theory of focal infection, i.e., the idea that oral conditions can significantly influence diseases elsewhere in the body, first became popular during the late 19th and early 20th centuries, but started to fall out of favor by the 1930s due to lack of scientific support. Interestingly, increasing evidence over the past 3 or so decades suggests that, due to dental bacteremia, the oral cavity can indeed serve as a reservoir for systemic dissemination of pathogenic bacteria and their toxins, leading to infections and inflammation in distant body sites. With the application of culture-independent technologies for microbial detection and identification, a diverse group of oral species has been found to be directly involved in infections at extra-oral sites. Advances in the human microbiome research have also facilitated a shift of focus from oral diseases to oral bacteria in systemic infections. In this review, we will summarize recent advances in the identification and detection of oral bacteria in extra-oral infections, using PubMed searches with key words such as oral bacteria, systemic disease, atherosclerosis, pregnancy, pneumonia, inflammatory bowel disease, colorectal cancer, rheumatoid arthritis, etc. The pathogenic mechanisms of select species in these infections are also reviewed.

Systemic Diseases Implicated by Oral Bacteria

Summarized in Table 1 are representative oral species that have been detected in various systemic infections and inflammation. More detailed descriptions are below, with an emphasis on the most recent findings.

Table 1.

Summary of Oral Species Implicated in Extra-oral Infections and Inflammation

Extra-oral Infections Complicated by Oral Bacteria	Oral Species Detected*
Cardiovascular diseases (CVD)	Aggregatibacter actinomycetemcomitans
	Campylobacter rectus Chlamydia pneumoniae
	Eikenella corrodens
	Fusobacterium necrophorum
	Fusobacterium nucleatum
	Porphyromonas gingivalis
	Prevotella intermedia
	Streptococcus mitis
	Streptococcus mutans
	Streptococcus oralis
	Tannerella forsythia
	Treponema denticola
Adverse pregnancy outcomes (APO)	Begeyella spp.
	Campylobacter rectus
	Capnocytophaga spp.
	Eikenella corrodens
	Fusobacterium nucleatum
	Peptostreptococcus micros
	Porphyromonas gingivalis
	Prevotella intermedia
	Prevotella nigrescens
	Rothia dentocariosa
	Streptococcus mutans
	Tannerella forsythia
	Treponema denticola
Rheumatoid arthritis (RA)	Fusobacterium nucleatum
	Porphyromonas gingivalis
	Prevotella intermedia
	Prevotella melaninogenica
	Serratia proteamaculans
	Tannerella forsythia
Inflammatory bowel disease (IBD) and colorectal cancer (CRC)	Campylobacter concisus
	Fusobacterium nucleatum
	Streptococcus mutans
Respiratory tract infections (RTI)	Staphylococcus aureus
	Pseudomonas aeruginosa
	Acinetobacter species
	Candida albicans
	Enteric species
Meningitis or brain abscesses	Campylobacter rectus
	Fusobacterium necrophorum
	Fusobacterium nucleatum
	Porphyromonas gingivalis
	Streptococcus intermedius
Lung, liver, or splenic abscesses	Fusobacterium necrophorum
	Fusobacterium nucleatum
	Porphyromonas gingivalis
	Prevotella spp.
	Treponema denticola
Appendicitis	Fusobacterium necrophorum
	Fusobacterium nucleatum

^*References are provided in the Appendix Table.

Atherosclerotic Disease (AD)

Atherosclerotic disease, including myocardial infarction and stroke, is the leading cause of death worldwide. Although conditions such as smoking, obesity, high blood pressure, and diabetes are well known to contribute to AD, studies show that up to half of individuals with AD may not have any of these traditional risk factors (Seymour et al., 2007), suggesting the presence of additional causes. The implication of oral bacteria in the initiation and progression of atherosclerosis is now widely accepted. Detection of oral bacterial DNA in human atheromatous plaques was first reported in 2000 (Haraszthy et al., 2000). More recently, Ford et al. detected oral bacterial DNA in coronary artery biopsies from patients diagnosed with coronary artery disease and in endarterectomy specimens from patients undergoing surgical treatment of atherosclerosis (Ford et al., 2005; Ford et al., 2006). Collectively, the following species have been detected: Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia, with some of the samples containing more than one type of bacteria.

Given the association between periodontal disease and AD, it is not surprising to find periodontal bacterial DNA in AD lesions (Table 1). The finding of cariogenic Streptococcus mutans in AD, however, was rather intriguing. In a large-scale study of 203 consecutive patients, which included 82 aortic valve specimens, 35 mitral valve specimens, and 86 aortic aneurysmal wall specimens, Nakano et al. found that S. mutans was the most frequently detected species, with a detection rate of 42.7% in the heart valves and 62.8% in the aneurysm walls, respectively. S. mutans DNA was also detected in dental plaque from most of the patients (Nakano et al., 2009). Furthermore, while the majority of cariogenic S. mutans belongs to serotypes c, e, and f, the AD-associated S. mutans belong to a novel serotype, k, characterized by a drastic reduction in the amount of glucose side-chains and reduced cariogenicity (Nakano et al., 2007a; Nakano et al., 2007b; Nakano et al., 2010).

Adverse Pregnancy Outcomes (APO)

The readers are referred to a recent review with regard to epidemiologic and interventional studies on the link between periodontal infections and APO (Han, 2011b). APO is a broad term including preterm labor, preterm premature rupture of membranes, pre-eclampsia, miscarriage, intra-uterine growth retardation, low birthweight, stillbirth, and neonatal sepsis. The oral species implicated in APO are summarized in Table 1.

F. nucleatum is by far the most prevalent oral species in APO and has been detected in a wide variety of placental and fetal tissues, including amniotic fluid, fetal membranes, cord blood, neonatal gastric aspirates, and fetal lung and stomach, associated with preterm birth, stillbirth, and early-onset neonatal sepsis (Gonzales-Marin et al., 2011; Han et al., 2009, 2010; Wang et al., 2013). A case report of term stillbirth caused by oral F. nucleatum provided the first human evidence that the bacterium originated from the mother’s subgingival plaque and translocated to the placenta and fetus, causing acute inflammation leading to the fetal demise (Han et al., 2010). While F. nucleatum has been detected as the most prevalent species in amniotic fluid from pregnancies complicated by preterm birth (Han et al., 2009), its recent detection in cord blood associated with early-onset neonatal sepsis is rather novel (Wang et al., 2013). The high invasiveness of F. nucleatum, and thus its ability to spread to different placental and fetal compartments likely underlies its simultaneous detection in the matching amniotic fluid and cord blood samples (Wang et al., 2013). There are currently 5 subspecies (sbsp) of F. nucleatum, i.e., sbsp nucleatum, sbsp polymorphum, sbsp fusiform, sbsp vincentii, and sbsp animalis. Only 2 of these 5 subspecies have been detected in intra-uterine infections, with the overwhelming majority belonging to sbsp animalis, and an occasional few belonging to sbsp polymorphum (Table 2).

Table 2.

F. nucleatum Subspecies Identified in Intra-uterine Infection

		Fn Subspecies Identified*	Source**	References
Cultivated strains	YWH7052	F. nucleatum ss. animalis	PTB AF	Han et al., 2004
	YWH7055	F. nucleatum ss. animalis	PTB AF	Han et al., 2004
	YWH7053	F. nucleatum ss. animalis	PTB placentas	Han et al., 2004
	YWH7056	F. nucleatum ss. animalis	PTB placentas	Han et al., 2004
	YWH7199	F. nucleatum ss. animalis	Stillbirth placenta	Han et al., 2010
Accession numbers	EU644455	F. nucleatum ss. animalis	PTB AF	Han et al., 2009
	EU644461	F. nucleatum ss. animalis	PTB AF	Han et al., 2009
	EU644463	F. nucleatum ss. animalis	PTB AF	Han et al., 2009
	EU644464	F. nucleatum ss. animalis	PTB AF	Han et al., 2009
	EU644466	F. nucleatum ss. animalis	PTB AF	Han et al., 2009
	EU644478	F. nucleatum ss. animalis	PTB AF	Han et al., 2009
	EU644480	F. nucleatum ss. animalis	PTB AF	Han et al., 2009
	JN546083	F. nucleatum ss. animalis	PTB AF (with EONS)	Wang et al., 2013
	JN546084	F. nucleatum ss. animalis	PTB AF	Wang et al., 2013
	JN584646	F. nucleatum ss. polymorphum	PTB AF	Wang et al., 2013
	JN546087	F. nucleatum ss. animalis	PTB AF	Wang et al., 2013
	JQ901488	F. nucleatum ss. animalis	PTB CB (with EONS)	Wang et al., 2013
	JN546096	F. nucleatum ss. animalis	PTB CB	Wang et al., 2013
	JN546097	F. nucleatum ss. animalis	PTB CB	Wang et al., 2013
	JN546100	F. nucleatum ss. animalis	PTB CB	Wang et al., 2013
	N/A	F. nucleatum ss. polymorphum	neonatal gastric aspirates	Gonzales-Marin et al., 2013

^*Those with accession numbers were detected by PCR and clone analysis. Species and subspecies identification was performed based on the full-length 16S rRNA gene sequences from the Human Oral Microbiome Database (HOMD). ss, subspecies.

^**AF, amniotic fluid; PTB, preterm birth; EONS, early-onset neonatal sepsis; CB, cord blood.

P. gingivalis was first detected in the amniotic fluid in a case of threatened premature labor and periodontitis (Leon et al., 2007). P. gingivalis antigens were detected by immunocytochemistry in the placental syncytiotrophoblasts, chorionic trophoblasts, decidual cells, amniotic epithelial cells, and vascular cells, with a substantial increase in the intensity of immunostaining of the tissues obtained from women with chorioamnionitis compared with those with normal-term pregnancy (Katz et al., 2009).

Uncultivated oral Bergeyella spp. is a novel genus only recently associated with APO. It was first detected in the amniotic fluid from a case of preterm birth, owing to the use of 16S rRNA gene-based culture-independent technology (Han et al., 2006). The clone identified in the amniotic fluid was detected in the mother’s subgingival plaque but not in her vaginal flora. Since then, Bergeyella has been repeatedly detected in amniotic fluid associated with preterm birth, and most recently in cord blood associated with neonatal sepsis (Han et al., 2009; Wang et al., 2013). Similar to F. nucleatum, Bergeyella sp. was detected in paired amniotic fluid and cord blood samples suggesting its ability to invade the fetal compartment (Wang et al., 2013). Oral bacteria are often detected in clinical samples as mixed infections, indicating co-translocation from the oral cavity (Han et al., 2009; Wang et al., 2013). These observations are supported by animal studies in which a variety of cultivated and uncultivated oral species were found to co-translocate to the mouse placenta (Fardini et al., 2010). The majority of oral species translocated to the murine placenta were oral commensals, suggesting that commensals in the oral cavity may become pathogens elsewhere. Identification of species/subspecies involved in APO will make it plausible for early and accurate diagnosis to identify individuals at risk (Han, 2011b).

Rheumatoid Arthritis (RA)

RA is an autoimmune disease characterized by chronic inflammation of the joints and the surrounding tissues, leading eventually to destruction of the joint architecture and impaired function. Numerous clinical studies point toward a potential association between RA and chronic periodontitis (Martinez-Martinez et al., 2009; Dissick et al., 2010). Two studies reported that treatment of periodontal infections reduced the severity of active RA, suggesting periodontitis as a causal factor in the initiation and maintenance of the autoimmune inflammatory responses (Al-Katma et al., 2007; Ortiz et al., 2009). Periodontal bacterial DNA was detected by PCR in the synovial fluid of patients with both RA and periodontal disease, with Prevotella intermedia and P. gingivalis as the most prevalent (Martinez-Martinez et al., 2009). In a recent study, two patients with RA and periodontitis had identical bacterial clones (Fusobacterium nucleatum and Serratia proteamaculans, respectively) detected in both the synovial fluid and the matching dental plaque samples (Temoin et al., 2012a). The finding of oral bacterial DNA in the synovial fluid suggests the possibility of organisms translocating from the oral cavity to the synovium.

Inflammatory Bowel Disease (IBD) and Colorectal Cancer (CRC)

IBD represents a group of chronic disorders of the gastrointestinal (GI) tract, including ulcerative colitis (UC) and Crohn’s disease (CD). Studies have revealed that both genetic and environmental factors are involved in the development of IBD. Dysbiosis of intestinal microbiota has been shown to play a pivotal role in the pathogenesis of IBD.

Oral bacteria, e.g., Fusobacterium nucleatum and Campylobacter concisus, have recently been associated with IBD (Strauss et al., 2011; Ismail et al., 2012). F. nucleatum strains originating from inflamed biopsy tissues from IBD patients were significantly more invasive than those isolated from healthy tissues from either IBD or control patients (Strauss et al., 2011). Enteric invasive C. concisus oral strains were detected in 50% IBD patients, but none in the normal healthy controls. Multilocus sequence typing (MLST) showed that individuals with Cluster I C. concisus had significantly higher incidence of IBD and/or bloody diarrhea compared with those without (Ismail et al., 2012).

IBD has been recognized as a risk factor for CRC. Thus, it is not surprising that the same micro-organisms are implicated in both diseases. Two recent studies reported detection of significantly elevated F. nucleatum levels in colorectal carcinomas, compared to the normal colon tissues from the same patients (Castellarin et al., 2012; Kostic et al., 2012). The overabundance of Fusobacterium in CRC was positively associated with lymph node metastasis. These findings were supported by a subsequent study in which Fusobacterium, Porphyromonas, Peptostreptococcus, and Mogibacterium were found to be enriched in the mucosa-adherent microbiota in CRC (Chen et al., 2012). Fusobacterium is rarely detected in the luminal flora (stools), but it is readily detected in mucosal specimens (Chen et al., 2012). This is likely due to its adherence properties (see below). Whether F. nucleatum is a cause or a consequence of CRC is currently under investigation. If a causative link is proven, antibiotics and/or vaccines could be potential therapies to treat and/or prevent CRC.

Respiratory Tract Infections (RTI)

Oral colonization by respiratory pathogens, fostered by poor oral hygiene and periodontal diseases, has been associated with nosocomial pneumonia. Particularly among patients on ventilators in intensive care units, dental plaque has been shown to serve as a reservoir for respiratory pathogens. Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter species, Candida albicans, and enteric species recovered from plaque were indistinguishable from isolates recovered from tracheo-bronchial sites in the same patient, whereas strains from different patients usually separated into different genotypes (Heo et al., 2008, 2011).

Other Organ Inflammation and Abscesses

Oral bacteria have been found to be associated with organ abscesses in the human body (Table 1). For example, Fusobacterium is the most common oral genus detected in brain, lung, liver, and splenic abscesses (Han, 2011a). One study reported that 4 periodontopathic bacterial species, including F. nucleatum, P. intermedia, P. gingivalis, and T. denticola, were detected in a pyogenic liver abscess site of a female patient (Ohyama et al., 2009). Two recent studies reported infective appendicitis cases in which oral Fusobacterium was shown to play a major role (Swidsinski et al., 2011).

Potential Virulence Mechanisms of Oral Bacteria in Extra-oral Infections

Regardless of their pathogenic potentials in the oral cavity, once colonized in the extra-oral sites, oral bacteria often become bona fide pathogens, especially in immune-compromised individuals, causing disease manifestation. Here we summarize the recently uncovered virulence mechanisms of F. nucleatum, P. gingivalis, S. mutans, and C. rectus in extra-oral infections and inflammation.

Fusobacterium nucleatum (Fn)

Fn is ubiquitous in the oral cavity and is the most prevalent species in extra-oral infections (Han, 2011a). It has the ability to adhere to and invade different types of host cells, including human epithelial and endothelial cells (Han, 2011a). These are essential virulence mechanisms because they provide means for colonization, evasion of host defense, and spread to deeper tissues. For example, Fn colonizes the murine placentas by binding and crossing the endothelium (Han, 2011a). Once colonized in the placenta, Fn proliferates quickly and eventually spreads to amniotic fluid, fetal membranes, and the fetus.

The surface adhesin FadA expressed by Fn plays a major role in the cell attachment and invasion processes (Han, 2011a). This adhesin is unique to and highly conserved among Fn, but is absent in non-oral fusobacteria (Han, 2011a). Fn colonization in the murine placenta requires FadA (Ikegami et al., 2009), which exists in 2 forms: the intact pre-FadA, consisting of 129 amino-acid (aa) residues and the secreted mature FadA (mFadA), consisting of 111 aa residues. Both pre-FadA and mFadA are required to form an active complex, FadAc, for binding and invasion (Xu et al., 2007). The crystal structure of mFadA reveals a predominantly alpha-helical hairpin structure, with the monomers linked together in a head-to-tail pattern via a novel leucine-chain motif (Nithianantham et al., 2009). While the long and thin filaments formed by mFadA alone had no cell-binding activity, the heterogeneous filaments formed by FadAc did (Temoin et al., 2012b). We have recently identified vascular endothelial (VE)-cadherin as the endothelial-cell receptor for FadA. FadA binding to VE-cadherin caused the latter to translocate from the cell-cell junctions to intracellular compartments, increasing endothelial cell permeability and allowing bacteria to penetrate (Fardini et al., 2011). Thus, FadA allows both direct invasion into the host cells and pericellular invasion via loosened cell-cell junctions. We postulate that this is the mechanism used by Fn for systemic dissemination. Fn is not only invasive by itself, but has also been shown to facilitate both intra- and inter-cellular invasion by other species, such as Streptococcus cristatus and E. coli (Edwards et al., 2006; Fardini et al., 2011). This phenomenon explains why Fn is often found in mixed infections (Han et al., 2009; Wang et al., 2013).

Following colonization, Fn stimulates TLR4-mediated inflammatory responses in the fetal-placental unit. Fetal loss was significantly reduced in TLR4 knock-out mice and in wild-type mice treated with a TLR4-antagonist, despite Fn colonization in the placenta. These results demonstrate that TLR4-mediated localized inflammation, rather than the bacterium per se, is the cause of fetal death (Liu et al., 2007). Fetal death occurs after 2 to 3 days of hematogenous infection. The pattern and duration of infection in the pregnant mice were consistent with those observed in humans (Han et al., 2010).

Additional virulence factors recently identified from Fn include GroEL, which induces factors predisposing to atherosclerosis in human microvascular endothelial cells (HMEC-1) and apolipoprotein E-deficient (ApoE^-/-) mice (Lee et al., 2012), and 2 outer-membrane protein adhesins of Fn, Fap2 and RadD, which share homology to autotransporter secretion systems (type Va secretion systems) and induce apoptosis of human lymphocytes (Kaplan et al., 2010). RadD has been shown to mediate co-aggregation with Streptococcus sanguinis (Kaplan et al., 2009).

Porphyromonas gingivalis (Pg)

Readers are referred to a recent review on the mechanisms of Pg in extra-oral infections, especially those involved in CVD (Hayashi et al., 2010). Here we summarize a few recent findings.

Although Pg is a long-recognized and well-studied pathogen, novel virulence mechanisms continue to emerge. Pg has been postulated as a keystone pathogen in periodontal disease because of its low abundance and high virulence in the oral flora. Pg could disrupt host-microbe homeostasis and cause inflammatory responses by modulating the complement system (Hajishengallis et al., 2011). A study revealed that immunoreactivity to Pg heat-shock protein was predominant in patients with autoimmune disease with ongoing periodontal disease, suggesting a role for Pg heat-shock protein in infection-triggered autoimmune diseases (Jeong et al., 2012). Pg is the only prokaryote described to date to possess peptidylarginine deiminase (PAD), which converts arginines within a peptide (peptidylarginine) into peptidylcitrulline (Liao et al., 2009; Mangat et al., 2010). Thus, Pg can induce anti-citrullinated peptide antibodies and trigger autoimmune inflammation in the host. It has been previously shown that Pg PAD plays an important role in the pathogenesis of periodontitis-associated RA (Liao et al., 2009; Mangat et al., 2010). In susceptible individuals, host protein citrullination by Pg PAD in the joint likely induces antibody response, paving the way for the development of chronic arthritis (Mangat et al., 2010). Inactivation of arginine gingipains, but not lysine gingipains, resulted in decreased citrullination (Wegner et al., 2010), suggesting that Pg virulence factors function synergistically in inducing systemic infection and inflammation.

Numerous animal models have been established to investigate the virulence mechanisms of Pg in systemic infections. A recent study showed that the subcutaneous infection with Pg at different gestation periods of the experimental rat model resulted in lower maternal weight gain, lower placenta/fetus weight, and more fetal-placental resorptions than in controls. The infection at mid-gestation was found to cause more severe consequences with the induction of pro-inflammatory cytokines expression in the fetal compartment (Michelin et al., 2012). As observed with S. mutans, C. concisus, and F. nucleatum, not all Pg strains exhibit the same level of virulence. In a mouse periodontitis model, it was shown that different Pg strains elicited various systemic responses and various degrees of alveolar bone loss (Marchesan et al., 2012). The infectivity of Pg in murine placentas was also strain-dependent (Belanger et al., 2008).

Streptococcus mutans (Sm)

Oral streptococci, major members of the oral flora, frequently cause bacteremia and systemic diseases, such as infective endocarditis (IE) (Que and Moreillon, 2011). Among them, Sm is a primary etiological agent of human dental caries. Sm can be divided into 4 different serotypes (c/e/f/k), based on its rhamnose-glucose polymers (RGPs) (Nakano et al., 2007). Serotype c is the most common in the oral cavity. Serotypes e and f have been found to invade endothelial cells (Abranches et al., 2011). Serotype k, with a defect of the glucose side-chain in RGPs, was found to be more frequently detected in cardiovascular specimens (Nakano et al., 2007a; Nakano et al., 2007b; Nakano et al., 2010).

Collagen-binding proteins (CBP), have been shown to be directly involved in hemorrhagic stroke (Nakano et al., 2011). The detection frequency of CBP-expressing Sm in patients with hemorrhagic stroke is significantly higher than in the control individuals. Infection of Sm of the k serotype, which expresses CBP, aggravates cerebral hemorrhage in mice (Nakano et al., 2011).

Two novel CBPs, Cnm and Cbm, have been demonstrated to play a major role in Sm attachment and invasion of human endothelial cells (Nomura et al., 2013). Cnm is encoded by invasive Sm strains and is required for Sm adherence and invasion of human coronary artery endothelial cells (Abranches et al., 2011). Most of the Cbm-positive strains showed higher levels of binding to type I collagen, a major collagen expressed in human heart valves, and higher rates of adhesion and invasion to human umbilical vein endothelial cells (HUVEC) compared with the Cnm-positive strains (Nomura et al., 2013). Cbm was detected significantly more frequently in heart valve specimens from IE patients than from non-IE patients (Nomura et al., 2013).

Campylobacter rectus (Cr)

Studies have shown that oral infection with Cr induced fetal growth restriction (IUGR), placental inflammation, and activation of TLR4 in a murine pregnancy model (Arce et al., 2009). Cr infection in TLR4-deficient mice did not lead to IUGR, confirming the role of TLR4 in placental infection, as in the case of Fn infection (Arce et al., 2012). Cr was able to invade and significantly up-regulate IL-6 and TNFα in human trophoblastic cells BeWo (Arce et al., 2010). So far, no specific virulence factors from Cr have been identified.

Concluding Remarks and Recommendations

Oral bacteria have developed aggressive mechanisms to invade and persist in the host cells, to escape host immune surveillance, to adapt to niches at extra-oral sites, and to induce inflammatory responses leading to adverse systemic effects. Based on the knowledge gained thus far, some recommendations are justified.

First, periodontal infection, as a trigger of dental bacteremia, likely plays a facilitating role in enabling the systemic dissemination of oral bacteria. Thus, it must be emphasized that good oral hygiene, especially for immune-compromised patients, is crucial for controlling total bacterial load to prevent bacterial dissemination.

Second, development of accurate bacterial detection techniques is important for targeted patient management. The study of microbes has traditionally focused on bacterial species isolated in culture. Because a great many bacteria are uncultivated, traditional microbiological cultures provide an incomplete spectrum of the human pathogens. For instance, 16S rRNA gene profiling revealed a far greater microbial diversity in amniotic fluid and cord blood than appreciated based on culture-dependent methods. In pregnancies where there was evidence of an inflammatory host response, approximately two-thirds of the microbes detected by culture-independent methods were not isolated by routine cultures (Han et al., 2006, 2009; Wang et al., 2013). Furthermore, it is becoming apparent that not all oral bacteria are created equal. Certain subtypes, such as serotypes e, f and k of S. mutans, Cluster I C. concisus, F. nucleatum sbsp animalis, and certain strains of P. gingivalis, are more prevalent in extra-oral infections than others. Thus, bacterial detection methods are needed for accurate identification of individuals with the truly virulent subtypes. Since each detection method has its flaws, it is necessary to develop multiple detection methods for cross-validation.

Third, many of the studies linking oral bacteria to extra-oral infections are still at the stage of association, and detailed mechanistic studies are needed to elucidate the role of these bacteria in systemic health. Novel virulence components and mechanisms continue to emerge, even from well-studied oral pathogens. Only when we have a clear understanding of the mechanisms of oral bacteria in extra-oral infections and inflammation can effective therapies be designed.