Oral Commensal Streptococci: Gatekeepers of the Oral Cavity

ABSTRACT

Oral commensal streptococci are primary colonizers of the oral cavity. These streptococci produce many adhesins, metabolites, and antimicrobials that modulate microbial succession and diversity within the oral cavity. Often, oral commensal streptococci antagonize cariogenic and periodontal pathogens such as Streptococcus mutans and Porphyromonas gingivalis, respectively. Mechanisms of antagonism are varied and range from the generation of hydrogen peroxide, competitive metabolite scavenging, the generation of reactive nitrogen intermediates, and bacteriocin production. Furthermore, several oral commensal streptococci have been shown to alter the host immune response at steady state and in response to oral pathogens. Collectively, these features highlight the remarkable ability of oral commensal streptococci to regulate the structure and function of the oral microbiome. In this review, we discuss mechanisms used by oral commensal streptococci to interact with diverse oral pathogens, both physically and through the production of antimicrobials. Finally, we conclude by exploring the critical roles of oral commensal streptococci in modulating the host immune response and maintaining health and homeostasis.

INTRODUCTION

The oral cavity is remarkably heterogeneous. The teeth, gingiva, and tongue are composed of vastly different tissue types, and nutrients are both transient and variable. As an auxiliary opening for the respiratory system, both oxygen and carbon dioxide are present at different concentrations depending on host behavior and location within the oral cavity. This diverse environment is conducive to microbial colonization, allowing many different microbes to colonize various regions of the oral cavity. Indeed, more than 700 bacterial species, 100 fungal species, and hundreds of viral genotypes are found within the human oral cavity (1–4). Microbe-host interactions within the oral cavity have been well documented, with substantial efforts being made to characterize diseases of bacterial etiology such as caries, endodontic infections, gingivitis, and periodontitis. While single-pathogen–single-disease models, such as Koch’s postulates, explain some diseases within the oral cavity, these models do not incorporate microbe-microbe interactions that can dampen or exacerbate disease progression.

To accommodate the impact of microbe-microbe interactions, Sigmund Socranksy developed a complex theory where bacteria within the oral cavity were broadly grouped into green, orange, and red complex organisms (5, 6). Green complex organisms such as streptococci are early colonizers of the mouth that bind well to the tooth pellicle and produce antimicrobial agents, such as hydrogen peroxide, that prevent colonization by other bacteria (7–15). Bacteria within the orange complex are so-called “bridging species” that physically associate with both green and red complex members (6, 16). Bacteria within the red complex were thought to be overt pathogens that cause disease (6). Interestingly, subsequent microbiome studies indicate that red complex pathogens are present in people free of disease in the oral cavity, illustrating that this complex theory does not integrate all factors that lead to disease (17). Thus, the polymicrobial synergy-dysbiosis model was put forth, which incorporates the importance of the microbial community in health and disease (18). Polymicrobial interactions within the oral cavity are exceedingly complex, with interactions characterized as physical, chemical, synergistic, or antagonistic. Of particular importance, however, are the interactions between oral streptococci and other bacteria. Oral commensal streptococci play a variety of roles within the oral cavity as both early colonizers and accessory pathogens, in which case they may enhance the colonization or metabolic functions of pathogens under certain conditions. As the largest bacterial group within the oral cavity, streptococci are a significant component of oral health (19, 20).

Streptococci are heterogeneous, with some streptococci being cariogenic, others being accessory pathogens of periodontitis, others inhibiting colonization by pathogens, and some that are known to modulate the host immune response. The genus Streptococcus is large, with approximately 72 identified species, of which at least 36 have been isolated from the human body. Of those within the human body, 18 species reside within the oral cavity (Table 1). The genus has been broken into 8 groups, mitis, sanguinis, anginosus, salivarius, downei, mutans, pyogenic, and bovis, although taxonomic changes are continuous where nomenclature, species, and subspecies designations have been variable for the genus (19, 21–28). The genus harbors many commensal organisms as well as pathogens such as S. pneumoniae, S. pyogenes, and S. agalactiae. It is important to note that many oral streptococci that are regarded as commensals have the ability to cause disease, namely, bacteremia and endocarditis, which is seen with several oral species such as S. mitis, S. parasanguinis, and S. salivarius.

TABLE 1.

Classification and species of human-associated streptococci

Group	Species	Body site(s)	Reference(s)
Anginosus	S. anginosus	Oral cavity, GIb	160, 161
	S. constellatus	Oral cavity	162, 163
	S. intermedius	Oral cavity	160, 164
Bovis	S. equinus	GI	161
	S. gallolyticus (subsp. bovis)	Blood	165
	S. infantarius	GI	166, 167
Downei	S. criceti	Oral cavity	168
	S. downei	Oral cavity	169
Mitis	S. australis	Oral cavity	170
	S. dentisani	Oral cavity	28, 171
	S. infantis	Oral cavity	172
	S. massiliensis	Blood	173
	S. mitis	Oral cavity	161, 174
	S. oralis	Oral cavity	174, 175
	S. parasanguinis	Oral cavity	176
	S. peroris	Oral cavity	172
	S. pneumoniae	Nasopharynx	177
	S. pseudopneumoniae	Blood	178
Mutans	S. mutans	Oral cavity	179
	S. sobrinus	Oral cavity	180
Pyogenic	S. agalactiae	Urogenital	181
	S. dysgalactiae	Urogenital	182
	S. equi	Urogenital	183
	S. iniae	Zoonotic (cellulitis)	184
	S. porcinus	Urogenital	185
	S. pseudoporcinus	Urogenital	186
	S. pyogenes	Throat	187
	S. urinalis	Urogenital	188
Salivarius	S. salivarius	Oral cavity	161
	S. vestibularis	Oral cavity	189
Sanguinis	S. cristatus (S. oligofermentans)a	Oral cavity	190, 191
	S. gordonii	Oral cavity	174
	S. sanguinis	Oral cavity	174, 192
Unresolved	S. acidominimus	Blood	193
	S. pluranimalium	Blood	194
	S. sinensis	Heart	195
	S. suis	Zoonotic (meningitis)	196

^aS. oligofermentans is a later synonym of S. cristatus.

^bGI, gastrointestinal.

Given the heterogeneity of tissue, metabolites, and nutrients within the oral cavity, the microbes that reside in the oral cavity are similarly heterogeneous and vary by site within the oral cavity (29, 30). Additionally, person-to-person variation is relatively high within the oral cavity, with some harboring significantly more or fewer microbial species than others. Despite this, core oral microbiota members have been identified. Hall et al. identified 26 core microbes that were present across all tissue sites, time points, and individuals sampled, indicating that while the community composition within the oral cavity can be transient, certain microbes are ubiquitously present (29). Importantly, mitis/oralis group streptococci are constituents of the core oral microbiota.

STREPTOCOCCI OF THE ENAMEL

Streptococci have been shown to be primary colonizers of the tooth surface. These colonizers adhere to the acquired salivary pellicle, which is a thin proteinaceous film that is important for calcium and phosphorous resorption, protects against acid erosion, and initiates the adherence of oral microbes to the tooth surface (10, 31, 32). S. sanguinis, S. gordonii, S. parasanguinis, S. mitis, and S. oralis have been shown to bind to the salivary pellicle that forms on the tooth surface with high affinity (33–39). These streptococci possess adhesins that enable fast and stable attachment to the tooth surface. Once bound, these primary streptococci can act as anchor points for secondary colonizers to bind (19, 40). Importantly, oral streptococci are hardy competitors and are capable of competitive exclusion through the production of hydrogen peroxide and various bacteriocins, nutrient uptake, and competitive binding (41). Thus, the prevalence of certain oral streptococci on the tooth surface can select for secondary colonizers that may shift the biosis of the oral cavity.

The species of streptococci that are present in the biofilm on the enamel surface can dramatically alter tooth health. Cariogenic streptococci such as Streptococcus mutans and S. sobrinus are capable of binding to other microbes, including other streptococci (42, 43). S. mutans establishes binding through the production of glucan, an extremely sticky exopolysaccharide that is produced by extracellular glucosyltransferase (Gtf) enzymes. S. mutans is very aciduric and acidogenic, and this acid lowers the pH and promotes enamel degradation, leading to caries (44, 45). Interestingly, the presence of commensal streptococci has been shown to inhibit S. mutans in both a hydrogen peroxide- and peroxynitrite-dependent manner (46, 47).

In addition to the direct antagonism of S. mutans through hydrogen peroxide activity, commensal-generated hydrogen peroxide has also been shown to inhibit Candida albicans, a fungal pathobiont that synergizes with S. mutans and is widely found in carious lesions with S. mutans during early childhood caries (48, 49). Falsetta et al. first demonstrated that (i) dual-species biofilms with S. mutans and C. albicans are composed of increased biomass compared to the single-species biofilms alone; (ii) C. albicans increases the abundance of S. mutans cells, and C. albicans mannans provide sites for S. mutans GtfB binding and activity; (iii) coculture with C. albicans induces the expression of S. mutans virulence factors such as Gtfs; and (iv) dual infection in a rat model leads to increased caries compared to single-species-infected animals (48). Interestingly, oral commensal streptococci such as S. parasanguinis can inhibit S. mutans and C. albicans synergy independently of both contact and hydrogen peroxide (50). Huffines and Scoffield demonstrated that S. parasanguinis can restrict sucrose utilization by S. mutans, perhaps through inhibiting GtfB binding to C. albicans. Additionally, metabolomics indicated that S. parasanguinis alters metabolites available to both S. mutans and C. albicans, further underscoring that oral commensal streptococci can restrict colonization by other microbes through metabolic competition (50).

STREPTOCOCCI ON THE TONGUE AND GINGIVA

Streptococci have been shown to adhere to the tongue, with S. parasanguinis and S. salivarius being the most dominant streptococci. Both S. parasanguinis and S. salivarius have been cultured from tongue scrape samples. S. mitis can be found on both the tongue surface and the enamel (51). Interestingly, regular tongue scraping has been shown to reduce S. mutans prevalence in saliva (52, 53). Furthermore, streptococci that colonize the tongue routinely mediate interactions with C. albicans, a pathobiont that causes oral candidiasis (54). In a mouse model of oral candidiasis, S. salivarius K12 inhibits hyphal growth and the adhesion of C. albicans to mucosal surfaces (55). However, there are instances where streptococci can potentiate C. albicans pathogenicity. For example, S. oralis has been demonstrated to enhance oral candidiasis disease severity and the dissemination of C. albicans in oral mucosal models (56). Similarly, S. oralis, S. sanguinis, and S. gordonii display enhanced biofilm development during coculture with C. albicans (57). These observations illustrate that interactions between commensal streptococci and other resident oral microbes are often synergistic and dynamic.

Although streptococci are prevalent on the tongue, they have not been demonstrated to bind to gingival cells in vivo; however, they are present in gingival swabs in high numbers (29). Additionally, when cultured with gingival cells, S. sanguinis grows to high numbers and forms a robust biofilm that can complex with other microbes such as Corynebacterium durum and Porphyromonas gingivalis (58). Interestingly, oral commensal streptococci have also been shown to be accessory gingival pathogens that can synergize with the gingival and periodontal pathogens P. gingivalis and Fusobacterium nucleatum. S. gordonii has been shown to bind P. gingivalis through two mechanisms: through surface polypeptides (SspA and SspB) on S. gordonii that bind P. gingivalis short fimbriae (59) and through glyceraldehyde 3-phosphate dehydrogenase on S. gordonii binding FimA on the long fimbriae of P. gingivalis (60). These instances of adherence have been demonstrated both in biofilms and in invasive disease where S. gordonii has been coisolated with P. gingivalis from periodontal abscesses (59, 61). The presence of S. gordonii has been shown to upregulate P. gingivalis adhesin expression through streptococcal 4-aminobenzoate/para-amino benzoic acid (pABA), and the presence of S. gordonii increases periodontal damage (62, 63). Conversely, S. gordonii has also been shown to alter cell signaling to modulate the host immune response to P. gingivalis (64, 65). Both S. sanguinis and S. gordonii have been found to physically associate with F. nucleatum to form so-called corncob aggregates (66, 67). F. nucleatum is a bridging species that is known to adhere to both early commensal streptococci as well as periopathogens such as P. gingivalis, Treponema denticola, and Tannerella forsythia (68). Interestingly, the presence of F. nucleatum and its production of autoinducer-2 (AI-2) have been shown to increase biofilm formation by S. gordonii as well as P. gingivalis, indicating that F. nucleatum can modulate the activity of both streptococcal commensals as well as periodontal pathogens (69, 70). Further, cross-feeding between oral commensal streptococci and periodontal pathogens has also been shown; for example, Aggregatibacter actinomycetemcomitans is capable of metabolizing streptococcus-generated lactic acid (71).

MODULATION OF pH IN THE ORAL CAVITY

As the formation of caries is acid dependent, the role of oral commensal streptococci in altering the pH within the oral cavity and on the tooth surface has been explored. pH is site dependent within the oral cavity, where the pH in healthy individuals ranges from 6.28 to 7.34 on the buccal mucosa and the hard palate, respectively (72). While the mechanism behind these differences in pH between these sites is unknown, proximity to salivary glands, the thickness of saliva that accumulates at each of these sites, and the amount of local blood flow may all play roles. In contrast to healthy sites within the oral sites, carious lesions have a pH value of between 5.5 and 6.2 (44). Many oral streptococci are capable of converting arginine into ammonia via the arginine deiminase system (ADS), which raises the local pH of biofilms and facilitates the antagonism of S. mutans. Arginolytic streptococci such as Streptococcus sp. strain A12, S. salivarius, S. gordonii, and S. dentisani have been shown to raise the local pH, thus restricting acid-induced enamel demineralization that leads to tooth decay (73, 74). Due to the pH-increasing capabilities of these ADS-harboring streptococci, their utility as potential probiotics has been examined. Interestingly, ADS activity is highly heterogeneous among oral commensal species, and high activity does not always correlate with increased S. mutans antagonism (75). Nonetheless, these studies reveal that probing antimicrobial or anti-infection mechanisms by commensal streptococci offers a unique opportunity for developing effective probiotics that target cariogenic pathogens.

EFFECTS OF STREPTOCOCCUS-PRODUCED HYDROGEN PEROXIDE ON MICROBIAL COMMUNITIES

Given that oral commensal streptococci are among the first bacteria to colonize the salivary pellicle and are abundant throughout the oral cavity over time and across individuals, understanding their roles in polymicrobial interactions within the oral cavity is paramount to understanding oral health and disease. One major mechanism through which oral commensal streptococci modulate the activity and colonization of other microbes is through the production of hydrogen peroxide. A potent, nonspecific antimicrobial, hydrogen peroxide is a reactive oxygen species (ROS) that can damage microbial DNA through the formation of hydroxyl radicals (41). When hydrogen peroxide reacts with iron III, extremely reactive hydroxyl radicals are formed through the Fenton reaction. These radicals can damage proteins, reduce protein function, and damage DNA to kill bacterial cells. While it is known that oral commensal streptococci produce hydrogen peroxide, hydrogen peroxide has never been detected in saliva, primarily due to ROS scavengers such as thiocyanate, hypothiocyanite, salivary peroxidase, and/or myeloperoxidase, all of which have been shown to be produced in the oral cavity (76, 77). Interestingly, while hypothiocyanite can sequester ROS, its combination with ROS and salivary peroxidase is also antimicrobial (78). While these ROS scavengers are present in saliva, it is clear that there are likely local effects within the biofilm and in proximity to streptococci through which hydrogen peroxide is produced at concentrations high enough to inhibit colonization and/or growth of other microbes. This phenomenon was first described by Thompson and Johnson where inhibition of Corynebacterium by oral streptococci was noted (13). Subsequent studies have shown robust hydrogen peroxide production by streptococci grown in biofilms, where Liu et al. found concentrations of up to 1.4 mM 100 μm away from S. gordonii biofilms (79). Hydrogen peroxide production is known to shape the microbial community within the oral cavity and has even been shown to inhibit nonoral bacteria such as Escherichia coli from colonizing the oral cavity in the first place (80, 81). Furthermore, E. coli lipopolysaccharide (LPS) was shown to induce hydrogen peroxide production, indicating that the presence of specific microbes or microbial components can influence hydrogen peroxide production, suggesting that oral commensal streptococci can fine-tune hydrogen peroxide production to ward off foreign invaders. Additionally, the presence of hydrogen peroxide can induce extracellular DNA (eDNA) release from these streptococci, contributing to both biofilm formation and the uptake and exchange of DNA, allowing dynamic genetic and physiological changes on the community level (82).

All streptococci within the mitis group, which is the most prevalent streptococcal group within the oral cavity, produce hydrogen peroxide. Hydrogen peroxide production is largely attributed to pyruvate oxidase activity through which pyruvate, oxygen, and inorganic phosphate are converted to acetyl phosphate, hydrogen peroxide, and carbon dioxide (Fig. 1). However, S. oligofermentans uses lactate oxidase for hydrogen peroxide production. Lactate oxidase catalyzes the conversion of lactate, oxygen, and l-amino acids into pyruvate and hydrogen peroxide, and this alternate pathway of hydrogen peroxide production has been demonstrated to influence other microbes (83, 84) (Fig. 1).

FIG 1.

Metabolic pathway for hydrogen peroxide production in streptococci. Lactate is converted into pyruvate through lactate oxidase activity, producing hydrogen peroxide. Pyruvate is converted into acetyl phosphate through pyruvate oxidase activity, producing hydrogen peroxide (S. gordonii, S. sanguinis, S. parasanguinis, and S. pneumoniae). The regulation of pyruvate oxidase is species dependent. S. gordonii represses pyruvate oxidase expression through catabolite control protein A (CcpA) in the presence of carbohydrates such as galactose, maltose, and lactose. S. sanguinis basally represses pyruvate oxidase activity via CcpA regardless of the carbohydrates present. Pyruvate oxidase has been shown to be regulated by SpxR in S. pneumoniae. The role of SpxR in oral commensal streptococci has not been elucidated.

REGULATION OF HYDROGEN PEROXIDE PRODUCTION

The mitis and sanguinis streptococcal groups are heterogeneous, with species within the groups having varied conservation of metabolic and enzymatic processes. While hydrogen peroxide production has not been explored in every streptococcus capable of producing hydrogen peroxide within the oral cavity, efforts to understand the mechanisms and regulation of hydrogen peroxide production in S. gordonii, S. mitis, S. oralis, S. sobrinus, S. oligofermentans, S. sanguinis, and S. parasanguinis have been undertaken. Hydrogen peroxide production has been found to differ not only among strains within species but also in solid versus liquid media, with different carbohydrate sources, and with various oxygen concentrations. García-Mendoza et al. found that S. sanguinis and S. oralis produced more hydrogen peroxide on solid media than on liquid media and that the addition of carbohydrates to base media reduced hydrogen peroxide production in general (85). Additionally, multiple groups have demonstrated that the addition of carbohydrates to media reduces hydrogen peroxide production in S. gordonii (86, 87). Zheng et al. demonstrated that this is due to catabolite repression in S. gordonii, where the pyruvate oxidase gene’s promoter region has a binding site for catabolite control protein A (CcpA), resulting in the reduced expression of pyruvate oxidase in the presence of many carbohydrates (88). This mechanism has also been demonstrated in S. pneumoniae, where there is an increase in hydrogen peroxide production during colonization of the nasopharynx, where carbohydrates are scarce, and a decrease in hydrogen peroxide production in the blood, where glucose is readily available (89, 90). Pyruvate oxidase expression is not modulated by the presence of carbohydrates in S. sanguinis; however, the deletion of CcpA increases pyruvate oxidase expression and hydrogen peroxide production (91). Additionally, SpxA1, a global regulator of S. sanguinis, has been found to positivity regulate pyruvate oxidase and is crucial for tolerance to both oxidative and acid stress (92). However, the regulation of pyruvate oxidase in S. pneumoniae has been better characterized where the regulator of pyruvate oxidase is SpxR (93). The regulation and behavior of SpxR as well as pyruvate oxidase remain to be elucidated in the majority of oral streptococcal species. To resist hydrogen peroxide damage, oral commensal streptococci secrete pyruvate through Nox, an NADH dehydrogenase, to (i) reduce the substrates available to generate hydrogen peroxide and (ii) sequester ROS (94).

HYDROGEN PEROXIDE-MEDIATED ANTAGONISM OF S. MUTANS

Observational studies indicate that a high prevalence of oral commensals such as S. sanguinis is correlated with a lower abundance of S. mutans (95, 96). Conversely, a high prevalence of S. mutans has been correlated with reduced colonization by commensal streptococci, suggesting that oral commensal streptococci and S. mutans antagonize one another to compete for space and nutrients on the enamel surface (45). Animal studies demonstrate that if S. sanguinis is dosed before S. mutans, S. sanguinis dominates; however, if S. mutans is given before S. sanguinis, S. mutans outcompetes S. sanguinis (97). More recent in vitro studies have indicated that S. sanguinis and S. oligofermentans antagonize S. mutans through the production of hydrogen peroxide, while S. mutans antagonizes commensal streptococci through the production of bacteriocins (mutacins), and that whichever species is established first can inhibit the other through the respective mechanisms (Fig. 2) (98, 99). Additional studies indicate that S. sanguinis isolated from individuals with carious lesions has reduced hydrogen peroxide production compared to that in individuals without caries (100). This antagonism of S. mutans by hydrogen peroxide can be altered by Veillonella species expressing catalase (101). Veillonella, a bridging species, is known to bind to both commensal streptococci as well as oral buccal cells and the periopathogens F. nucleatum and P. gingivalis (102, 103).

FIG 2.

Microbial mechanisms of caries inhibition and formation. (Left) Oral commensal bacteria colonize the salivary pellicle. The production of hydrogen peroxide prevents colonization by cariogenic microbes. Commensal streptococci, such as S. parasanguinis, dominate the metabolomic profile, reducing the concentration of sucrose that is available, blunting S. mutans growth and Gtf activity. Arginolytic streptococci produce ammonia to raise the pH, preventing caries formation. (Right) S. mutans establishes glucan-rich biofilms to promote colonization. Once the biofilm is established, copious amounts of lactic acid are produced, which decreases the local pH, leading to caries formation. Mutacin production prevents colonization by oral commensal streptococci (OCS). The presence of C. albicans allows increased glucan binding and production and increased microbial biomass.

IMPACT OF COMMENSAL-MEDIATED HYDROGEN PEROXIDE PRODUCTION ON PERIODONTAL PATHOGENS

Periodontal pathogens vary in their sensitivities to oxidative stress and hydrogen peroxide. As many periodontal pathogens are anaerobic bacteria, oxygen is toxic and often leads to cell death; however, these pathogens have evolved various strategies to tolerate oxidative stress. Nonetheless, hydrogen peroxide antagonism has been documented to reduce periodontal pathogen viability.

P. gingivalis is an obligate anaerobic bacterium that is widely associated with adult periodontal disease. P. gingivalis infection is associated with increased bone resorption and nonproductive inflammation, which often leads to tooth loss and subsequent infection. During infection, P. gingivalis often encounters atmospheric oxygen as well as ROS from the host immune response. Additionally, P. gingivalis encounters hydrogen peroxide from host commensal streptococci, where the hydrogen peroxide antagonism of P. gingivalis has been documented for a variety of mitis group streptococci (65, 104–107). Thus, mechanisms to tolerate oxidative stress are paramount for the survival of P. gingivalis in the oral cavity. P. gingivalis is known to tolerate oxidative stress by (i) acquiring heme and sequestering it on its cell surface and (ii) upregulating the expression of iron-binding proteins that limit the Fenton reaction (108–111). While oxidative stress by hydrogen peroxide does not seem to inhibit P. gingivalis directly, hydrogen peroxide from S. gordonii has been shown to decrease gingipain activity (65).

Another periodontal pathogen, F. nucleatum, has differing sensitivity to hydrogen peroxide that is dependent on binding with oral commensal bacteria. When F. nucleatum is introduced into an oral community, hydrogen peroxide production increases (68). However, this hydrogen peroxide production is decreased if F. nucleatum is allowed to form aggregates with S. sanguinis, indicating that oral commensal streptococci can modulate hydrogen peroxide production depending on the microbes present within that community and that F. nucleatum can mask this phenomenon if it binds directly to S. sanguinis. Furthermore, F. nucleatum has been found to synergize with Veillonella spp., where the presence of Veillonella increases the viability of F. nucleatum. When grown in a trispecies coculture containing Veillonella parvula, F. nucleatum, and S. gordonii, catalase produced by Veillonella parvula protects F. nucleatum from death by S. gordonii hydrogen peroxide (112).

Aggregatibacter actinomycetemcomitans has also been found to be sensitive to commensal streptococcus-generated hydrogen peroxide (40). Coculture of A. actinomycetemcomitans with S. parasanguinis revealed that A. actinomycetemcomitans decreases the protein levels of pyruvate oxidase and consequently reduces the amount of hydrogen peroxide generated by the commensal (113). Furthermore, the presence of A. actinomycetemcomitans increased S. parasanguinis cell numbers, but the loss of hydrogen peroxide production altogether decreased the biofilm biomass, indicating that the presence of subinhibitory concentrations of hydrogen peroxide is beneficial for the ecology of these two organisms. While A. actinomycetemcomitans is sensitive to streptococcus-generated hydrogen peroxide, it encodes a catalase gene, where catalase activity has been associated with reduced concentrations of hydrogen peroxide in dual culture with S. sanguinis (107). Trispecies cultures with S. sanguinis, P. gingivalis, and A. actinomycetemcomitans indicate that the presence of A. actinomycetemcomitans protects P. gingivalis from S. sanguinis hydrogen peroxide-dependent killing through the expression of its catalase. Additionally, Stacy et al. demonstrated that the viability of A. actinomycetemcomitans is increased in an abscess model in the presence of S. gordonii due to the ability of A. actinomycetemcomitans to aerobically respire using hydrogen peroxide (114). Furthermore, Ramsey and Whiteley demonstrated that streptococcus-generated hydrogen peroxide induces the expression of the A. actinomycetemcomitans oxidative stress regulator OxyR, which induces the expression of ApiA, a complement resistance protein, promoting A. actinomycetemcomitans growth in serum (115). Taken together, A. actinomycetemcomitans modulates the production and availability of hydrogen peroxide and can aerobically respire using hydrogen peroxide but is sensitive to oxidative stress at high concentrations of hydrogen peroxide.

EFFECTS OF STREPTOCOCCUS-GENERATED REACTIVE NITROGEN SPECIES ON MICROBIAL COMMUNITIES

Nitrate and its derivatives are found in the oral cavity at various concentrations. Nitrate is taken in through the consumption of food with nitrate, such as leafy green vegetables. While human cells are not capable of reducing nitrate, many anaerobic bacteria within the oral cavity, such as Prevotella, Veillonella, Neisseria, and Rothia, are capable of reducing nitrate to nitrite, nitric oxide, and nitrous oxide (Fig. 3). This nitrate reduction has been shown to increase the pH of oral community biofilms (116–118). Ex vivo studies have indicated that the addition of nitrate to saliva biofilm communities increases ammonium concentrations while decreasing lactate concentrations, raising the overall pH. The addition of nitrate was also found to reduce the prevalence of both cariogenic and periodontal pathogens while increasing the number of nitrate-reducing commensals in the oral cavity (117, 119).

FIG 3.

Microbial nitrate reduction and hydrogen peroxide production contribute to RNS production. Dietary nitrates (NO₃) are reduced by oral bacteria into nitrite (NO₂) and nitric oxide (NO). Salivary nitric oxide enters the bloodstream and regulates vascular tone and blood pressure. The remaining nitrite is available to react with streptococcus-generated hydrogen peroxide to form RNS such as peroxynitrite (ONOO⁻). Hydrogen peroxide, nitric oxide, and peroxynitrite are antimicrobials that inhibit oral pathogens.

The reduction of nitrate also leads to the generation of nitric oxide, a reactive nitrogen species (RNS) that has been shown to disperse bacterial biofilms, alter bacterial physiology, and reduce bacterial viability. As nitric oxide is a short-lived but potent antimicrobial, nitric oxide-releasing nanoparticles have been developed to reduce periodontal burden, where nitric oxide reduces the viability of A. actinomycetemcomitans and P. gingivalis (120). Nitric oxide alone is not effective at reducing streptococci, including S. mutans, indicating that nitric oxide treatments would be less effective for caries prevention and/or treatment. In addition to nitric oxide acting as an antimicrobial RNS, the hydrogen peroxide generated by oral commensal streptococci can react with nitrite, an intermediate in nitrate reduction, to form other RNS such as peroxynitrite (121) (Fig. 3). This generation of RNS by oral commensal streptococci has been shown to inhibit S. mutans, A. actinomycetemcomitans, and Enterococcus faecalis. In rat infection studies, the presence of S. parasanguinis and nitrite significantly reduced S. mutans viability, biofilm formation, as well as caries (47). Furthermore, in the presence of nitrite in a trispecies coculture model with S. parasanguinis, S. mutans and C. albicans were both inhibited within biofilms (122).

The effects of RNS generated in the oral cavity are not restricted to the oral cavity. The reduction of nitrate leads to available nitrite and nitric oxide, which are important regulators of blood pressure. Multiple studies have indicated that the use of mouthwash increases blood pressure due to the loss of nitrate reduction by oral commensals (123–127). Additionally, the effect of oral commensals is not restricted to the oral cavity. Multiple studies have indicated that oral commensal streptococci can translocate to the lung in people with cystic fibrosis (CF). The presence of these oral commensal streptococci has been associated with increased lung function (128, 129). The generation of RNS through streptococcus-produced hydrogen peroxide has been shown to inhibit the major CF pathogen Pseudomonas aeruginosa, with increased inhibition being seen in CF isolates of P. aeruginosa compared to acute non-CF or environmental isolates (121, 130).

BACTERIOCIN PRODUCTION BY ORAL STREPTOCOCCI

Specific Streptococcus species in the oral cavity produce bacteriocins, which are antimicrobial peptides that inhibit the growth of genetically related species in many cases. These can be classified into lantibiotic and nonlantibiotic bacteriocins. Lantibiotic bacteriocins are small (<5-kDa) peptides that are characterized by containing the unusual amino acids lanthionine and β-methyllanthionine as well as a number of dehydrated amino acids (131, 132). This class of bacteriocins is produced by Gram-positive bacteria and inhibits the growth of other Gram-positive species by targeting the cell wall (133). A high-BLIS (bacteriocin-like inhibitory substance)-producing S. salivarius strain, K12, produces lantibiotics known as salivaricin A2 and salivaricin B (134). Through numerous studies and clinical trials, S. salivarius strain K12 has been shown to inhibit the growth of Streptococcus pyogenes, the causative agent of streptococcal pharyngitis and tonsillitis, through bacteriocin production. Additionally, S. salivarius strain K12 is used as a commercially developed probiotic against S. pyogenes infection (135). Another BLIS strain of S. salivarius, M18, produces the lantibiotics salivaricin A2, salivaricin 9 (136), and salivaricin M (137) and the nonlantibiotic salivaricin MPS the non-lantibiotic bacteriocin (salivaricin-MPS) (138). Salivaricin 9 has an inhibitory effect against Corynebacterium sp. strain GH17 as well as S. pyogenes at higher concentrations (139). Salivaricin D, a lantibiotic identified in S. salivarius strain 5M6c, isolated from a healthy infant, displays a broad spectrum of inhibition against members of the genera Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Micrococcus, Bacillus, and Clostridium (140). While many S. salivarius bacteriocins have been identified, further research into their target species and effects on the oral polymicrobial community is warranted. S. dentisani (designated S. oralis subsp. dentisani in this study) produces bacteriocins that inhibit the growth of many Streptococcus mutans strains (141). Streptococcus anginosus, a commensal of the oral cavity and other bodily sites, produces a bacteriocin called angicin, which is inhibitory against Gram-positive species, including the oral commensal Streptococcus constellatus, other strains of S. anginosus, vancomycin-resistant E. faecalis, and multiple Listeria species (142).

S. mutans produces multiple bacteriocins, termed mutacins, which have demonstrated inhibitory effects against many other Streptococcus species, including species belonging to the pyogenic, mitis, salivarius, anginosus, and bovis subgroups (143). Mutacins have been categorized into lantibiotic and nonlantibiotic mutacins. Specifically, S. mutans has been shown to inhibit the growth of S. sanguinis through the production of the lantibiotic mutacin I and the nonlantibiotic mutacin IV (98). This inhibition of S. sanguinis is contingent upon initial colonization by S. mutans before S. sanguinis; simultaneous colonization by both species leads to coexistence, while colonization by S. sanguinis before S. mutans leads to the inhibition of S. mutans growth via S. sanguinis-mediated hydrogen peroxide production (98). In a trispecies model with S. mutans, S. sanguinis, and S. gordonii, mutacin IV gene expression was shown to be coordinated with S. mutans competence genes, and the expression of both sets of genes was elevated under aerobic conditions (46). The coordination of these two systems is a proposed mechanism by which S. mutans acquires transforming DNA from the surrounding environment; mutacin IV production is increased, causing S. sanguinis and S. gordonii cells to lyse and release eDNA, which can be more effectively taken up by S. mutans competent cells under aerobic conditions (144). The novel oral commensal Streptococcus sp. strain A12 inhibits S. mutans mutacin production via the protease degradation of competence-stimulating peptide (CSP), which is essential for bacteriocin production by S. mutans (74).

MODULATION OF IMMUNE RESPONSES BY ORAL STREPTOCOCCI

Inflammatory responses in the oral cavity play a critical role in tissue damage associated with periodontitis. Periodontal pathogens stimulate the release of proinflammatory cytokines, including interleukin-1α (IL-1α), IL-1β, IL-6, and IL-8, which facilitate immune cell recruitment, tissue destruction, and bone resorption that contribute to periodontitis (145). Many commensal streptococci residing within the oral cavity have been shown to downregulate proinflammatory pathways in host cells and are associated with a healthy state of the oral cavity (Fig. 4). This modulation of the host immune response by commensal streptococci may help sustain their own colonization while modulating the growth of pathogens.

FIG 4.

Oral commensal streptococci decrease inflammation. The production of both hydrogen peroxide (H₂O₂) and small molecules by oral commensal streptococci has been shown to modulate NF-κB activity both basally and during pathogen induction of NF-κB activity. The presence of oral commensal streptococci also promotes the induction of β-defensin 2, to which oral pathogens are sensitive.

Modulation of the proinflammatory NF-κB pathway by commensal streptococci has been extensively studied. Several studies have demonstrated the ability of S. salivarius to downregulate NF-κB activity and downstream proinflammatory cytokines in gingival fibroblasts, human bronchial epithelial cells, primary keratinocytes, and intestinal epithelial cells (135, 146, 147). This phenomenon is consistent among multiple strains of S. salivarius (135, 148). One study concluded that S. salivarius downregulates NF-κB activation by inhibiting the NF-κB transcription factor p65 from entering the nucleus for transcription initiation. This inhibition is dependent on an S. salivarius protein with a molecular weight of less than 3 kDa (146). Transcriptomics performed in the same study on bronchial epithelial cells incubated with S. salivarius revealed an overall upregulation of host genes involved in homeostatic activities and anti-inflammatory responses as well as a general downregulation or baseline maintenance of inflammatory gene expression (146). Therefore, S. salivarius may ensure tolerance by the host while also potentially protecting the host against pathogen-induced inflammation.

While hydrogen peroxide produced by oral streptococci has been shown to modulate the growth of polymicrobial communities in the oral cavity, including the inhibition of S. mutans, hydrogen peroxide also has a complex role in the development of the host immune response. Hydrogen peroxide-producing oral streptococci, including S. mitis and S. oralis, have been shown to downregulate the NF-κB response in a hydrogen peroxide-dependent manner. Hydrogen peroxide produced by S. mitis and S. oralis activates nuclear factor erythroid 2-related factor 2 (Nfr2), which subsequently inhibits NF-κB transcription (149). Additionally, hydrogen peroxide produced by the oral commensals S. sanguinis and S. mitis has been shown to induce cell death of a THP-1 human macrophage cell line despite being associated with a healthy state in the oral cavity. This killing of macrophages by hydrogen peroxide is dependent on the pyruvate oxidase gene spxB, which is critical for H₂O₂ production in many streptococcal species (150).

A unique mechanism of pathogen inhibition by an oral streptococcus has been described, in which host cells are stimulated to produce antimicrobial products. Streptococcus mitis colonization of gingival epithelial cells induces the production of human β-defensin 2 (hβd-2), an antimicrobial peptide known to kill oral pathogens, including S. mutans and S. sobrinus (151). S. mitis is highly tolerant to hβd-2 and other antimicrobial peptides (152); therefore, S. mitis stimulation of hβd-2 produced by host cells is a mechanism by which S. mitis can persist in the oral cavity while modulating the oral polymicrobial community, including the inhibition of oral pathogens.

MODULATION OF PATHOGEN-INDUCED INFLAMMATION

Oral streptococci have also been shown to modulate inflammation induced during pathogenic infection. S. salivarius colonizing human bronchial epithelial cells and primary keratinocytes inhibits IL-8 production that is induced by the respiratory pathogen P. aeruginosa (146). S. salivarius strains K12 and M18 downregulate the proinflammatory cytokines IL-6 and IL-8 in gingival fibroblasts that have been challenged with oral pathogens, including P. gingivalis, A. actinomycetemcomitans, and F. nucleatum (147). While the anti-inflammatory factor produced by S. salivarius that downregulates IL-6 and IL-8 was not identified, it was demonstrated to be a small molecule of <10 kDa that is heat stable and proteinaceous (147). Another oral commensal, Streptococcus cristatus, was shown to downregulate IL-8 production induced by F. nucleatum in an oral epithelial cell infection model via the inhibition of NF-κB transcription (153, 154). F. nucleatum induced the degradation of the NF-κB-inhibitory protein IκB-α, while the presence of S. cristatus during F. nucleatum infection stabilized IκB-α (154). S. gordonii was shown to significantly impact mitogen-activated protein kinase (MAPK) and Toll-like receptor pathway genes, which are involved in host cell proliferation as well as cytokine expression. Additionally, S. gordonii was shown to downregulate gingival epithelial cell IL-6 and IL-8 secretion induced by the oral pathogens F. nucleatum, P. gingivalis, and A. actinomycetemcomitans (104). Conversely, S. sanguinis was shown to induce IL-6 and IL-8 expression in oral keratinocytes and primary periodontal ligament cells despite being a health-associated commensal (58). In a trispecies biofilm model with P. gingivalis and the commensal species Corynebacterium durum in the same study, the cytokine expression profiles resembled that of single-species S. sanguinis-colonized cells, demonstrating the ability of oral streptococci to govern the host response to polymicrobial communities (58). Finally, S. salivarius was shown to alleviate radiation-induced oral mucositis in a mouse model by preventing the overgrowth of oral anaerobes commonly observed in oral mucositis (155).

THE PATHOBIONT STATUS OF ORAL STREPTOCOCCI

Oral streptococci tend to behave as commensals at steady state, producing mediators such as hydrogen peroxide and bacteriocins that can antagonize other bacteria and prevent colonization. However, in the presence of pathogenic bacteria that have successfully colonized, oral streptococci can act as accessory pathogens and develop associations with these pathogens. For example, S. gordonii binds to and is commonly coisolated with P. gingivalis and has been shown to increase bone loss when present with P. gingivalis (63). Furthermore, outside the oral cavity, oral streptococci can initiate inflammatory immune responses in the bloodstream and the heart. In rare cases, S. mitis, S. oralis, S. gordonii, S. parasanguinis, and S. sanguinis can cause infectious bacteremia and endocarditis (156, 157). This entry into the bloodstream and the heart is typically associated with poor dental health, i.e., periodontitis, which provides a leaky, inflammatory environment that promotes translocation into the bloodstream (158, 159). Overall, it is important to note that while oral commensal streptococci are generally associated with health, there are unique instances where these microbes influence or potentiate the virulence of pathogens or directly cause disease themselves.

CONCLUSION

Commensal streptococci comprise the vast majority of the oral polymicrobial community and therefore play a complex but important role in oral health. In this review, we discuss mechanisms by which commensal streptococci modulate the homeostasis of polymicrobial communities within the oral cavity. Some commensal streptococci are thought to be accessory periodontal pathogens by interacting with and forming robust biofilms with these pathogens. However, commensal streptococci also employ multiple mechanisms to inhibit pathogen growth and maintain oral health. Many streptococcal species produce hydrogen peroxide, which can directly kill a wide variety of oral and nonoral pathogens. Additionally, several oral commensals can reduce dietary nitrate in the oral cavity down to nitric oxide, an RNS that has antimicrobial properties and has been shown to inhibit the growth of specific periodontal pathogens. Nitrite, another nitrate reduction intermediate, can react with hydrogen peroxide produced by commensal streptococci to form the RNS peroxynitrite, which has been shown to kill the oral pathogens S. mutans and A. actinomycetemcomitans. Certain streptococcal species, such as A12, S. salivarius, S. gordonii, and S. dentisani, can counteract the acidification caused by S. mutans that leads to the progression of dental caries via the conversion of arginine to ammonia, thereby raising the oral cavity pH. S. salivarius produces a variety of bacteriocins that have the potential to influence the microbial makeup of the oral cavity and have been shown to effectively inhibit S. pyogenes growth in the oropharynx. Finally, S. salivarius, S. gordonii, S. mitis, and S. oralis downregulate inflammatory cytokines that have been shown to contribute to bone loss and periodontal disease progression during infection with periodontal pathogens. In summation, oral commensal streptococci play dynamic roles in the maintenance of oral health and homeostasis by structuring the composition of the oral microbiota through microbial competition and the regulation of the host immune response.