Coaggregation is a process whereby two or more species of bacteria adhere to each other via specific adhesin-receptor interactions. Coaggregation is highly selective and dependent on interaction of multiple binding receptors in order to build the structured and complex multispecies community that resides in the oral cavity. Coaggregation allows for colonization of microbial species that lack pellicle receptor sites. This colonization is based on cell-cell interactions that were found to be mediated by existing surface molecules from both viable and dead cells attached to the salivary pellicle or floating in free saliva [54, 57, 58]. These interactions are dependent on multiple factors, including physical proximity, nutrient sources, bacterial factors, and host factors. The spatial location of adhesins and their receptors has been identified using antibodies and microscopy [115]. Confocal microscopy revealed that bacterial cells with complimentarily binder/receptors were found to be adjacent to each other within the biofilm structures [86, 116]. Further studies with electron microscopy demonstrated that there appears to be a competition for binding sites between common partners when using the same binding mechanisms. A classic example of this is seen when coccoid cells (streptococci) bind to long rods such as Fusobacterium nucleatum or Corynebacterium matruchotii forming a “corncob structure” [117].

If different mechanisms are used in coaggregation, then some organisms may serve as a bridge to connect other species. Indeed, Prevotella loescheii was found to link Streptococcus oralis to Actinomyces israelii [118–120]. The presence of simple sugars in the local environment is also able to change the coaggregation dynamic of several interspecies pairs [63, 119, 121–124]. Dextrans are produced by streptococci, encouraging coaggregation and forming part of the intracellular matrix in the biofilms [121]. The enhanced binding of multiple bacterial partners also has the potential to prevent colonization of pathogenic species as the binding sites are blocked [124]. Collectively, the ability of multiple species to coaggregate with each other plays a key role in the spatial architecture and volume of oral biofilms (Fig. 5.4).

Bacterial metabolic products and food sharing also have the ability to shape the biofilm, encouraging the growth of some species while deterring others. For example, lactic acid is produced by a number of microorganisms in the mouth as a result of carbohydrate fermentation. Some of the most notable acid producers are from Streptococcus and Actinomyces. This lactic acid can then be used by Veillonella allowing for the production of menadione that enriches for growth of Porphyromonas and Prevotella. Fusobacterium creates fatty acids that are used by Treponema. Porphyromonas gingivalis then works together with Treponema to generate products used by Mogibacterium timidum [169].

Cooperative nutritional behaviors have also been studied for bacteria growing in saliva as its sole nutrient source [54, 57]. Some species are able to be grown individually with saliva as the sole food source, but many common oral bacteria are not. Veillonella parvula, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum are unable to grow in saliva by themselves. However, pairwise and all together, these three species are able to thrive in saliva. Actinomyces oris paired with Porphyromonas gingivalis, V. parvula paired with A. oris, and V. parvula paired with P. gingivalis all show increased growth in saliva; yet, together the three-species group refuses to grow. Streptococcus oralis, P. gingivalis, and V. parvula grow together, while S. oralis paired with P. gingivalis does not. This dependency on the presence of certain species before others can survive leads to a defined spatial architecture within the oral biofilms. This spatial dependence is thought to influence the survival of certain microorganisms within these dynamic oral biofilms, and coaggregation used in conjunction with saliva represents a key mechanism of group bacterial clearance [88, 170] and facilitates symbiotic relationships [58].

In contrast to coaggregation and food webs, oral bacteria may also have antagonistic relationships. Thus, production of certain by-products by established microbiota may act to exclude or inhibit growth of other incompatible microbes. A classic example is the production of acid by S. mutans, which eliminates competitors that are less tolerant of low pH [171–174]. Streptococcal species may also compete by production of hydrogen peroxide or bacteriocins in an effort to establish certain bacterial species as dominant in a particular host.

Some of the most well-documented relationships between health, disease, and the oral flora can be observed with the common dental ailments of tooth decay (caries) and periodontitis, including gingivitis. These diseases represent two of the most common chronic diseases seen worldwide [178–180]. Several decades of research by dental scientists have clearly illustrated that dental caries and periodontitis are caused by ecological changes in the homeostatic balance between host and bacterial flora (Table 5.2). While initial work assumed that the presence or absence of a single species would be linked to cause and effect for these diseases, it was quickly discovered that these diseases develop along a more complex story line. These oral diseases involve regulation by many sources such as multispecies bacterial connections, host-bacterial interactions, and behavioral or environmental shifts. This body of work begins to demonstrate that changes in the microbial balance can both lead to and result from active oral disease profiles in humans. Oral biofilms have been studied extensively in oral diseases. There is a general progression seen in the buildup of the oral composition as early/primary colonizers attach to the salivary pellicle, additional bacteria bind to the primary colonizers, and finally late colonizers join the group only after a complex community is present. These later colonizers include a number of key oral disease-linked bacteria.

Dental caries lesions are localized demineralization of tooth enamel as a result of acid production by dental plaque biofilms. Caries progression has been well documented from superficial white spot lesion to deep-dentin cavitations [56]. This decay is due to chronic acid production from bacterial fermentation of carbohydrates in the vicinity of the tooth enamel [56]. Previous work looking for the causative agent of dental caries showed that mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) appeared to be the primary acid producers associated with enamel caries [56, 134–136, 171, 181]. These bacteria were consistently shown to create the extreme acidic environment necessary for caries conditions, a pH <5.5 [182–184]. More recent research has shown that Lactobacillus spp. are also prevalent at active caries site and appear to create the necessary cariogenic conditions in the absence of the S. mutans or S. sobrinus [137, 138, 185]. These species are all aciduric, meaning that they thrive in low pH environments, while other members of the oral flora do not survive long exposures to acid conditions. The loss of competing species during low pH events creates a feedback loop that ultimately yields more acid production, due to the enrichment of aciduric species in the population, leading to the progressive destruction of enamel and dentin, if left untreated. Thus, caries patients display an increasing accumulation of these cariogenic bacteria. Modern techniques using sequencing technology have allowed for further characterization of not just a few select species but analysis of the entire microbiome at the disease sites. More detailed microbiome sequencing reveals an apparent distinct overall microbial shift as the tooth deterioration progresses from mild white spot lesion to caries dentin involvement and finally to severe disease with deep-dentin lesions [21, 46]. The attachment of specific oral flora to the salivary pellicle was directly tied to the particular binding partners that were present or absent during multispecies biofilm formation [51, 116]. Organisms that would normally be able to bind to the pellicle may be outcompeted for binding sites by other common oral species, while others are completely dependent on “helper” bacteria to mediate any active binding to the pellicle surface. These multispecies interactions appear to significantly influence the makeup of the biofilm composition as well as the circulating bacteria in saliva. These interactions must be constantly reestablished following removal from common oral practices such as swallowing, mechanical abrasion from mastication, and toothbrushing.

Lapses in oral hygiene and increased access to fermentable carbohydrates correlate with increased acid production from certain oral species leading to a localized drop in pH at sites with high levels of dental plaque [83, 186, 187]. This drop in pH then starts an environmental feedback cycle shifting some of the oral flora in the mouth. The decrease in pH starts to kill off susceptible oral bacterial species and selects for acid-tolerant organisms while simultaneously dissolving mineral from the tooth enamel. As the caries disease progresses, the environment has been shown to enrich growth of acid-producing and acid-tolerant bacteria including S. mutans, S. sobrinus, Propionibacterium acidifaciens, Lactobacillus spp., Veillonella spp., and Actinomyces spp. [138, 188]. Allowed to persist, this localized loss of mineral from of tooth enamel can lead to significant structural deterioration and pain.

While these acid-tolerant species increase in numbers, it should be emphasized that the remaining oral species do not completely disappear, but instead are less prevalent at the disease site. It was mentioned earlier that the oral environment normally undergoes a number of environmental shifts throughout the day; nonetheless, the overall composition of the oral flora remains constant in healthy individuals [177, 189]. Thus, bacterial and host biomarker profiles can then be used to infer the risk of developing dental disease as well as monitor dental treatment effectiveness. For example, caries risk has been assessed by the amount of lactobacilli, mutans streptococci, and Actinomyces spp. in the patient’s saliva [46, 135, 137, 139, 140, 185]. Young children with high levels of S. mutans and S. sobrinus were more than five times as likely to develop dental caries than individuals with lower levels [135, 136, 141, 190]. Sweden has used salivary levels of lactobacilli (>100,000/ml saliva) and mutans streptococci (>1 million/ml saliva) to determine caries risk for more than 30 years [142–144]. The progression of dental caries was also studied by looking at the microbial composition in white spot lesions, dentin lesions, and deep-dentin lesions [46]. They saw a continuous shift in the bacterial composition as the caries sites progress through the stages of disease. Together, these works indicate that while the presence of key bacterial species is necessary for caries development, a microbial community is crucial to supporting the persistence of those pathogenic species in the oral cavity. This change appears to be driven by changes in the local environmental conditions, such as a lower pH and changes in the host response. Therefore, monitoring known caries pathogens as well as the rest of the microbial community should provide a global picture of the oral disease state and can act as a tool to predict caries risk and treatment outcomes.

Periodontal diseases range from basic inflammation of the gum tissue to major damage of oral connective tissue and bone. Gingivitis is mild to moderate inflammation of the gum tissue, and diseased tissue can normally be reversed to a healthy state with a proper oral hygiene regimen [191, 192]. In contrast, periodontitis is diagnosed when inflammation begins to cause permanent damage to the connective tissue surrounding a tooth or the teeth. As the body tries to respond to the presence of bacterial biofilms on the tooth, the host’s immune system actually starts to damage oral tissue. Left untreated, this host-pathogen interaction can lead to loss of gum tissue, loss of attachment of connective tissue to the tooth and bone, and can potentially lead to the loss of teeth [193]. Extensive research has linked the pathology of gingivitis and periodontitis to certain microbial species such as Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Prevotella intermedia/nigrescens, and Campylobacter rectus [146, 147, 194, 195]. These etiological agents are dependent on other members of the oral flora to allow for colonization, and many times there is also an increase in salivary viral load (see later). These bacteria are late colonizers of the biofilm and are not often allowed access to gum tissue when excellent oral hygiene standards are followed.

Reminiscent of dental caries, periodontitis progression is underpinned by a global shift in microbial species [13, 23, 196]. Extensive work has looked at outlining the composition of the periodontal microbiota in order to help develop more effective treatments and diagnostic testing [12, 13, 193]. The predicted periodontitis microbiome shift was analyzed using the microarray technology HOMIM to discover the major differences between healthy control patients and those patients that responded well to treatment versus those that suffered from refractory periodontitis [23]. More species of bacteria in total were detected in diseased patients in comparison to healthy subjects, where 28 % of the 300 species were not detected in healthy individuals. The putative periodontal pathogens were found in much higher numbers than those with healthy periodontium. Those patients with treatable periodontitis were also distinguishable from those with refractory periodontitis based on their unique bacterial profiles. Significantly higher accumulations of common perio-pathogens such as T. forsythia, P. gingivalis, and Prevotella spp. were found within patients with refractory periodontitis compared to treatable but also more “unusual” species that were present, such as Brevundimonas diminuta, Mycoplasma salivarium, and Filifactor alocis [23]. The microbial profile of the healthy individuals revealed that certain bacterial species were more prevalent in subgingival plaque samples and have been found consistently associated with oral health in other studies, including Actinomyces spp. [16, 23], Capnocytophaga sputigena [23, 46, 138], Cardiobacterium hominis [16, 23], Haemophilus parainfluenzae [16, 23], Rothia dentocariosa/mucilaginosa [16, 23], and Streptococcus sanguinis [23, 46, 138].

Periodontal pathogens have been found to colonize the tongue and other non-periodontal sites [38, 197, 198]. The whole saliva provides the immediate source of bacteria for oral biofilm formation and is therefore predicted to have bacterial profiles that are coupled to the biofilm composition. P. gingivalis, P. nigrescens T. denticola, and P. intermedia were found to have a statistical relationship when comparing saliva and periodontal pocket samples [199]. Additional research has shown both genetic and environmental factors influence bacterial colonization in saliva and the periodontal pocket [200–202]. Several groups have assessed the severity of periodontal disease by using levels of both pathogenic bacteria and host-response biomarkers in saliva [199, 203–206]. Interestingly, investigations have started to show that the bacterial composition shift paired with host biomarkers is correlated with detecting disease stability or periodontitis disease progression [27, 204]. Those patients who showed low bacterial and host biomarker levels fared better than those with high bacterial and host biomarker amounts. Kinney et al. [27] showed that the elevated presence of Fusobacterium nucleatum, C. rectus, and P. intermedia predicted disease progression in 82 % of patients. Low levels of host biomarkers, matrix metalloproteinases 8 and 9 (host-derived enzymes involved with tissue and bone degradation), osteoprotegerin (inhibitor of differentiation and activation of osteoclasts involved in bone resorption), and IL-1 (inflammatory cytokine) predicted periodontal site stability 78 % of the time. These studies present some of the novel work that is starting to link microbiome composition to disease progression and treatment outcomes [13, 193]. Overall, patients who were successfully treated for periodontitis showed a shift back toward a healthy oral microbiome [13].

Defining the oral microbiome has allowed researchers to start to understand the normal commensal organisms that are consistent with health. Key work looking at oral-specific disease supported the hypothesis that the oral microbiome usually harbors a core microbiome, and changes in the core composition are directly related to oral disease progression due to dysbiosis. However, it was still unclear if other disease states were linked to the oral microbiome. This shift from homeostasis to dysbiosis reflects a common theme seen across many microbiome profiles that a disease state may not show complete removal and/or enrichment of certain species but instead results in a dramatic shift in the overall proportions of the total microbiome. The microbiome acts like a community that is influenced by the overall presence of key players that may aid in attachment, acquiring nutrients, surviving fluctuations in the environment, etc. Work outlined in the next section will focus on how the oral microbiome has ties to other systemic diseases such as diabetes, autoimmune disorders, hematologic diseases, vitamin deficiencies, and cancer.

Numerous studies have started to uncover the link between oral health and type 2 diabetes [211]. High, uncontrolled blood sugar levels are associated with excess bleeding and poor healing overall [212–214]. Diabetic patients have been found to have a significantly higher risk for gingivitis and periodontitis [211, 212, 215]. One large-scale study that looked at 4,343 adults revealed that diabetics have an odds ratio of 2.9 for periodontitis compared to those not suffering from diabetes [216]. Diabetic patients who better controlled their glycemic levels saw their odds ratio fall to approximately 1.56 [216]. When patients are failing to control their diabetes, they show a consistent flare in oral diseases [215, 216]. Systemic problems with increased blood glucose levels lead to chronic inflammatory immune response leading to excess production of inflammatory proteins, which may help to keep the microbiome in check [12, 211, 217]. While the subgingival bacterial profile showed the same type of species in periodontal patients with diabetes in comparison to those periodontal patients without diabetes, the relative abundance was different between diabetic and nondiabetic [152, 153]. The diabetic patients show higher numbers overall of assessed perio-pathogens (Table 5.3). Diabetic patients appear to show an exaggerated inflammatory response to the same oral periodontal pathogens and in some cases increased apoptosis (programmed cell death). This enhanced inflammation and apoptosis is thought to be involved with delayed wound healing and likely explains some of the excess periodontal tissue damage seen in diabetic patients [218]. Interestingly, a key relationship was found between one particular oral pathogen, P. gingivalis, and insulin. Lipopolysaccharide released from P. gingivalis has been shown to be toxic to some cytokine proteins that regulate insulin levels in the body [219, 220]. Proinflammatory cytokines, including interleukins 1 and 6 along with tumor necrosis factor-alpha, are produced in inflamed periodontal tissue [221–223]. These factors gain access to the body’s general circulation via the periodontal microcirculation [224, 225], which then allows them to systemically antagonize insulin [226]. Whole-body insulin resistance was also found to be triggered by bacterial infection [154].

Research has demonstrated that treatment for periodontitis, which also shifts the microbial microbiome, improves the overall health outcomes for these patients [12, 211, 222]. However, diabetes can also lead to additional oral disorders including fungal infections, salivary functional disorders, dental caries, and burning mouth syndrome [154, 227, 228]. Together, this systemic disorder leads to major changes in the body and importantly the oral environment. Diabetes and oral infections have a bidirectional relationship where the presence of one seems to further provoke the other and leads to a global increase in symptoms and decrease in quality of life. Here we see that a systemic disorder can significantly advance the periodontitis infection resulting in a shift in the microbial profiles with increased frequency of perio-pathogens consistent with diabetes plus periodontitis.

Autoimmune disorders represent a wide-ranging group of diseases that can impact the human body with varied severity. Oftentimes, autoimmune disorder patients suffer cyclical disease presentation where “flares” coincide with severe disease symptoms and times when symptoms are manageable to nonexistent [162, 229, 230]. There are some autoimmune diseases that show consistent oral presentations in addition to other symptoms [230]. In some cases, the oral symptoms have been known to precede more classic symptoms [230]. This is sometimes seen with Crohn’s disease oral lesions. Crohn’s disease is an autoimmune disorder that primarily impacts the intestines, but secondary symptoms are seen with the joints, skin, and liver [229]. Oral manifestations have been used for Crohn’s diagnosis [78, 231, 232]. These symptoms include mucosal tags; gingival, labial, or mucosal swelling; and ulcers. The patterns of oral symptoms and histology mimic those seen in the intestine with inflammation, ulcers, and swelling. The severity of the oral lesions has been shown to predict the extent of disease in the intestine [230]. In some cases, the oral presentation will precede the systemic symptoms and could be used as an indicator for further confirmatory testing [230, 231, 233]. Microbial shifts were seen in Crohn’s disease patients including an overall decrease in Firmicutes and increases in Prevotella melaninogenica and Neisseria mucosa [158, 159]. Patients with Crohn’s disease show an increased risk for caries development [159]. In addition, inflammation in the intestine has been shown to lead to malabsorption [234, 235]. This nutritional deficiency can lead to significant changes in the oral environment (see later).

Sjögren’s syndrome is an autoimmune disease that attacks the endocrine glands, including the salivary glands that lead to a significant decrease in the production of saliva [113, 162]. The dry mouth conditions means there is considerable risk for severe dental diseases [62, 162]. Predictably, patients with primary Sjögren’s syndrome were found to have increased levels of S. mutans, Lactobacillus spp., and Candida albicans within their supragingival plaque samples [81]. The smooth mucosa and tongue samples also showed increased prevalence of Staphylococcus aureus and Candida albicans [80, 81, 160, 163]. The greatest shift in microbial differences was observed by patients who had a stimulated saliva secretion rate of <0.5 ml/min [81]. The primary risk factor that seems to be associated with this shift in the bacterial profile is the reduced saliva production. There is, however, a change seen in the protein levels of Sjögren’s syndrome patients as well [114, 236]. This suggests that reduced salivary flow combined with changes in the salivary composition shapes the microbial accumulation in the mouth. These shifts may therefore be used to predict “flares” along with assessing treatment progress.

Changes in the diet are known to alter the overall composition of some species within the oral flora [177, 237–239]. Malnutrition has been linked to reduced salivary flow rates, changes in bacterial numbers, and shifts in salivary composition [82, 164, 239–241]. There has been a documented increase in the prevalence of oral pathogens that are involved with caries development as our modern human diets have increased in refined sugar consumption [83, 242]. Scarcity of certain foods and nutrients can dramatically alter the oral environment. The mucous membranes in the mouth show a high cell turnover rate (3–7 days) versus the skin (up to 28 days) [243, 244]. The oral cavity may, therefore, exhibit signs and symptoms more quickly from direct nutritional deficiencies or systemic diseases that lead to nutritional/vitamin depletion. Vitamin deficiencies can lead to generalized issues in the oral cavity [164]. Many deficiencies are associated with oral-specific symptoms including caries, delayed dental eruption, enamel defects, and oral lesions [164, 235, 239, 241]. Dentists may be involved with diagnosis of vitamin deficiencies based on presenting oral symptoms when no other obvious cause is present. For example, megaloblastic anemia, due to a lack of the vitamin B12, displays oral mucosal membrane change in approximately 50–60 % of the patients [195, 245, 246]. In general, low iron levels are known to contribute to diminished bactericidal activity from leukocytes, impaired cellular immunity, epithelial abnormality, and inadequate antibody response. These changes in the host lead to generalized oral problems including inflammation, higher prevalence of oral candidiasis, and predicted shifts in oral flora that are sensitive to low iron levels [239, 247–252]. Vitamin A deficiency leads to dry mouth, impaired tooth growth, and increased susceptibility to infection [164, 253, 254]. Vitamin C is important for healthy collagen formation and serves to enhance iron absorption [164, 255–257]. Vitamin C deficiency has been shown to contribute to the severity of periodontal disease [165, 241]. Patients that were suffering from vitamin C deficiency displayed more attachment loss than those with normal vitamin C levels. Together, these missing nutrients and many others may indicate the start of systemic disease that impacts the oral cavity early on and left untreated can result in severe damage [112, 164, 214, 235, 239].

There is a new and novel work that is following potential links between the oral microbiome and various cancers (Table 5.4). This field is just starting to highlight microbiome differences observed in cancer patients versus healthy individuals. A majority of the work, so far, has looked at the oral flora composition and its link to oral and other head-/neck-associated cancers (e.g., esophagus, pharyngeal, etc.). Oral, head, and neck cancers are the sixth most common cancer seen worldwide [258]. Despite many advances in cancer diagnosis and treatment, in general, the overall prognosis for oral cancer patients has not improved much in recent years [259–261]. Improving early detection of many different types of cancers remains a key objective of salivary diagnostics, and here we will emphasize some of the promising research in that field.

A few groups have started to survey the bacteria that colonize different regions in the mouth, focusing on both malignant and healthy sites in oral and upper gastrointestinal locations [47, 127, 128, 133, 262, 263]. These groups found that the microbial composition differed significantly for at least some of the bacterial species when comparing normal, healthy tissue to malignant sites. For example, Hooper et al. [125, 126] conducted two studies that looked at the bacterial composition of oral squamous cell carcinoma tissue samples. The oral cancer tissues showed a distinct profile of several oral species including Fusobacterium naviforme and Staphylococcus aureus. In contrast, analysis of various upper gastrointestinal tract carcinomas found a distinct link with one particular species, Streptococcus anginosus [128]. S. anginosus DNA was also found in carcinoma tissue from head and neck squamous cell biopsies but not in those from other cancer types, including oral cancer [127, 264, 265]. One potential explanation given for these observed differences in bacterial composition between cancer and cancer-free sites is that certain oral bacterial cells may actually be influencing signals that initiate and advance oral and head-/neck-associated cancers [266], as well as other cancers [267, 268]. There are several cases where microorganisms are considered the primary source of cancer, including the classic bacterial example of Helicobacter pylori infection leading to gastric cancer formation [269, 270] plus numerous viral-associated cancers [271–276]. In these cases, the microorganisms were directly involved with influencing the development of specific cancers. Intriguingly, preliminary studies have found a link between H. pylori infection and oral cancer diagnosis [263, 277, 278]. In addition, several normal oral bacterial residents have been shown to have a direct influence on stimulating an inflammatory response in the mouth [153, 211, 279]. Although many of the exact mechanisms leading from bacterial infection to carcinogenesis remain largely unclear, there is a general link between upregulation of cytokines and other inflammatory mediators that leads to an increased risk of carcinogenesis [280–282].

Because the aforementioned studies imply that the presence of certain bacterial species may actually increase the likelihood of developing certain cancers, these and other bacterial species can potentially be used as a portent for diagnosing cancer early. One particular study looked at comparing the bacterial salivary composition from 229 healthy controls versus 45 patients with oral cancer [47]. This work found principal increases in Streptococcus mitis, Prevotella melaninogenica, and Capnocytophaga gingivalis among patients with oral squamous cell carcinoma in comparison to the controls. Further analyses showed that these species could be used as a diagnostic biomarker for oral cancer. The three-species diagnostic marker predicted 80 % of the cancer cases correctly while excluding the controls approximately 82 % of the time. Importantly, this research group found that the soft tissue bacterial composition was similar to that of the saliva composition. As mentioned earlier, different locations in the mouth may have large differences in the oral microbiome structure. This represents a key feature of testing since the cancerous or precancerous oral lesions may not be obvious in patients and will have to be diagnosed from a shift in saliva alone.

Several groups have also found an interesting link between the oral microbial composition and other cancers [47, 262, 263, 268, 283, 284]. Gastric precancerous lesion risk was found to positively correlate with high levels of periodontal bacterial pathogens [131, 132]. Gastric cancer has the second highest mortality rate worldwide and is the fourth most common cancer [285]. Patients with periodontitis that showed an increased colonization by Actinobacillus actinomycetemcomitans were more likely to have precancerous gastric lesions. Of interest, A. actinomycetemcomitans is associated with systemic infections along with being a general cause of periodontitis; therefore, its association with the oral flora may have been directly influenced by additional signals from the host resulting in favorable colonization. Fusobacterium nucleatum was recently shown to have a link to colorectal cancer [129, 130]. Colorectal adenomas, colorectal cancer tissue, and feces from colorectal cancer patients all showed significantly higher rates of Fusobacterium spp. in comparison to healthy controls. A mouse model of intestinal tumorigenesis demonstrated a rise in proinflammatory markers and increased infiltration of myeloid immune cells in F. nucleatum treated animals [129]. Human colon tissue samples also showed a strong correlation between the F. nucleatum levels and the expression of proinflammatory markers (COX-2, IL-1β[beta], IL-6, IL-8, and TNF-α[alpha]) [129, 286, 287]. A proposed mechanism for Fusobacterium involvement in colorectal cancer is based on the cell-surface Fusobacterium adhesion molecule (FadA) binding to E-cadherin on the surface of host epithelial cells [288]. Binding activates β(beta)-catenin signaling, F. nucleatum invasion, and inhibition of tumor suppressor activity promoting colorectal cancer formation. FadA levels are presented as a proposed marker for early diagnosis of colorectal cancer. However, it is still unclear whether Fusobacterium is acting as a commensal to modify the forming tumor microenvironment or instead it is an opportunistic pathogen and directly responsible for the promotion of colorectal cancer.

Finally, HOMIM was used to look at the oral microbiota in patients with various pancreatic diseases. The salivary microbiome was observed to be significantly different in patients with pancreatic disease, including cancer, in comparison to the healthy controls [133]. Pancreatic cancer patients had an increase in 31 bacterial clusters/species and decrease in 21 bacterial clusters/species. Further analysis, testing 16 species/clusters as a pancreatic disease biomarker, showed that combining Neisseria elongata and Streptococcus mitis were the most valid biomarkers. The combination biomarker resulted in a 96.4 % sensitivity and 82.1 % specificity in determining pancreatic cancer patients from healthy individuals. Novel recent work from the same lab has presented evidence for a mechanism connecting systemic disease with salivary changes. Lau et al. [210] found that pancreatic cancer-derived exosomes, “cell-specific lipid vesicles,” provide a link between the primary cancer site and saliva biomarker production in mice. These exosomes that are able to travel through the vascular systems are involved with intercellular communication and are thought to be associated with a number of functions including immune response regulation and tumor invasion promoters [289, 290]. A number of host factors were shown to be upregulated based on saliva comparison from healthy and diseased mice using microarray analysis. Some of these host factors have the potential to influence the bacterial compositions observed in the cancer patients versus cancer-free individuals (Table 5.5). The presence of human serum was also shown to influence the adhesion and coaggregation phenotypes in a number of oral species [55]. This leads to a tempting mechanism that exosomes have the potential to travel from newly forming cancer sites to deliver disease-specific products directly to saliva, which can then shape the oral microbial environment through direct messages or trigger secondary messages such as hormones and cytokines (Fig. 5.5). These oral changes can, therefore, presumably be used to monitor distant body sites throughout disease progression.

Candida albicans has been consistently seen as an indicator of oral microbiome imbalance and other oral diseases [303, 304]. This overgrowth of Candida albicans is commonly known as oral thrush. Thrush is seen in the immunocompromised and immunosuppressed patients, including patients with human immunodeficiency virus (HIV), cancer patients, infants, elderly, etc. As many as 90 % of HIV patients will present with reoccurring oral candidiasis [305–308]. Diabetic patients with high blood sugar and patients with Sjögren’s are also more prone to candidiasis infections [160, 163, 309]. The generalized presentation of candidiasis is a good visual indictor that something is systemically wrong with the patient, though infant oral thrush does not indicate a problem unless the infection continues for several weeks. Unfortunately, because a variety of conditions can lead to candidiasis, it is not specific and thus provides limited information about the potential systemic issue without further testing. Just like the bacterial composition, a defined oral mycobiome will need to be established to determine differences between conditions of health and disease. One group has started this work by looking at the oral mycobiome of 20 healthy individuals [207]. They were able to detect 74 culturable and 11 unculturable fungal genera in the oral cavity. As few as 3 and as many as 39 genera were found in these healthy volunteers. Candida spp. were present in more than 75 % of the individuals, followed by Cladosporium at 65 % and Saccharomycetales and Aureobasidium at 50 % each. Intriguingly, a cluster analysis showed that mycobiome profiles of males and females were separate from one another. This study represents the first survey of the fungal species present in the oral cavity and only surveyed a small number of people. Similar to the bacterial microbiome surveys, the oral mycobiome is predicted to reveal a core set of fungal organisms present within healthy populations along with unique species specific to each individual. In fact, efforts have shown that the total composition of an individual’s human microbiome is just as unique as a fingerprint with no two people alike [310].

The persistent presence of viruses in saliva can be observed for a number of reasons. Viruses can be transferred by direct contact with infected individuals, a blood-borne viral infection can allow for salivary gland involvement, or the oral cavity itself may be infected in the mucosa or diseased periodontal sites [148]. Like with candidiasis patients, those with compromised or suppressed immune systems will also likely display a viral load increase in saliva. The presence of viruses in saliva presents another noninvasive disease diagnosis opportunity. In light of this, a number of groups have developed saliva-based viral detection tests for common human viruses including HIV [311], hepatitis viruses [312], herpes viruses [312, 313], and measles virus [314]. Researchers are starting to characterize the human virome; however, this field is still emerging. One key problem lies in detecting the presence of unknown viruses. Unlike bacteria that have a conserved 16s ribosomal region within their DNA, viruses have little DNA sequence in common. Plus, the majority of viruses in the human body are specific for bacteria hosts (bacteriophage) and not humans [315, 316]. Those bacteriophages are able to indirectly impact humans by changing the abundance or function of our resident bacteria. With the advent of shotgun sequencing, we can now look at all the DNA whether it is human, bacterial, fungal, or viral. Studies using this approach on the gut virome found that mothers and children, and identical twins have similar viromes [317, 318]. Another study looked at the viral composition present inside the lungs of cystic fibrosis (CF) patients in comparison to non-CF patients [319]. They found some differences in the viral composition but significant changes in metabolic functions as a result of these viruses in the lung. These variations are thought to influence the severity of cystic fibrosis; however, this was a small-scale study with five CF and five non-CF patients. A larger analysis used nasal swabs from 176 children, some healthy and some with unexplained fevers. The children with unexplained fevers had more viruses on average than those that were healthy, suggesting the fevers may have been viral in nature and not bacterial [320].

New human viruses and bacteriophages are discovered every day; nonetheless, new viruses that posses DNA or RNA that is unlike any other known sequence will likely be missed in these large-scale sequence surveys until a more universal method of identifying viruses becomes available. Much of the work to date has, therefore, focused on quantifying the viral load of known human viruses, mainly those belonging to the herpes-type viruses.

Herpes viruses are the most common viral type found in human saliva [321–323]. There are eight known herpes viruses that infect humans: herpes simplex types 1 and 2, cytomegalovirus, Epstein-Barr virus, varicella zoster virus, herpes virus 6, herpes virus 7, and herpes virus 8 (Kaposi’s sarcoma) [324]. Herpes viral infections can lead to diseases of the periodontium and oral mucosa or may present asymptomatically but still with active viral shedding into saliva [322, 324